WO2014132100A1 - Mobilizing agents and uses therefor - Google Patents

Mobilizing agents and uses therefor Download PDF

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Publication number
WO2014132100A1
WO2014132100A1 PCT/IB2013/003118 IB2013003118W WO2014132100A1 WO 2014132100 A1 WO2014132100 A1 WO 2014132100A1 IB 2013003118 W IB2013003118 W IB 2013003118W WO 2014132100 A1 WO2014132100 A1 WO 2014132100A1
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Prior art keywords
hydroxy
acid
hif
amino
mobilizer
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PCT/IB2013/003118
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French (fr)
Inventor
Jean-Pierre Jose Rene LEVESQUE
Ingrid Gabriele WINKLER
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Mater Medical Research Institute Limited
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Application filed by Mater Medical Research Institute Limited filed Critical Mater Medical Research Institute Limited
Priority to CN201380076305.4A priority Critical patent/CN105377251A/en
Priority to US14/772,022 priority patent/US20160015786A1/en
Priority to EP13876687.8A priority patent/EP2961398A4/en
Publication of WO2014132100A1 publication Critical patent/WO2014132100A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4418—Non condensed pyridines; Hydrogenated derivatives thereof having a carbocyclic group directly attached to the heterocyclic ring, e.g. cyproheptadine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Definitions

  • This invention relates general iy to the use of a HIF-a potentiating agent and a mobiiizer of hematopoietic stem ceils and/or progenitor cells in methods and compositions for mobilizing hematopoietic, stem ceils and progenitor cells from the bone marro into the peripheral blood.
  • the present invention is particular relevant for stem ceil transplantation as well as treating or preventing immune deficiencies.
  • HSC hematopoietic stem eel!
  • CD34 + HSPCs are robustly mobilized after 4-5 days of G-CSF treatment and blood aphaeresis from day 5 is sufficient to reach the minimum threshold of 2 x 10 6 CD34 ⁇ cells / kg body weight to ensure rapid reconstitution.
  • up to 30-60% of chemotherapy-treated patients fail to reach this minimal threshold in response to G-CSF, precluding transplantation (1), Most at-risk are patients who have undergone chemotherapy with purine analogs sueh as fiudarabine for more than three chemotherapy cycles (2).
  • the present invention is related in part to the discovery that mobilization of
  • I S FiSPCs by mobilizing agents also referred to herein as “niobHizers” or “mobiiizer of
  • hematopoietic stem cells and/or progenitor cells such as G-CSF or Pierixafor, or
  • hypoxia-inducible factor a also referred to herein as a "HIF-a potentiating agent”
  • stem cell mobili ers alone, Concurrent administration of a HIF-a potentiating agent and a mobiiizer of hematopoietic stem cells and/or progenitor cells is useful in compositions and methods for stimulating or enhancing mobilization of
  • hematopoiesis j or for stem cell transplantation, as described hereafter.
  • compositions that comprise, consist or consist essentially of a HIF-a potentiating agent and at least one mobiiizer of hematopoietic stem cells and/or progenitor cells.
  • compositions that comprise, consist or consist essentially of a HIF-a potentiating agent and at least one mobiiizer of hematopoietic stem cells and/or progenitor cells.
  • composition thai comprises a HIF-a potentiating agent for use in
  • the HIF-a potentiating agent is selected, without limitation, from agents that stabilize HIF-a, agents that stimulate or enhance expression of HIF-a HIF-a polypeptide or coding sequences, and combinations thereof.
  • the HlF-a potentiating agent inhibits the activity of a HiF hydroxylase, e.g., a HIF prolyl hydroxylase (PHD) (e.g., PHDL PHD2 and/or PHD3).
  • PHD inhibitors is a selective inhibitor of a HIF-3 ⁇ 4 PHD.
  • the PHD inhibitor is an inhibitor of two or more PHD enzymes.
  • the at least one mobilizer is selected from a growth factor, a cytokine, a chemokine or a polysaccharide.
  • the at least one mobilizer is characterized by its abilit to decrease or block the expression, synthesis or function of CXCL12 or is characterized by its ability to block or antagonize CXCR4.
  • the mobilizer is selected from a colony stimulating factor such as G-C5F or a variant, derivative or analog thereof, a CXC 4 antagonist such as Plerixafor, or a combination thereof.
  • the compositions further comprise a pharmaceutically acceptable carrier.
  • compositions of the present invention are useful for stimulating or enhancing mobilization of hematopoietic stem cells and/or progenitor cells, or for stimulating or enhancing hernatopoiesis, or for stem cell transplantation.
  • the present invention provides a use of a HlF-a potentiating agent for stimulatin or enhancing mobilization of hematopoietic stem cells and/or progenitor cells, or for stimulating or enhancing hematopoiesis. or for stem cell transplantation, or for treating or preventing an immunocompromised condition (e.g.,. neutropenia,, agranulocytosis ⁇
  • an immunocompromised condition e.g. neutropenia,, agranulocytosis
  • the use is in subjects that have been, are, or will be administered at least one mobilizer of hematopoietic stem cells and or progenitor cells.
  • the HlF-a potentiating agent and the at least one mobilizer are prepared or manufactured as medicaments for those applications.
  • Another aspect of the present invention provides method for enhancing a hematopoietic function of a mobilizer of hematopoietic stem cells and/or progenitor cells in a subject.
  • These methods generally comprise, consist or consist essentially of administering to the subject a HlF ⁇ a potentiating agent in an effective amount tp enhance an hematopoietic function of the mobilizer ( .g., increasing the number of hematopoietic stem cells and/or progenitor celis including granulocytes/macrophage progenitors and/or
  • Yet another aspect of the present invention provides methods for mobilizing hematopoietic stem ceils and/or progenitor cells from bone marrow into peripheral blood of a donor subject These methods generally comprise, consist or consist essentially of:
  • HIF-a potentiating agent in an effective amount to mobilize hematopoietic stem cells and/or progenitor cells from the bone marrow into the peripheral blood of the subject.
  • the donor subjects in these embodiments are selected from subjects tha have been, are, or will be administered at least one mobilizer of hematopoietic stem cells and/or progenitor cells.
  • a related method generally comprises, consists or consists essentially of: administering concurrently to the donor subject a HlF-a potentiating agent and at least one mobilizer of hematopoietic stem cells and/or progenitor cells in effective amounts to mobilize hematopoietic stem cells and/or progenitor cells from the bone marrow into the peripheral blood of the subject.
  • a HlF-a potentiating agent is administered concurrently to the donor subject.
  • at least one mobilizer of hematopoietic stem cells and/or progenitor cells in effective amounts to mobilize hematopoietic stem cells and/or progenitor cells from the bone marrow into the peripheral blood of the subject.
  • the HIF-ot potentiating agent is
  • the ⁇ - ⁇ potentiating agent and the at least one mobilizer are administered in synergisticaliy effective amounts.
  • the subject has an immunocompromised condition or is at risk or acquiring an
  • the subject has a hyperproHferative cell disorder e.g., cancer, which can be a primary cancer or a metastatic cancer, or an autoimmune disease), and has been, is or will be subjected to a encompassai treatment in specific embodiments, the hyperproHferaiive cell disorder is cancer (e.g., leukemia, multiple myeloma, lymphoma, etc.).
  • the encompassai treatment targets rapidly dividing cells or disrupts the cell cycle or cell division (e.g.,. chemotherapy and/or radiation therapy).
  • the immunocompromised condition is selected from neutropenia, agranulocytosis, thrombocytopenia, and anemia.
  • the methods further comprise collecting or
  • the methods further comprise cultur ing and/or storing the collected or harvested mobilized hematopoietic stem cells and/or progenitor cells (e.g., to maintain or expand the collected or harvested mobilized hematopoietic stem cells and/or progenitor ceils).
  • the methods furthe -comprise infusing or transplanting the collected or harvested mobilized hematopoietic stem cells and/or progenitor cells, which have been optionally cultured or stored, into a recipient subject.
  • the donor subject and the recipient subject may be the same subject or may be different subjects, in some embodiments, the subject is both donor and a recipient" of the collected, or harvested mobilized hematopoietic stem cells and/or progenitor cells, which have been optionally cultured or stored, and is suitably in need of a stem cell transplantation.
  • the stem cell transplantation in these embodiments is autologous with respect to the recipient
  • the subject has an immunocompromised condition or has been exposed to a medical treatment that results in an immunocompromised condition.
  • the methods further comprise infusing or
  • the. donor subject and the recipient subject are different subjects.
  • the subject from which the mobilized hematopoietic stem ceils and/or progenitor cells are collected or harvested is a donor and the other subject is a recipient that is suitably in need of a stem cell transplantation.
  • the stem cell transplantation in these embodiments is allogeneic or xenogeneic with respect to the recipient
  • the other (recipient) subject has an immunocompromised condition or has been exposed to a medical treatment that results in an immunocompromised condition.
  • the methods further comprise administering to the recipient prior to. simultaneously with, or after the stem ceil transplantation a HIF-a potentiating agent and a mobilizer of ' hematopoietic stem cells and/or progenitor ceils in effective amounts to mobilize hematopoietic stem cells ' and or progenitor cells from the bone marrow into the peripheral blood of the subjec
  • HIF- ⁇ potentiating agents are useful for enhancing a hematopoietic function (e.g. , increasing the number of hematopoietic stem cells and or progenitor cells, including ⁇ granulocytes/macrophage progenitors and/or niegakaryocyte/eiytlmwjyte progenitors in the peripheral blood) of at least one mobilizer of hematopoietic stem cells and/or progenitor celis.
  • the HIF-a potentiating agent may be known, or identified using any suitable screening assay.
  • the present invention provides screening methods for identifying agents that are useful for enhancing a hematopoietic function of the mobilizer. These methods generally comprise testing whether a test agent potentiates HIF ⁇ a increasing the accumulation of, orstability of, HIF-a; directly provide HIF- activity; or increase expression of FRF- 1) and determining whether the test agent stimulates or enhances mobilization of hematopoietic stem cells and/or progenitor cells on the basis that it tests positive for the potentiation,
  • the present invention provides methods of producing an agent that enhances a hematopoietic function of at least one mobil izer of hematopoietic stem celis and/o progenitor cells. These methods generally comprise:
  • the method further comprises derivatizing the agent, and optionally formulating the derivatized agent with a pharmaceutically acceptable carrier, to improve the efficacy of the agent for enhancing the hematopoietic function of the mobilizer.
  • the mohiiizerfs) and the HIF-a potentiating agent are suitably administered in the form of one or more compositions each comprising a pharmaceutically acceptable carrier.
  • the compos ition(s) may be administered by injection, by topical application or by the oral rente including sustained-release modes of .administration, over a period of time and in amounts which are effecti ve for increasing the number of hematopoietic stem cells and/or progenitor cells including granulocytes/macrophage progenitors and or
  • the mobi!ker(s) and the HiF ⁇ a potentiating agent are administered simultaneously to the subject
  • the HIF-a potentiating agent is adminis tered to the subject prior to administration of the mobilizer.
  • the HIF-a potentiating agen t is administered after administration of the mobilizer to the subject.
  • the methods are useful for treating or preventing an immunocompromised condition in a subject (e.g.., a condition resulting from exposure of the subject to a medical treatment).
  • the mobilizer(s) and the HIF-s potentiating agent are concurrently administered i amounts effective for treatment or prevention of the immunocompromised condition (e.g., neutropenia, agranulocytosis, thrombocytopenia, or anemia).
  • the methods further comprise identifying a subject having or at risk of acquiring the immunocompromised condition.
  • the HIF-a potentiating agent and the mobilizer(s) may be administered to the subject simultaneously, .sequentially or separately with the medical treatment, in some embodiments, the concurrent administration of the HIF-a potentiating agent and the mobilizer(s) is a prophylactic treatment (e.g., the subject is preparing t undergo chemotherapy or radiation ⁇ treatment). In- others, it is a therapeutic treatment (e.g., the subject has received at least one dose of chemotherapy or at least one radiation treatment),
  • the methods may further comprise exposing ibe subject to an ancillary treatment that treats or prevents an immunocompromised condition.
  • the immunocompromised condition is anemia and the ancillary treatment may comprise administering to the subject an anemia.
  • medicament selected from recombinant erythropoietin (EPQ), ferrous iron, ferric iron, vitamin B 12, vitamin B6, vitamin C, vitamin D, calcHrioi, aipbacaicidol, folate, androgen, and carnitine.
  • the immunocompromised condition is thrombocytopenia and the ancillary treatment may comprise administering to the subject a thrombocytopenia medicament selected from a glucocorticoid- recombinant thrombopoietin (TPO), recombinant megakaryocyte growth and development factor (MGDF), PEGylated recombinant MGDF and ItsophylUne.
  • TPO glucocorticoid- recombinant thrombopoietin
  • MGDF recombinant megakaryocyte growth and development factor
  • ItsophylUne ItsophylUne
  • the immunocompromised condition is neutropenia and the ancillary treatment suitably comprises administering to the subjeet a neutropenia medicament selected from glucocorticoid, immunoglobulin, androgens, recombinant IFN-y, and uteroferrin.
  • the ancillary treatment is administered to the subjeet simultaneously, sequentially or separately with the HIF-a potentiating agent and/or the mobilizer ⁇ s).
  • the medical treatment is likely to expose the subject to a higher risk of infection.
  • the methods may further comprise administering simultaneously, sequentially or separately with the HIF-a potentiating agent and/or me mobilizer(s) at least one anti-infective agent that is effective against an infection that develops or that has an increased risk of developing from the immunocompromised condition, wherein the anti-infective agent is selected from antimicrobials, antibiotics, arrtivirals, antifungals, anthelmintics, antiprotozoals and nematocides.
  • one or both of the HIF-a potentiating agent and the at least one mobilizer are administered on a routine schedule, or example, every day, at least twice week, at least three times a week, at least four times a . week, at least five times a week, at least six times a week, every week, every other week, every third week, every fourth week, every month, every two months, every three months, every four months, and eye* ⁇ ' sis months.
  • the concurrent administration of a ⁇ - ⁇ potentiating agent and at least one mobilizer of hematopoietic stem cells and/or progenitor ceils is useful for treating or preventing hematopoietic disorders such as neutropenia, agranulocytosis, thrombocytopenia, and anemia, which may result, for example, from myeiosuppressive, myeloablative or cytoredtsctive treatments that target rapidly dividing cells or that disrupt the cell cycle or cell division (e.g., chemotherapy or radiation therapy).
  • the present invention provides methods for treating a hyperproliferative cel l disorder (e.g., a cancer or a uto immune disorder) in a subject.
  • a hyperproliferative cel l disorder e.g., a cancer or a uto immune disorder
  • these methods generally comprise administering concurrently to the subject a medical treatment (e.g. * a chemotherapeutic agent or radiation) for the disorder, which targets rapidly dividing cells or disrupts the cell cycle or cell division, together with at least one mobiiizer of hematopoietic stem cells and-'or progenitor cells and a HIF- potentiating agent in amounts effective for stimulating or enhancing mobilization of hematopoietic stem cells and/or progenitor cells, or ' for stimulating or enhancing hemalopoiesis.
  • a medical treatment e.g. * a chemotherapeutic agent or radiation
  • the preseM invention provides methods for increasing the dose in a subject of a medicament for treating a hypetproliferattve ceil disorder (e.g., cancer or an autoimmune, disease), wherein the medicament results or increases the risk of developin an immunocompromised condition.
  • a hypetproliferattve ceil disorder e.g., cancer or an autoimmune, disease
  • These methods comprise administering concurrently the medicament to the subject in a dose mat ordinarily induces side effects (e.g., the development of the immunocompromised condition), together with at least one mobiiizer of hematopoietic stem cells and or progenitor cells and a ' HIF-a potent iating agent i n amounts effecti ve for inhibiting or preventing the induction of those side effects (e.g., in amounts effective for increasing the number of hematopoietic stem cells and/or progenitor cells including gianu i oc te s macrophage progenitors and/or
  • the present invention provides pharmaeeuticai compositions for treating or preventing a hyperproliferative celt disorder (e.g., cancer or an autoimmune d isease) that is treatable or preventable by a medical treatment that targets rapidly dividing cells or thai disrupts the cell cycle or cell division (e.g., chemotherapy or radiation therapy).
  • a hyperproliferative celt disorder e.g., cancer or an autoimmune d isease
  • a medical treatment that targets rapidly dividing cells or thai disrupts the cell cycle or cell division (e.g., chemotherapy or radiation therapy).
  • compositions generally comprise, cons ist or consist essentially of a HIF-a potentiating agent, at least one mobiiizer of hematopoietic stent ceils arid/or progenitor cells and at least one other agent selected from a .chemotherapeutic agent (e.g. ⁇ a cytotoxic agent), a radiosensitrang agent, an anemia medicament, a thrombocytopenia medicament, a neutropenia medicament, a agranulocytosis medicament and an anti- infective agent, and optionall a pharmaceutically acceptable carrier.
  • a chemotherapeutic agent e.g. ⁇ a cytotoxic agent
  • a radiosensitrang agent e.g. ⁇ a cytotoxic agent
  • anemia medicament e.g. ⁇ a cytotoxic agent
  • thrombocytopenia medicament e.g. ⁇ a cytotoxic agent
  • neutropenia medicament e.g. ⁇ a thrombocytopenia medicament
  • FIGS !A, IB, IC, and ID are graphical representations showing the effect of Compound X and G-CSF on HiF-ta protein and CFC mobilization.
  • I A Western blot analysis of bone marrow (BM) cell y sates from mice treated with saline (Saline), with Compound X for 3 days (X3) ⁇ with G-CSF for 2 days (G2) or with both Compound X and G- CSF (G2X3) for presence of HIF-la and ⁇ -actin. Each lane represents a different mouse.
  • mice were treated with Plerixafor for 1 hour (PI ) together with Compound X for 1, 2, 3 or 4 days (P1X1- P1X4). Data are mean ⁇ SD of 6 mice per condition. * p ⁇ Q.05; ** p ⁇ 0.01 *** p ⁇ 0.001.
  • FIG. 2A, 2B, 2C, and 2D present graphical representations showing synergistic effect of Compound X with G-CSF,of in combination with G-CSF and Plerixafor.
  • 2A Treatment groups used.
  • (2B) Graphs shovv the number of CFC per ml blood and per spleen of treated mice, GCSF only (filled circles); GCSF and Compound X (open squares); GCSF and Plerixafor (filled triangles); and the combination of GCSF, Plerixafor and Compound X (filled diamonds),
  • FIGs 3 A and 3B are graphical representations showing that Compound X synergizes with G-CSF and Plerixafor to enhance mobilization of competitive repopu!ating HSCs.
  • GD45,2 + mice were mobilized with G-CSF for 2 or 4 days with G-CSF alone (filled circles), with G-CSF in combination with Plerixafor for 1 hr (filled triangles), with
  • Figures 4A, 4B, 4C, and 4D are graphical representations showing that deletion of Hifla gene in HSPCs compromises HSPC mobilization in response to- -G-CSF. CreER was activated in mutant mice in which both Hifla alleles are floxed to delete the Hifl genes and the mice were mobilized for 3 days with G-CSF.
  • Figures 5 A, 5B, and 5C are graphical representations showing that deletion of Hifla gene in osteoprogenitors delays HSPC mobilization in response to G-CSF,
  • SB Number of CFCs and Lin Seal + Kit + CD48 ' HSCs, Lin Scal + it + HSPCs per femur
  • R20R YmYFP OsxCreER mice (HIF la wt open circles, HIF 1 a fl/fL solid circles).
  • 5C Number of CFCs mobilized in blood and spleen in Hi la m R26R yFP/YFP OsxCreER mice following 2-4 days treatment with G-CSF (P1IF la wt, open circles, tUF l a fi/fl, solid circles).
  • Figures 6A, and 6B are graphical representations demonstrating the effect of treatment with Compound A, Compound B, or Compound C in combination with G-CSF on the number and phenotypic distri ution of hematopoietic stetn cells and progenitor cells in the bone marrow. Treatment with G-CSF and vehicle control served as control. (6A)
  • LPS- Myeloid progenitors
  • LMS+ hematopoietic stem and progenitor cells
  • Figures 7A. and 7B ate graphical representations demonstrating the -effect of treatment with Compound A, Compound 6 or Compound C in combination with G-CSF on mobilization to the blood of hematopoietic stem ceils and progen itor cells.
  • Figures 8 A and 8B are graphical representations demonstrating the effect of treatment with Compound A, Compound B or Compound C in combination with G-CSF on mobilization to the spleen of hematopoietic stem cells and progenitor ceils.
  • Treatment with G-CSF and vehicle control served as control (8A) phenotypic myeloid progenitors (LKS-)- Top Pane hematopoietic stem and progenitor cells (L S+)- Middle Panel, and L S+
  • Figures 9A. and 9B are graphical representations demonstrating the effect of treatment with Compound A, Compound B or Compound C in combination with G-CSF on total mobilization per moose (blood and spleen) .of hematopoietic stem cells and progenitor cells.
  • Figure 10 is a graphical representation demonstrating the effect of treatment with Compound A.
  • G-CSF and vehicle control served as control, * p 0,05; . ** p ⁇ 0.01 ; ***p ⁇ G.QQl.
  • Figure 1 1 is a graphical representation demonstrating the effect of treatment with Compound A, Compound B or Compound C in combination with G-CSF on white bloodtiti l (WBC) number per ml of blood- Top Panel, and spleen weight- Bottom Panel Treatment with O-CSP and vehicle control served as control * p ⁇ Q,05; ** p ⁇ 0,01;
  • Figures 12A and 12 B are graphical representations dem nstrating the effect of treatment with Compound D, Compound E or Compound F in combination with G-CSF on total mobilization to blood and spleen of hematopoietic stem cells and progenitor cells.
  • Figure 13 is a graphical representation demonstrating the effect of
  • FIGS I4A and 14B are graphical representations demonstrating the effect of treatment with Compound H, Compound J or Compound in combinatio with G-CSF on the total mobilization to blood and spleen of hematopoietic stem cells and progenitor cells.
  • Treatment with G-CSF and vehicle control served as control.
  • LKS+ phenotypic myeloid progenitors
  • LKS+ hematopoietic stem and progenitor ceils
  • Figure 15 is a graphical representation demonstrating the effect of treatment with Compound H, Compound I or Compound K in combination with G-CSF on colony forming unit (CFU) mobilization to the blood- To Panel spleen- Middle Panel, and combined total (blood and spleen) ⁇ Bottom Panel * p ⁇ 0.05; ** p ⁇ 0.01; ***p ⁇ 0.OGl
  • a mobilizer of hematopoietic stem cells and/or progenitor cells means one mobilizer of hematopoietic stem ceils and/or progenitor cells or more than one mobilizer of hematopoietic stem cells and/or progenitor ceils.
  • administered concurrently or “administering concurrently” or “eo-admintstering” and the like refer to the administration of a single composition containing two or more active agents, or the administration of each active agent as separat
  • a HI -ec potentiating agent may be administered together with a mobilizer of hematopoietic stem cells and/or progenito cells in order to increase the numbers of hematopoietic stem cells, progenitor ceils and/or differentiated cells thereof in peripheral bipod.
  • a HIF-a potentiating agent and a mobilizer of hematopoietic ster ce l ls and/or progenitor cells are administered together with another agent to enhance their effects or to ameliorate the effects of a medical treatment that gives rise or contributes to an
  • a HIF-a potentiating agent is administered at a later point in time than a. mobilizer of hematopoietic stem cells and/or progenitor cells but within the time period during which the mobilizer of hematopoietic, stem ceils and/or progenitor cells is still exerting an effec
  • sequential administration is meant a time difference of f om seconds, minutes, hours or days between the administration of the two types of molecules or active agents. These molecules or active agents may he administered in any order.
  • Simultaneously is meant that the active agents ar administered at substantially the same time, and desirably together in the same formulation.
  • the active agents are administered closely in time, e.g. , one agent is administered within from about one minute to within about one day before or after another. Any contemporaneous time is useful. However, i ⁇ will often be the case that when not administered simultaneously, the agents will he administered within about one minute to with in about eight hours and preferably within less than about one to about four hours.
  • the HiF-a potentiating agent and the mohilizer are administered within about 60 minutes, about 50 minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes, about 5 minutes, or about 1 minute of each, other or separated in time by about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, or about 72 hours, Or more.
  • the agents may suitably be administered at the same site on the subject.
  • the term "same site" includes the exact location, but can be within about 0.5 to about 15 centimeters, usually from within about 0.5 to about 5 centimeters.
  • the term “same site” includes the exact location, but can be within about 0.5 to about 15 centimeters, usually from within about 0.5 to about 5 centimeters.
  • “separately” as used herein means that the agents are administered at an interval, for example at an interval of about a day to several weeks or months.
  • the active agents may be administered in either order.
  • the term “sequentially” as used herein means that the agents ar admin istered i n sequenc e, for example at an in terval or intervals of minutes, hours, days o weeks. If appropriate the active agents may be administered in a regular repeating cycle.
  • agent includes a compound, composition, or molecule that induces a desired pharmacological and/or physiological effect.
  • the term also encompasses pharmaceutically acceptable and pharmacologically active ingredients of those compounds specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like.
  • salts esters, amides, prodrugs, active metabolites, analogs and the like.
  • agent is not to be construed narrowly but extends to small molecules, proteiiiaeeous molecules such as peptides, polypeptides and proteins as well as compositions comprising them and genetic molecules such as RNA, DNA and mimetics and chemical analogs. thereof as .well -as cellular agents.
  • agent includes a cell which is capable of producing and secreting polypeptides referred to herein as well as a polynucleotide comprising a nucleotide sequence that encodes this polypeptide-.
  • the term “agent” extends to nucleic acid constructs including vectors such as viral or non-viral vectors, expression vectors and piasniids for expression in and secretion in a range of ceils.
  • Preferred acyl includes C(0 ⁇ -R, wherein R is hydrogen or an alkyl, alkenyl alkynyl, aryl, cyeioalkyl, heterocyclyL arylalkyl,
  • acyl include formyi; straight chain or branched alkanoyl such as, acetyl, propanoyi, biitanoyl, 2-rnethylproparioyI, pentanoyl, 2 3 ⁇ 4 2-d1 ⁇ 2ethyipropanoyL hexanoy , heptanoyl, octanoyL nonanoyl- decanoyl, undecanoyl, dodeeanoyl, tridecanoyl, tetradecanoyl,
  • peniadecanoyi hexadecaaoyL heptadeeanoy ' i, oetadeeanoyl, nonadeeanoyl and ieosarsay!; pheny!carbonyl; eyeioalkyfcarbonyl such as eyclopropylmethyl(or etliyi)carboiiyl cyclobutylmethyi(or etxtyl)carbonyL cyclopentylraeihyl(or ethyl)carbonyl and
  • cyclohexyi ethyl or e ⁇ hyl)earbonyi; aikanoyl such as phenySaikanoyl (e.g., phenylaeefyl, ?. ⁇ ?,, benzoyl, plien lpropanoyl, pheny !butanoyl, phenylpentanoyL phenylhexanoyl) and naphthylalfcanoyl (e.g., naphthylacetyL naphtihyipropanoyl and naphthytbytanoyl), and phenylalkenoyl (e.g., pheny lhex-4-en-oyl, phenylhex-3-en-oy.l, phenylheptanoyl, phenyihept- 4-en-oy 1, phen lhept ⁇ 3 -en-o i).
  • An "agranulocytosis medicament” as used herein refers to a composition of matter which reduces the symptoms related to agranulocytosis, prevents the development of agranulocytosis, or treats existing agranulocytosis.
  • alkenyl denotes groups formed from straight chain or branched hydrocarbon residues coniainmg at least one carbon to carbon double bond including ethylenieally mono-, di ⁇ or poly-unsaturated alkyl groups as defined herein, suitably Gj.io or Ci-s).
  • alkenyl include ⁇ vinyl, allyl, 1- met y!vinyl. butenyl, iso-botenyl, 3-meihyl-2-butsnyl, 1 -p nteny ' l, cyclopentenyl, 1-niethyl- cyclopentenyk 1-hexenyi.
  • alkenyl group may be optionally substituted by one or more optional sobstituents as herein defined. Accordingly, "alkenyl" as used herein is taken to refer to optionally substituted alkenyl.
  • alkyl when used alone or in words such as “arylalkyl " “heterocyclylalkyl” and “cyeloalky lalfcyl,” denotes straight chain or branched hydrocarbon residues;, suitabl Ci-ao ' .alkyl, e.g. , Cvio or Cj .6.
  • straight chain and branched alkyl include methyl ethyl, propyl,, isopropyl, butyl, isobutyL sec-butyl ten-butyl amyL isoamyl see-amy!.
  • alkynyl denotes groups formed from straight chain or branched hydrocarbon residues containing at least one carbon to carbon triple bond including ethynyically mono-, di- or poiy-unsatufated alkyl or cycloalkyl groups as defined herein, or Cj. f i); Examples, include ethynyL propynyi, butynyl, pentynyl.
  • An alkynyl grou may be optionally substituted by one or more optional substituents as herein defined. Accordingly, ⁇ alkytiyl" as used herein is taken to refer to optionall substituted aSkyny l,
  • arylalkyP and aryloxy denotes single, -polynuclear, conjugated or fused residues of aromatic hydrocarbons. Examples o aryl included phenyl, bi phenyl and naphthyl. In specific embodiments, aryi groups include phenyl and naphthyl An aryl. roup may be opiionaily substituted by one or more optional substituents as herein defined. Accordingly, "aryi” as used herein is taken to refer to aryl that may be opiionaily substituted, such as optionally substituted phenyl and optionally substituted naphthyl.
  • arylaikyl refers to an alkyl group substituted (suitably terminally) by an aryl, cycloalkyl or heterocyclyl group, respectively.
  • anemia medicament refers to a composition of matter which reduces the symptoms related to anemia, prevents the development of anemia, or treats existing anemia.
  • antigen-binding molecule is meant a molecule that has binding affinity for a target antigen, it will b understood that this term extends to immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity.
  • Antigenic or immunogenic activity refers to the ability of a polypeptide, fragment, variant or derivative according to the invention to produce an antigenic or immunogenic response in an animal, suitably a mammal, to which it is administered, wherein the response includes the production of elements which specifically bind the polypeptide or fragment thereof,
  • bacteria or "bacterial infection " ' includes any bacterial pathogen including emerging bacteria! pathogen of vertebrates.
  • Representative bacterial pathogens include without limitation species of: Aciwtobaefer, Actinobacillus,
  • Salmonella Serraiia, Shigella, Staphylococcus, Stenotrophomonas, Streptococcus,
  • Treponema Vibrio (cholera) and Yersinia (plague).
  • coding sequence is meant an nucleic acid sequence that contributes to the code for the poly peptide product of a gene.
  • the terra “non-codin sequence'' refers to any nucleic acid sequence that does not contribute to the code fo the polypeptide product of a gene
  • colony stimulating factor refers to a secreted glycoprotein that binds to receptor proteins on the su face of hematopoietic cells acti vating intracellular signallin pathways that cause the cells to proliferate and differentiate into different types of blood ceils.
  • CSF-1 macrophage colony stimulating factor
  • CSF-2 granulocyte macrophage colony stimulating factors; GM-CSF; sargramostim
  • arid C.SF-3 granulocyte colony stimulating factors; G-CSF; filgrastim
  • promegapoietio are examples of colony stimulating factors.
  • a nucleic acid sequence that displays substantial sequence identit to a reference nucleic acid sequence (e.g., at least about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 6.1, 62, 63, 64, 65, 66, 67, 68, 6% 70, 71., 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 » 84, 85, 86, 97, 88, 89, 90, 1, 2, 93, 94, 95, 6, 7, 98, 99% or e ven up to 100% sequence identity to all or a portion of the refefence nucleic acid sequence) or an amino acid sequence that displays substantial sequence similarity or identity, to a reference amino acid sequence (e.g., at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
  • cycloa!koxy refers to cyclic hydrocarbon residues, including mono- or polycyclic alkyl groups. Exemplary cycloaikyl are C4.7 a!kyL A "cyeloalkyi” group may contain one or more double or triple bonds to form a cyc!oalkenyl or cycloaikynyl group and accordingly, “eyeloalkyP also refers to non-aromatic unsaturated as well as saturated cyclic hydrocarbon residues.
  • cycloaikyl examples include cyc!opropyl, cyc!obuiyl, eyclopeniyl, cyclohexyi, cycioheptyi, cyelooetyL cyciononyl, cyclodecyi, 1,.3-cyclo.hexadienyl, 1 yl-eyelohexadieny!, l s 3 ⁇ eycloheptadieny1, ] ,3,5-eycioheptatrienyl and 1 ,3,5,7-cyclooctatetraenyl.
  • a eycloalkyl group may be optionally substituted ' by one or more optional substituents as herein defined. Accordingly ; , "cycioalkyl” as used herein is taken to refer to optionally substituted cycloalkyl.
  • the term "derivatize,” “derivatizing” and the like refer to producing or obtaining a cpmppimd from another substance b chemical eac ion e.g., by adding one or mor reactive groups to the compound by reacting the compound with a functional group- adding reagent, etc.
  • derivative in the context of polypeptide derivatives, refers to a polypeptide that has been derived from the basic seqiience by modification, for example by conjugatio or complexing with other chemical moieties, or by post-transiational modification techniques as would be understood in the art.
  • derivative 5' also includes within its scope alterations that have been made to a parent sequence including additions or deletions that provide for functional equivalent molecules,
  • the term "differentiation" of hematopoietic stem ceils and/or hematopoietic progenitors as used herein refers to both the change of hematopoietic stem ce lls into hematopoietic progenitors and the change of hematopoietic progenitors into umpotent hematopoietic progenitors and or cells having characteristic functions, namely mature cells including erythrocytes, leukocytes (e.g. , neutrophils) and megakaryocytes.
  • Differentiatio of hematopoietic stem, ceils i to a variety of blood eel! types involves sequential activation or silencing of several sets of genes.
  • Hematopoietic stem cells typically choos either a ly mpho i d or myelo id lineage pa thway at an early stage of differentiation.
  • an effective amount in the context of treating or preventing a condition is meant the administration, of an amount of an agent or composition to an. individual in need of such treatment or prophy laxis, either in a single dose or as part of a series, that is effective for the prevention of incurring a symptom, holding in check such symptoms, and/or treating existing symptoms, of that condition.
  • the effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomie grou of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range thai can be determined through routine trials.
  • expression refers to transcription, of the gene and, as appropriate, translation of th resulting mKNA transcript to a protein.
  • expression of a coding sequence results from transcription and translation of the coding sequence.
  • expression of a non- coding sequence results from the transection of the non-coding sequence.
  • the term "function" refers to a biological, enzymatic, or therapeutic' function.
  • the term "gene” as used herein refers to any and all discrete coding regions of the cell's genome, as well as associated non-coding and regulatory regions.
  • the terra is intended to mean the open reading frame encodin specific polypeptides, introns. and adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression, in this regard, the gene may further comprise control signals such as promoters, enhancers, termination and or poiyadenylation signals that are naturally associated with a given gene, or heterologous control signals.
  • the DNA sequences may be cDNA or genomic KN A or a fragment thereof, The gene may be introduced into an appropriate vector for
  • group refers to a set of atoms mat forms a portion of a molecule.
  • a group can include two or more atoms that are bonded to one another to form a portion of a m olecule.
  • a group can be mo novalent or polyvalent (e.g. , bivalent) to allow bonding to one or more additional groups of a molecule.
  • a monovalent group can be envisioned as a molecule with one of its hydrogen atoms removed to allow bonding to another group of a molecule, A group can be positively or negatively charged.
  • a positivel charged group can be envisioned as a neutral group with one or more protons -(/.e., IT " ) added, and a negatively charged group can be envisioned as a neutral group with one or more protons removed.
  • groups include, but are not limited to, alkyl groups, alkylene groups, alkenyl groups, alkenylene groups, alkynyl groups, alkyny!ene groups, aryl groups, arylene groups, immyl groups, iminylene groups, hydride groups, halo groups, hydroxy groups, aikoxy groups, carbox groups, thio groups, alkylthio groups, disulfide groups, cyano groups, nitre groups, amino groups, alky lam ino groups, dialkylamino groups, siiyl groups,, and siloxy groups.
  • Groups such as alkyl alkenyl alkynyl. aryl, and heterocyclyl, whether used alone or in a compound word or in the definition of a group may be optionally substituted by one or more substitueuts.
  • 'Optionally substituted refers to a group may or may not be further substituted with one or more groups selected from alkyl, alkenyi, alkynyL aryl, halo, haloalkyl, halpalkenyl, haloa!kyny!, haloaryl, hydroxy, aikoxy, aikenyloxy, aryloxy, benzyloxy, haleaikoxy, haloalkenyloxy, haloary loxy, nitro, nitroalkyi, nitroalkenyk nitroaikynyl, .nitrparyl, nitroheteroeyclyl, amino.
  • aSkylamino diafkyianiino, alkeny!amino, alkynyfemiao, ary ⁇ amnio, diarylamino, phenylamiao, diphenyiamino, benzylamino, dibensykmino, hydrazmo, aeyl, acyiamino, diaeylamioo, acyloxy, heteroeyelyl,
  • heterocycioxy may also refer to the replacement of a Ci3 ⁇ 4 group with a carbonyi (OO) group.
  • Non-limiting examples of -optional, substituents include alkyl, preferably C f -a alkyl (e.g., butyl, cyclopropyi, cyclobutyl, cyeiopentyl or eydohexyi), hydroxy Gs.g alkyl (e.g., hydroxymethyl, hydro.xyeth I, hydroxypropyi), alkoxyalkyl (e.g., methoxymethyl, rneihox ethyl, methoxypropyi, ethoxy meth l, ethoxyethyL
  • phenyl which itself may be further- substituted, by an optional substituent as described herein, e.g., hydroxy, halo, methyl, ethyl propyl, butyl, methoxy, ethoxy, acetoxy, amino), benzyl (wherein the C3 ⁇ 4 and/or phenyl group ma be further substituted as described herein), phenoxy (wherem the CS3 ⁇ 4 and/or phenyl group may be further substituted as described herein), benzyloxy (wherein the CH?
  • phenyl group may be further substituted as described herein), amino, Cj.g alkylamino ( .g. ) C] ⁇ alkyl, such as methyiamino, ethylamino, propylamine), di alkylamino (e.g., G .
  • alkyl such as diniethyianino, diethylamino, dipropy!amino
  • acyiamino e.g., NHC(Q)CH 3 .
  • phenylamino wherein phenyl itself may he further substituted as described herein
  • nitro, formyj, -C(0)-Ci-ij alkyl e.g., alkyl such as acetyl
  • 0-C(G) ⁇ alkyl e.g. , Cw alkyl, such as acetyloxy
  • benzoyl whe ein the CH?
  • phenyl .group itself may be fiirther substituted), replacement of C3 ⁇ 4 with C ⁇ 0, C(3 ⁇ 4H, CG2 C].
  • alkyl e.g., Cs-g alkyl such as methyl ester, ethy! ester, propyl ester, butyl ester
  • CONH2 CONHphenyl (wherein phenyl itself may be further substituted as. described herein)
  • CONH C alkyl
  • C14 alkyl such as methyl amide, ethyl amide, propyl amide, butyl amide
  • CONHdi C 1-8 alkyl e.g , Chalky!
  • Hematopoiesis refers to the highly orchestrated process of blood cell development and homeostasis. Prenatally, hematopoiesis occurs in the yolk sack, then liver, and eventually the bone marrow. In normal adults it occurs in bone marrow and lymphatic tissues. Ail blood cells develop from pluripotent stem cells. Pluripotent cells differentiate into stem cells that are committed to three,, two or one hematopoietic differentiation pathway, Norte of these stem ceils are morphologically .distinguishable, however.
  • hematopoietic stem cells or “BSC” as used herein refers to muftipoient stem cells that are capable of differentiating into all blood ceils including erythrocytes, leukocytes and platelets.
  • BSC hematopoietic stem cells
  • tire term “hematopoietic stem cells” includes and encompasses those contained not only in bone marro but also in umbilical cord blood derived cells.
  • hematopoietic progenitors or “hematopoietic progenitor cells”, which are used interchangeabl with the term “hematopoietic precursors,” refers to those progenitor or precursor cells which are differentiated furthe 'than hematopoietic stem ceils but have yet to differentiate into progenitor.; or precursors of respective blood cell lineages (unipotent precursor cells).
  • progenitor cell( ⁇ p or "precursor ce!i ' (s) are defined as cells that . are lineage-committed, i.e., an individual cell can give rise to progen limited to a single lineage such as the myeloid or lymphoid lineage.
  • progenitor cells do not have self-renewal properties. They can also be stimulated by lineage-specific growth factors to proliferate, if activated to proliferate, progenitor cells have life-spans limited to 50-70 cell doublings before programmed cell senescence and death occurs.
  • hematopoietic progenitors include granulocyte/macrophage associated progenitors (colony- forming unit granulocyte, macrophage, CFU-GM), erythroid associated progenitors (burst- forming unit erythroid, BFU-E), megakan'ocyie associated progenitors (colony-forming unit megakaryocyte, CFU-Mk), and myeloid associated stem cells (col ny-fonning unit mixed, CFU-Mix).
  • granulocyte/macrophage associated progenitors colony- forming unit granulocyte, macrophage, CFU-GM
  • erythroid associated progenitors burst- forming unit erythroid, BFU-E
  • megakan'ocyie associated progenitors colony-forming unit megakaryocyte, CFU-Mk
  • myeloid associated stem cells col ny-fonning unit mixed, CFU-Mix
  • HSPC refers to both hematopoietic progenitor ceils and hematopoietic stem cells.
  • HIF-a potentiating agents include agents that increase the accumulation of, or stability of, HIF-a: directly provide HIF-a activity; or increase expression of HT.F-1. Such: agents are known in the art, or may be identified through art- recognized screening methods.
  • HIF ⁇ a potentiating agents include compounds that increase the accumulation .aad or stability of HiF-a by inhibiting the activity of one or more HIF hydroxylase enzymes, e.g., one or more HIF prolyl hydroxylase enzymes. Inhibitors of HIF hydroxylase enzyme activity are well known, readily identified, and are further described herein.
  • Homolog is used herein to denote a gene -or its product which is related to another gene or product by decent from a common ancestral D A sequence.
  • hyperproliferative cell disorder refers to a disorder in which cellular hyperproliferatioii causes or contributes to the pathological state or symptoms of the disorder
  • illustrative hyperproliferaiive cell disorders include, but are not limited to, neoplasms, benign tumors, malignant tumors, pre-cancerous conditions, in situ tumors, encapsulated tumors, metastatic tumors, liquid tumors, solid tumors, immunological tumors, hematological tumors, cancers, carcinomas, leukemias, lymphomas, sarcomas, and rapidly dividin cells.
  • rapidly di viding cell as used herein is defined as any cell that divides at a rate that exceeds or is greater than what is expected or observed among neighboring or juxtaposed cells within the same tissue.
  • hyperproliferaiive cell disorders include: cancers; blood vessel proliferative disorders such as restenosis, atherosclerosis, in-stent stenosis, vascular graft restenosis, ete,; fibrotic disorders; psoriasis; inflammatory disorders, e.g..
  • the hyperproliferative cell disorder is a precancer or a precancerous condition.
  • a "precancer ceil” or “precancerous -cell” is a cell manifestin a hyperproiiferative cell disorder that is a precancer or a precancerous condition, in other embodiments, the hyperproliferative cell disorder is a cancer.
  • cancer includes primary and metastatic cancer and is used
  • Neoplastic refers to a disease or condition involving cells that metastasize or have the potential to metastasize to distal sites and exhibit phenofypic traits that diffe from those of non-neoplastic cells, for example, formation of colonies in a three-dimensional substrate suc as soft agar or the formation- f tubular networks or web-like matrices in a three-dimensional basement membrane or extracellular matrix preparation * such as MatrigelTM.
  • Non-neoplastic cells do not form colonies in soft agar and form distinct sphere-like structures in three-dimensional basement membrane or extracellular matrix, preparations.
  • Neoplastic cells acquire a characteristic set of functional capabilities during their development, albeit through various mechanisms.
  • non-neoplastic means that the condition, disease, or disorder does not involve cancer cells.
  • exemplary cancers includes solid tumors, as we ' ll -as, hematologic tumors. and/or malignancies,
  • a "cancer cell,” “cancerous cell” or “neoplastic cell” is a cell manifesting a hypsrproliferative cell disorder that is a cancer. Any reproducible means of measurement ma be used to identii cancer cells or precancerous cells.
  • Cancer cells or precancerous cells can be identified by histological typing or gradin of a tissue sample (e.g. , a biopsy sample). Cancer cells or precancerous cells can be identified through the use of appropriate molecular markers, in some embodiments, the h erpro I i ferati ve cell disorder is a non-neop!astk disorder in which cellular hyperproliferation causes or contributes to the pathological state or symptoms of the disorder.
  • Hybridization is used herein to denote the pairing of complementary nucleotide sequences to produce a DNA-DNA hybrid or a DN A-RNA hybrid.
  • Complementary base sequences are those sequences that are related by the base-pairing rules.
  • RNA U pairs with A and C pairs with G hi this regard, the terms "match” and -"mismatch” as used herein refer to the hybridization potential of paired nucleotides in complementary nucleic acid strands. Matched nucleotides hybridize, efficiently, such as the classical A-T and G-C base pair mentioned above.
  • the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteeii or reversed Hoogsteen hydrogen bonding s between complementary nucleoside or nucleotide base (micleobases) of the strands of oligomeric compounds.
  • adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
  • Hybridization can occur under varying
  • hybridizing specifically to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • H ' lF hypoxia-inducible factor
  • HIF ' is a heterodimerie protein consisting of a constitutively expressed HIF- ⁇ ⁇ subun.it and one of three subunfts HIF- (HIF-1 a, HIF-2a or HIF-3a), The most widely studied, and seemingly major, RTF isoform is the HIF-1 isoform.
  • the stability and activity of HTF- subunits are regulated by 5 various post-txarssiationai modifications, including hydroxy lation, acetylation, and
  • vHL von Hlppel- Lindau tumor suppressor gene product
  • ODDD ox genic' dependent degradation domain
  • HIF-pro!yl hydroxylases HPH1 -3 also referred to as PHD 1 -3
  • PHD 1 2-oso.gIutarate
  • HIF-1 acts as a master regulator of -ftumerous. ' hypoxia-inducible genes under hypoxic conditions. The heterodimer HIF-1 binds to the hypoxic response elements (HREs). of target gene regulatory sequences, resulting in the transcription of genes implicated in the control of cell proliferation/survival, glucose iron metabolism and angiogenesis, as well as apoptosis and DC!uiar stress.
  • HREs hypoxic response elements
  • Some of these direct target genes 20 include glucose transporters, the glycolytic enzymes, erythropoietin, and angiogenic factor vascular endothelial growth factor (VEGF).
  • VEGF vascular endothelial growth factor
  • HIF-a is the oxygen-responsive component of HIF-1 and may refer to any mammalian or non-mammalian HIF-a polypeptide or fragment thereof, e.g., HIF- 1, , HIF ⁇ 2a, or HIF-3 a.
  • the term refers to the human form of HIF- la, as set forth for example in GenPept Accession os. AAC50152. 25 P_0Q152.1, NP_8S 139? and NPJOO 1230.013, HIF-a coding or gene sequences are also encompassed, as discussed for example mfi-a.
  • a fragment of HIF-a of interest is any fragment retaining at least one functional or structural characteristic of HIF-a
  • Non-limiting fragments of HIF-1 a suitably include proline residue 402 and/or 564 (as set forth in GenPept Accession No. AAC50152), which are hydrox lated by PHD polypeptides.
  • Suitable 30 fragments may include or consist of residues 344-698. particularl residues 364-678, more particularly residues 364-638 or 384-638 and still more particularly residues 364-598 or 394- 598.
  • Other suitable fragments may include or consist of residues 549-652 and even more particularly the N-terminal region thereof which interacts with the vHL protein. C-terminal.
  • fragments may include residues 549 to 582 and in particular residues 556-574, Other suitable fragments comprise or consist of residues 3 -4 i 7 » more suitably 380-417.
  • Such a region, or its equivalent in other HiF-a sirbimit proteins, is desirabiy present in HiF-a substrates used in assays for screening PHD inhibitors.
  • Exemplary HiF-a fragments may typically comprise residues 549 to 582 of the human H IF- l sequence.
  • T e term "immunocompromised” as used herein refers to a subject with an innate, acquired, or induced inability to develop a normal immune response.
  • immunocompromised subject therefore, has a weakened or impaired immune system relative to one of a normal subject.
  • a subject with a weakened or impaired immune system ' has- n "imtmmodeficiency" or “immunoeompiOtnised condition" which is associated with a primar or secondary deficiency, induced or non-induced, in one or more of the elements of the normal immune defense system.
  • An immunocompromised condition is commonly due to a medical treatment, e.g., radiation therapy, chemotherapy or other immunosuppressing ⁇ treatment, such as induced by treatment with steroids, cyclophosphamide, ai3 ⁇ 4thioprine.
  • methotrexate cyclosporme or rapamycin, in particular in relation to Cancer treatment or the treatment or prevention of rransplaot rejection
  • risk of acquiring an immunocompromised condition resulting from a medical treatment refers only to medical treatments that leads to or confers an immunocompromised condition, especially chemotherapy or other ' immunosuppressing treatment, such as . induced by treatment with radiation, steroids, cyclophosphamide, azamioprine, methotrexate,
  • an immunocompromised condition in a subject can be diagnosed by any suitable technique known to persons of skill the art. Strong indicators that an immunocompromised condition may be present is when rare diseases occur or the subject gets ill from organisms that do not normally cause diseases, especially if th subject gets repeatedl infected. Other possibilities are typically considered, such as recently acquired infections— for example, HIV, hepatitis, tuberculosis, etc. Generally, however, definitive diagnoses are based on laborator tests that determine the exact nature of the immunocompromised condition. Most tests are performed on blood samples. Blood contains antibodies, iymphocytes, phagocytes, and complement components— all of the major immune components that might cause immunodeficiency.
  • a blood ceil count wiii determine if the number of phagocytic cells or iymphocytes is below normal. Lower than normal counts of either of these two cell types correlates with an immunocompromised condition.
  • the blood cells are also checked for their appearance. Occasionally , a subject may have normal ceil counts, but the ceils are ⁇ structurally defective. If the lymphocyte ceil, count is low, further testing is usually conducted . to determine whether any particular type of lymphocyte is lower than normal.
  • a lymphocyte. roliferation, test may be conducted to determine if the lymphocytes can respond to stimuli- " The: failure to respond to stimulants correlates with an immunocompromised condition.
  • Antibody levels and complement levels can also be determined for diagnosing the presence of an immunocompromised condition. However, it shall be understood that the methods of the present invention are not predicated upon diagnosing the absence of an immunocompromised condition in the subjects to be treated.
  • immuno-interactive includes reference to any interaction, reaction, or other form of association between moiecuies and in particular where one of the molecules is, or mimics, a component of the immune system.
  • the present invention is particularly suited to treating or preventing mixed infections by more than one microbe.
  • Pathogenic algae include
  • Protothec and Pfiesieria also includes within the scope of these terms are prion proteins causing conditions such as Creutzfeidt- Jakob disease.
  • pathogenicity or the ability of a classieaily non-pathogenic agent to infect a subject and cause pathology can vary with the genotype and expression profile of the infectious agent, the host and the environment.
  • Fungal pathogens include without limitation species of the following genera: Absidia ⁇ Aeremom m, Aspergillus, Bg$idiohoh&s; s Bipolaris, Blastomyces, Candida (yeast), Cl dophialophora, Coccidioides ⁇ Cryptococc s, CunmnghameUa, C rvirfaria, Epidermophyt n, Exophial , Exsero i!um, Fonsecaea, Fus rium, Geotrichum, Histoplasm , Hori ea, Lac zi .
  • Lasiodiplodi Leptosphaeria, Madurella, Malassezia, Microsporum, MIICG Neotestudina, Onychocola, PaecUomyces, Paracoccidioides, FenieUivum,
  • Pathogenic conditions include any deleterious condition that develops as a result of infection with an infectious organism.
  • the term "interact” includes clos contact between molecules that results in a measurable effect, e.g., the binding or associatio of one molecule to another or a reaction of one molecule with another,
  • lower alky refers to straight and branched chain alky groups having from 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, iso-propyl, n-butyi, tert- bistyl, see-butyl, n-pentyl,. n-hexyi, 2-raethyl eniyl, and the like.
  • the lower alky! grou is methyl, or ethyl.
  • lower alkoxy refers to straight and branched chain alkoxy groups having from 1 to 6 carbon .atoms* " such as methoxy, ethoxy, n-propoxy,. iso-propoxy, n-hutoxy, rert-butoxy, sec-butoxy, n-pentoxy, ii-hexoxy, 2-methyl-pentoxy, and the like.
  • th tower alkoxy group is methoxy or ethoxy.
  • a "mobilizer of hematopoietic stem cel ls and or progenitor ceils,” “mobilizing agent” or “mobilized are used interchangeabl to refer to any compound, whether it is a small organic -molecule, synthetic or naturally derived, or a polypeptide, such as a growth factor or colony stimulating factor or an active fragment or mimic thereof, a nucleic acid, a carbohyd ate, an antibody, or any other agent that acts to enhance the migration of stem
  • modulating is meant increasing or decreasing, either directly or indirectly, the level or functional activity of a target molecule.
  • an agent may indirectly modulate the level/activity by interacting with a molecule other than the target molecule.
  • indirect modulation of a gene encoding a target polypeptide includes within its scope modulation of the expression of a First nucleic acid molecule, wherein an expression product of the first nuc leic acid molecule modulates the expression of a nucleic acid molecule encoding the target polypeptide,
  • a ⁇ neutropenia medicament refers to a composition of matter which reduces the symptoms related to neutropenia, prevents the development of neutropenia, or treats existing neutropenia.
  • oligonucleotide' refers to a polymer composed of a multiplicity of nucleotide residues (deoxyribonueleotides or ribonucleotides, or related structural variants or synthetic analogues thereof) linked via phosphodiester bonds (or related structural variants or synthetic analogues thereof).
  • oligonucleotide typically refers to a nucleotide polymer !rt which the nucleotide residues and linkages between them are naturally occurring, it will be understood that the terra also includes within its scope various analogues including, but not restricted to,, peptide nucleic acids (FNAs), phosphorasTi ates, phospkjrothioates, methyl phosphonates, 2-O-methyI ' ribonucleic acids, and the like. The exact size of the molecule can var '' depending on the particular application.
  • An oligonucleotide is typically rattier short irs length, generally from about 10 to 30.
  • nucleotide residues can refer t molecules of any length, although the term “polynucleotide” or “nucleic acid” is typically used for large oligonucleotides.
  • operbiy connected or "operabiy linked” as used herein refers to a j uxtapos ition where in the components so described are in a relationship permitting them to function in their intended manner.
  • a transcriptional control sequence "operabiy linked" to a coding sequence or non-coding sequence refers to positioning and/or orientation of the transcriptional control sequence relati e to the cod in or non-codin sequence to permit expression of the coding or non-coding sequence under conditions compatible with the transcriptional control sequence.
  • pharmaceutically acceptable refers to a compound or combination of compounds that will not impair the physiology of the recipient human or animal to the extent that the viability of the recipient is compromised.
  • the administered compound or combination, of compounds - will elicit, at most a temporary detrime ntal effect on the health of the recipient human, or animal.
  • pharmaceutically acceptable carrier a pharmaceutical vehicle or solvent comprised of a material thai is not biologically of otherwise undesirable, i.e. the materia! ma be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction.
  • Carriers ma include exeipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.
  • Illustrative vehicles or solvents include ' without limitation water, saline, physiological saline, ointments, creams, oil-water emulsions, geis, or any other ⁇ vehicle/solvent or combination vehicles/solvents and compounds known to one of skill in the art that is pharmaceutically and physiologically acceptable to the recipient human or animal.
  • a "pharmacologically acceptable" salt, ester, amide, prodrug or derivative of a compound as pro vided herein is a salt, ester, am ide, prodrug or derivative that this not biologically or otherwise undesirable.
  • Pathogenic "protozoa” include, without limitation, Trypanosoma,
  • pathogenic "parasites” include those from the phyla Cestoda (tapeworms), ematoda and Trematoda (flukes).
  • Pathogenic trematodes are, for example, species of the followi ng genera; Schistosoma, Echinostoma, Fasciolapsis, Clonorchis, Fascfola, OpisiharcMs and Paragonimus.
  • Cestode . pathogens include, without limitation, species from the following orders; Pseudophyllidea ⁇ e.g., Diph ohofhrmm) and
  • CyclophyUidea e.g., Taenia
  • Pathogenic nematodes include species from the orders; Rhabditida (e.g., .Strongyloieks), .Sirongylida (e.g., Ancy stvma), Asmridia (e..g, s Asmris, Toxoc ra , Spir rida (e.g., Dracim l , Brugia, Onchocerca, W cheria) and Adenophorea (e.g. , Trichuris tmd TrichineUa).
  • polynucleotide variant and “variant” refer to polynucleotides displaying substantial sequence identity wit a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions as known in the art (see for example Sanibrook e? al , Molecular -Cloning. A Laboratory Manual", Cold Spring Harbor Press, 1989), These terms also encompass polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides.
  • polypeptide proteinaeeous molecule
  • peptide and protein are used interchangeably herein to refer to a poly mer f amino ac id residues and to variants and synthetic analogues of the same.
  • these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-natnra-Hy-occmring amino acid, such as a chemical analogue of a corresponding naturally-occurring amino acid, as well as to naturally-occurring amino acid polymers.
  • These terms do not exclude mod ificai ions, for example, glycosyiaiions. acetyiations, phosphorylations and the like.
  • Soluble forms of the subject protemaeeous molecules are particularly useful. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids or polypeptides with substituted linkages.
  • polypeptide variant refers to polypeptides in which one or more amino acids have been replaced by different amino acids. It is well understood in the art that some am in® acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide (conservati ve substitutions) as described hereinafter. These terms also encompass polypeptides in which one or .more amino acids have been added or deleted, or replaced with different am ino acids,
  • the terms “prevent, 5'”pre vented,' or “preventing, 5 ' when used with respect to the treatment of a immunocompromised condition refers to a prophylactic treatment which increases the resistance of a subject to developing the immunocompromised condition or, in other words, decreases the likelihood that the subject will develop the immunocompromised condition as well as a ⁇ treatment after the immunocompromised condition has begun in order to reduce or eliminate it altogether or prevent it from becoming worse
  • pro-drug is used in its broadest sense and encompasses these deri vatives that are converted in vivo to the compounds of the in vention. Such derivatives would readily occur to those skilled in the art and include, for example, compounds where a free hydroxy group is converted into an ester derivative,
  • racemate refers to a mixture of enantiomers
  • reporter gene refers to any gene or DNA that expresses product that is detectable b spectroscopic, photochemical, biochemical, enzymatic, immunochemical, electrical, optical or chemicai means.
  • Other reporter genes for use for this purpose include, for example, ⁇ -galactosidase gene ( ⁇ -gal) and ..chloramphenicol acetyl transferase gene (CAT) Assays for expression produced in conjunction with each of these reporter gene elements are well-known to those skilled in the art.
  • Suitable pharmaceutically acceptable salts include salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulfuric, phosphoric, nitric, carbonic, boric, sulfamic and hydrobromic scids, or salts of pharmaceutically acceptable organic acids such. as acetic, propionic, butyric, tartaric, nialeie.
  • Base salts include, but are not limited to, those forme with pharmaceutically acceptable cations, such as ⁇ : sodium, potassium, lithium, calcium, magnesium, ammonium and alky lamraonium.
  • basic nitrogen -containing groups may be quatemized with such agents as lower alfcyi haiides, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyi sulfates like dimethyi and diethyl sulfate; and others.
  • lower alfcyi haiides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides
  • dialkyi sulfates like dimethyi and diethyl sulfate
  • uon-pharoiaeeuticaliy acceptable salts also fall within the scope of the invention since these may be useful in the preparation of pharmaceutically acceptable salts.
  • salts and prodrugs and derivatives can be carried out by methods known in the art.
  • metal salts can be prepared by reaction of a compound of the invention with a metal hydroxide.
  • An acid salt can be prepared by reacting an appropriate acid with a compound of the invention.
  • the term "selective" refers to compounds that inhibit or display antagonism towards a PHD (e.g., PI-ID 1, PHD2, or PHD3) (e.g., a prolyI-4-hydroxylase) without displaying substantial inhibition or antagonism towards another PHD- Accordingly, a compound that is selective for a ⁇ particular PHD (e.g., proIyl-4-hydroxyiase) exhibits inhibition or antagonism of that PHD that is greater than about 2-fold, 5 -fold, ! 0 -fold, 20- fold, 50-fold or greater than about 100-fold ' with respect to inhibition or antagonism of another PHD.
  • a PHD e.g., PI-ID 1, PHD2, or PHD3
  • a prolyI-4-hydroxylase e.g., a prolyI-4-hydroxylase
  • selective compounds display at least 50-fold greater inhibition or antagonism towards a particular PHD (e.g., a ptoly I-4-hydroxyiase) than towards another PHD.
  • selective compounds inhibit or display at least 100-fold greater inhibition or antagonism towards.
  • a particular PHD e.g., a prolyI-4- hydroxyjase
  • selective compounds display at least 500-fold greater inhibition or antagonism towards a particular PHD (e.g., a pro1y f-4-hydiOxylase) than, to wards another PHD, in still other embodiments, selective compounds display at least 1000-fold greater inhibition or antagonism towards a particular PHD (e.g., & proiyl-4-hydroxylase) than towards another PHD
  • a particular PHD e.g., & proiyl-4-hydroxylase
  • sequence identity refers to the extent that sequences are identical on a nucleotide-by ⁇ nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a "percentage of sequence identity” is calculated by compar ing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base ( .g., A, T, C, G, I) of the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val., Leu, lie, Phe, Tyr s Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys arid Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the iota! number of positions i the window of. comparison (Le.
  • sequence identity will be understood to mean the "match percentage” calculated by an appropriat method.
  • sequence identity analysis ma be carried out using the .DNASIS computer program (Version 2,5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software,
  • sequence comparison programs such as GAP (Deveraux el at 1984, Nucleic Acids Research 12. 387-395). In this way, sequences of a similar or substantially different length to those cited herein might be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.
  • Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference .sequence”, “comparison window”, “sequence identity”, “percentage of sequence identity” and “substantial identity”,
  • a "reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two
  • polynucleotides may each comprise. (I), a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence mat is divergent between the two ' polynucleotides, seq uence com par isons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a. "comparison window" to identif and compare local regions of sequence similarity.
  • a “comparison window” refers to a -conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is- compared to a reference sequence of the same num er, of .contiguous positions after the two. sequences are optimally aligned.
  • the comparison windo may comprise additions or deletions (i.e., gaps ⁇ of about 20% o less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequence for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFiT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive- Madison, I, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison ⁇ window) generated by any of the various methods selected.
  • GAP Garnier-FiT
  • FASTA Altschul et ⁇
  • TFASTA TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive- Madison, I, USA
  • the best alignment i.e., resulting in the highest percentage homology over the comparison ⁇ window
  • a "small molecule” refers to a composition that has a molecular weight of less than 3 ktlodaltons (kDa and typically less than 1.5 kiiodaitQns, and more preferably less than about 1 Modal ton. Small molecules may be nucleic acids, peptides, polypeptides* peptidoroknetics, carbohydrates, lipids or other organic (carborj- containing) or inorganic molecules.
  • a "small organic molecule” is an organic compound (or organic compound eoraplexed with an inorganic compound (e.g., metal)) that has a molecular weight of less than 3 kilodaitons, less than 1 ,5 kiledaltons, or even less than about 1 kDa.
  • Stem cells refer to cells, which are not terminally differentiated and are therefore able to produce cells of other types. Stem cells are generally divided into three types, including totipotent, pluripotent, and mult ipotent. "Totipotent stem cells” can grow and differentiate into any ceil in the body, and thus can grow into an entire organism. These cells are not capable of self-renewal. In mammals, only the zygote and early embryonic cells are totipotent. "P!uripotent stem, cells” are true stem cells, with the potential to make any differentiated cell in the body, but cannot contribute to making the extraembryonic membranes (which are derived from the trophobiast).
  • Multipotent stem cells are clonal cells that self-renew as well as differentiate to regenerate adult tissues, “Multipoteiit stem cells” are also referred to as “unipotenf ' and can only become particular types of cells, such • as blood cells or bone cells.
  • stem ceils refers to plnripotertt stem cells capable of self-renewal.
  • stringency refers to the temperature and ionic strength conditions, and presence or absence of certain organic solvents, during hybridization. The higher the stringency f the higher will be the observed degree of complementarity between sequences.
  • stringency conditions refers to temperature and ionic conditions under which only polynucleotides having a high proportion of complementary bases, preferably having exact complementarity, will hybridize. The stringency required is nucleotide sequence dependent and depends upon the various components present during hybridisation, and is greatly changed when nucleotide analogues are used.
  • stringent conditions are selected to be about 10° C to 20° C less than the thermal melting point (Tm for the specific sequence at a defined ionic strength and pH.
  • Tm is. the temperature (under defined ionic strength and pH) at which 50% of a target sequence hybridizes to a complementary probe-, It will .be understood that a polynucleotide will hybridize to a target sequence under at least low stringency conditions, preferably under at least medium strmgency conditions and more preferably under high stringency conditions.
  • Reference herein to low stringency conditions include and encompass from a least about .1 % v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization at 42° C, and at least about 1 M to at least about 2 M salt for washing at 42° C.
  • Lo stringency conditions also ma inciude 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHP04 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2 SSC, 0.1 % SDS; r (ii) 0.5% BSA, 1 rftM EDTA, 40 mM NaHP04 (pH 7.2), 5% SDS for washing at room temperature.
  • BSA Bovine Serum Albumin
  • Medium stringency conditions include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from, at least about 0.5 M to at least about 0.9 M salt for hybridization at 42° C, and at least about 0.5 M to at least about 0.9 M salt for washing at 42° C.
  • Medium stfingeney conditions also may include 1% Bo ine Serum Albumin (BSA), 1 tiiM EDTA, 0,5 M NaHP04 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2 X SSC, 0.
  • BSA Bo ine Serum Albumin
  • 1 tiiM EDTA 1,5 M NaHP04 (pH 7.2)
  • SDS for hybridization at 65° C
  • 2 X SSC 0.
  • High stringency conditions inciude and encompass from at least about 31% v/V to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization at 42° C, and at least about 0.01 M to at least about 0.15 M salt for washing at 42° C.
  • High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHPQ4 (jpH 7.2), 7% SDS for hybridization at 65° C, and (i) 0.2 x SSC,
  • Subjects 1 ' contemplated in the present invention include any animal of commercial,, humanitarian, or epidemiological interest includin conveniently, primates, livestock animals (such as sheep, cows, horses, donkeys, pigs, fish and birds), laboratory test animals (such as mice, rabbits, guinea pigs and hamsters and the- like), companion animals (such as dogs and cats), or capti ve wild animals, Avian species include poultry birds and caged avian species, i some embodiments the subject is a mammalian animal in other embodiments, the .
  • a donor subject is the subject in which the mobilization of hematopoietic stem and/or progenitor cells occurs and/or from which the mobilized stern and/or progenitor cells are harvested, if any
  • a recipient subject is the subject to which the harvested stem and/or progenitor cells ' are transplanted.
  • the donor subject and the recipient subject may be the same subject or may be different subjects.
  • B ''substantially .complementary it is meant that an oligonucleotide or a subsequence thereof is sufficiently complementary to hybridize with a target sequence. Accordingly, the nucleotide sequence of the oligonucleotide or subsequence need not reflect the exact complementar sequence of the target sequence, in a preferred embodiment, the oligonucleotide contains no mismatches and with the target, sequence.
  • the term “synergistic” means that the therapeutic effec t of a HIF-a potentiating agent when administered in combination with at least one mobilizer of hematopoietic stem cells and/or progenitor cells (or viee-versa) is greater than the predicted additive the rapeutic effects of the HIF-a potentiating agent and the at l east one mobilizer when administered alone.
  • the term "synergistically effective amounf as applied to a HIF-a potentiating agent and at least on e mobilizer of hematopoietic stem cells and/or progenitor cells refers to the amount of each component in a composition (generally a pharmaceutical ⁇ composition), , which is effective for stimulating or enhancing mobilization of hematopoietic stem ceils and/or progenitor ceils from the bone marro into th peripheral blood, and which prod uces an effect which does n ot intersect, in a dose-respons e plot of the dose of HIF- potentiating agent versus a dose of the at least one mobilizer versus stimu l ating or enhancing mobilization of hematopoietic, stem cells and/or progenitor cells from the bone marrow into the peripheral blood, either the dose HIF-a potentiating agent axis or the dose at least one mobilizer axis.
  • Th dose response curve used to determine synergy in the art is described for example by Sande et al. (see, p. 1080-1 105 in A. Goodman ei at., ed., the Pharmacological Basis of Therapeutics, MacMillan Publishing Co,, Inc. New York . (1980)).
  • the optimum synergistic amounts can be determined, using a 95% confidence limit b varying factors suc as dose level, schedule and response, and using a computer-generated model that generates isoboktgrams from the dose response c urves for various combinations of the HIF-a potentiating agent and the at least one mobilizer.
  • a "thrombocytopenia medicament?' as used herein refers to a composition of matter which reduces the symptoms related to thrombocytopenia, prevents the development of thrombocytopenia, or treats existing -thromboc topenia.
  • transcriptional control sequence refers to nucleic acid sequences, such as initiator sequences, enhancer sequences and promoter sequences, which induce, repress, or otherwise control the transcription of protein encoding nucleic acid sequences to which they are operably-Iinked.
  • treatment ** "treat,” ' “treated,” “treating” and the like is meant to include both therapeutie and prophy lactic treatment, including the administration of medicine or the performance of medical procedures with respect, to a patient, for either prophylaxis (prevention) or to cure or reduce the extent of or likelihood of occurrence of the infirmity or malady or condition or event in the instance where the patient is afflicted.
  • the treatments usin the agents described may be provided to treat patients suffering from a hyperproliierattve eel!
  • the treatment of the disorder with a cytoreductive or myeloablative therapy results in a decrease in bone marrow ce!iularity, thus making the patient more immunocompromised and more prone therefore -to acquiring infectious agents or diseases.
  • a cytoreductive or myeloablative therapy e.g., chemotherapy or radiation therapy
  • the administration of the agents of the invention allows for enhanced mobilization of hematopoietic stem cells and/or progenitor cells from the bone marrow to the peripheral blood.
  • the treating is for the purpose of reducing or diminishing the symptoms or progression of a hyperproliferative cell disorder by allowing for the use. of accelerated doses of chemotherapy or radiation therapy.
  • vector is meant a polynucleotide molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast or virus, into which a polynucleotide can be inserted or cloned.
  • a vector preferably contains one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or- tissue or a progenitor cell or tissue thereof, o be integrable with the genome of the defined host such that the cloned sequence is reproducible.
  • the vector can be an autonomously replicating vector, .e,, a vector that exists as an extrachroroosomal e n tity, the repl ication of whic h is independent of chromosomal replication, e.g. , a linear -or closed circula plasmid, an extrachromosomal element, a m inichromos me, or an artificial chromosome.
  • the vector ca contain any means for assuring self-replication.
  • the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the ehromosonie(s) into which it has been integrated.
  • a vector system can comprise a single vector or plasmid, two or more vectors or piasmids, whic together contain the total DNA to be introduced into the genome of the host ceil, or a transposon.
  • the choice of the vector will typically depend on the compatibility of the vector with the host sell into which the vector is to he introduced-
  • the vector is preferably a vtral or viral-derived vector, which is opsrably functional in animal and preferably mammalian cells.
  • Such vector may be derived from a poxvirus, art adenovirus or yeast.
  • the vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformatits.
  • resistance genes examples, of such resistance genes are known to those of skill in the art and include the nptll gene that confers resistance to the antibiotics .kanamycm and G418 (Geneticin®) and the hph gene which confers resistance to the antibiotic hygrarnycin B.
  • a virus includes any virus or viral pathogen or emerging viral pathogen.
  • Viral families contemplated include Adenoviridae, African swine fever-like viruses, Arenaviridae (such as viral hemorrhagic fevers, Lassa fever), Astroviridae (astro viruses) Birayaviridae (La Crosse), Calicivsridae (Norqvirus), Coronaviridae ⁇ Corona virus), Filoviridae (such as Ebola virus, Marburg virus), Parvoviridae (B 1 virus),
  • Flaviviridae such as hepatitis C virus, Dengue viruses), Hepadnaviridae (such as hepatitis B vires, Delta virus), Herpesviridae (herpes simple vims, varicella zoster vims),
  • Qrthomyxoy dae influenza vims
  • Papovaviridae papilloma virus
  • Paramyxoviridae such as human parainfluenza viruses, mumps virus, measles virus, human respiratory syncytial virus, Ntpah virus, Hertdra virus
  • Pieomayiridae common cold virus
  • Poxviridae small pox virus, orf virus, monkey poxvirus
  • Reoviridae rotavirus
  • Retroviridae human
  • Parvoviridae parvoviruses
  • Papillomaviridae Papillomaviridae
  • .(papillomaviruses) alphaviruses papillomaviruses) alphaviruses
  • Rhabdoviridae rabies virus
  • underscoring or ital cizing the name of a gene shall indicate the gene, in contrast to its protein product, which is indicated by the name of the gene in the absence of any underscoring or italicizing.
  • "fflF-la” shall mean the HJF-la gene
  • "HIF- ⁇ ” shall indicate the protein product or products generated from transcription and translatio and alternative splicing of the "EiF-la” gene
  • HSC hematopoietic stem cells
  • HSPC hematopoietic stem and progenitor ceils
  • the present invention is based in part on the surprising discovery that mobilization of hematopoietic stem ceils and or progenitor celis by mobilizing agents such as G-CSF and Pierixafor, and by combinations thereof, is sigtviflcantly enhanced in the presence of HIF-ct potentiating agents.
  • This increased mobilization in turn results in higher numbers of hematopoietic stem and progenitor cells (HSPCs) migrating from the bone marrow into the peripheral blood when compared to the number resulting from administration of stem cell mpbilizers alone.
  • HSPCs hematopoietic stem and progenitor cells
  • the increased mobilization may also result in increased HSPCs mobilizin from the peripheral blood to particular tissues or organs such as the lymph nodes, the heart, the lung, the liver, the skin, the spleen, small and large intestines, the stomach, or the pancreas.
  • HSPCs may al so inc rease the rate of differentiation of HSPCs into various cell lineages.
  • the HSPCs may also be capable of differentiation or starting a path to becoming a mature hematopoietic cell. For example ⁇ the differentiation of the HSPCs. may lead to an increase in the number of common myeloid progenitor cells in the bone marrow or the peripheral blood.
  • the differentiation of HSPCs may also lead to an increase in the number of granulocyte/macrophage progenitor cells or megakaryocyte/erythrocyte progenitor cells in the bone marrow or peripheral blood.
  • the HSPCs may differentiate into a common lymphoid precursor, line increase in number of common myeloid progenitor cells may lead to a differentiation into granulocyte/macrophage progenitor cells or megaka ⁇ o ' cyte/erythrocyte progenitor cells,
  • the granulocyte/macrophage progenitor cells may further differentiate into granulocytes such as neutrophils, eosinophils, basophils, tissue precursor cells, monocytes, and immature dendritic ceils.
  • megakaryocyte/erythrocyte progenitor ceils may differentiate into megakaryocytes and erytliroblasis,
  • the common lymphoid precursor cell may differentiate into B lymphocyte cells and T lymphocyte cells.
  • Th ' e.B lymphocyte ceils may differentiate into anttbody- seereting cells, wherein T lymphocytes, may differentiate Into effector T cells.
  • granulocyte may further differentiate into tissue mast ceils, macrophages, and immature dendritic cells.
  • the megakaryocyte may differentiate into platelets.
  • the erythro blast may differentiate into erythrocytes, .
  • HSPCs may also be capable of differentiating into ceils such as muscle (skeletal myocytes and cardiomyoeytes), brain, liver, skin, lung, kidney, intestinal, and pancreatic.
  • the number or proportion of cells presenting particular molecular or cell surface markers may be indicative of an HSPC or HSPC population.
  • 1 in every 10,000 to i 5,000 bone marrow cells may normally be a stem cell. In the bloodstream, the proportion may fall to 1 to 300,000 blood cells.
  • Administering a HIF-a potentiating agent and at least one mobiilzer of hematopoietic stem cells and/or progenitor ceils in vivo may Increase the number of ail stem eel!
  • populations in the bloodstream in about 1 hr or less, 2 hrs or less, 3 hrs or less, 4 hrs or less, 6 S rs or less, 8 hrs or less, 10 hrs or less, 12 hrs or less, 14 hrs or less, 16 hrs or less, 18 hrs or less, 20 hrs or less, 22 hrs or less, 24 hrs or less. 26 hrs or less, 28 hrs or less, or 30 hours or less after administration, and accumulation -of .
  • stem cells including HSPC in the blood may peak m about 65 hrs or less, 66 hrs or less, 67 hrs or less, 68 hrs or less, 69 hrs or less, 70 hrs or less, 71 hrs or less, 72 hrs or less, 73 hrs or less, 74 hrs or less, 75 hrs or less, 76 hrs or less, 77 hrs or less, 78 hrs or less, 79 hrs or less, 80 hrs or less.
  • methods and compositions that take advantage of a HIF-a potentiating agent and at least one mobii tzsr of hematopoietic stem cells and/or progenitor cells for stimulating or enhancing mobilization of hematopoietic stem cells and/or progenitor cells from the bone marrow into the peripheral blood, for stimulating or enhancing hematopotesis, for ' the. treatment or prophylaxis of immunocompromised conditions, including ones resulting from medical treatments that target rapidly dividing ceils or that disrupt the cell cycle or cell division (e.g., myetoablative therapy), or for stem cell transplantation.
  • a HIF-a potentiating agent and at least one mobii tzsr of hematopoietic stem cells and/or progenitor cells for stimulating or enhancing mobilization of hematopoietic stem cells and/or progenitor cells from the bone marrow into the peripheral blood, for stimulating or enhancing hematopotesis,
  • the HIF-a potentiating agent includes and encompasses an active agent that increases the accumulation of, or stability of, HIF-a; directly provides HIP-s activity; or increases expression of HlF-a, including without limitation, small molecules and macromoiecules such as nucleic acids, peptides, polypeptides, peptidomimetks, carbohydrates, polysaccharides, lipopolysaecrsarides, lipids or other organic (carbon containing) or inorganic molecules.
  • HIF-a refers to one or more of HIF- la, HIF-2 HlF-3a.
  • the HIF-a potentiating agent can be anything that results, in an increase in the amount or activity of a- HIF-a polypeptide.
  • Non-limiting examples include agents; that improve the stability (e.g., half-life) of the protein; that block or reduce deactivation of the HIF-a polypeptide, for example by preventing the
  • the HIF-a potentiating -agent can increase the activity of the HIF-a polypeptide by acting directly or indirectly on the HIF-a polypeptide to stabilize the protein, protect it from inhibition, or to increase the activity of the protein.
  • the substance can increase the activity of the HTF ⁇ ot polypeptide by inhibiting or otherwise blocking the act ivity of compound ' s or enzymes that inhibit the acti vity or reduce the stability of the- HIF-a polypeptide
  • the method includes introducing into at least one cell of the subject, such as a hematopoietic stem cell or progenitor cell, a nucleic acid construct that comprises at least one HIF-a coding, sequence operable connected to a transcriptional control sequence, and permitting the eeii to express the encoded HIF-a polypeptide.
  • a nucleic acid construct that comprises at least one HIF-a coding, sequence operable connected to a transcriptional control sequence, and permitting the eeii to express the encoded HIF-a polypeptide.
  • HIF-a coding sequences include: huma ⁇ ' ⁇ coding sequences as disclosed for example in GenBank Accession N >s.
  • mouse f ilF-a coding sequences as disclosed for example in GeaBaiik Accession Nos. AF003695, AAC52730, AFO5730S and Q61221 ; squirrel HIF-a coding sequences as disclosed for example in GenBank Accession No.
  • HIF-a may also be an mammalian or non-mammalian protein or fragment thereof.
  • HIF-a gene sequences may also be obtained by- routine cloning techniques, for example by using all or part of a HIF-a gene sequence described above as a probe to recover and determine the sequence of a HIF-a gene la another species.
  • a fragment of HIF-a of interest is any fragment retaining at least one functional or structural characteristic of HIF-a.
  • Fragments of HIF-a include, e.g., th regions defined by human HIF-a from amino acids 401 to 603 (Fluang et al,, (1998) Proc Natl Ami ScL USA 95:7987-7992), amino acid 531 to 575: (Jiang &t at ( ⁇ W!) JBiol Chem. 272:19253-19260 ⁇ , amino acid 556 to 57-5 (Tanrmoto et al (2000) EMBO J, 19:4298-4309), amino acid 557 to 5 1 (Srimvas et al. (1999) Bioekem Biop ys Res Commirn.
  • HIF-a fragments include any fragment containing at least one occurrence of the motif LXXLAP, e.g. , as occurs in the human HIF-a native sequence at i 3 s7 LL P and LssetE LAP.
  • HIF-a potentiating agents stimulate or enhance expression of ffIF- , representative examples of which include metallothlonein and zinc (see, e.g., Xue -et al (2012) Am J Physiol Heart Ore Physiol 302: H2528-F12535).
  • HIF-a potentiating agents inhibit the level or activit of a HIF-a ' interacting protein that inhibits the acti vity of a HIF-a polypeptide.
  • HIF-I interacting proteins of this type include: the von Hippel-Lindau tumor suppressor protein (vHL, Hon et al (20.02) Nature 41.7:975-8; Mi et al (2002) Science 296: 1886-9); hydroxylases including prolyl hydroxylases (e.g, pjroly-4 -hydroxylases) (also referred herein as H1F hydroxylases ' such as the HIP prolyl hydroxylases P.HDL FFID2 and PHD3, as described for example by Epstein et aL (2001) Cell 107:43-54, Kaelin (2005) Annu Rev BiQchem, 74:1 15-28; Schmid ei-af: (2004) J Ceil Mai Med, , 8:423
  • inhibitory PAS domain protein (IPAS, Makino et L (2002) Nature 414:550-4) and the like, which interact with one or more proteins comprising the HIF-1 heteradtmer and/or modulate the activity thereof.
  • HIF- interacting proteins see, e.g. , Accession Nos. P40337, HP 000542, NP937799,
  • HIF-e activity or protein levels can be increased by using small molecules to disrupt ' the rapid degradation of HIF- ' (Hewitson, K S and Schofield, C J. (2004) Drug Discovery-. Today 9(1 ⁇ ):704-711).
  • FIH factor inhibiting HIF
  • dihydrobeazoic acids e.g.. 3,4-dihydrobenz town
  • proteasomal inhibitors that affect degradation of the HIF-a sybunit
  • small molecules or antibodies that would block vHL .eorri lex:HIF-ct interaction
  • small molecule inhibitors of ubiquitination and inhibitory nucleic acid molecules such as small interfering RNAs (siR As) targeting PHD 1 -3 and/or FIH.
  • the HIF-a potentiating agent is an antagonistic nucleic acid molecule that functions to inhibit the transcription or translation of PHD, FIH- 1 or vHL encoding transcripts.
  • Representative transcripts of this type include nucleotide sequences corresponding to any one the following sequences: (1) human PHD1 nucleotide sequences as set forth for example in GenBank Accession Nos.
  • nucleotide sequences that share at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, S3, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% sequence identity with any one of the sequences referred to in (l); (3) nucleotide sequences that hybridize under at least low, medium or high stringency conditions to the seq uences referred to in (1 ): (4) nucleotide sequences th at encode any one of the following amino acid sequences: human PHD I.
  • nucleotide sequences that encode an amino acid sequence that shares at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similarity with an one of the sequences referred to in (4); and nucleotide sequences thai encode an amino acid sequence that shares at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 1 , 2, 93, 94, 95, 96, 97, 98, 99% sequence identity with an one of the sequences referred to in (/I).
  • illustrative antagonist nucleic acid molecules include antisense molecules, aptamers, ribozymes and triplex forming molecules, RNAI and external guide sequences.
  • the nucleic acid molecules can act as effectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule., or the functional nucleic acid molecules can possess a de novo activity independent of any other m olecules.
  • Antagonist nucleic acid molecules can interact with any maerorooiecole, such as DNA, RNA, polypeptides, or carbohydrate chains.
  • antagonist nucleic acid molecules can. interact with PHD, FIH-l or vHL mRNA or the genomic DMA of FED, FIH- 1 or v.HL or the can interact with the PHD, FIH-1. or vHL polypeptide.
  • antagonist nucleic acid molecules are designed to interact with other nucleic acids based on sequence homology between the target molecule and the antagonist nucleic acid molecule.
  • the specific recognition between the antagonist nucleic acid molecule and the target molecule is not based on sequence homology between the antagonist nucleic acid molecule and the target moleeuie, ' but rather is based on the formation .of tertiary structure that allows specific recognition to take place.
  • anti-sense RNA or DNA moleeules are used to directly block the translation of PHD, FlH-1 or vHL .mRNA by binding to targeted ra&NA and preventing protein ' translation;
  • Antisense molecules are designed to interact with a target nucleic aeid moleeuie through either canonical or non-canonical base pairing.
  • the interaction of the antisense molecule and the target moleeuie may be designed to promote the destruction of the target molecule through, for example, RNAseH mediated RNA-DNA hybrid degradation.
  • the antisense molecule may be designed to interrupt a processing , function that normally would take place on the target molecule, such as transcription or replication
  • Antisense molecules can be designed based on the sequence of the target molecule. Numerous methods for optimization of antisense efficiency by Finding the most accessible regions of the target molecule exist Non-limiting methods include in vitro selection experiments and DN A modification, studies using diniefhyl ' sulfate (DMS) and d ethylpyrocarbonaie (DEPC).
  • the antisense molecules bind the target molecule with a dissociation constant (Kd) less than or equal to 10 " °, 10 "8 , !0 ⁇ !0 , or 10 ' .
  • Kd dissociation constant
  • antisense oligodeoxyribonucieotides derived from the translation initiation site e.g., between. -10.and -HQ regions are employed.
  • Aptamers are molecules that interact with a target molecule, suitably in a specific way, Aptamers are generally small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets. Aptamers can bind small molecules, such as ATP and theophiline, as well as large molecules, such as reverse transcriptase and thrombin. Aptamers can bind very tightly with Kds from the target molecule of less than I0 ' M.
  • the aptamers bind the target molecule with a Kd less than 10 ⁇ 10 "s , 10 "l °, or !0 '!2
  • Aptamers can bind the target molecule with a very high degree of specificity.
  • aptamers have been isolated that have greater than a 10,000 fold difference in binding affinities between the target molecule and another molecule that differ at only a single position on the molecule, it is desirable that an aptamer have a K d with the target molecule at least 10-, 100-, 1000-, 10,000-, or 100,000- fo!d lower than the « with a background-binding molecule.
  • a suitable method for generating an aptamer to a target of interest is the "Systematic Evolution of Ligands by Exponential Enrichment" (S.EL.EXTM).
  • S.EL.EXTM Systematic Evolution of Ligands by Exponential Enrichment
  • the SELEXTM method is described in U.S. Pat No. 5,475,096 and U. S, Pat. No, 5,270,163 (see also WO 91/198 ⁇ 3), Briefly, a mixture of nucleic acids is contacted with the target molecule under conditions favorable for binding. The unbound nucleic ac ids are partitioned from the bound nucleic acids, and.
  • nucleic acid-target complexes are dissociated, Then the dissociated nucleic acids are amplified to yield a ligand-enriehed mixture of nucleic acids, which is subjected to repeated cycles of binding, partitioning, dissociating and amplifying as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
  • anti-PHD, -FIH-1 or ⁇ vHL ribozymes are used for catalyzing the specifie cleavage of PHD, FIH-l or vHL RNA.
  • the mechanism ofribozyme action involves sequence spec ific hybridization of the nbozym molecule to complementary target RNA, followed by a endonucleolytic cleavage.
  • ribozymes that catalyze .nuclease or nucleic acid polymerase type reactions, which are based on ribozym.es found in natural systems, such as hammerhead ribozymes, hairpin ribozymes, and tetrahytnena ribozymes.
  • ribozymes that are not found in natural systems, but which have been engineered to catalyze specific reactions de novo, Representauve ribozymes cleave RNA or DN A- substrates. In some embodiments;, ribozymes that cleav RNA substrates are employed.
  • RNA targets Specific ribozyme cleavage sites within potential RNA targets are initiall identified b scanning the target molecule for ribozyme cleavage siteSj which include the following sequences, GUA, GUU and GUC.
  • ribozyme cleavage siteSj which include the following sequences, GUA, GUU and GUC.
  • short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable.
  • the suitability; of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ri ' bomiclease protection assays,
  • Triplex forming functional nucleic acid molecules are molecules that can interact with either doub!e-siranded or single-stranded nucleic acid.
  • triplex molecules When triplex molecules interact with a target region, a structure called a triplex is formed, in which there are three strands of DNA forming a complex dependent on both Watson-Crick and Hoogsteen base pairing, Triplex molecules are preferred because they can bind target regions with high affinit and specificity. It i generally desirable that the triplex forming molecules bind the. target molecule with, 3 ⁇ 4 less than W ( W ⁇ ⁇ 0 , or 10 ⁇ ' 2 .
  • External guid sequences are molecules that bind a target nucleic acid molecule forming a complex, and this complex is recognized by RNAse P, which cleaves the target molecule.
  • EGSs can be designed to specifically target a RNA molecule of choice.
  • RNAse P aids in processing transfer RNA (1RNA) within a cell.
  • Bacterial RNAse P can be recruited to cleave virtually any RNA sequence fay using an EGS that causes the target R ArEGS complex to mimic the natural . tRNA substrate.
  • EGS/RNAse P-directed cleavage of RNA can be utilized to cleave desired targets within eukaryotie cells.
  • RNA molecules that mediate RNA interference are RNA molecules that mediate RNA interference '
  • RNAi of a PHD, FiH- 1 or vNL gene or PHD, FIH- J or vHL transcript can be used to reduce or abrogate gene expression.
  • RNAi refers to interference with or destruction of the product of a target gene by introducing a single-stranded or usually a double-stranded RNA (dsR A) that is homologous t the transcript of a target gene.
  • dsR A double-stranded RNA interference
  • siRNA small interfering RNA
  • RNAi can e triggered by 21- to 23-nucleotide (nt) duplexes of small interfering RNA (siRNA) (Chili et at, 2002, Mol. Cell.
  • dsRNA per se and especially dsRNA-producing constructs corresponding to at least a portion of a PHD, FIH-1 or vBL gene are used to reduce or abrogate its expression.
  • RNAi-mediated inhibition of gene expression may bs accomplished using any of the techniques reported in the art, for instance by trahsfecting a nucleic acid construct encoding a stem-loop or hairpin RNA structure into the genome of the target cell, or by expressing a ixansfected nucleic acid construct having homology for &PHD,- FlH-l or vHL gene from between convergent promoters, or as a head to head or tail to tail duplication from behind a single promoter.
  • Any similar construct may be used so long as it produces a single RNA having the abilit to fold back oft itself and produce a dsRNA, or so long as it produces two separate RNA transcripts, which then anneal to form a dsR A having homology to a target gene,
  • R A-encodjng constructs that express a single RNA transcript designed to anneal to a separately expressed KNA, or single constructs expressing separate transcripts from convergent promoters * are suitably at least about 1.00 nucleotides m length.
  • RNA-encoding constructs that express a single RNA designed to form a dsRNA via internal folding ar usually at least about 200 nucleotides in length.
  • the promoter used to express the dsRN A- forming construct may be any type of promoter if the resulting dsRNA is .specific for a gene product in the cell lineage targeted for destruction.
  • the promoter m y be lineage specific in that it is onl expressed in ceils of a particular- development lineage. This might be advantageous where some overla in homolog is observed with a gene that is expressed in a non-targeted cell lineage.
  • the promoter may also be inducible by externally controlled factors,, or by intracellular environmental factors.
  • RN A molecules of about 21 to about 23 nucleotides which direct c leavage of specific mRNA to- which they correspond, as for example descri bed by Tuschl et al in U.S. 2002/0086356, can be utilized for mediating RNAi.
  • Such 2,1- to 23-nt RNA molecules can comprise a 3'- ydroxy! group, can be single -stranded or double stranded (as two 21- to 23-nt RNAs) wherein the .dsRNA molecules can be blunt ended or comprise overhanging ends (e.g., 5 3').
  • the antagonist nucleic acid molecule is a siRNA
  • siRNAs can be prepared by any suitable method.
  • Sequence specific gene silencing can be achieved in mammalian cells using synthetic, short double-stranded RNAs that mimic the siRNAs produced by the enzyme dicer, siRNA can be chemically or in v/fro-synthesized or can be the result of short double-stranded hai ⁇ in-like RNAs (shRNAs) that are processed into siRNAs inside the cell.
  • Synthetic siRNAs are generally designed using algorithms and a conventional D A/RN A synthesizer. Suppliers include Arnbion (Austin, Tex.), ChemGenes
  • siRNA can also be synthesized in vitro using kits such as Ainfaion's
  • siRN short hairpin RNAs
  • Kits for the production of vectors comprising shRNA are available, such as, for example, Imgenex's GENESUPPRESSORTM Construction Kits and Jnvitrogen's BLOCK-ITTM inducible RNAi plasmid and lentiviras vectors.
  • RNAi molecules e.g., PHD., FIE-l or vEL siRNA and. stiRNA are available commercially from Santa Cruz Biotechnology, Inc. (Santa Cru3 ⁇ 4 CA, USA).
  • the HIF-a potentiating agent is an inhibitor of HIF hydrox lase enzyme, particularly an inhibitor of a HIF prolyl hydroxylase enzyme.
  • a compound that inhibits the activity of a HIF hydroxylase enzyme refers to any compound that reduces, eliminates, or attenuates the activity of at least one HIF hydroxylase enzyme (e.g., PHD ! -3).
  • the HIF-a potentiating agent is an inhibitor of a HIF proly l hydroxylase enzyme. Methods for determining whether a compound inhibits HIF hydroxylase activity are well known in the art.
  • HIF hydroxylase inhibitors for use in the methods of the present invention are defined by their ability to inhibit an activity of a 2-Qxogluiarate dioxygenase enzyme, wherein the enzyme has specific activity toward hypoxia inducible factor.
  • Such compounds are often referred to as HIF hydroxylase i hibitors, HIF prol l hydroxylase inhibitors, HIF prolyl-4-hydroxylase inhibitors, prolyl hydroxylase inhibitors or "PHF's.
  • the PHls for use in the invention are small molecule compounds.
  • a compound that inhibits the activity of a HIF hydroxylase enzyme may additionally show inhibitory activity toward one or more other 2-oxoglutarate- and iron- dependent dioxygenase enzymes, e.g., FIH (GenBanfc Accession No, AAL27308), procollagen prolyl 4-hydroxyiase (CP4H), eta
  • FIH GeneBanfc Accession No, AAL27308
  • CP4H procollagen prolyl 4-hydroxyiase
  • compounds used in the present methods and medicaments provided herein are structural miraeties of 2-oxoglutarate, wherein the compound inhibits the target HIF prolyl hydroxylase enzyme competitively with respect to 2-oxoglutarate and -noncompetitively with respect to irom PHIs are typically heterocyclic car bpxamide compounds, especially heterocyclic carbonyl glycine derivatives, and may be, for example, a heterocyclic carboxamide, including pyridine, pyrimidine, pyridazine, naphthyridine, pytrolop ridine * thlazo ' lopyridine, isot iazolopyridine, quinoline, isoquinoHne, e noline, beta-earholine, quinolone, thienopyridiiie, ehromene, or
  • the inhibitor s-nay be a ' heterocyclic carbonyl glycine.
  • Patent No, 7,608,621 U.S. Patent No. 7,728,130, U.S. Patent No. 7,63.5,715, U.S. Patent
  • die HIF-ct potentiating agent is selected from the group consisting of [(4-Hydroxy-7 ⁇ henylsuifany ' I-isoq' inoline-3-earbonyl)- aminoj-aeetic acid (Compound X), ⁇ [5-(4-Chioro-phen xy)-l-cyano-4-h)'droxy- isoquinoline-3-cai ' bonyl]-amiiio ⁇ ac&tie aeid (Compound A), [(1 -Cyano-4-hydroxy-5- p enoxy-isoquinoline-3-earbony!-amino]-aeetic acid (Compound.
  • the HIF-a potentiating agent is selected from the group consisting of [(4 ⁇ Hydroxy-7- phenylsirlfany -isoqumoline-3-carbonyi)-anjino]-acetic acid (Compound X), ⁇ [5 ⁇ (4-Chloro- phenoxy)-I -cy&no ⁇ 4 ⁇ hydroxy-isaqiHno1 ⁇ 2 ⁇ acid (Compound A),
  • Methods of determining if a «y particular compound, inhibits HIF prolyl hydroxylase are well known, illustrative examples of which include the methods described in U.S. Patent No. 7,323.475.
  • the inhibitory activity of any particular compound can he conveniently evaluated arid compared by determining the ICso for one or more of the HIF prolyl hydroxylase enzymes.
  • the IC 0 for any compound for each of the HIF prolyl hydroxylase enzymes can be determined using assays known in the art.
  • the IC50 values for compounds that inhibit HIF prolyl hydroxylase will be in the ⁇ range or less, typically in the nM range, for one or more of the HIF prolyl hydroxylase enzymes.
  • the ICso for inhibition of the PHD2 enzyme of Compounds A, B, C, D, E, F, H, J, K, and X range from 0.05- 1 .5 ⁇ .
  • the ICjo of the exemplified compounds for the PH ' Dl and PHD3 enzymes are in similar ranges.
  • Illustrative small molecule PHls include, for example, the nitrogen- containing heteroaryl compounds disclosed in U.S. 2004/02542] 5 (WO 2004/410868.1) and in U.S. Patent Nos, 7,323,475; 7.629,357; 7,863,292; and 8,017,625, each of which is expressly incorporated herein by reference in its entirety.
  • Exemplary compounds of this type are represented by formula ⁇ :
  • p is zero or one
  • R a is -COOH or -WR S ; provided that when R a is -COOH then p is zero and when R a is— WR* then p is one;
  • W is selected from the group consisting of oxygen, -S(0) K - and— Nowhere, n is zero, one or two,
  • y is selected from the grou consisting of hydrogen, alk l, substituted alkyi, acyl, aryi, substituted aryi, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic and.
  • R s is selected tem the group, consisting of hydrogen, alkyi, substituted alkyl, aryl, substituted aryl, freteroaryL substituted heteroaryl, heterocyclic and. substituted heterocyclic, or when W is -MR 9 - then R 8 and R 3 ⁇ 4 , together with the nitrogen atom, to which they are bound, can be joined to " form a ' heterocyclic or a substituted
  • R ! is selected from the group consisting, of hydrogen, alkyl , substituted alkyl, alkoxy, substituted alkoxy, amino, substituted amino, arainoacyl, ary!, substituted aryl, halo, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, and -XR 6 where X is oxygen, - (0) - or -NR.
  • R 7 is hydrogen, alkyl or aryl or, when X is -MR? - then R' and R 8 , together with the nitrogen atom to which they are bound, can be joined to fo rm a .heterocyclic or substituted heterocyclic group;
  • R 2 and J are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryf, heteroaryl, substituted heteroaryl, halo, hydroxy, cyano, ⁇ S(0) n ⁇ (R. 6 ⁇ R 6 where n is 0, 1 , or 2, -NR. 6 C(0)NR 6 R 6' , -XR 8 where X is oxygen, ⁇ S(0) suit- or --MR'- where rs is zero, one or two, each R s is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl.
  • R 6 is not hydrogen
  • R 7 is selected from the group consisting of hydrogen, aikyi, aryl, or R 2 , R ! together with the carbon atom pendent thereto, form. an aryl substituted aryl, heteroaryl, or substituted heteroaryl;
  • R. 4 and R 5 are independently selected from the group consisting of hydrogen,, halo, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl and -XR e where X is oxygen, ⁇ S(0) «- or -MR 7 - where n is zero, one or two, R 6 is selected from the grou consisting of alky l, substituted " alkyi, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and ..substituted heterocyclic, and R 7 is hydrogen, -alkyl or aryl or, when X is -MR 7 - then R' and R 8 , together with the nitrogen atom to which they are bound, can be joined to form a heterocyclic or substituted heterocyclic group; [00165] R is selected ftom the grou consisting of hydrogen, deBteriurn and methyl
  • R' is selected from the group consisting of hydrogen, deuterium, alky] and substituted alky I; alternatively- R and R' and the carbon pendent thereto can be joined to form eycloalkyl. substituted eycloalkyl, heterocyclic or substituted heterocyclic group;
  • R" is selected from the group consisting of hydrogen and alkyi or R' together with R' and the nitrogen pendent thereto can be joined to form a heterocyclic or substituted heterocyclic group;
  • '" is selected from the group consisting of hydroxy, alkoxy, substituted alkoxy, acyloxy, eyc!oalkoxy, substituted eyeloalkoxy, ary!oxy, substituted aryloxy, heteroaryioxy, substituted heteroaryioxy, aryl,TMS(0 ⁇ , -R 10 wherein R'° is selected from the grou consisting of a!kyl, substituted alkyl, eycloalkyl substituted eycloalkyl, aryl, substituted aryl, heteroaryl and substituted heterqaryi and n. is zero, one or two;
  • R 1 , R 2 , R 3 , R , R 5' , R, R', R", R"', WR 8 and q are as defined above; and pharmaceutically acceptable salts, esters, prodrugs thereof,
  • the invention is directed to compounds represented by the formula. ID:
  • Exemplary .compounds according to the above formulae include ⁇ [4- Hydroxy ⁇ l -(naphthaien-2-yloxy)-!soquinoiine-3-carbonyl]-amin ⁇ acid; ⁇ [4-
  • succinic ac (S)-2-[(l-Chloro-4-hydroxy-isoquino!in acid; (R)-2-[ ⁇ 1 -Ch!of o-4-hydfoxy-6-!Sopr opoxy-isoquinoliae-3-earbon ! ⁇ -am ino] -succinic acid; ⁇ S)-2-[(l-Chloi"0-4-hyd]Oxy ⁇ 6-isoprGpoxy-!SO acid; (R)-2-[(i-Chloro-4-hydroxy-7-isopropoxy-isoquinolrae-3-car 1- [(l ⁇ Cfalofo-4-hydroxy ⁇ sequinonne ⁇ acid;
  • small molecule PH!s may be selected from eyanoisoqinno!ine compounds disclosed in. U.S. Patent No. 7,928,120, which is expressly incorporated herein by reference in its entirety. These compounds can be represented by formula li:
  • R is selected from the group consisting of hydrogen, alkyi, and substituted alkyl
  • R s , R 3 , R 3 and 4 are independently selected from the group consisting of hydrogen, halo, cyans, hydroxy!, alkyf. substituted alkyl, aryL substituted aryl, heteroaryi. • substituted heteroaryi, amino, substituted amino-, -OR', -SR , -SGR', and -SO2 wherein R' is selected from the group consisting of alkyi, substituted alkyl, cycloalkyl, substituted cyeloalkyL aryl, substituted aryi, heteroaryi, and substituted heteroaryi; and
  • R 5 and R* are independently selected from the group consisting of hydrogen or Cy.j alkyl
  • Representative compounds of Formula II include;
  • sniaii molecule PH is may be selected from pyrrolo- and thiazoSo-pyridiiie compounds disciosed in U.S. Patent No. 7,696,223, which is expressly incorporated herein by reference in its entirety. These compounds can be represented by formula 111:
  • q is O or 1;
  • a and B are independently selected from the grou consisting -C( 7 )- » -NCR 8 )-,TMN-, and -S- with the proviso that one of the following is presen
  • A C(R 7 ) ⁇ and B is -N(R 8 )-;
  • A N- and B is-S-; or
  • one of ' -A C(R 6 )- or -B - » C(R 6 )- is a double bond and the other is a single bond;
  • R 1 is selected from the group consisting of hydroxyl, alkoxy, substituted aikoxy, aeyloxy, cyc!oaikoxy, substituted cycloalkoxy, aryloxy, substituted aryloxy, heteroaryloxy, substituted heteroaryloxy, heteroeyciytoxy, substituted heterocyclyioxy, mercapto, thioether, swstifuted alk ithio, arylsulfanyi, heteroarylsulfanyl, amino, .substituted amino, acylamsno and aniinoacyi;
  • R "' is selected from the group consisting of hydrogen ⁇ deuterium, and methyl
  • [00201] 3 is selected from the group consisting of hydrogen, deuterium, alkyi. and substituted alkyi;
  • R 4 is selected from the group consisting of hydrogen,, alkyi, and substituted alkyi;
  • R 5 is selected from the group consisting of hydrogen, halo, eyano, hydroxy I, alkyi, substituted alkyi, cycioalkyl, substituted cycioalkyl, alkenyl, substituted aikenyl, alkynyl, substituted alkynyl, a!koxy, substituted alkoxy, cyc!aalkoxy, substituted cycioaikoxy, aryl, substituted aryl, aryloxy, substituied arytoxy, beteroaryl, substituted heteroaryl, heterocyciyj, substituted heterocyciyj, heterocyclyioxy, substituted
  • heterocyclyioxy heteroaryloxy, substituted heteroaryloxy, aeyl, aminoacyl, nitro, amino, substituted amino, ac iamino, sulfan l, sislfonyl thioether, arylthip, and substituted atylthio:
  • R° and R 7 are each independently selected from the grou consisting. of hydrogen, halo, eyano, hydrox I, alkyi, substituted alkyi, cycioalkyl, substituted cycioalkyl, alkenyl, substituted alkenyl, alkynyi, substituted alkynyi, alkoxy, substituted alkoxy, cycioaikoxy. substituted cycioaikoxy, aryl, substituted aryl, aryloxy, substituted aryloxy, substituted aryloxy,.
  • heteroaryl substituted, heteroaryl, h teroc arbor substituted heteroeyclyl, heterocyclyioxy, substituied heterocyclyioxy,. heteroaryloxy, substituted heteroaryloxy, acyl, aroinoacyi, nitra, amino, substituted amino, acylamino, sulfanyl, sulfony ' I, thioether, aryithio, and substituted aiySthio;
  • R is selected from the group consisting of hydrogen, hydroxyl, alkyi, substituted alkyi, aikenyl, substituted alkenyl, alkynyi, substituted alkynyi, aryl, substituted atyi, heteroaryl, arid substituted heteroaryl;
  • small molecule PHIs may be selected from those disclosed in WO 2004/308121 (U.S. 2005/020487), which can be represented by formula IV:
  • 00212 ⁇ A is 1 ,2-arylidene. 1.,3-arylid.ene, 1 , -aiyUdene ' ; or (C C ⁇ -a ' ikylene, optionally substituted by one or two halogen, cyano, nitro, trifluororaethy!, (Ci-Q -alkyl, (C i -C ⁇ i)-h droxyalky 1, (C i -Ce)-aikoxy , -0- CH 2 ] .x ⁇ CiH (2f ⁇ i- g) Hal g , (d -C6)-fli!oroalkoxy J: (C r C 8 >fiuoroalkenylexy 9 (d-3 ⁇ 4-fltioroalkynyloxy, ⁇ QCF 2 C1, -GM ⁇ F -CHFCl; (CrC 6 )- alkyimercapto, (d-d)-alky
  • B is -C0 2 H, -N3 ⁇ 4, ⁇ HS0 2 CF 3l tetrazolyl, imidazolyl, 3- hydroxyisoxazoiyl, -CO HCQR'', -GONHSOR"', CONHS ⁇ 3 ⁇ 4R" ⁇ where R'" is aryl, heteroaryl by (Cs-d2) ⁇ at l, heterpary], (d-dMMoalkyl, (d-C «)-sulfi yl, (Ci- C ⁇ salfonyl, CF 3 , CI, Br, F, 1, NG3 ⁇ 4 -GQQH, (C 2 -.C $ ) ⁇ alkoxycarbony!, H3 ⁇ 4, rao.n.o-(C C - aikyl)-amino, di-(C f -C ⁇ (-alkyl)-amino f or or wherein B is a 03 ⁇
  • alkenynyl radicals contain one or more multiple bonds;
  • Ci2 Ci2)-aryIoxy ⁇ (C-]-Ci ⁇ ))-alkyl)-carbaifnoy.i, N- ⁇ Cf-Ci())-aiky!-N-- ⁇ C7-'Ci6.)-aralkyloxy-(CrC 1 - sub.1 Q) ⁇ alky l)-earbar» oy 1, carbamoy lox , H-(C j -C j stalk icarbam oyloxyv N.N-di-(C 3 ⁇ 4 -C n aJky!carbaxnoyloxy, N- ⁇ C3-C 3 ⁇ 4 )-cycioalky3carbamoyloxy N ⁇ C6-C]2)-atylcarban o loxy 5 N- (C ?
  • Q is Cv S, NR ⁇ or a bond
  • R 4 is hydrogen, (C;-C 5 o)-aikyl radical, (C 2 - Cio)-alkenyl radical, ⁇ Cs-Gtt -alkynyl radical, wherein aikenyl or alkynyl radical contains one or two C-C multiple bonds; umubsiituted iluoroa!ky!
  • E is a heteroaryl radical, a (C3--C «)-cycioalkyi radical, or a phenyl radical of the formula F
  • v is 0-6,
  • t is 0-3
  • R 7 , R 8 , R 9 , R !0 , and R l ! are identical or different and are hydrogen,, halogen, cyano, nitre, trifluoromethyi, (C-rC 6 )-aikyl, ⁇ 3 ⁇ 4-Cs ⁇ -cycIoaikyI, (Cj-GgJ-alkoxy, - ⁇ OC ⁇ 2 -Ch -O-CFa-CRFCl, (Ci-Qs)-alkylmercapto, (d-C*)- hydroxyaSkyi, (Ci- e)-allcoxy (Ci-C6)-alkoxy, (Ci-Ce -a?koxy-(Cj ⁇ Cc -aikyl, (Ci-CV,)- a!kylsuifiny 1, (Cs-C$)-a3kylsulfonyl, (Cj -C ⁇ -alkykarbonyi
  • alkoxycarbonyliniino, and h is 3 to ?; phertylmercapto, phenylsulfonyl, phenylsulfmyl, sulfarnoyl, -(Gi-Cs)-alkyIstJlfamoyi, or ' N,N-di-(Ci-Cs)-alfcylsuifamoyl or alternatively R 7 and R s , R 3 and 9 , 9 and R i0 , or R i0 and R !
  • R is alternatively R", where R' and R" are identical or different and are hydrogen, (C,;-Cji) ⁇ ary ⁇ , (C T -Ci -a alk l, (Cs-C ⁇ -aikyl, (Gi-Cg)-alkoxy- (Ci-Cg ' kyl iC7-Gi 2)-araIkoxy-(Gi-C 8 ;)-alky1, (C 6 -C 2 )-an'!oxy-(C (Ci-do)- alkyfcarbonyi, optionally substituted (C 7 -Ci6)-aralkyIcai'bonyl, or optionally substituted (Cg- C ⁇ -aryiearbonyl; or R' and R" together are -[CH3 ⁇ 4jh » in which a CFfe group can be replaced by O, S, N-aeylimlno, or N-lC
  • Y is " or CR 3 ;
  • R 1 , R 1 and R' are identical or different and are hydrogen, hydrox I halogen, cyano, ixifluoromethyl. nifro, carboxyl, (CrC_o)-ajkyl (C3-C8)-c cioalk l, (C-3-C3 ⁇ 4) ⁇ cyc ⁇ oalkyi- ⁇ Ci-C!2)-alkyl, ⁇ Cs-Cg ⁇ eycioalkoxy, (C3-Cs)-cycioaikyl ⁇ (C f -Ci2) ⁇ alkoxy, ⁇ Cr .C s >cyc.oaikyloxy-(C> -Ci ⁇ alk l, (C 3 -C 8 )-cycIoalIty xy-(Ci-C»2)-alkpxy, (C ⁇ Q s
  • N- C6-Cia)-arylimino N-(C7-CK ⁇ araik limino J N- ⁇ C] ⁇ i)-aikox ⁇ Gj-C 6 )-a!k li!T!mo ⁇ and h s from 3 to 7; a carbamoyl radical of the formula
  • R* and R v are each independently ' selected from hydrogen, (Gi-Cy-alk l, (C3-C7)-cycloalkyi, aryl, or the substitueni of an a-carbon of an ct-amino acid, to which the L- and D-amino acids belong,
  • T is OH, or NR*R**, and R.*, R** and R*** are identical or different and are selected from hydrogen, (Cg-CnJ-aryl, (C7 ⁇ Cu)-aralky!, (CH3 ⁇ 4)-alkyL .
  • aralkylsulfonamido where an aryl radical may be substituted by 1 to 5 substituents selected from hydroxyl, halogen, cyano, trifiuoro methyl, nitro, carboxyl.
  • substituents selected from hydroxyl, halogen, cyano, trifiuoro methyl, nitro, carboxyl.
  • Ci2 Ci2-ai' 5ox ? (C 6 -C!(s)-aralkyloxy 5 (C6-Ci2)-afylo - ⁇ Cj ⁇ C6)-alkox , (C7-Cs .6 )-ara!koxy-(Cj- CftJ-alkoxy, (GrCs ⁇ -hydroxyalkyL (C Ci ⁇ $) ⁇ ar lo Ci: 8) ⁇ -kyI,. ⁇
  • R ! and R 2 > or R 2 and R 3 form a carbocyclic or heterocyclic 5- or 6- membered aromatic ring;
  • R ! and R 2 , or R 2 and R J together with the pyridine or pyridazine carrying them, form an optionally substituted heterocyclic ring systems selected from thsenopyridines, furanopyridines, pyridopyfidines, pyrimidinopyridines, imidazopyridijies, thiazoiopyridisies.
  • V is S, O, or NR k
  • R k is selected from hydrogen, (Cj-Qj-alkyl, aryl, or benzyl;
  • R 24 , R ⁇ 5 , R.” 6 , and m each case independently of each other have the meaning of R 1 , R ⁇ and R 3 :
  • f is 1 to 8;
  • [002421 g is 0 or 1 to (2f+l);
  • x is Q to 3;
  • h is 3 to 7;
  • Additional compounds according to formula (IV) are substituted heterocyclic carboxyamides described in U.S. Patent No. 5.620,995; 3 ⁇ hydroxypyridine-2- carboxamidoesters described in U.S.. Patent No. 6,020,350; suifonaimdocarbonyIpyridine-2- carboxasnides described in U.S. Patent No, 5,607,954 ; : and sulfo «amidocarbonyl»pyridine-2- carboxamides aud su!fonamjdosarbony!-pyrid!ne-2-carboxaniid.e esters described in U.S. Patent Nos. 5,610, 72 and 5,620,996, All compounds listed in these patents, in particular, those compounds listed in the compound claims and the final products of the working examples, are hereby incorporated herein by reference,
  • b is an integer from 0 to 4.
  • c is an integer from 0 to 4.
  • Z ?s selected from the group consisting of (Cs-Gioj-cycloalkyi, .(CS-CTO)-. • cycloalkyl independently substituted with one or more Y s , 3-30 membered heteroeycloalkyl and 3- 30 membered heieroeycloaikyl independently substituted with one or more Y 1 ; (C 5 - ⁇ GaoJ-aryl, (Cs-Caol-aryl independently substituted with one or more Y ! , 5-20 membered heteroaryl and 5-20 membered heteroary! independently substituted with one or more Y ! ;
  • Ar l is selected from the group, consisting of (Cj-QoJ-a l, (C5-C20) aiyl independently substituted with one or ore Y " , 5-20 membered heteroaryl and 5-20 membered heteroaryl independently substituted with on or more Y 2 :
  • each Y ! is mdependentiy selected from the group consisting of a lipophilic functional group, (C Cso ⁇ ar i, iQ- ⁇ 3 ⁇ 46) ⁇ aikaryJ- 5-20 membered heteroaryl and 6-26 membered alk-lieteroaryl;
  • each Y 2 is independently selected from the group consisting of -it', -OR', - OR", -SR', ⁇ SR", -NR'R', -NO3 ⁇ 4 -CN, -halogen, -trihalomethyl, nihaiomeihoxy, -C(0)R ⁇ - C(0)OR' ; ⁇ C(0)NR'R', -C(0)NR'OR'.
  • each R 1 is independently selected from the group consisting of-H ' , (Ci-Cg)- alkyl, (C2-Cg)-a3kenyl, and (C 2 -!3 ⁇ 4)-aikynyI; and .
  • each R" is independently selected from the grou consisting of (Cs-C2o)-ar l and (Cs-G2o ar l independently substituted with one or more OR', -SR', -NR'R', ⁇ N ⁇ 3 ⁇ 4 > - CN, halogen or trmalomethy! groups, [00262] or wherein c is 0 and Ar is an 1ST substituted urea-aryl, the compound has the structural formula Ya):
  • 3s and W 6 are each independently selected from the group consisting of hydrogen, (Cj-CgJ-aikyS, (C 2 -Cg)-a!kenyI s (Cs-CsJ-alkynyl, (Cj-C l0 )-cycloalkyl (C5-C 20 )- aryl, 5-20 merobered heteroaryl, 5-20 raembered substituted heteroaryl, 6-26 membered alk-heteroaryl, and 6-26 membered substituted alk-heteroaryi; and
  • B ' is independently selected from the group consisting of hydrogen, (Gi- CsJ-alkyl, and (CrCs)-alkynyl.
  • [00269] 28 is hydrogen, nitro, amino, cyano, halogen, (Cj-C4) ⁇ alkyl, carboxy or a metaboHcaliy labile ester derivative thereof; (Gi-C4)-allcyiaierino, di-(GrC4)-aIkylamino, (Cr C ⁇ -alkoxyearboayl, (Cs-C ⁇ -alkanoyl, hydroxy-(CrC4)-alkyl s carbamoyl, M-(Cj- €4) ⁇ alkylcaf amoyf, (Cj-C4) «alkyithio, (Ci-C4)-aIkylsul6nyJ, (C ⁇ Q ⁇ alkylsulfonyl, phenyltfaio, phenylsulfinyl, phenylsulfonyl, said phenyl or phenyl groups being optionally substituted with i to 4 identical or different hal
  • R 29 is hydrogen, hydrox 5 amino, eyano, halogen, (Ci-C -alkyL carboxy or roetaholioaliy labile
  • aikylcarbamoyl ammo-(Ca-Cg)-alkyi3-carb8m yl, N-[(C j -Gt)-alky lam ino-(Ci-Gs)-aik ⁇ !]- carbamoyl, N-[di ⁇ (Ci ⁇ .
  • R 30 is hydrogen, (Ci-CL -alkyI, (C2 ⁇ C4)-alko , halo, nitro, hydroxy, fiuoro- (Ci-G4)-a!kyi- or py idinyi;
  • R 3 ' is hydrogen, (Ci ⁇ C 4 )-aikyi, (C;2 ⁇ C 4 )-alkoxy, halo, nitro, hydroxy, f!uoro-
  • R J ⁇ is hydrogen, hydroxy, amino
  • alkyia ino halo, (Cj-C ⁇ j)-aIkoxy ⁇ (CrC4)-a!koxy,. fluoro-(Gi-C6)-alkoxy, pyrrol id in- 1-yi, piperidino, piperazin-l-yi. or raorpholino, wherein the heterocyclic group is optionall substituted with 1 to 4 identical or different (Ci-C4 alkyi or benzyl; and
  • R 33 and R 34 are individually selected from hydrogen, (Ci ⁇ Gj)-alkyI, and (Cr
  • PH ' is selected from quinazoUnone compounds as disclosed for example in U.S. 2010/0204226, which are represented by formula VII:
  • n 0-3;
  • [00281] 3 is a member independently selected from the group consisting of fraio, - Ci.4alk.yl, -Q.4alky.nyI. -CMaiken ! optionally substituted with halo, ..-GFj, -OCF3, -SCFs, S(0)CF 3 ⁇ 4 -C(0)- ° s -C(0)N- -OH, -NOa, -CN, -OC i alkyl, -SCMalkyl, -S(0) ⁇ C alkyk -S0 2 , -C a1kyl, ⁇ S-R C , -S(0) ⁇ R c , -SQHl c , -SOaN-R 6 , -G ⁇ R C , -NR 3 R fc , 2,3» dihydro-b:enzo[I f 43dioxme, benzo[l,3]dioxole, IH-indoie
  • gheterocycloalkyl optionally substituted with one or more R c members, and two adjacent R ! groups may be joined to form an optionally substituted 3-8 member ring optionally containing one or more O, S or N;
  • R a and R b are independently selected from the group consisting of H, C-j.. 4 alkyi, -C(0)C, ⁇ alkyl, -C(0)-R c , -C(0)NW-R? -SOs-R 0 , ⁇ SQ 2 -C w aSkyl ? phenyl optionally substituted with R d , benzyl optionally substituted with R d or monocyclic heteroaryl ring optionally substituted .with R d ; or
  • R a and b can be taken together with the nitrogen to which they are attached to form an optionally substituted monocyclic heteroeycioaikyl ring containing one or more O- S or N;
  • R* is a -member independently selected from the group consisting of -C3. gcycloalkyl, -Cs.ghetefocycloaikyl, biphenyl, phenyl optionally substituted with one or more R d members, benzyl optioftally substituted with R d , naphthyl, indanyl, 5,6,7,8-tetrahydro- naphthyl. and pyridyl optionally substituted with one or more R d members:
  • R d is a member independently selected from the group consisting of-H, halo, -OH, -C h alky!, -S0 2 -C M aIkyl, ⁇ CN, or-CF 3 , -OCF3, -OCM lk l, -C(0)N3 ⁇ 4 -G ⁇ phenyl, and -0-benzyl; and
  • R 1 i s a member independently selected from the group consisting -of halo, - C M alkyl, -OCF 3 ⁇ 4 -CF 3j -OH, -NO 2 , ⁇ CN, -OC alkyl, -SC H aikyl -SiOK ⁇ alk !, - S0 2 -C M alkyl, -S-R c , -S(0)---R c , -S0 2 -R c , -G-R c , -NR e R b , benzylo y optionally
  • R d substituted with R d , phenyl or mouocyclic heteroaryl optionally substituted with one or more R d members, and -C3.scyci0a.kyl optionally containing O, S or N wherein said -G 3 .
  • scycloalkyl is optionally substituted with R d ;
  • R ! groups are joined to form an aromatic 3-8 membered ring optionally containing one or more O, S or N:
  • two adjacent R J groups are joined to form an optionally substituted 3-8 raembered ring containing one or more O, S or N;
  • n 1, 2 or 3:
  • R a R b is a member -independently selected from th group consisting of -E, ⁇ CH3 f -ClfeCHs, benzoyl, 2,6-diniethylbeozoyl, acetyl, -C(0)NH-phenyl, benzenesulfonyl, methariesu!fonyl, benzyl, 2-met.hyibenzyl, 2-chlonObenzyi,.2,6-dimethylbenzyl, 2,6- difhtorohenzyl, 2-eyanobenzy , 3'C . yanobenzy , 3-carbamoyi-benzyl, 2,6-dtc orobenzy!, 3- cklorobenzyL and 4-methyibenzy
  • R s and R b can be taken together wit the nitrogen to which they are attached to form an optionally substituted N-methytpiperazm- 1 -yl, 3,4-dihydro-iH-isoquinolin-2-yl, piperidinyl, morpholin-4-yl, and pyrro!idinyl;
  • R c is a ' member independently selected from the group consisting of phenyl, cyclohexyl,.4-tert-butyi-ph.enyl, 3 3 4 ⁇ diraethoxy-phenyl- 2,6-dimethy!-phenyl, 3,4,5- trimetftoxy-phenyl, naphihale.n-1- l, 3-ch.lpio ⁇ ph.enyI, 4-chioro-phenyl, 3-raethpxy ⁇ phenyl, : 4- fluoro-pheivyl, 2-fluoro-phenyl, 3-f!uoro-phenyL 3,5-dI-tert ⁇ hutyi-phenyi, 4-oxa-5 ⁇ m ⁇ toly!, 4- oxo-S-o-tolyi, 2,6-dichloro-phenyl, 2,4 ⁇ dichloro-phenyl, 2,5-dichIoro-phenyl,
  • R d is a .member independently selected from the group consisting of ⁇ H, cbloro, fluoro, bromo, iodo, -C M alk l, ⁇ CF 3 ,— QCF 3> -QC walk 1, phenyl, -0-pheny.l, or ⁇ O-benzyi; and
  • R 1 is independentl selected from the group consisting of chioro, fiuoro, bromo, iodo, -N0 2 , -OH, ⁇ CF 3 , -CH 3 , -CH 2 CH.3 ⁇ 4 ⁇ CH 2 CH 2 CH3, -OCF 3 , -OCR-?, - QCH 2 GH 3 , -SCH 3 , -SGF3 ⁇ 4 -S(0)CF 3s -S0 2 CH 3 , -NH3 ⁇ 4 -N(CH 3 ) 2s -NH(CH 2 01 ⁇ 4).
  • PHis are selected from benzp imidazole compounds as disclosed for example in U.S. 2611/0046132, which is expressly incorporated herein by reference in its entirety.
  • benzo imidazole compounds are represented by formula Vill:
  • n is 2-4;
  • each R 1 is independently selected from H, halo, -C ⁇ alkyl, -Cwjeycloaikyl-
  • gcyeloaJkyi optionally containing one or more O, S or N wherein said -C3.gcycloaikyi is optionally substituted with and two adjacent R ! groups may be joined to form an optionally substituted 3-8 member ring optionally containing one or more O. S or N;
  • R a and R are each independently H, C M aiky! 5 - ⁇ C(0)C 3- alkyl, -C(0) ⁇ R c , - G(0)CHr-R s , C M alky:l-R*, .-.S02-R c , - SOr € 3 ⁇ 4 , phenyl optionally substituted with R d 5 benzyl optionally substituted with e or monocyclic heteroaryl ring optionall substituted with R d ; or
  • R a and R fa can be taken together with the nitrogen to wh ich they are attached to form an optionally substituted monocyclic heterocycloalkyl ring optionally containing one or more hete oaioms;
  • R c is -Cs-scycioalkyL phenyl optionally substituted with R d , benzyl optionally substituted with R d , or a monocyclic heteroaryl ring optionally substituted with R*;
  • 11 ⁇ is independently -H, halo, » OFL -CMalk i or -C -pef aloalk l, trifluoroC alkoxy. . -OCwalkyl, -O-pheny!, or -Q-benzyi;
  • R ⁇ and R 3 are both H, -CF 3 , or Cj-jalkyl; each Z is C or N. provided that no more that*, two Z's 5 can simultaneously be N; and
  • Some embodiments of the compounds of formula VI 0 have any one or more of the following: '
  • R 2 and R 3 are each -H;
  • R f is independently selected from the group consisting of H, halo, ⁇ €F;, - QCF. ⁇ phenyl (optionally substituted or unsubstituted with up to three ⁇ - €3 ⁇ 4 halo, -OH, Ci_ 4alkyl, arid -OCF3), phenoxy (optionally substituted or imstibstituted with up to three halo.
  • CM alk t, CM alkoxy, and -OCFj besnzyloxy-phenyl (optionally substituted or
  • phenylrnethauesulfoiiyi (optionall substituted or unsubstituted with up to three halo, C 3 .gcycloalkylmethyi, -CF 3 , and -OCF3).
  • the optionally substituted 3-8 mernbered ring is aromatic:
  • each R ! is independently selected from FT, halo, -CF-5, -C3.
  • gcycloalkyl, ⁇ M3 ⁇ 4 30 -NH-CMalkyl, - M-SOj-Cs-scycloalkyl, -NH-S0 2 -C alkyl, -NH-C(03-C 1 .
  • PHIs are suitably selected from triazolopyridine compotmds as disclosed for example in U.S. 2011 /0077267, which are represented by formula ⁇ , or a pharmaceutically acceptable salt thereof, or a solvate thereof:
  • [00315] is a grou represented by any of the following formulas :
  • R 1 is (1) a hydrogen. atom, (2) a 0 ⁇ ,..3 ⁇ 4 aikyi group. (3) a Ce-i4 ar i gr up. (4) a C3..g cycioaikyl group, (5) a C 6 -R aryl-C]. 6 alkyi. group, or (6) a C3-8 cycloalfcyl-Ci.6 alky I group;
  • R 2 is ⁇ 1 ) a hydrogen atom, (2) a Cwo a3 ⁇ 4 l group, (3) a C s . ! aryl group optionally substituted by the same or different 1 to 5 substituent ' s selected from the followin grou B, (4) a Cj.g eycloalkyi group optionally substituted by the same or different 1 to 5 substttuents selected from the following group B.
  • R 3 is (0 * hydrogen atom, (2) a halogen atom, (3) a C ⁇ alkyl group,. (4) a CM* aryi group, (5) a Qj-g eyeloaikyl group, or (6) a Cs-t* aryl-Cikos6 alkyl group; and R ** and R 5 are each independently (1) a hydrogen atom, or (2) a Cus alkyl group, grou 8; (a) a halogen atom, (b) a eyano group, and (e) a aio-C 1-6.alkyl groiip,
  • Fi lls are suitably selected from the pyrimidinyl compounds as disclosed in IX S. Paten No.8324208, which are represented by foraiala XI (003201
  • Rl and R4 are each independently selected from the group consisting of hydrogen,— NK5R6, CI -CIO alky.L C2-C10 alkenyl, C2-C10 alkynyl, C3-C8 eyeloaikyl, C3-C8 eycIoa!kyl-Cl ⁇ C10 alkyl, C5-C8 cycloalkenyL C5-C8 cyeloalkenyl-Cl-ClO alkyL C3-C8 heteroeycioalkyl, C3-C8 heterocycioalkyl-Cl-ClQ alkyl, aryi, aryl-CI-CiO alkyl, heteroaryl and heterparyI-Cl . -CI.O alkyl;
  • R2 is— -NR7R8 or— OR9;
  • R3 is H or C1-G4 alkyl
  • R5 and R6 are each independently selected from the group consisting of hydrogen, CI -CI O alkyl C3-C8 eyeloaikyl, C3-C8 eycloa!kyl-Cl-CI 0 alkyl, C3-CS heterocyeloalkyl, C3-C8 heterocycloalkyl-Cl-CIO alkyl, aryi, aryl-CI-Cl O alkyl, heteroaryl, heteroaryl-Cl-ClO alkyl, - » -C ⁇ 0)Cl-C4afky!,— C(0)C3-C6 eyeloaikyl,— C(0)C3-C6 h terocycloa!kyl,— €(G)aryl, ⁇ C(Q) heteroaryi and— S(0)2Cl-C4aIkyl, or, when RS and R6 are attached to the same nitrogen, 5 and R6 taken together with the nitrogen to which they are attached form
  • R7 and R8 are each independently selected from the group consisting of hydrogen, Cl-Cl 0 alkyl, C2-C10 aikeny!, C2-C ! 0 aikyn L G3-C8 cycloalkyl, C3-CS hetemeycloaikyl, ar l and heteroaryl, and R9 is H or a cation, or C l-Cl 0 alkyl which is unsubstittrted or substituted with one or more substituents, suitably from 1 to 6 substi uents, suitably from. 1 to 3 substituents, independently selected from the group consisting of C3 ⁇ C6 cycloalkyl * heterocycloaikyl, ar l and heteroaryl;
  • X is O or 3 ⁇ 4
  • Rl , R2, R3, R4, R5, R S R7, R8, R9 is unsubstrtuted or, where possible, is substituted with one or more substitueuts, suitably from 1 to 6 substituents, suitably from 1 to 3 substitnents, independently selected from C1-C6 alkyl,. C1 -C6 haioalkyl, halogen,—OR 10,— NR5R6, oxo, oyano, nitro,— C(O)R10 5 —
  • heterocycloaikyl aiy 3, C1 -C6 alkyl-aryl, heteroaryl and C1-C6 alkyl-heteroaryl, wherein R5 and R6 are the same as defined above and R10 is selected from hydrogen, Cl-Ci Oalky!, G2 ⁇ Cl Oalkenyl, C2-C10 alkynyl,— C(G)C1-C4 alkyl,— C(0)aryl, ,— (0) heteroaryl,—
  • PHIs are suitably selected from the pyridine compounds as disclosed in U.S. Patent Mo.781 1595, which is represented by formula .XII;
  • R and Rl are each independently: i) hydrogen; «) substituted or unsubstituted phenyl; or iii) substituted or imsubstituted heteroaryl; said substitutions being; i) C1-C4 linear, branched, or cyclic alkyl; ii) C1-.C4 linear, branched, or cyclic alkoxy; iii) CI-C4 linear, branched, or cyclic haloa!kyl; iv) halogen; v)— --CN; vi)— NHC(0)R4 vit)— G(0)NR5aRSb; or viii) heteroaryl; or ix) two substitutions are taken together to form a .fused ring having from .5 to 7 atoms;
  • R.4 is C1 -C4 linear, branched, or cyclic -alkyl
  • R5 and R5 are each independently; i) hydrogen; ii) C1-C4 linear, branched, or cyclic alkyl; or ili) R5a and R5b .are taken together to forirt a ring having from 3 to 7 atoms;
  • R2 is: i) - » -OR6; o rii)— NR7aR7b;
  • R6 is hydrogen or C1.-C4 linear, branched, or cyclic alky!
  • R7a and R7b are each independently: i hydrogen; or ii) C1-C4 linear, branched, or cyclic alkyl; or iii) R?a and R7b are taken together to form a ring having from 3 to 7 ring atoms; R3 is hydrogen, methy l, of ethyl;
  • L is a linking unit having the formula: -[C(RSaRSb)] n -
  • R8a and R8b are each independently hydrogen, methyl, or ethy l;
  • the index n is from 1 to 3;
  • R9 is hydrogen or methyl; or a pharmaceutically acceptable salt thereof; provided R and R 1 are not both hydrogen,
  • Pffls are selected from the compounds disclosed in U.S. Patent No. 7608621. which are represenetd by formula XJIi:
  • R1 is selected from the group consisting of hydrogen,— NR5R6, Cl- Cl 0alky C2-Cl0alkBn l, C2-C 0 alkyny!, C3-C8 eycloalkyl, C1-G 10 alkyl-C3-C8 cycloalkyl, C5-C8 cycloalken l. CI -C10 alkyi-CS-Cg eyeloalkenyl, C3-C8
  • heteroeyelqalkyl Cl-CIO- alkyl-C3-C8 h ⁇ teiocydoalkyi, aryl Cl-Cl 0 aikyi-aryL heteroaryl and Cl -CI 0 alkyl-heteroaryi;
  • [00347] 4 is selected from the group consisting- of hydrogen, COQR9,,CQ R7R8, — NR5R.6, C1 -C30 alkyl C2-CI0 alkenyl, C2-C1Q alkynyl, C3-C8 cycloalkyl.
  • R2 is "- R7R8 or—OR9;
  • R3 is H or Cl-C4a!kyl
  • R5 and R6 are each independently selected from the group consisting of hydrogen, C1 -C10 alky I, C3-C8 cycloalkyl, Cl-Cl 0 aIkyl-C3-C8 cycloalkyl, C3-C8 fieterocyetoalkyL Ci -CIO alkyl-C3-C8 heterocycloalky aryl, Cl -Cl 0 alky!-aryl, heteroaryl, C l -Cl 0 alkyi- etefoaryl,— CO(Cl-C4 alkyl),— CO(C3-C6 cycloalkyl),— CO(C3-C6 heterocycloalkyl),— CO(aryl), - ⁇ CO(heteroaryl), and— SG2.(C1-C4 alkyl); or R5 and R6 taken together with the nitrogen to which they are attached form a 5- or 6- or 7-membered saturated ring optionally containing
  • R7 and R8 are each independently selected from the group consisting of hydrogen, Cl-CIO alk l, C2-C10 alkenyl, C2-C1Q alkynyl, C3-C8 cycloalkyl, C3-C8 heieroeycloalkyi, .-aryl and heteroaryl:
  • R9 is H or a cation, or Cl -C 1 Oaikyl which is unsubstituted or substituted with , one or more substittients- independently selected from the group consisting of C3-C6 .cycloalkyl, heterocycloalkyl, aryl, and heteroaryl;
  • R9 is imsubstituted or, where possible, is- -substituted with one or more substituents independentl selected from the group consisting of C1-C6 alkyl, aryl, heteroaryl, halogen, OR I 0,— NR5R6, cyan ⁇ , nitro,— C(0)R10, -TMC(0)0R10,— SR10,— S(0> 10,—
  • FHis include without limitation substrate-based inhibitors, such as 3- e omethylenepro!ine peptide like compounds (Tandon et l (1998) Biaorg, Me Chem. Lett. 8:1 139-44), derivatives of proline, derivatives of 4(S)hydroxy proline, and derivatives of 4-keto proline.
  • substrate-based inhibitors such as 3- e omethylenepro!ine peptide like compounds (Tandon et l (1998) Biaorg, Me Chem. Lett. 8:1 139-44), derivatives of proline, derivatives of 4(S)hydroxy proline, and derivatives of 4-keto proline.
  • Suitable compounds include cofaetor-based inhibitors such as 2-oxogkitarate analogues, ascorbic acid analogues and iron chelators such as desferoxamine (DFO) and the hypoxia mimetic cobalt chloride (CoClj), or other factors that may mimic hypoxia.
  • cofaetor-based inhibitors such as 2-oxogkitarate analogues, ascorbic acid analogues and iron chelators such as desferoxamine (DFO) and the hypoxia mimetic cobalt chloride (CoClj), or other factors that may mimic hypoxia.
  • DFO desferoxamine
  • CoClj hypoxia mimetic cobalt chloride
  • prolyl hydroxylase inhibitors such as defers prone, 2,2' ⁇ dipyridyS 5 eielopirox, dimethy!oxali l glycine (DMOG), L-Miraosii e (Mlm) and 3 -Hydroxy- 1,2-dimethy 1-4(1 H)- Pyridone (OH-pyridone), DMOG is a ceil permeable, competitive inhibitor of PHDs, It acts to stabilize HIF-a expression at normal oxygen tensions in cultured cells, at concentrations ' between 0,1 and 1 raM.
  • DMOG 2,2' ⁇ dipyridyS 5 eielopirox, dimethy!oxali l glycine
  • Mlm L-Miraosii e
  • OH-pyridone 3 -Hydroxy- 1,2-dimethy 1-4(1 H)- Pyridone
  • DMOG is a ceil
  • PHD inhibitors encompassed by the present invention include, but are not limited to, oxoglutarates, heteroeye lie carboxamides, phenanihrolines, hydroxamates, and heterocyclic carbony 1 glycines (including, but not limited to, pyridine carboxamides, quinoline carboxamides, isoqtiirtoline carboxamides, cirmolirie carboxamides, beta-earboiuie carboxamides, including substituted qumo!ine-2-carboxamides and esters thereof; substituted isoquinoline-3-carboxamides-and N-subsiititted ary Isn!fonyiam ino hydroxar c acids (see, e.g.
  • HiF-a potentiating agents include compounds which interact or modulate the RTF- J pathway, A general report of sueh compounds and the pathways associated with HIF- ⁇ levels and H1F- la activity are .-disclosed in Semenza (2003, Nature Rev. Cancer 721) RatciifFe et al (2003, Nature Medicine 677) and W outers .et al (2004, Drug Resistance Updates 25) (each of which is incorporated herein by reference in its entirety), illustrative such compounds include without limitation rapamyc in (see, e.g., Abraham (2004) Current Topics in
  • resveratrol see,, e.g., Cao et al, (2004) Clin Cancer Res. 10(15):5253 ⁇ 63
  • Flavonoids see, e.g., Rasebe et al (2003) Biol Pharm Bulletin 26(1 0):1379-1383: Fan et al (2003) Eur J Fharm. 4Sl(l);33-4G); Majamaa et al. ( ⁇ 9 ) -Eur J Biochem 138:239-245; and Majamaa et al (1985; Biochem J.
  • the HJF-a potentiating agents include the following compounds or derivatives or analogs thereof: Quercetm, 2,4-Diethyipyridme dicarboxylat (2,4-DPD), Dimeihy loxaloy iglyci i>e (DMOG), 2 ⁇ (oxaiyl-amino)-propiome acid, N-oxalyl glycine (NOG), [2,2*] Bipyridinyl, Dihydroxy benzoic acid, Pyridine 2,4- dicarboxylic acid- 4-Hydix>xy s quinoline-3-carbo ' nyi glycine, and 8- tro-7-Qxo- 48,7,8,1 Ob-tetrahydro-p J jphenanthro!ine-S-carboxyiic acid
  • growth factors and cytokines are known to stabilize HiF-a under normoxic conditions, including insulin, insulin-like growth factor, epidermal growth factor, mterleukin- ⁇ (Zelzer df. ( ⁇ 99%) EMBO J 17:5085-94; Feidser eiei (1999) Cancer Res 59:391 5-8); Richard et. al. (20 Q0) J Biol Chem 275:26765-71 ; Goriac* et al. (2001) Ore Res 89:47-54; Haddad et al. (2001) FEBSLeti 505:269-74; Stieh .
  • hypoxia-inducible factor-a HIF-a stabilizers for enhancing erythropotesis.
  • a novel proline hydroxylase inhibitor BP049-33 and its manufacture with Ophioboltis Furui, Megumi; Takashima, Junko; Mikawa, Takasht; Yoshikawa, Nobuji; Ogishi, Haruyuki. (Mitsubishi Kasei . ., Japan).
  • iP 04074163 A2 (each herein incorporated by reference).
  • HIF-a contains an oxygen dependent degradation domain OD.DD), which has both an ⁇ erminai portion (NODDD) and a C-terrninal portion (CODDD).
  • HIF-a subunit Hydroxy lation at any of the prolyl residues in the ODDD targets the HIF-a subunit is the vHL protein for degradation; therefore,, blocking the interaction of vHL with HIF-a. leads to build-up of HlF-a.
  • peptides encoding the HIF-a NODDD or CODDD are capable of ' up-regulating: HIF- regulated transcripts in vitro (William, ei at. (2002) Proc Natl Acad S i, USA 99(16): 10423-10428) either by saturating the PHD enzymes or vHL binding, indicating that peptide therapy may also be efficacious.
  • HIF- ⁇ mRNA by increasin its transcription.
  • Compounds useful in increasing HIF-ct transcription include, for example, o- substituted carbamoyl-phensxyacetic acids, as disclosed for example by Agani et at. (1998, Mel Pharmacol 54:749-754).
  • the invention not only encompasses known HIF-a potentiating agents but also HlF-a potentiating agents identified by any suitable screening assay. Accordingly, the present invention extends to methods of screening for modulatory agents that are useful for potentiating HlF-a and, in turn, enhancing a hematopoietic function of a mobilizer of hematopoietic stem cells and/or progenitor cells.
  • the screening methods comprise (1) contacting preparation with a test agent, wherein the preparation comprises (I) a polypeptide comprising an amino acid sequence corresponding to at least a fragment of a HlF-a-inhibitor interacting polypeptide selected from a PHD (e.g., a HIF-a PHD) polypeptide, a ⁇ - 1 polypeptide, a vHL polypeptide, or a variant or derivative of any one of these); or (ii) a polynucleotide comprising at least a portion of a genetic sequence
  • a PHD e.g., a HIF-a PHD
  • a transcriptional control sequence that reulates the expression of a gene selected from a PHD gene, a FI -I gene or a vHL gene, wherein the gen etic sequence is opera ' b ly linked to a reporter gene; and (2). detecting a chang in the level or functional activity of the polypeptide, or an expression product of the reporter gene, relative to a reference level o functional activity in the absence of the test agent.
  • a detected reduction in the level or activity of polypeptide, or expression product, relative to the reference level or functional activity indicates that the test agent is useful for enhancing a hematopoietic function of a mobilizer of hematopoietic stem cel ls and/or progen itor cells.
  • this is confirmed by analyzing or determining whether the test agent enhances a hematopoietic function of ' a niobilizer of hematopoietic stem cells and/or progenitor cells.
  • the test agent inhibits the prolyl hydrolase activity of the PHD,, as determined by: contacting a.
  • the test agent inhibits the activity of FIH-1, as determined by: contacting a preparation comprising (i) the polypeptide tha comprises an amino acid sequence corresponding to at least a fragment of a FIH- 1 or variant, or derivative thereof and (ii) a HfF- 1.
  • the test agent inhibits the activity of vHL, as determined by: contacting a preparation comprising (A) the polypeptide that comprises an amino acid sequence corresponding t at least a fragment of a vHL or variant or derivative thereof and (B) a HIF-a polypeptide or fragment thereof with the test agent and measuring whether the test agent reduces de radation of the HIF-a polypeptide or fragment thereof.
  • the test agent may inhibit binding between HIF-a.
  • test agents may bind to the HIF-a or to the polypeptide and test positive when they reduce or abrogate the binding of the HIF-s with the polypeptide.
  • Modulators falling -within the scope of the present invention include antagonists of the level or functional activity of HEF-a-inhibitory. interacting polypeptides (e.g., PHD FIH-1 or vHL), including antagonistic antigen-binding molecules, and inhibitor peptide fragments, antisense molecules, ribozymes, RNAi molecules and co-suppression molecules as well as polysaccharide and lipQpolysac.chari.de inhibitors ofHIF-a-inbibitoiy interacting polypeptide function.
  • interacting polypeptides e.g., PHD FIH-1 or vHL
  • Candidate agents encompass, numerous, chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 Dalton.
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding,, and typically include at least an amine, carbonyl, hydroxyl or carboxy! group, desirably at least two of the function onal chemical groups.
  • the candidate agent often comprises cyclical carbon or ⁇ heterocyclic structures or aromatic or polyaiOmatic structures substitoted with one or more of the above functional groups.
  • Candidate agents are also found among biomolecul.es including, but not limited to: peptides, saccharides, tatty acids, steroids, purines, pyrimidines, derivatives, structural analogues or combinations thereof.
  • Small (non-peptide) molecule modulators of a HIF-a-inhibitory interacting polypeptides are particularly advantageous.
  • small molecules are desirable because such molecules are more readily absorbed after oral administration, have fewer potential antigenic determinants, or are more likely to cross the cell membrane than larger, protein-based pharmaceuticals.
  • Small organic molecules may also have the ability to gain entry into an appropriate cell and affect the expression of a gene (e.g. , by interacting with the regulatory region or transcription factors involved in gene expression); or affect the activity of a gene by inhibiting or enhancing the binding of accessory molecules,
  • libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced.
  • natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries.
  • pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alfcylation, esterifkation, amidifieation, etc. to produce structural, analogues.
  • Screening may also b directed to known pharmacologically active compounds and chemical analogues thereof.
  • Screening for modulatory agents according to the invention can be achieved by any suitable method.
  • the method may include contacting a cell expressing a polynucleotide corresponding to a gene thai encodes a HlF-a-inhtbitory interacting polypeptide with an agent suspected of having the modulatory activity and screening for the modulation of the level or functional activity of the HTF-c - inhibitory interacting polypeptide, or the modulation of the level of a transcript encoded by the polynucleotide ⁇ or the modulation of the activity or expression of a downstream cellular target of the polypeptide or of the transcript (hereafter referred to as target molecules).
  • Detecting such modulation can be achieved utilizing techniques including, but not restricted to, ELISA, cell-based ELiSA, inhibition ELISA, Western blots, immunoprecipitation, slot or dot blot assays,
  • irnmunostafnmg, RiA scintillation proximity assays
  • fluorescent immunoassays using antigen-binding molecule conjugates or antigen conjugates of fluorescent substances such as fluorescein or rhodamine, Ouchterlony double diffusion analysis
  • immunoassays employing an avidin-biotin or a sireptavidm-bibtiri detection system and nucleic acid detection assays including reverse transcriptase polymerase chain reaction (RT-PCR).
  • RT-PCR reverse transcriptase polymerase chain reaction
  • a polynucleotide, from which a HIF-a-inhibitpry interacting polypeptide is regulated or expressed may be naturall occurring in the celi which is the subject of testing or it may have been introduced into the host cell for the purpose of testing.
  • the naturally-occurring or introduced polynucleotide may be const itutiveiy expressed - thereb providing a model useful in screening for agents which down-regulate expression of an encoded product of the sequence wherein the down regulation can be at the nucleic acid or expression product level.
  • polynucleotide may comprise the entire coding sequence that codes for the HIF-a-inhibitory interacting polypeptide or it may comprise a portion of that coding sequence (e.g., the I igand-binding. domain of the HIF-a-inhibitory interacting polypeptide) or a portion that regulates expression of the corresponding gene that encodes the ⁇ HIF-a-inhibitory interacting polypeptide e.g., & PHD promoter, & FI -1 promoter, or a vHL promoter).
  • the promoter that is naturally associated with the polynucleotide may be introduced into the cell that is the subject of testing.
  • detecting dulation of the promoter activity can be achieved, for example, by operabi linking the promote to a suitable reporter polynucleotide including, but not restricted to, green fluorescent protein (GFP), fueiferase, -ga!actosidase and catecholamine acetyl transferase (CAT), Modulation of expression ma be determined by measuring the activity associated with the reporter polynucleotide.
  • GFP green fluorescent protein
  • fueiferase fueiferase
  • CAT catecholamine acetyl transferase
  • These methods provide a mechanism for performing high throughput screening of putative modulatory agents such as prole inaceous or non-proteinaceotis agents comprising synthetic, combinatorial, chemical and natural libraries. These methods will also facilitate the detection of agents which bind either the polynucleotide encoding the target molecule or which modulate the expression of an upstream molecule, which subsequently modulates the expression of the polynucleotide encoding the target molecule. Accordingly, these methods provide a mechanism of detecting agents that either directly or indirectly modulate the expression or activity of a target molecule according to the invention.
  • compounds are screened for hydroxylase activity.
  • Assays for hydroxylase activity are standard in the art. Such assays can directly or indirectly measure hydroxylase activity.
  • an assay can measure hydroxyiaied residues (e.g.. proline, eic.) present in the enzyme substrate, e.g.., a target protein, a synthetic peptide mimetic, or a fragment thereof (se , e.g. , Palmer ini ef al. (3985) J Chm atogr 339:285- 292.)
  • a reduction in hydroxy lated residue (e.g. , proline, etc.) in the presence of a compound is indicative of a compound that inhibits hydroxylase activity.
  • assays can measure other products of the hydroxylation reaction (e.g., formation of succinate from. 2- oxoglutarate (see, e.g, T Cun!iffe ei l. ( ⁇ 9 ) Biockem 7240:61 7-619; and auie and Gunzter (1 90) Anal Biac em 184:2 1 -297),
  • Procedures such as those described above can be used to identify eompoimds that inhibit HIP hydroxylase activity.
  • Target protein used in the assa may include HIFa or a fragment thereof, e.g., HIF(556-575).
  • Enzyme ma include, e.g., HIF prolyl hydroxylase (see, e.g., GenBank Accession No, AAG33965, etc.) obtained from arty source. Human HIF prolyl hydroxylase is preferred.
  • Enz me may also be present in a crude cell lysate or in a partially purified form. For example, procedures that measure HIF hydroxylase activity are described in Ivan et al (2001.
  • a. suitable compound is one that stabilizes HlF-a.
  • HIF prolyl hydroxylase prevent or reduce the hydroxylation of the HIF subunit of the HIF protein.
  • This lack of hydroxy iated proline leads to the stabilization (often referred to as activation) of HIF
  • Determination of the stabilization of HIF by a compound can be used as an indirect measure of the ability of the compound to inhibit HIF prolyl hydroxylase.
  • the ability of a compound to stabilize or activate HTF-a can be measured, for example, b direct measurement of HIF-1 ct in a sample, indirect measurement of HIF- l a, e.g. , by measuring a decrease in HIF- 1 a associated with the vHL protein (see, , e.g., International Publication No.
  • WO 200Q/699Q8 or activation of HIF responsive target genes or reporter constructs (see, e.g., U.S. Patent No. 5,942,434). Measuring and comparing levels of HIF and/or HIF-responsive target proteins in the absence and presence of the compound will identif eompoimds that stabilize .HIF-1 a and/or activate HIF.
  • Suitable compounds for use in the present methods may be identified and characterized using the assay described in International Publication No, WO 2005/1 18836, or in Example 10 of International Publication I3 ⁇ 4Q. WO 2003/049686, both of which are incorporated herein by reference in their entirety. Compounds identifiable by these assays are specifically envisaged for use in the present invention.
  • test agents are screened using the assays disclosed for example in U.S. 2004/0146964, U.S. 2005/0214894, U.S. 2008/0213404, U.S. 2010/0272726 and U.S. 201 103:0.10.95, each of which are . incorporated by reference herein in their entirety.
  • Compounds may be further tested in the animal models to identify those compound having the most potent in vivo effects. These molecules may serve as 'iead compounds" for the further development of pharmaceuticals by, for example, subjecting the compounds to sequential modifications, molecular modeling, and other routine procedures employed in rational drug design.
  • agents that decrease the expression or function of a chemokine (the function being the binding of the chemokine to its receptor and further signaling), particularly
  • CXCL12 as well as those that block or antagonize the chemokine receptor, CXCR4.
  • the mobilization agent ma be an agent that decreases the expression or function of a chemokine, more particularly, CXCL12, also known as SDF-L
  • CXCL12 also known as SDF-L
  • the human amino acid sequence of SDF-1 corresponds to GenBank accession number NP_000600.
  • the alpha isoform has GenBank accession number
  • the beta isoform has GenBank accession number P_000600, The gamma isoform. has GenBank accession number P_001029058.
  • the mobilization agent may be an agent that blocks or antagonizes a chemokine receptor, in particular, CXC 4.
  • CXC 4 The human amino acid sequence of CXCR4 corresponds to GenBank accession number CAA 12166.
  • Chemokines are a superfamily of chemoattractant proteins. Chemokines regulate a variety of biological responses and they promote the recruitment of multiple lineages of leukocytes and lymphocytes to a body organ tissue. Chemokines may be classified into two families according to the relative position of the first two cysteine residues in the protein. In one family, the first two cysteines are separated b one amino acid residue, the CXC chemokines, and in the other family the. first two cysteines are adjacent, the CC chemokines. Two minor subgroups contain only one of the two cysteines (G) or have three amino acids between the cysteines (CX3C). In.
  • the genes of the CXC ehemokin s are clustered on chromosome 4 (with the exception of SDF-l gene, which has been localized to chromosome 10) and those of the CG ehemokines on chromosome 17.
  • CXC ehemokine receptor 4 CXC ehemokine receptor 4
  • G 1 cell surface receptor 4
  • HUMSTR Federsppiel, B., Duncan, A. M. V. s Deianey, A., Schappert, K,, Clark-Lewis, L and Jirik, F. R. (1993) Genomics 16, 707-71
  • Fusin Feng, Y,, Breeder, C. C, ;
  • CXCR4 is widely expressed on cells of hematopoietic origin, and is a major co-receptor with CD4 fo human immunodeficiency virus 1 (HiV-1) (Feng, Y., Broeder, C. C, Kennedy, P. E., and Befger, E. A. (1996) HIV- 1 entry cofaetor: Functional eDN A cloning of a seven- transmembiane Q protein-coupled receptor. Science 272, . 872-877).
  • HiV-1 human immunodeficiency virus 1
  • Ghemokines are thought to mediate their effect by binding to seven transmembrane G protein-coupled receptors, and to attract leukocyte subsets to sites of inflammation (Baglionmi et at (1998) Nature 392; S65-56S). Many of the ehemokines have been, shown to be constitutivejy expressed in lymphoid tissues, indicating that they may have a homeostatic function in regulating lymphocyte trafficking between and within lymphoid organs (Kim and Broxmeyer (1999) J. Leuk. Biol 56: 6-15).
  • Stromal ceil derived factor one also known as CXCL12 is a member of the CXC family of ehemokines that has been found to be constitutively secreted from the bone marrow stroma (TasMro, (1993) Science 261 , 600-602).
  • the human and mouse SDF-l predicted protein sequences are approximately 92% identical.
  • Stromal cell derived factor- la (SDF- la) and stromal cell derived faetor-l p (SDF-l ⁇ ) are closely related (together referred to herein as SDF-l).
  • SDF-l a and SDF-l ⁇ are known, as are the genomic sequences encoding these proteins (see U.S. Pat. No. 5,563,048 issued 8 Oct. 1996, and U.S. Pat. No. 5,756,084 issued 26 May 1998).
  • Biological effects of SDF-1 may be mediated by the chemokine receptor CXCR4 (also known as fwsin or LESTR). which is expressed on mononuclear leukocytes including hematopoietic stem cells.
  • CXCR4 also known as fwsin or LESTR
  • SDF- 1 is thought to be the natural ligand for CXCR4
  • CXCR4 is thought to be the natural receptor for SDF-1 -(Nagasawza et at (1997) Proc. Natl Acad.
  • SDF-1 is functionally distinct from other chemokines in that it is reported to have a fundamental roie in the traffickmg, export and homing of bone marrow progenitor cells (Aiuri, A., et at (1 96) X Exp. Med, 1 85, 1 1 1-120 and Nagasawa, T., et al (1996) Nature 382, 635-638). SDF-1 is also structurally distinct in that it has only about 22% am ino acid sequence identity with other CXC chemokines.
  • CXCR4 may be selected from small organic molecules, poly peptides, nucleic acids and carbohydrates.
  • the polypeptides that decrease the expression of CXCL 1.2 may be selected from the group consisting of a cytokine, a colony stimulating factor, a protease or & chemokine other than CXCL 12,
  • the cytokine ma be selected from the group consisting of interleukin-l (IL-1), interleakin-3 (IL-3), interfeukfn-6 (IL-6), interieukin-l 1 (IL- ' i l), interleukin-7 (IL-7) and interteukln-12 (I.L12).
  • the protease ma be selected from the group consisting of a metal loproteinase (like MMP2 or MM.P9) a serine protease, (like cathepsin G, or elasta.se) a cysteine protease (like cathepsin K) and a dipeptidy! peptidase- 1 (DDP-1 OR CD26),
  • the chemokine other than CXCL 12 may be selected from the group consisting of IL-8, MIP-1 a and Gro$
  • the colony stimulating- factor may be selected from the group consisting of granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony
  • the nucleic acid may be a DNA or an RNA molecule.
  • the nucleic acid may be a small interfering RNA (siRNA) molecule or an antisense molecule specific for CXCL 12 or
  • the carbohydrate may be a sulfated carbohydrate selected from the group consisting of Fucoidan and sulfated dextran.
  • the mo Uizer(s) is(are) are selected from colony-stimulating factors such as G-CSF and GM-CSF.
  • erythropoietin which is now commonly used among cancer patients undergoing chemotherapy to maintain hemoglobin in the near normal range, also has some abilit to mobilize CD34* cells
  • SCF stem cell factor
  • polysaccharides such as zymosan
  • chemokiiies such as IL-8 and Gro- ⁇
  • growth factors such as vascular endothelial growth factor (VEGF), and CX.C 4 antagonists.
  • the mobilizer or at least one of the mobiiizers used in combination wit a HlF-a potentiating agent is G-CSF or GM-CSF, or their variants, derivatives or analogs.
  • G-CSF G-CSF
  • GM-CSF GM-CSF
  • Examples of functional G-CSF variants include any proteins, peptides or fragments thereof that are at least 70, 75, 80, 85, 90 or 95% sequence identity or similarity to full-length human G-CSF amino acid sequence or its nucleotide sequence.
  • Modifications of G-CSF to improve functionality or resident serum clearance include .but are not limited to poly ethylenegly col and poly ethyl enegly col derivatives thereof, glycosylated forms
  • G-CSF or the drug name Filgrastim, is currently bein sold as Neupogen® and its polyethylene glycol modified or pegylated form, with the drug name Pegfilgrastim, sold as NeulastaTM.
  • GM-CSF The coding sequence and amino acid sequence of GM-CSF are known as well as var ious methods employed to produce recombinant proteins (U.S. Pat. No. 5,641,663, incorporated herein by reference).
  • Examples of functional GM-CSF variants include any proteins, peptides or fragments thereof that are at least 70, 75, 80, 85, 90 or 95% sequence identity or sim larity to full- length human GM-CSF amino a id sequence or its coding sequence.
  • Modifieations of GM-CSF to i mprove functionality or resident serum clearance include but are not limited to pqlyethyleneglycol and polyethyleneglycol derivatives thereof, glycosylated forms, norleucine analogs, addition of amino acids at either terminus to improve folding, stability or targeting, and fusion proteins.
  • An increase in biological or functional activity over the native peptide may reduce the amount of dose ⁇ and/or the time period required for treatment.
  • Any chemical or biological entity thai functions similar to GM-CSF can also be employed.
  • Examples of GM-CSF, or the drug name Sargramostim, which are currentl being sold include Leukine®, Leucoinax.® and Leucotropin®.
  • G-CSF or a variant, derivative or analog thereof, is used either alone or in combination with another mobilizer of HSPCs
  • the HTF- potentiating agent is a PHI (e.g., a small molecule PHI including ones selected from compounds represented by any one of formulae J- IX supra).
  • the mobilizer or at least one of the rnobilizers used in combination with a HIF-a potentiating agent is a CXCR4 antagonist
  • illustrative CXCR4 antagonist ' s include aromatic-linked polyamine macrocyclic compounds, as described for example in U.S. Patent No. 5,583, 131, reissued as U.S. RE 42,152, which is expressly incorporated herein by reference in its entirety.
  • the CXCR4 antagonist is ⁇ , ⁇ - [ 1 ,4 ⁇ phenyknebis(methy lene)]-bis ⁇ 1 ,4,8, 1 1 -tetra-azacyclotetradecane (A DS 100;
  • small molecule CXCR4 antagonists may be selected from macrocyclic compounds disclosed in U.S. Pat Appl. Pub.. No. 2012/0301427, which is expressly incorporated herein by reference in its entirety. These compounds comprise a "core" nitrogen atom surrounded by three pendant groups, wherein two of the three pendant groups are suitably henzimidazoiyi methyl and tetTahydroquinolmyl, and the third is a pendant group contains an additional nitrogen.
  • small molecule CXCR4 antagonists include compounds disclosed in U.S. Pat. Appl. Pub. No. 2012/0101280, which is expressly incorporated herein by reference in its entirety.
  • CXC 4 antagonists are selected from p-hair in peptidomimeiics as disclosed for example in U.S. Pat. Appl. Pub, No. 2012/0283196, which is expressly incorporated herein b reference in its entirely.
  • a CXCR4 antagonist as described in the foregoing patents and patent applications, for example, P!erixafor is used either alone or in combination with another mobi!izer of HSPCs for concurrent administration with a HIF-a potentiating agent.
  • the HIF-a potentiating agent is a PHI
  • the at least two mobilizers are used for concurrent administration with the HIF-a potentiating agent.
  • the at least two mobilizers comprise a CXC 4 antagonist, and a colony stimulating factor such as G-CSF or GM-CSF, or variants, derivatives or analogs thereof.
  • the CXCR4 antagonist is Plerixafor, or similar compounds
  • the colony stimulating factor is G-CSF or a variant, derivative or analog thereof.
  • the HIF-a potentiating agent is suitably a PHI (e.g., a small molecule PHI including, but not limited to, ones selected from compounds according to any one of formulae MX).
  • HIF-a potentiating agents are useful as actives for enhancing the hematopoietic properties of mobilizers of hematopoietic stem cells and/or progenitor ceils.
  • a HIF-a potentiating agent can be administered to an individual concurrently (e.g., in the same composition or in separate compositions) with at least one mobilize.
  • *- "combination treatmenf ), and optionally with a pharmaceutically acceptable carrier, to stimulate or enhance hematopoiests including the mobilization of hematopoietic stem cells and/or progenitor cells, including
  • the present invention thus provides a method for mobilizing hematopoietic stern cells and/or progenitor sells from bone marrow into peripheral blood of a dono subject, the method comprising, administering to the subject a HIF-a potentiating agent in an effective amount. to mobilize hematopoietic stem cells and/or progenitor cells from the bone marrow into the peripheral blood of the subject.
  • a suitable donor subject in this embodiment is one that has been, is, or will be administered a raobilizer.
  • the present invention provides a method for mobilizing hematopoietic stem ceils and/or progenitor cells from bone marrow into peripheral blood of donor subject, the method comprising, consisting or consisting essentially of: administering concurrently to the subject a HIF- ⁇ potentiating agent and at least one mobiiizerof ' hematopoietic stem cells and/or progenitor cells in effective amounts to mobilise hematopoietic stem celts and/or progenitor cells from the bone marrow into the peripheral blood of the subject,
  • the above methods may further comprise collecting or harvesting mobilized hematopoietic stem cells and/or progenitor cells from the subject, and optionally eultoring and or storing the collected or harvested mobilized hematopoietic stem cells and/or progenitor cells, and further optionally transplanting the collected or harvested mobilized hematopoietic stem ceils and/or progenitor ceils into a recipient subject.
  • T he dosages of HI F-ct potentiatin agent and the at least one mobilizer to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof
  • the dosages will also tak into consideration the binding affinity or modulatory activity of the HIF-a potentiating agent to its target molecule, the hematopoietic capaci ty of the mobifizer s) > their bioavailability and their in vivo and pharmacokinetic properties.
  • precise amounts of the agents for administration can also depend on the judgment of the practitioner.
  • the physicia or veterinarian may evaluate the progression of the disease or condition over time. In any event, those of skill in the art may readily determine suitable dosages of the agents of the invention without undue experimentation.
  • the dosage of the active agents administered to a patient should be sufficient to effect a beneficial response in the patient over time such as enhanced hematopoiesis or a reduction, in the symptoms associated with an
  • the dosages may be administered at suitable intervals to boost hematopoiesis or ameliorating the symptoms of the immunocompromised condition.
  • Such intervals can be ascertained using routine procedures known to persons of skill in the art and can vary depending on the type of active agent employed and its formulation. For example, the interval may be daily, every other day, weekly, fortnightly, monthly, bimonthly, quarterly, half-yearly or yearly.
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active agent which are sufficient to maintain HIF-a potentiating agent modulatory effects and hematopoietic functiono enhancing effects.
  • Usual patient dosages for systemic administration range from 1 -2000 mg/day, 10-1000 mg day, 50-500 mg/day, 100- 800 mg day, commonly from 1 -250 rag/day, and typically from 10-150 mg/day. Stated in terms of patient body weight, usual dosages range from 0,02-25 mjg%g/day, 0.5-15 mg/kg/day, 1.0-10 mg/kg/day, 1-5 mg kg/day, commonly from 0.02-3 mg/kg day, typically firora 0.2-1.5 mg kg/day.
  • Pharmaceutically . ' acceptable -carriers will generally include water, saline, buffers, and other compounds described, e.g., in the Merck index, Merck & Co.. Railway, NJ.
  • T hus, the HIF-a potentiating agent and the mobilizer(s) may be provided in effective amounts to stimulate or enhance hematopoiesis.
  • This process may involve administering the. HIF-a potentiating agent separately, simultaneously or sequentially with the mobilizer(s).
  • this may be achieved by administering a single composition or pharmacological formulation that includes both types of agent, or by administering two or more sepajaie compositions or formulations at the sasne time, wherein one composition includes the HIF-a potentiating agent and the other(s), the mobilizer(s).
  • the mobilizers may ' be administered separately, simultaneously or sequentially,
  • the treatment with the HIF-a potentiating agent may precede or follow the treatment with the mobilfeer(s) by Intervals . ranging from minutes to days.
  • the HIF-a potentiating agent is applied separately to the mobUizer(s)
  • HTP-ft potentiating agent is "A”
  • mobilizer(s) is(are) "B”
  • both . ' agents are delivered to a subject's immune system in a combined amount effective to enhance hematopoiesis as compared to the adm inistration of the same amount of mobiiizer alone.
  • the HIF-ct potentiating agent and the mobilizer(s) may be administered directly to a subject or it may b desirable to conjiigate one or both to carrier proteins such as ovalbumin or serum albumin prior to administration. While it is possible for the active agent to be admi istered alone, it is generally desirable to present it as a pharmaceutical composition.
  • Such compositions typically comprise at least one active agent or ingredient, together with one or more acceptable carriers thereof. Each carrier should be both
  • compositions include those suitable for oral, rectal, nasal, topical, -or -parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.
  • the compositions may conveniently be presented in unit dosage form and may be prepared by many methods we ' ll known in the art of pharmacy. See, e.g., Oilman* et al (eds.) (1990) Goodman and Oilman's; The Pharmacological Bases -of Therapeutics, 8th Ed. s Pergamon Press; and Remingtoivs Pharmaceutical Sciences., 17th ed. (1 90), Mack Publishing Co., Easton, Pa.;.
  • cyclic neutropenia post-infective neutropenia
  • myelodys lasia syndrome or a medical treatment such as induced by treatment with cytoreductive, tnyeioab!ative or
  • immunosuppressive therapies e.g. , chemotherapy, radiation therapy and imniunosuppressi ve drugs such as steroid
  • immunosuppressive therapies in particular in relation to the treatment of transplant rejection and the treatment of hyperpro!iferative cell disorders such as cancer and autoimmune disease.
  • the subjects are individuals who may serve as allogeneic, syngeneic or xenogeneic donors of HSPCs and the treatment is used to mobilize and collect HSPCs for subsequent delivery to a recipient who has an immunocompromised condition or has an increased risk of acquiring an immunocompromised condition.
  • the treatment can be used for patients or donors who are "difficult to mobilise" because, for example, they are not sensitive to gro wth factors.
  • the treatment can further be used to cause tolerance of a recipient for organ transplantation.
  • the treatment can also be used in cancer therapy methods and in methods for Inhibiting, ameliorating,, or -ablation of cancer ceils and/or tumors.
  • leukemia initiating cells LICs
  • BM bone marrow
  • LICs are resistant to traditional cancer therapy e.g, cytoreductive or myeloablative therapy.
  • a cancer therapy e.g. , cytoreductive or myeloablative therapy.
  • Detachment of LTCs from their niche b inducing mobilization of LICs can be a used in combination with traditional cancer therapies (e,g., eytoreduciive or myeloablative therapies) to provide more effective or improved cancer therapy methods and methods for inhibiting, ameliorating, or ablation of cancer cells and/or tumors,
  • traditional cancer therapies e.g., eytoreduciive or myeloablative therapies
  • the treatment can additionally be used for gene therapy. Because
  • pluripotent hematopoietic stem cells are self-renewing, and give rise to cell progenitors as well as mature blood eeiis, the stem ceils are an appropriate target for gene therapy.
  • HSPCs can be collected.
  • the HSPCs can be modified to deliver gene products upon reinrroduction to the individual. After modification, the cells are reimused into the affected individual, [00419]
  • the treatment is administered to a patient to stimulate or enhance mobilization of HSPCs from the bone marrow into the peripheral blood and the mobilized HSPCs are then collected or harvested from the patient.
  • Blood containing mobilized HSPC may be collected from the donor by means well known in the art. In a typical protocol, the mobilized ceils are collected from the donor by. for
  • the levels HSPCs can be monitored by methods well known to those of skill in the art, and collection timed to coincide with HSPC peaks.
  • the donor ceils can be enriched ex vivo by treating them with factors that stimulate the TNFa and GM-CSF receptors.
  • factors that stimulate FLT3 and the G-CSF receptor such as FL and G-CSF, may be used.
  • hematopoietic tissues such as bone marrow and blood can be harvested from a donor by methods well ' known to those skilled in the art, and treated with NFa, GM-CSF, ' FL, SCF, IL-7, IL-12, and G-CSF, either singularly or in combination, to enrich selectively for HSPCs,
  • the cells harvested from the donor are typically cultured ex vivo for several days in medium supplemented with TNFa, GM-CSF, FL, SCF, IL-7, 1L-I 2, and G-CSF, either singularly or in -combination.
  • concentration of GM-CSF administered would typically be in the range of 1,000 U/m ' L.
  • TNFa may be administered, typically at a concentration of 200 U niL.
  • Appropriate concentrations of G- CSF, SCF, IL-7, IL-12, and FL can be readi ly determined by those of skill in the art, as by titration experiments or by reference to the working examples provided herein.
  • graft versus host disease GVHD
  • the enriched HSPCs may then be selectively collected from the culture using techniques known to those of skill in the art, as for example discussed below.

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Abstract

Disclosed is the use of a HIF-α potentiating agent and a mobilizer of hematopoietic stem cells and/or progenitor cells in methods and compositions for mobilizing hematopoietic stem cell and progenitor cells from the bone marrow into the peripheral blood. The compositions and methods are particularly useful for stem cell transplantation and for treating or preventing immune deficiencies.

Description

TITLE OF THE INVENTION
"MOBILIZING AGENTS AND USES THEREFOR"
CROSS REFERENCE TO RELATED APPLIC ATIONS
[0001] This application claims the benefit under 35 U.5.C. § 1 19(e) of United States Provisional Application 61/77 ! ,373 filed March 1, 2013 and is hereby incorporated by reference in. its entirety,
FIELD OF THE INVENTION
[0002] This invention relates general iy to the use of a HIF-a potentiating agent and a mobiiizer of hematopoietic stem ceils and/or progenitor cells in methods and compositions for mobilizing hematopoietic, stem ceils and progenitor cells from the bone marro into the peripheral blood. The present invention is particular relevant for stem ceil transplantation as well as treating or preventing immune deficiencies.
{0003} Bibliographic details of certain publications numerically referred to in this specification are■.collected at the end of the description.
BACKGROUND OF THE INVENTION
[0004] Autologous hematopoietic stem eel! (HSC) transplantation is a curative treatment for various hematologic malignancies including leukemias, lymphomas and multiple myeloma. During the last decade, hone marrow aspiration has been progressively replaced b m obilized peripheral blood as a source of transplantable HSCs. The cytokine granulocyte colony-stimulatin factor (G-CSF) is the main mobilizing agent used in the clinic. Adm inistered subcutaneousl daily at doses of 10 g/kg, it promotes the forced egress of hematopoietic stem and progenitor cells (HSPCs) from the bone marrow into the circulation. In the vast majority of healthy allogeneic donors, CD34+ HSPCs are robustly mobilized after 4-5 days of G-CSF treatment and blood aphaeresis from day 5 is sufficient to reach the minimum threshold of 2 x 106 CD34÷ cells / kg body weight to ensure rapid reconstitution. However, in the autologous setting, up to 30-60% of chemotherapy-treated patients fail to reach this minimal threshold in response to G-CSF, precluding transplantation (1), Most at-risk are patients who have undergone chemotherapy with purine analogs sueh as fiudarabine for more than three chemotherapy cycles (2). The chemotaciic interaction between th ehernokine CXCLl 2 and its receptor CXCR4 are pivotal to HSFC retention within the bone marrow (3.-4) and mobilization in response to G-CSF (5-6). Consequently, additional i nh ibition of the chemotaciic interaction between CXCL12 and GXCR.4 with specific small synthetic inhibitors such as Pierixafor (AMD3100) enhances syneigisticai!y HSPG mobilization in response to G-CSF in humans and mice (7), The synergistic effect of Pierixafor has been confirmed in at least two large phase 3 clinical trials with multiple myeloma and rion-Hodgkin's lymphoma patients eligibie for autologous HSC transplantation 5 who previously failed to mobilize adequately in response to G-CSF alone. Pierixafor injected daily 1 hr prior to blood aphaeresis from day 4 of G-CSF administration enables
approximately 60% of patients who previously failed to mobilize in response to G-CSF alone to reach the minimal threshold of 2 10° CB34* cells / kg (8-9). However, the remaining 40% of patients who failed to mobilize in response to G-CSF alone, still fail to mobilize 10 adequatel wit G-CSF and Pierixafor (8-9).
[0005] Accordingly, there is a pressing need for more effective approaches for mobilizing HSPCs from the bone marrow int the peripheral blood.
SUMMARY OF THE INVENTION
[0006] The present invention is related in part to the discovery that mobilization of
I S FiSPCs by mobilizing agents (also referred to herein as "niobHizers" or "mobiiizer of
hematopoietic stem cells and/or progenitor cells") such as G-CSF or Pierixafor, or
combinations thereof, is significantly enhanced by co-administration of an agent that increases th activity of hypoxia-inducible factor a (HIF-a) (also referred to herein as a "HIF-a potentiating agent"). This in turn results in higher numbers of hematopoietic stem
2 cells and/or progenitor ceils including granulocytes/macrophage progenitors and/or
megakaryocyte/erythrocyte progenitors in peripheral blood, when compared to
administration of stem cell mobili ers alone, Concurrent administration of a HIF-a potentiating agent and a mobiiizer of hematopoietic stem cells and/or progenitor cells is useful in compositions and methods for stimulating or enhancing mobilization of
25 hematopoietic stem cells and/or progenitor cells or for stimulating or enhancing
hematopoiesisj or for stem cell transplantation, as described hereafter.
10007] Accordingly, i one aspect, the present invention provides compositions that comprise, consist or consist essentially of a HIF-a potentiating agent and at least one mobiiizer of hematopoietic stem cells and/or progenitor cells. Is another aspect, the
30 invention provides a composition thai comprises a HIF-a potentiating agent for use in
mobilizing hematopoietic stem cells and/or progenitor cells. The HIF-a potentiating agent is selected, without limitation, from agents that stabilize HIF-a, agents that stimulate or enhance expression of HIF-a HIF-a polypeptide or coding sequences, and combinations thereof. In some embodiments, the HlF-a potentiating agent inhibits the activity of a HiF hydroxylase, e.g., a HIF prolyl hydroxylase (PHD) (e.g., PHDL PHD2 and/or PHD3). Such agents are also referred to he ein as "PHD inhibitors" or "PHI." In specific embodiments, the PHD inhibitor is a selective inhibitor of a HIF-¾ PHD. In some embodiments, the PHD inhibitor is an inhibitor of two or more PHD enzymes. In some embodiments, the at least one mobilizer is selected from a growth factor, a cytokine, a chemokine or a polysaccharide. Suitably, the at least one mobilizer is characterized by its abilit to decrease or block the expression, synthesis or function of CXCL12 or is characterized by its ability to block or antagonize CXCR4. In. specific embodiments, the mobilizer is selected from a colony stimulating factor such as G-C5F or a variant, derivative or analog thereof, a CXC 4 antagonist such as Plerixafor, or a combination thereof. In some embodiments, the compositions further comprise a pharmaceutically acceptable carrier.
[OOOSJ The compositions of the present invention are useful for stimulating or enhancing mobilization of hematopoietic stem cells and/or progenitor cells, or for stimulating or enhancing hernatopoiesis, or for stem cell transplantation. Accordingly, in related aspect, the present invention provides a use of a HlF-a potentiating agent for stimulatin or enhancing mobilization of hematopoietic stem cells and/or progenitor cells, or for stimulating or enhancing hematopoiesis. or for stem cell transplantation, or for treating or preventing an immunocompromised condition (e.g.,. neutropenia,, agranulocytosis^
thrombocytopenia, or anemia). In this aspect, the use is in subjects that have been, are, or will be administered at least one mobilizer of hematopoietic stem cells and or progenitor cells. In some embodiments, the HlF-a potentiating agent and the at least one mobilizer are prepared or manufactured as medicaments for those applications.
[0009] Another aspect of the present invention provides method for enhancing a hematopoietic function of a mobilizer of hematopoietic stem cells and/or progenitor cells in a subject. These methods generally comprise, consist or consist essentially of administering to the subject a HlF~a potentiating agent in an effective amount tp enhance an hematopoietic function of the mobilizer ( .g., increasing the number of hematopoietic stem cells and/or progenitor celis including granulocytes/macrophage progenitors and/or
niegakaryocyte/eryoHrocyte progenitors in the peripheral blood).
[0010] Yet another aspect of the present invention provides methods for mobilizing hematopoietic stem ceils and/or progenitor cells from bone marrow into peripheral blood of a donor subject These methods generally comprise, consist or consist essentially of:
administering to the subject a HIF-a potentiating agent in an effective amount to mobilize hematopoietic stem cells and/or progenitor cells from the bone marrow into the peripheral blood of the subject. The donor subjects in these embodiments are selected from subjects tha have been, are, or will be administered at least one mobilizer of hematopoietic stem cells and/or progenitor cells. A related method generally comprises, consists or consists essentially of: administering concurrently to the donor subject a HlF-a potentiating agent and at least one mobilizer of hematopoietic stem cells and/or progenitor cells in effective amounts to mobilize hematopoietic stem cells and/or progenitor cells from the bone marrow into the peripheral blood of the subject. Suitably, the HIF-ot potentiating agent is
adm inistered in an amount that is effective t enhance the hematopoietic function of the at least one mobilizer. In specific embodiments, the ΗΪΡ-α potentiating agent and the at least one mobilizer are administered in synergisticaliy effective amounts. In some embodiments, the subject has an immunocompromised condition or is at risk or acquiring an
'immunocompromised condition (e.g., the subjec will b exposed to an agent or treatment that gives rise or is likely to give rise to an immunocompromised condition). In illustrative examples of this type, the subject has a hyperproHferative cell disorder e.g., cancer, which can be a primary cancer or a metastatic cancer, or an autoimmune disease), and has been, is or will be subjected to a medieai treatment in specific embodiments, the hyperproHferaiive cell disorder is cancer (e.g., leukemia, multiple myeloma, lymphoma, etc.). Suitably, the medieai treatment targets rapidly dividing cells or disrupts the cell cycle or cell division (e.g.,. chemotherapy and/or radiation therapy). Suitably, the immunocompromised condition is selected from neutropenia, agranulocytosis, thrombocytopenia, and anemia.
[0011] in some embodiments, the methods further comprise collecting or
harvesting mobilized hematopoietic stem cells and/or progenito cells from the donor subject (e.g., from the subject's peripheral circulation). In some of these embodiments, the methods further comprise cultur ing and/or storing the collected or harvested mobilized hematopoietic stem cells and/or progenitor cells (e.g., to maintain or expand the collected or harvested mobilized hematopoietic stem cells and/or progenitor ceils). In some embodiments, the methods furthe -comprise infusing or transplanting the collected or harvested mobilized hematopoietic stem cells and/or progenitor cells, which have been optionally cultured or stored, into a recipient subject. The donor subject and the recipient subject may be the same subject or may be different subjects, in some embodiments, the subject is both donor and a recipient" of the collected, or harvested mobilized hematopoietic stem cells and/or progenitor cells, which have been optionally cultured or stored, and is suitably in need of a stem cell transplantation. The stem cell transplantation in these embodiments is autologous with respect to the recipient Suitably, the subject has an immunocompromised condition or has been exposed to a medical treatment that results in an immunocompromised condition.
[0012] In other embodiments, the methods further comprise infusing or
transplanting the collected or harvested mobilized hematopoietic stem cells and/or progenitor cells, which have been optionally cultured or stored, into another subject. In thes embodiments the. donor subject and the recipient subject are different subjects. In these embodiments, the subject from which the mobilized hematopoietic stem ceils and/or progenitor cells are collected or harvested is a donor and the other subject is a recipient that is suitably in need of a stem cell transplantation. The stem cell transplantation in these embodiments is allogeneic or xenogeneic with respect to the recipient Suitably, the other (recipient) subject has an immunocompromised condition or has been exposed to a medical treatment that results in an immunocompromised condition.
[OOOj I som embodiments, the methods further comprise administering to the recipient prior to. simultaneously with, or after the stem ceil transplantation a HIF-a potentiating agent and a mobilizer of 'hematopoietic stem cells and/or progenitor ceils in effective amounts to mobilize hematopoietic stem cells 'and or progenitor cells from the bone marrow into the peripheral blood of the subjec
{0014] In accordance with the present invention, HIF-α potentiating agents are useful for enhancing a hematopoietic function (e.g. , increasing the number of hematopoietic stem cells and or progenitor cells, including■granulocytes/macrophage progenitors and/or niegakaryocyte/eiytlmwjyte progenitors in the peripheral blood) of at least one mobilizer of hematopoietic stem cells and/or progenitor celis. The HIF-a potentiating agent may be known, or identified using any suitable screening assay. Accordingly, in a related aspect, the present invention provides screening methods for identifying agents that are useful for enhancing a hematopoietic function of the mobilizer. These methods generally comprise testing whether a test agent potentiates HIF~a increasing the accumulation of, orstability of, HIF-a; directly provide HIF- activity; or increase expression of FRF- 1) and determining whether the test agent stimulates or enhances mobilization of hematopoietic stem cells and/or progenitor cells on the basis that it tests positive for the potentiation,
[0015] In another related aspect, the present invention provides methods of producing an agent that enhances a hematopoietic function of at least one mobil izer of hematopoietic stem celis and/o progenitor cells. These methods generally comprise:
identifying a HIF-a potentiating agent by a screening assay as broadly described above; and synthesizing the agent on the basis that it tests positive for enhancing a hematopoietic function of the mobilizer. Suitably, the method further comprises derivatizing the agent, and optionally formulating the derivatized agent with a pharmaceutically acceptable carrier, to improve the efficacy of the agent for enhancing the hematopoietic function of the mobilizer.
[0016) The mohiiizerfs) and the HIF-a potentiating agent are suitably administered in the form of one or more compositions each comprising a pharmaceutically acceptable carrier. The compos ition(s) may be administered by injection, by topical application or by the oral rente including sustained-release modes of .administration, over a period of time and in amounts which are effecti ve for increasing the number of hematopoietic stem cells and/or progenitor cells including granulocytes/macrophage progenitors and or
megakaryocyte/erythrocyte progenitors in the peripheral blood.
1001 J In some embodiments, the mobi!ker(s) and the HiF~a potentiating agent are administered simultaneously to the subject In other embodiments the HIF-a potentiating agent is adminis tered to the subject prior to administration of the mobilizer. In still other em bodiments, the HIF-a potentiating agen t is administered after administration of the mobilizer to the subject.
[0018] In a related aspect, the methods are useful for treating or preventing an immunocompromised condition in a subject (e.g.., a condition resulting from exposure of the subject to a medical treatment). In these embodiments, the mobilizer(s) and the HIF-s potentiating agent are concurrently administered i amounts effective for treatment or prevention of the immunocompromised condition (e.g., neutropenia, agranulocytosis, thrombocytopenia, or anemia). In some of these embodiments, the methods further comprise identifying a subject having or at risk of acquiring the immunocompromised condition. Suitably , the HIF-a potentiating agent and the mobilizer(s) may be administered to the subject simultaneously, .sequentially or separately with the medical treatment, in some embodiments, the concurrent administration of the HIF-a potentiating agent and the mobilizer(s) is a prophylactic treatment (e.g., the subject is preparing t undergo chemotherapy or radiation■treatment). In- others, it is a therapeutic treatment (e.g., the subject has received at least one dose of chemotherapy or at least one radiation treatment),
1001 J In some embodiments, the methods may further comprise exposing ibe subject to an ancillary treatment that treats or prevents an immunocompromised condition. In illustrative examples of this type, the immunocompromised condition is anemia and the ancillary treatment may comprise administering to the subject an anemia. medicament selected from recombinant erythropoietin (EPQ), ferrous iron, ferric iron, vitamin B 12, vitamin B6, vitamin C, vitamin D, calcHrioi, aipbacaicidol, folate, androgen, and carnitine. In other illustrative examples, the immunocompromised condition is thrombocytopenia and the ancillary treatment may comprise administering to the subject a thrombocytopenia medicament selected from a glucocorticoid- recombinant thrombopoietin (TPO), recombinant megakaryocyte growth and development factor (MGDF), PEGylated recombinant MGDF and ItsophylUne. In s'till other illustrative examples, the
immunocompromised condition is neutropenia and the ancillary treatment suitably comprises administering to the subjeet a neutropenia medicament selected from glucocorticoid, immunoglobulin, androgens, recombinant IFN-y, and uteroferrin. In some embodiments, the ancillary treatment is administered to the subjeet simultaneously, sequentially or separately with the HIF-a potentiating agent and/or the mobilizer{s).
[0020] In some embodiments, the medical treatment is likely to expose the subject to a higher risk of infection. Accordingly, in these embodiments, the methods may further comprise administering simultaneously,, sequentially or separately with the HIF-a potentiating agent and/or me mobilizer(s) at least one anti-infective agent that is effective against an infection that develops or that has an increased risk of developing from the immunocompromised condition, wherein the anti-infective agent is selected from antimicrobials, antibiotics, arrtivirals, antifungals, anthelmintics, antiprotozoals and nematocides.
50021} Typically, one or both of the HIF-a potentiating agent and the at least one mobilizer are administered on a routine schedule, or example, every day, at least twice week, at least three times a week, at least four times a. week, at least five times a week, at least six times a week, every week, every other week, every third week, every fourth week, every month, every two months, every three months, every four months, and eye*}' sis months.
[0022] In some ad vantageous em bodiments, the concurrent administration of a ΗϊΒ-α potentiating agent and at least one mobilizer of hematopoietic stem cells and/or progenitor ceils) is useful for treating or preventing hematopoietic disorders such as neutropenia, agranulocytosis, thrombocytopenia, and anemia, which may result, for example, from myeiosuppressive, myeloablative or cytoredtsctive treatments that target rapidly dividing cells or that disrupt the cell cycle or cell division (e.g., chemotherapy or radiation therapy). Accordingly, in yet another aspect, the present invention provides methods for treating a hyperproliferative cel l disorder (e.g., a cancer or a uto immune disorder) in a subject. These methods generally comprise administering concurrently to the subject a medical treatment (e.g.* a chemotherapeutic agent or radiation) for the disorder, which targets rapidly dividing cells or disrupts the cell cycle or cell division, together with at least one mobiiizer of hematopoietic stem cells and-'or progenitor cells and a HIF- potentiating agent in amounts effective for stimulating or enhancing mobilization of hematopoietic stem cells and/or progenitor cells, or 'for stimulating or enhancing hemalopoiesis.
[0023] Since administration of the combination treatment of the present invention will reduce the risk of having or developing a hematopoietic disorder as a side effect of the myeioabiative, myelosuppressive or cytoreductive treatment, it is possible to administer higher therapeutic doses of a chemotherapeutic agent or radiation to a subject in order to kilt or inhibit the growth or proliferation of a tumor or to treat or prevent an autoimmune disease in the subject. Accordingly, in yet another aspect, the preseM invention provides methods for increasing the dose in a subject of a medicament for treating a hypetproliferattve ceil disorder (e.g., cancer or an autoimmune, disease), wherein the medicament results or increases the risk of developin an immunocompromised condition. These methods generall comprise administering concurrently the medicament to the subject in a dose mat ordinarily induces side effects (e.g., the development of the immunocompromised condition), together with at least one mobiiizer of hematopoietic stem cells and or progenitor cells and a 'HIF-a potent iating agent i n amounts effecti ve for inhibiting or preventing the induction of those side effects (e.g., in amounts effective for increasing the number of hematopoietic stem cells and/or progenitor cells including gianu i oc te s macrophage progenitors and/or
megakaryocyte/erythrocyte progenitors in the peripheral blood).
[0024] In yet another aspect, the present invention provides pharmaeeuticai compositions for treating or preventing a hyperproliferative celt disorder (e.g., cancer or an autoimmune d isease) that is treatable or preventable by a medical treatment that targets rapidly dividing cells or thai disrupts the cell cycle or cell division (e.g., chemotherapy or radiation therapy). These compositions generally comprise, cons ist or consist essentially of a HIF-a potentiating agent, at least one mobiiizer of hematopoietic stent ceils arid/or progenitor cells and at least one other agent selected from a .chemotherapeutic agent (e.g.^ a cytotoxic agent), a radiosensitrang agent, an anemia medicament, a thrombocytopenia medicament, a neutropenia medicament, a agranulocytosis medicament and an anti- infective agent, and optionall a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figures !A, IB, IC, and ID are graphical representations showing the effect of Compound X and G-CSF on HiF-ta protein and CFC mobilization. (I A) Western blot analysis of bone marrow (BM) cell y sates from mice treated with saline (Saline), with Compound X for 3 days (X3){ with G-CSF for 2 days (G2) or with both Compound X and G- CSF (G2X3) for presence of HIF-la and β-actin. Each lane represents a different mouse. (I B) Graph shows the numbers of CFC per mL blood and per spleen for mice treated with G~ CSF (open diamonds) or with Compound X for 3 days and G-CSF (filled squares) for indicated numbers of days. (iC) Graph shows the numbers of CFC per mL blood and per spleen for mice treated with G-CSF for 2 days and with Compound X for 0 to 4 days. (ID) Time-course n umbers of CFC per mL blood or per spleen for mice treated wit h Plerixafor with or without Compound X. Mice were treated with Plerixafor for 1 hour (PI ) together with Compound X for 1, 2, 3 or 4 days (P1X1- P1X4). Data are mean ± SD of 6 mice per condition. * p<Q.05; ** p< 0.01 *** p < 0.001.
{0026 Figures 2A, 2B, 2C, and 2D present graphical representations showing synergistic effect of Compound X with G-CSF,of in combination with G-CSF and Plerixafor. (2A) Treatment groups used. (2B) Graphs shovv the number of CFC per ml blood and per spleen of treated mice, GCSF only (filled circles); GCSF and Compound X (open squares); GCSF and Plerixafor (filled triangles); and the combination of GCSF, Plerixafor and Compound X (filled diamonds), (2C) Timecourse of the number of Lin- Seal* Kit* HSPCs and (2D) Lin- Seal * Kir CD48-CD1 0*HSCs per mL of blood or per spleen following treatments with combinations of G-CSF, Plerixafor and Compound X as in 2A, GCSF only (filled, circles); GCSF and Compound X (open squares); GCSF and Plerixafor (filled triangles); and the combination of GCSF. Plerixafor and Compound X (filled diamonds). Data are mean ± SD of 6 mice per time-point per treatment group. *: p<0.05; ** 0.001<p<0.01 ; *** p<0.001 ,
[0027] Figures 3 A and 3B are graphical representations showing that Compound X synergizes with G-CSF and Plerixafor to enhance mobilization of competitive repopu!ating HSCs. GD45,2+ mice were mobilized with G-CSF for 2 or 4 days with G-CSF alone (filled circles), with G-CSF in combination with Plerixafor for 1 hr (filled triangles), with
Compound X for 3 days (filled squares) or with both Plerixafor, 1
Figure imgf000010_0001
and Compound X, 3 days (filled inverted triangles). Biood (20^iL) was transplanted in lethally irradiated recipients together with 200,000 competitive BM cells from CD45.1 f mice. Proportion of blood CD45.2+ leukocytes at 16 weeks post-transplantation and number of repoptslatin units per mL of mobilized blood calculated from donor chimerism at .16 weeks post- transplantation from (3 A) 2 days treatment with G-CSF and (3B) 4 days treatment with G- CSF. Each dot is a result from one recipient mouse, bars are average. '*: p<0.G5; ** Q.001<p<0,01 ; *** p<0.001.
|0028J Figures 4A, 4B, 4C, and 4D are graphical representations showing that deletion of Hifla gene in HSPCs compromises HSPC mobilization in response to- -G-CSF. CreER was activated in mutant mice in which both Hifla alleles are floxed to delete the Hifl genes and the mice were mobilized for 3 days with G-CSF. (4A) Proportion of Lin" Scal 'Kif' CD4.8" CDl 50* HSCs, Lin Scal^Kit* HSPCs, Lin" ScaFRif myeloid progenitors and Lin* leukocytes in which yellow fluorescent protein (YFP) is induced by Cre ER following a 3- day tamoxifen treatment in ifla 1 R26RYi"mFP SclCreER mice. Number of (4B) CFCs and (4C) Lin Seal ÷Kit+ CD48' C.D150+ HSCs, Lin Seal * it* HSPCs mobilized in blood and spleen in Hiflam R26RYl'pnf > SclCreER mice (solid circles or squares) and control tfifl:a >m R26RYFP,YFP SclCreER mice (open circles or squares) , in 4.B each dot represents a separate mouse. p<0.05; ** O.OOKpO.OI; *** p<Q.00L (4D) Proportion of YFP" (Hifl^ non-deleted) and YFP" ( ifl^ deleted) HSCs mobilized to the blood or spleen from the bone marrow versus YFP- or YPP-r FISCs remaining in the bone marrow following a CreER induction with tamoxifen and a 3 day G-CSF treatment Pairs of dots show results for YFP- and YFP+ HSC within the same mobilized Hiflam R26RyHVYFP SclCreER moiise. Significance were calculated with a paired t-test
{0029] Figures 5 A, 5B, and 5C are graphical representations showing that deletion of Hifla gene in osteoprogenitors delays HSPC mobilization in response to G-CSF, (5A) Proportion of CD45-Lin-CD31 -Seal-51 + osteoblasts and CD45-Lm-CD31+ endothelial cells i which YFP is induced by Cre ER in Hiflam R26RYFP Yfp OsxCreER mice. (SB) Number of CFCs and Lin Seal +Kit+ CD48' HSCs, Lin Scal+ it+ HSPCs per femur
Figure imgf000011_0001
R20RYmYFP OsxCreER mice, (HIF la wt open circles, HIF 1 a fl/fL solid circles). (5C) Number of CFCs mobilized in blood and spleen in Hi lam R26RyFP/YFP OsxCreER mice following 2-4 days treatment with G-CSF (P1IF la wt, open circles, tUF l a fi/fl, solid circles).
J0030] Figures 6A, and 6B are graphical representations demonstrating the effect of treatment with Compound A, Compound B, or Compound C in combination with G-CSF on the number and phenotypic distri ution of hematopoietic stetn cells and progenitor cells in the bone marrow. Treatment with G-CSF and vehicle control served as control. (6A)
Myeloid progenitors (LKS-)-Top Panel, hematopoietic stem and progenitor cells (LKS+)-
Middle Panel, and LKS - CD4JH- lineage-restricted progenitors- Bottom Panel. (6B).LKS+ CD48-CD15Q- midiipoteni progenitors-Top Baseb nd LS + CD4S- GDI 50+ hematopoietic stem cells-Bottom Panel
f 0031] Figures 7A. and 7B ate graphical representations demonstrating the -effect of treatment with Compound A, Compound 6 or Compound C in combination with G-CSF on mobilization to the blood of hematopoietic stem ceils and progen itor cells. Treatment with G-CSF and vehicle control served as control (7 A) phenotypic myeloid progenitors (LKS-)~ Top Panel, hematopoietic stem and progenitor cells (L S+)- Middle Panel, and LKS* CD48+ lineage-restricted progenitors- Bottom Panel (7B) L S+ CD48-CD150- multipotent progenitors- Top Panel, and L .S+ CD48- CD15CH" hematopoietic stem cells- Bottom Panel * p<0.GS: ** p<0.Ql; ***p<Q.O01.
[0032] Figures 8 A and 8B are graphical representations demonstrating the effect of treatment with Compound A, Compound B or Compound C in combination with G-CSF on mobilization to the spleen of hematopoietic stem cells and progenitor ceils. Treatment with G-CSF and vehicle control served as control (8A) phenotypic myeloid progenitors (LKS-)- Top Pane hematopoietic stem and progenitor cells (L S+)- Middle Panel, and L S+
CD4S+ lineage-restricted progenttors-Bottom Panel (SB) L 5+ CD48-CD 150- mn!tipotent progenitors- To Panel, and LKS- CD 8- CD! 50+hematopoietie stem cells- Bottom Panel * p<0.05; ** p<0.01; ***p<0.001.
[0033] Figures 9A. and 9B are graphical representations demonstrating the effect of treatment with Compound A, Compound B or Compound C in combination with G-CSF on total mobilization per moose (blood and spleen) .of hematopoietic stem cells and progenitor cells. Treatment with G-CSF and vehicle control served as control (9 A) phenotypic myeloid progenitors (L S-)- Top Panel, hematopoietic stem and progenitor cells (LKS+)- Middle Panel, and LK.S+ CD48+ lineage-restricted progenitors- Bottom Panel (9B) LKS+ CD48- CD 15Θ- multipotent progenitors- Top Panel, and L S+ CD4&- CD 15 G÷ hematopoietic stem cells-Bottom Panel * p<0.QS; ** p<Q,01 ; *«*p<Q.001.
10034] Figure 10 is a graphical representation demonstrating the effect of treatment with Compound A. Compound B or 'Compound C in combination .with G-CSF on colony forming unit (CFU). mobilization to the blood- Top Panel, spleen- Middle Panel, and combined total mobilized (blood and spleen)- Bottom Panel Treatment with G-CSF and vehicle control served as control, * p 0,05; . ** p<0.01 ; ***p<G.QQl.
[0035] Figure 1 1 is a graphical representation demonstrating the effect of treatment with Compound A, Compound B or Compound C in combination with G-CSF on white blood cei l (WBC) number per ml of blood- Top Panel, and spleen weight- Bottom Panel Treatment with O-CSP and vehicle control served as control * p<Q,05; ** p<0,01;
***p<0J30 i .
[00363 Figures 12A and 12 B are graphical representations dem nstrating the effect of treatment with Compound D, Compound E or Compound F in combination with G-CSF on total mobilization to blood and spleen of hematopoietic stem cells and progenitor cells. Treatment with G-CSF and vehicle control served as control (12A) phenotypic myeloid progenitors (LKS-)- Top Panel, hematopoietic stem and progenitor cells (L S+)- Middle Panel, and LRS+ CD48+ lineage-restricte progenitors- Bottom Panel (12B) L S+ CD4S- GDI 50- multipotent progenitors- Top Panel, and LKS+ CD48- CD150+ .hematopoietic stem cells- Bottom Panel. * p<0.05; ** p<0.0L; ***p«0.001.
[0037] Figure 13 is a graphical representation demonstrating the effect of
Compound D, Compound E or Compound F in combination with, G-CSF on colony forming unit (CFU) mobilization to the blood- Top Pane!? spleen- Middle Panel, and combined total (blood and spleen)- Bottom Panel Treatment' ith G-CSF and vehicle control served as control. * p<0.05; ** p<0.01 ; ***p<0<0Ql .
(0038] Figures I4A and 14B are graphical representations demonstrating the effect of treatment with Compound H, Compound J or Compound in combinatio with G-CSF on the total mobilization to blood and spleen of hematopoietic stem cells and progenitor cells. Treatment with G-CSF and vehicle control served as control. (14A) phenotypic myeloid progenitors (LKS-)- Top Panel, hematopoietic stem and progenitor ceils (LKS+)- Middle Panel and LKS+ CD48+ lineage-restricted progenitors- Bottom Panel, (14B) L S+ CD48-CD 15.0- multipotent progenitors- Top Panel, and LKS+ CD4S- CDl 50÷
hematopoietic stem cells- Bottom Panel * p<0.05; ** p<0.01; ***p<0.001.
|O039j Figure 15 is a graphical representation demonstrating the effect of treatment with Compound H, Compound I or Compound K in combination with G-CSF on colony forming unit (CFU) mobilization to the blood- To Panel spleen- Middle Panel, and combined total (blood and spleen)~Bottom Panel * p<0.05; ** p<0.01; ***p<0.OGl
BET AILED DESCRIPTION OF THE INVENTION
2, Definitions
[0040] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill i the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in. the practice or testing of the present invention, preferred raethods and materials are described. For the purposes of the present mvention, the following terms are defined below.
[0041] The articles "a" and "an" are used herein to. refer to one or to more than one (-i. e.. to at l east one) of the grammatical obj ect of the article. By way of example, "a mobilizer of hematopoietic stem cells and/or progenitor cells" means one mobilizer of hematopoietic stem ceils and/or progenitor cells or more than one mobilizer of hematopoietic stem cells and/or progenitor ceils.
[0042] By "about" is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 10 % to a reference quantify, level, value, number, frequency, percentage, dimension., size, amount, weight or length to which it refers.
[0043] The terms "administered concurrently" or "administering concurrently" or "eo-admintstering" and the like refer to the administration of a single composition containing two or more active agents, or the administration of each active agent as separat
compositions and/or delivered by separate routes either contemporaneously or
simultaneously or sequentially within a short enough period of time that the effective result is equivalent to that obtained when all such active agents are administered as a single composition, or that the result achieved is the combined effect of agents. For example, a HI -ec potentiating agent may be administered together with a mobilizer of hematopoietic stem cells and/or progenito cells in order to increase the numbers of hematopoietic stem cells, progenitor ceils and/or differentiated cells thereof in peripheral bipod. In another example, a HIF-a potentiating agent and a mobilizer of hematopoietic ster ce l ls and/or progenitor cells are administered together with another agent to enhance their effects or to ameliorate the effects of a medical treatment that gives rise or contributes to an
immunocompromised condition. In another example, a HIF-a potentiating agent is administered at a later point in time than a. mobilizer of hematopoietic stem cells and/or progenitor cells but within the time period during which the mobilizer of hematopoietic, stem ceils and/or progenitor cells is still exerting an effec By "sequential" administration is meant a time difference of f om seconds, minutes, hours or days between the administration of the two types of molecules or active agents. These molecules or active agents may he administered in any order. By "simultaneously is meant that the active agents ar administered at substantially the same time, and desirably together in the same formulation.
By '"contemporaneously" it is meant that the active agents are administered closely in time, e.g. , one agent is administered within from about one minute to within about one day before or after another. Any contemporaneous time is useful. However, i† will often be the case that when not administered simultaneously, the agents will he administered within about one minute to with in about eight hours and preferably within less than about one to about four hours. Irs certain embodiments, the HiF-a potentiating agent and the mohilizer are administered within about 60 minutes, about 50 minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes, about 5 minutes, or about 1 minute of each, other or separated in time by about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, or about 72 hours, Or more. When administered contemporaneously, the agents may suitably be administered at the same site on the subject. The term "same site" includes the exact location, but can be within about 0.5 to about 15 centimeters, usually from within about 0.5 to about 5 centimeters. The term
"separately" as used herein means that the agents are administered at an interval, for example at an interval of about a day to several weeks or months. The active agents may be administered in either order. The term "sequentially" as used herein means that the agents ar admin istered i n sequenc e, for example at an in terval or intervals of minutes, hours, days o weeks. If appropriate the active agents may be administered in a regular repeating cycle.
(0044) The term "agent" includes a compound, composition, or molecule that induces a desired pharmacological and/or physiological effect. The term also encompasses pharmaceutically acceptable and pharmacologically active ingredients of those compounds specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the above term is used, then it is to be understood that this includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc. The term "agent" is not to be construed narrowly but extends to small molecules, proteiiiaeeous molecules such as peptides, polypeptides and proteins as well as compositions comprising them and genetic molecules such as RNA, DNA and mimetics and chemical analogs. thereof as .well -as cellular agents. The term "agent" includes a cell which is capable of producing and secreting polypeptides referred to herein as well as a polynucleotide comprising a nucleotide sequence that encodes this polypeptide-. Thus, the term "agent" extends to nucleic acid constructs including vectors such as viral or non-viral vectors, expression vectors and piasniids for expression in and secretion in a range of ceils.
[0045] The term "acyP denotes a group containing the moiety C=0 (and not being a carboxylic acid, ester or amide or thioester). Preferred acyl includes C(0}-R, wherein R is hydrogen or an alkyl, alkenyl alkynyl, aryl, cyeioalkyl, heterocyclyL arylalkyl,
cycloalkylalkyl or heterpcy'clylalkyl residue, suitably a C^g residue. Non-limiting examples of acyl include formyi; straight chain or branched alkanoyl such as, acetyl, propanoyi, biitanoyl, 2-rnethylproparioyI, pentanoyl, 2¾2-d½ethyipropanoyL hexanoy , heptanoyl, octanoyL nonanoyl- decanoyl, undecanoyl, dodeeanoyl, tridecanoyl, tetradecanoyl,
peniadecanoyi. hexadecaaoyL heptadeeanoy'i, oetadeeanoyl, nonadeeanoyl and ieosarsay!; pheny!carbonyl; eyeioalkyfcarbonyl such as eyclopropylmethyl(or etliyi)carboiiyl cyclobutylmethyi(or etxtyl)carbonyL cyclopentylraeihyl(or ethyl)carbonyl and
cyclohexyi ethyl (or e†hyl)earbonyi; aikanoyl such as phenySaikanoyl (e.g., phenylaeefyl, ?.<?,, benzoyl, plien lpropanoyl, pheny !butanoyl, phenylpentanoyL phenylhexanoyl) and naphthylalfcanoyl (e.g., naphthylacetyL naphtihyipropanoyl and naphthytbytanoyl), and phenylalkenoyl (e.g., pheny lhex-4-en-oyl, phenylhex-3-en-oy.l, phenylheptanoyl, phenyihept- 4-en-oy 1, phen lhept~3 -en-o i).
[0046| An "agranulocytosis medicament" as used herein refers to a composition of matter which reduces the symptoms related to agranulocytosis, prevents the development of agranulocytosis, or treats existing agranulocytosis.
[0047] The terra "alkenyl" as used herein denotes groups formed from straight chain or branched hydrocarbon residues coniainmg at least one carbon to carbon double bond including ethylenieally mono-, di~ or poly-unsaturated alkyl groups as defined herein, suitably
Figure imgf000016_0001
Gj.io or Ci-s). Examples of alkenyl include■vinyl, allyl, 1- met y!vinyl. butenyl, iso-botenyl, 3-meihyl-2-butsnyl, 1 -p nteny'l, cyclopentenyl, 1-niethyl- cyclopentenyk 1-hexenyi. 3-hexenyl, eyeiohexenyl, i-heptenyL 3~he.pte.nyl, 4-heptenyl, S- heptenyL 3-hexenyl, 4-hexenyl, l-oetenyl, eyciooetenyl, 1-rio.nenyl, 2-nonenyl, 3-nonenyl, 1- deceny!, 3-decenyl, 1 ,3-butadieny!, I -4>pentadienyi, 1,3-cycIopentadienyl, 1,3-hexadienyl, and 1,4-hexadienyl, An alkenyl group may be optionally substituted by one or more optional sobstituents as herein defined. Accordingly, "alkenyl" as used herein is taken to refer to optionally substituted alkenyl.
{0048] As used herein, the term "alkyl." when used alone or in words such as "arylalkyl " "heterocyclylalkyl" and "cyeloalky lalfcyl," denotes straight chain or branched hydrocarbon residues;, suitabl Ci-ao'.alkyl, e.g. , Cvio or Cj .6. Examples of straight chain and branched alkyl include methyl ethyl, propyl,, isopropyl, butyl, isobutyL sec-butyl ten-butyl amyL isoamyl see-amy!. 1 ,2~dimethylpropyi, 1, -dimethyl -propyl, hexyi, 4-meth)ipeniyiI 1 - methylpentyl, 2-methySperityl, 3-methyIpefttyl, i,i-dimethylbutyis 2,2-diraethylbutyl, 3,3- dimethyibuty 1.2-dimethylbutyl, 1 ,3-dimethyibut l l,2s2,-trimethylpro.pyl, 1, 1 ,2- trimeihylpropyl,. foeptyl. 5-meihoxyhexyl, 1-roethylhexyi, .2,2-dime.thylpe.rityl, 3,3- dimethyipentyl, 4,4-diraethyl entyl, 1 ,2-dimethylpenty 1$ 1 ,3-dimethyipentyl, 1,4- dimemyl-pentyl, 152t3^rrimethylbatyl, ] ,] ,2-trimetbylbutyl IJ,3 nmethylburyl, ociyl, 6- methylheptyi, i-methylheptyJ, 1,1 ^^etrameth lbu !, nonyl, 1-, 2-, 3-» 4-, 5-, 6- or 7- methyl-ociyl, I-, 2-, 3-, 4- or 5-etbyl.hep†yi> 1-, 2- or 3-propylhexyl, deeyl, 1-, 2-, 3-, 4-, 5~, 6-, 7- and 8-methylnony!, 1-, 2-, 3~5 4-5 5~ or 6-eihyloctyl, I -, 2-, 3- or 4-propylbept i, undecyl, I-, 2-, 3-, 4-, 5-, 6-. 7-, 8- or 9-methyl ecyL 1-, 2-, 3-, 4-, 5-, 6- or 7-ethylnonyl, 1-, 2-, 3-, 4- or 5-propyloeytl, I-, 2- or.3-buiylheptyl, l-peniylhexyl., dodeeyl, 1-, 2-, 3-, 4-, 5-, 6- , 7-, 8-, 9- or 10-methyiuiidecyi, 1-, 2-, 3-, 4-, 5-, 6-, 7- or S-ethyldeeyl, 2-, 3-, 4-, 5- or 6- propylnonyt, I-, 2-, 3- or 4-butyIoctyI, 1 -2- entylheptyl and the like. An alky! group raay.be opiionaily substituted by one or more optional substituents as herein defined. Accordingly, "alkyl" as used herein is takes to refer to optionally substituted alkyl
{0049] The term "alkynyl" denotes groups formed from straight chain or branched hydrocarbon residues containing at least one carbon to carbon triple bond including ethynyically mono-, di- or poiy-unsatufated alkyl or cycloalkyl groups as defined herein,
Figure imgf000017_0001
or Cj.fi); Examples, include ethynyL propynyi, butynyl, pentynyl. An alkynyl grou may be optionally substituted by one or more optional substituents as herein defined. Accordingly, ^alkytiyl" as used herein is taken to refer to optionall substituted aSkyny l,
[0050] The term "aryl" used either alone or in compounds words such as
"arylalkyP and "aryloxy," denotes single, -polynuclear, conjugated or fused residues of aromatic hydrocarbons. Examples o aryl includ phenyl, bi phenyl and naphthyl. In specific embodiments, aryi groups include phenyl and naphthyl An aryl. roup may be opiionaily substituted by one or more optional substituents as herein defined. Accordingly, "aryi" as used herein is taken to refer to aryl that may be opiionaily substituted, such as optionally substituted phenyl and optionally substituted naphthyl.
[0051] The terms "arylaikyl," "cycloaiky!alkyi" and "heterocyclyialkyl" refer to an alkyl group substituted (suitably terminally) by an aryl, cycloalkyl or heterocyclyl group, respectively..
[0052] The terms "aryloxy,'' "cycloalkylo y" and "heterocyciyloxy" denote aryl, cycloalkyl and heterocyclyl groups, respectively, when linked fay an oxygen atom. [0053] An "anemia medicament" as used herein refers to a composition of matter which reduces the symptoms related to anemia, prevents the development of anemia, or treats existing anemia.
[0054] By "antigen-binding molecule" is meant a molecule that has binding affinity for a target antigen, it will b understood that this term extends to immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity.
[0055] "Antigenic or immunogenic activity" refers to the ability of a polypeptide, fragment, variant or derivative according to the invention to produce an antigenic or immunogenic response in an animal, suitably a mammal, to which it is administered, wherein the response includes the production of elements which specifically bind the polypeptide or fragment thereof,
[0056] Reference herein to "bacteria" or "bacterial infection"' includes any bacterial pathogen including emerging bacteria! pathogen of vertebrates. Representative bacterial pathogens include without limitation species of: Aciwtobaefer, Actinobacillus,
Actmomycetes, Actinomyces, Aeromonas, Bacillus, Bacteroides, Bordetella, Borrelia,.
Brucella (brucellosis), Burkholderia, Campylobacter, CHrobactsr, Clostridium,
Car nebeicterium, Enterobaefer, Enteracoecus, Etysipelothrix, Escherichia, Fr neisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Micrococcus, Moraxella> Morganella, Mycobacterium (tuberculosis), Nocardla, Neisseria, Pasteurella, Plesiomonas, Propionibacterium, Proteus, Providencia, Pseudomc as, Rhodococcw,
Salmonella, Serraiia, Shigella, Staphylococcus, Stenotrophomonas, Streptococcus,
Treponema, Vibrio (cholera) and Yersinia (plague).
fOOSTf Throughout this■.specification,, unless the context requires otherwise^the words "comprise," "comprises" and "comprising-' will be understood to imply the inclusio of a stated step or element or group of Steps or elements but not (he exclusion of any- other step or element or group of steps or elements. Thus, use of the term "comprising'' and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of. Thus, the phrase "consisting o indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of is meant: including any element's listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the liste elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.
[0058] By "coding sequence" is meant an nucleic acid sequence that contributes to the code for the poly peptide product of a gene. By contrast, the terra "non-codin sequence'' refers to any nucleic acid sequence that does not contribute to the code fo the polypeptide product of a gene,
00S9J The term "colony stimulating factor' refers to a secreted glycoprotein that binds to receptor proteins on the su face of hematopoietic cells acti vating intracellular signallin pathways that cause the cells to proliferate and differentiate into different types of blood ceils. CSF-1 (macrophage colony stimulating factor), CSF-2 (granulocyte macrophage colony stimulating factors; GM-CSF; sargramostim), arid C.SF-3 (granulocyte colony stimulating factors; G-CSF; filgrastim), and promegapoietio are examples of colony stimulating factors.
[0060] By "corresponds to'5 or "corresponding to" is meant a nucleic acid sequence that displays substantial sequence identit to a reference nucleic acid sequence (e.g., at least about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 6.1, 62, 63, 64, 65, 66, 67, 68, 6% 70, 71., 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83» 84, 85, 86, 97, 88, 89, 90, 1, 2, 93, 94, 95, 6, 7, 98, 99% or e ven up to 100% sequence identity to all or a portion of the refefence nucleic acid sequence) or an amino acid sequence that displays substantial sequence similarity or identity, to a reference amino acid sequence (e.g., at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 7L 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84,. 85, 86, 97, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even up to 100% sequence similarity or identity to all or a portion of the reference amino acid sequence).
[0061] "CycioaikyP when used alone or in compound words such a$
"cycloa!koxy," refers to cyclic hydrocarbon residues, including mono- or polycyclic alkyl groups. Exemplary cycloaikyl are C4.7 a!kyL A "cyeloalkyi" group may contain one or more double or triple bonds to form a cyc!oalkenyl or cycloaikynyl group and accordingly, "eyeloalkyP also refers to non-aromatic unsaturated as well as saturated cyclic hydrocarbon residues. Examples of "cycloaikyl" include cyc!opropyl, cyc!obuiyl, eyclopeniyl, cyclohexyi, cycioheptyi, cyelooetyL cyciononyl, cyclodecyi, 1,.3-cyclo.hexadienyl, 1 yl-eyelohexadieny!, l s3~eycloheptadieny1, ] ,3,5-eycioheptatrienyl and 1 ,3,5,7-cyclooctatetraenyl. A eycloalkyl group may be optionally substituted 'by one or more optional substituents as herein defined. Accordingly;, "cycioalkyl" as used herein is taken to refer to optionally substituted cycloalkyl.
[0062] The term "derivatize," "derivatizing" and the like refer to producing or obtaining a cpmppimd from another substance b chemical eac ion e.g., by adding one or mor reactive groups to the compound by reacting the compound with a functional group- adding reagent, etc.
[0063] The term "derivative, in the context of polypeptide derivatives, refers to a polypeptide that has been derived from the basic seqiience by modification, for example by conjugatio or complexing with other chemical moieties, or by post-transiational modification techniques as would be understood in the art. The term "derivative5' also includes within its scope alterations that have been made to a parent sequence including additions or deletions that provide for functional equivalent molecules,
[0064] The term "differentiation" of hematopoietic stem ceils and/or hematopoietic progenitors as used herein refers to both the change of hematopoietic stem ce lls into hematopoietic progenitors and the change of hematopoietic progenitors into umpotent hematopoietic progenitors and or cells having characteristic functions, namely mature cells including erythrocytes, leukocytes (e.g. , neutrophils) and megakaryocytes. Differentiatio of hematopoietic stem, ceils i to a variety of blood eel! types involves sequential activation or silencing of several sets of genes. Hematopoietic stem cells typically choos either a ly mpho i d or myelo id lineage pa thway at an early stage of differentiation.
[0065] By "effective amount", in the context of treating or preventing a condition is meant the administration, of an amount of an agent or composition to an. individual in need of such treatment or prophy laxis, either in a single dose or as part of a series, that is effective for the prevention of incurring a symptom, holding in check such symptoms, and/or treating existing symptoms, of that condition. The effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomie grou of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range thai can be determined through routine trials.
[0066] The term "expression" with respect to a gene sequence refers to transcription, of the gene and, as appropriate, translation of th resulting mKNA transcript to a protein. Thus, as will be clear from the context, expression of a coding sequence results from transcription and translation of the coding sequence. Conversely , expression of a non- coding sequence results from the transection of the non-coding sequence.
£0067] As used herein, the term "function" refers to a biological, enzymatic, or therapeutic' function.
|0068] The term "gene" as used herein refers to any and all discrete coding regions of the cell's genome, as well as associated non-coding and regulatory regions. The terra is intended to mean the open reading frame encodin specific polypeptides, introns. and adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression, in this regard, the gene may further comprise control signals such as promoters, enhancers, termination and or poiyadenylation signals that are naturally associated with a given gene, or heterologous control signals. The DNA sequences may be cDNA or genomic KN A or a fragment thereof, The gene may be introduced into an appropriate vector for
extrachromosomal maintenance or for integration into the host,
[0069] The term "group" as applied to chemical species refers to a set of atoms mat forms a portion of a molecule. In some instances, a group can include two or more atoms that are bonded to one another to form a portion of a m olecule. A group can be mo novalent or polyvalent (e.g. , bivalent) to allow bonding to one or more additional groups of a molecule. For example, a monovalent group can be envisioned as a molecule with one of its hydrogen atoms removed to allow bonding to another group of a molecule, A group can be positively or negatively charged. For example, a positivel charged group can be envisioned as a neutral group with one or more protons -(/.e., IT") added, and a negatively charged group can be envisioned as a neutral group with one or more protons removed. Non-limiting examples of groups include, but are not limited to, alkyl groups, alkylene groups, alkenyl groups, alkenylene groups, alkynyl groups, alkyny!ene groups, aryl groups, arylene groups, immyl groups, iminylene groups, hydride groups, halo groups, hydroxy groups, aikoxy groups, carbox groups, thio groups, alkylthio groups, disulfide groups, cyano groups, nitre groups, amino groups, alky lam ino groups, dialkylamino groups, siiyl groups,, and siloxy groups. Groups such as alkyl alkenyl alkynyl. aryl, and heterocyclyl, whether used alone or in a compound word or in the definition of a group may be optionally substituted by one or more substitueuts. 'Optionally substituted," as used herein, refers to a group may or may not be further substituted with one or more groups selected from alkyl, alkenyi, alkynyL aryl, halo, haloalkyl, halpalkenyl, haloa!kyny!, haloaryl, hydroxy, aikoxy, aikenyloxy, aryloxy, benzyloxy, haleaikoxy, haloalkenyloxy, haloary loxy, nitro, nitroalkyi, nitroalkenyk nitroaikynyl, .nitrparyl, nitroheteroeyclyl, amino. aSkylamino, diafkyianiino, alkeny!amino, alkynyfemiao, ary {amnio, diarylamino, phenylamiao, diphenyiamino, benzylamino, dibensykmino, hydrazmo, aeyl, acyiamino, diaeylamioo, acyloxy, heteroeyelyl,
heterocycioxy, heterocycl&mmQt haloheterocyclyl, carboxy ester, carboxy, carboxy amide, mercapto, alk ithio, ben^lthio, acylthio and phosphorus-containing groups. As used herein, the term "optionally substituted" may also refer to the replacement of a Ci¾ group with a carbonyi (OO) group. Non-limiting examples of -optional, substituents include alkyl, preferably Cf-a alkyl (e.g.,
Figure imgf000022_0001
butyl, cyclopropyi, cyclobutyl, cyeiopentyl or eydohexyi), hydroxy Gs.g alkyl (e.g., hydroxymethyl, hydro.xyeth I, hydroxypropyi), alkoxyalkyl (e.g., methoxymethyl, rneihox ethyl, methoxypropyi, ethoxy meth l, ethoxyethyL
Figure imgf000022_0002
such as metfaoxy. ethoxy, propoxy, botoxy, cyelopropoxy, cyclobutoxy), halo (fiuoro, chksro, bromo, iodo), trifluoromethyl, trichloromethyl, tribrorrtomethyi, hydroxy, phenyl (which itself may be further- substituted, by an optional substituent as described herein, e.g., hydroxy, halo, methyl, ethyl propyl, butyl, methoxy, ethoxy, acetoxy, amino), benzyl (wherein the C¾ and/or phenyl group ma be further substituted as described herein), phenoxy (wherem the CS¾ and/or phenyl group may be further substituted as described herein), benzyloxy (wherein the CH? and/or phenyl group ma be further substituted as described herein), amino, Cj.g alkylamino ( .g. ) C]^ alkyl, such as methyiamino, ethylamino, propylamine), di alkylamino (e.g., G. 5 alkyl, such as diniethyianino, diethylamino, dipropy!amino), acyiamino (e.g., NHC(Q)CH3.), phenylamino (wherein phenyl itself may he further substituted as described herein), nitro, formyj, -C(0)-Ci-ij alkyl (e.g.,
Figure imgf000022_0003
alkyl such as acetyl), 0-C(G)~alkyl (e.g. , Cw alkyl, such as acetyloxy), benzoyl (whe ein the CH? and/or phenyl .group itself may be fiirther substituted), replacement of C¾ with C~0, C(¾H, CG2 C].g alkyl (e.g., Cs-g alkyl such as methyl ester, ethy! ester, propyl ester, butyl ester),
Figure imgf000022_0004
(wherein phenyl itself may be further substituted),. CONH2, CONHphenyl (wherein phenyl itself may be further substituted as. described herein), CQNHberc_yl
(wherein the CI¾ and/or phenyl group ma be further substituted as described herein), CONH C],s alkyl (e.g., C14 alkyl such as methyl amide, ethyl amide, propyl amide, butyl amide), CONHdi C1-8 alkyl (e.g , Chalky!).
[0076] "Hematopoiesis" refers to the highly orchestrated process of blood cell development and homeostasis. Prenatally, hematopoiesis occurs in the yolk sack, then liver, and eventually the bone marrow. In normal adults it occurs in bone marrow and lymphatic tissues. Ail blood cells develop from pluripotent stem cells. Pluripotent cells differentiate into stem cells that are committed to three,, two or one hematopoietic differentiation pathway, Norte of these stem ceils are morphologically .distinguishable, however.
[0071] The term "hematopoietic stem cells" or "BSC" as used herein refers to muftipoient stem cells that are capable of differentiating into all blood ceils including erythrocytes, leukocytes and platelets. For instance,, tire term "hematopoietic stem cells" includes and encompasses those contained not only in bone marro but also in umbilical cord blood derived cells.
[0072] The term "hematopoietic progenitors," or "hematopoietic progenitor cells",, which are used interchangeabl with the term "hematopoietic precursors," refers to those progenitor or precursor cells which are differentiated furthe 'than hematopoietic stem ceils but have yet to differentiate into progenitor.; or precursors of respective blood cell lineages (unipotent precursor cells). Thus, "progenitor cell(¥p or "precursor ce!i'(s),s are defined as cells that. are lineage-committed, i.e., an individual cell can give rise to progen limited to a single lineage such as the myeloid or lymphoid lineage. They do not have self-renewal properties. They can also be stimulated by lineage-specific growth factors to proliferate, if activated to proliferate, progenitor cells have life-spans limited to 50-70 cell doublings before programmed cell senescence and death occurs. For example, "hematopoietic progenitors" as used herein include granulocyte/macrophage associated progenitors (colony- forming unit granulocyte, macrophage, CFU-GM), erythroid associated progenitors (burst- forming unit erythroid, BFU-E), megakan'ocyie associated progenitors (colony-forming unit megakaryocyte, CFU-Mk), and myeloid associated stem cells (col ny-fonning unit mixed, CFU-Mix). Hematopoietic progenitor ceils possess the ability to differentiate into a final cell type directly or indirectly through particular developmental lineage, Undifferentiated, piuripotent progenitor cells that are not committed to any lineage are referred to herein as "stem cells." All hematopoietic cells can in theory be derived from a single stem cell, which is also able to perpetuate the stem cell lineage, as daughter cells become differentiated. The isolation of populations of mammalian bone marrow cell populations wh ich are enriched to a greater or lesser extent, in piuripotent stem cells has been reported (see for example, C.
Verfaillie si l, J, Exp. Med., 172, 509 (1990)).
[0073] "HSPC" as used herein refers to both hematopoietic progenitor ceils and hematopoietic stem cells.
[0074] As used herein, "HlP-a potentiating agents" include agents that increase the accumulation of, or stability of, HIF-a: directly provide HIF-a activity; or increase expression of HT.F-1. Such: agents are known in the art, or may be identified through art- recognized screening methods. HIF~a potentiating agents include compounds that increase the accumulation .aad or stability of HiF-a by inhibiting the activity of one or more HIF hydroxylase enzymes, e.g., one or more HIF prolyl hydroxylase enzymes. Inhibitors of HIF hydroxylase enzyme activity are well known, readily identified, and are further described herein.
[0075] "Homolog" is used herein to denote a gene -or its product which is related to another gene or product by decent from a common ancestral D A sequence.
[0076] As used herein, the term hyperproliferative cell disorder" refers to a disorder in which cellular hyperproliferatioii causes or contributes to the pathological state or symptoms of the disorder, illustrative hyperproliferaiive cell disorders include, but are not limited to, neoplasms, benign tumors, malignant tumors, pre-cancerous conditions, in situ tumors, encapsulated tumors, metastatic tumors, liquid tumors, solid tumors, immunological tumors, hematological tumors, cancers, carcinomas, leukemias, lymphomas, sarcomas, and rapidly dividin cells. The term "rapidly di viding cell" as used herein is defined as any cell that divides at a rate that exceeds or is greater than what is expected or observed among neighboring or juxtaposed cells within the same tissue. Exemplary hyperproliferaiive cell disorders include: cancers; blood vessel proliferative disorders such as restenosis, atherosclerosis, in-stent stenosis, vascular graft restenosis, ete,; fibrotic disorders; psoriasis; inflammatory disorders, e.g.. arthritis, etc ; glomerular nephritis; endometriosis macular degenerative disorders; benign growth disorders such as prostate enlargement and lipomas; autoimmune disorders; and scarring disorders such as post-operative scarring, hypertrophic scarring, keloid scarring and glial scarring. In some embodiments, the hyperproliferative cell disorder is a precancer or a precancerous condition. A "precancer ceil" or "precancerous -cell" is a cell manifestin a hyperproiiferative cell disorder that is a precancer or a precancerous condition, in other embodiments, the hyperproliferative cell disorder is a cancer. The term "cancer" includes primary and metastatic cancer and is used
interchangeably herein with the term "neoplastic" to refer to a disease or condition involving cells that metastasize or have the potential to metastasize to distal sites and exhibit phenofypic traits that diffe from those of non-neoplastic cells, for example, formation of colonies in a three-dimensional substrate suc as soft agar or the formation- f tubular networks or web-like matrices in a three-dimensional basement membrane or extracellular matrix preparation* such as Matrigel™. Non-neoplastic cells do not form colonies in soft agar and form distinct sphere-like structures in three-dimensional basement membrane or extracellular matrix, preparations. Neoplastic cells acquire a characteristic set of functional capabilities during their development, albeit through various mechanisms. Such capabilities include evading apoptosls, self-sufficiency in growth signals, insensitivity to anti-growth signals, tissue invasion metastasis, limitless replica ive- potential, and sustained, angiogenesis. Thus, "non-neoplastic" means that the condition, disease, or disorder does not involve cancer cells. Exemplary cancers includes solid tumors, as we'll -as, hematologic tumors. and/or malignancies, A "cancer cell," "cancerous cell" or "neoplastic cell" is a cell manifesting a hypsrproliferative cell disorder that is a cancer. Any reproducible means of measurement ma be used to identii cancer cells or precancerous cells. Cancer cells or precancerous cells can be identified by histological typing or gradin of a tissue sample (e.g. , a biopsy sample). Cancer cells or precancerous cells can be identified through the use of appropriate molecular markers, in some embodiments, the h erpro I i ferati ve cell disorder is a non-neop!astk disorder in which cellular hyperproliferation causes or contributes to the pathological state or symptoms of the disorder.
[0077] "Hybridization" is used herein to denote the pairing of complementary nucleotide sequences to produce a DNA-DNA hybrid or a DN A-RNA hybrid.
Complementary base sequences are those sequences that are related by the base-pairing rules. In DNA, A pairs with T and C pairs with G. In RNA U pairs with A and C pairs with G, hi this regard, the terms "match" and -"mismatch" as used herein refer to the hybridization potential of paired nucleotides in complementary nucleic acid strands. Matched nucleotides hybridize, efficiently, such as the classical A-T and G-C base pair mentioned above.
Mismatches are other combinations of nucleotides that do not hybridize efficiently. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteeii or reversed Hoogsteen hydrogen bondings between complementary nucleoside or nucleotide base (micleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying
circumstances as known to those of skill m the art.
|0O?8] The phrase "hybridizing specifically to" and the like refer to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
10079] "Hypoxia-inducible factor a" or "HIF-a" is a subunit of the oxygen- dependent transcriptional activator, hypoxia-inducible factor (H'lF), which plays crucial roles in the angiogenesis of tumors and mammalian development HIF' is a heterodimerie protein consisting of a constitutively expressed HIF-Ι β subun.it and one of three subunfts HIF- (HIF-1 a, HIF-2a or HIF-3a), The most widely studied, and seemingly major, RTF isoform is the HIF-1 isoform. The stability and activity of HTF- subunits are regulated by 5 various post-txarssiationai modifications, including hydroxy lation, acetylation, and
phosphor lation. Under normoxia, the HIF-a subunit is rapidly degraded via fe von Hlppel- Lindau tumor suppressor gene product (vHL)-mediaied ubiquitin-proieasorne pathway. The association of vHL and HIF-a under normoxic conditions is triggered by the hydroxyktion of prolines and the acetylation of lysine within a polypeptide segment known as the ox genic' dependent degradation domain (ODDD). The hydroxylattpn of 'proline residues (e.g.,
■specifically proline 402 and 564 in human HIF-1 a polypeptide) within the ODDD is carried out by specific HIF-pro!yl hydroxylases (HPH1 -3 also referred to as PHD 1 -3) in the presence of iron, oxygen, and 2-oso.gIutarate, During hypoxic conditions HIF-a. subunit becomes stable and interacts with co-activator such as p300/CBP to modulate its
15 -transcriptional activity. HIF-1 acts as a master regulator of -ftumerous. 'hypoxia-inducible genes under hypoxic conditions. The heterodimer HIF-1 binds to the hypoxic response elements (HREs). of target gene regulatory sequences, resulting in the transcription of genes implicated in the control of cell proliferation/survival, glucose iron metabolism and angiogenesis, as well as apoptosis and cei!uiar stress. Some of these direct target genes 20 include glucose transporters, the glycolytic enzymes, erythropoietin, and angiogenic factor vascular endothelial growth factor (VEGF). "HIF-a"' is the oxygen-responsive component of HIF-1 and may refer to any mammalian or non-mammalian HIF-a polypeptide or fragment thereof, e.g., HIF- 1, , HIF~2a, or HIF-3 a. In specific embodiments, the term refers to the human form of HIF- la, as set forth for example in GenPept Accession os. AAC50152. 25 P_0Q152.1, NP_8S 139? and NPJOO 1230.013, HIF-a coding or gene sequences are also encompassed, as discussed for example mfi-a. A fragment of HIF-a of interest is any fragment retaining at least one functional or structural characteristic of HIF-a, Non-limiting fragments of HIF-1 a suitably include proline residue 402 and/or 564 (as set forth in GenPept Accession No. AAC50152), which are hydrox lated by PHD polypeptides. Suitable 30 fragments may include or consist of residues 344-698. particularl residues 364-678, more particularly residues 364-638 or 384-638 and still more particularly residues 364-598 or 394- 598. Other suitable fragments may include or consist of residues 549-652 and even more particularly the N-terminal region thereof which interacts with the vHL protein. C-terminal. fragments may include residues 549 to 582 and in particular residues 556-574, Other suitable fragments comprise or consist of residues 3 -4 i 7» more suitably 380-417. Such a region, or its equivalent in other HiF-a sirbimit proteins, is desirabiy present in HiF-a substrates used in assays for screening PHD inhibitors. Exemplary HiF-a fragments may typically comprise residues 549 to 582 of the human H IF- l sequence.
[00803 T e term "immunocompromised" as used herein refers to a subject with an innate, acquired, or induced inability to develop a normal immune response. An
immunocompromised subject, therefore, has a weakened or impaired immune system relative to one of a normal subject. A subject with a weakened or impaired immune system 'has- n "imtmmodeficiency" or "immunoeompiOtnised condition " which is associated with a primar or secondary deficiency, induced or non-induced, in one or more of the elements of the normal immune defense system. An immunocompromised condition is commonly due to a medical treatment, e.g., radiation therapy, chemotherapy or other immunosuppressing ■treatment, such as induced by treatment with steroids, cyclophosphamide, ai¾thioprine. methotrexate, cyclosporme or rapamycin, in particular in relation to Cancer treatment or the treatment or prevention of rransplaot rejection, However, it will be understood that the phrase "risk of acquiring an immunocompromised condition resulting from a medical treatment" refers only to medical treatments that leads to or confers an immunocompromised condition, especially chemotherapy or other 'immunosuppressing treatment, such as .induced by treatment with radiation, steroids, cyclophosphamide, azamioprine, methotrexate,
cyclosporin© or rapamycin. The presence of an immunocompromised condition in a subject can be diagnosed by any suitable technique known to persons of skill the art. Strong indicators that an immunocompromised condition may be present is when rare diseases occur or the subject gets ill from organisms that do not normally cause diseases, especially if th subject gets repeatedl infected. Other possibilities are typically considered, such as recently acquired infections— for example, HIV, hepatitis, tuberculosis, etc. Generally, however, definitive diagnoses are based on laborator tests that determine the exact nature of the immunocompromised condition. Most tests are performed on blood samples. Blood contains antibodies, iymphocytes, phagocytes, and complement components— all of the major immune components that might cause immunodeficiency. A blood ceil count wiii determine if the number of phagocytic cells or iymphocytes is below normal. Lower than normal counts of either of these two cell types correlates with an immunocompromised condition. The blood cells are also checked for their appearance. Occasionally , a subject may have normal ceil counts, but the ceils are■structurally defective. If the lymphocyte ceil, count is low, further testing is usually conducted. to determine whether any particular type of lymphocyte is lower than normal. A lymphocyte. roliferation, test may be conducted to determine if the lymphocytes can respond to stimuli- "The: failure to respond to stimulants correlates with an immunocompromised condition. Antibody levels and complement levels can also be determined for diagnosing the presence of an immunocompromised condition. However, it shall be understood that the methods of the present invention are not predicated upon diagnosing the absence of an immunocompromised condition in the subjects to be treated.
[0081] Reference herein to "immuno-interactive" includes reference to any interaction, reaction, or other form of association between moiecuies and in particular where one of the molecules is, or mimics, a component of the immune system.
[0082] Reference herein to an "infectious agent," "infectious, organism," "microbe" or "pathogen" inc ludes any one or more species or subspecies of bacterium, fungus, virus, algae, parasite, (including ecto-or endo-parasiies) prion, oorayceies, slime, moulds, nematodes, mycoplasma and the like. The present invention is particularly suited to treating or preventing mixed infections by more than one microbe. Pathogenic algae include
Protothec and Pfiesieria. Also includes within the scope of these terms are prion proteins causing conditions such as Creutzfeidt- Jakob disease. As the skilled artisan will appreciate, pathogenicity or the ability of a classieaily non-pathogenic agent to infect a subject and cause pathology can vary with the genotype and expression profile of the infectious agent, the host and the environment. Fungal pathogens include without limitation species of the following genera: Absidia^ Aeremom m, Aspergillus, Bg$idiohoh&s;s Bipolaris, Blastomyces, Candida (yeast), Cl dophialophora, Coccidioides^ Cryptococc s, CunmnghameUa, C rvirfaria, Epidermophyt n, Exophial , Exsero i!um, Fonsecaea, Fus rium, Geotrichum, Histoplasm , Hori ea, Lac zi . Lasiodiplodi , Leptosphaeria, Madurella, Malassezia, Microsporum, MIICG Neotestudina, Onychocola, PaecUomyces, Paracoccidioides, FenieUivum,
Phi lophora, Piedraia, Piedra, Pityriasis, Pneumocystis? Pseudallescheria, Pyrenoch ta,
RJmomimon Rhizopm, Rhodotonrfa, Scedospori m, Scopidariopsis, Scytalidium^ Sporothrix, Trichophyton, T ickosporon and Zygomycete. Pathogenic conditions include any deleterious condition that develops as a result of infection with an infectious organism.
[0083] As used herein, the term "interact" includes clos contact between molecules that results in a measurable effect, e.g., the binding or associatio of one molecule to another or a reaction of one molecule with another,
[0084] By "isolated" is meant material that is substantially or essentially free from components that normall accompany it in its native state. [0085] The term "lower alky!" refers to straight and branched chain alky groups having from 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, iso-propyl, n-butyi, tert- bistyl, see-butyl, n-pentyl,. n-hexyi, 2-raethyl eniyl, and the like. In some embodiments, the lower alky! grou is methyl, or ethyl.
[0086] The term "lower alkoxy" refers to straight and branched chain alkoxy groups having from 1 to 6 carbon .atoms* "such as methoxy, ethoxy, n-propoxy,. iso-propoxy, n-hutoxy, rert-butoxy, sec-butoxy, n-pentoxy, ii-hexoxy, 2-methyl-pentoxy, and the like. Usually, th tower alkoxy group is methoxy or ethoxy.
[008.7] As used herein, a "mobilizer of hematopoietic stem cel ls and or progenitor ceils," "mobilizing agent" or "mobilized are used interchangeabl to refer to any compound, whether it is a small organic -molecule, synthetic or naturally derived, or a polypeptide, such as a growth factor or colony stimulating factor or an active fragment or mimic thereof, a nucleic acid, a carbohyd ate, an antibody, or any other agent that acts to enhance the migration of stem cei ls from the bone marrow into the peripheral blood. Such a "mobilizef may increase the number of hematopoietic stem ceils or hem topoietic progenitor/precursor cells in the peripheral blood.
(0088} By '"modulating" is meant increasing or decreasing, either directly or indirectly, the level or functional activity of a target molecule. For example, an agent may indirectly modulate the level/activity by interacting with a molecule other than the target molecule. In this regard, indirect modulation of a gene encoding a target polypeptide includes within its scope modulation of the expression of a First nucleic acid molecule, wherein an expression product of the first nuc leic acid molecule modulates the expression of a nucleic acid molecule encoding the target polypeptide,
[0089] A ^neutropenia medicament" as used herein refers to a composition of matter which reduces the symptoms related to neutropenia, prevents the development of neutropenia, or treats existing neutropenia.
[0090] The term "oligonucleotide'" as used herein refers to a polymer composed of a multiplicity of nucleotide residues (deoxyribonueleotides or ribonucleotides, or related structural variants or synthetic analogues thereof) linked via phosphodiester bonds (or related structural variants or synthetic analogues thereof). Thus, while the term "oligonucleotide" typically refers to a nucleotide polymer !rt which the nucleotide residues and linkages between them are naturally occurring, it will be understood that the terra also includes within its scope various analogues including, but not restricted to,, peptide nucleic acids (FNAs), phosphorasTi ates, phospkjrothioates, methyl phosphonates, 2-O-methyI' ribonucleic acids, and the like. The exact size of the molecule can var '' depending on the particular application. An oligonucleotide is typically rattier short irs length, generally from about 10 to 30.
nucleotide residues, but the term can refer t molecules of any length, although the term "polynucleotide" or "nucleic acid" is typically used for large oligonucleotides.
[0091 j The term "operabiy connected" or "operabiy linked" as used herein refers to a j uxtapos ition where in the components so described are in a relationship permitting them to function in their intended manner. For example, a transcriptional control sequence "operabiy linked" to a coding sequence or non-coding sequence refers to positioning and/or orientation of the transcriptional control sequence relati e to the cod in or non-codin sequence to permit expression of the coding or non-coding sequence under conditions compatible with the transcriptional control sequence.
[0092] The term "pharmaceutically acceptable" as used herein refers to a compound or combination of compounds that will not impair the physiology of the recipient human or animal to the extent that the viability of the recipient is compromised. Suitably, the administered compound or combination, of compounds -will elicit, at most a temporary detrime ntal effect on the health of the recipient human, or animal.
10093) By "pharmaceutically acceptable carrier" is meant a pharmaceutical vehicle or solvent comprised of a material thai is not biologically of otherwise undesirable, i.e. the materia! ma be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction. Carriers ma include exeipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like. Illustrative vehicles or solvents include' without limitation water, saline, physiological saline, ointments, creams, oil-water emulsions, geis, or any other■vehicle/solvent or combination vehicles/solvents and compounds known to one of skill in the art that is pharmaceutically and physiologically acceptable to the recipient human or animal.
[00941 Similarly, a "pharmacologically acceptable" salt, ester, amide, prodrug or derivative of a compound as pro vided herein is a salt, ester, am ide, prodrug or derivative that this not biologically or otherwise undesirable.
[0095] Pathogenic "protozoa" Include, without limitation, Trypanosoma,
Leishm ma,-Giardia^ Trichomonas, Entamoeba, Naeglertei, AcanthamoebiX, Plasmodium, Toxoplasma, Cryptosporidium hospora and Balantidium, [0096] Larger pathogenic "parasites" include those from the phyla Cestoda (tapeworms), ematoda and Trematoda (flukes). Pathogenic trematodes are, for example, species of the followi ng genera; Schistosoma, Echinostoma, Fasciolapsis, Clonorchis, Fascfola, OpisiharcMs and Paragonimus. Cestode. pathogens include, without limitation, species from the following orders; Pseudophyllidea {e.g., Diph ohofhrmm) and
CyclophyUidea (e.g., Taenia), Pathogenic nematodes include species from the orders; Rhabditida (e.g., .Strongyloieks), .Sirongylida (e.g., Ancy stvma), Asmridia (e..g,sAsmris, Toxoc ra , Spir rida (e.g., Dracim l , Brugia, Onchocerca, W cheria) and Adenophorea (e.g. , Trichuris tmd TrichineUa).
[0097] The terms "polynucleotide," "genetic materia!." "genetic forms." "nucleic acids" and "nucleotide sequence" include R A, cDNA, genomic DNA, synthetic- forms and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, -as will be readily appreciated by those skilled In the art.
f 0098] The terms "polynucleotide variant" and "variant" refer to polynucleotides displaying substantial sequence identity wit a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions as known in the art (see for example Sanibrook e? al , Molecular -Cloning. A Laboratory Manual", Cold Spring Harbor Press, 1989), These terms also encompass polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides. In this regard, it is well' understood in the art that certai alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains a biological function or activity of the reference polynucleotide. The -terms "polynucleotide variant" and "variant" also include naturally-. occurring allelic variants.
f 0099] The terms "polypeptide," "proteinaeeous molecule," "peptide" and "protein" are used interchangeably herein to refer to a poly mer f amino ac id residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-natnra-Hy-occmring amino acid, such as a chemical analogue of a corresponding naturally-occurring amino acid, as well as to naturally-occurring amino acid polymers. 'These terms do not exclude mod ificai ions, for example, glycosyiaiions. acetyiations, phosphorylations and the like. Soluble forms of the subject protemaeeous molecules are particularly useful. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids or polypeptides with substituted linkages.
|09100] The terra "polypeptide variant" refers to polypeptides in which one or more amino acids have been replaced by different amino acids. It is well understood in the art that some am in® acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide (conservati ve substitutions) as described hereinafter. These terms also encompass polypeptides in which one or .more amino acids have been added or deleted, or replaced with different am ino acids,
I'OOIOi] As used herein, the terms "prevent,5' "pre vented,' or "preventing,5' when used with respect to the treatment of a immunocompromised condition (e g-, anemia, thrombocytopenia, agranulocytosis or neutropenia), refers to a prophylactic treatment which increases the resistance of a subject to developing the immunocompromised condition or, in other words, decreases the likelihood that the subject will develop the immunocompromised condition as well as a■treatment after the immunocompromised condition has begun in order to reduce or eliminate it altogether or prevent it from becoming worse,
[00102] The term "pro-drug" is used in its broadest sense and encompasses these deri vatives that are converted in vivo to the compounds of the in vention. Such derivatives would readily occur to those skilled in the art and include, for example, compounds where a free hydroxy group is converted into an ester derivative,
[00103] As used herein, "racemate" refers to a mixture of enantiomers,
[00104] As used herein; a "reporter gene" refers to any gene or DNA that expresses product that is detectable b spectroscopic, photochemical, biochemical, enzymatic, immunochemical, electrical, optical or chemicai means. The preferred reporter gene to which a promoter element is ligated i lueiferase. Other reporter genes for use for this purpose include, for example, β-galactosidase gene (β-gal) and ..chloramphenicol acetyl transferase gene (CAT) Assays for expression produced in conjunction with each of these reporter gene elements are well-known to those skilled in the art.
[0010S] The terms "salts," ''derivatives" and "prodrugs* includes any
pharmaceutically acceptable salt, ester, hydrate, or any other compound which, upon administration to the recipient is capable of providing (directly or indirectly) a compound of the invention, or an active metabolite or residue thereof. Suitable pharmaceutically acceptable salts include salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulfuric, phosphoric, nitric, carbonic, boric, sulfamic and hydrobromic scids, or salts of pharmaceutically acceptable organic acids such. as acetic, propionic, butyric, tartaric, nialeie. hydroxymaleie, fumarie, citric, lactic, mueie, gluconic, benzoic, succinic, oxalic, phenylaeetic, methanesulfbnic, tolu/enesulfonic, benzenesulfome, sa!icyc!ic, sulfani!ic, aspartic, glutam ic, edetie, stearic, palmitic, oleic, laurie, pantothenic, tannic, ascorbic and valeric acids. Base salts include, but are not limited to, those forme with pharmaceutically acceptable cations, such as ·: sodium, potassium, lithium, calcium, magnesium, ammonium and alky lamraonium. Also, basic nitrogen -containing groups may be quatemized with such agents as lower alfcyi haiides, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyi sulfates like dimethyi and diethyl sulfate; and others. However, it will be appreciated that uon-pharoiaeeuticaliy acceptable salts also fall within the scope of the invention since these may be useful in the preparation of pharmaceutically acceptable salts. The preparation of salts and prodrugs and derivatives can be carried out by methods known in the art. For example, metal salts can be prepared by reaction of a compound of the invention with a metal hydroxide. An acid salt can be prepared by reacting an appropriate acid with a compound of the invention.
1130106] The term "selective" refers to compounds that inhibit or display antagonism towards a PHD (e.g., PI-ID 1, PHD2, or PHD3) (e.g., a prolyI-4-hydroxylase) without displaying substantial inhibition or antagonism towards another PHD- Accordingly, a compound that is selective for a■particular PHD (e.g., proIyl-4-hydroxyiase) exhibits inhibition or antagonism of that PHD that is greater than about 2-fold, 5 -fold, ! 0 -fold, 20- fold, 50-fold or greater than about 100-fold' with respect to inhibition or antagonism of another PHD. In some embodiments, selective compounds display at least 50-fold greater inhibition or antagonism towards a particular PHD (e.g., a ptoly I-4-hydroxyiase) than towards another PHD. in still other embodiments, selective compounds inhibit or display at least 100-fold greater inhibition or antagonism towards. a particular PHD (e.g., a prolyI-4- hydroxyjase) than towards another PHD. In still ether embodiments, selective compounds display at least 500-fold greater inhibition or antagonism towards a particular PHD (e.g., a pro1y f-4-hydiOxylase) than, to wards another PHD, in still other embodiments, selective compounds display at least 1000-fold greater inhibition or antagonism towards a particular PHD (e.g., & proiyl-4-hydroxylase) than towards another PHD
|00107] The term "sequence identity" as used her in refers to the extent that sequences are identical on a nucleotide-by~nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by compar ing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base ( .g., A, T, C, G, I) of the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val., Leu, lie, Phe, Tyrs Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys arid Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the iota! number of positions i the window of. comparison (Le. , the windo size), and multiply tag the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by an appropriat method. For example, sequence identity analysis ma be carried out using the .DNASIS computer program (Version 2,5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software,
100108] "Similarity" refers to the percentage number of amino acids that are identical or constitute conservative substitutions as defined in Table 1 below.
TABLE 1
ORIGINAL SE WDE ExEMPLA RY SUBSTITUTIONS
Ala Ser
Arg Lys
Asn Gin, His
Asp Glu
Cys Ser
Gin Asn
Glu Asp
Gl Pro
His Asn, Gin
lie Leu, Val
Leu He, Val
Lys Arg, Gin, Glu
Met Leu, lie.
Piie Met, Leu, T r
Ser Thr
Thr Ser
Figure imgf000035_0001
100109] Similarit may be determined using sequence comparison programs such as GAP (Deveraux el at 1984, Nucleic Acids Research 12. 387-395). In this way, sequences of a similar or substantially different length to those cited herein might be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP. Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference .sequence", "comparison window", "sequence identity", "percentage of sequence identity" and "substantial identity", A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two
polynucleotides may each comprise. (I), a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence mat is divergent between the two 'polynucleotides, seq uence com par isons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a. "comparison window" to identif and compare local regions of sequence similarity. A "comparison window" refers to a -conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is- compared to a reference sequence of the same num er, of .contiguous positions after the two. sequences are optimally aligned. The comparison windo may comprise additions or deletions (i.e., gaps} of about 20% o less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequence for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFiT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive- Madison, I, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison■window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed b Attschul et αί, 1997, NucL Acids Res. 25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausube! et al, "Current Protocols in Molecular Biology", John Wile & Sons fnc, 1 94-1998, Chapter 1 . [OOilO] As used herein a "small molecule" refers to a composition that has a molecular weight of less than 3 ktlodaltons (kDa and typically less than 1.5 kiiodaitQns, and more preferably less than about 1 Modal ton. Small molecules may be nucleic acids, peptides, polypeptides* peptidoroknetics, carbohydrates, lipids or other organic (carborj- containing) or inorganic molecules. As those skilled in the art will appreciate, based on the present description, extensive libraries of chemical and/or biological mixtures, often fungal, bacterial, or algal extracts, may be screened with any of the assays of the invention to identify compounds that modulate a bioactivity. A "small organic molecule" is an organic compound (or organic compound eoraplexed with an inorganic compound (e.g., metal)) that has a molecular weight of less than 3 kilodaitons, less than 1 ,5 kiledaltons, or even less than about 1 kDa.
[60111] "Stem cells" refer to cells, which are not terminally differentiated and are therefore able to produce cells of other types. Stem cells are generally divided into three types, including totipotent, pluripotent, and mult ipotent. "Totipotent stem cells" can grow and differentiate into any ceil in the body, and thus can grow into an entire organism. These cells are not capable of self-renewal. In mammals, only the zygote and early embryonic cells are totipotent. "P!uripotent stem, cells" are true stem cells, with the potential to make any differentiated cell in the body, but cannot contribute to making the extraembryonic membranes (which are derived from the trophobiast). "Multipotent stem cells" are clonal cells that self-renew as well as differentiate to regenerate adult tissues, "Multipoteiit stem cells" are also referred to as "unipotenf ' and can only become particular types of cells, such •as blood cells or bone cells. The term "stem ceils", as used herein, refers to plnripotertt stem cells capable of self-renewal.
[00112] "Stringency" as used herein refers to the temperature and ionic strength conditions, and presence or absence of certain organic solvents, during hybridization. The higher the stringency f the higher will be the observed degree of complementarity between sequences. "Stringent conditions" as used herein refers to temperature and ionic conditions under which only polynucleotides having a high proportion of complementary bases, preferably having exact complementarity, will hybridize. The stringency required is nucleotide sequence dependent and depends upon the various components present during hybridisation, and is greatly changed when nucleotide analogues are used. Generally, stringent conditions are selected to be about 10° C to 20° C less than the thermal melting point (Tm for the specific sequence at a defined ionic strength and pH. The Tm is. the temperature (under defined ionic strength and pH) at which 50% of a target sequence hybridizes to a complementary probe-, It will .be understood that a polynucleotide will hybridize to a target sequence under at least low stringency conditions, preferably under at least medium strmgency conditions and more preferably under high stringency conditions. Reference herein to low stringency conditions include and encompass from a least about .1 % v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization at 42° C, and at least about 1 M to at least about 2 M salt for washing at 42° C. Lo stringency conditions also ma inciude 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHP04 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2 SSC, 0.1 % SDS; r (ii) 0.5% BSA, 1 rftM EDTA, 40 mM NaHP04 (pH 7.2), 5% SDS for washing at room temperature. Medium stringency conditions include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from, at least about 0.5 M to at least about 0.9 M salt for hybridization at 42° C, and at least about 0.5 M to at least about 0.9 M salt for washing at 42° C. Medium stfingeney conditions also may include 1% Bo ine Serum Albumin (BSA), 1 tiiM EDTA, 0,5 M NaHP04 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2 X SSC, 0. i % SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPCM (pfi 7.2), 5% SDS for washing at 42° C. High stringency conditions inciude and encompass from at least about 31% v/V to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization at 42° C, and at least about 0.01 M to at least about 0.15 M salt for washing at 42° C. High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHPQ4 (jpH 7.2), 7% SDS for hybridization at 65° C, and (i) 0.2 x SSC,
0.1 % SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPG4 (pH 7.2), 1% SDS for washing at a temperature in excess of 65° C. Other stringent conditions are well known in the art. A skilled addressee will recognise that various factors can be manipulated to optimize the specificity of the hybridization. Optimization of the stringency of the final washes can serve to ensure a high degree of hybridization. For detailed examples, see CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY (supra) at pages 2.10.1 to 2.10.16 and MOLECULAR CLONING. A LABORATORY MANUAL (Sambrook, et al, eds.) (Cold Spring Harbor Press 1989) at sections 1.101 to 1 , 104.
[00113] "Subjects1' contemplated in the present invention include any animal of commercial,, humanitarian, or epidemiological interest includin conveniently, primates, livestock animals (such as sheep, cows, horses, donkeys, pigs, fish and birds), laboratory test animals (such as mice, rabbits, guinea pigs and hamsters and the- like), companion animals (such as dogs and cats), or capti ve wild animals, Avian species include poultry birds and caged avian species, i some embodiments the subject is a mammalian animal in other embodiments, the . subject is a human■ subject The present composition 'and methods have applications in human and veterinary medicine, domestic or wild animal husbandry, cosmetic or aesthetic treatments for the skin after injury or surgery, A donor subject is the subject in which the mobilization of hematopoietic stem and/or progenitor cells occurs and/or from which the mobilized stern and/or progenitor cells are harvested, if any, A recipient subject is the subject to which the harvested stem and/or progenitor cells' are transplanted. The donor subject and the recipient subject may be the same subject or may be different subjects.
[00114] B ''substantially .complementary" it is meant that an oligonucleotide or a subsequence thereof is sufficiently complementary to hybridize with a target sequence. Accordingly, the nucleotide sequence of the oligonucleotide or subsequence need not reflect the exact complementar sequence of the target sequence, in a preferred embodiment, the oligonucleotide contains no mismatches and with the target, sequence.
[00115] A used herein, the term "synergistic" means that the therapeutic effec t of a HIF-a potentiating agent when administered in combination with at least one mobilizer of hematopoietic stem cells and/or progenitor cells (or viee-versa) is greater than the predicted additive the rapeutic effects of the HIF-a potentiating agent and the at l east one mobilizer when administered alone. The term "synergistically effective amounf as applied to a HIF-a potentiating agent and at least on e mobilizer of hematopoietic stem cells and/or progenitor cells refers to the amount of each component in a composition (generally a pharmaceutical ■composition),, which is effective for stimulating or enhancing mobilization of hematopoietic stem ceils and/or progenitor ceils from the bone marro into th peripheral blood, and which prod uces an effect which does n ot intersect, in a dose-respons e plot of the dose of HIF- potentiating agent versus a dose of the at least one mobilizer versus stimu l ating or enhancing mobilization of hematopoietic, stem cells and/or progenitor cells from the bone marrow into the peripheral blood, either the dose HIF-a potentiating agent axis or the dose at least one mobilizer axis. Th dose response curve used to determine synergy in the art is described for example by Sande et al. (see, p. 1080-1 105 in A. Goodman ei at., ed., the Pharmacological Basis of Therapeutics, MacMillan Publishing Co,, Inc. New York .(1980)). The optimum synergistic amounts can be determined, using a 95% confidence limit b varying factors suc as dose level, schedule and response, and using a computer-generated model that generates isoboktgrams from the dose response c urves for various combinations of the HIF-a potentiating agent and the at least one mobilizer. The highest mobilization of hematopoietic stem cells and/or progenitor cells on the dose response curve correlates with the optimum dosage levels. A "thrombocytopenia medicament?' as used herein refers to a composition of matter which reduces the symptoms related to thrombocytopenia, prevents the development of thrombocytopenia, or treats existing -thromboc topenia.
{00116] As used herein, the term "transcriptional control sequence" refers to nucleic acid sequences, such as initiator sequences, enhancer sequences and promoter sequences, which induce, repress, or otherwise control the transcription of protein encoding nucleic acid sequences to which they are operably-Iinked.
(00117] By "treatment, ** "treat,"' "treated," "treating" and the like is meant to include both therapeutie and prophy lactic treatment, including the administration of medicine or the performance of medical procedures with respect, to a patient, for either prophylaxis (prevention) or to cure or reduce the extent of or likelihood of occurrence of the infirmity or malady or condition or event in the instance where the patient is afflicted. In some embodiments of the present invention, the treatments usin the agents described may be provided to treat patients suffering from a hyperproliierattve eel! disorder, whereby the treatment of the disorder with a cytoreductive or myeloablative therapy (e.g., chemotherapy or radiation therapy) results in a decrease in bone marrow ce!iularity, thus making the patient more immunocompromised and more prone therefore -to acquiring infectious agents or diseases. Thus, the administration of the agents of the invention allows for enhanced mobilization of hematopoietic stem cells and/or progenitor cells from the bone marrow to the peripheral blood. In some embodiments, the treating is for the purpose of reducing or diminishing the symptoms or progression of a hyperproliferative cell disorder by allowing for the use. of accelerated doses of chemotherapy or radiation therapy.
[00118] By "vector" is meant a polynucleotide molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast or virus, into which a polynucleotide can be inserted or cloned. A vector preferably contains one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or- tissue or a progenitor cell or tissue thereof, o be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector can be an autonomously replicating vector, .e,, a vector that exists as an extrachroroosomal e n tity, the repl ication of whic h is independent of chromosomal replication, e.g. , a linear -or closed circula plasmid, an extrachromosomal element, a m inichromos me, or an artificial chromosome. The vector ca contain any means for assuring self-replication. Alternatively, the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the ehromosonie(s) into which it has been integrated. A vector system can comprise a single vector or plasmid, two or more vectors or piasmids, whic together contain the total DNA to be introduced into the genome of the host ceil, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the host sell into which the vector is to he introduced- In the present case, the vector is preferably a vtral or viral-derived vector, which is opsrably functional in animal and preferably mammalian cells. Such vector may be derived from a poxvirus, art adenovirus or yeast. The vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformatits. Examples, of such resistance genes are known to those of skill in the art and include the nptll gene that confers resistance to the antibiotics .kanamycm and G418 (Geneticin®) and the hph gene which confers resistance to the antibiotic hygrarnycin B.
[00119] Reference herein to "a virus" includes any virus or viral pathogen or emerging viral pathogen. Viral families contemplated include Adenoviridae, African swine fever-like viruses, Arenaviridae (such as viral hemorrhagic fevers, Lassa fever), Astroviridae (astro viruses) Birayaviridae (La Crosse), Calicivsridae (Norqvirus), Coronaviridae {Corona virus), Filoviridae (such as Ebola virus, Marburg virus), Parvoviridae (B 1 virus),
Flaviviridae (such as hepatitis C virus, Dengue viruses), Hepadnaviridae (such as hepatitis B vires, Delta virus), Herpesviridae (herpes simple vims, varicella zoster vims),
Qrthomyxoy dae (influenza vims) Papovaviridae (papilloma virus) Paramyxoviridae (such as human parainfluenza viruses, mumps virus, measles virus, human respiratory syncytial virus, Ntpah virus, Hertdra virus), Pieomayiridae (common cold virus), Poxviridae (small pox virus, orf virus, monkey poxvirus) Reoviridae (rotavirus) Retroviridae (human
immunodeficiency virus) Parvoviridae (parvoviruses) Papillomaviridae, .(papillomaviruses) alphaviruses and Rhabdoviridae (rabies virus).
{0 120] As used herein, underscoring or ital cizing the name of a gene shall indicate the gene, in contrast to its protein product, which is indicated by the name of the gene in the absence of any underscoring or italicizing. For example, "fflF-la" shall mean the HJF-la gene, whereas "HIF-Ια" shall indicate the protein product or products generated from transcription and translatio and alternative splicing of the "EiF-la" gene,
[00121] Each embodiment described herein is to be applied, mutatis mutandis to each and every embodiment unless specifically stated otherwise. , Abbreviations
CFC = eoiony-forraing cells
HIF ~ hypoxia-inducible factor
HlFa ~ hypoxia-inducible factor-a
HSC = hematopoietic stem cells
HSPC = hematopoietic stem and progenitor ceils
d = day
h = hour
s = seconds
i.v. = intravenous
i.p. = intraperitoneal
rliu = recombinant human
s.c. = subcutaneous . Composit m mid methods fat enhancing hemampoieik function
[00122] The present invention is based in part on the surprising discovery that mobilization of hematopoietic stem ceils and or progenitor celis by mobilizing agents such as G-CSF and Pierixafor, and by combinations thereof, is sigtviflcantly enhanced in the presence of HIF-ct potentiating agents. This increased mobilization in turn results in higher numbers of hematopoietic stem and progenitor cells (HSPCs) migrating from the bone marrow into the peripheral blood when compared to the number resulting from administration of stem cell mpbilizers alone. The increased mobilization may also result in increased HSPCs mobilizin from the peripheral blood to particular tissues or organs such as the lymph nodes, the heart, the lung, the liver, the skin, the spleen, small and large intestines, the stomach, or the pancreas.
[00123] Increasing the number or mobil ity of HSPCs may al so inc rease the rate of differentiation of HSPCs into various cell lineages. The HSPCs may also be capable of differentiation or starting a path to becoming a mature hematopoietic cell. For example^ the differentiation of the HSPCs. may lead to an increase in the number of common myeloid progenitor cells in the bone marrow or the peripheral blood. The differentiation of HSPCs may also lead to an increase in the number of granulocyte/macrophage progenitor cells or megakaryocyte/erythrocyte progenitor cells in the bone marrow or peripheral blood. The HSPCs may differentiate into a common lymphoid precursor, lire increase in number of common myeloid progenitor cells may lead to a differentiation into granulocyte/macrophage progenitor cells or megaka^o'cyte/erythrocyte progenitor cells, The granulocyte/macrophage progenitor cells may further differentiate into granulocytes such as neutrophils, eosinophils, basophils, tissue precursor cells, monocytes, and immature dendritic ceils. The
megakaryocyte/erythrocyte progenitor ceils may differentiate into megakaryocytes and erytliroblasis, The common lymphoid precursor cell may differentiate into B lymphocyte cells and T lymphocyte cells. Th'e.B lymphocyte ceils may differentiate into anttbody- seereting cells, wherein T lymphocytes, may differentiate Into effector T cells. The
granulocyte may further differentiate into tissue mast ceils, macrophages, and immature dendritic cells. The megakaryocyte may differentiate into platelets. The erythro blast may differentiate into erythrocytes,. HSPCs may also be capable of differentiating into ceils such as muscle (skeletal myocytes and cardiomyoeytes), brain, liver, skin, lung, kidney, intestinal, and pancreatic. The number or proportion of cells presenting particular molecular or cell surface markers may be indicative of an HSPC or HSPC population.
[00124] Although not wishing to be bound by any theory or mode of operation, 1 in every 10,000 to i 5,000 bone marrow cells may normally be a stem cell. In the bloodstream, the proportion may fall to 1 to 300,000 blood cells. Administering a HIF-a potentiating agent and at least one mobiilzer of hematopoietic stem cells and/or progenitor ceils in vivo may Increase the number of ail stem eel! populations in the bloodstream in about 1 hr or less, 2 hrs or less, 3 hrs or less, 4 hrs or less, 6 S rs or less, 8 hrs or less, 10 hrs or less, 12 hrs or less, 14 hrs or less, 16 hrs or less, 18 hrs or less, 20 hrs or less, 22 hrs or less, 24 hrs or less. 26 hrs or less, 28 hrs or less, or 30 hours or less after administration, and accumulation -of. stem cells including HSPC in the blood may peak m about 65 hrs or less, 66 hrs or less, 67 hrs or less, 68 hrs or less, 69 hrs or less, 70 hrs or less, 71 hrs or less, 72 hrs or less, 73 hrs or less, 74 hrs or less, 75 hrs or less, 76 hrs or less, 77 hrs or less, 78 hrs or less, 79 hrs or less, 80 hrs or less. 81 lirs or less, 82 hrs or less, 83 hrs or less, 84 hrs or less, 85 hrs or less, 86 hrs or less, 87 hrs or less, 88 hrs. or less, 89 hrs or less, 90 hrs or less, 91 hrs or less, 92 rs or less, 93 hrs or less, 94 hrs or less, 95 hrs or less, 96 hrs or less, 97 hrs or less, 98 hrs or less, 99 hrs or less, 100 hrs or less, 101 hrs or less, 102 hrs or less,. 103 hrs or less, 104 hrs or less, 105 hrs or less, 106 hrs or less, 1.07 hrs or less, 108 hrs or less, 1 09 hrs or less, and i 10 h s or less after administration,
[00 25] Thus, in accordance with the present invention, methods and compositions are provided that take advantage of a HIF-a potentiating agent and at least one mobii tzsr of hematopoietic stem cells and/or progenitor cells for stimulating or enhancing mobilization of hematopoietic stem cells and/or progenitor cells from the bone marrow into the peripheral blood, for stimulating or enhancing hematopotesis, for 'the. treatment or prophylaxis of immunocompromised conditions, including ones resulting from medical treatments that target rapidly dividing ceils or that disrupt the cell cycle or cell division (e.g., myetoablative therapy), or for stem cell transplantation.
Figure imgf000043_0001
[00126] The HIF-a potentiating agent includes and encompasses an active agent that increases the accumulation of, or stability of, HIF-a; directly provides HIP-s activity; or increases expression of HlF-a, including without limitation, small molecules and macromoiecules such as nucleic acids, peptides, polypeptides, peptidomimetks, carbohydrates, polysaccharides, lipopolysaecrsarides, lipids or other organic (carbon containing) or inorganic molecules. HIF-a refers to one or more of HIF- la, HIF-2 HlF-3a.
[00127] Thus, in some embodiments, the HIF-a potentiating agent can be anything that results, in an increase in the amount or activity of a- HIF-a polypeptide. Non-limiting examples include agents; that improve the stability (e.g., half-life) of the protein; that block or reduce deactivation of the HIF-a polypeptide, for example by preventing the
hydroxylation and/or acetylatioh; and agents that increase the amount of HIF-α polypeptide in a. sample under consideration, for example by increasing the amount expressed from a HIF-a coding sequence or by introducin multiple copies of HIF-a codin sequence. Accordingly, without limiting the present invention to an particular mechanism, with regard io an exemplary mode of action, the HIF-a potentiating -agent can increase the activity of the HIF-a polypeptide by acting directly or indirectly on the HIF-a polypeptide to stabilize the protein, protect it from inhibition, or to increase the activity of the protein. Alternatively, the substance can increase the activity of the HTF^ot polypeptide by inhibiting or otherwise blocking the act ivity of compound's or enzymes that inhibit the acti vity or reduce the stability of the- HIF-a polypeptide
[00128] In certain embodiments, the method includes introducing into at least one cell of the subject, such as a hematopoietic stem cell or progenitor cell, a nucleic acid construct that comprises at least one HIF-a coding, sequence operable connected to a transcriptional control sequence, and permitting the eeii to express the encoded HIF-a polypeptide. Non-limiting examples of HIF-a coding sequences include: huma ΗΉ 'α coding sequences as disclosed for example in GenBank Accession N >s. NMQG1530, Q9NWT60, U2243 ί , ABO-73325, AF2084S7 and AF304431 ; bovine HIF-a coding sequences as disclosed for example in GenBank Accession Hos. Q9XTA5, AB01-8398 and BAA78675; rat HiF-α coding sequences as disclosed for example in GenBank Accession Nos.
AF057308, 035800 and CAA70701 ; mouse f ilF-a coding sequences as disclosed for example in GeaBaiik Accession Nos. AF003695, AAC52730, AFO5730S and Q61221 ; squirrel HIF-a coding sequences as disclosed for example in GenBank Accession No.
AY7I347S; avian HIF-a coding sequences as disclosed for exampl in GenBank Accession No, Q9YIB9; amphibian HIF- coding sequences as disclosed for example in GenBank Accession No. Q98SW2; antelope HIF-a coding sequences as .disclosed for example n GenBank Accession No. AY?? 1808; Xenapus l vis HIF-a coding sequences as disclosed for 'example- in GenBank Accession No. CAB96628; DrosapMla mekmogaster HIF-a coding sequences as disclosed for example in GenBank Accession No, JC4S5 \ ; zebra fish HIF-a coding sequences. as disclosed for example in GenBank Accession No. AY326951 : chicken HIF-a coding sequences as disclosed for example in GenBank Accession Nos. ABA02179 and BAA34234, and the l ike. Others species of interest would be dogs, cats, and other domesticated and farm animals, such as pigs and horses, HIF-a may also be an mammalian or non-mammalian protein or fragment thereof. HIF-a gene sequences may also be obtained by- routine cloning techniques, for example by using all or part of a HIF-a gene sequence described above as a probe to recover and determine the sequence of a HIF-a gene la another species. A fragment of HIF-a of interest is any fragment retaining at least one functional or structural characteristic of HIF-a. Fragments of HIF-a include, e.g., th regions defined by human HIF-a from amino acids 401 to 603 (Fluang et al,, (1998) Proc Natl Ami ScL USA 95:7987-7992), amino acid 531 to 575: (Jiang &t at (\W!) JBiol Chem. 272:19253-19260}, amino acid 556 to 57-5 (Tanrmoto et al (2000) EMBO J, 19:4298-4309), amino acid 557 to 5 1 (Srimvas et al. (1999) Bioekem Biop ys Res Commirn. 260: 557-561), and. amino acid 556. to 575 (Ivan and aeiin (2001) Science 292:464-468). Further, HIF-a fragments include any fragment containing at least one occurrence of the motif LXXLAP, e.g. , as occurs in the human HIF-a native sequence at i3s7 LL P and LssetE LAP.
[001:29] In other embodiments, HIF-a potentiating agents stimulate or enhance expression of ffIF- , representative examples of which include metallothlonein and zinc (see, e.g., Xue -et al (2012) Am J Physiol Heart Ore Physiol 302: H2528-F12535)..
[00130] in some embodiments, HIF-a potentiating agents inhibit the level or activit of a HIF-a 'interacting protein that inhibits the acti vity of a HIF-a polypeptide. Non-limiting HIF-I interacting proteins of this type include: the von Hippel-Lindau tumor suppressor protein (vHL, Hon et al (20.02) Nature 41.7:975-8; Mi et al (2002) Science 296: 1886-9); hydroxylases including prolyl hydroxylases (e.g, pjroly-4 -hydroxylases) (also referred herein as H1F hydroxylases 'such as the HIP prolyl hydroxylases P.HDL FFID2 and PHD3, as described for example by Epstein et aL (2001) Cell 107:43-54, Kaelin (2005) Annu Rev BiQchem, 74:1 15-28; Schmid ei-af: (2004) J Ceil Mai Med, , 8:423-31 ; Huang et aL (2002) / Bio! Chem. 277:39792.-800· and Metzen et al (2003) JCeil Sel 1 16: 131 -26),
dehydroxylases, ubiquftylaiion and deubiquitylatiort enzymes, ARDl acetyltransferase (as described for example by Jeong et al. (2002) Cell 11 1 :709-20), factor inhibiting HIF-1 (FIH- 1; as described for example by Hewitson et al (2002) J Biol Chem. 277(29):2635.I -5; ndo et ai (2002) Genes Dev. 16: 1 66-71 ; PCX Application Publication Nos. WO03.Q28663, WO04035812, WO02074981) inhibitory PAS domain protein (IPAS, Makino et L (2002) Nature 414:550-4) and the like, which interact with one or more proteins comprising the HIF-1 heteradtmer and/or modulate the activity thereof. Of particular interest are human HIF- interacting proteins (see, e.g. , Accession Nos. P40337, HP 000542, NP937799,
NP005154, NPQ60372, NP0Q3363, and the like) and homoiogues. .analogues and isoforms thereof (including animal homoiogues). Those of skill in the art will readily be able to identify additional HIF-a interacting proteins suitable In the present invention.
[00131] Thus, in the HIF-a pathway in which the HIF-a polypeptide interacts with several HIF-a Interacting proteins, there are several possible points of .therapeutic
intervention. First, HIF-e activity or protein levels can be increased by using small molecules to disrupt 'the rapid degradation of HIF- '(Hewitson, K S and Schofield, C J. (2004) Drug Discovery-. Today 9(1 β):704-711). This would include* for example, inhibitors of PHD 1-3 (prolyl hydroxylase domain-containing enzymes 1-3), which include proly!-4-hydroxylase inhibitors, illustrative examples of which include oxalamic acid alkyl esters (e.g.,
dimethyloxaliyl glycine) and disubstituted pyridines (e.g., dieihylpyridine dicarboxylate); inhibitors of FIH (factor inhibiting HIF) such as, for example, dihydrobeazoic acids (e.g.. 3,4-dihydrobenzoaie); proteasomal inhibitors that affect degradation of the HIF-a sybunit; small molecules or antibodies that would block vHL .eorri lex:HIF-ct interaction; small molecule inhibitors of ubiquitination; and inhibitory nucleic acid molecules such as small interfering RNAs (siR As) targeting PHD 1 -3 and/or FIH.
[00132] In some embodiments, the HIF-a potentiating agent is an antagonistic nucleic acid molecule that functions to inhibit the transcription or translation of PHD, FIH- 1 or vHL encoding transcripts. Representative transcripts of this type include nucleotide sequences corresponding to any one the following sequences: (1) human PHD1 nucleotide sequences as set forth for example in GenBank Accession Nos. A 10544, BC036051, NMJ353046 and NM_080732; human PHD2 nucleotide sequences as set forth for example in GenBank Accession Nos, NM_022O5I and NG_Q-15865; human PHD3 nucleotide sequences as set forth for example in GenBank Accession Nos. NMJ322073 and AJ310545; human FIH-l nucleotide sequences as set forth for example in GenBank Accession No, NM_017902; and human vHL nucleotide sequences as set forth for example in GenBank Accession os . N „000551 and NM„198156;· (2) nucleotide sequences that share at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, S3, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% sequence identity with any one of the sequences referred to in (l); (3) nucleotide sequences that hybridize under at least low, medium or high stringency conditions to the seq uences referred to in (1 ): (4) nucleotide sequences th at encode any one of the following amino acid sequences: human PHD I. am ino acid sequences as set forth for example in GenPept Accession Nos. CAC4251Q, AAH36051, .NP_ 44274 and NP_542770; human PHD2 amino acid sequences as set forth for example in GenPept Accession Nos, NPJJ71334 and NP_071334; human PHD3 amino acid sequences as set fort tor example in GenPepi Accession Nos. NP_071356 and CAC4251 1 ; human . FIH-1 amino acid sequences as set forth for example in GenPept Accession No. NPJ36G372; and human vHL amino acid sequences: as set forth for example in GenPept Accession Nos. NP_000542 and NP_937799; (5) nucleotide sequences that encode an amino acid sequence that shares at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similarity with an one of the sequences referred to in (4); and nucleotide sequences thai encode an amino acid sequence that shares at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 1 , 2, 93, 94, 95, 96, 97, 98, 99% sequence identity with an one of the sequences referred to in (/I).
[0003] illustrative antagonist nucleic acid molecules include antisense molecules, aptamers, ribozymes and triplex forming molecules, RNAI and external guide sequences. The nucleic acid molecules, can act as effectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule., or the functional nucleic acid molecules can possess a de novo activity independent of any other m olecules.
[00134] Antagonist nucleic acid molecules can interact with any maerorooiecole, such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, antagonist nucleic acid molecules can. interact with PHD, FIH-l or vHL mRNA or the genomic DMA of FED, FIH- 1 or v.HL or the can interact with the PHD, FIH-1. or vHL polypeptide. Often antagonist nucleic acid molecules are designed to interact with other nucleic acids based on sequence homology between the target molecule and the antagonist nucleic acid molecule. In other situations, the specific recognition between the antagonist nucleic acid molecule and the target molecule is not based on sequence homology between the antagonist nucleic acid molecule and the target moleeuie, 'but rather is based on the formation .of tertiary structure that allows specific recognition to take place.
[00135] In some embodiments, anti-sense RNA or DNA moleeules are used to directly block the translation of PHD, FlH-1 or vHL .mRNA by binding to targeted ra&NA and preventing protein 'translation; Antisense molecules are designed to interact with a target nucleic aeid moleeuie through either canonical or non-canonical base pairing. The interaction of the antisense molecule and the target moleeuie may be designed to promote the destruction of the target molecule through, for example, RNAseH mediated RNA-DNA hybrid degradation. Alternatively the antisense molecule may be designed to interrupt a processing, function that normally would take place on the target molecule, such as transcription or replication, Antisense molecules can be designed based on the sequence of the target molecule. Numerous methods for optimization of antisense efficiency by Finding the most accessible regions of the target molecule exist Non-limiting methods include in vitro selection experiments and DN A modification, studies using diniefhyl'sulfate (DMS) and d ethylpyrocarbonaie (DEPC). in specific examples, the antisense molecules bind the target molecule with a dissociation constant (Kd) less than or equal to 10"°, 10"8, !0~!0, or 10' . In specific embodiments;, antisense oligodeoxyribonucieotides derived from the translation initiation site, e.g., between. -10.and -HQ regions are employed.
[00136] Aptamers are molecules that interact with a target molecule, suitably in a specific way, Aptamers are generally small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets. Aptamers can bind small molecules, such as ATP and theophiline, as well as large molecules, such as reverse transcriptase and thrombin. Aptamers can bind very tightly with Kds from the target molecule of less than I0' M. Suitably, the aptamers bind the target molecule with a Kd less than 10^ 10"s, 10"l°, or !0'!2 Aptamers can bind the target molecule with a very high degree of specificity. For example, aptamers have been isolated that have greater than a 10,000 fold difference in binding affinities between the target molecule and another molecule that differ at only a single position on the molecule, it is desirable that an aptamer have a Kd with the target molecule at least 10-, 100-, 1000-, 10,000-, or 100,000- fo!d lower than the « with a background-binding molecule. A suitable method for generating an aptamer to a target of interest (e,g., PHD, FlH-1 or vHL) is the "Systematic Evolution of Ligands by Exponential Enrichment" (S.EL.EX™). The SELEX™ method is described in U.S. Pat No. 5,475,096 and U. S, Pat. No, 5,270,163 (see also WO 91/198Ϊ3), Briefly, a mixture of nucleic acids is contacted with the target molecule under conditions favorable for binding. The unbound nucleic ac ids are partitioned from the bound nucleic acids, and. the nucleic acid-target complexes are dissociated, Then the dissociated nucleic acids are amplified to yield a ligand-enriehed mixture of nucleic acids, which is subjected to repeated cycles of binding, partitioning, dissociating and amplifying as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
£90137] In other embodiments, anti-PHD, -FIH-1 or ~vHL ribozymes are used for catalyzing the specifie cleavage of PHD, FIH-l or vHL RNA. The mechanism ofribozyme action involves sequence spec ific hybridization of the nbozym molecule to complementary target RNA, followed by a endonucleolytic cleavage. There are several different types of ribozymes that catalyze .nuclease or nucleic acid polymerase type reactions, which are based on ribozym.es found in natural systems, such as hammerhead ribozymes, hairpin ribozymes, and tetrahytnena ribozymes. There are also a number of ribozymes that are not found in natural systems, but which have been engineered to catalyze specific reactions de novo, Representauve ribozymes cleave RNA or DN A- substrates. In some embodiments;, ribozymes that cleav RNA substrates are employed. Specific ribozyme cleavage sites within potential RNA targets are initiall identified b scanning the target molecule for ribozyme cleavage siteSj which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable. The suitability; of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ri'bomiclease protection assays,
[00138] Triplex forming functional nucleic acid molecules are molecules that can interact with either doub!e-siranded or single-stranded nucleic acid. When triplex molecules interact with a target region, a structure called a triplex is formed, in which there are three strands of DNA forming a complex dependent on both Watson-Crick and Hoogsteen base pairing, Triplex molecules are preferred because they can bind target regions with high affinit and specificity. It i generally desirable that the triplex forming molecules bind the. target molecule with, ¾ less than W( W\ ΗΓ0, or 10~'2.
[00139] External guid sequences (EGSs) are molecules that bind a target nucleic acid molecule forming a complex, and this complex is recognized by RNAse P, which cleaves the target molecule. EGSs can be designed to specifically target a RNA molecule of choice. RNAse P aids in processing transfer RNA (1RNA) within a cell. Bacterial RNAse P can be recruited to cleave virtually any RNA sequence fay using an EGS that causes the target R ArEGS complex to mimic the natural . tRNA substrate. Similarly, eukaryotie EGS/RNAse P-directed cleavage of RNA can be utilized to cleave desired targets within eukaryotie cells.
[00140] In other embodiments, RNA molecules that mediate RNA interference'
(RNAi) of a PHD, FiH- 1 or vNL gene or PHD, FIH- J or vHL transcript can be used to reduce or abrogate gene expression. RNAi refers to interference with or destruction of the product of a target gene by introducing a single-stranded or usually a double-stranded RNA (dsR A) that is homologous t the transcript of a target gene. RNAi methods, including double-stranded RNA interference (dsR i) or small interfering RNA (siRNA), have been extensively documented in a number of organisms, including mammalian ceils and the nematode C. e!egam (Fire et el, 1998, Nature, 391 , 806-81 1 ). In mammalian cells, RNAi can e triggered by 21- to 23-nucleotide (nt) duplexes of small interfering RNA (siRNA) (Chili et at, 2002, Mol. Cell. 10:549-561; Elbashir et el, 2001, Nature 41 1 :494-498), or by micro-RMAs (miRKA), functional small-hairpin RNA (shRNA), or other dsRNAs which ar expressed in vivo using D A templates with RNA polymerase III promoters (2¾ng et al, 2002, Mol Cell 9:1327-1333 ; Paddison ei oL, 2002, Genes Dev. 16:948-958; Lee et al, 20.02, Nature Biatechnoi. 20:500-505; Paul et ai. Nature BiotechnoL 2002, 20:505-508; Tuschi, T., 2002, Nature BiotechnoL 20:440-448; Yu et aL 2002, Proe, Natl. Acad. Sci. USA 99(9):604?-605¾ cManus ei al, 2002, RNA 8:842-850; Surer aL 2002, Proc. Natl Acad. Sci. USA 99(6):5515-5520).
[00141] in specific embodiments, dsRNA per se and especially dsRNA-producing constructs corresponding to at least a portion of a PHD, FIH-1 or vBL gene are used to reduce or abrogate its expression. RNAi-mediated inhibition of gene expression may bs accomplished using any of the techniques reported in the art, for instance by trahsfecting a nucleic acid construct encoding a stem-loop or hairpin RNA structure into the genome of the target cell, or by expressing a ixansfected nucleic acid construct having homology for &PHD,- FlH-l or vHL gene from between convergent promoters, or as a head to head or tail to tail duplication from behind a single promoter. Any similar construct may be used so long as it produces a single RNA having the abilit to fold back oft itself and produce a dsRNA, or so long as it produces two separate RNA transcripts, which then anneal to form a dsR A having homology to a target gene,
[001-42] Absolute homology is not required for RNAi, with a lower threshold being described at about 85% homology for dsRNA of about 200 base pairs (Plasterk and Kettmg, 2000, Current Opinion in Genetics and Dev.10: 562-67). Therefore, depending on the -length of the dsRNA, the RNAi-encoding. nucleic ..acids .can -vary in the level of homology they contain toward the target gene transcript, i.e., with dsRN As of 100 to 200 base -pairs having at least about 85% homolog with the target gene, and longer dsKNAs. i.e., 300 to 100 base pairs, having at least about 75% homology to the target gene, R A-encodjng constructs that express a single RNA transcript designed to anneal to a separately expressed KNA, or single constructs expressing separate transcripts from convergent promoters* are suitably at least about 1.00 nucleotides m length. RNA-encoding constructs that express a single RNA designed to form a dsRNA via internal folding ar usually at least about 200 nucleotides in length.
[00143] The promoter used to express the dsRN A- forming construct may be any type of promoter if the resulting dsRNA is .specific for a gene product in the cell lineage targeted for destruction. Alternatively, the promoter m y be lineage specific in that it is onl expressed in ceils of a particular- development lineage. This might be advantageous where some overla in homolog is observed with a gene that is expressed in a non-targeted cell lineage. The promoter may also be inducible by externally controlled factors,, or by intracellular environmental factors.
[0G144] in some embodiments, RN A molecules of about 21 to about 23 nucleotides, which direct c leavage of specific mRNA to- which they correspond, as for example descri bed by Tuschl et al in U.S. 2002/0086356, can be utilized for mediating RNAi. Such 2,1- to 23-nt RNA molecules can comprise a 3'- ydroxy! group, can be single -stranded or double stranded (as two 21- to 23-nt RNAs) wherein the .dsRNA molecules can be blunt ended or comprise overhanging ends (e.g., 5 3').
[001451 In some embodiments, the antagonist nucleic acid molecule is a siRNA, siRNAs can be prepared by any suitable method. For example, reference may be made to International Publication WQ 02 443 1, which discloses siRNAs capable: of sequence- specific degradation of target niRNAs when base-paired with 3' overhanging ends, which is incorporated by reference herein. Sequence specific gene silencing can be achieved in mammalian cells using synthetic, short double-stranded RNAs that mimic the siRNAs produced by the enzyme dicer, siRNA can be chemically or in v/fro-synthesized or can be the result of short double-stranded hai^in-like RNAs (shRNAs) that are processed into siRNAs inside the cell. Synthetic siRNAs are generally designed using algorithms and a conventional D A/RN A synthesizer. Suppliers include Arnbion (Austin, Tex.), ChemGenes
(Ashland, Mass.), Dharmacon (Lafayette, Colo.), Glen Research (Sterling, Va.), MWB Biotech (Esbersberg, Germany), ProUgo (Boulder, Colo.), and Qiagen (Vento, The
Netherlands). siRNA can also be synthesized in vitro using kits such as Ainfaion's
SILENCER™ siRNA Construction Kit
[00146] The production of siRN from a vector is more commonly done through the transcription of a short hairpin RNAs (shRNAs). Kits for the production of vectors comprising shRNA are available, such as, for example, Imgenex's GENESUPPRESSOR™ Construction Kits and Jnvitrogen's BLOCK-IT™ inducible RNAi plasmid and lentiviras vectors.
[00147] Illustrative RNAi molecules (e.g., PHD., FIE-l or vEL siRNA and. stiRNA) are available commercially from Santa Cruz Biotechnology, Inc. (Santa Cru¾ CA, USA).
(00148] In some embodiments, the HIF-a potentiating agent is an inhibitor of HIF hydrox lase enzyme, particularly an inhibitor of a HIF prolyl hydroxylase enzyme. A compound that inhibits the activity of a HIF hydroxylase enzyme refers to any compound that reduces, eliminates, or attenuates the activity of at least one HIF hydroxylase enzyme (e.g., PHD ! -3). In some embodiments, the HIF-a potentiating agent is an inhibitor of a HIF proly l hydroxylase enzyme. Methods for determining whether a compound inhibits HIF hydroxylase activity are well known in the art.
[00149] Functionally, HIF hydroxylase inhibitors for use in the methods of the present invention are defined by their ability to inhibit an activity of a 2-Qxogluiarate dioxygenase enzyme, wherein the enzyme has specific activity toward hypoxia inducible factor. Such compounds are often referred to as HIF hydroxylase i hibitors, HIF prol l hydroxylase inhibitors, HIF prolyl-4-hydroxylase inhibitors, prolyl hydroxylase inhibitors or "PHF's. In specific embodiments, the PHls for use in the invention are small molecule compounds. A compound that inhibits the activity of a HIF hydroxylase enzyme may additionally show inhibitory activity toward one or more other 2-oxoglutarate- and iron- dependent dioxygenase enzymes, e.g., FIH (GenBanfc Accession No, AAL27308), procollagen prolyl 4-hydroxyiase (CP4H), eta
[00150] In particular embodiments, compounds used in the present methods and medicaments provided herein are structural miraeties of 2-oxoglutarate, wherein the compound inhibits the target HIF prolyl hydroxylase enzyme competitively with respect to 2-oxoglutarate and -noncompetitively with respect to irom PHIs are typically heterocyclic car bpxamide compounds, especially heterocyclic carbonyl glycine derivatives, and may be, for example, a heterocyclic carboxamide, including pyridine, pyrimidine, pyridazine, naphthyridine, pytrolop ridine* thlazo'lopyridine, isot iazolopyridine, quinoline, isoquinoHne, e noline, beta-earholine, quinolone, thienopyridiiie, ehromene, or
thioehromene carboxamides. More paiticularly, the inhibitor s-nay be a 'heterocyclic carbonyl glycine.
[00151] 'Compounds' that inhibit HIP prolyl hydroxylase are known in the art and are described, inter alia, m U.S. Patent Nos. .5,658,933; 5,620,995; 5,719,164; 5,726,305;
6,093,730; 7,323,475; U.S. Application Serial No. 12/544,861 ; U.S. 2006/0199836; U.S.
2007/0298104; U.S. 2008/0004309; and WO 2009/073669; WO 2009/089547; WO
2009/100250; WO 02/089799; WO 02/089809; U.S. 2003/0176317, U.S. 2003/083351 ; U.S.2003/0153503, U.S, 2004/0053977; U.S. Patent No. 7,323,475, U.S. 2006/0199836, U.S.
Patent No. 8,324,208; U.S. Patent No. 8,323,671 ; U.S. Patent No.8,343, 952; U.S. Patent No.
8,269,008; U.S. Patent Application Publication No. 201 /0309977; U.S. 2012/0 S36; U.S.
2012/0316204; U.S. Patent Application Publication No, 2011 /0305776; U.S. Patent No.
7,928,120, US. PatenlNo. 7,696,223, U.S. 2010/0303928, U.S. 2010/0330199, U.S.
2010/0331400, U.S. 201 /0047367, PC U 2009/06406S, U.S. Patent No, 7,897,612, U.S..
Patent No, 7,608,621 , U.S. Patent No. 7,728,130, U.S. Patent No. 7,63.5,715, U.S. Patent
No.7,569,726, U.S. Patent No.7,811,595; U.S.. 2007/0299086; U.S. 201 1/01 1 1058 U.S.
201 1/0110961; U.S, Patent No. 8,309,537; WO 2003/049686; U.S. 2003/1 6317; US.
2004/0254215; WO 2004/4108681 ; WO 2005/034929; WO 2005/007192; WO
2004/108121; U.S. 2005/020487; WO 2003/053997; U.S, 2003/153503; WO 2007/070359;
U.S.. 2009/0111806; U.S. Patent No, 8,124,775; U.S. 2009/0093483 ; U.S, 2009/0156605;
U.S. 2009/0088475; U.S. 2009/0099171 WO 2008/137060; U.S. 2009/0156633; U.S.
2010/0035906; WO 2008/049538; WO 2008/067871 ; U.S. 2010/0093803; U.S.
2009/269420; WO 201 1/006355; WO 201 17106226; US. 201.1 /028507; WO 2010/018458; WO 201 1/056725; WO 201 1/049126; WO 201 1/049127; WO 2007/038571; U.S.
2009/0082357; WO 07/136990; WO 09/039323; US. 2009/0176825; U.S. 2010/0113444;
WO 08/08905 J; U.S. 08/0171756; WO 08/089052; WO 2009/039321 ; WO 2009/039322;
U.S. 2009/0170825; WO 09/0491 12; U.S. 2010/0305154; U.S. 2010/0305.133; U.S.
2010/0298324; WO 2009/134847; US. 201 1/0039895; U.S, 201 1/0098324; U.S.
201 1/0160227; WO 2010/022308; U.S. 20.11/0144167; WO 2010/059549; WO
2010/059552; WO 2010/059555; U.S. 2011/0046132; WO 2009/134754; U.S.
2010/0204226; WO 2012/021830; U.S. 2011/0077267; U.S. 2012/004197; U.S.
2010/0056563; U.S. 2010/0137297; US. .2010/0331358; U.S, 201 1/009425; U.S.
201 1 /009406; U.S. 2009/0239876; U-S. 2011/0152304; WO .2010/147776; WO 201 1/002623; WO 201 1/002624; WQ 201 1/133444; WO 2011/130908; WO 2010/076524; WO 2010/076525; WO 201 1/04581 1 ; U.S.. 2011/0130414; WO 201 1/04861 1 ; WO
2012/106472; WO 2013/013609; WO 2013/017063; JP 04/083570; WO 09/131127; U.S. 201 1 /112103; L S. 2012/220609; U.S. 2006/040986; U.S. 2004/00539 8; U.S.
2012/0108581 ; U.S. 89471,024; U.S. 5,985,913; and U.S. 2009/0048294, The foregoing patents and patent appiieations are incorporated herein by reference in their entireties. In one aspect, the present invention specifically contemplates the use of one of more of the compounds -that are described and/or specifically exemplified or claimed in an of the foregoing patents and patent appiieations.
fOOlSJj in one embodiment of the invention, die HIF-ct potentiating agent is selected from the group consisting of [(4-Hydroxy-7^henylsuifany'I-isoq' inoline-3-earbonyl)- aminoj-aeetic acid (Compound X), {[5-(4-Chioro-phen xy)-l-cyano-4-h)'droxy- isoquinoline-3-cai'bonyl]-amiiio}~ac&tie aeid (Compound A), [(1 -Cyano-4-hydroxy-5- p enoxy-isoquinoline-3-earbony!)-amino]-aeetic acid (Compound. S), {[7-Cyano-i~(2- fluoro-benzyl)-4-lrydroxy-1 H-pyn-oloiS^-eJpyrtdine-S-earbonyll-atnino} -acetic acid (Compound C), [(I -Dicyelohsxyi-6-hydra^
carbonyl)-amino]-acetic aeid (Compound D), {r2-(3t4'-Diflooro-biphenyi-4-yta
hydroxy-6-isGpropyl~3-oxo-2 -d^
(Compound E), 2-(6-Morpholin-4-yi-pyn½idin^
pyrazol-S-one (Compound F), [{4~Hydroxy~7-p!ienoxy~isoqu5nofe
acetic acid. (Compound E {[4-Hydroxy-7-(4-meihoxy-phenoxy)-jsoquinolme-3-carboriyI]- amino} -acetic aeid (Compound J), and {[5~(3~Fluoro-plienyl.)-3-ltydroxy-pyridine-2- carbon l'l-amino} -acetic acid (Compound ). In another embodiment of "the invention the HIF-a potentiating agent is selected from the group consisting of [(4~Hydroxy-7- phenylsirlfany -isoqumoline-3-carbonyi)-anjino]-acetic acid (Compound X), {[5~(4-Chloro- phenoxy)-I -cy&no~4~hydroxy-isaqiHno½^ acid (Compound A),
{( I -C ano-4-hydroxy-5 -phenoxy-iso mna 1 ine-3 -earbony l)-am ino] -acetic acid (Compound B), f[7-Cyano- -(2-f1aoro-benzyr}-4 iydroxy H-pyrrolo[2,3-ejp^^
amino}-acetic aeid (Compound G), [(4-Hydfoxy-7-phenoxy-i.soquinoIi:ne-3~carbonyl)- aminoj-aeetic acid (Compound H)} and {[4-Hydroxy-7-(4-metboxy-phenoxy)-isoquIno!ine- 3-earbonyi]-ainino}-acetic aeid (Compound J),
[001531 Other prolyl hydroxylase inhibitors are well known and have been described, inter alia, Bioorg Med Chem Lett. 16(2 !):5616-20 ( 2006); Bioorg Med Chem Lett. 16(21 ):55 7-22 (2006); Bioorg Med Chem Lett. J 6(2l):S598-601 (2006); Bioorg Med Cheai Lett. 16(21):5687-90 (2006); Analytical Biochemistry (2008) 384(2):213-23, 2009; J. Comb. Chem (2010) 12(5):676-86; J Cardiovase Pharmacol 2010 Aug 56(2): 147-55
[00154] Methods of determining if a«y particular compound, inhibits HIF prolyl hydroxylase are well known, illustrative examples of which include the methods described in U.S. Patent No. 7,323.475. The inhibitory activity of any particular compound can he conveniently evaluated arid compared by determining the ICso for one or more of the HIF prolyl hydroxylase enzymes. The IC 0 for any compound for each of the HIF prolyl hydroxylase enzymes can be determined using assays known in the art. In general, the IC50 values for compounds that inhibit HIF prolyl hydroxylase will be in the μΜ range or less, typically in the nM range, for one or more of the HIF prolyl hydroxylase enzymes. The ICso for inhibition of the PHD2 enzyme of Compounds A, B, C, D, E, F, H, J, K, and X range from 0.05- 1 .5 μΜ. The ICjo of the exemplified compounds for the PH'Dl and PHD3 enzymes are in similar ranges.
|0OI55] Illustrative small molecule PHls include, for example, the nitrogen- containing heteroaryl compounds disclosed in U.S. 2004/02542] 5 (WO 2004/410868.1) and in U.S. Patent Nos, 7,323,475; 7.629,357; 7,863,292; and 8,017,625, each of which is expressly incorporated herein by reference in its entirety. Exemplary compounds of this type are represented by formula Ϊ:
Figure imgf000054_0001
[00156] wherein:
[00IS7] q is zero or one;
p is zero or one;
[00159] Ra is -COOH or -WRS; provided that when Ra is -COOH then p is zero and when Ra is— WR* then p is one;
[00160] W is selected from the group consisting of oxygen, -S(0)K - and— Nowhere, n is zero, one or two,
[00161] y is selected from the grou consisting of hydrogen, alk l, substituted alkyi, acyl, aryi, substituted aryi, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic and. Rs is selected tem the group, consisting of hydrogen, alkyi, substituted alkyl, aryl, substituted aryl, freteroaryL substituted heteroaryl, heterocyclic and. substituted heterocyclic, or when W is -MR9- then R8 and R¾ , together with the nitrogen atom, to which they are bound, can be joined to "form a 'heterocyclic or a substituted
heterocyclic group, provided that when W is -3(0.),,·--' and n is one or two, then Rs is iot hydrogen;
[00162] R! is selected from the group consisting, of hydrogen, alkyl , substituted alkyl, alkoxy, substituted alkoxy, amino, substituted amino, arainoacyl, ary!, substituted aryl, halo, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, and -XR6 where X is oxygen, - (0) - or -NR.7- where- a is zero, one or two, * is selected from the group consisting, of alkyl, substituted alkyi, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic,, and R7 is hydrogen, alkyl or aryl or, when X is -MR? - then R' and R8 , together with the nitrogen atom to which they are bound, can be joined to fo rm a .heterocyclic or substituted heterocyclic group;
[00163] R2 and J are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryf, heteroaryl, substituted heteroaryl, halo, hydroxy, cyano, ~S(0)n~ (R.6 }~R6 where n is 0, 1 , or 2, -NR.6 C(0)NR6 R6', -XR8 where X is oxygen, ~S(0)„- or --MR'- where rs is zero, one or two, each Rs is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl. substituted aryl, eyc!oalkyi, substituted cyeioajkyi, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic provided that when X is -SO- or -SOj— then R6 is not hydrogen, and R7 is selected from the group consisting of hydrogen, aikyi, aryl, or R2 , R! together with the carbon atom pendent thereto, form. an aryl substituted aryl, heteroaryl, or substituted heteroaryl;
[00164] R.4 and R5 are independently selected from the group consisting of hydrogen,, halo, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl and -XRe where X is oxygen, ~S(0)«- or -MR7- where n is zero, one or two, R6 is selected from the grou consisting of alky l, substituted "alkyi, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and ..substituted heterocyclic, and R7 is hydrogen, -alkyl or aryl or, when X is -MR7 - then R' and R8 , together with the nitrogen atom to which they are bound, can be joined to form a heterocyclic or substituted heterocyclic group; [00165] R is selected ftom the grou consisting of hydrogen, deBteriurn and methyl;
[00166] R' is selected from the group consisting of hydrogen, deuterium, alky] and substituted alky I; alternatively- R and R' and the carbon pendent thereto can be joined to form eycloalkyl. substituted eycloalkyl, heterocyclic or substituted heterocyclic group;
{00167] R" is selected from the group consisting of hydrogen and alkyi or R' together with R' and the nitrogen pendent thereto can be joined to form a heterocyclic or substituted heterocyclic group;
[00168] '" is selected from the group consisting of hydroxy, alkoxy, substituted alkoxy, acyloxy, eyc!oalkoxy, substituted eyeloalkoxy, ary!oxy, substituted aryloxy, heteroaryioxy, substituted heteroaryioxy, aryl,™S(0}, -R10 wherein R'° is selected from the grou consisting of a!kyl, substituted alkyl, eycloalkyl substituted eycloalkyl, aryl, substituted aryl, heteroaryl and substituted heterqaryi and n. is zero, one or two;
[00169} and pharmaceutical iy acceptable salts, esters and prodrugs thereof;
[00170] In an aiternative embodiment, the compounds of formula I ate represented by formula lA:
Figure imgf000056_0001
[00171] wherein R! , R2 , R3 , R4 , K5 , R, R', R", R'" and q are as defined above; and
[00172] phamiaceuticall acceptable salts, esters, prodrugs thereof.
[00173]' In an another alternative embodiment, the compounds of formula I are represented by the formula IB:
Figure imgf000056_0002
[00174] wherein R! , R2 , 3 , R4 , R5 , R", R"'? WR8 and are as defined above; and
[00175] phannaceiiticaily acceptable salts, esters, prodrugs thereof. [G0176] In an another alternative embodiment, the invention is directed to compounds represented b the formula iC:
Figure imgf000057_0001
[00177] wherein R1 , R2 , R3 , R , R5', R, R', R", R"', WR8 and q are as defined above; and pharmaceutically acceptable salts, esters, prodrugs thereof,
[00178] In yet another alternative. embodiment, the invention is directed to compounds represented by the formula. ID:
Figure imgf000057_0002
[001.79] wherein R! , R" , R\ R , R 5 , R, R\ R"t R"' and q are as defined above;
[00 ISO] and pharmaceutically acceptable salts, esters, prodrugs thereof.
[00181] Exemplary .compounds according to the above formulae include {[4- Hydroxy~l -(naphthaien-2-yloxy)-!soquinoiine-3-carbonyl]-amin^ acid; {[4-
Hydroxy- 1 -{pyridin~3~ytoxy)~isoqmnolme-3-carbonyl]rainino}-acetiC 'acid; {[4-Hyd.roxy-l - (4-methoxy-phenoxy) tsoquinoline-3-carbonyl]-amino-}-acetic acid; {[4-Hydroxy-i-(3- methoxy~phenoxy)-.isQquinolme-3-carbonyl]-amtno}-acetic acid; {[l-{3-Fkioro-pbenoxy)-4- hyd.roxy-isoqusnoiine-3-carbonyl}-aiiiino} -acetic acid; {[i-(4-Fluora-phenoxy)-4-hydrQxy- isoquiiioi!iie-3-carbony]]-amino- } -acetic acid; { [1 -(2-Fluoro-phenoxy)-4~hydroxy- !Soquino]ine~3-carbony!]-araino}-acetic acid: {[4-Hydroxy-l-(2-m.ethoxy-phendxy)- isoquino i ine-3 -carbon ij-ami.no} -acetic ac id ; { [ i-(4- Acety lam ϊηο-phenox )-4-hydroxy- iSGquinolme~3-earbonyi]-amino}-acetic acid; {[4-Hydroxy-l-(4-roetiianesni&nylamino~ phe!ioxy)~i:Soquinoline-3-carbony.l]-aminQ}-acetic acid; [(4-Hydroxy-l-phenylamino- isoqiiinoline-3-carbonyi)~amino]-acetic acid; {[4-iIydroxy-6-(pyridin-3-yloxy)-isoqiiinoline- 3 -carbonyi]-amine} -acetic acid;
Figure imgf000057_0003
amino} -ace- tic acid; [(l-Chloro^4-methoxy-isoquinolme-3^carbony1)-amino]-acettc" acid; [(1 -Chloro-4-ethoxy-isoquieoiine-3-cai¾onyi)-amino]-aceiic acid; [(4-Hydroxy- 1-methoxy- isequino!ine-3-carbonyI)-amino]-acetic acid; [(l~Efhoxy-4~hyd.roxy-isoquinoliiie-3- carbony l)-amino3~acetic acid; [(4-Aeetoxy-l -ptenyi-isoquinoline~3-carbonyl)-ainTJi( ]-acetic acid; [{4-Hydroxy- -p en l-isoqulnof ine~3-carbon !)-am:ino]-acetic acid; [(1 ~Ethoxy-4- pIienyi-is0qumoiiiie-3-carb «iyi)-amir!o]-aceiic acid; [(i -C lofo~4~phenyl-isoqiimo!ine~3- carbG.nyl)-amino]-acetie acid; [(4-P enyl-isoqu{noline-3-carbonyl)-amisiQ]--aeetic acid; [(4- Hydroxy-l-methyI-isoquinoline-3-carbonyl)-amino]-acetic acid; '[(4- Hydroxy*! - meiHoxymethyl-isoquinoiiner3-carbofiyl)-amino]-acetic acid; [(i -Dimet ylcarbamoyi-4- hydroxy-isQquinoi3ne-3-earbonyl)-aramoJ-acetic acid; [(4-Hydroxy- l-raethy 1-6-phenoxy- isoqu ino iiji e-3 -carbony l)-am too] -acetic acid; [(4-Hydroxy- .1 -raethy i-?-phenoxy- isoqujnoiine~3-earbonyl)-amifto]-acetic acid; [(4-Benzyloxy-l-methyl~7-phenoxy- isoqainolme-3-earbonyl)-amino-]-acetiQ acid;
Figure imgf000058_0001
3-carbonyt)-ammo]-a,cetic acid; [(l -Dimethyicarbmnoyl-4-hydroxy-7-phenoxy-isoqymoline- 3 carbonyl)- mino] -acetic acid; [(4-Hydroxy- l-methQxymet y1-7-phenoxy-LsoqyinGline-3- carbony1)-aminoJ-acetic acid; [(4~Hydrexy-l-p-tQlyi-i8oquin iine-3-carbonyl)-amino]~acetic acid; {[7-i4~FItioro~phenoxy)-4-hydro amino) -acetic acid; {[l-Chloro-4~hydr©xy*7~(4-m^
acid; {[4-Hydroxy-7-(4-methoxy~ph noxy)-isoquinoime aeid (Compound J); {[ l-Gh!oro-4- ydroxy--6-(4-i¾'iethoxy-pIienoxy}-isoquinoline-3-carbony]]- am ino } -acetic acid; { [4~Hy droxy-6-(4-metboxy-pheao3cy)-isoquinoli ne-3 -carbony 1] -amino } - acetic acid; {[l-€hl0ro-4-hydroxy~7-i4-tri^^
amino} -acetic acid; { 44rlydroxy-7-(4-tritluoromet^
amino} -acetic acid; { [ 1 -Chioro~4-hydroxy-6-{4 n¾oro^
carbony-l]-araino}-aeetie acid; {[4-Hydroxy-6-(4itrifluoromethyl~phenoxy)-isoquinQ.line-3''- carbonylj-aminoj-acetic acid; {[l-Chlorc~7-(4-fiuoro-phenQxy)-4-hydroxy-isoq inoli¾c-3- carbonyTJ-arnmo) acetic acid; {[7-(4-Fluoro-ph8noxy)-4-hydroxy-isoquinoliiie-3-C8('boriy!]- am ino) -acetic acid; {[l-Ch!Gro-6-(4-fluoro-pheiioxy
amino) -acetic acid; {[6-(4-F]uoro-phenoxy)-4-hydix xy-tso^uinoIine-3-carbonyl]-amino}* acetic acid;
Figure imgf000058_0002
acid; {[4-HydrGxy-6-(pyridin-4^
[(7-Benzenesuffiny]-4-hydtQxy-te^ [(7~
BerjzejiesuIfony]-4~hydroxy~isoquino1ine-3-carbonyi) acid; [{6-
Benze.oesuifmyl~4-hy acid; [(6-
Figure imgf000058_0003
[(6-Amino-4- bydroxy-isoquiooIiHe-3-carbonyi)-arniao3-acetic acid; {[4-Hydroxy-7-(4-meihoxy-
Figure imgf000058_0004
acid; {[4-Hydroxy-7-(3* phenyl-ureido)-isoquihoiine-3-carboiiyl}-^ina}-aeetic acid; {[4-Hydroxy~6-(3~phenyl- «re o)-isoquii oline-3-carbenyi]-amino}-acetie acid; [(4-Hydroxy-l -phenylsoIfanyt isoqumoime-3~carbQny1)-arairio]-acei lc acid; { [ 1 -(4-Cli!oro-phen isulfanyl)-4-hydroxy- isoquinoiine-S-caitonylJ-amtnol-acetic acid; [(4-Hyd!O y-l -p Giylsi!ifany1-igoquinoHne-3- cai'banyi}-afflino]-aceiic acid; { [^.Hydroxy-i -(pyrldin~2-y]suifanyl)-isoquinolifie-3- carbonylj-amino- }-acetic acid;
Figure imgf000059_0001
carbo.nyi]-ammo}- acetic acid; {[4-Hydroxy- i '(t-metho - heo lsulfenyO-isQquinoMne-S- carbonylj-ammo}- acetic acid; {[4-Hydroxy- 1 -(naphthaien-2-ylsuIfanyl)-i.soquisoiine-3- carbanylj-ammo} -acetic acid; [(l-BenzenesuIFinyl-4-iwdroxy~i,soquinoiiiie-3-carbonyi)- amino] -acetic
Figure imgf000059_0002
acid; { 4-Hydroxy-7-(pyrid½-2-y!stdf^ acid; { [4~Hydroxy-0~(p ridin-2-yls uifany ί )- isoquino Une-3 -carbGnyl]-amii.io}~acetic acid; |(i- Cliioro-4-hydroxy-6J-dip enoxy-isoqisiftolisie-3-carbonyl)-a∞ino- ] -acetic acid; [(4-
Figure imgf000059_0003
(†o!uene-4-suifonyiamino)-pb^ acid; {[4-
Hyd:roxy~7~(4-nitro-phenGxy)-¾^ acid; [(4-Mereapto-7- phenoxy-isoquinoiirie-3-carbony1)--amiiio]-acetic acid; [(4-Mercapio-7-trifluororaetl'!.yi- isoquffioline-S^carbonyQ-aminol-acetic acid* {[7-(4-Beiizef."esuIfonyiamin0~phenoxy)«4- hydroxy-isoquinoline-3-carbonyI]-amino} -acetic acid; { [4-Hydroxy- 7-(4- methanesulf0nylami G-phsnoxy)-isoq inoline -3-carbonyl]-amino}-acetic acid; [7-(4-
C Ioix>~p enoxy)-4-hydroxy- isoquino line-3 -carbony I}-ammo } -acetic acid; { [ 6-(4-Chloro- phenoxy) 4-hydroxy-isoquinolrae-3-carb nyl]~ammo)-acetic acid;: {[6-(3"FI ero-5-
Figure imgf000059_0004
acid; {[?-(3-Fluoro-
5-methoxy~phenoxy)-4-hydrc^ acid; {[7-{354- Difii!Qro-phenoxy)-4 -hydroxy- ^ {[6-(3,4-
Difiirem-phenQxy}~4-hydrGxy-is^ -aceti acid; { [4-Hydraxy-7-
Figure imgf000059_0005
(4-trinuoforaethoxy-phenoxy)-!Soq«taoiine-3-carbony^ acid; 2-(S {[7-(4-
Ch'loro-phenoxy)-4-hy.droxy-iseq uin olin e-3 -c arbony 1] -am ino } -propionic acid; 2-{ S)~ { [6-{4- Chjoro-phenoxy)-4-hydroxy-iso uinoiine-3 -carbony 1] -ammo} -propionic acid; 2- {[7-(3,4- Difl oro-phenoxy)-4-hydrox acid; 2-(S)-[(4-,
Hydroxy~7-phenyisu1i¾nyl-isoqt*inoline-3-carbonyi)-am acid; 2-(R)~[{4-
Hydroxy^7-phenylsu!fanyl-isoquinolme-3-carbonyI)-ainino]-propionic acid; 2-( )-[{4- Hydroxy-7»p enoxy-isoquinoline-3-carbonyl)-amino3-propioiiic acid; 2~(S)~[4-Hydroxy-{(4- ffietboxy-phenoxyj-isoqu oim^ acid; 2-(S)-[(7~
BenzenesuIfonyl-4-hydroxyHSoquinoline^ acid; (R)-2-[(4-
Hydroxy-l- methoxymeibyl-7-p ooxy-isoqoinoline-3-carbo«y acic; (S)~
2- [{4 -Hydroxy- 1 -methoxyrae hy i- 7-phenoxy-isoqu i no!ine r3-cart>onyl)-amino]-propionic acid; <S)-2-f (4-MercaptG-7-phenoxy-isoq inoi me-3-earboo l)-amino]-propionic acid; (S)-2- {[l-(4-CMoro~phenyisl fanyl)^
(R)-2-{[l-(4 ¾loro-phe^
acid; [{4-Hydraxy^7-phenytsuHk>yi-i^^ acid (Compound
X); [{4-H drox -6^ heny1sulfa^^^ acid; [(1 -Ch!oro-
Figure imgf000060_0001
hydroxy-6-phenyisidfa^ ae d; (l-Broroo-4- hydi-oxy-7-pheny lsulf¾nylrisoquirioli:ne-3-carbonyi)-amino --aGetic acid; [( 1 -Bromo-4- hydroxy-6~phenyisulfaiiyl-IsoqiHnoltne-3-carbonyI)-araino]-aeetie acid; [(4-HydrGxy-7- phenoxy~iSoqisinoiine-3-carbonyi)-amino]-acetiq acid (Compound H); [(4-Hydroxy~6- phenoxy-isoquinoiirie-S-carboiiy -aminol-acetic acid; [(i-Chioro-4-hydroxy-7-phenoxy- isoquinolme-3-carbonyi)--ara!i'io]-acetic acid; [(i-CIiIoro-4-hydiOxy-6-pl'5ei oxy-isoquraoiine-
3- carbonyi)-ainiiio]-acetic acid; E(I"Bromo-4-hydfOxy~7-phenoxy-isoqiM>noline-3-carbonyi)- aminoj-ace- tic acid; [(1 -Bromo-4-bydraxy-6-phenoxy~isoqiii^
etic acid; {£7-(2,6-Dtffieihyl-pheimy)-4^
acid; { [ 1 -Chlor ^7"(2,6-dimetliyi-phen^
acetic add; {[Ϊ ~Bromo-7~(2>6-dimethyi-phenoxy)-4^drqxy-isoqu
aminoj-acetic acid; [(l~Brorao-7-ehioro-4-hydroxy^
Figure imgf000060_0002
acid;
Bromo-4-hy droxy-7-trifimtfGm ef h i-isoqumo iine-3 -e arbon l)-am into] -acetic acid; [( 1 - Bix>mo^~hydroxy~6 rifluoromethyM^^ [{4-
Hydroxy- l.-phenoxy^oqatnolme-3^rbonyl)-atnino] iacetTC acid, [(l,7-dibromo-4-hydroxy- isoqumol ine-3 -carbon i )-am ino] -acetic acid; [(7-B m a~ 1 -eh 1 oro-4 -hydroxy- isoqu inoiins-3- carbonyi3-anHno]-acetic acid; {(6-Bromo-4-hydroxy-isoqirinoline-3-carbonyl)-amii'io}-acetic
Figure imgf000060_0003
{(7- F]»oro-4~.hydroxy~i5oqusm i!B^ acid; [(l-Chloro-7-fIuoro-4- iiydroxy-isoqumoime-3-carbonyl)-ainino -acetic acid; [(l-ChloFo-4-hydroxy- benzo[g3isoqumoline-3-earbonyl)~a!i¾itio]~ac tic acid; [(l-Brftmo-4-bydroxy-isoq molii5e-3- carbonyi3-amino]-acetic acid; (4-Hydroxy-6-pheiiyl-isoquinoIiiie~3-carbonyi)-amino]-aeetic acid; [(4~Hydroxy-7~phsnyl-isoquinoHne-3-carbonyl)-amiiio] [(l-ChIoro- -
Figure imgf000061_0001
p enyl--isoquin9ime-3-carbotiyi)-araino]-acefic acid; [(l-Brom.o-4-hydroxy-6-phenyl- isoquino3ine^3-carbonyl)-ain!noj-acetic aeid; f(l-Bromo-4-hydTQxy-7-phenyl--isoquinoline~3- caTboiiyl)-atniiro] -acetic acid [(4-Hydjx>xy-5~phenyl-isc^iHnoline-3-carbonyl)-ammo3 acetic acid; [{4-Hydroxy-8-phe.oyMsoqm^ acid; [(l-Chioro-4- hydroxy-5-phenyi-!SGqidnoiine-3-c8rbony -araino3-aceiic acid; [(i-Chloro-4-i¾ydroxy-8~ phenyi-isoquinoline-3-earbonyl)-amino]-acetic acid; [(l-BromOr4-faydroxy-5-phenyl- tsoqinno!ine-3-carbonyl)~amino]-acetic acid; [(i-Brorno-4-hydroxy-8-p enyHsoqu½oHne-3- carbonyl)-am.siio}-aceti~ c acid; [(1 -BthyIsulfanyl-4rhydTOxy-isoq.uinoUne-3-carb.onyl)- amino] -acetic acid; {[4-Hydroxy-l-(4-inethoxy-phei^
amino} -acetic acid; [( 1 -Chloro-4-hydroxy-7-iodo-isoq inoiine-3-carbonyl)-am.ino]-acetic acid; [(i -Chloro~4 \ydrQxy-6-iodo-isoq^ [(4- Hydroxy-7-iodo-isoqiuftoHne-3-carbony!)-amiao]-acetic acid; [(4.-BiOmo-4-hydroxy-7- metliyi-isoqiiinoline-3-caFbonyl)-aminD]-acetic acid; [(l -Bromo-7-bt o y~4-hydroxy- !soquinolir!ie-3-carbonyI)-atn:in ]-acetic acid; [(l-Broino-6-biitoxy-4- ydroxy-isoquinoline-3- carbonyi)-amino]-aeetic acid; [{6-Benzy1oxy-6-cliioro-4-bydroxy-isoqa!no]ine-3-cafbonyi)- raethyl-aminoj-acetic acid; [(l-chJoro-4-hydroxy-isoqumoiine-3-carbonyl)-raethyl^ara!no]- aeetic acid; [(l^CWoro-4-hydroxy-6~i$Dprx>poxy^
acetic acid; [(l-Chloro-4-hydTOxy-7-isopr po
acetic aeid; [GarbQxymeihyi-(l-chioro-4~hydroxy-isoquinoSine-3-carbonyl)-amino]-acetic aeid;
Figure imgf000061_0002
acetic acid; l-Chloro-4-hydroxy-isoqiiinoiine-3-caivboxyiic acid (2~amino~ethyl)- amide(trifiuoro -acetic acid salt); !-ChJoro^-hydroXy-isoquiooline-S-cafboicylic acid (2- methox -eth i)-amide ; 1 -Chloro-4-hydro.xy-isoq uinoline-3 -ear oxy 1 k acid (2-hydroxy- ethyt)-amide; l-CWo.ro-4-hydroxy-isoquino-Hne-3-carb.05-ylic.acid (2--diinethylamino-ethy!)- amide; l -Chloro-4-hydiOxy'-isoquinoline-3-carboxyii acid (2-acetyiamino-ethyl)-amtde; 1- -Chiorc^ -hydroxy~6~iSQpropoxy-isoquinoline-3-carboxylic.aci-d (2-hydToxy-ethyl -am i- Chioro-4-hydroxy-6-isopropoxy-isoquinoline-3-carboxylic acid (2-methoxy-ethyf)-amide; i- Chloro-4-hydroxy-6-tsopropoxy-isoquinoiiTie~3-carboxylic acid (2-amino~ethyl)-ar de (trifluoro-acetic acid salt); 1 -Cbloro-4-hydroxy-6-isopropoxy-isoquiiio! ine~3-carboxy!ic acid (2^dimethylamino-'ethyI)-amide; l -ChIoro-4-hydroxy-7-isopropoxy-isoquinoline-3- .carboxylie acid (2-ammo-ethyl)-aoitde {trif!uoro-aceiic acid gait); l -ChioTO~4-hydroxy-7- isopropoxy-isoquinoHne-3~carbox lic acid (2-methoxy-ethyi)-amide; 1 -ChIoro-4-Iiydroxy-7- isopropoxy~isoquinoline-3-ca-rboxylic. acid (2-dimethy Iamino~ethy i)~amide: 1 -Chloro-4- hydmxy-7-isopropaxy-isQquwQline-3-c^^ (S)~2-[(6-
Figure imgf000062_0001
C ioro-4-iiydroxy"iaoquinoiine-3-earbonyl}-amlnG]- acid; (S)-2-[(I-
Figure imgf000062_0002
Chloro-4-hydroxy-6~isQpropoxy-!Soqtnno!ine-3-carbony!}- acid; (S)-2."[{l-Clii rG-4-hydrQxy-6-isQpropoxy-!SoqainoSiae- propionie acid; (R)-2-[(l C Ioro^ hydroxy-7-isopropoxy-isoqumo1ine-3-oarbonyl)-aniino]-
3-hydroxy-propIonie ae d; (S)-2- {l-Ch|oro--4»hydroxy-7-isopropoxy-isoquiiioime-3- carbonyl)~aTnino]-3~hydroxy-propioi3ic acid; 2-[(l-Chloro-4-hydroxy-isoqi!inoIine-3~ carbonyl)-an).ino]-2-methyl-propionic acid; '2-[(l-ChlQro-4- ydroxy-6-isopr0poxy- isoqumoIine-S-carbony l)-amino]-2-meth l-propiomc acid; (R)-2-(C I -C:hjoro-4-hydroxy- isoqu inol ine-3-carbony Q-araino] ~3-( I H- iro idazo !~4~y 1)~ props on ic ac i d(irifluoro- acetic aeid salt); (S)-2-[( i-Ciiloro-4-hydroxy-isoq unoline-3-carbony!)^
propionic, acid trifluoro-acetic acid salt); (R)-2-[{l -Cb 1 oro-4 - by droxy- isoqu ino ! ine-3 - carbonyl)-afnin;0]-3-nieihyI-biiiyric aeid; (S)"2^[(i~Chiofo-4-hydroxy-isoqiiinQiine-3~ carbonyI)-amino]-3-methyi-biityrk acid; (R)-2-[(l-Chloro-4-hydroxy-6-isopmpoxy- isoqumoiine-3-carbonyl)-aniino]--3-ineihy[-bi!tync acid; (S)~2-((l-C.hlotO~4-hydroxy-6-
Figure imgf000062_0003
h dro y^^so ro Q ^soqumo^ (S)-2-[(l- C ioro-4 iydroxy-7-i;so
{S)-2-[(0-8eri;?ylQxy ~ehforo^
acid; (R)-2-[(l-Chlofo-4-hydxOxy-isoqumoiine-3-carbonyl)-amino]-3-
Figure imgf000062_0004
2-[(l -Chioro-4-hydroxy-6-isopropoxy-isoquinoline-3-cafboriyi)-^
acid; (S)-2~[{l-Oh!of0-4-hy^
propionic acid; {RV2-[(l-Ch!oro~4-hydroxy-7^isopropoxy-i3oquinoIin^
S-phenyl-propioRic acid; (S)-2-[(l-Chloro-4-hydroxy-7-isopropoxy4soquinoline-3- carbonyi)-amino]-3-pbenyi-propionic acid; (R)-2-[(l-ChioiO-4-hydroxy~isoquino!ine-3- earbonyl)-araino]:-3-(4-bydfoxy-phenyi)-prop:iortic acid; (S)-2~[( 1 -Ch ro-4-hydroxy-
Figure imgf000062_0005
aeid; (R)-2-[(l-Chl0ro-4- hydroxy-6 sopropoxy4soquinoline-3-ea^^
acid; (S)-2-[(l-Chioro-4-hydroxy"6-isopropexy-isQquinolme-3-cafbonyl)-amH
bydroxy-pheRy -propionic acid; (R)-2-{{ 1 -Chloro-4-hydf0xy-7 sopropoxy-isoqi!inoline-3'i' earbonyi)-am:ino]-3-(4-hydroxy-phenyl)--pf'opionic acid; (S)-2-i(l -Chloi -4-hydroxy~7-
Figure imgf000063_0001
acid; (R)-2-
[(l -Chloro-4~ hy ro y^
[(1-ChlQro-4~hydrQxy-6-isoprQpoxy-is0^^ acid; (R)-'l-
(l-Ch!oro-44iydroxy-isQquino!m^ (S)»3-(l- Chtoro^ ydrQxy-tsoquinollne-3-carbQnyl)-pyri¾Udine-2- ear oxylic- acid; (R)- I -(l-CWoro- 4-hydroxy-6-isopropoxy-isoquinoiine-3-carbonyI)-pyrfoiidin (S)-]-(l- €h3oro-44iydroxY-6 sopropo
(R)-6-Amino-2-[(i -cl)ioro-4-hydroxy-isoqutnolme-3-earbon I) acid (txifiuoro-acetic acid salt) (S)-6-Amino-2-[(I -ehloro-4-hydroxy-isoq«moline-3.- carbonyl)- arainoj-hexanoic acid (trifluoro-aeeiic acid salt); (R)-6-A-m!no-2-[(l~Ghloro-4-hydroxy-6- isopropQxy-isoquirioime-3-carbonyl)- aminoj-hexanoic acid; trifiiioroaeetfe acid salt; (S)-6-
Figure imgf000063_0002
ani!Hoj-hexaRoic acid (trifluoro-acetlc acid salt); (R.)-6-Amino~2~[(l -chioro-4~hyd{Oxy-7-isopropoxy- isoq inoiine-3-carbonyi)~ amino] -hexanoie acid; trffluoroacetic acid salt; (S)-6~Ainrao-2-
Figure imgf000063_0003
aromo] iexanoic acid
(trifluoro-acetic acid salt}; (R)-2-[{l-ChlorG-4-hydrox
succinic ac ; (S)-2-[(l-Chloro-4-hydroxy-isoquino!in acid; (R)-2-[{ 1 -Ch!of o-4-hydfoxy-6-!Sopr opoxy-isoquinoliae-3-earbon !}-am ino] -succinic acid; {S)-2-[(l-Chloi"0-4-hyd]Oxy~6-isoprGpoxy-!SO acid; (R)-2-[(i-Chloro-4-hydroxy-7-isopropoxy-isoquinolrae-3-car 1- [(l~Cfalofo-4-hydroxy^sequinonne^ acid;
CIi3oro-4-hydroxy-6-isopJOpoxy~ Isoq o inoIme-3 -carbony i)~am mo]-ey c lopropartecarboxylic
Figure imgf000063_0004
[(6-Benzy3oxy-l-eh!oro-4-hydroxy-!Soq inolin -3-carbQny])~amino] acid; (S)-2- [(7-Benzyloxy -chiero-4-iiydra acid; {R)-2~
[(7~Ben2y1oxy-i~eldoro^ acid; (S)-2-
[(l -Gh!oK>-4 yd! XY-isoqu^ acid; (R)-2-[{1 -CMoro--4- hydroxy~isoqtrino3me-3-eafbonyi)-amino]-propionic acid; (S)~2-[(6-fsopropoxy~l -chioro-4~ hydroxy-isoquinol:ine-3-carbonyl)-aini«o]-propIonic aeid; (R)-2-[6-IsopiOpoxy-l-cliloro-4- hydroxy-isoquinoline-3-carboBy1)"amino]-p:"opionic acid; (S)-2-[(7-Isop! poxy-i-cliioro-4- hydiOxy-isGquinolme-3-carbony1)-araino-propionic acid; (R}-2-[(7-lsoprppt!xy-I-chioro-4- hydrGxy-isoquinoliiie-3-carbony!)~amino]~propjonie acid; l-Chloro-4-bydroxy-6- isopropoxy-isoquinoiine-S-earboxyi tc acid (2-bydroxy-l~hydroxyn)ethyl-ethy])-amide; 1 - Cbloro-4-hydroxy-7-isopropoxy-isGqainoiine-3-carbGx lic acid (2-hydroxy-l~ hydroxymethy i -et¾yl)-am ide; 1 -C loro-4-hydroxy- tsoquinol i e-3-carbox lie ac id (2- hydroxy-l-hydfOx.ymeiky!-ethyi)~aiTiic!e; {[7-(355-Difluoro-phenoxy)-4- ydrosy- isoquinoiine-3-carbonyi]-amino}-acetic acid; {[e-CBjS-Difly^iO- heaoxy^-hydro - isoquinoUne-3-carbonyl]-amrao}-acetic acid; ({7-[4-(4-Fluoro-pheRoxyVphenox%']-4- hydroxy-isoquinoline~3-carbonyi } -amlno)-acetic acid; ( {6-[4-(4-Fl sofO-phenoxy)-phenoxy]- 4-hydroxy-isoqainoline-3-carboiiyi}-aiT!jiio}-aeetic acid; {[7-{3-ChlDro-4-fliroro-phe.noxyJ-4- hydroxy-isoquiiioline-3-caTbony!]-araino}-aceiic acid; {[6-(3-Ch]oro-4-fi«orQ-phenox'y)-4~ hydiOxy-isoquino!ine-3-caxbonyI]-amtno}-acetic acid; (S)-2-{[7-(3-Fkioro-5-methoxy- phenoxy)-4-hydroxy-isoquinoUne-3-carbony!]-amino}-piOpionic acid; 2-(S)-[(7- Cyciohexyioxy-4-hydroxy-isoquino1me-3-carbo.nyi)-a5Binoi-propioi-iic acid; 2-(S)-{[7-{4- -
Figure imgf000064_0001
acid; 2-(S)-{[4-Hydrsxy-7-(4-trifluoromei^^ amino}- propionic acid; {[7-(4-Chioro-phenoxy)-4~hydroxy- 1 -metbyl-tsoqui;noline-3-carbony]]» amino} -acetic acid; {[0-{4-Chlora-phenoxy)-4^^
ammo}-aeetic acid; {[7~(3s5~Difluoro-phenoxy^
carbonyll-aiTiinoj-acetie acid; {[4-iiydroxy-7-(4-roethoxy-phenoxy)-l -niethyi-isoquiiioiine- 3-carfaonyl]-amino- }-acetic acid; { [4-Hydroxy-6 4-raethoxy--pbenojQif5-l -methyl- isoqumoline-3- arbonyI]-ai'nmo-}-aceiic acid; [(6-Cyclohexyjoxy-4-hydtoxy--isoqLiinoiine-3~ carbonyl)-amino]-acette acid; f(7-Cyciobexyl.oxy-4-hydroxy-isoquinoline-3-carbonyl)- ammoj-acetic acid; (7-Cycfohexylsxy-4- ydroxy-l-raeihyl-isoq«!noiirie-3-carboriyl)- aniitioj-aeetie acid; [{7-Cyelohexyistiifanyi~4~hydro^
acetic acid; [(7-Cyclohexanesulfonyl-4-hyd
acid; ((4-Hydroxy-l-isobuty4-i5oqu!no!ine-3~ca?boiiyi)-ainino]-acetic acid; [(4-Hydroxy- 1- pyridiii-2-yi-isoquinoiine-3-carbony!)~amino]~acet!c acid; [(1 -Eihyl-4-hydiOxy-7-phenoxy- isoquinolme-3-carbonyl)-ariiino]-acetic acid; [(l -DimethylaramometIiyl-4-hydroxy-7- phenylsulfanyiHsoqumolm^ acid; [(4~FIydroxy~l~methyi-7- phenyisulfanyl-isoquiiioiine-S-carbony -arainoj-ace- tic acid: {[4-Hydroxy-l-methyS-7-(4- trifhioro methy i-phenox )-is oqu oline-- 3 -carbonyl] -amino } -acetic acid; and
pharmaceutically acceptable salts, esters and prodrugs thereof.
[00182] In some embodiments, small molecule PH!s may be selected from eyanoisoqinno!ine compounds disclosed in. U.S. Patent No. 7,928,120, which is expressly incorporated herein by reference in its entirety. These compounds can be represented by formula li:
Figure imgf000065_0001
[00183] wherein:
100184] R is selected from the group consisting of hydrogen, alkyi, and substituted alkyl;
[00185] Rs, R3, R3 and 4 are independently selected from the group consisting of hydrogen, halo, cyans, hydroxy!, alkyf. substituted alkyl, aryL substituted aryl, heteroaryi. •substituted heteroaryi, amino, substituted amino-, -OR', -SR , -SGR', and -SO2 wherein R' is selected from the group consisting of alkyi, substituted alkyl, cycloalkyl, substituted cyeloalkyL aryl, substituted aryi, heteroaryi, and substituted heteroaryi; and
[00186] R5 and R* are independently selected from the group consisting of hydrogen or Cy.j alkyl;
[00187] or pharmaceutically acceptable salts, tautomers, stereoisomers, solvates, and/or prodrugs thereof.
[00188] Representative compounds of Formula II include;
[001.89] { [ 1 -cyan0-4~hydroxy-i3oquiRolIne-3-carbonyI]-amino } -acetic acid, 2-{ S)- (l.-cyano-4-hydrp y~isoquihonne-3~earb ny{)- acid, {[l-cyai o-4-hydroxy-
7~phenoxy-isoquinoline-3-carbonyl]-arri!no}-aceiic acid, 2~(S}-{{l -c.yaoo-4-hydroxy-7- phenoxy-isoqu{noline-3-carbonyl)-ami:no]-propioHic acidj^-i ^f l-cya o^-hydrox -?- phenoxy-isoquinoline-S-carbonyl^aminoJ-propioriic acid, {[l-cyano-7-(4-fluorophenoxy)-4- hydroxy-isoq«inoltne-3-carbonyi]-amino}-acet!c acid, {[l -cyano-4-'hydroxy-7- (tri fluoromethy i)-isoquino 1 ine-3 -carbonyl] -amino } -acetic acid, .{ [ 1 -eyaoo-4-hy droxy-7- chloro-isoquBJoline-S-carbooylj-aminQ^c^tie-.acid, {[1 -qyario-4-fiydroxy -S-phenoxy- isoquinoiine-B-carbon i] -amino} -acetic acid, {[1 -eyaflo-S-(4-fliioro-phenoxy)-4-hydroxy~ isoq«inoline-3-carbonyl]-amino} -acetic acid, [( ί -cyano-4-hydroxy-6-methoxy-isoquinoline- 3 -carbonyI)-ammo] -acetic acid, [{l-cyanQ-4-hydroxy~ -phenoxy-isoquinolirie-3-carbonyi)- amino]-acetic acid, {[l -cyano-6-(4-fluoro-phenoxy)-4~hydroxy~isoqujnQ]ine-3~carboriy!j- araino}-acetic acid. {[l -cyano-4-hydrQxy-6~(4-metl 5^
amino) -acetic acid. [{l-cyano-4-hydroxy-6-phenyistsifanyi-isoqu!noiine-3-carbonyl)-amino]- acetic acid, [(I ~cyanG-44)ydroxy~7-phenyisulfanyi-^
acid, {[l -cyano-0-(2,0-dim6A^
acetic acid, [(] -cyano-4-hydroxy-5~phenoxy-!soq^ acid (Compound B),
Figure imgf000066_0001
ainiiioj-acetic acid. {[l -cyano-44iydrexy-8~^
amino}~acetic acid, {[l -cyanLO-4-hydrOxy-8-(2-methoxy-phenoxy)-isoquinoliRe-3-caiboi¾yl]- amino} -acetic acid, [{7~ en^l.- -e ano^-^ dro y-isa ^
ac id, { [ 1 -cyano~5-(4-f!iioro-pher)oxy)-4- y droxy- soquinoi ine-3 -earbony 1] -amino } -acetic acid, {[l~eyaoo~7-(2,6-di et^
acetic acid, {[ ί 'Cyano-6-(2-ethyl-6-methyl-phen^
amino}-acetic acid, []^ n©~44ydro -6^2,4^^
earbonyl]-araino}-aceiic acid, {[6-(4-chSoro-2,6-dimeibyi-plienoxy)-l-cyanx")~4-1iydroxy~ isoquiRoHne-3-carbonyi]-ammo}--acetic acid, { [ i -cyano-4-hydroxy-7-{4-metfioxy-phenoxy)- isoquiaoUHe-3-carbonyi]-arninQ}-acetic acid, [(i -cyan.o-6-eycloi!exyioxy~4-hydroxy- isoquinolme-3-cafbofiyl)-airsino]-acetic acid, [(6-ben2enesiiIfonyi~l-cyano-4-hydroxy- isoquinoline-3-carbo!iyi)-ammo]--acetic acid, { L-cyanG-4-hydroxy-6-(4-propoxy-pheiioxy)- isoquihoi:ine-3-carbonyl]-amiiso}-ace{ic acid, {[7-{ber!zo[i;3]dtoxoi-5-yloxy)-l -cyano-4- hy droxy-is quinoline-3~carbony SJ-amir.o j-acei ie ac id, { [6-(benzo 1 r3]dioxoI-5-yloxy)- i ~ cyano-4-hydroxy-isoqtnnoHBe"3-carbonyl]-amif!o}-acetfc acid, { i-cyano-6-(2.3-dihydro- henzofuran~5-yioxyj-4-l^ acid, [(l~cyano-4- methoxy-&-phenoxy-isoquino^ acid methyl ester, [(l-cyasi -4- methoxy-8rph.enoxy-isoqyinoline-3-c-arbonyl)-ainino]-aGe.fic acid, (S)-2-¾l -cyano-4- hydroxy-8-pheiioxy-isoquinoljne-3-cafbonyi)-ammo]-pr pionic acid, (jR)-2-[(l-Qyauo-4^ hydToxy-8-phenoxy-isoquinoiioe-3 -earbon i)-amirio]-propionie add, { [ 1 -cyano-4-hydfoxy-
0~(2-methyI-ben;Mfto {[1-cyano-
6~(2-d3mei kmino-benzooxazol-5-yloxy)-4~hydroxy-!S quinoline-3~carbon^
acetic acid, { [ 1 -cyano-7-(2-d imetnyJamino-benzooxazoi-S-y lQxy)-4-hydroxy-isoquinol ine-3 - ■earbon l]-amino} -acetic acid, {[i-eyano-4~hydraxy~6-i2-morphQ^
yIoxy)-isGquinoiine-3-carfaoByl]-anTino}-acetic acid, {[l~cyano-4^hyidroxy-6.^(2-raetiiylr
Figure imgf000066_0002
[(6-chloto-l -cyano»4- hydroxy-isoquinoline-3-carbonyl)-amin.oj~acetic acid, [(7-butoxy-l -cyano-4-hydroxy- isoqi!inoiii}e-3-carbonyl}-araino]:-acetic acid, [( 1 -cyano-4-h.ydroxy-6,7-dipheiioxy- isoqinnoline-3-carbonyi)^a!Tiiiio]~acetic acid, [(i-cyano-4-hydi¾xy-7-iiiethoxy-isoquinoline^. 3-carbony1)~amino]~acetic acid, [(l-CYano~4-hydroxy-7-isoprapoxy-isGquino1ine--3- earbony i)-am ino] -acetic ac id, [( 1 -cyaiio-4-h droxy-6-isopiOpoxy- isoquino iine-3 -carboo l)- aminoj-acetic acid, [(I-cyano-4-hydr xy-5-p enyl-:jsoqHinDiin -3-carbonyl)-aminoj-acetic acid, [( I -cyano-4-hydroxy~8-phenyl-is^ ac d, [(7- be«2yloxy-i-eyano~4 ]ydro^^ [{S-(4-.e oro- phenoxy)- H5yano-4-hydroxy-isoqi»noime-3-carbony{ -amino)-acetic acid (Compound A), and [( 1 -cyano-4,7 -d ihy droxy-isoqiu noline-3-carbony 1;)- am ino] -acetic acid.
[00190 In other embodiments, sniaii molecule PH is may be selected from pyrrolo- and thiazoSo-pyridiiie compounds disciosed in U.S. Patent No. 7,696,223, which is expressly incorporated herein by reference in its entirety. These compounds can be represented by formula 111:
Figure imgf000067_0001
[00591 J wherein:
[00192] q is O or 1;
100193] A and B are independently selected from the grou consisting -C( 7)- » -NCR8)-,™N-, and -S- with the proviso that one of the following is presen
[00194] A is =C(R7)~ and B is -N(R8)-;
[00195] A is ~S- and B is = -;.
[00196] A =N- and B is-S-; or
[001971 A. is -NCR8)- and B is =C(R7 ;
[00198] one of'-A C(R6)- or -B -» C(R6)- is a double bond and the other is a single bond;
f 001 9] R1 is selected from the group consisting of hydroxyl, alkoxy, substituted aikoxy, aeyloxy, cyc!oaikoxy, substituted cycloalkoxy, aryloxy, substituted aryloxy, heteroaryloxy, substituted heteroaryloxy, heteroeyciytoxy, substituted heterocyclyioxy, mercapto, thioether, swstifuted alk ithio, arylsulfanyi, heteroarylsulfanyl, amino, .substituted amino, acylamsno and aniinoacyi;
[00200} R"' is selected from the group consisting of hydrogen^ deuterium, and methyl;
[00201] 3 is selected from the group consisting of hydrogen, deuterium, alkyi. and substituted alkyi;
[00202] R4 is selected from the group consisting of hydrogen,, alkyi, and substituted alkyi;
[00203] R5 is selected from the group consisting of hydrogen, halo, eyano, hydroxy I, alkyi, substituted alkyi, cycioalkyl, substituted cycioalkyl, alkenyl, substituted aikenyl, alkynyl, substituted alkynyl, a!koxy, substituted alkoxy, cyc!aalkoxy, substituted cycioaikoxy, aryl, substituted aryl, aryloxy, substituied arytoxy, beteroaryl, substituted heteroaryl, heterocyciyj, substituted heterocyciyj, heterocyclyioxy, substituted
heterocyclyioxy , heteroaryloxy, substituted heteroaryloxy, aeyl, aminoacyl, nitro, amino, substituted amino, ac iamino, sulfan l, sislfonyl thioether, arylthip, and substituted atylthio:
[00204] R° and R7 are each independently selected from the grou consisting. of hydrogen, halo, eyano, hydrox I, alkyi, substituted alkyi, cycioalkyl, substituted cycioalkyl, alkenyl, substituted alkenyl, alkynyi, substituted alkynyi, alkoxy, substituted alkoxy, cycioaikoxy. substituted cycioaikoxy, aryl, substituted aryl, aryloxy, substituted aryloxy,. heteroaryl, substituted, heteroaryl, h teroc clic substituted heteroeyclyl, heterocyclyioxy, substituied heterocyclyioxy,. heteroaryloxy, substituted heteroaryloxy, acyl, aroinoacyi, nitra, amino, substituted amino, acylamino, sulfanyl, sulfony'I, thioether, aryithio, and substituted aiySthio;
[00205] or where when A or B is=C(R7)- , then R& and R7 together with the carbon atoms bound thereto join to form a cycioal keny I, substituted c eloalkenyl, ar l, substituted .aryl, heteroaryl, or substituted heteroaryl; and
[00206] R is selected from the group consisting of hydrogen, hydroxyl, alkyi, substituted alkyi, aikenyl, substituted alkenyl, alkynyi, substituted alkynyi, aryl, substituted atyi, heteroaryl, arid substituted heteroaryl;
[00207] or pharmaceutically acceptable salts, single stereoisomers, mixtures of stereoisomers, esters, or prodrugs thereof. [00208] Representative compounds of Formula ill include:
[00209] [(2-hro o-4-hydr0xy~l -pta
aminoj-acetic acid, [(4-hydroxy- 1 -phenyl-l H-pyrroIof2 -c]pyridine-5-carboii !}-amino]- acet c acid* [(2,3-dibromo-4-hydr0xy~I^
amino] -acetic acid, {[3-bromo-2-(4-i1isoro-pher!yl)-4-hydfoxy-i-ph.enyl-1 H-pyrrolo[2s3- c] n dine~5-carbony i]-amino } -acetic acid, [( 1 -benzy 1-2 -rd ihromo-4- ydroxy- 1 H- py.rro i o [2,3 -c] yridine- 5 -carbony 1] -amino } -acetic ac id, { [2-{4-f! uoro-pheny l)-4-bydf oxy-'l - phenyi-lH-pyrroio[2J3-c]p> idine-5~caxbony1]-amino}-aceiic acid, [(l~benz>'l~4-hydroxy- ΐ H-pytTo|o[2 -<∑]py dine-S-csrbonyl)-amiao]-a!Bet!c acid, { [3-bromo- 1 ,2-bis-(4-fluoro- p'he ny i)-4-hydroxy- 1 H-pyrro lo[2,3-c] pyridme-S-sarbon l]-amin o } -acetic acid, { [ 1 ,2~bi s-(4- fluor0~pheny!)-4-hydroxy-IH~pyrc^^ acid, {[3- chloTO-l,2-bss-(4-flijoro-p.henyI)-4-hydroxy~l H-pyn-o
acetie acid. {[3-brQmo- ~(4-f¾Qro- .hsn^^^^
pyrrolo [2,3 -c] pyridine-5-earbony f ]-amme} -acetic sci d, { [2- (4-fl uoro-pheny l)-4~bydroxy- 1 -
Figure imgf000069_0001
{[2-bromo- l~(4-fl«Qfa-piienyi)-4-hydroxy- acetic acid, {[l-(4-f1uoro^henyl -4-hydroxy-3>-phenyi-lH~p> roto[2^ pyridin€-÷5- earborsyl]-mniiio}-a.cetic acid, {[7-chioro-l -{4-fl uoro-pheny I)-4-Iiy droxy-3 -phenyl- 1 H- pyrroJ o [2,3 -cjpyrid ine- 5 -carbon l]-amino -acetic ac d, { [7-methyl- 1 ~(4-fIuoro-pheny'i)-4- hydroxy-3-pheny)-lH-pyrrolo[2,3-c]pyridine-5-carbonyi]-amino}-acetic aeid, {[3-b.rprn.o-2- tert-butyl-1 -{4-flu.oro-plieny i.)-4-hydroxy- iH-py.rrolo[23-e]pyridine-5-carbonyl]-aramo}- acetic acid, {[2~teri-buryl -(4-†lup!X>-p^
earbonyl]-amrao}-acetie aeid, [(l-benzyl-4-hydroxy-2!3-dimeth.yi-l H-pyrrolo[2s3- e]pyridine-5-carhonyl)-ainiRo]-acetic acid, [(2,3-dibroiBo-4-hydroxy-l-metbyi-lH- pyriOlo[2,3-e] yridine-5-carbonyi)-amino]-acetic acid, [(4-hydroxy- 1 ,2:,3-tj'imethy1-:l H- pyrrO;!o[2,3-c]pyri.dine-5-carbonyi)-afflino]-acetie aeid, :{[2~bromo-3-tert~butyH~(4-fiuoro- phenyj)-4-hydroxy-i
Figure imgf000069_0002
acid, ([3-teit- butyl— 1 -(4-flLsoro~phenyl)-4-hydroxy~lH-pyrrolo[2,3-e]pyridme-S-carbony
acid, [(1 -beTizyi-4~hydr0xy-2,3-d!propy
acetic acid, [(! -benzyl-3 ,,?-dicWoro-4-.hydroxy-1 H-pyrrolo[2.3-e}pyndine-5-carbonyl)- aK»no]-aeette
Figure imgf000069_0003
[(4-hydroxy- 1 -meite^
Figure imgf000069_0004
[{!- benzyl-3-chl0ro-4-hydroxy-7-phenyl-.lH^
acid, [( I -benzyi-3-chloro~7-eihy]~4-hydroxy- lH-pyrroio[2^-c]pyridine-5-earbonyi)-aittmo]-- acetic acid, {[2-(4-fluor o-phen l)-4-hydroxy- 1 ,3-dfphenyi- 1 H-pyitoioj^S-c] pyridi ne-5- carbany1]-amino}-acetic acid, [(3-ch{oro-4-hydroxy-i-phenyl-lH.-pyrrofo[2,3-c]pyndiii£'S- 5 earbony!)~srnrao]-aceiic acid, [(3-chloro-4-hydmxy-7-methy!-l-p eiiy! H-pyrro1o[2!3- c3pyr.idme-5-carboi5yi)-amino]-acefic acid, {p.-(benzo[I,3]dioxQl--5-yimeihyl)-3~bromo-2~(4- chioro-pfieny])-4-hydroxy-!H^ aeid, {{3-
Figure imgf000070_0001
amino} -acetic aeid, (I^ben^i Sjdioxpl-S-ylme^ ^h drox ^- hen j-iH- yrrolpPjS-
J 0 c]pyridir!e-5-carbony!}-aiTiino]-aceiic acid, {[ 1 -(benzo[I,3]dioxol-5~yimeihyl)~2-{4-chloro- he x ])^h droxy H^yrrolo[2-^ acid, {(1 - beftzof 1 ,3] icsxoI-5-yImeihy l-2-(4-chloro-phenyl)-4-hydroxy-3-methyj- 1 H-pyrroio[2,3- c]pyr!diiie~3-carbooy!3-ainirio}-acetic acid, [(4-hydrQxy-l ,2- ii.pbenyl-lH-pyrrolo 2,3~ c]pyndine-5~carbony!)-a iiiio]-acetie aeid, {[2-(4-Gh.loro-ph nyl)-4~hydroxy-3-Tnet yH-
•15 phenyl-lH-pyn,olQ[2,3-c]pyridiBe-5.'<aitonyl3-araino}-acetic.acid, [(7-hydroxy-2-phenyi- thiazoio[4J5-c]pyridine~6-carbonyl)-arnino]-acetic. acid, [(7-hydroxy-2,4-dipbenyi- ihiazoio[4,5-c p}^idine~6- arbonyl)-a!ns:n ]-acetic acid, [(7-hydroxy-4-methyl-2-phenyl- ihia2oio[4,5-c]pyridme-6-carbonyl)-amino]~acetic acid, .(S 2'[(7-hydJO.xy-4-methyl-2- phenylrthia2olo[4}S-c]pyridine-6^carb«nyl)-araino]-propiGnk acid, {[7-hydroxy-2-(4-
Figure imgf000070_0002
chIora.-phenyI)-7-hydroxy-W aeid, .' hydroxy-2-(4-methoxy-pheoyI)-tbia¾o1o^ aeid. {[2-
(4 1uoro-phenyl)-7-hydroxy-ite acid, [(4-
Figure imgf000070_0003
{methyi-phenyi-aroino)^ {[?-
Figure imgf000070_0004
hydroxy-2-phen I-thiazelo [ 5 ,4-c] pyr i d e~6-earbonyl)~arn inpj-aeetie acid, { [2- (5 -brorn o- pyridin-3 -y ί )-7-hydrQxy-thia2p So [4,5-c] pyr id me -6-carbony I]-ara ino} -acetic acid, [ (7-
30 hydroxy-2-pyTidin-3-yi-thiazolo[4,5-c]py^ acid, (4-butyi-7- hydroxy-2-phenyl bia¾oio[4?5^^ acid, [{7-hydroxy-i2- pyridin-2-yi hia2olo[4}5-c]py^din'e~6-carbonyi)-animoJ-acetic acid, {|2~(4-fluoro-phenyl)-
Figure imgf000070_0005
phenyl-4-propyl hiazoio[4,5-c3pyridme-6-earboiiyl)-a»mo]-acetic acid, {[7-hydroxy-2- (4- phenoxy-p e yi)-thiazo!o[4,5^ acid, [(4-cyano-7- hydroxy~2-ph& vi-thiazo!G[4s5-c]pyridme-6-carbonyi)-am¾no]-aceti.e acid, (7~hydroxy-4- isp:bulyIr2- henyHhiazol [4s5-c]pyridia^ .acid, {[?-hydroxy-2*(3- snethoxy-phenyi)-tiijazolo[4,5-e ^^^ acid, [(4~furan~2-yi-7~ hydrQxy-2-pheny! hiazQio 4,5-e]pyr^^ acid, [(7-hydroxy-2- phenyl-44 jazoI--2-yl Maz^^ acid, {[7-hydroxy-.
2-(2-methoxy-phenyi)~tl)I zoio[4J.5~c]pyndin acid, [(7~ ydroxy-
4~methyl-2-phenyl4hiazol0[5,4-c]pyr^ {[2-(4~qyanc~
Figure imgf000071_0001
acid, (7-hydroxy-2,4- diphenyl lviazalo[5,4-c]pyridte acid, {[2-(3-ciilo!O--4-fl.uQ!O- phenyi)~7 iydroxy hiazoio[45,5~e]pyr.id3ne-6~cart yl]-arajno}-aeeiic acid, [(4-benzy!-7~ hydr:oxy-2-phenYi hiazoIe[5I4-c]pyridlae-6-earbony!)-a acid, {[7 iydroxy-4-(4~ morph0iin~4~yl-phenyl)-2~phenyl^ acid, [4-(4-cy an :o-phenyl)-7-hy droxy-2-pheiiy 1-thiazQ.lp [4, 5-e]pyridir!e-6-earboriyi]-amino}- acetic acid, {[4-cyaiio-2-(4-fluoro-phenyl}-74iydroxy-diia2oto
amino}-aeeiic acid, {[4-eyanQ-7-hydroxy~2-(3-me¾^^^
carbonyI]-araino}-acetic acid, [(4-cyano-7-bydroxy-2-phenyl hiazolo[5,4-c]pyridin.e-6- carbonyl)-amiii ]-acetic acid, ^-ethyayl-T-hydmxy^-p enyl-thiazoio^S-ejpyridine-S- carbonyi)-ammo]~aeetic acid, [(4-aeetyl-7-hy^^xy-2-phe^^
carbQnyl)-ammo]-acetic acid, [(7- -droxy-2-ph nyI-4-pjpefidso-! -yI-tbiazob 4,5-- c]pyridiiie~6-earboayi)-artiiBo]-acetie acjd, {[2-(4-feri-butjd-phenyi)-7-hydiOxy-tiiia2olo{4, 5-c]pyr3dirte-6-carbony]-amino}-acetic actd, {[2-(2,3-dihydro-benzo[1 ,4]dioxin-6-y!)-7~
Figure imgf000071_0002
[(2-benzo[(¾]ihiopher¾-3-yl-
7-hydroxy-th!azoio[4,5-e]pyridine-6-carbonyl}-ami!'!oj~acetie acid, {(2~bipbenyi-4-yi-7-
Figure imgf000071_0003
7-hydroxy4 iazoio[4,S-c]^ acid, [(7-hydroxy-2-quinolin-
3~yHhiazolo[4,5~i]pyridine-6-carbonyi)~aniin.o]-ace acid, [(2-benzoiltran-2-yI-.7-hydroxy- ihiazolo[4J5-c]pyridine-6-carbonyl)-ami.Oo)-acetic acid, [ 2-diben2ofuraii-4-yl"7-hydroxy- thiazoio[4s5-c]pyridine-6-carboH> )-a!njno3-acetic acid, f[2-(2j3~dihydro-benzofuran-5-yi)-
Figure imgf000071_0004
acid, [(7-hydroxy-2-pynmidfn-
5-yi-ihiazolo[4,S "]pyridine-6-carbonyl)'arainG]-ac.eiic acid, {[2-(l -benzyl- lH-pyrazol-4- !)- 7 iydroxy-ihia2olo[4,5^e]pyrf^^^ acid, {{2-(6--c 0ro-pyridm-3- yl^T^b drQ -thiazolo^S-cJ yridine-e^ar o i ll^nimoj-acefie aeid. {[2-{6-btitoxy~
Figure imgf000071_0005
acid, {[7- hydroxy-2-(6-phenyisulfanyl^
acetic
Figure imgf000072_0001
carbonyl]~ammo}~acetic cid, { 2,3-diehloi$-7-c ano^hy^^
p}o:ro1o[2,3-c]pyndine-5-carbonyl]-animof -acetic acid, {[?»cyano-4-hydroxy-.l -{3~m.etliyl~ bu y1)-lH-pyn'o!o[2s3~i? pyridme-5-carboEy1 ]-at«irio}-acetie acid, { [3<-dhloro-7-cyarioHi- hydroxy- ! -(3-methy!~b.styi)-iH-py^
{ [2!3-d)chloi' -7-cyano- 1 -cyclo exylnrethy !-4-hydroxy- 1 i -pyiToio[2;3-e]pyridine~5- carbony i]-am mo } -acetic acid. { [7-c ario~4-h droxy- 1 -eye tohexy !methy 1- 1 H-pyrrGlo{2, 3 - c]pyrjdine-5-caFboByl]-an-sino}~a.cet5c acid, [(!-bei\¾ -3-chloro-4-hydiOxy- iH~pyrroio[2i3- c}pyridine"5-c.arbonyi)-aiiiinoj-acetic acid, [(4-hydroxy-9-meihyl-9H-beta-carboline-3- carbonyi)-a.min0]-aeetic acid, ^^ydfoxy-l^-dimetliyl-^H-beta-carboiine-j-carbony!)- aminoj-aceiic acid, [(4 iydroxy-9-methy!~1 -phenyl^^
acetic acid, [(l-cyano-4~hydroxy-9-niethyl-9H-beta-ear^^
acid, { [3~bromo~7-cyatto»2~(4-f] uoro-phe.n -4-hydroxy-l -phenyl- 1 H-pyrre io [2,3 - cjpyri dme-5-carbonyI]-axnit5o } -acetic acid, { [7-cyano-2-{4-fl uoro-pheny 3)-4-hydroxy- 1 - pheRyi»iH-pyrroio[2s3-c]p^ acid, [(4-bydiOxy-5-phenyi-
SH-pyrido^^-bjjndole-S-carboiiyli-ammoj-acetic acid, [(l -cyano-4-hydroxy-5-phenyI-5H- pyrido^^-bjmdoIe-S-carboflyO-aminoj-acetic acid, [(4-hydroxy- 1 -methyl-5-phenyl-5H- pyrido^^S-bjaidole-B-carboiiylJ-anifnoJ-acetic acid, [(i-benz>'f-3-ch1oro-7-eyano-4-hydiOxy- lH.-pyrroiGi2,3~c]pyridine^5~carbony!)-:araiiio]-acetic acid, { 3-cyano-2-(4~.fiuoro~phenyi)-4~ hydroxy- 1 -phenyl- Ϊ ft {[3-cyano-2- (4~fI«or o-pheiiyl)~4-hydr xy-7-ffiethyl- I -phenyl- Ί H-pyrrolo 2.,3-c]pyridine-5-carbGnylj- am.ioo}-aeetic acid, { 3,7-dicyan -2 {4"fiuoro-phenyl)-4-hydroxy-l -phenyl- 1 H-pyrrolo[2,3- c]pyridine-5-carboayi]-amino} -acetic acid, [(7-cyano-4-hydroxy-L-phen 1-lH-pyrrol0[2,3- e]pyridiiie-5-carbonyi)-am!no]-acetic add, {3-chloro-7-cyanG-4-.byd]Oxy-l -phenyi-]H- pyfrolo[2,3-c]pyridme-5-carbonyi)-ait!ino]-aceiic acid, {P -dibroino-l-(4-lluoro~ben2yi)-4- hydroxy-lH-pyrJO!oi2,3-e]pyridiae-5-earbonyi]-amino}-acetie acid, [(4-hydroxy- 1- pltenethyi-IH-pyrfolo[2,3~c^ acid, {[¾3-d;ibro o-7- cyano-l-( -Ruoro-be:nzyl)-4-h^
acid, [(3-bromQ-7-e ano-4«hydroxy-l -phenyl- l H-pyiTofo[2;3-c3pyridine-5-carbonyl)- aminol-acetic acid, { 7-cyano-i-(4-fluoro-benzyi)-4-hydroxy-lH-pynOio[2!3-e]pyridine-S carbo!iyl]-amino}~acetic acid,. [(3-diIoro-7-cyano-4-hydraxy-l-phen€thyl-! H-pyrro.to c]pyridme-5-carbonyl)-ainir!oj-acetic acid, {p,3-dibroi'nQ-4- ydroxy-] -(! (S)-phe!iyl-ethy!)- iH-pyrmio[2:,3-e]pyridine-5-carbonyl]-arnino}~acetic acid {[3-chloro~7-eyano~.l-(4-fiuoro- benzyi)-4- hydroxy- 1 H-p rrolo[2 J-c]pyridin.e-5-carboRyl]~am mo}~acetic acid, i( 1-betHyi- 2,3 -dich toro-7-cy aoo-4-hydroxy- 1 H-py rroio[2, 3-e] py rid me-S-earbony I)-am moj-acetic acid, {[4-hydrQxy- .l-(lS-phenyI-e^
[(2J-dichIoro-?-cya«o-4-hydroxy-l-phe«yl-i.H-pyrrolo[253-e]pyridi
acetic acid, (2,3-<ϋοίϊΙθ£·ο-7-ε>¾ηο- -ίι>^ίΌχ -- ! -phenet yl- 1 H-pyTFploPJ-cjpyr idi0e-5~ carbonyl)-amino]-aeetic aeid, {[2t3-dichlpro-7-cya o-4--hydroxy~l-(l S-phenyl-ethyl 1 H- pyrroio[2,3-c]pyrid!ne-5~carboriyl -amir!o}-acetie acid, [(l-bejizyl-3~bromo-7-cyano-4- hydr0xy~lH~pyfroI [2;3-c]pyridine-5- arbonyl)-amiiTO]-acetic acid, {[4-hydroxy-l-(lR- pheny1-ethyl)-l Fi-pyrrolo[2,3-c]pyridine-5-carbony!]-amin.o}-aceiic acid, {[4-hyflroxy-l-(4- rnethoxy-benzyi)- 1 H-pym lo[2 -c]pyrid!ne-5-earbQnyl]-aramo} -acetic acid. {[7-cyano-4- hydroxy-l-(4-ffiethoxy-beri2yI)-l H-pyrroIo[2,3-c]pyrid{ne-5~carb acid, [{ I -benzyi-7-qyano-4~hydrox.y-3-metbyl-l,H-pyfrolo[2,3
acetic acid, .{[2 '-dichl6ro-7-eyano^^
c]pyridine-5-carbo.nyi]-amino}-aceiic acid:, {[2?3-d:ichIo!O-7-cyano-4-hydroxy- l -(lR-phenyl- ethyl)- ! H-pyriOlo[2,3-c]pyrid.iRe~5-carbon^ {[3-cliioro~7-cyano-4- hyd:roxy-l-(4~methaxy~be^ acid, {[7-cyano-4-hydroxy- 1 -(4~methoxy-pheny H-pyrr o!o[2i3-c]pyrid¾e-5-earbonyI]-amino}- aeetie acid, {[2,3^ichloFO-7~cyano-4~hydroxy-l-(4-m tho.xy-phenyl)-IFi~pyrfolo[2,3- c]pyridi e-5-carbonyl]-amino}-acetic acid, {[3-chIoro-7-cyaiio-4~hydroxy- l-(4-methoxy- phenyl)- ! H-pyrrolo[2!3-c]pyridine-S-carbonyl]-a«iirio J -acetic acid, {[l-(4-fli!oro-benzyI)-4- hydiOxy -2!3-diraethyi~iH-pynOio[2!3-c]pyridme~ {[?-cyanp- ί -(4-fliioiO-pheny!)-4-bydroxy- 1 H~pyrro!o[2,3-c]pyTfdiiie-5-carboiiyl]-amino}-a;cetic aeid, {[2,3-djcftloro-7-eyanQ~4-hydr0xy
carbonyi]-am'{no}-acetie acid, {[3-chloro~7-cyano-4-hydroxy-]-(4-fiiioro-phenyl)-lH- pyrro!:o[2i3~€]pyridine-5-carboiiyi]--ammo} -acetic acid, { [ 1 -(4-fiuoro-benzyl)-4-hydroxy-lH- pynOio[2,3-c]pyridlne-5-cafboayl]-ainino}-acetic acid, [(2-cyano-4-hyd'roxy- 1 -phenyl-1 H- · pyrrolo[253-c]pyridine-5-carbotiyi)-aniir!o]-acetic acid, { l-(2-fluoro-benzyi)-4-hydroxy-IH- pyrrolo[2,3-c]pyridiiie~5-earboiiyl]-amirio}-aceric acid, {[4-hydroxy-l -(2-methoxy-benzyl)- i H-pyrrolp[2:,3-c]pyTid:ine-5-cafbonyl]-amino}-aceiic acid, .{[4-hydroxy-l-(3-methoxy- beiizyi)-lH-pyrToio[2s3-c]pyridi e-5-carbon !]~araino}-acetic acid, { [7-cyaiio-l -(4-f uorOr pheiiyi -hydroxy-3-phe^^^ acid, { 7r
Figure imgf000073_0001
acid (Compound C), {[7-cyano-l-(2-methoxy-benzyl)-4 iydroxy-iH-pyrroIo[2,3-c]pyfi 5 -earbo rsy 1] '-amino } -acetic ae id, :{ [7~cyano- 1 -(3-raeihoxy-be zy i)-4-hydroxy- 1 H- pyrrolo[2,3-e]pyridine-5-earbonyl]-amino} -acetic acid, {[2-cyauo~i-(3~fluoro-benzyi)-4- hydroxy- 1 H-pyiTOlo[253-c]pyridine-5^art>onyl]-amtec>}^aoefie acid, {[2,3-did)loro-7-tsyaiJ< l-(2 TuGro-benzyl)-4-hydroxy H-pyrroio^^ acid, {[l -{3 1iioro-benzyl)-4-hYdroxy-lH-pyrro!o[2,3-c]pyridine-5~carbonylj acid, {[3-c !orp-7-cy¾no-H2-fluoro jea2yl)-4-hydr¾xy-lH-pyrro^
amino}-acetie acid, {[3~ciiiQro-7-eyano-4~hydrox.y~I -(j-methaxy-beiizyi)-! H-pynx>lo[25.3~ c]pyridin -5~earbonyl]-amino}-aeeik acid, {[7-cyano~l-(3-fluoro-beii¾.'i)~4-hydroxy-l.H~ pyrrolo[2t3-c]pyridine-5-carbon.yi]-am.tno} -acetic acid, {[7-cyano-l-(3i4-dif!uoro-berszy!)-4- h droxy- 1 H-pyrro !o [2,3 -c] p ridine-5 -earbon i]-am ino } -acetic ac d, { [3 -ehloro- 7-cyano- 1 -
Figure imgf000074_0001
{[2,3~dichlQro-7-cyano-1.~(3-fiuoro-ben.^
carbonylj -amino} -acetic acid, {[3-chloro-?-cyano~] -(3-fluoro-ben2yi)-4-hydFoxy-l H- pyrrolo[2 -c]pyridine-5-carb ny!]~amino}-acetic aeidi {[2,3-dic.hloro~7~cyano-l~(3,4- dii¾oro~ben2y!)-4-hyd^ acid, [(1- benzyl-2,3«dic.hlaro-7-hy^ acid, [(2-ier^biJ{y1-7-hydiOxy-thfazo1o[4,5-c]pyridine~6-c-«rbonyl)-ajriino -aGetic acid, [(2-tert- butyI-7-hydroxy-4~methyI-thia^ acid, [(2-tert- bu^l-4-cyano-7-hydroxy-thiazoJor4,5-c]pyridine-6-C bQnyl)-amino]-acetic'acid!! [{4-butyS- 2~iari-buiyt-7-hydrQxy-thiazo^ acid, [(2-teri- buiyI~7 rydroxy-4-((E)-siyryi^ acid, [(2- fer^butyi-7-hydroxy-4-phenyl-thia^^ acid, [(2~ f ^bui l-7 i drox -4-pherieft^^ acid, [(2-fert-bi^l~7'hydroxy-4-isopropyIsu¾^
a inoj-aceiic acid, '[(7-hydroxy-2-methyl-4^1ienyl-ihiazolo[4j5-c]pyridine-6>carbonyl)- aminoj-acetie acid, [{7-hydroxy~2-meth> hiazolo|4,5-c]pyi ^
acid, [(74iyd«sxy-2-naphihalfen-2^^ acid,
Figure imgf000074_0002
acid, and:
[(2-furan~2~yi-7~hydroxy^ acid.
[00210] In some embodiments, small molecule PHIs may be selected from those disclosed in WO 2004/308121 (U.S. 2005/020487), which can be represented by formula IV:
Figure imgf000075_0001
[00211] wherein
00212} A is 1 ,2-arylidene. 1.,3-arylid.ene, 1 , -aiyUdene'; or (C C^-a'ikylene, optionally substituted by one or two halogen, cyano, nitro, trifluororaethy!, (Ci-Q -alkyl, (C i -C<i)-h droxyalky 1, (C i -Ce)-aikoxy , -0- CH2].x~CiH(2f† i-g)Halg, (d -C6)-fli!oroalkoxyJ: (CrC8>fiuoroalkenylexy9 (d-¾-fltioroalkynyloxy, ~QCF2C1, -GM^F -CHFCl; (CrC6)- alkyimercapto, (d-d)-alkylsulfh yi,
Figure imgf000075_0002
(Ci-¾)- lkoxycarbony!, carbamoyl. -iCs-C^-a!ky!carbamoyl, N.N-dKd-C^aikylearbamoyl, (Ci-Cs)-alkylcarbonyioxy, (C3-Cs)-cye.Ioa!kyl phenyl, benzyl, phenoxy, beozyloxy, anilfiio, -methylanilino, phenylmercapto, phenylsulfooyi, pheriylsul ra i, sulfampyi, N~(d-C " alkylsuifamoyl, N,N-dir(Ci-d)-alkylsulfamoyl; or by a substituted
Figure imgf000075_0003
(C?- Ci i)~aralkylpxy,
Figure imgf000075_0004
which carries in the aryl m.oisry one to five identical or different substftuente selected from halogen, cyano, nitre, trifluoromethyl, (CrC6)-aIkyL (d-C6 *JkQxy, -0-j;C¾}x-CjH(2fti,g)HaIg, -OCFaCI, -0-CFr HFCL (Cr
Figure imgf000075_0005
(Crd)-alky!mlforryl, .(Cr¾)-alkykar onyl} {d- Cs)-aikoxycar bony I carbamoyl, T» --(Ci-d}-alk {carbamoyl. N,N-di-(Ci-C )-aiky!carbamoy1, (Ci-Gf -aikyiearbotiyloxy, (d-C8 eyeloa!kyi, sitlfamoyi, N~-(Ci-G4}-alkyIsul¾i¾oyl, N,N- di-(Ci-C4)-alky]sulfamoyI; or wherein A is -CR5 R6 and JR.* and R6 are each independently selected from hydrogen, (d-Q>]ha]kyi,
Figure imgf000075_0006
or a substitisent of the a- earbon atom of an -a-amino acid, wherein the araiiio acid is a natural L-amino acid or its D- isomer.
[00213] B is -C02H, -N¾, ~ HS02CF3l tetrazolyl, imidazolyl, 3- hydroxyisoxazoiyl, -CO HCQR'', -GONHSOR"', CONHS<¾R"\ where R'" is aryl, heteroaryl
Figure imgf000075_0007
by (Cs-d2)~at l, heterpary],
Figure imgf000075_0008
(d-dMMoalkyl, (d-C«)-sulfi yl, (Ci- C^salfonyl, CF3, CI, Br, F, 1, NG¾ -GQQH, (C2-.C$)~alkoxycarbony!, H¾, rao.n.o-(C C - aikyl)-amino, di-(Cf-C<(-alkyl)-aminof or
Figure imgf000075_0009
or wherein B is a 0¾-G earboxyl radical, where G is a radical of an alcohol G-OH in which G is selected from (C r C2o)- ikyl radical, (Ca-Cs) eycloalkyl radical (CrC2a)-aikenyl radical, (C3-Cs)-cycloalkenyi radical, retinyi radical, (C2-C2o)~alfcynyl radical, (C4-C2o)-alkenynyl radical, where the alkenyl, cyeloalkeiiyl. aikyn l, and alkenynyl radicals contain one or more multiple bonds; (Cs-Ci(i)"CarbQcyclic ary! radical,. (CT-CigJ-carbocyelic araiky! radical, heteroaryl radical, or beteroafaikyl radical, wherein a heteroary'i radical or heterparyl moiety of heferoaraltyl radical contains 5 or 6 ring atoms- and wherein radicals defined for G are sabstituted by one or more hydroxy!,
Figure imgf000076_0001
{C Cs)- cycloalk l, (C5-C«>eycloa1kenyi, (Ck-C&j-aryi, (C7-Ci6)-aralkyi, (Ca-C^alkenyl, (<¼-(¾- alkynyl, CrC^-alkpx , (Ci~Ci2)-alkoxy-{Ci~Ci2)-alkyl)■(Ci-C]2)-alkoxy-(Ct-Cj3)-alkoxy, (<¾-Gia)-aryl xy, (C7-Cjs)-araikyloxy, (CrCs)½'droxyalkyl,
OCF2Cl, -OCF HFCl, ( t^i2)^lkyl6arbqnyl, (C3-Cs)~cyeIoa!kylcarbonyi, (Q-Ct¾>- arytearbonyl, (C7-Ct6)-aTalkylcarbonyl, cinnamoyl,
Figure imgf000076_0003
(Ca-Cn)- alkynylcarbonyli (CrCnJ-alkoxycarbonyL {Ci-C i)"alkox -(C C j2)- lk )x car on L (Q- Ci2)-aryiQxycarbonyl, (C7-Cj«)-aralkoxycarbonyl,
Figure imgf000076_0004
(C2-C12}- alkenyloxycarbonyl, (<¾-C f 2)-alkynyJoxycatbonyl, . acyloxy, (C Ci2 aikoxycarbonyIaxy, (Ci-Ci2)-alkpxy-<Cj-Qi2)-aIkoxycarbonyloj{y,
Figure imgf000076_0005
.(CrCj$)- araikyloxycarbonyloxy, (C3~Cs)-cycloalkoxyearbonyloxy, (Cs-CiaJ^alkenyioxycarboiiyloxy, (C?-C]2)-alkyiiyloxycarbor!yioxy, carbamoyl,
Figure imgf000076_0006
N.N-d.-(Ci~<¼2 - alkylcarbamoyl, N-CCs-Cs^yeloalkylearbamoyl, ¾i-(G6-Cis)-arylcarbamoyk N-(CrCj[«)- araiky lcarbamoy 1, N-(G 1 *€ j o)~alky l-N-(Cfi-C Ί 6)-ar lcarbamoyl, N-(Ci ~C 3 o)-alkyl-N-{Cr Ci«)-aralkytearba oyI, N^^
aryioxy-(Ci-Cio)alkyi}-carbmnoy^^ -
Figure imgf000076_0007
Cii0~ rylo y Cf~Cio)-alky1Harba^
sub.10)-aIky i.)-carbaraoyls carbamoyloxy, M-(C1-Ct2)-alky!carbamoy loxy, N,N-di-(Cj-Cn)- alkylcarbainoyloxy, N-{C3-Cs)-cycloalkylcarbainoyloxy, N^Cs-Cf^-arykarbamoyioxy, N- (C Ci6)-af ik l carbampylpxy, N-{C r C 1 o)-aikyI-N- C C)«ary learbampy loxy, N(C■ -C 50)- aikyl-N- G7-Ci¾}-araf.kylcarbamGyl{3xy, N-((CrCjo}-alkyl)-carbamoyloxy5 N~((C,s-Ci )- ary loxy-(C j -Q o)-a!ky i)~carbamo 3oxy, >J-((C7-Ci$)-¾ralkylQxy-(C 1 -C io)-aikyl)- earbarad loxy,
Figure imgf000076_0008
C^Q)-a!k l-^ (Q~C.i2)-aiyloxy-(Ci-Cio)-
Cj(5)-aralkyloxy-(C]-G.s- ubA0)-alkyl)-carbamoylPxy¾ amino, (CrCt2)-alkylamuid, di-(Ci- Ci2)-alkylamino,
Figure imgf000076_0009
N-^C&-C 12)-arylam ino, H-iC -C i^-aralkylamino, N-alkyl-aralky lam o, N-alky ί-arylaraino, (Ci-C|2>aIkoxyamtno, (C1-Ci2)-alkG3sy-N-{Cj-C)o)-alkylamino- (Cj~C32)-
Figure imgf000077_0001
C j 6)-araik Icarbony lamiiio, (C ¾ -C s2)- iky Icarbon i-N--{C i~C 1 o)-aik lamiiio, (C.5-C¾)- cy eloalky icarbony l- -(Ct -C uj)-aiky iam i no, (Cs-C t 2)-ar lca Aonyi-N-~{C j -C io)-a Iky lam mo, (C6-C j )~ara ik icarbon l-M-(C < -C 1 i>)-alky iam im, (C 1 -C 12)-alk Icarbony lamino~(C j -C«)-alky], (C3-C8)-cycloaIky!carbonyia!iiin&-(CrC8)a!!iyi> (Cs-Cn^afylc r onylamino-iGrCgJ-aikyl, (C7-Ci2)-a alk icarbon kmino-{C C8)-a{lt l5 amino-(C]-Cio)-a}kyi5 N-<Ci-Cio)-aikylatnino- (G!-Cio)-alkyi, N.N-d!-(ej-Gio)-aikylaraino-(CrCio aIkyl, (C3-Cs)-cycloalkylamino-(Ci- C ] o)-alky 1, (C i -€ i2)~alk imereapto, (C j-C 12)-alky Isulf ¾y ! , (C 1 -C j 2)-alkylsulfony 1 , (C&-C a)- atylmercapto,
Figure imgf000077_0002
C s6)-ara!kyisulfmyl, {G7~Ci.6}-aralk lsuifo!i i! su!farooyL N- Ci-Cio}~alkyIsulfamoyk NfN- di-(Cs-Cio)- lkylsulfamoyi, (Cj-CsKycioaikylsuIfamoyl, N^Cs-C^-alkyisulf moyi, . N- (C Ci^-araIk lsulfamay1, N^C Cio)- l^^ N~<CpGjo)-alk l- N-(C7-Ci6)-ara'lkyisulfar«oyl, (Ci-CioJ-alkylsulfonamido, N-{{Ci~Cio)-alkyi)~iCi-Cio)- alkyisulfonamido, (C?-C:i$ ara1kylsulfonaraido, or -((Cj-Cio)-alkyl-(C7-Ci6)- araiky!siiifonarnido; wherein radicals hich are aryi or contain an aryS moiety, may he substituted' .on the aryl by one to five identical or different hydroxyl, halogen, cyano, trifluoromethyl, nitro, earfaoxy!, (C!-Cn)-a]kyl, (Ci-C3)-cycloalkyL (Cs-Ci2}-aryl, (ΟΗ¾«>·.
Figure imgf000077_0003
(C$-C{2)-aryloxy, (C7-C j s)-aralkyioxy5 (C t -Cg)~hy droxyaiky!, (C t-G] 2)~alky lcarbonyl, (C>-
Figure imgf000077_0004
(C7- C i6)-aralkoxycarbonyL (CH¾}~eydoalkoxycarbQnyi5 (Cs-CiaJ-aikenyloxycarbonyi, (C2- C^-aikynyloxyearbonyl, (Gi-Cn)-alkyicarbony!oxy? (C3-Gs)-eycl.oaIkyiearbonyloxy; (C$- Cv2)-aiylcarbGa loxy, (Cr-C½)-8ralkylcarbonyloxy, .c.mriam-oylQxy> (C2-Q2)- aikeny Icarbony ioxy,
Figure imgf000077_0005
aIkoxy-(Ci-Ci2)-alkoxycarbonyloxy5 (C6-Ci2)-aryIoxycarbonylo y, (Cj-Cig)- araikyipxycarbonyioxy, .(Gj-GgJ-cycloalkoxycarbonyloxy, (Cs-GisJ-alkeayloxycarbOiiyloxy,. (•C2-.Ci2)-alkynylo-xycarbanyloxy, carbamoyl, N- C:s-Ci2)-aIky1carbamoy1s N. N-th~(Ci-C52)- alkylcarbamoyI. iS-(C3-Cs)-cyc!oalkylcarbampyi}
Figure imgf000077_0006
-{CVCi.<s)- aralkylcarbarrtoyl ^Ci-CiQ)-alk i-NH^C6-Cs )~ r lcar ^
C e)-araikylcarbamoyl, N-((Ci-C|D)-alkoxy-(Ci^C! )-alky.i)-carbaraoy!, N-~((CfrCi.2)~ atyloxy-CCi-Ci ^alky'O-carbamoyl, N- (C7-Crg)-atalk loxy-(Ci-C10>a kyl)-earbamoyl, N~
Figure imgf000077_0007
Ci2)-aryIoxy^(C-]-Ci{))-alkyl)-carbaifnoy.i, N-~{Cf-Ci())-aiky!-N--{{C7-'Ci6.)-aralkyloxy-(CrC1- sub.1 Q)~alky l)-earbar» oy 1, carbamoy lox , H-(C j -C j stalk icarbam oyloxyv N.N-di-(C ¾ -C n aJky!carbaxnoyloxy, N-{C3-C¾)-cycioalky3carbamoyloxy N^C6-C]2)-atylcarban o loxy5 N- (C?~C j 6)-aral ky Icarbam oy loxy, N-(C ·. -C io)-dkyi-N-(C¼-C , 2)-ary lcarbamoy lox , N-(C j - CiQ)-aIky]-N -(C7-Cie)-aralkyicarbamoyioxy; N-<(C7-Ci o)-a1k l carbamoyloxy, N-((C(5- C f2)-ary loxy-(C] -C}o)-alkyl)-carbamo loxy. N-((C7-C < e)-aralky loxy-(Ci-Cso)-alkyl)-
Figure imgf000078_0001
Gi6)-aralkyloxy-(C|.-Cr- sub.10)-aIkyl)-caibamoyIoxy, amino, (Cj-<¾)-a3kyIamin0, di-(C[- Cj2)-alkylammo, (Q-CsJ-cycloalkylamino. (Cj-Csj alken l roino, (C3-Ct2)-alkynylaramo, N-(Ci>-Ci2)-ar amino; N~(C7-C[i)-aralkySamiiio, N-alky aralky amino, N-alkyl-arylaramo, (C T-C 52)-alkoxyam ins, (C i-Cj 2)-alkoxy-N-(C i ~Q o)~alk iamino, (C i -C 1 ¾)- aikylearbony iamino,
Figure imgf000078_0002
C½ a!kylcarbonylamin©, (Ci-Ci2)-alkyleaibonyl-N-(C].-Cio)-alkylamino- (Ca-Cs)- ■cycjoatkylcarbonyl-N-iCrCmJ-alkylainino, (C6-C!2)-aryicarbonyi-N-(C;--C]o.Va ky!ami:no) (C7-Cn. aralkylcarbQnyi-N^CrCio)-alkylamino, {Ct-C32)-aIkyicarbonylamino-iCrCs)- alkyl, (Cj-CgJ-cycloalkylcarbonylamino-iCrCsJ-alky!, (Cg-Cr^-ar lcarbon lamino-CCi-Gs)- alkyl, (G7-Cts)-ara!kylearbonyiamino-(C!-Cs)-alkyL amino-(Cs~Cio)-alkyl, N-(C Cjo)- alkylaraino-(CrCio>a1kyl, .N^
eycloalkylajnjno-(Gj-Cto)-aIkyl, (CrC^J-alkylmercapto. (Cj-CiiValkyisulfmyi, (C1-C.12)-
Figure imgf000078_0003
(C.5-Cs2)-aiy!suIfinyL (C6-Cj2)-arylsulfonyls {C7-Ct&)- aralkylrnercapto, (G7-C]6)-araikylsul.ftnyl, or (C7-Cjs)-araikylsu!fonyl;
Figure imgf000078_0004
j00215] Q is Cv S, NR\ or a bond;
[00216j where, if Q is a bond, R* is halogen, nitrite, or rifluoromethyl;
[00217 or where, if Q is G, S, or NR1, R4 is hydrogen, (C;-C5o)-aikyl radical, (C2- Cio)-alkenyl radical, {Cs-Gtt -alkynyl radical, wherein aikenyl or alkynyl radical contains one or two C-C multiple bonds; umubsiituted iluoroa!ky! radical of the formula -[&¾<- CfH(2M-gHPs- (Ci-Ca)-alkoxy-(Ci-C6)-aIkyi radical (Cs~C6)-alkoxy~(CrC4)-aikoxy.(Cr C4)-alkyl radical, aryl radical, heteroaryl radical, (C7-C1 i)-ar ikyi radical or a radical of the formula Z
-[CH2]v-[Q]*-[C¾]rE (Z) 00218] where [00219] E is a heteroaryl radical, a (C3--C«)-cycioalkyi radical, or a phenyl radical of the formula F
Figure imgf000079_0001
[00220] v is 0-6,
[00221] w is Q o h
00222] t is 0-3, and
[00223] R7 , R8 , R9 , R!0 , and Rl ! are identical or different and are hydrogen,, halogen, cyano, nitre, trifluoromethyi, (C-rC6)-aikyl, <<¾-Cs}-cycIoaikyI, (Cj-GgJ-alkoxy, -
Figure imgf000079_0002
~OC¥2-Ch -O-CFa-CRFCl, (Ci-Qs)-alkylmercapto, (d-C*)- hydroxyaSkyi, (Ci- e)-allcoxy (Ci-C6)-alkoxy, (Ci-Ce -a?koxy-(Cj~Cc -aikyl, (Ci-CV,)- a!kylsuifiny 1, (Cs-C$)-a3kylsulfonyl, (Cj -C^-alkykarbonyi (G(-Ca)-aIkoxycarbonyl( carbamoyl, N~(Ci-Cs)-alkylcarbamoyi, N:,N-di-(CrC8)-a!kylcarbamoyl, or (Cy-Cu)- aralkyicarbamoy!, optionally substituted by fluorine, chlorine, bromine, trifluoromethyi, (C(- C6)-ai kox , N-(CrCs)-eyeIoalky icarbamo !, N~(C3-C8)-cycloalky 1-(C ] -C4)-al kylearbaraoyl, (Ci-Cfj-a!k lcarbony!oxy, phenyl, 'benzyl, phenox , benzy!oxy, N R2 wherein RY and R^ are independently selected from hydrogen, (Ci-C^-atkyL. (C Ca)-aikoxy C;-Cs)-alliyl, <G
Figure imgf000079_0003
alkeriyl,
Figure imgf000079_0004
(CrCnJ-aralkyi, (Cj-C12)-alkoxy, (C7~Ci2 aralkoxy. (Ci-Cj-zJ-alkylearbonyl, (Cs-Cgj-cycloaikylcarbonyl, (C Cs2)-ary?earbonyl, (C7- Ci6)-aJ¾lkylcarbonyi; or further wherein Y and R2 together are ~[C¾]h, in which a€% group can be replaced by O,
Figure imgf000079_0005
or N-(Ci-C4
alkoxycarbonyliniino, and h is 3 to ?; phertylmercapto, phenylsulfonyl, phenylsulfmyl, sulfarnoyl, -(Gi-Cs)-alkyIstJlfamoyi, or 'N,N-di-(Ci-Cs)-alfcylsuifamoyl or alternatively R7 and Rs , R3 and 9 , 9 and Ri0 , or Ri0 and R! 1 .together are a chain selected from -[CHj]h- or ~ XH=CH-CH=CH----, where a Cl¾ group of the c ha in is optionally replaced by O, S, SO, SQ¾ o l RY; and n is 3, 4, or 5; and if E is a heteroaryl radical, said radical can carry 1 -3 substitaente selected from those defined for R7-R" , or if E is a eycloalky! radical, the radical can carry one substituent selected from those defined for R7-R! i ; [00224] or where, if Q Is NR', R is alternatively R", where R' and R" are identical or different and are hydrogen, (C,;-Cji)~ary{, (CT-Ci -a alk l, (Cs-C^-aikyl, (Gi-Cg)-alkoxy- (Ci-Cg 'kyl iC7-Gi 2)-araIkoxy-(Gi-C8;)-alky1, (C6-C 2)-an'!oxy-(C (Ci-do)- alkyfcarbonyi, optionally substituted (C7-Ci6)-aralkyIcai'bonyl, or optionally substituted (Cg- C^-aryiearbonyl; or R' and R" together are -[CH¾jh» in which a CFfe group can be replaced by O, S, N-aeylimlno, or N-lCi-CioJ-alkOxycarbonySimino, and h is 3 to 7.
100225] Y is " or CR3 ;
[00226] R1 , R1 and R' are identical or different and are hydrogen, hydrox I halogen, cyano, ixifluoromethyl. nifro, carboxyl, (CrC_o)-ajkyl (C3-C8)-c cioalk l, (C-3-C¾)~ cyc{oalkyi-{Ci-C!2)-alkyl, {Cs-Cg^eycioalkoxy, (C3-Cs)-cycioaikyl~(Cf-Ci2)~alkoxy, {Cr .Cs>cyc.oaikyloxy-(C> -Ci^alk l, (C3-C8)-cycIoalIty xy-(Ci-C»2)-alkpxy, (C^Qs
cycloatkylr(CrQ)ralkyHGrC6 alkoxy, (C3-Cg}-eycloalkyi-(CrCiiVaikoxy-(CrC6}-alkyi, (Cj-Cs)-cyclQalkyloxy-(Ci-C8)-aik0xy-(Ci-C6)-aIkyl? (Q-C^-cycloalkoxy-iCi-Cs koxy- (d-Gsj- aikoxy, (C6~Ci2)-aryl,, <G7-Cj$)-aralkyl, (C Ci6 anUkenyL (G7-C|6)-araIkynyl, (C3~
Figure imgf000080_0001
retinyloxy, (Gj-G2o)-alkoxy-(C»-Ci2)-alkyl, (CrCS2)-alkoxy-(CrCt2)-alkoxy, (Cj-Ci2> alkoxy-(C C8)-alkoxy Ci-C8)-aiky- 1, (C¼-C{2)~arylaxy? (C7-Ci&)-aralkyloxy$ (C6-C32)- aryloxy-CCrCej-alkoxy, (C7-Cjs)-araikoxy-(CrCs)-alkoxy5 (Cj-Ct6)-hyd.roxyalkyl, Ce-C^)- ary loxy-(C \ ~Cg aIky 1 , (C?-C j-e)-aralkoxy-(C r Cs)-alky i, (C§-Ci2)-ary 1 oxy-(C i -Cg)-alkoxy- (C C0)-a]kyl (C7-Ci2)-aralkyloxy-(C£-Cg)-alkGxy-(Ci-C6)-a!kyl, (Ca-C2o)-aIkenyloxy-(Ct- . Cf -alkyl, (C3-C2o)-alkynylox -{C!~C6)-alkyl:,
Figure imgf000080_0002
g)-Fg, ~OCF2CI, -OCFs-CHFGL (Ci-Csoj-alkylcarbonyi, (C3-C3)-cycloalkylcarbonyl; (C6- Ci2 -aiylcarbonyl, ^-C^-aralfcyiearbonyl, cinnaraoyi, (p2-C2o)-alkenylcarbonyI, (Ca-C^)- aikynyicarbonyl, (Ci-Cjoi-alkoxycarbonyi, iC]-C3 )--alkox ~(C Cn}-aIko ea bony (C&- C:i2)-ary1oxycarbonyl, (G7-Cf«)-ara1koxycarborjyi, (Cs-C^-eyeloalkoxycarhonyi, (C2-C20)- alkenyloxycarbonyl, retinyloxyearbonyl, (Ca-C^-alkynyloxycarbonyL (Ce-C^-aryloxy- <CrC6)-alkoxycarbonyI, (C7-C½ -atalkoxy-(CrC6)-alkoxycafbonyl, (C3-Cs)-cycloalkyl-(C j - C^alkoxycarbon l, iC3-Cs)~eycloaikexy-{C C6)-a1koxyearbonyL (Cj-Ca)- alkylearbonyloxy, {C:rCs)-cycIoalkylcarbonyioxy5 (Cs-Cijj-ai lcarhonyioxy, (Cj-Cij,)- ar Iky learbony loxy, cinnamo lox„ (C2~C j 2)-aIken } carboti lox , (C2-G f ?)- alkynylcarbottyioxy, (C;-Ci2)-aikoxycarbonyloxyi (Ct-Cs2)-aikoxy~(Ci-C]2
alkoxycarbonyloxy, (Q-CixVary'toxycarbonyloxy, (Cr-Ci6)-aralkyloxycarbonyloxy, (C3-C3)- cycioalkoxycarbonyloxy, (C2-C12)-alkenyloxycarboftyi.Gxys. (CrCi2)-alkynyloxycarbonyloxy4 carbamoyl,
Figure imgf000080_0003
N,N-di-(G:i-C)2)-a1kykarbaraoyi3 - C3-Cs)- cycloalky .carbamoyl, N! -dicyc!o-(Cj-Cs)-alkyfcarbamoyL N-(Ci-Cio)-al:kyi-N-{C3-C8)-
Figure imgf000081_0001
Figure imgf000081_0002
b.6)-aikyi)-carbamoyi N-(+)~dehydroabieiyicarbamoyi, N-
Figure imgf000081_0003
araikylcarbamoyl,
Figure imgf000081_0004
C ] §)-aralk icarbamoyi ? N-((C Ct s)-alkoxy-(C -C io)-aikyl)-carbam oyi, -((Cg-C j e)~ ary loxy-(C j-C i «)-aIky l^carbamo I, N-«C6-G i6)-aral k i oxy- (C j -C i a)-a!kyl)-c arbamoy 1, N-
Figure imgf000081_0005
G3j aryIoxy-{Cj-Cso)-alkyi)-carbamoyl, N^Ci-GsoJ-alk i-N-^fC Ci^-ar lk lox - Ci-C.- sub.10)-alk i)- carbamoyl; CON(C-¾)h,. in which a C-hh group can be- replaced by 0, S, N- (Cs-Cg:}-alkylim.ixiQ, N-{C3^C¾)-c c]Qalk liffi^^
N- C6-Cia)-arylimino, N-(C7-CK ~araik liminoJ N-{C]~ i)-aikox ~{Gj-C6)-a!k li!T!mo} and h s from 3 to 7; a carbamoyl radical of the formula
Figure imgf000081_0006
[00227] in which
[00228] R* and Rv are each independently 'selected from hydrogen, (Gi-Cy-alk l, (C3-C7)-cycloalkyi, aryl, or the substitueni of an a-carbon of an ct-amino acid, to which the L- and D-amino acids belong,
[00229] s s 1 -5,
[00230] T is OH, or NR*R**, and R.*, R** and R*** are identical or different and are selected from hydrogen, (Cg-CnJ-aryl, (C7~Cu)-aralky!, (CH¾)-alkyL. (Cj-Cg)- cycloalkyl, (+)-dehydroabietyl, (Ci-CsJ-alkoxy-CCi-CgJ-alky!, (C7-C;2 arai:koxy-(C!-Cg alkyl, (C6-C12)-ar>f!oxy-(Ci-C¼)-alkyk (C{-Cio)ralkanoyis optionally substituted (CT-GIS)- aralkanoyl. optionally substituted (C&-C;¾haro I; or R* and R** together are -[CHsjh, in hieh a CH2 group can be replaced by 0„ S, SO, SO¾ -acy lamina,
Figure imgf000081_0007
Figure imgf000081_0008
NH¾~Cs)-cycIoa3kyHminc>, N-(C3-C8)- cycloalkyl-(GrC4>alkylitnino, N~(C6-Ci2 arylin mo,
Figure imgf000081_0009
N-{Ci-G aikoxy-(Ci-C6)-alkylimino:, and h is from 3 to 7; [00231] carbamoyloxy, N~(Ci -Ci¾)-aIkylcartenoyloxy, N,N-di-(Ci-Cf2)- aikyicarbamoyloxy, N-^Ci-Cg^cyeloaJkylcarbamoyioxy, N-^Q-C^^arylcar amoyloxy, -
Figure imgf000082_0001
C ] o aIky i-N-(C7-C■■ 6)-aralky learbamoy 1 oxy, N-((Ct -C i o)-aiky 3)-carbamoy ioxy s V iQr Ci2)-aryloxy-(Ci~Cio)-a.lkyi)-carbamoyiexy, NH'(C7-Gi6)-ara!k loxy-(C CKt)-^ l)- carbamcyloxy, N^'C!-Cio)-aikyi-N-{(CrCi }-aikoxy-(Cs-Cio)-alkyr) carbarn.pyl.Dxys N~(Cj- C io)-alkyi- -«C6-Cj 2)-aryloxy« G] ~C i o)-alky l)-carbanioyioxy, N~(Ci-CH))-alkyl- H{C7- Cj6)-ara1kyioxy-(C -C.- sub.10)~a!kyl)-carbamoyioxyamino; (Cr-Ci2)-alkylaminos di-(Ci - C j3)-alky lam-tno, (Cj-CsJ-cycIoalky!amino, (C3-C1 ¾ -aIkersyIamuio, (C3-Ci2)- lk .nyI mirio> N-(C6~Ci2)-ar iaisiino;
Figure imgf000082_0002
(Gi-Gv2)-alkoxyanrtno, (C Gs2)-aSfco -N-(Ci-Cio)-alk lamir!o, (Cj-Cj2).-aikanOylaniin0, (CrCgj-cyctoalkanoylamino, (Cs-CsiJ- ro i inino, (C7-Cj6)--araIkanoylamino, (CrCi?)- alkanoyl-N ft-QoHlkylaaiino, {CrC^-cycloalkanoyi-N-iCj-CioJ-aikylamino, (C^Cn)- aroy 1-N~(C 5 -C 1 o)-alkyiam ino, (C7-C $ 3 )-araikanoyl-N-{C 5 -C j o)-alky lam ino5 (C 1 -C 52)- alkanoylarainp-(Ci-C8)-alkyl, (C3-Cs)-eycioaSkanoylamiTio-(CrCs)-alkyl, (Ce-C^)- aroylarnino-{G.i-Cs}-alkyl,. (C7-Ci5)-ar8lkanoylainino-(C!~Cg)-alkyl, amino-(Gi-Cjg)-alkyii N^;Ci~C5o)-aiky] mmo C Cto)~alkyl ,]^-d
cye!oalkylaraiiw(CrCi i)-alky
Figure imgf000082_0003
(Gi-Cjo)- a!kylsulfonyl (Cg-CnJ- r lrnercapto, (Cg-CisJ-ary!siiffinyl, (CrCnJ-aiylsulfonyl, (C6-Cid -
Figure imgf000082_0004
(C7-Ci6)-aralkylsalfonyls (Ci-Cssi-alkyimercapto- (C,-C6)-alkyIs (CrC!2)-a{kySsu]finyl-{C5-C6)-alkyi> (C{-Ci2 a1kyIsulfonyHCt-C6 alkyl, (C C i 2)-aryimereapto-(¾ -C6)-aikyL (e<H¾ j)-ary isuffiny 1-(C 1 -QHlkyl, (C6-Ci2)- atylsulfonylr(Ci-G6>alkyi» (G7-Gi6}"ara(kylmercapto-(C1-C1)~alkyi> (CrC'igJ-araikylsulfinyl-
Figure imgf000082_0005
N-(C7-C s §)~aralk lsulfamoyl 5 N-(C j -C io)-alky 1-N-(C¾-C j 2)-ar Isul fani oy I, N-(C 5 -C io)- alkyi-N~(C7-Cis)-aralkyls«lfamoyi! {Ci-Cio}-aiky!s lfonamido; N~((Ci-Cio)-a;]lcyl)-(Ci-Ci0)- aikylsulfonamido, {C7-Ct6)'-araikylsu]foaamidof and
Figure imgf000082_0006
aralkylsulfonamido; where an aryl radical may be substituted by 1 to 5 substituents selected from hydroxyl, halogen, cyano, trifiuoro methyl, nitro, carboxyl. {G CieJ-alk l. (Cs-Gs)- cyefoaJkyl, (C GsHycIoaikyKC CnHl i . (C3-Cg)-cycloa!koxy. (C3-Cs)-cyeioaikyl-.(C]-
Figure imgf000082_0007
(C3-iGg)-cycloalkyloxy-(Cj-Gi2 ralkoxys (C3- Cs)-cyc!oaIkyi-(CrC8)-amyl-(CrGf;)-al- koxy, (Cs-Csi-c ctoalk i-CCi-CgJ-alko -CCi- C.syb- .6)-alkyL. {C3~C8)-cycloa:lky]oxy-{CrCg)-aSkoxy-( cycIoalkoxy-(Ci-Cg)-alkoxy-(CrCs)~ alkoxy, '(C¼-Ct2)-aryL (GrCi<&>aralkyL iC2'Ci6}- alkenyl, {C2-Cj2)-aikyii j.5 (Ci-Cje)-a]kpx , (Ci-Cj6}-alkenylQxy5 (Cj-Gj2)-alko y-(Ci-Ci2)- alkyl, (C]-Ci2)-aikoxy-(CjOi2 alko»xy, (Ci.-Cj2)-alk.oxy-(G^G8)-alkoxy~(C]-Cfe)~alky- 1, (Ο,
Ci2)-ai' 5ox ? (C6-C!(s)-aralkyloxy5 (C6-Ci2)-afylo -{Cj~C6)-alkox , (C7-Cs.6)-ara!koxy-(Cj- CftJ-alkoxy, (GrCs}-hydroxyalkyL (C Ci<$)^ar lo Ci: 8)^-kyI,. {^
Cs ikyl,
Figure imgf000083_0001
Figure imgf000083_0002
-OCE2CI, -OGFr-CHFCl, (CrCl2 alkylcarbony (C3-C8)~cyeloaikylcarbGny.l5
Figure imgf000083_0003
(C7-Ct6)-ara!kylcarbonyL (C i ~C i2)-aIkoxyearbony 1,
Figure imgf000083_0004
(Cg-Cja)- aiyloxycar onyl, (C7~Ci6)-aralkoxycarbony!, (C3-Cs)~cycioaikoxycarbooyl, (Ca-Cn)- alkenyloxycarbonyi, (Ci-CisJ-aikynyloxycarhonyi, (Cg-Ci2)-ar>'l.oxy-(C|-C¾)- aikoxy carbony I. (C.7-Ci$3-aralkQxy-(C i rCs)-alkoxycar onyI- (C CsJ-cyc ί oaiky 1-(C t -Ce)- alkoxycarbonyl, (Ca-CgJ-cycloa!koxy-CCi-CijJ-alkoxycarbonyl, (Cj-Ci2)-aIkylcafbosiyloxyJ: (Ga-CsJ-Gycloalkylcarbonyloxy, (G6-Ci2>-afyfcarbonyloxy, (CrG½)-ai¾lkylearbonyloxys cmnamoyloxy, (Ca-C^-alkenylcarbony!oxy, (Ca-C ^-alfcynylcarbonyloxy, (Ci -C 53)- alkoxyearbony lox , (C 1 -C !2)-aIkoxy-(C r C 5 s)-alkoxycarbony loxy, ( ¾-C 12)- aryloxycarbonyloxy, (C7-C ei-araikyioxycarbonyloxy, (C3-C«)-cycloalkoxycarbonyloxy, (C2 Cs2)-aIkeiTyioxycarboiiyloxy; (C2-Ci2)-aIkynyloxyGafbonyloxy, carbamoyl, N~(CfCi2)- alkylcarbamoyl, N,N-di-(C j -Ct2)-aIkylicarbamoyls -{C3-C3)~cycioa[kylcarbamoyl, N,N- dicyclo-(G3-C¾)-aIky!carbar!ioyi, H-(CrGio a|kyl-N-^CrC8)-cycIoalkyicarbainoyl, N-({Cr Cg)-cy cloalkyl-(C t -C^-alky l)carbam oy I, N-(C t-C<s a1 ky !~N~((C3-Cs)-ey cloaik l-(C] -C.su- fa.6)-aIkyi}carbamoyi, -{-i-)-debydroabi!e¾'iearbamoyl, N~(Ci-Cc)-aikyl-N-(+)- dehydroabietylcarbamoyl N~(C«-Cj2)raiylcarbamoyl,
Figure imgf000083_0005
C ! o)-alky i-N-(C6-C ; &)-my lcarbamoy 1, N-(C 1 ~C \ o)-aiky i- -(C7-C g)-aralky lcarbamoyi, N-
Figure imgf000083_0006
N-({CrC[6)-aralkylGxy-(C, -Cio aikyl)carbamoyl, N~{C C.io)-aikyl-N-{(C Cio}-alkoxy- (Ci-CH))-alkyl)carbaiiiCfyi, N~^CrC!o)-aikyI-'N^(Gi-C!2)-afyioxy-(Ci-Cio)-alky!)earbaiBoyi, N~(C!-Ci0)-aIkyi-N-((C7-Ci6)--aralkyioxy-{Cl-C.-- sub,IO)-alkyl>carbaraoy!, CON(CH2)hs in whic a C¾. group can be replaced by,
Figure imgf000083_0007
cye loalkyiim ino, N-(C¾-Ce cyclo.aikyHC s ~Gj)-alky 1 im mo, N-(C Ci a)~aryl im ino, Ν-ξΟτ·
Figure imgf000083_0008
carbam oyloxy, N~(C -C 12)- alky lcarbam oy loxy , N-N-di-CCj-Cj lcarbamoy loxy, N-iGs CgJ-cydoalkykarbamoy!cxy, N~(C<5-Ci6)-arylcarbaiiioylo y, N-(C6-Ci<i)- amikvicarbaraoyloxy- N-^Gi-C 6)-alkvl-N- C6-C|2 aryktarbamoyloxy, - Cj-Cto -alkvl-N (C7~Ci6)-aralkyIearhamoytoxy, N-((CrCio)-alkyl)cai¼rnoylQxy,
Figure imgf000084_0001
Cto)-alk l)carbamo lox . N-^(C7-^^^
C }-N (C C to)-a Ikoxy- (C ί -C ro)-alkyl)carbamoy to
ary|oxy-{Ci-Cjo)-alkyl)carbanioyioxys N CrC^o)-8lk i~ ^(C7-Ci6)-ara¾yiox -(C C,--' sub,10)-aikyl)carbamoyloxy, amine, (Ci-C]2)-alk iamino, di-{Gi-C!2}-a!kylaniino, (Cj-Cg)- cyeloalkylamino, (Cs-Cni-aikenylamino, (C3~C;2)-aikyny!amino,, N-{C6-Ci2)-arylamjno, N- (Cj-Ci ^-aralkylamino, N-alkyl-aralkylammo, N-alkyl-aiylamino, (Ci-Cia)-alkQxyamin (C j ~C J j)-aikoxy-N~(C I -C s o)-aIk lamino, (C i -Q 2)- aikano iamino, (C3-C3)- eycioa!kanoylaraino, (Q^^- ro lamino, CC7-C½)-aralkanoylarnma, {Ci-Ci2)-alkanoyl-N- (C j -Cjo)-alky lam ino? (Cj-Cs)-cycloalkanoyl-N-(Ci -Ci a)-a1k lam ino, (Gs-GjaJ-aro i-N-*(C s - C io)-aiky lamino. (C7-C 1 1 }-aralkanoyl- -(C 1 -C io)-alky larai no, (C 1 -C 12)-alkarsoy iamirio-(C j - Cs)-aikyi, (C3-C8>cycloaikanpylamino-(Cj-C8)-alkyl, (C -C u)-aroyia ino-(Ci -C&Hlk l, (C7-Ci6)-aralkanoyiafnino-{Ci~Cs)-aiky!, amino-(C)-C.io)-alkyl, N~(C]-Cio)-alkyiamino-(C!- Cio)-alkyl, N5 -di-(Ci-Cs(i)-aikylammo-(CrCio)-alkyis (G3-Gs)-cyc Qalkylarai.iTO~.(C!-Ci )- aikyl,
Figure imgf000084_0002
aiylmeroapto, (C6-Gi¾ -aiylsulfinyl, (C<s-Ci6 aiylsuIfooyl- (C7-Ci6)-aralkylmefcapto, :(€?- Ci6)-aralkyisulf3nyi, or (Cy-Ciij-aralkylsulfony!;
[00232] or wherein R! and R2 , or R2 and IV form a chain [C¾]fl, which is saturated or unsaturated by a C:=C double bond, in which 1 or 2 C¾ groups are optionally replaced by G, S, SO, S02s or NR', and R is hydrogen, (Ce-C^ai l, {Ci-C3)-alkyI, (C,~C8)-aikqxy-(Cr C« alkyl, (C TCraVaralko -CCi-CeJ-alk (C¾-C,2>-afylo}iy Ct-C8>-alkyl, CC-Cie)*
Figure imgf000084_0003
or optionally substituted (Cs-C)2)-aroyI; and o is 3, 4 or 5;
(00233] or wherein the radicals R1 and R2 , or R2 and R3 , together with the pyridine or pyridazine carrying them, form a 5,6.7,8-tetrahydroisoqumoIine ring, a 5,6,7.8- tetrahydroqis!Bo ne ring, or a 5s6,7s8-ietrahydroeinnoline ring;
[00234] wherein R! and R2 > or R2 and R3 form a carbocyclic or heterocyclic 5- or 6- membered aromatic ring;
[00235] or where R! and R2 , or R2 and RJ , together with the pyridine or pyridazine carrying them, form an optionally substituted heterocyclic ring systems selected from thsenopyridines, furanopyridines, pyridopyfidines, pyrimidinopyridines, imidazopyridijies, thiazoiopyridisies. oxazoiopyridines, qumoiine, isoqisinolme, and einnoline; where quinoline, isoquinoime or ciiinolins preferably satisfy the formulae !Va, lYb and !Vc:
Figure imgf000085_0001
[00236] and the.subsiituents R to R23 in each case independently of each other have the meaning of R! . R2 and R3 ;
[00237] or wherein the radicals R! and R2 ; together with the pyridine carrying thera, form a compound of form ula IVd:
Figure imgf000085_0002
[00238| where V is S, O, or NRk, and Rk is selected from hydrogen, (Cj-Qj-alkyl, aryl, or benzyl;
[00239] where an aryl radical may be optionally substituted by 1 to 5 substituents as defined above; and [00240] R24 , R~5, R."6, and m each case independently of each other have the meaning of R1 , R~ and R3 :
[00241] f is 1 to 8;
[002421 g is 0 or 1 to (2f+l);
[00243] x is Q to 3; and
[00244] h is 3 to 7;
[00245] including the physiologically activ salts and prodrugs derived therefrom.
[00246] Exemplary: compounds according to formula (IV) are described in European Patent Nos. EP 0650960 and EP 065096 L Ail compounds listed in EP 0650960 and EP 065096 ϊ , in particular, those listed in the compound claims and the final products of the working examples, are hereby incorporated herein by reference.
[00247] Additionally, exemplary compounds according to formula (IV) are described in U.S. Patent No. 5,658,933. Ail compounds listed in U.S. Patent No. 5,658,933, in particular, those listed in the compound claims and the final products of the working examples, are hereby incorporated herein by reference.
[00248] Additional compounds according to formula (IV) are substituted heterocyclic carboxyamides described in U.S. Patent No. 5.620,995; 3~hydroxypyridine-2- carboxamidoesters described in U.S.. Patent No. 6,020,350; suifonaimdocarbonyIpyridine-2- carboxasnides described in U.S. Patent No, 5,607,954 ;: and sulfo«amidocarbonyl»pyridine-2- carboxamides aud su!fonamjdosarbony!-pyrid!ne-2-carboxaniid.e esters described in U.S. Patent Nos. 5,610, 72 and 5,620,996, All compounds listed in these patents, in particular, those compounds listed in the compound claims and the final products of the working examples, are hereby incorporated herein by reference,
[00249] Exemplar compounds according to formula (IVa) are described in U.S. Patent Nos, 5,719,164 and 5,726,305, All compounds listed in the foregoing patents, in particular, those listed in the compound claims and the final products of the working examples, are hereby incorporated herein by reference.
[00250] Exemplar compounds according to formula (I.Vb) are described in U.S. Patent No. 6,093,730. AH compounds listed in U.S. Patent No. 6,093,730, in particular, those listed in the compound claims and the final products of the working examples, are hereby incorporated herei by reference. [00251] Compounds disclosed in WO 2004/108121 (U.S, 2005/020487} can be represented by formula V:
Figure imgf000087_0001
[00252] or pharmaceutically acceptable salts thereof, wherein:
[00253] a ts an integer from 1 to 4;
[00254] b is an integer from 0 to 4;
[00255] c is an integer from 0 to 4;
[00256] Z ?s selected from the group consisting of (Cs-Gioj-cycloalkyi, .(CS-CTO)-. •cycloalkyl independently substituted with one or more Ys , 3-30 membered heteroeycloalkyl and 3- 30 membered heieroeycloaikyl independently substituted with one or more Y 1 ; (C5- ■GaoJ-aryl, (Cs-Caol-aryl independently substituted with one or more Y! , 5-20 membered heteroaryl and 5-20 membered heteroary! independently substituted with one or more Y! ;
[00257] Arl is selected from the group, consisting of (Cj-QoJ-a l, (C5-C20) aiyl independently substituted with one or ore Y" , 5-20 membered heteroaryl and 5-20 membered heteroaryl independently substituted with on or more Y2 :
[00258] each Y! is mdependentiy selected from the group consisting of a lipophilic functional group, (C Cso^ar i, iQ-<¾6)~aikaryJ- 5-20 membered heteroaryl and 6-26 membered alk-lieteroaryl;
[00259] each Y2 is independently selected from the group consisting of -it', -OR', - OR", -SR', ~SR", -NR'R', -NO¾ -CN, -halogen, -trihalomethyl, nihaiomeihoxy, -C(0)R\ - C(0)OR'; ^C(0)NR'R', -C(0)NR'OR'. -CCNR '^NOR, - ^-C(0) '} -SOjR', -S<¼R", - NR'-SQr-R', -NR'-CiOJ-NR' 1, tetrazoI-S-yl, -NR'-C hOR', ~C(NR'R')=NR!, -$(0)-^ S(0>-Rn, and -NR'-C^S^-NR'R ; and
[00260] each R1 is independently selected from the group consisting of-H', (Ci-Cg)- alkyl, (C2-Cg)-a3kenyl, and (C2-!¾)-aikynyI; and .
[00261] each R" is independently selected from the grou consisting of (Cs-C2o)-ar l and (Cs-G2o ar l independently substituted with one or more OR', -SR', -NR'R', ~N<¾> - CN, halogen or trmalomethy! groups, [00262] or wherein c is 0 and Ar is an 1ST substituted urea-aryl, the compound has the structural formula Ya):
Figure imgf000088_0001
[00263] or- harmaceutically acceptable salts thereof, wherein:
[002643 as b, and Z are as defined above; and
[00265] 3s and W6 are each independently selected from the group consisting of hydrogen, (Cj-CgJ-aikyS, (C2-Cg)-a!kenyIs (Cs-CsJ-alkynyl, (Cj-Cl0)-cycloalkyl (C5-C20)- aryl,
Figure imgf000088_0002
5-20 merobered heteroaryl, 5-20 raembered substituted heteroaryl, 6-26 membered alk-heteroaryl, and 6-26 membered substituted alk-heteroaryi; and
[00266] "B ' is independently selected from the group consisting of hydrogen, (Gi- CsJ-alkyl,
Figure imgf000088_0003
and (CrCs)-alkynyl.
[00267] Additional compounds disclosed in WO 2003/053997 (U.S. 2003/153503) can be represented by formula VI:
Figure imgf000088_0004
[002681 wherein
[00269] 28 is hydrogen, nitro, amino, cyano, halogen, (Cj-C4)~alkyl, carboxy or a metaboHcaliy labile ester derivative thereof; (Gi-C4)-allcyiairiino, di-(GrC4)-aIkylamino, (Cr C^-alkoxyearboayl, (Cs-C^-alkanoyl, hydroxy-(CrC4)-alkyls carbamoyl, M-(Cj-€4)~ alkylcaf amoyf, (Cj-C4)«alkyithio, (Ci-C4)-aIkylsul6nyJ, (C^Q^alkylsulfonyl, phenyltfaio, phenylsulfinyl, phenylsulfonyl, said phenyl or phenyl groups being optionally substituted with i to 4 identical or different halogen, (Cj-C -a!kyoxy, (Ci-QJ-alkylj cyano, hydroxy, trifluoromethyl, f1tioi -(Cj-C4)-aikylthio.5 fluoro-fCs-C^-aikylsuifmyl, -fluoro-(Cj-C4}~ alkylsuifonyl. {C[-C )-alkoxy-(C2-C4)--aIkoxy'carbohyI, . N,N-di-[(CrC4)-alkyl]catban).oyl- (Ci-C4)-a!koxyearbo.nyI, (Ci~C4)-alkyiainii'!o-(C2-Q)-alkoxycarfaoiiyl, di-'(Ci-C.j)- alkylataitiQ-iCa-Gi^aikoxycarbonyi, . (Ct-C^-a!koxy-CC^-C^alko y-CCa^Q)- alkoxycarfaonyl, (C2-C4)-alkanoyloxy-(C] -C4)-alkyl, or N~[amtno-(C2"Cs)-aikyl]-carbaraoy!;
[00270] R29 is hydrogen, hydrox 5 amino, eyano, halogen, (Ci-C -alkyL carboxy or roetaholioaliy labile
Q)-alkoxycarboriy
a!koxycarbonyi-(Ci
Figure imgf000089_0001
aikylcarbamoyl, ammo-(Ca-Cg)-alkyi3-carb8m yl, N-[(C j -Gt)-alky lam ino-(Ci-Gs)-aik ■!]- carbamoyl, N-[di^(Ci^.|)-a!kylamino^(Ci.-Cs)-alkyl)]-carbainoyL N-eyclohexylearbamoyl, -feyclopentylJ-earbaraoyl, N— (Ci-Gt)-alkylcyclohfexylcarbamQyl, !-(Ci-C4}- ajkylcy lopentyicar'bamoy , N-phenylearhampyl,: ~(Gi-G4)-aIky1-N-phenylcarbamoyl,. NSN- diphenylcarbamoyl, N~[phenyl-{CrC4)-aikyi]-carbamoyl, N-<Cj-C4)-alky!-N-[phenyKCi- C4)-aikyl]~carbaraoyif or N, -di-[phenyi-(Ci-C4)-aikyI]-carbarnoylJ said phenyl or phenyl groups being optionally substituted with
[00271]' 1
Figure imgf000089_0002
(Gj -Chalky 1, cyano, hydroxy, trifluoromethyl, N-[(C2'-C4)-alkanoyl]-Garbamoyl, N-[(C! -C4)-aikoxycajboii I]- earbam o N- [flu0ro-(C2-Ce)-alk ^carbamo l, N,M- f! uoro-(G2- Csj-alkyi] -N-{0 \ -C,?)- aikylcarbamoyl, ,M- [di-fl ooro-(C;_- Cs}~alk I] carbamoyl, pyrrol idin- 1 - learbonyi , piperidinocarbonyl. piperazin-l-ylcarbonyl. morphoiinocarbonyL wherein the heterocyclic. group, is optionally substituted with 1 to 4, (C C4)~alkyl, benzyl, 142,3»4~tetrah dro-< isoqufflolm-2~yicarbonyI, N!N-[di-(Ci-C4)-alkyi]-tl)iocarbamoyl, N-(C2-C4)-alkanoylanu!io, or N~{{Gi-C4)"alkoxycarbo!iyi| -amino;.
[00272] R30 is hydrogen, (Ci-CL -alkyI, (C2~C4)-alko , halo, nitro, hydroxy, fiuoro- (Ci-G4)-a!kyi- or py idinyi;
[00273] R3' is hydrogen, (Ci~C4)-aikyi, (C;2~C4)-alkoxy, halo, nitro, hydroxy, f!uoro-
(Ci -C^-aiky i, pyridinyl, or methoxy;
[00274] RJ~ is hydrogen, hydroxy, amino,
Figure imgf000089_0003
alkyia ino, halo, (Cj-C<j)-aIkoxy~(CrC4)-a!koxy,. fluoro-(Gi-C6)-alkoxy, pyrrol id in- 1-yi, piperidino, piperazin-l-yi. or raorpholino, wherein the heterocyclic group is optionall substituted with 1 to 4 identical or different (Ci-C4 alkyi or benzyl; and
[00275] R33 and R34 are individually selected from hydrogen, (Ci~Gj)-alkyI, and (Cr
C4)-alkoxy; {00276] including pharmaceuticaliy-aceeptabie salts and pro-drugs derived therefrom.
100277] Exemplary compounds disclosed in WO 2005/034929, WO 2005/007192, WO 2004/108121 (U.S. 2005/020487), WO 2003/053997 (U.S. 2003/1 S35Q3), and WO 2003/049686 (U.S. 2003/176317) include [(7-chlot -3-hydro y-quinoline-2-carbonyl)- arainoj-acet!c acid; (l -chloro- -h /droxy-isoquii >line-3-carbonyI)-aiiiino]-aceisc acid; 4- oxo-l,4~dihydro~[1 , 10] phsnanthroline-S-carboxy lie add, [(3-hydroxy-6-isopropox>'- qiti!ioiine-2-carbonyi}-amino]-aceiic acid; [(l -bromo-4~ ydroxy-7-trifIuorGmethyl- isQquinoline-3-carbonyI)-amino]-acetic acid;•4-hydroxy-5-methoxy-[ls103p enanthroline -3- earboxyltc acid ethyl
Figure imgf000090_0001
sodium salt; 3-{[4-(3,3-dtben2yl-areidoJ~benzene5id
amiriol-N-hydroxy-propioiiamide; [(4-hydroxj'-7-phenylsultanyl-isoquinoiine-3-carbonyl)- a iiio] -acetic acid;
Figure imgf000090_0002
acid; [ ί -chlof o-44iydroxy-7-phenoxy-isoqirinoline-3-carbonyl)-amino]-acetic acid; [(1 - bionTO-4-hydroxy~7-phewxy-isoqM^^ [(4-hydroxy-7- p enoxy-isoqumolijie-3 carb0nyl)~amioo]-acetie acid; [(1 -c i].oro-4-hydK>xy-isoquinoline-3- carbonyl)-aminG]-acetiC acid; 3-carboxy-5-hydroxy^l-oxo-3,4-dIhydro- phenanthrojine 3- carboxy-S-meihoxy^-oxo-S^-dihydfo-ljl O- pfienanlhroline; 5-me hoxy-4-oxo~ls4~dihydro- [L10] phenanthro ine-S-carboxyiic acid ethyl ester; S-n¾ethoxy-4-oxo-l,4-dihydiO- [L 10]phenaRthro!ine-3-carboxy.lic acid; 3-carboxy-8-hydroxy-4-oxo~354~dihydro-iii G- phenanthroline; [(3-hydroxy-pyrid!ne-2-carbo{iyl)-aTrifflo]-acetic acid; [(3-methoxy-pyridine- 2-carbonyl)-amino]-i-cetic acid; 3~metlioxy-pyridine-2 -earboxyltc acid N-(((hexadecyioxy)- earbony -methyi)- amide hydrochloride; 3-methoxypyridine-2-carboxyltc acid N-(((l - ocryloxy)-carbonyl)-methyi)-a{riide" 3-met¾Qxypyndine~2-carboxylic acid N-(((hexyloxy)- carbonyl)-methyl)-amide; 3-metlioxypyridine-2-carboxylic acid'N-{((butyloxy)-carbonyl)- mei¾yl)-amide; 3-methoxypyridine~2-carboxylic acid N-(((2-noriyioxy)-carbonyi)~rnethyI)- amtde racemate; 3-niethoxypyridine-2-carboxyiic acid N-(((hepiyloxy)-carboi3yi)-roethyl)- amide; 3-benzyloxypyridme-2-carboxyIic acid N-(((octy]oxy)-carbonyl)-methyl)-ajriide- 3- benzylQxypyridifie-2-carboxyiic acid N~(((buiyi0xy)-carbonyl)-'methyi)-amids; 5-{((3-(i -
Figure imgf000090_0003
((ben2y,joxycaibony!)-rnethyl)--amsde; 5~(({3-(l-fautyl y)-propyl)-ammo)
methoxypyridine-2~carboxyiic acid N-(((l-bulyloxy)carbonyl)-roethyi)-iimide; 5-(((3- laisryloxy)-propyi)a in0) ar^^ acid M-(((benzy1oxy)- carbmiyi)'methy])-amide; N-((6-(l-butyloxy)-34iydroxyquinoIin-2-yl)~carboriy])-giyci.n8; [{3-hydraxy-6-tfrfiuorom^ N-((6-c'hloro-3- hydroxyqai«olin-2-yl)-carbohyl)-glycme; N-((7-ciitoro-3- ydroxyqiunGlin^2-y!)~carbonyi)- giycine; [(6-Ghioro-3-hydroxy-c{uinoIine~2-carbonyl)-amino]-aceti.c acid; N-((l-ehloiO-4- hydroxy-7-(2-propyto)^}feoquinolin-3-yl)-carbony!)-glyciae-- ; N-((l-cliloro-4-hydrGxy-6-(2- propyloxy)isoqumoItn-3-y!)-carbo:nyl}-glycine; N~((l-chioro-4-hydroxy-isoquinoltne-3- carbo:nyl}-ammo)-acerse acid N-((l-ch!oro-4 )ydroxy-7~raethoxyisoquiT}oHn-3~yl)-- carbonyi)-glycine; N-(il~ehloro-4 iydroxy~5-meth^^
N-((7-batyloxy)- --coIoro-4-hydroxyisoqiiinoiiii-3-y N-((6-ben¾'loxy-l - cliloro-4-hydroxy-isoquinoliiie-3-carbonyl)-ammo)-aeeiic acid; ((7-benzyloxy-i-chloro~4- ydroxy-isoqi»r!oiine-3-carbonyl)-amino)-acette acid methyl ester; N-((7-benz loxy-l- c 1oro-4-hydroxy-isoqt!ino3iiie~3-carbonyi)-amino)-acei!c acid; N-({8-chlO;ro-4- irydroxy isoqu inoliri~3-yl)-earbony !)-glyeme; N-((7-butoxy-4-hydroxy-isoquinciiine-3- carbooyl)-am!no)-aeetiC: acid; 6-cyclohexyl-l ~hydrGxy~4-roeftj'l- l-H-pyridm-2-one; 7-{4- methylijipetazin- 1 -ylmeft^ 4- itfo-quiripiin-8-ol; S-butoxymethyl-quinctlin-fc-ol; 3-;{{4-[3-(4-chloro-phenyl)-ureido]-ben2enesuli nyl.}-[2r(4- methoxy~p¾enyl)-ethyl]-amm^
ufejdoJ-^snzefiestilfonyl}~[2~(4-methoxy-pheriyl)-ethyl]-a^m
and. pharmaceutically acceptable salts; esters; and prodrugs thereof.
[00278] In other embodiments, PH'is are selected from quinazoUnone compounds as disclosed for example in U.S. 2010/0204226, which are represented by formula VII:
Figure imgf000091_0001
[00279] wherein:
[00280] n is 0-3;
[00281] 3 is a member independently selected from the group consisting of fraio, - Ci.4alk.yl, -Q.4alky.nyI. -CMaiken ! optionally substituted with halo, ..-GFj, -OCF3, -SCFs, S(0)CF¾ -C(0)- °s -C(0)N- -OH, -NOa, -CN, -OCi alkyl, -SCMalkyl, -S(0)~ C alkyk -S02, -C a1kyl, ~S-RC, -S(0)~Rc, -SQHlc, -SOaN-R6, -G~RC, -NR3Rfc, 2,3» dihydro-b:enzo[If43dioxme, benzo[l,3]dioxole, IH-indoie. benzyl, biphenyl optionally substituted with one or more Rd members, benzyloxy optionally substituted with one or more Rd members, phenyl or monocyclic heteroaryl optionally substituted with one or more Rd members. ~Cj.scycloalk.yl optionally substituted with one or more Rd members, -C3.
gheterocycloalkyl optionally substituted with one or more Rc members, and two adjacent R! groups may be joined to form an optionally substituted 3-8 member ring optionally containing one or more O, S or N;
100282] Ra and Rb are independently selected from the group consisting of H, C-j.. 4alkyi, -C(0)C,^alkyl, -C(0)-Rc, -C(0)NW-R? -SOs-R0, ~SQ2-CwaSkyl? phenyl optionally substituted with Rd, benzyl optionally substituted with Rd or monocyclic heteroaryl ring optionally substituted .with Rd; or
00283] Ra and b can be taken together with the nitrogen to which they are attached to form an optionally substituted monocyclic heteroeycioaikyl ring containing one or more O- S or N;
[00284] R* is a -member independently selected from the group consisting of -C3. gcycloalkyl, -Cs.ghetefocycloaikyl, biphenyl, phenyl optionally substituted with one or more Rd members, benzyl optioftally substituted with Rd, naphthyl, indanyl, 5,6,7,8-tetrahydro- naphthyl. and pyridyl optionally substituted with one or more Rd members:
[00285] Rd is a member independently selected from the group consisting of-H, halo, -OH, -Chalky!, -S02-CMaIkyl, ~CN, or-CF3, -OCF3, -OCM lk l, -C(0)N¾ -G~ phenyl, and -0-benzyl; and
f00286] enantiomers, diastereomers, raeemates, and pharmaceutically acceptable salts thereof.
[00287] Some embodiments of the compounds of formula VII have any one or more of the following:
[00288] R1 i s a member independently selected from the group consisting -of halo, - CMalkyl, -OCF¾ -CF3j -OH, -NO2, ~CN, -OC alkyl, -SCHaikyl -SiOK^alk !, - S02-CMalkyl, -S-Rc, -S(0)---Rc, -S02-Rc, -G-Rc, -NReRb, benzylo y optionally
substituted with Rd, phenyl or mouocyclic heteroaryl optionally substituted with one or more Rd members, and -C3.scyci0a.kyl optionally containing O, S or N wherein said -G3.
scycloalkyl is optionally substituted with Rd;
[00289] too adjacent R! groups are joined to form an aromatic 3-8 membered ring optionally containing one or more O, S or N: [00290] two adjacent RJ groups are joined to form an optionally substituted 3-8 raembered ring containing one or more O, S or N;
100291] n is 1, 2 or 3:
[00292] »RaRb is a member -independently selected from th group consisting of -E, ~CH3f -ClfeCHs, benzoyl, 2,6-diniethylbeozoyl, acetyl, -C(0)NH-phenyl, benzenesulfonyl, methariesu!fonyl, benzyl, 2-met.hyibenzyl, 2-chlonObenzyi,.2,6-dimethylbenzyl, 2,6- difhtorohenzyl, 2-eyanobenzy , 3'C.yanobenzy , 3-carbamoyi-benzyl, 2,6-dtc orobenzy!, 3- cklorobenzyL and 4-methyibenzy
[00293] Rs and Rb can be taken together wit the nitrogen to which they are attached to form an optionally substituted N-methytpiperazm- 1 -yl, 3,4-dihydro-iH-isoquinolin-2-yl, piperidinyl, morpholin-4-yl, and pyrro!idinyl;
[00294] Rc is a 'member independently selected from the group consisting of phenyl, cyclohexyl,.4-tert-butyi-ph.enyl, 334~diraethoxy-phenyl- 2,6-dimethy!-phenyl, 3,4,5- trimetftoxy-phenyl, naphihale.n-1- l, 3-ch.lpio~ph.enyI, 4-chioro-phenyl, 3-raethpxy~phenyl,: 4- fluoro-pheivyl, 2-fluoro-phenyl, 3-f!uoro-phenyL 3,5-dI-tert~hutyi-phenyi, 4-oxa-5~m~toly!, 4- oxo-S-o-tolyi, 2,6-dichloro-phenyl, 2,4~dichloro-phenyl, 2,5-dichIoro-phenyl, 4-ine hoxy- phenyl, 2,6-dimethyl-phenyl, naphthaien-2-y), 5,6,7,8-tetrahydro-nap thalen-l-yl, 4-chioro- phenyi, p-tolyl, indan-5-yI, 2,3-dichloro-pheny!, and pyridin~3-yi;
[00295] Rd is a .member independently selected from the group consisting of~H, cbloro, fluoro, bromo, iodo, -CMalk l, ~CF3,— QCF3> -QC walk 1, phenyl, -0-pheny.l, or ~ O-benzyi; and
[00296] R1 is independentl selected from the group consisting of chioro, fiuoro, bromo, iodo, -N02, -OH, ~CF3, -CH3, -CH2CH.¾ ~CH2CH2CH3, -OCF3, -OCR-?, - QCH2GH3, -SCH3, -SGF¾ -S(0)CF3s -S02CH3, -NH¾ -N(CH3)2s -NH(CH20¼). cyano, isopropoxy, isopropyl, sec-butyl, ierf-buiyl, ethynyl, I,-ch.ioro-vinyl, .4-memy -piperazinyl, triorphoim-4-y!, pyrroiidinyi. pyrrolidine-! -earbonyl, pipendinyl, phenyl, benzyl, bipbenyl, tolyl, phenoxy, cyc{op.rqpyIs cyclohexyl, phenyisulfanyS, 3t4-dimethp y-phenylsalfanyl, 4- tert-butyl-phenylsu!fanyl, 7-piperidinyl, 2,6-dimethyl-phenoxy, 3-,4,5-trimethQxy-phenoxy* aphthalen- 1 -yloxy, naphthalen-2-yloxy, 5f6,7,8-tetrahydrp-naphthaien-i-yfoxy5 mdan-5- yloxy, 3-chlorophenoxy, 4-ehlorophenoxy, 2,3-dichioro-plienoxy. 3-methoxy-phenaxy, 4- fluorophenoxy, 2-fiuorophenoxy, 3-fluorophenoxy, 3,5*di-tert~butyl-phenoxyt 3- rnethylphenoxy, 2,6-dichloro-phenoxyi 2,5-dich.iorophenoxy, 4-raeihoxyphenoxy, pyridih-3- ylojty, tetrahydro-pyian-4-yl 3,4-dihydrp- Ϊ H-isoquinoiin-2~yl}. ?-br0mo-3,4-dihydro-lH- feoquinolm-2 yi..3-rnethoxyphenyl-piperidinylf and benzenesulfonyl.
[00297] in still other embodiments. PHis are selected from benzp imidazole compounds as disclosed for example in U.S. 2611/0046132, which is expressly incorporated herein by reference in its entirety. In representative examples, the benzo imidazole compounds are represented by formula Vill:
Figure imgf000094_0001
{00298] wherein:
[00299] n is 2-4;
[00300] each R1 is independently selected from H, halo, -C^alkyl, -Cwjeycloaikyl-
Cwperhaloalkyl, trifluoroCMalkoxy, ·~ΟΗ, ~NC¾, ~CN,
Figure imgf000094_0002
S<GM?ik l -Re > ~S(0)2{CMalkyl)~Rc, -SCpK-Malk L -S02-C1. s1kyl! ~ $~R\ ~-S(Q)~Rc 5 - ~SO¾-N.H-Rc, -0-R*, -CH -O- *, -C(0)NH-Rc, -NRaRb, benzyloxy optionally substituted with: Rd 5 phenyl or monocyclic heteroaryl optionally substituted with Rd, -C¾. gcyeloaJkyi optionally containing one or more O, S or N wherein said -C3.gcycloaikyi is optionally substituted with and two adjacent R! groups may be joined to form an optionally substituted 3-8 member ring optionally containing one or more O. S or N;
[00301] Ra and R are each independently H, CMaiky!5 -~C(0)C3- alkyl, -C(0)~Rc, - G(0)CHr-Rs, CMalky:l-R*, .-.S02-Rc, - SOr€ ¾ , phenyl optionally substituted with Rd 5 benzyl optionally substituted with e or monocyclic heteroaryl ring optionall substituted with Rd; or
|00302] Ra and Rfa can be taken together with the nitrogen to wh ich they are attached to form an optionally substituted monocyclic heterocycloalkyl ring optionally containing one or more hete oaioms;
[00303] Rc is -Cs-scycioalkyL phenyl optionally substituted with Rd, benzyl optionally substituted with Rd, or a monocyclic heteroaryl ring optionally substituted with R*; [00304] 11ύ is independently -H, halo, »OFL -CMalk i or -C -pef aloalk l, trifluoroC alkoxy.. -OCwalkyl, -O-pheny!, or -Q-benzyi;
[00305]
Figure imgf000095_0001
containing one or more O, S orN;. R~ and R3 are both H, -CF3, or Cj-jalkyl; each Z is C or N. provided that no more that*, two Z's 5 can simultaneously be N; and
[00306] enantiomers- diastereoraers, racemates, and .pharmaceutically acceptable salts thereof.
[00307] Some embodiments of the compounds of formula VI 0 have any one or more of the following:'
I Q [00308] R2 and R3 are each -H;
[00309] Rf is independently selected from the group consisting of H, halo, ~€F;, - QCF.^ phenyl (optionally substituted or unsubstituted with up to three■-€¾ halo, -OH, Ci_ 4alkyl,
Figure imgf000095_0002
arid -OCF3), phenoxy (optionally substituted or imstibstituted with up to three halo. CM alk t, CM alkoxy, and -OCFj besnzyloxy-phenyl (optionally substituted or
15 unsubstituted with up to three halo), benzyloxy, beiizyloxymethyl, phenyisulfairyl (optionally substituted or unsubstituted with up to three -Ci^lk 'l, halo, ~CF¾ -OCFj, and -C^
^a koxy), benzy!sulfanyi (optionally substituted or unsubstituted with up to three halo,, Cj. 4alk L
Figure imgf000095_0003
-CF3, and -OCF3), phenethylsuifanyl, benzenesulforiyi
(optionally substituted or unsubstituted with up to three Ch lk !, Ct_<$aikoxy5 halo, -CFj,
20 and -OCF3), phenylrnethauesulfoiiyi (optionall substituted or unsubstituted with up to three
Figure imgf000095_0004
halo, C3.gcycloalkylmethyi, -CF3, and -OCF3). pheayi-ethanesulfonyl, benzenesi!ifiny!. cyano-hiphenyl--4-y3methyisulfanyl, cyano-biphenyI-4-y!methanesuifonyL pheny .carbamoyl, benzyicarbaraoyl, benzyiammo, phenylsulfamoy!, phenylaraino, berizoylarmno, and benzenesulfonylamino;
25 [00310] two adjacent Rf groups are joined to form an optionall substituted 3-8 mernbere ring containing one or more Q, S or N;
[00311 ] the optionally substituted 3-8 mernbered ring is aromatic: and
[00312] each R! is independently selected from FT, halo,
Figure imgf000095_0005
-CF-5, -C3. gcycloalkyl,
Figure imgf000095_0006
~M¾ 30 -NH-CMalkyl, - M-SOj-Cs-scycloalkyl, -NH-S02-C alkyl, -NH-C(03-C1.4alkyl, -CN, -CG2H, - CMalkyI, -NH-(CM2)2-morpholine,™NH(CO)CH2rmorphoiine, -NHC(0>-CH2- ptperidlne, ---MHC(0)~CH'H^"∞®thylpipera2ine), -HH-C alkyknorpholine, -S" CH?)2- morpho!ine, -C(0)- H-morpholine, pyrrolidine, piperidine, arid morpholme.
[00313] Other embodiments of PHIs are suitably selected from triazolopyridine compotmds as disclosed for example in U.S. 2011 /0077267, which are represented by formula ΪΧ, or a pharmaceutically acceptable salt thereof, or a solvate thereof:
Figure imgf000096_0001
[00315] is a grou represented by any of the following formulas :
Figure imgf000096_0002
[00316] R1 is (1) a hydrogen. atom, (2) a 0·,..¾ aikyi group. (3) a Ce-i4 ar i gr up. (4) a C3..g cycioaikyl group, (5) a C6-R aryl-C].6 alkyi. group, or (6) a C3-8 cycloalfcyl-Ci.6 alky I group;
[0031.7] R2 is {1 ) a hydrogen atom, (2) a Cwo a¾ l group, (3) a Cs.! aryl group optionally substituted by the same or different 1 to 5 substituent's selected from the followin grou B, (4) a Cj.g eycloalkyi group optionally substituted by the same or different 1 to 5 substttuents selected from the following group B. (5) a G3-g cycloalkenyi group optionally 'substituted by the same or differeiit 1 to 5 substituents selected from the following group BP (6) a heteroaryl group optionally substituted by the same or different 1 to 5 substituents selected from the following grou B (wherein the heteroaryl has. besides carbon atom, 1 to 6 heiero atoms selected from nitrogen atom, oxygen atom and sulfur atom), (7) a Ce-w aryl-C}.6 alkyl group (wherein C^ aryi is optionally substituted by .'the same or different 1 to 5 substi.tue.nts selected from the following grou B), or (8) a C>g cyc]oalk.yl-C'i,6 alkyl grou (wherein Qj.g eyeloaikyl is optionall substituted by the same or different 1 to 5 substituents selected from the following group B); and
[003181 R3 is (0 * hydrogen atom, (2) a halogen atom, (3) a C^ alkyl group,. (4) a CM* aryi group, (5) a Qj-g eyeloaikyl group, or (6) a Cs-t* aryl-Ci„6 alkyl group; and R** and R5 are each independently (1) a hydrogen atom, or (2) a Cus alkyl group, grou 8; (a) a halogen atom, (b)
Figure imgf000097_0001
a eyano group, and (e) a aio-C 1-6.alkyl groiip,
[00319] Other embodiments of Fi lls are suitably selected from the pyrimidinyl compounds as disclosed in IX S. Paten No.8324208, which are represented by foraiala XI (003201
Figure imgf000097_0002
100322] Rl and R4 are each independently selected from the group consisting of hydrogen,— NK5R6, CI -CIO alky.L C2-C10 alkenyl, C2-C10 alkynyl, C3-C8 eyeloaikyl, C3-C8 eycIoa!kyl-Cl~C10 alkyl, C5-C8 cycloalkenyL C5-C8 cyeloalkenyl-Cl-ClO alkyL C3-C8 heteroeycioalkyl, C3-C8 heterocycioalkyl-Cl-ClQ alkyl, aryi, aryl-CI-CiO alkyl, heteroaryl and heterparyI-Cl.-CI.O alkyl;
100323] R2 is— -NR7R8 or— OR9;
100324] R3 is H or C1-G4 alkyl;
100325] where R5 and R6 are each independently selected from the group consisting of hydrogen, CI -CI O alkyl C3-C8 eyeloaikyl, C3-C8 eycloa!kyl-Cl-CI 0 alkyl, C3-CS heterocyeloalkyl, C3-C8 heterocycloalkyl-Cl-CIO alkyl, aryi, aryl-CI-Cl O alkyl, heteroaryl, heteroaryl-Cl-ClO alkyl, -»-C{0)Cl-C4afky!,— C(0)C3-C6 eyeloaikyl,— C(0)C3-C6 h terocycloa!kyl,—€(G)aryl, ~~C(Q) heteroaryi and— S(0)2Cl-C4aIkyl, or, when RS and R6 are attached to the same nitrogen, 5 and R6 taken together with the nitrogen to which they are attached form a 5- or 6- or 7-mern.bered saturated ring optionally containing one other heteroatom selected from oxygen, nitrogen and sulphur,
[00326] R7 and R8 are each independently selected from the group consisting of hydrogen, Cl-Cl 0 alkyl, C2-C10 aikeny!, C2-C ! 0 aikyn L G3-C8 cycloalkyl, C3-CS hetemeycloaikyl, ar l and heteroaryl, and R9 is H or a cation, or C l-Cl 0 alkyl which is unsubstittrted or substituted with one or more substituents, suitably from 1 to 6 substi uents, suitably from. 1 to 3 substituents, independently selected from the group consisting of C3~C6 cycloalkyl* heterocycloaikyl, ar l and heteroaryl;
[00327] X is O or ¾ and
[00328] Y ls Q o S;
[00329] where any carbon or heteroatom of Rl , R2, R3, R4, R5, R S R7, R8, R9 is unsubstrtuted or, where possible, is substituted with one or more substitueuts, suitably from 1 to 6 substituents, suitably from 1 to 3 substitnents, independently selected from C1-C6 alkyl,. C1 -C6 haioalkyl, halogen,—OR 10,— NR5R6, oxo, oyano, nitro,— C(O)R105—
C(O)OR10,— SRI G S(O)R i 0,—S(0)2R1 Q9— R5R6,— CO R5R6,— N(R5)C(O)R1 , — N(R5)C{O)OR10,— OC(0)NR5R6,— K(R5)C(0)NR5R6, --S02NR5R6,—
N(R5)SO2R10, C2-C1G aikenyl, C2-C10 alkynyl, C3-C6 cycloalkyl, C3-C6
heterocycloaikyl, aiy 3, C1 -C6 alkyl-aryl, heteroaryl and C1-C6 alkyl-heteroaryl, wherein R5 and R6 are the same as defined above and R10 is selected from hydrogen, Cl-Ci Oalky!, G2~ Cl Oalkenyl, C2-C10 alkynyl,— C(G)C1-C4 alkyl,— C(0)aryl, ,— (0) heteroaryl,—
C(0)C3-C cycloalkyl,— <XO)C3-G6 heterocycloaikyl, ~S(0)2C1-C4 alkyl, C3-C8 cycloalkyl, C3-C8 heterocycloaikyl, C6-CI4 aryl, aryi-Cl-ClO alkyl, heteroaryl and heteroaryl-Cl -CI 0 alk l; and/or a pharmaceutically acceptable salt or solvate the eof.
[00330] Still other embodiments of PHIs are suitably selected from the pyridine compounds as disclosed in U.S. Patent Mo.781 1595, which is represented by formula .XII;
[00331] (XII)
Figure imgf000098_0001
[00333] wherein R and Rl are each independently: i) hydrogen; «) substituted or unsubstituted phenyl; or iii) substituted or imsubstituted heteroaryl; said substitutions being; i) C1-C4 linear, branched, or cyclic alkyl; ii) C1-.C4 linear, branched, or cyclic alkoxy; iii) CI-C4 linear, branched, or cyclic haloa!kyl; iv) halogen; v)— --CN; vi)— NHC(0)R4 vit)— G(0)NR5aRSb; or viii) heteroaryl; or ix) two substitutions are taken together to form a .fused ring having from .5 to 7 atoms;
[003341 R.4 is C1 -C4 linear, branched, or cyclic -alkyl
[00335] R5 and R5 are each independently; i) hydrogen; ii) C1-C4 linear, branched, or cyclic alkyl; or ili) R5a and R5b .are taken together to forirt a ring having from 3 to 7 atoms;
[00336] R2 is: i) -»-OR6; o rii)— NR7aR7b;
[00337] R6 is hydrogen or C1.-C4 linear, branched, or cyclic alky!;
[00338] R7a and R7b are each independently: i hydrogen; or ii) C1-C4 linear, branched, or cyclic alkyl; or iii) R?a and R7b are taken together to form a ring having from 3 to 7 ring atoms; R3 is hydrogen, methy l, of ethyl;
[00339] L is a linking unit having the formula: -[C(RSaRSb)]n-
[00340] R8a and R8b are each independently hydrogen, methyl, or ethy l;
[00341] the index n is from 1 to 3; and
[00342] R9 is hydrogen or methyl; or a pharmaceutically acceptable salt thereof; provided R and R 1 are not both hydrogen,
[00343] Other suitable embodiments of Pffls are selected from the compounds disclosed in U.S. Patent No. 7608621. which are represenetd by formula XJIi:
[00344]
[00345] (ΧΙΠ)
Figure imgf000099_0001
[00346} R1 is selected from the group consisting of hydrogen,— NR5R6, Cl- Cl 0alky C2-Cl0alkBn l, C2-C 0 alkyny!, C3-C8 eycloalkyl, C1-G 10 alkyl-C3-C8 cycloalkyl, C5-C8 cycloalken l. CI -C10 alkyi-CS-Cg eyeloalkenyl, C3-C8
heteroeyelqalkyl, Cl-CIO- alkyl-C3-C8 h^teiocydoalkyi, aryl Cl-Cl 0 aikyi-aryL heteroaryl and Cl -CI 0 alkyl-heteroaryi;
[00347] 4 is selected from the group consisting- of hydrogen, COQR9,,CQ R7R8, — NR5R.6, C1 -C30 alkyl C2-CI0 alkenyl, C2-C1Q alkynyl, C3-C8 cycloalkyl. Cl -ClO alkyl-C3-C8 cycloalkyl, C5-C8 cycloalkenyl, Cl -C 10 aik.yl-C5-C8 cycloalkenyl, C3-C8 heteroeycioaikyi, Cl-Cl 0 alkyl~C3~C8 heterocyetealkyl, aryl, C i -C! Oalkyl-aryl, heteroaryl and Cl-Cl 0 alk l-heteroary 1;
R2 is "- R7R8 or—OR9;
[00348] R3 is H or Cl-C4a!kyl;
[00349] R5 and R6 are each independently selected from the group consisting of hydrogen, C1 -C10 alky I, C3-C8 cycloalkyl, Cl-Cl 0 aIkyl-C3-C8 cycloalkyl, C3-C8 fieterocyetoalkyL Ci -CIO alkyl-C3-C8 heterocycloalky aryl, Cl -Cl 0 alky!-aryl, heteroaryl, C l -Cl 0 alkyi- etefoaryl,— CO(Cl-C4 alkyl),— CO(C3-C6 cycloalkyl),— CO(C3-C6 heterocycloalkyl),— CO(aryl), -~CO(heteroaryl), and— SG2.(C1-C4 alkyl); or R5 and R6 taken together with the nitrogen to which they are attached form a 5- or 6- or 7-membered saturated ring optionally containing one other heteroatorri selected from the group consisting of oxygen, nitrogen and sulphur:
(00350] R7 and R8 are each independently selected from the group consisting of hydrogen, Cl-CIO alk l, C2-C10 alkenyl, C2-C1Q alkynyl, C3-C8 cycloalkyl, C3-C8 heieroeycloalkyi, .-aryl and heteroaryl:
[00351] R9 is H or a cation, or Cl -C 1 Oaikyl which is unsubstituted or substituted with, one or more substittients- independently selected from the group consisting of C3-C6 .cycloalkyl, heterocycloalkyl, aryl, and heteroaryl;
[003S2] and wherein any carbon or heteroatorri of Rl , R2, R3, R4S R5, R6, R7, R8,
R9 is imsubstituted or, where possible, is- -substituted with one or more substituents independentl selected from the group consisting of C1-C6 alkyl, aryl, heteroaryl, halogen, OR I 0,— NR5R6, cyan©, nitro,— C(0)R10, -™C(0)0R10,— SR10,— S(0> 10,—
S(O)2R105— NR5R6,— CONR5R6,— N(R5)C(O)R10, --N{R5)C(O)OR10s—
OC(0} .R5R6, -- {R5)C(G)NR5R6,— SG2NR5R6,— N(R3)S02R]O; Cl-Cl 0 alkenyl,
Cl-ClO alkynyl, C3-C6■cycloalkyl, C3-C6 beterocye!oalkyL aryi and heteroaryl group; wherein RS and R6 are the same as defined above and RIO is hydrogen, Cl-Gi O alkyl C2- C10 alkenvi, C2-C 10 alkyn l,— C0(C1-G4 afkyl),— CQ(aryl),™~CQ(heteroaryi),— CO(C3~C6 cycloalkyl),— CO{C3-C6 heterGcyeioaikyl),— S02(C.l-C4 alkyl), C3-CS cycloa!kyi, C3-C8 heterocycioalkyl C6-C14 atyl, Cl -CI O -alk l-aryi, heteroaryL or C1-C10 aSkyl-heieroaryl;
or a pharmaceutically .acceptable salt or solvate thereof.
f 00353] Other FHis include without limitation substrate-based inhibitors, such as 3- e omethylenepro!ine peptide like compounds (Tandon et l (1998) Biaorg, Me Chem. Lett. 8:1 139-44), derivatives of proline, derivatives of 4(S)hydroxy proline, and derivatives of 4-keto proline. Furthermore, in view of the fact that the activity of PHD polypeptides is iron, 2-oxo glutarate and ascorbic acid dependent ( ivirikko and Pihkjaniemi (1998) T, A v Emymol Relai Areas Mol Biol 72:325-98) and the activity of HIF-a targeting PHDs such as those discussed above is also dependent on these co-factors (Bruick and McKnight (2001) Science 294(5545): 1337-40), examples of suitable compounds include cofaetor-based inhibitors such as 2-oxogkitarate analogues, ascorbic acid analogues and iron chelators such as desferoxamine (DFO) and the hypoxia mimetic cobalt chloride (CoClj), or other factors that may mimic hypoxia. Also, of interest as compounds suitable for the present invention, are prolyl hydroxylase inhibitors, such as defers prone, 2,2'~dipyridyS5 eielopirox, dimethy!oxali l glycine (DMOG), L-Miraosii e (Mlm) and 3 -Hydroxy- 1,2-dimethy 1-4(1 H)- Pyridone (OH-pyridone), DMOG is a ceil permeable, competitive inhibitor of PHDs, It acts to stabilize HIF-a expression at normal oxygen tensions in cultured cells, at concentrations' between 0,1 and 1 raM. Other PHD inhibitors encompassed by the present invention include, but are not limited to, oxoglutarates, heteroeye lie carboxamides, phenanihrolines, hydroxamates, and heterocyclic carbony 1 glycines (including, but not limited to, pyridine carboxamides, quinoline carboxamides, isoqtiirtoline carboxamides, cirmolirie carboxamides, beta-earboiuie carboxamides, including substituted qumo!ine-2-carboxamides and esters thereof; substituted isoquinoline-3-carboxamides-and N-subsiititted ary Isn!fonyiam ino hydroxar c acids (see, e.g. ,. WO 05/007192, WO 03/049686 and WO 03/053997), and the like. Also of interest are compounds described or identified using the methods described in the art, including U. S. Patent No, 6,787,326, and 6,767,705, and 6,436,654; U.S.
2004/01 61794, 2004/0152655, 2004/01.46964, 2004/0096848, 2004/0087556, 2003/022 108 and 2002/0048794; and WO 04/066949, WO 04/047852, WO 04/043359, WO 04/000328, WO 03/100438, WO 03/0851 10, WO 03/080566, WO 03/074560, WO 03/049686, WO 03/01 8014, WO 02/12326, and WO 0.2/0749:81 (each herein incorporated by reference). [003S4 Other compounds of interest encompassed by the present invention as HiF-a potentiating agents include compounds which interact or modulate the RTF- J pathway, A general report of sueh compounds and the pathways associated with HIF-Ιά levels and H1F- la activity are .-disclosed in Semenza (2003, Nature Rev. Cancer 721) RatciifFe et al (2003, Nature Medicine 677) and W outers .et al (2004, Drug Resistance Updates 25) (each of which is incorporated herein by reference in its entirety), illustrative such compounds include without limitation rapamyc in (see, e.g., Abraham (2004) Current Topics in
Microbiology and immunology 279:299-319; Arsham et al (2003) J. Biol Chem. 27.8(32), 29655-29660), curcumm see* e.g., Sukhatme, V P, WO 03/094904), fibrostatin (see, e.g., ishimaru et al (1988) J Antibiotics, 41(11): 1668-74), mimosine (see, e.g., 'Warnecke et al (2003) FASEB J. 17(9):1186- 1 18.8; Park, WO03/018014; Clement et al (2Q02) ./wiJ Cancer 10O(4):491~498), 3 hydroxy, 1.2 dimethyl 4-pyridone (see, e.g., Weldmano el al, WO 97/41 103; Weidmann et al, EP/65096 ; Iyer et al. (1998) Exp. Lung Res. 24(1): 1 1 - 32), camptothecin (see, e.g., Rapisarda et al (2002) Cancer Res. 62(15):4316-4324), resveratrol (see,,, e.g., Cao et al, (2004) Clin Cancer Res. 10(15):5253~63), Flavonoids (see, e.g., Rasebe et al (2003) Biol Pharm Bulletin 26(1 0):1379-1383: Fan et al (2003) Eur J Fharm. 4Sl(l);33-4G); Majamaa et al. (\9 ) -Eur J Biochem 138:239-245; and Majamaa et al (1985; Biochem J. 229:127-133; ivirikko and Myllyharjv (1998> ώ&¾ε Biol 16:3.57- 368: Bickel et al (1998) Hepatohgy 28:404-41 1 ; Friedman et al (2000) Proc Natl Acad Set USA 97:4736-4741 : Franklin (\99l) Biochem Soc Tram 19):812~815; and Franklin et at. (2001) B 'he hem «7353:333-338; and the like (each of which is incorporated herein by reference in its entirety), in some embodiments, the HJF-a potentiating agents include the following compounds or derivatives or analogs thereof: Quercetm, 2,4-Diethyipyridme dicarboxylat (2,4-DPD), Dimeihy loxaloy iglyci i>e (DMOG), 2~(oxaiyl-amino)-propiome acid, N-oxalyl glycine (NOG), [2,2*] Bipyridinyl, Dihydroxy benzoic acid, Pyridine 2,4- dicarboxylic acid- 4-Hydix>xy s quinoline-3-carbo'nyi glycine, and 8- tro-7-Qxo- 48,7,8,1 Ob-tetrahydro-p J jphenanthro!ine-S-carboxyiic acid.
[00355] In addition, many growth factors and cytokines are known to stabilize HiF-a under normoxic conditions, including insulin, insulin-like growth factor, epidermal growth factor, mterleukin-ΐβ (Zelzer df. (\99%) EMBO J 17:5085-94; Feidser eiei (1999) Cancer Res 59:391 5-8); Richard et. al. (20 Q0) J Biol Chem 275:26765-71 ; Goriac* et al. (2001) Ore Res 89:47-54; Haddad et al. (2001) FEBSLeti 505:269-74; Stieh.l et al (2002) FEBS Lett 512: 15-62; Thornton et, al (2000) Biochem J 350 Pt 1» 307; H llwig-Burgel et a (1999) Blood 94:1561; Saadau et al. (2.001) ?<¾/ 97: 1009; Zhou et al. (2003) Am J Physiol Celt Physiol 284:C439; Zhou et al (2003) Mol Bid Cell 14:2216; Kasurto et al, (2004) J Biol Che 279:2550) (each of which is incorporated herein by reference in its entirety). Similarly, NO and other certain reactive oxygen species are reported to stabilize HlF-1 a under normaxia (Brune & Zhou (2003) Cwr Med Chem 10(10):845-55 Palmer el al. (2000) kM Phwm ol.58: 1 1.97-203 (each of which is incorporated herein by reference in its entirety). Suitably, such compounds could be utilized as potential lead compounds, to develop additional HIF-a potentiating agents.
[00356] Representative compounds and generic structures for deriving other suitable compounds in elude those described t's the following:
[00357] Preparation of 3-hydroxypyridine-.2-carboxamides for treatment of fibrotie disease. Weidmann, Klaus; Baringhaus, Kari-hemz; Tschank, Georg; Bickei, Martin.
(Hoeehst A.-G., Germany), EP 900202 Al (which is 'incorporated herein by reference in its entirety),
|00358] Use of hypoxia-inducible factor-a (HIF-a) stabilizers for enhancing erythropotesis. Klaus, Stephen J.i Mohneaux, Christopher 1; 'Neff. Thomas B.; Guersz!er- Pukall, Voikmar; Lansetrao Parobok, ingrid; Seele , Todd W.; Stephenson, Robert C.
(Fibrogen, inc., USA). WO 2004108121 Al (which is incorporated herein by reference in its entirety).
[00359] Preparation of substituted 3-hydroxyq«inQ.line-2-carbo¾ami(ies as prolyi- - hydroxylase inhibitors. Weidmann, Klaus; Baringhaus, Karl-Heinz; Tsebank, Georg; Bickei, Martin. (Hoeehst A.-G,, Germany; Fibrogen inc.), EP 765871 Al (which is incorporated herein b reference in its entirety).
[00360] Pyridinecarboxamides and related c m pounds for treating fibrotic disease. Weidmann, Klaus; Baringhaus, Karl-Heinz; Tschank, Georg; Bickei, Martin. (Hoeehst A.~ G,s Germany), EF 673929 Al (which is mcorporaied herein by reference in its entirety),
100361} Novel inhibitors of prolyl 4- ydroxy lase. 5. The intriguing structure-activity relationships seen with 2,2>-bipyridine and its 555'-dicarbox lie acid derivatives. Hales, Neii J.; Beattie, John F. Infect. Res. Dep., Zeneca Pharrn., Macclesfield/Cheshire, UK. Journal of Medicinal Chemistry (1993), 36(24). 3853-8 (which is incorporated herein by reference in its entirety).
[00362] Beneficial effects of inhibitors of prolyl 4-hydroxylase in carbon tetrachloride-indiiced fibrosis of the liver in rats. Bickei, M.; Baader, E.; Brocks, D. G.; Enge!barf, K.; Gyenzler, V.; Schmidts, H. L.; VogeL G. H. Hoechst A--G.s Frankfurt,. Germany, journal of Hepatology (1991 ), 13(SuppI, 3), S26-S34 (which is incorporated herein by reference in its entirety),
[00363] inhibition of prolyl hydroxylase activity and collagen biosynthesis by fibrostatm C, a novel inhibitor produced by Streptomyces catenulae sisbsp, griseospora No, 23924, Ishimaru, Takenori; Kauamaru, Tswneo; Takahashi, Toshiyuki; Okazaki, Hisayoshl. Cent, Res, Div., Takeda Chera. Ind.,. Ltd., Osaka, Japan. Journal of Antibiotics (1-988), 41(1 .1), 1668-74 (which is incorporated herein by reference in its entirety),
[003641 MBP039-06 as proline hydroxylase inhibitor and its manufacture with Phaeosphaeria, Furui, Megumi; Takashima, Junko; Sudq, Keiko; Chiba, Noriko; Mikawa, Takashf, (Mitsubishi Chemical Industries Co., Ltd., Japan). JP 05239023 A2 (which is incorporated herein by reference in its entirety).
[00365] The absolute configuration of P-I894B. A potent prolyl hydroxylase inhibitor. Ohta} Razuhiko; Mizuta, Eiji; Okazaki, Hisayoshl; KishL Toyokazu. Cent. Res. Div., Takeda Chem. Ind., Ltd., Osaka, Japan, Chemical & Pharmaceutical Bulletin (1984), 3-2(11), 4350-9 (which is incorporated herein by reference in its entirety).
|00366] Preparation of Novel Cufcumin/Tetrahydrocurcumin Derivatives for Use in cosmetics, pharmaceuticals and for nutrition. Rid s, Andre; Kaehier, Markus; irchner, Ulrike; Wiggenhorn, erstin; Kinzer, Mona. (Andre Rieks-Labor fiser Enzyffitechnoiogie G.ru.b.h., Germany). WO 04/031 122 (which is incorporated herein by reference in its entirety).
[00367] Review on pharmacology in lithospermrc acid B. Peng, Zonggen; Chen, Hongshan. Department of Virology, Institute of Medicinal Biotechnology.. Chinese Academy of Medical Sciences arid Peking Union Medical College, Beijing, Peop, Rep. China.
Zhongguo Yaoxue Zazhi (Beijing, China) (2003), 38(10), 744-747 (which is incorporated herein by reference in its entirety).
[00368] Proline hydroxylase-iahibiting tetracyclines and their manufacture with Streptomyces species. Furui, Megumi; Takashima, Junko; Sudo, Keiko; Chiba, Noriko: Sashita, Reiko. (Mitsubishi Chemical Industries Co., Ltd., Japan), JP 06339395 A2 (which is incorporated herein b reference in its entirety).
[00369] A novel proline hydroxylase inhibitor BP049-33 and its manufacture with Ophioboltis. Furui, Megumi; Takashima, Junko; Mikawa, Takasht; Yoshikawa, Nobuji; Ogishi, Haruyuki. (Mitsubishi Kasei . ., Japan). iP 04074163 A2 (each herein incorporated by reference).
[00370] HIF-a contains an oxygen dependent degradation domain OD.DD), which has both an ^erminai portion (NODDD) and a C-terrninal portion (CODDD).
Hydroxy lation at any of the prolyl residues in the ODDD targets the HIF-a subunit is the vHL protein for degradation; therefore,, blocking the interaction of vHL with HIF-a. leads to build-up of HlF-a. Also, peptides encoding the HIF-a NODDD or CODDD (see, e.g.. Figure 3 of U.S. 2006/0216295, which is incorporated by reference herein in its entirety) are capable of ' up-regulating: HIF- regulated transcripts in vitro (William, ei at. (2002) Proc Natl Acad S i, USA 99(16): 10423-10428) either by saturating the PHD enzymes or vHL binding, indicating that peptide therapy may also be efficacious.
[00371] An alternative strategy is to increase HIF-α mRNA by increasin its transcription. Compounds useful in increasing HIF-ct transcription include, for example, o- substituted carbamoyl-phensxyacetic acids, as disclosed for example by Agani et at. (1998, Mel Pharmacol 54:749-754).
[00372] The invention not only encompasses known HIF-a potentiating agents but also HlF-a potentiating agents identified by any suitable screening assay. Accordingly, the present invention extends to methods of screening for modulatory agents that are useful for potentiating HlF-a and, in turn, enhancing a hematopoietic function of a mobilizer of hematopoietic stem cells and/or progenitor cells. In some embodiments, the screening methods comprise (1) contacting preparation with a test agent, wherein the preparation comprises (I) a polypeptide comprising an amino acid sequence corresponding to at least a fragment of a HlF-a-inhibitor interacting polypeptide selected from a PHD (e.g., a HIF-a PHD) polypeptide, a ΡΪΗ- 1 polypeptide, a vHL polypeptide, or a variant or derivative of any one of these); or (ii) a polynucleotide comprising at least a portion of a genetic sequence
(e.g., a transcriptional control sequence) that re ulates the expression of a gene selected from a PHD gene, a FI -I gene or a vHL gene, wherein the gen etic sequence is opera'b ly linked to a reporter gene; and (2). detecting a chang in the level or functional activity of the polypeptide, or an expression product of the reporter gene, relative to a reference level o functional activity in the absence of the test agent. A detected reduction in the level or activity of polypeptide, or expression product, relative to the reference level or functional activity indicates that the test agent is useful for enhancing a hematopoietic function of a mobilizer of hematopoietic stem cel ls and/or progen itor cells. Suitably , this is confirmed by analyzing or determining whether the test agent enhances a hematopoietic function of 'a niobilizer of hematopoietic stem cells and/or progenitor cells. In some embodiments, the test agent inhibits the prolyl hydrolase activity of the PHD,, as determined by: contacting a. preparation comprising (a) the polypeptide that comprises an amino acid sequence corresponding to at least .a fragment of a PHD or variant or derivative thereof and (fa) a substrate of the PHD wi th the test age nt and measuring whether the test agent inhibits hydroxy!ation of proline residues of the■■substrate (e.g., proline residues of HIF-a). in other embodiments, the test agent inhibits the activity of FIH-1, as determined by: contacting a preparation comprising (i) the polypeptide tha comprises an amino acid sequence corresponding to at least a fragment of a FIH- 1 or variant, or derivative thereof and (ii) a HfF- 1. polypeptide with the test agent and measuring whether the test agent enhances the transcriptional activity of the B!F-J polypeptide, in still other embodiments, the test agent inhibits the activity of vHL, as determined by: contacting a preparation comprising (A) the polypeptide that comprises an amino acid sequence corresponding t at least a fragment of a vHL or variant or derivative thereof and (B) a HIF-a polypeptide or fragment thereof with the test agent and measuring whether the test agent reduces de radation of the HIF-a polypeptide or fragment thereof. Suitably, in the above embodiments, the test agent may inhibit binding between HIF-a. and the polypeptide, as determined by: contacting a preparation comprising the HIF-a and the polypeptide with the test agent and measuring the binding of the HIF-a with the polypeptide.. In these embodiments, the test agents may bind to the HIF-a or to the polypeptide and test positive when they reduce or abrogate the binding of the HIF-s with the polypeptide.
[00373] Modulators falling -within the scope of the present invention include antagonists of the level or functional activity of HEF-a-inhibitory. interacting polypeptides (e.g., PHD FIH-1 or vHL), including antagonistic antigen-binding molecules, and inhibitor peptide fragments, antisense molecules, ribozymes, RNAi molecules and co-suppression molecules as well as polysaccharide and lipQpolysac.chari.de inhibitors ofHIF-a-inbibitoiy interacting polypeptide function.
[QQ374J Candidate agents encompass, numerous, chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 Dalton. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding,, and typically include at least an amine, carbonyl, hydroxyl or carboxy! group, desirably at least two of the functi onal chemical groups.. The candidate agent often comprises cyclical carbon or■heterocyclic structures or aromatic or polyaiOmatic structures substitoted with one or more of the above functional groups. Candidate agents are also found among biomolecul.es including, but not limited to: peptides, saccharides, tatty acids, steroids, purines, pyrimidines, derivatives, structural analogues or combinations thereof.
[00375] Small (non-peptide) molecule modulators of a HIF-a-inhibitory interacting polypeptides are particularly advantageous. In this regard, small molecules are desirable because such molecules are more readily absorbed after oral administration, have fewer potential antigenic determinants, or are more likely to cross the cell membrane than larger, protein-based pharmaceuticals. Small organic molecules may also have the ability to gain entry into an appropriate cell and affect the expression of a gene (e.g. , by interacting with the regulatory region or transcription factors involved in gene expression); or affect the activity of a gene by inhibiting or enhancing the binding of accessory molecules,
[00376] Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known, pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alfcylation, esterifkation, amidifieation, etc. to produce structural, analogues.
[00377] Screening may also b directed to known pharmacologically active compounds and chemical analogues thereof.
[00378] Screening for modulatory agents according to the invention can be achieved by any suitable method. For example, the method may include contacting a cell expressing a polynucleotide corresponding to a gene thai encodes a HlF-a-inhtbitory interacting polypeptide with an agent suspected of having the modulatory activity and screening for the modulation of the level or functional activity of the HTF-c - inhibitory interacting polypeptide, or the modulation of the level of a transcript encoded by the polynucleotide^ or the modulation of the activity or expression of a downstream cellular target of the polypeptide or of the transcript (hereafter referred to as target molecules). Detecting such modulation can be achieved utilizing techniques including, but not restricted to, ELISA, cell-based ELiSA, inhibition ELISA, Western blots, immunoprecipitation, slot or dot blot assays,
irnmunostafnmg, RiA, scintillation proximity assays, fluorescent immunoassays using antigen-binding molecule conjugates or antigen conjugates of fluorescent substances such as fluorescein or rhodamine, Ouchterlony double diffusion analysis, immunoassays employing an avidin-biotin or a sireptavidm-bibtiri detection system, and nucleic acid detection assays including reverse transcriptase polymerase chain reaction (RT-PCR).
[00379] it will be understood that a polynucleotide, from which a HIF-a-inhibitpry interacting polypeptide is regulated or expressed may be naturall occurring in the celi which is the subject of testing or it may have been introduced into the host cell for the purpose of testing. In addition, the naturally-occurring or introduced polynucleotide may be const itutiveiy expressed - thereb providing a model useful in screening for agents which down-regulate expression of an encoded product of the sequence wherein the down regulation can be at the nucleic acid or expression product level. Further, to the extent that a polynucleotide is introduced into a cell, that polynucleotide may comprise the entire coding sequence that codes for the HIF-a-inhibitory interacting polypeptide or it may comprise a portion of that coding sequence (e.g., the I igand-binding. domain of the HIF-a-inhibitory interacting polypeptide) or a portion that regulates expression of the corresponding gene that encodes the■ HIF-a-inhibitory interacting polypeptide e.g., & PHD promoter, & FI -1 promoter, or a vHL promoter). For example, the promoter that is naturally associated with the polynucleotide may be introduced into the cell that is the subject of testing. In this instance, where only the promoter is utilized, detecting dulation of the promoter activity can be achieved, for example, by operabi linking the promote to a suitable reporter polynucleotide including, but not restricted to, green fluorescent protein (GFP), fueiferase, -ga!actosidase and catecholamine acetyl transferase (CAT), Modulation of expression ma be determined by measuring the activity associated with the reporter polynucleotide.
[00380] These methods provide a mechanism for performing high throughput screening of putative modulatory agents such as prole inaceous or non-proteinaceotis agents comprising synthetic, combinatorial, chemical and natural libraries. These methods will also facilitate the detection of agents which bind either the polynucleotide encoding the target molecule or which modulate the expression of an upstream molecule, which subsequently modulates the expression of the polynucleotide encoding the target molecule. Accordingly, these methods provide a mechanism of detecting agents that either directly or indirectly modulate the expression or activity of a target molecule according to the invention.
[00381] In specific embodiments, compounds are screened for hydroxylase activity.
Assays for hydroxylase activity are standard in the art. Such assays can directly or indirectly measure hydroxylase activity. For example, an assay can measure hydroxyiaied residues (e.g.. proline, eic.) present in the enzyme substrate, e.g.., a target protein, a synthetic peptide mimetic, or a fragment thereof (se , e.g. , Palmer ini ef al. (3985) J Chm atogr 339:285- 292.) A reduction in hydroxy lated residue (e.g. , proline, etc.) in the presence of a compound is indicative of a compound that inhibits hydroxylase activity. Alternatively, assays can measure other products of the hydroxylation reaction (e.g., formation of succinate from. 2- oxoglutarate (see, e.g,T Cun!iffe ei l. (}9 ) Biockem 7240:61 7-619; and auie and Gunzter (1 90) Anal Biac em 184:2 1 -297),
[00382] Procedures such as those described above can be used to identify eompoimds that inhibit HIP hydroxylase activity. Target protein used in the assa may include HIFa or a fragment thereof, e.g., HIF(556-575). Enzyme ma include, e.g., HIF prolyl hydroxylase (see, e.g., GenBank Accession No, AAG33965, etc.) obtained from arty source. Human HIF prolyl hydroxylase is preferred. Enz me may also be present in a crude cell lysate or in a partially purified form. For example, procedures that measure HIF hydroxylase activity are described in Ivan et al (2001. Science 292:464-468; and 2002, Froc Natl Acad Sci USA 99:1.3459- 13464) and Hirs !a et l. (2003, JBM CHem 2.78.:30772- 30780); additional methods are described in international Publication No. WO 03/049686. Measuring and comparing enzyme activity in the absence and presence of the compound will identify compounds that inhibit hydroxylation of HIF-ct,
[00383] In■certain aspects, a. suitable compound is one that stabilizes HlF-a.
Compounds that inhibit HIF prolyl hydroxylase prevent or reduce the hydroxylation of the HIF subunit of the HIF protein. This lack of hydroxy iated proline leads to the stabilization (often referred to as activation) of HIF, Determination of the stabilization of HIF by a compound can be used as an indirect measure of the ability of the compound to inhibit HIF prolyl hydroxylase. The ability of a compound to stabilize or activate HTF-a can be measured, for example, b direct measurement of HIF-1 ct in a sample, indirect measurement of HIF- l a, e.g. , by measuring a decrease in HIF- 1 a associated with the vHL protein (see,, e.g., International Publication No. WO 200Q/699Q8), or activation of HIF responsive target genes or reporter constructs (see, e.g., U.S. Patent No. 5,942,434). Measuring and comparing levels of HIF and/or HIF-responsive target proteins in the absence and presence of the compound will identif eompoimds that stabilize .HIF-1 a and/or activate HIF. Suitable compounds for use in the present methods may be identified and characterized using the assay described in International Publication No, WO 2005/1 18836, or in Example 10 of International Publication I¾Q. WO 2003/049686, both of which are incorporated herein by reference in their entirety. Compounds identifiable by these assays are specifically envisaged for use in the present invention. [00384] In alternative embodiments, test agents are screened using the assays disclosed for example in U.S. 2004/0146964, U.S. 2005/0214894, U.S. 2008/0213404, U.S. 2010/0272726 and U.S. 201 103:0.10.95, each of which are .incorporated by reference herein in their entirety.
[00385] Compounds may be further tested in the animal models to identify those compound having the most potent in vivo effects. These molecules may serve as 'iead compounds" for the further development of pharmaceuticals by, for example, subjecting the compounds to sequential modifications, molecular modeling, and other routine procedures employed in rational drug design.
3 ,2 MobiHzsrs of hematopoietic stem cells and/or progenitor cells
f 00386] Several classes of agents have been shown to increase the circulation of progenitor and stem cells by "mobilizing" them from the marrow into the peripheral blood. These include agents that decrease the expression or function of a chemokine (the function being the binding of the chemokine to its receptor and further signaling), particularly
CXCL12, as well as those that block or antagonize the chemokine receptor, CXCR4.
[00387] Accordingly, in some embodiments, the mobilization agent ma be an agent that decreases the expression or function of a chemokine, more particularly, CXCL12, also known as SDF-L The human amino acid sequence of SDF-1 corresponds to GenBank accession number NP_000600. The alpha isoform has GenBank accession number
P 954637. The beta isoform has GenBank accession number P_000600, The gamma isoform. has GenBank accession number P_001029058.
[00388] Alternatively, the mobilization agent may be an agent that blocks or antagonizes a chemokine receptor, in particular, CXC 4. The human amino acid sequence of CXCR4 corresponds to GenBank accession number CAA 12166.
[00389] Chemokines are a superfamily of chemoattractant proteins. Chemokines regulate a variety of biological responses and they promote the recruitment of multiple lineages of leukocytes and lymphocytes to a body organ tissue. Chemokines may be classified into two families according to the relative position of the first two cysteine residues in the protein. In one family, the first two cysteines are separated b one amino acid residue, the CXC chemokines, and in the other family the. first two cysteines are adjacent, the CC chemokines. Two minor subgroups contain only one of the two cysteines (G) or have three amino acids between the cysteines (CX3C). In. humans, the genes of the CXC ehemokin s are clustered on chromosome 4 (with the exception of SDF-l gene, which has been localized to chromosome 10) and those of the CG ehemokines on chromosome 17.
[00390] The molecular targets for ehemokines are cell surface receptors. One such receptor is CXC ehemokine receptor 4 (CXCR4), which is a 7 transmembrane protein, coupled to. G 1 and was previously called LESTR {Loetscher, M., Geiser, T., O'Reilly, T,, Zwahlen, R., Bagg nlmi, M., and Moser, B., (1994) J, Biol. Chem, 269, 232-237), HUMSTR (Federsppiel, B., Duncan, A. M. V.s Deianey, A., Schappert, K,, Clark-Lewis, L and Jirik, F. R. (1993) Genomics 16, 707-71 ) and Fusin (Feng, Y,, Breeder, C. C,;
Kennedy, P. an Berger, E, A. (1996) HIV- 3 entry cofaetor; Functional cDN A cloning of a seven-transmerabrane G protein-coupled receptor, Science 272, 872-877). CXCR4 is widely expressed on cells of hematopoietic origin, and is a major co-receptor with CD4 fo human immunodeficiency virus 1 (HiV-1) (Feng, Y., Broeder, C. C, Kennedy, P. E., and Befger, E. A. (1996) HIV- 1 entry cofaetor: Functional eDN A cloning of a seven- transmembiane Q protein-coupled receptor. Science 272,. 872-877).
[003911 Ghemokines are thought to mediate their effect by binding to seven transmembrane G protein-coupled receptors, and to attract leukocyte subsets to sites of inflammation (Baglionmi et at (1998) Nature 392; S65-56S). Many of the ehem okines have been, shown to be constitutivejy expressed in lymphoid tissues, indicating that they may have a homeostatic function in regulating lymphocyte trafficking between and within lymphoid organs (Kim and Broxmeyer (1999) J. Leuk. Biol 56: 6-15).
[00392] Stromal ceil derived factor one (SDF-l), also known as CXCL12 is a member of the CXC family of ehemokines that has been found to be constitutively secreted from the bone marrow stroma (TasMro, (1993) Science 261 , 600-602). The human and mouse SDF-l predicted protein sequences are approximately 92% identical. Stromal cell derived factor- la (SDF- la) and stromal cell derived faetor-l p (SDF-l β) are closely related (together referred to herein as SDF-l). The native amino acid sequences of SDF-l a and SDF-l β are known, as are the genomic sequences encoding these proteins (see U.S. Pat. No. 5,563,048 issued 8 Oct. 1996, and U.S. Pat. No. 5,756,084 issued 26 May 1998).
Identification of genomic clones has shown that th alph and beta isofbrms are a consequence of alternative splicing of a single gene. The alpha form is derived from exons I- 3 while the beta form contains an additional sequence from exon 4. The entire human gene is approximately 10 kb. SDF-l was initially characterized as a pre-B cell-stimulating factor and
- Π 0- as highly efficient ehemotaetie factor for B cells and monocytes (Bieul el at (1996) J. Exp. Med. 184: 1 101 -1110).
[00393] Biological effects of SDF-1 may be mediated by the chemokine receptor CXCR4 (also known as fwsin or LESTR). which is expressed on mononuclear leukocytes including hematopoietic stem cells. SDF- 1 is thought to be the natural ligand for CXCR4, and CXCR4 is thought to be the natural receptor for SDF-1 -(Nagasawza et at (1997) Proc. Natl Acad. Scl USA 93 :726-732), Genetic eltrainatjon of SDF-1 is associated with perinatal lethality, including abnormalities in cardiac development, B-eeil lymphopoiesis, and bone marrow myeiopoies is {Nagasawa el: at (1996) Nature 382:635-637). SDF-1 is functionally distinct from other chemokines in that it is reported to have a fundamental roie in the traffickmg, export and homing of bone marrow progenitor cells (Aiuri, A., et at (1 96) X Exp. Med, 1 85, 1 1 1-120 and Nagasawa, T., et al (1996) Nature 382, 635-638). SDF-1 is also structurally distinct in that it has only about 22% am ino acid sequence identity with other CXC chemokines.
[00394] Agents that decrease the expression of CXCL 12 or that block or antagonize
CXCR4 may be selected from small organic molecules, poly peptides, nucleic acids and carbohydrates. In more particular embodiments, the polypeptides that decrease the expression of CXCL 1.2 may be selected from the group consisting of a cytokine, a colony stimulating factor, a protease or & chemokine other than CXCL 12, The cytokine ma be selected from the group consisting of interleukin-l (IL-1), interleakin-3 (IL-3), interfeukfn-6 (IL-6), interieukin-l 1 (IL-'i l), interleukin-7 (IL-7) and interteukln-12 (I.L12). The protease ma be selected from the group consisting of a metal loproteinase (like MMP2 or MM.P9) a serine protease, (like cathepsin G, or elasta.se) a cysteine protease (like cathepsin K) and a dipeptidy! peptidase- 1 (DDP-1 OR CD26), The chemokine other than CXCL 12 may be selected from the group consisting of IL-8, MIP-1 a and Gro$, The colony stimulating- factor may be selected from the group consisting of granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony
stimulating factor (M-CSF), stem cell factor, FLX-3 ligand or a combination thereof. The nucleic acid may be a DNA or an RNA molecule. The nucleic acid may be a small interfering RNA (siRNA) molecule or an antisense molecule specific for CXCL 12 or
CXCR-4. The carbohydrate may be a sulfated carbohydrate selected from the group consisting of Fucoidan and sulfated dextran.
- I l l - [00395] Suitably, the mo Uizer(s) is(are) are selected from colony-stimulating factors such as G-CSF and GM-CSF. erythropoietin (which is now commonly used among cancer patients undergoing chemotherapy to maintain hemoglobin in the near normal range, also has some abilit to mobilize CD34* cells), stem cell factor (SCF), polysaccharides such as zymosan, chemokiiies such as IL-8 and Gro-β, growth factors such as vascular endothelial growth factor (VEGF), and CX.C 4 antagonists.
[00396} Jn some embodiments, the mobilizer or at least one of the mobiiizers used in combination wit a HlF-a potentiating agent is G-CSF or GM-CSF, or their variants, derivatives or analogs. The nucleic acid sequence and encoded amino acid sequence of G- CSF, as■well as chemically synthesized polypeptides sharing its biochemical and immunological properties, have been previousl disclosed (U.S. Pat. Nos. 6,379,661 ;
6,004,548; 6,830,705; 5,676,941 , 6,027,720; 5,994,518; 5,795,968; 5,214,1 2; 5,218,092; 6,261 ,550; 4,810,643; 4,810,321 , each of which is incorporated herein by reference in its entirety). Also encompassed are analogs of G-CSF molecules which retain their three- dimensional structures and hybrid molecules maintaining their biological and structural integrity, as described for example by Qssiund (U.S. Patent No. 6,261,550, incorporated herein by reference). Examples of functional G-CSF variants include any proteins, peptides or fragments thereof that are at least 70, 75, 80, 85, 90 or 95% sequence identity or similarity to full-length human G-CSF amino acid sequence or its nucleotide sequence. Modifications of G-CSF to improve functionality or resident serum clearance include .but are not limited to poly ethylenegly col and poly ethyl enegly col derivatives thereof, glycosylated forms
(Lenogastrtm™) (WO 2000/44785, incorporated herein by reference), norleucine analogs (U.S. Pat. No. 5,599,690, incorporated herein by reference), addition of amino acids at either terminus to improve folding, stability or targeting, and fusion proteins, such as G-CSF and albumin fusion protein (Albugra in™) (U.S. Pat No. 6,261,250, incorporated herein by reference). An increase in biological or functional activity over the native peptide may reduce the amount of dose and/or the time period required for treatment. Any chemical or biological entity that functions similar to G-CSF can also be employed. G-CSF, or the drug name Filgrastim, is currently bein sold as Neupogen® and its polyethylene glycol modified or pegylated form, with the drug name Pegfilgrastim, sold as Neulasta™.
(00397] The coding sequence and amino acid sequence of GM-CSF are known as well as var ious methods employed to produce recombinant proteins (U.S. Pat. No. 5,641,663, incorporated herein by reference). Examples of functional GM-CSF variants include any proteins, peptides or fragments thereof that are at least 70, 75, 80, 85, 90 or 95% sequence identity or sim larity to full- length human GM-CSF amino a id sequence or its coding sequence. Modifieations of GM-CSF to i mprove functionality or resident serum clearance include but are not limited to pqlyethyleneglycol and polyethyleneglycol derivatives thereof, glycosylated forms, norleucine analogs, addition of amino acids at either terminus to improve folding, stability or targeting, and fusion proteins. An increase in biological or functional activity over the native peptide may reduce the amount of dose■ and/or the time period required for treatment. Any chemical or biological entity thai functions similar to GM-CSF can also be employed. Examples of GM-CSF, or the drug name Sargramostim, which are currentl being sold, include Leukine®, Leucoinax.® and Leucotropin®.
[00398] In specific embodiments, G-CSF, or a variant, derivative or analog thereof, is used either alone or in combination with another mobilizer of HSPCs For concurrent administration with a HIF-a potentiating agent, in illustrative examples of this type, the HTF- potentiating agent is a PHI (e.g., a small molecule PHI including ones selected from compounds represented by any one of formulae J- IX supra).
[00399] in some embodiments, the mobilizer or at least one of the rnobilizers used in combination with a HIF-a potentiating agent is a CXCR4 antagonist, illustrative CXCR4 antagonist's include aromatic-linked polyamine macrocyclic compounds, as described for example in U.S. Patent No. 5,583, 131, reissued as U.S. RE 42,152, which is expressly incorporated herein by reference in its entirety. In one aspect, the CXCR4 antagonist is Ι,Γ- [ 1 ,4~phenyknebis(methy lene)]-bis~ 1 ,4,8, 1 1 -tetra-azacyclotetradecane (A DS 100;
Pierixafor; MoKobil®),
[00400] In other embodiments, small molecule CXCR4 antagonists may be selected from macrocyclic compounds disclosed in U.S. Pat Appl. Pub.. No. 2012/0301427, which is expressly incorporated herein by reference in its entirety. These compounds comprise a "core" nitrogen atom surrounded by three pendant groups, wherein two of the three pendant groups are suitably henzimidazoiyi methyl and tetTahydroquinolmyl, and the third is a pendant group contains an additional nitrogen.
[00401] Still other embodiments of small molecule CXCR4 antagonists include compounds disclosed in U.S. Pat. Appl. Pub. No. 2012/0101280, which is expressly incorporated herein by reference in its entirety.
[00402] Irs other embodiments, CXC 4 antagonists are selected from p-hair in peptidomimeiics as disclosed for example in U.S. Pat. Appl. Pub, No. 2012/0283196, which is expressly incorporated herein b reference in its entirely. [00403] in specific embodiments, a CXCR4 antagonist as described in the foregoing patents and patent applications, for example, P!erixafor, is used either alone or in combination with another mobi!izer of HSPCs for concurrent administration with a HIF-a potentiating agent. In Illustrative examples of this type, the HIF-a potentiating agent is a PHI,
[00404) Suitably, at least two different mobilizers are used for concurrent administration with the HIF-a potentiating agent. In specific embodiments, the at feast two mobilizers comprise a CXC 4 antagonist, and a colony stimulating factor such as G-CSF or GM-CSF, or variants, derivatives or analogs thereof. In illustrative examples of this type, the CXCR4 antagonist is Plerixafor, or similar compounds, and the colony stimulating factor is G-CSF or a variant, derivative or analog thereof. In these embodiments, the HIF-a potentiating agent is suitably a PHI (e.g., a small molecule PHI including, but not limited to, ones selected from compounds according to any one of formulae MX).
, Therapeutic and Prophylactic Use
[0040S] In accordance with the present invention, it is proposed that HIF-a potentiating agents are useful as actives for enhancing the hematopoietic properties of mobilizers of hematopoietic stem cells and/or progenitor ceils. Thus, a HIF-a potentiating agent can be administered to an individual concurrently (e.g., in the same composition or in separate compositions) with at least one mobilize.*- ("combination treatmenf ), and optionally with a pharmaceutically acceptable carrier, to stimulate or enhance hematopoiests including the mobilization of hematopoietic stem cells and/or progenitor cells, including
granulocytes/macrophage progenitors and/or megakaryocyte/erythrocyte progenitors, fro the bone marrow, and more particularly to increase the number of hematopoietic stem cells, progenitor cells and .granulocytes such as neutrophils in a patient, particularly in the peripheral blood,
[00406] In one aspect, the present invention thus provides a method for mobilizing hematopoietic stern cells and/or progenitor sells from bone marrow into peripheral blood of a dono subject, the method comprising, administering to the subject a HIF-a potentiating agent in an effective amount. to mobilize hematopoietic stem cells and/or progenitor cells from the bone marrow into the peripheral blood of the subject. A suitable donor subject in this embodiment is one that has been, is, or will be administered a raobilizer.
[00407] in another aspect, the present invention provides a method for mobilizing hematopoietic stem ceils and/or progenitor cells from bone marrow into peripheral blood of donor subject, the method comprising, consisting or consisting essentially of: administering concurrently to the subject a HIF-α potentiating agent and at least one mobiiizerof ' hematopoietic stem cells and/or progenitor cells in effective amounts to mobilise hematopoietic stem celts and/or progenitor cells from the bone marrow into the peripheral blood of the subject,
[00408] The above methods may further comprise collecting or harvesting mobilized hematopoietic stem cells and/or progenitor cells from the subject, and optionally eultoring and or storing the collected or harvested mobilized hematopoietic stem cells and/or progenitor cells, and further optionally transplanting the collected or harvested mobilized hematopoietic stem ceils and/or progenitor ceils into a recipient subject.
[00409] T he dosages of HI F-ct potentiatin agent and the at least one mobilizer to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof The dosages will also tak into consideration the binding affinity or modulatory activity of the HIF-a potentiating agent to its target molecule, the hematopoietic capaci ty of the mobifizer s)> their bioavailability and their in vivo and pharmacokinetic properties. In this regard, precise amounts of the agents for administration can also depend on the judgment of the practitioner. In determining the effective amount of the agents to be administered in the treatment of an immunocompromised condition, the physicia or veterinarian may evaluate the progression of the disease or condition over time. In any event, those of skill in the art may readily determine suitable dosages of the agents of the invention without undue experimentation. The dosage of the active agents administered to a patient should be sufficient to effect a beneficial response in the patient over time such as enhanced hematopoiesis or a reduction, in the symptoms associated with an
immunocompromised condition, including a reduction in anemia, thrombocytopenia, agranulocytosis and/or neutropenia. The dosages may be administered at suitable intervals to boost hematopoiesis or ameliorating the symptoms of the immunocompromised condition. Such intervals can be ascertained using routine procedures known to persons of skill in the art and can vary depending on the type of active agent employed and its formulation. For example, the interval may be daily, every other day, weekly, fortnightly, monthly, bimonthly, quarterly, half-yearly or yearly.
[004103 Dosage amount and interval may be adjusted individually to provide plasma levels of the active agent which are sufficient to maintain HIF-a potentiating agent modulatory effects and hematopoietic functio enhancing effects. Usual patient dosages for systemic administration range from 1 -2000 mg/day, 10-1000 mg day, 50-500 mg/day, 100- 800 mg day, commonly from 1 -250 rag/day, and typically from 10-150 mg/day. Stated in terms of patient body weight, usual dosages range from 0,02-25 mjg%g/day, 0.5-15 mg/kg/day, 1.0-10 mg/kg/day, 1-5 mg kg/day, commonly from 0.02-3 mg/kg day, typically firora 0.2-1.5 mg kg/day. Stated in terms of patient body surface areas, usual dosages range from 9-5-1200 mg/nr/day, 10-800 mg nr/day. 50-500 mg/nr/day, 75-200 mg/m*Vday commonly From 0.5- 150 mg/nr/day, typically from 5-100 mg m2/day, Animal testing of ' effective doses for treatment of particular disorders will provide .further predictive indication of human dosage. Various considerations are described, e.g.,. in Oilman et a!., (eds.) (1990) Goodman and Oilman's: The Pharmacological Bases of Therapeutics, gfli Ed., Pergamon Press, and Remington's Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co., Baston, Pa, Methods for administration are discussed therein, e.g., for oral, intravenous, intraperitoneal, or intramuscular administration, transdermal diffusion, and others.
Pharmaceutically .'acceptable -carriers will generally include water, saline, buffers, and other compounds described, e.g., in the Merck index, Merck & Co.. Railway, NJ.
[004.11] "T hus, the HIF-a potentiating agent and the mobilizer(s) may be provided in effective amounts to stimulate or enhance hematopoiesis. This process may involve administering the. HIF-a potentiating agent separately, simultaneously or sequentially with the mobilizer(s). In some embodiments, this may be achieved by administering a single composition or pharmacological formulation that includes both types of agent, or by administering two or more sepajaie compositions or formulations at the sasne time, wherein one composition includes the HIF-a potentiating agent and the other(s), the mobilizer(s). It will be appreciated that if more than one raobiiizer is used, the mobilizers may' be administered separately, simultaneously or sequentially,
[004121 In other embodiments, the treatment with the HIF-a potentiating agent may precede or follow the treatment with the mobilfeer(s) by Intervals .ranging from minutes to days. In. embodiments where the HIF-a potentiating agent is applied separately to the mobUizer(s), one would generally ensure that a significant period of time did not expire between, the time of each delivery, such that the HIF-a potentiating agent would still be able to exert an advantageously combined effect on hematopoiesis with the mobilizer(s), in particular, to' maintain or enhance a subject's capacity to mobilize hematopoietic stem cells and/or progenitor cells inc luding an increase in the number of granulocy tes sueh as neutrophils. In such instances, it is contemplated that one would administer both modalities within about 1-12 hours of each other and, more, suitably, within about 2-6 hours of eac other. In some situations, it ma be desirable to extend the time period, for treatment. Significantly, however, where several hours (2, 3, 4, 5, 6 or 7) to several days .(1, 2, 3, 4f 5. 6, 7 or 8) lapse between the respective administrations.
[G0413| it is conceivable that more than one administration of either the HlF-e potentiating agent or mofaihzer(s) will be desired.. Various combinations may be employed, where the HTP-ft potentiating agent is "A" and the mobilizer(s) is(are) "B", as exemplified below:
00414] A B/A B/A B B/B/A/ A/A./B/ B/A./A A/B/B B/B/B/A B/B/A/B A/A B B A/B/A/B A B B A B/B A A B/A B/A B/A/A B B/B B/A A/A/ ® B/A/A/A A B/A/ A/A/B/A A/B/B/B B/A B B B/B/A/B, Alternately, A B/A B A B/A B, etc.: B B/A/ A/B/ B/A/A B/B . etc.; B/B/B/A B/B/B/A/B/B/B, etc, A/A B/B ..A B A B/ A, etc. B B/A B B/A A B/B/A/A, etc. B/A BB/A/A/B B B/A/ A/A, etc.; and A/A/A/B/A/B/A/A /A/B/A/B, etc.Giher combinations are contemplated. Again, both .'agents are delivered to a subject's immune system in a combined amount effective to enhance hematopoiesis as compared to the adm inistration of the same amount of mobiiizer alone.
[00415) The HIF-ct potentiating agent and the mobilizer(s) may be administered directly to a subject or it may b desirable to conjiigate one or both to carrier proteins such as ovalbumin or serum albumin prior to administration. While it is possible for the active agent to be admi istered alone, it is generally desirable to present it as a pharmaceutical composition. Such compositions typically comprise at least one active agent or ingredient, together with one or more acceptable carriers thereof. Each carrier should be both
pharmaceutically and. physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient. Compositions include those suitable for oral, rectal, nasal, topical, -or -parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The compositions may conveniently be presented in unit dosage form and may be prepared by many methods we'll known in the art of pharmacy. See, e.g., Oilman* et al (eds.) (1990) Goodman and Oilman's; The Pharmacological Bases -of Therapeutics, 8th Ed.s Pergamon Press; and Remingtoivs Pharmaceutical Sciences., 17th ed. (1 90), Mack Publishing Co., Easton, Pa.;. Avis, et l. (eds.) (1993) Pharmaceutical Dosage Forms, Parenteral Medications Dekker, N.Y.; Lieberman, et at (eds.) (1990) Pharmaceutical Dosage Forms; Tablets Dekker, Ν,Υ,; and Lieberman, et al. (eds.) (1 90) Pharmaceutical Dosage Forms: Disperse Systems Dekker, N. Y. The methods of the invention may be combined with or used in association with other therapeutic agents. [00416] The methods and uses of the present Invention are useful for mobilizing HSPCs in subjects. The subjects may have an immunocompromised condition or have an increased risk of acquiring an imraimoeomprormsed condition, which may result for example from a congenital disorder (e.g. , congenital leukopenia), childhood or adult
cyclic neutropenia, post-infective neutropenia, and myelodys lasia syndrome or a medical treatment such as induced by treatment with cytoreductive, tnyeioab!ative or
immunosuppressive therapies (e.g. , chemotherapy, radiation therapy and imniunosuppressi ve drugs such as steroid), in particular in relation to the treatment of transplant rejection and the treatment of hyperpro!iferative cell disorders such as cancer and autoimmune disease.
Alternatively, the subjects are individuals who may serve as allogeneic, syngeneic or xenogeneic donors of HSPCs and the treatment is used to mobilize and collect HSPCs for subsequent delivery to a recipient who has an immunocompromised condition or has an increased risk of acquiring an immunocompromised condition. In addition, the treatment can be used for patients or donors who are "difficult to mobilise" because, for example, they are not sensitive to gro wth factors. The treatment can further be used to cause tolerance of a recipient for organ transplantation.
[0043 ?j The treatment can also be used in cancer therapy methods and in methods for Inhibiting, ameliorating,, or -ablation of cancer ceils and/or tumors. For example, like normal HSPCs, their malignant counterparts, leukemia initiating cells (LICs) reside in their bone marrow (BM) niches that provide the structural and physiologic l conditions supporting their survi val and growth, LICs are resistant to traditional cancer therapy e.g, cytoreductive or myeloablative therapy.) by interacting with their BM microenvironment, which can be the culprits of leukemia relapses after a period of remission induced by a cancer therapy (e.g. , cytoreductive or myeloablative therapy). Detachment of LTCs from their niche b inducing mobilization of LICs (e.g., by administering the treatment) can be a used in combination with traditional cancer therapies (e,g., eytoreduciive or myeloablative therapies) to provide more effective or improved cancer therapy methods and methods for inhibiting, ameliorating, or ablation of cancer cells and/or tumors,
[00418] The treatment can additionally be used for gene therapy. Because
pluripotent hematopoietic stem cells are self-renewing, and give rise to cell progenitors as well as mature blood eeiis, the stem ceils are an appropriate target for gene therapy.
After mobilization, HSPCs can be collected. The HSPCs can be modified to deliver gene products upon reinrroduction to the individual. After modification, the cells are reimused into the affected individual, [00419] In some embodiments, the treatment is administered to a patient to stimulate or enhance mobilization of HSPCs from the bone marrow into the peripheral blood and the mobilized HSPCs are then collected or harvested from the patient. Blood containing mobilized HSPC may be collected from the donor by means well known in the art. In a typical protocol, the mobilized ceils are collected from the donor by. for
example, apheresis and then stQred/eulfured/expande&'fraeuonated as desired. In order to ensure capture of a re-populating quantity of cells, it is generally desirabl to collect the donor' s blood when the levels of mobilized HSPCs peak, in order to optimize the number of HSPCs harvested from mobilised blood, the levels HSPCs can be monitored by methods well known to those of skill in the art, and collection timed to coincide with HSPC peaks.
[00420] If desired, the donor ceils can be enriched ex vivo by treating them with factors that stimulate the TNFa and GM-CSF receptors. Alternatively, or in addition, factors that stimulate FLT3 and the G-CSF receptor, such as FL and G-CSF, may be used. More particularly, hematopoietic tissues such as bone marrow and blood can be harvested from a donor by methods well' known to those skilled in the art, and treated with NFa, GM-CSF, 'FL, SCF, IL-7, IL-12, and G-CSF, either singularly or in combination, to enrich selectively for HSPCs,
[00421] The cells harvested from the donor are typically cultured ex vivo for several days in medium supplemented with TNFa, GM-CSF, FL, SCF, IL-7, 1L-I 2, and G-CSF, either singularly or in -combination. The concentration of GM-CSF administered would typically be in the range of 1,000 U/m'L. in an alternative embodiment, TNFa may be administered, typically at a concentration of 200 U niL. Appropriate concentrations of G- CSF, SCF, IL-7, IL-12, and FL can be readi ly determined by those of skill in the art, as by titration experiments or by reference to the working examples provided herein.
[00422] In some applications, it may be desirable to treat the cultured cells to remove graft versus host disease (GVHD) causing cells, using routine methods known in the art, as for example discussed below. The enriched HSPCs may then be selectively collected from the culture using techniques known to those of skill in the art, as for example discussed below.
[00423] In order to ensure enrichment of HSPCs t a repopulating quantity, it is generally desirable to collect the cultured cells when the levels of HSPCs peak. As with in vivo mobilization, ex vivo enrichment of cultured hematopoietic cell produces peak levels of HSPCs on different days depending on the cytokine administration protocol used. In order to
- 11.9 - optimize tile number of HSPGs collected from cultured cells, the levels of HSPCs ears be monitored by methods well/known to those of skill in the art, and collection timed to coincide with HSPCs peaks.
[00424] Following collection, the HSPCs can be resuspended, stored, expanded and/or fractionated and administered to a recipient The recipient may be the original donor and thus the administration of HSPCs is for an autologous stem cell transplantation.
Alternatively, the recipient is not the donor and the administration of HSPCs is for an allogeneic syngeneic or xenogeneic stem ceil transplantation.. In some erabodirnents. the allogenic or xenogenic stem cells are transplanted after the receipient undergoes a non- rayeloablative conditioning regimen (a "mini-aliogentc" or "niini-xenogenic'' stem cell transplantation).
{00425] Once the HSPCs have been mobilized into a subject's peripheral blood or enriched i the cultured cells, they may be used as donor ceils in the form of total white blood ceils or peripheral blood mononuclear cells, or selectively enriched by various methods which utilize specific antibodies which suitably bind specific markers to select those cells possessing or lacking various markers. These techniques may include, for example, flow cytometry using a fluorescence activated cell sorter (FACS) and specific fiuorochromes, biotin-avidm or biotin-streptavidin separations using biotin conjugated to cell surface marker-specific antibodies and avidin or streptavidio bound to solid support such as affinity column matrix or plastic surfaces, m gnetic separations using antibody-coated magnetic beads, destructive separations such as antibod and complement or antibody bound to cytotoxins or radioactive isotopes.
(00426] If the mobilized blood is used for an autologous transplant, the peripheral blood mononuclear cells (PBMC) may be re-infused into the patient without modifications, with the exception that in the case of a cance patient, the ceil preparation is generally first purged of tumor cells. In contrast, if the mobilized blood is transferred into an allogeneic or xenogeneic recipient, the PBMC may first be depleted of GHVD-produciiig cells, leaving the HSPCs enriched in the PBMC population. In that connection, the PBMC may be treated with aiiti-o TC and anti»-y8TC. antibodies to deplete T cells, anti-CD 19 to deplete B cells and anti-CD56 to deplete N cells. It is important to note that anti-Thy-1 antibodies should not be used to deplete OVHD producing cells, as they would deplete T cells and HSPCs.
Therefore, it is important to choose carefully the appropriate markers as tar gets for selecting the cells of interest and removing undesirable cell types. [00427] Separation via antibodies for specific markers may be by negative or positive selection, procedures. In' negative separation, antibodies are used which are specific for markers present on tmdesired cells. Cells bound by an antibody may be removed or lysed and the remaining desired mixture retained. In positive separation, antibodies specific for markers present on the desired cells are usedL Cells bound by the antibody are separated and retained. It will be understood that positive and negative separations may be -used substantially simultaneously or in a sequential (Banner.
[00428] The most -common technique for antibody based separation has been the use of flow cytometry such as by a FACS. Typically, separation by flow cytometr is performed as follows. The suspended mixture of hematopoietic cells are centrifuged and resuspended in media. Antibodies which are conjugated to fluoroc'hrome are added to allow the binding of the antibodies to specific cell surface markers. The cell mixture is then washed by one or more eentrifttga'tion and resuspension steps. The mixture is run through a FACS which separates the ceils based on different fluorescence characteristics. FACS systems are available in varying levels of performance and ability, .including ..multi-color analysis. The H SPCs can be identified by a characteristic profile of forward and side scatter which is influenced by size and granularity, as well as by positive and/or negative -express ion of certai cell surface markers,
[00429] Other separation techniques besides flow cytometry may provide for faster separations. One such method is biotin-avidin based separation by affinity chromatography. Typically, such a technique is performed by incubating the washed bone marrow with biotin- coupled antibodies to specific markers followed by passage through an avidin column. Bioiin-aniibody-eell complexes bind to the column via the biotin-avidin interaction, while other ceils pass through the column. Finally, the column-bound cells may be released by perturbation or other methods. The specificity of the biotin-avidin system is well suited for rapid positive separation.
[00430] Flow cytometry and biotin-avidin techniques provide highly specific means of cell separation, if desired,, a separation may foe initiated by less specific techniques which, however, can remove a large proportion of non-HSPC from the hematopoietic cell source. It is generally desirable to lyse red blood cells from mobilized blood before use. For example, magnetic bead separations may be used to iuitially remove lineage committed, differentiated hematopoietic cell populations, including T-ceils, B-cel!s, natural killer (N ) cells, and macrophages (MAC), as well as minor cell populations including megakaryocytes, mast cells, eosinophils, and basophils, Desirably, at least about 70% and usually at least about 80% of the total hematopoietic cells present can be removed.
[00431] An exemplary initial separation technique is density-gradient separation. Here, the mobilized blood is centrifuged and the supernatant removed. The cells are resuspended in. for example, PMI 1.640 medium (Gibco) with 10% HSA and placed in a density gradient prepared with, for example, FieoH or PercoU or Buroeollins media. The separation may then be performed by eentrifugation or may be performed automatically with, for example, a Cobel & Cell Separator '2991 (Cobev, Lakewood, Colo,)- Additional separation procedures may be desirable depending on the source of the hematopoietic cell mixture and on its content,
[00432J The HSPCs contained in enriched cell cultures or mobilized blood may be used in the form of total mononuclear cells, or partially purified or highly purified cell populations. If these cellular compositions are separate compositions, they are suitably administered simultaneously, bu may be administered separately within a relatively close period of time. The mode of adm inistration is generally but not limited to intravenous injection.
[00433] Once administered, it is believed that the cells home to various
hematopoietic cell sites in the recipient's body, including bone marrow. The number of cells which should be administered is calculated fo a specific species of recipient. For example, in rats, the T-cell depleted bone marrow component administered is typically between about I x lO7 cells and 5x1 ? cells per recipient. In mice, the T-cell depleted bone marrow component administered is typically between about 1 * IG6 cells and 5 106 cells per recipient In humans, the T-cell depleted bone marrow component administered is typically between about I x lO8 cells and 3x10 s ceils per kilogram body weight of recipient For cross-species engraftment, larger numbers of cells may be required,
(00434] In mice, the number of HSPCs administered is suitably between about 100 and 300 HSPCs per recipient, in rats, the number of HSPCs administered is generally between about 600 and 1200 HSPCs per recipient in humans, the number of HSPCs administered is suitably between about I* 10* and 1 * 105 HSPC per recipient. The amount of the specific ceils used wiil depend on many factors, including the condition of the recipient's health. In addition, co-administration of cells with various cytokines may further promote engraftment. [00435] In addition to total body irradiation, a recipient may be conditioned by a medical, treatment that results in immunosuppression and myeioablation or eytoreduetion by the same techniques as are employed in substantially destroying a recipient's im une system, including, for example, irradiation, toxins, antibodies bound to toxins or radioactive isotopes, or some combination of these techniques. However, the level or amount of agents used is substantially smaller when immunosuppress g and cytoreducing than when substantially destroying the immune system. For example, substantially destroying a recipient's remaining immune system often involves lethally irradiating the recipient -with 950 rads (R) of total body irradiation (TBI). This level of radiation is fairly constant no matter the species of the recipient. Consistent xenogeneic (rat-wnouse) cliimerism has been achieved with 750 R TBI and consistent allogeneic (mouse) chimerism- with 600RTBT,. Chimerism was established by PB typing and tolerance confirmed by mixed lymphocyte reactions (MLR) and cytotoxic lymphocyte (CTL) response.
[00436] The mobilized blood and enriched cultured cells prepared in accordance with the present invention may be used for establishing both allogeneic chimerism and xenogeneic chimerism. Xenogeneic chimerism may be -established when the donor and recipient as recited above are different species. Xenogeneic chimerism between rats and mice, between hamsters and mice, and 'between .chimpanzees and baboons has been established. Xenogeneic cliimerism between humans and other primates is also possible, Xenogeneic chimerism between humans and other mammals, such as pig, is equall viable.
[00437] It will be appreciated that, though the methods disclosed above involve one recipient and one donor, the present invention encom asses methods in which HSPCs from two donors are engrafted in a single recipient.
[00438] In some embodiments, the mobilized cells and enriched cultured cells prepared ming-inter alia the combination, treatment of the present invention are useful in reestablishing a recipient's hematopoietic system by substantially destroying the recipient's immune system or immunosuppressing and myeloablating or cytoreducing the .recipient* s immune system, and then administering to the recipient syngeneic or autologous cell compositions comprising syngeneic or autologous purified HSPCs which are MHC-identical to the HSPCs of the recipient,
[00439] As noted above, the combination treatment of the present invention also finds utility inter alia in the treatment or prophylaxis of immunocomprom ised conditions, including those resulting from medical (Teatment that target hematopoietic stem cells, such as treatments that target rapidly dividing cells or that disrupt the cell cycle or cell division and that result in immunosuppression and myeioablatio or eyioreduction. The HIF-a potentiating agent and mobilizer(s) of hematopoietic stem cells and/or progenitor celts may be used therapeutically after the medical treatment or may be used prophyiacticaily before the treatment is administered or together with the medical treatment Accordingly, the present invention contemplates further combination therapies which employ both a medical treatment that induces- an immunocompromised condition and concurrent administration of an 'HIF-a potentiating agent antagonist and at least one mobiiizer of hematopoietic stem ceils and/or progenitor■■cells.,
[00440] It is well known that chemotherapy and radiation therapy target rapidly dividing cells and/or disrupt the cell cycle or cell division. These treatments are offered as part of the treating hyperproliferative cell disorders including cancers and autoimmune diseases, aiming either at slowing thei progression or reversing the symptoms of disease by means of a curati ve treatment. In some embodiments, therefore, the combination treatment is used for treating a hyperproliferative ceil disorder, including cancers and autoimmune diseases,
[00441] Representative cancers contemplated by the present invention include, but are not limited to, sarcomas, melanomas, adenomas, carcinomas of solid tissue (e.g., breast, ovary, prostate, colon, lung, skin, kidney, bladder, pancreas,, head and neck) including squamous cell carcinomas of the mouth, throat, larynx, and lung; hypoxic tumors;
hematopoietic cancers; nervous system cancers; benign lesions such as papillomas;
leukemias, and lymphom as, illustrative examples of which 'include carcinomas, such as squamous cell carcinoma, non-small cell carcinoma (e<g., non-small ceil lung carcinoma), small cell carcinoma (e.g., small cell lung carcinoma), basal cell carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocareinoma, medullary carcinoma, undifferentiated carcinoma, bronchogenic carcinoma, melanoma, renal cell carcinoma, hepatoma-liver cell carcinoma, bile duct carcinoma, cholangiocarcinoma, papillary carcinoma, transitional cell carcinoma, chorioc arcihoina, semonoma. embryonal carcinoma, mammary carcinomas, gastrointestinal carcinoma, colonic carcinomas, bladder carcinoma, prostate carcinoma, and squamous ceil carcinoma of the neck and head region; sarcomas, such as fibrosarcoma, myxosarcoma, li osarcoma, chondrosarcoma, osteogenic sarcoma, chordosarcoma, angiosarcoma, endotheiiosarcoma, lymphangiosarcoma, synoviosarcoma and mesotheiiosarcoina;
hematologic cancers, such as myelomas, leukemias {e.g. , acute myelogenous leukemia, acute rom elocyte leukemia, chronic myelogenous leukemia, chronic myelomonocytic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, granulocytic leukemia, hairy' cell leukemia, juvenile myelq onocyte leukemia, large granular lymphocytic leukemia, monocytic leukemia, T-cei! leukemia,T-eeli prolymphocyte leukemia), lymphomas (e,g. AipS-related lymphoma, Burkft s lymphoma, follicular lymphoma,, mantle cell lymphoma, diffuse large B-cel! lymphoma, malignant lymphoma, ALT lymphoma, mycosis fungoides,. piasmocytom , precursor T-ceil lymphoma, reticulum cell sarcoma. Thyroid lymphoma, or Hodgkin's disease including nodular sclerosing or mixed-celiularity subtypes), and tumors of the nervous system including glioma, meningoma, medu!lobiastoma, schwannoma and epidy oma, in specific embodiments, the cancer is leukemia, non-Hodgkin's lymphomas or multiple myeloma.
100442] Non-limiting examples of additionally hematologic disorders contemplated by the present invention include myelodysplasia syndromes (e.g., refractory anemia, refractory eytopema, chronic myelomonocytic leukemia or unclassifiable myeodfyspiastic syndrome), myeloproliferative disorders (e.g., polycythemia vera, essential thrombocytosis and primary or idiopathic myelofibrosis) and aplastic anemia.
|00443| Non-limiting examples of autoimmune diseases contemplated by the present invention include Chagas disease, chronic obstructive pulmonary disease, Crohn's disease (one of two types of idiopathic inflammatory bowel disease "IBD"), dermatomyositis, diabetes mellitus type I , endometriosis, Goodpasture's syndrome, Graves' disease, Guiiiam- Barre syndrome (GBS), Hashimoto's disease, hidradenitis suppurativa. Kawasaki disease, IgA nephropathy, idiopathic thrombocytopenic purpura, interstitial cystitis, lupus erythematosus, mixed connective tissue disease, morphea, . myasthenia gravis, narcolepsy, neuramyotonia, pemphigus vulgaris, pernicious anaemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis, rheumatoid arthritis, schizophrenia. Scleroderma, Sjogren's syndrome, stiff person syndrome, temporal arteritis (also known as "giant cell arteritis"), ulcerative colitis (one of two types of idiopathic' inflammatory bowel disease "IBD"), vasculitis, vitiligo, Wegener's granulomatosis, celiac disease, chronic thyroiditis (Hashimoto's thyroiditis), pernicious anemia, autoimmune hepatitis, Behcet's disease, uveitis, atherosclerosis, stroke, anti-phospholipid antibody syndrome, and the like.
[00444 Accordingly, in some embodiments, the treatment will additionall employ a ehemotherapeutsc agent, which is suitable selected from cytostatic agents and cytotoxic agents, on-Jimitiog examples of cytostatic agents are selected from: (1) microtubuie- stabiH'zing agents such as but not limited to taxanes, paeiitaxel, docetaxel, epothilones and lautimalides; (2) kinase inhibitors, illustrative examples of which include Iressa®, Gieevee, Tarceva™, (Erlotmib HCl), BAY-43-9006, inhibitors of the .split kinase domain receptor tyrosine kinase subgroup (e.g., ΡΤΚ7Ϊ57/ΖΚ 222584 and SUI 1248); (3) receptor kinase targeted antibodies, which include* but are not limited to, Trastazuraab (Herceptin®),
Cetuxirn.ab (Erbitux®), Bevaeizumab (Avasiiu™), Rjtuximab (ritusan®), Pertuzumab
(Omnitarg™); (4) m O'R. path way inhibitors, illustrative examples of which include ■rapamycin and CCl-778; (5) Apo2L Traii. anti-angiogenic agents such as but no limited to endostatin, eombrestatirv, .angiostatin, thrombpspondin and vascular endothelial growth inhibitor (VEGi); (6) antineoplastic, immunotherapy vaccines, representative examples of which include activated T-cells, non-specific immune boosting agents (ie., interferons, interleukins)" (7) antibiotic cytotoxic agents such as but not limited to doxorubicin,
bleomycin, dactinomycin, daunorubicin, epirubicirt, mitomycin and mitozantrone (8) alkylating agents, illustrative examples of which include elphalan, Carmustsne, Loroustine, Cyclophosphamide, Ifosfamide, Chlorambucil, Fotemustine, Busulfan, Temozolomide and Thiotepa; (9) hormonal antineoplastic agents, non-limiting examples of which include ihitarnide, Cyproterone acetate, Anastrozoie, Exemestane, Tamoxifen, Raloxifene, Biealutamide, Aminoglutethimide, Leuproreiin acetate, Toremifene citrate, Letrazoie, Fiutamide, Megestroi acetate and Goserelin acetate; (10) gonadal hormones such as but not limited to Cyproterone acetate and edoxyprogesterone acetate; (1 1 ) antimetabolites, illustrative examples of which, include Cytarabine, FluorouraciL Gemdtabiiie, Topotecao, Hydroxyurea, Thiogoanine, Methotrexate, Colaspase, Raltitrexed and Capicitabine (12) anabolic agents, such as but not limited to, Naadrolone; (13) adrenal steroid hormones, 'illustrative examples of which include ethylprednisolone acetate, Dexamethasone, Hydrocortisone, Prednisolone and Prednisone; (14) rteoplastie agents such as but not limited to irinotecan, Garhoplatin, Cisplatin, Oxalip latin, Etopqside and Dacarbazine; and (15) topoisomerase inhibitors, illustrative examples of which include topotecan and irinotecan.
[00445] illustrative cytotoxi agents can be selected from sertenef, cachectin, ifosfamide, tasonerrain, iomdamine, carbopiatin, altretami'ne, prednimustine,
dibromoduleitol, ranim stine, fotemustine, nedapiatin, oxaliplatin, teraozolomide
(TEMODAR™ from Schering-Plougli Corporation, Kenilworth, NX), cyclophosphamide, heptaplatin, estramustine, imptosulfan tosiiate, ..trofosfam.de» nimustine, dibrospidium chloride, purnitepa, lofcaplatin, satraplatin, profirornycm, cisplatin, doxorubicin,, irafuiyen, dexifosfamide, cis-am:inedichioro(2-niethyI-pyridine)plaiinum, benzylguanme, glufosfaniide, GPXl OO (trans, trans, trans)-b!S-mu hex^^ bis[dtamme(chIoro) platinum(ii)] tetrachloride, diarSzidinylsperrame, arsenic trioxide., i -(l Ϊ- dodecylamino-l O-hydroxyundecyl^JJ-dimethylxan&inej Zombiein, idarubicin, dauo.ofubic.in, bisantrene, raitoxanrxone. pirarubicirt, pinafide, val uhicin, amrabicin, aiitmeoplaston, 3'-deansi:nG-3'-mo^hoHiio-13-deoxo-10~hydroxycarminomycin. annanrycin, gaiarubicin, eimafide, MEN! 0755, -demethoxy-3-deammo-3-a2;irldiny!-4-metbyisnlph(>nyl- daunombicin (see Internationa! Publication WQ 00/50032), methoxtrexate, gemritabme, and mixture thereof.
[00446] In some embodiments, the concurrent administration of the HIF-a potentiating agent and the mobiiizef(S) is used in combination with radiotherapies, such as but not limited to, conformal external beam radiotherapy (10-100 Grey given as fractions over 4-8 weeks), either single shot or fractionated, high dose rate braeh herapy, permanent interstitial brachytherapy, systemic radio-isotopes (e.g., Strontium 89) or radiaoiabeied antibodies or peptides. In illustrative examples of this type, the radiotherapy is administered in combination with a radiosensitizing agent, illustrative examples of radiosensitizing agents include but are not limited to efaproxiral, etamdazole, fluosol, misonidazole, nimorazole, temoporfin and tirapazamme.
[0O447j immunocompromised conditions generally lead to pathogenic infections and thus the present invention also extends to the treatment and/or prophy laxis of infections in individuals suffering from an immunocompromised condition, or to treatment of individuals who are likely to contract such a condition due to treatraertt known to be associated with the occurrence of an immunocompromised condition. Accordingly, an immunocompromised condition arising from a medical treatment is likely to expose the individual in question to a higher risk of infection. It is possible according to the invention to prophylaeiieally treat an infection in an individual having the immunocompromised condition before or during treatments known to generate such a condition. B prophylacticaliy treating with concurrent administration of the HIF-a potentiating agent and one or more mobilizers (also referred to herein as an "HIF-a potentiating agent/mobilizer combination" or "combinatio treatment") before or during a treatment known to generate an immunocompromised condition it is possible to prevent a subsequent infection or to reduce th risk of the individual contracting an infection manifesting from that condition. In some embodiments*, therefore, the present invention extends to ancillary combination therapies, which employ both the HIF-a potentiating agent/mobilizer combination and an anti-infective agent that is effective against an infection that develops or that has an increased risk of developing from an immunocompromised condition resulting from a medical treatment a broadly described above.
[00448] The anti-infective agent is suitably selected from antimicrobials, which include -without limitation compounds that kill or inhibit the growth of microorgan isms such as viruses, bacteria, yeast,, fungi, protozoa, etc. and thus include antibiotics, amebicides, antifungals, antiprotozoals, antimalarials., ant tuberculotics and antivirals, Artti -infective agents also include within their scope anthelmintics and nematoeides. Illustrative■antibiotics include quinoiones (e.g.., amiflbxacio, cmoxacin, ciprofloxacin, enoxacin, fleroxacin, fhimequine, lomefloxacin, nalidixic acid, norfloxacin, ofloxacin, levof oxacio, lomefloxacin, oxoii ic acid, pefioxacin, rosoxacin, temafloxacin, tosufloxacin, sparfloxacai, ejmafloxacin, gatifloxacin, moxifloxacin; gemifloxaein; and garenoxaein), tetracyclines, glycylcyclines and oxazolidinones (e.g., ehiorietrae eiine, demeclocycline, dox eycline, lymecycline*
meinaeycline, minocycline, ox tetracycline, tetracycline, t gecyeime; linezoHde, eperozo!id), glyeopeptides, aminoglycosides (e,g., amikacin, arhekacm, butirosin, dibekacjri, fortimicms, gehtamiciri, kanamycin, tfteomycm, netilmicin, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin), β-lactams (e.g., imipenem, meropenem, biapenem, cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedorse, eefazoHn, cefixime, csfmenoxime.
eefodizime., cefonicid, cefoperaxone, eeforamde., cefotaxime, cefotiam, cefpiniizoie, cefpiramide, ceipodoxime, eefsulodin, ceftazidime, eefteram, ceftezoie, ceftibnten, ceftizoxime, ceftriaxone, cefuroxime, cefuzonam, cephaacetrlle, cephalexin, cephaloglycin, cephalondine, cephalothiri, cephapirin, cep radine, cefmetazole, cefoxitin, cefotetan, azthreonara, carumonam, fJomoxef, oxaiactam, smidinocii!m, amoxicillin, ampiciliin, azlocillin, carbenieiilin, benzylpemciilin, carteciilm, cioxaciilin, dicloxaeiilin, methiciliin, mezlocillin, nafcillin, oxacillin, penicillin Gs piperacillin, suIbenieiUm, teraocjllin, ticarciilin, cefditoren, SC004, KY-020, cefdi ir, eeftibuten, F -312, S-l 090, CP-Q467, BK-218, F - 037, DQ-25S6, F -5.S 8, cefozopran. ME1228, P-736, CP-6232, Ro 09-1227, OPC-20Q0G, LY206763), rifamycfns, macrolides (e.g., azithromycin, clarithromycin, erythromycin, oleandomycin, rokitamycin, rosaramrcin, roxithromycin, iToieandomycin), ketolides (e.g.., telithromycin, cethrorayein), coumermyems, lmcosamtdes (e.g.. clindamycin, Itncornycin) and chloramphenicol.
[00449] Illustrative antivirais include abacavir sulfate, acyclovir sodium, amantadine hydrochloride, ampre.navhy eidofbvir, de'lavirdine mesylate, didanosine, efavirenz, fameieiovir, foraivirsen sodium, foscarnet sodium, ganciclovir, indinavir sulfate, Samivudme, lamivudine/zidovadine, nelfioavir mesylate, nevtrapme, oseltamivir phosphate,, ribavirin, rimantadine hydrochloride, ritonavir,, saquinavir, saquinavir mesylate, stavudine, vajacyeiovir hydrochloride, zalcitabine, zanamivir, and zidovudine.
[00450] Non-limiting examples of amebic ides or antiprotozoals include atovaquone, chloroquine hydrochloride;, ehloroqume phosphate, metronidazole, metronidazole
hydrochloride, and pentamidine iseihionate. Anthelmintics can be at least one selected from mebendazole, pyrantel pamoate, albendazole, ivermectin and thiabendazole, illustrative antifungals can be selected from amphotericin B, amphotericin B cholesteryl sulfate complex, amphotericin B lipid complex, amphotericin B liposomal, fluconazole, flucytosine, griseofulvin mierosize, griseofulvin ultramic resize, itraconazole, keioconazole, nystatin, and terbinafme hydrochloride. Non-limiting examples of antimalarials include chloroqu ine hydrochloride, chioroquine phosphate, doxycycHne, hydroxychloroquine sulfate, mefloquine hydrochloride, primaquine phosphate, pyrimethamine, and pyrimethamine; with, sulfadoxine. Antituberculotics include but are not restricted to clofazimine, cycloserine, dapsone, ethambutoi hydrochloride, isomazid, pyrazinamide, rifabutin, rifampin, rifapentine, and streptomycin sulfate.
[00451] it is also known that medical treatments that target rapidl dividing cells and/or disrupt the cell cycle o cell division (e.g. , chemotherapy and radiation therapy) are immunocompromising since cells of the immune system including hematopoietic cells are destroyed or -substantially reduced in number, thus leading to a state of immunosuppression characterized by neutropenia, agranulocytosis, thrombocytopenia and/or anemia.
Accordingly, the present invention finds particular utility in the treatment or prophylaxis of any one or more of these conditions that manifest from a medical treatment as broadly noted above.
(00452] Anemia, thrombocytopenia, neutropenia and agranulocytosis are frequently defined in terms .of laboratory measurements indicating a reduced hematocrit (volume percent), a reduced platelet count (per mm"5},. a reduced neutrophil count (per mm3), a reduced total granulocyte (i.e., neutrophils, basophils and eosinophils) or white blood cell count (per mm3), respectively. Methods of determining these values ate well known in the art, including automated as well as manual methods. The lower limits of normal for hematocrits and platelet counts in healthy non-pregnant humans is somewhat variable, depending on th age and sex of the subject, method of determination, and the norms for the laboratory performing the measurements. Generally, however, an adult human subject is said to have anemia when the hematocrit is less than about 37-40%. Likewise, generally an adult human subject is said to have thrombocytopenia when the platelet count is below about 100,000 per mm3. Anemia is also frequently reported In terms of a reduced hemoglobin (g/d'L) or red blood ceil count (per mm3}. Typical lower limits of normal values for ihese in health adult humans are 12-13 g d'L and about 4.1 l O6 per ram" , respectively. Generally an adult human subject is said to have neiuropenia when the neutrophil count falls below 1000 per mm3. Additionally, an adult human is generally said to have agranulocytosis when the total granulocyte cell count falls below 500 eel!s/rrim*. Corresponding values for all these parameters are different for other species.
[004533 Hematopoietic disorders such as anemia, thrombocytopenia, neutropenia and agranulocytosis are also frequently associated with clinical signs and symptoms in relation to their degree of severity. Anemia may be manifested as pallor, generalized fatigue or weakness, reduced exercise tolerance, shortness of breath with exertion, rapid heart rate, irregular heart rhythm, chest pain (angina), congestive heart failure, and headache.
Thrombocytopenia is typically manifested in terms of spontaneous or imcontrolled bleeding, petechiae, and easy bruising. Neutropenia i associated with infections, including notably infections from endogenous microbial flora, and lack of inflammation.
[00454] Accordingly, the present invention: contemplates ancillary combination treatment which employ both the HIF-a potentiating agent mobiltzer combination and an ancillary treatment that treats a hernatopoieiic disorder as broadly described above. In some embodiments, the ancillary combination treatment will employ a HIF-a potentiating agen.t/mobilizer combination and a medicament selected from an anemia medicament, a thrombocytopenia medicament, an agranulocytosis medicament or a neutropenia medicament, illustrative examples of which include steroids, inducers of steroids, and immunomodulators.
[004555 The steroids include, but are not limited to, system ical!y administered corticosteroids including methy lprednisoJone, prednisolone and prednisone, cortisone, and hydrocortisone. Inducers of steroids include, but axe not limited to adrenocorticotropic, hormone ( ACTH),
[0045631 Corticosteroids inhibit cytokine production, adhesion protein activation, and mfiarnrnatory cell migration and activation. The side effects associated with systemic corticosteroids include, for instance, reversible abnormalities in glucose metabolism, increased appetite, fluid retention, weight gain, mood alteration, hypertension, peptic ulcer, and aseptic necrosis of bone. Some side effects associated with longer term use include adrenal axis suppression, growth suppression, dermal thinning, hypertension,, diabetes meliitus, Cus n.g's syndrome, cataracts, muscle weaitnesas, and in rare instances, impaire immune function. It is recommended that these types of compounds be used at their lowest effective dose,
[00457] Commonly used anemia drags which are currently on the market or in development include recombinant human EPO (EPOGEN; P OC 1T), preparations of iron {ferrous and ferric, CHROMAGEN; FEOSOL; INFED; IROSPAN; NEPFIRO-FER;
NEPH.RO- V ITE ; NIFEREX; NO- IRON; SLOW FE)5 vitamin B12, vitamin B6, folic acid (CHROMAGEN; FERRQ-FQLiC; NEPHRO-FER; NIFEREX), ascorbic acid, certain metabolites of vitamin- D. (calc triol and alphacalcidol; CALCIJEX: ROCALTROL), androgens, and anabolic steroids (AN ADROL), carnitine. In a specific embodiment the anemi medicament is recombinant EPO.
[08458} Drugs in common usage or development for the treatment of
thrombocytopenia include glucocorticoids (prednisolone; prednisone; meihy!prednisotone; SOLUMEDROL), recom inant TPO, recombinant JvlGDF, pegylated recombinant MGDFj and lisophylRne. In a specific embodiment the thrombocytopenia medicament is recombinant TPO.
[00459] Drugs in common usage or development for the treatment of neutropenia include glucocorticoids (prednisolone; prednisone; methy!predmsoione; SOLUMEDROL), immunoglobulin G {SANDOGLOBUtiN, IVEEGAM, GAMMAR-P, GAMIMNE N, GAMMAGARD S/D), androgens, recombinant IFN-y (AGTIMMUNE), and uteroferrin.
Antibiotics are frequently administei ed in association with neutropenia medicaments to treat or reduce the risk of infection,
[00460] As noted above, the present invention encompasses co-adniiriistration of a HIF'-a potentiating agentftnobilizer -combination in concert with an additional or ancillary agent, it will be understood that, in embodiments comprising administration of the HIF-a potentiating agent mobilizer combi nation with other agents, the dosages of the active agents in the combination may .on their own comprise an effective amount and the additional agent(s) may further augment the therapeutic or prophylactic benefit to the patient.
Alternatively, the HIF-a potentiating. agent mobiiizer combination and the additional agent(s) may together comprise an effective amount for preventing or treating the
immunocompromised condition or infection. It will also be understood that effective amounts may be- defined in the context of particular treatment regimens, including, e.g., timing and number of administrations, modes of administrations, formulations, etc,
- 1.31 - [00461] In some embodiments, the present 'invention contemplates administering a high dose of the -medical treatment that induces the immunocompromised condition, withou inducing side effects or inhibiting the induction of side effects, Ordinarily, when medical treatments such as chemotherapy and. radiotherapy are administered ¾ a high dose, a variety of side effects can occur, including the induct ion of the immunocompromised condition and infection. As a result of these side effects, the medical treatment is not administered in such high doses, in accordance with the present invention, such high doses of medical treatment (e.g., a higher dose of chemotherapeutic agent or radiation) which ordinarily induce side effects can be administered without inducing the side effects as long as the subject also rec ives concurrent administration of a HIF-a potentiating agent and at least one mobilizer of hematopoietic ste ceils and/or progenitor cells- The iype and extent of the side effects ordinarily induced by the medical treatment will depend on the particular treatment used,
[00462] Suitably, the HIF-α potentiating agent'mobiiizer combination, and optionally the ancillary treatment, are administered on a routine schedule. Alternatively, the ancillar treatment may be administered as symptoms arise, A "routine schedule" as used herein, refers to a predetermined designated period of time. The routine schedule may encompass periods of time which are identical or wh ich differ i n length, as long as the schedule is predetermined. For instanee, the routine schedule may involve administration of the HIF-a potentiating agent on a dail basis, every two days, every three days, even' four days, every five days, every six days, a weekly basis, a monthly basis or any set number of days or weeks there-between, every two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, 'etc. Alternatively, the predetermined routine schedule may involve concurrent administration of 'HIF-a potentiating agent and the mobilizer on a daily basis for the first week., followed by a monthl basis for several months, and then every three months after that. Any particular combination would be covered by the routine schedule as long as it is determined ahead of t ime that the appropriate schedule involves administration on a. certain day,
[00463] Additionally, the present invention provides pharmaceutical compositions for stimulating or enhancing mobilization of hematopoietic stem ceils and/or progenitor ceils, or for stimulating or enhancing hematopoiesis. o for stem cell transplantation or for treating or preventing an immunocompromised con ition, including one that results from a medical treatment as broadl described above. Tile pharmaceutical compositions include an HIF-a potentiating agent an d at least one m obilizer of hematopoietic stem cells and/or progenitor cells, optionally formulated in a pharmaceutically acceptable carrier. n specific embodiments, the pharmaceutieal compositions comprises a ?HY(e,g.t a small molecule PHI including ones selected from compounds according to any one of formulae I- FX) and one or both of a colony-stimulating Factor (e.g., G-CSF or.a variant, derivative or analog thereof) and a CXC 4 antagonist (e.g., a small molecule CXCR.4 antagonist)
[00464] The pharmaceutical composition may include an ancillary or additional medicament as broadly described above. n some embodiments, the HlF-a potentiating agent and the mobiiizer{s) will be present in the pharmaceutical composition in an effective amount for preventing or treating an immunocompromised condition (e.g., anemia., thrombocytopenia, or neutropenia), The effective amount for preventing or treating the immunocompromised condition is that amount which completely or partially prevents the development of, prevents the worsening of, or treats the established existence of, the immunocompromised condition. In some instances, the effective amount for preventing or treating immunocompromised condition completely or partially prevents or treats clinical symptoms of that condition.
[00465] In addition to clinical outcomes measured in terras of physiology, in vitro assays measurmg erythrocyte, platelet, granulocyte and total white blood cell counts may be used in determining a therapeutically effective amount of a particular H!F-a potentiating agent. These methods are standard medical laboratory techniques that are well known in the art- in -common practice such measurements may be made by automated ceil counting devices' -designed for that purpose, or they may be performed manually. Manual counts may be more accurate than automated counts when cell counts are particularly low.
[00466] The compositions of the invention are adm inistered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable
concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients. The HIF-α potentiating agent, in particular, may be formulated in pharmaceutically acceptable solid form, e.g., tablet, capsule, capiet, powder, pill, etc. Depending on the specific conditions being treated, the formulations may be administered systemically or locally.. Techniques, for fonrsuiation and administration may be found in Goodman and Oilman's: The Pharmacological Bases of Therapeutics, 8ih Ed.s supra; Remington's Pharmaceutical Sciences, 17th ed.. Mack Publishing Co., supra;
Pharmaceutical Dosage Forms, Parenteral Medications Dekker, N.Y. supra Pharmaceutieal'
Dosage Forms: Tablets Dekker, N.Y. supra; and Pharmaceutical Dosage Forms: Disperse
Systems Dekker, N.Y, supra. Suitable routes may, for example, include oral, rectal, transmucosai, or intestinal administration;, parenteral delivery, including intramuscular, ■subcutaneous, intramedullar injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. For injection, the active agents or drugs of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution. Ringer's solution, or physio.logical saline buffer. For transmucosai administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
[00467] The drugs can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the invention to be formulated in dosage forms such as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. These carriers may be selected from sugars, starches, celluiose and its derivatives, malt, .gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, poiyols, aiginic acid., phosphate buffered solutions, emuisifiers, isotonic saline, and pyrogen-free water,
[00468] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active agents in water-soluble form. Additionally, suspensions of the active agents may be prepared as appropriate oily injection suspensions, Suitabie lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxytnethyi cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitabie stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
[00469] Pharmaceutical preparations for oral use can be obtained by combining the active agents with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragaeanth, methyl cellulose, hydroxy propylmeth i- eeilu!ose, sodium carboxymethylcellulose, or polyvinylpyrrolidone (PVP), If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or aigin ic acid or a salt thereof such as sodium alginate. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more drugs as described above with the carrier which constitutes one or more necessary ingredients, in general, the pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means .of .conventional mixing, dissolving, granulating, dragee-making, .levigating, emulsifying, encapsul ting, entrapping or lyophilizmg processes.
[00470] Dragee cores are provided with suitable coatings. For this purpose,
concentrated sugar solutions may be used, which may optionall contain gam arable, talc, polyvinyl pyrrolidone, earbopol gei, polyethylene glycol, or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestyffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active agent doses,
[00471] Pharmaceutical which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a p!asiicizer, such as glycerol or sorbitol. The push-fit capsules, can contain the active agents in admixture with filler such as lactose, binders such as starches, or lubricants such as tale or magnesium siearaie and, optionally, stabilizers.- In soft capsules, the active agents may be dissol ved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.
[00472 ] Dosage forms of the drugs of the invention may also includ injecting or implandng controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of an agent of the invention may be effected b coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactie and polyglycolic acids and certain cellulose derivatives such as hydroxypropylraethyl cellulose. In addition, controlled release may be effected by using other polymer matrices; liposomes or microspheres,
[00473] The drugs of the invention ma be provided as salts with pharmaceutically compatible eounterions, Pharniaceuticaiiy compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc, Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding, free base forms.
Ϊ00474] For any compound used i the method of the invention, the therapeutically effective dose can be estimated initiall from ceil culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the ICS© as determined in cell culture (e.g., the concentration of an active agent which achieves a haif-rrsaximaf inhibition in activity' of a PHD polypeptide), Such information ca be used to more■accurately determine useful doses in humans.
[00475] Toxicity' and therapeutic efficacy of such drugs can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the EDS© (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic inde and it can be expressed as the ratio LD50/ED50, Compounds that exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosag of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may -vary withi this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician i view of the patient's condition. (See for example Fingl el t, 1975, 111 "The Pharmacological Basis of Therapeutics", Ch. 1 pi).
[00476] Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a tissue, which is preferably subcutaneous or omental tissue, often in a depot or sustained release formulation.
[00477] Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with tissue-specific antibody. The liposomes will be targeted to and taken up .selectively by the tissue.
[00478] in cases of local administration or selective uptake, the effective local concentration of the agent may not be related to plasma concentration.
[00479] in order that the invention may be readil understood and put into practical effect particular preferred embodiments will now be described by way of the following non- limiting examples. EXAMPLES
EXAMPLE 1
COMPOUND 'X SYNERG!ZES WITH G-CSF TO ENHANCE. HSC MOBILIZATION
[00480] To evaluate whether pharmacological stabilization of HIF-l . protein would enhance HSC mobilization in response to G-CSF, C.57B1 6 mice were treated with recombinant human G-CSF (rhuG-CSF) twice daily for 1-3 days together with a PHD inhibitor. Compound X, daily for 3 days. Pharmacological stabilization of HlF-1 protein was demonstrated in. bone marrow leukocytes treated with Compound X {Figure 1 A).
Additionally;, treatment with Compound X in combination with G-GSF significantly increased stabilization of HiF~l protein compared to G-CSF alone. Mobilization of eoiony- forming cells (C PCs) to the blood and spleen were measured (Figure 1 B). Addition of Compound X to G-CSF treatment resulted in a 2,5-fold increase in CFCs mobilized per mL blood following 2 days of G-CSF, and a 6 - fold increase of CFCs mobilized to the spleen following 3 days of G-CSF. Of note, Compound X alone for 3 days did not induce CFC mobilization into the blood. Thes results demonstrate that Compound X synergizes with G- CSF on HSPC mobilization. To assess the optimum duration of Compound treatment, Compound X was administered for 1-4 days and G-CSF for the last 2 days before tissue sampling (Figure ! C). CFC mobilization into blood and spleen in response to 2 days of G- CSF progressively increased with the duration of Compound X treatment peaking at day 3 of Compound X administration,
EXAMPLE 2
COMPOUND X .SYNERGIZES WITH PLERIXAFOR TO ENHANCE CFC MOBILIZATION [G0481j The present inventors next evaluated the ability of Compound X to synergize with Plenxafor to enhance mobilization. Since mobilization with P!erixafor peaks Ih post-injection (7), this time-point was used together with dally administration o
Compound X for 1 -4 days prior sacrifice (Figure ID). Similar to G-CSF, the number of CFC mobilized per mL blood in response to Plenxafor, was significantly increased by a 2 to 3 day treatment with Compound X demonstrating synergy betwee CXCR4 inhibition and PHD inhibition. As mobilization in response to Plerixafor is very rapid, there was no significant mobilization of HSPC to the spleen Ihr following Plerixafor treatment with or without Compound X (Figure I D). EXAMPLE 3
PHD INHIBITION SVNERGIZES WITH HUE- COMBINATION, OF G-CSF AND PL.ERIXAFOR
[06482] Since Compound X synergized -with both G-CSF and Plerixafor separately, the combined effects of G-CSF. Plerixafor and Compound X were next assessed on mobilization. This was divided into two experiments in which G-CSF was administered for 2 days in one experiment and 4 days in the other (Figure 2). C57BL/6 mice were in 4 treatment groups in both experiments: (G) 250 pg kg day G-CSF alone; (G+X3) G-CSF + 20 rag/kg/day Compound X for 3 days; (GPl) G-CSF together with 16 tng fcg Plerixafor ! hour prior harvest; (GH-P1X3) G-CSF together with Plerixafor and Compound X with same dosing as above. Mobilization of CFCs, phenotypic Lin*CD41 "Seal T it* HSPCs and Lin"CD41" Seal+KitrCD48"CD;l 50t HSCs were measured in blood and spleens. Mice in the G-hX3 group (G-CSF + Compound X) mobilized CFC to the blood 4-foid and the spleen. 47-fo!d more than mice mobilized with G-CSF alone for 2 days (p<0,00i ; Figure 2B). Fol Sowing 4 days treatment with G-CSF, the addition of Compound X resulted in a 4-fold increase in the number of CFC/mL blood (P<0,001 ) and a further significant increase in the number of CFC/spleen (P<0.01 - Figur 2B). Compound X alone had no mobilizing effect (data not shown). Expected ly, 'Plerixafor enhanced mobilization of CFC 10-fold in response to 2 days treatment with G-CSF (p<O,0O5)» Most interestingly, addition of Compound X further increased by '2,5-fold the mobilization . of CFC/mL into blood and by 4-fold the mobilization of CFC/spiee in response to 2 days G-CSF plus Plerixafor (p<O,0O5; Figure 2B), Addition of -Compound X also boosted by 2.5-fold the mobilization of CFC/mL blood in response to .4 days G-CSF plus Plerixafor (p<0,005; Figure 2 B).
[00483] Mice mobilized with G-CSF for 2 days together with Compound X increased the number of phenotypic HSCs 3-fold while the number of Lif Scal itT HSPCs doubled in the blood and tripled spleen compared to miee mobilized with G-CSF alone
(p<0.005 Figure 2c-d). Furthermore, the addition of Compound X together with Q-CSF and Plerixafor boosted mobilization, of phenotypic HSCs 3-fold and HSPCs 2,5-fold in the blood and 4-fold in the spleen following 2 days G-CSF plus Plerixafor (P<0.01 ; Figure 2C-D). The addition of Compound X to G-GSF and Plerixafor also dramaticall increased the number of phenotypic HSCs and HSPCs in the blood following 4 days treatment with G-CSF and Plerixafor (P<0.001 ; Figure 2C-D),
[00484] This synergistic increase in BSC mobilization was further confirmed in long-term competitive ^population assays following tt-ansplantation of 20 pL mobilized blood in competition with 2* 10s BM cells from congenk donors. GD45.2/CD45.1 ehinierism. showed that combination of 2 days G-CSF+Pierixafor+Compound X (G2P1 3) mobilized competitive repopulating HSC 6-fold more than G-CSF+Plerixafor (G2P1, p<0.01). The number of mobilized long-term competitive repopulating HSC (measured 16 weeks post-transplant) doubled after treatment with 2 days G-CSF + lh Pierixafor + 3 days Compound X (G2P1X3) compared to G-CSF+P rixafor (G2P1, p<0.05; Figure 3A). Most interestingly, this increase in the number of mobilized repopulating units with the addition of Compound X was evert further pronounced after treatment with G-CSF for 4 days prior to transplant. The addition of Compound X io G-CSF (G4X3) increased 6-fold the number of mobilized repopulating units compared to G-CSF (G4) alone (P<G.G01 ; Figure 3B). The -combination. -of Compound X with G-CSF and Pierixafor (G4P1X3) increased mobilization of repopulating units 3-foid compared to G-CSF+Plerixafor (G4P1 ) alone (P<G,Q0k Figure 3B). These data demonstrate that Compound X synergistica!ly enhances the mobilization of transplantable HSCs in response to the already potent combination of G--CSF with Pierixafor.
EXAMPLE 4
DELETION OF HI F 1 A GENE m. HSPCS IMPAIRS THEIR MOSILIZATLO
[004851 As Compound X was found by the present inventors to stabilize HlF-la protein in bone marrow HSPCs, it was decided to further investigated the role of HlF-l a in HSPC mobilization in response to G-CSF. A mutant strain was first established in which both Hifia gene alleles are floxed, together with a Cre-mducible YFP reporter knocked in the Rosa26 gene trap locus R26RyFr'( i 0) together with a tamoxifen-inducible Cre recomhinase (CreER fusion protein) specifically expressed, in HSC under the control of a HSC specific Scl gene enhancer fragment (SclCreER)(l 1). By measuring expression of the Cre-mdacibje YFP reporter by flow cytometry, a 3 day tamoxifen treatment of these Hiflam R26R¾FP,Yn>
SclCreER mice caused Cre activation in 30± % ©f Lin'Scal +Kit+GD48"CDl5.0* HSCs, 10± 5 % Un Scal+ i HSPCs (Figure 4A) but was virtually undetected in Lin'Scal " if myeloid progenitors or lineage-positive cells.
[00486] Cohorts of Hiflam R26RyFP/¥FP SclCreER mice and control Hifi ^1^ R26R >, VFP SclCreER mice were induced with tamoxifen for .3 days and then 3 days with G-CSF beginning on the last day of tamoxifen induction. Tissues were harvested 24 hours following the last G-CSF injection. Deletion of 'Hifia gene in HSPCs significantly reduced mobilization of CFCs (P<0.01 Figure 4B), HSPCs (P<Q.05) and HSCs (P<0.02) to the blood and spleen in Hifiam R26RYFF/Y?P SclCreER mice compared to control Hifiawm R26 'sTP Vip SclGreE mice treated -with tamoxifen and G-CSF (Figure 4C). As Hifla. was deleted from approximately 30% of HSCs in Eifl m R26RYmYW SelCreER mice treated with tamoxifen and G-CSF, we took advantage of this mosaic deletion to calculate within each individual mouse the proportion of YFP" (H¾f7».gene not deleted) and YFP* (Hifl deleted) LhvScal +Kit+ HSPCs mobilized to blood or spleen versus the number of H^Za- intaet or ift ia-deleted HSPCs remaining within the bone marrow. Within each individual mouse, the proportion ofYFP* /¾-deie†ed HSPCs that left the bone marrow to mobilize into blood or spleen was significantly lower than the proportion of YFP+ Hifla-int& t HSPCs (Figure 4.D). This further demonstrates thai deletion of the Hifla gen e in HSPCs severely impairs their mobilization in response to G-GSF,
EXAMPLE S
BIF-lA EXPRESSION IN-OSTEOPROGE ITORS IS NECESSARY FOR OPTIMAL HSPC
MOBILIZATION
[00487] As osteoprogenitors and the r progenies play an important role in HSC retention, within, the BM and mobilization in response to G-CSF (6, 12-15), Eiflam osxYFPCre mice ere generated with conditional deletion of the Hifla gene in
osteoprogenitors expressing osterix (Sp? gene) (16). Compared to control Hifia^m i osxYFPCre mice, Hiflam osxYFPCre mice had delayed CFC mobilization into blood and spleen at early time-points of G-CSF administration (Figures 3 A, SB, 5C). This demonstrates that the effect of HIF- 1 a in HSPC mobilization is not entirely HSPC autonomous but also requires expression in cells forming the niche such as osteoprogenitors and osteoblasts.
EXAMPLE 6
COMPOUNDS A, B AND C ENHANCE HSC MOBILIZATION
[00488] Additional HIF-ά potentiating agents in the form of PHD inhibitors were tested for their ability to enhance mobilization induced by G-CSF. Compound A, Compound B or Compound C, were tested in male, 7 weeks-old C57BL 6 mice. Mice were injected twice daily sub-cutaneously with rhuG-CSF at a dose of 125 μ-g/kg per injection for four days. Mice were gavaged with. 0 mg kg Compound A, Compound B, Compound Cor vehicle control once daily for three days beginning on day 3 of the experiment,
.[00489] At the end of treatment, blood was harvested by cardiac puncture, mice were sacrificed by cervical dislocation and spleens harvested. Femurs were removed, cleaned and the bone marro w was flushed with ImL PBS + 1.0% newborn calf serum (MC S), [00490] The number of phenotypic hematopoietic stem and progenitor cells remaining in the bone marrow following treatment was measured by flow cytometry using antibodies specific for lineage markers (CD3, CDS, CD45.R/B22Q, F4/S0, Grl , CD4L Terl 19), Sca-1, Kit, CD48 and CD 150. Similarly, the number of phenotypic hematopoietic stem cells mobilized into the blood and spleen were measured. Colony assays were also performed on blood and spleen to determine the number of colony forming cells per mi., of blood and per spleen. Ail results are presented as mean ± standard deviation with ti-6 per groups. Levels of significance for differe nces were calculated using the Student's t-test
[00491] As shown in Figures 6A-8, treatment with Compound A, Compound B, or Compound G in combination with G-CSF did not significantly alter the total number of phenotypic hematopoietic stem and progenitor cells (HSPCs) in the bone marrow compared to vehicle control in combination with G-CSF. These results demonstrate that the treatments did not change the phenotype of the mobilized HSCs and HPCs and suggest that the mobilized cells should engraft following transplantation.
[00492] As shown in Figures 7A-B, treatment with Compound A, Compound B, or
Compound C i n combination,■with G-CSF resu lted in a significant increase of approximately 3-fold or greater in the number of phenotypic HSCs (LSK48-150+) and a significant increase of approximately 2-fold or greater in the number of HSPCs (LSK48-150- and LSK) mobilized to the blood compared to vehicle control in combination with G-CSF. Treatment with Compound A, Compound B, or Compound C in combination with G-CSF did not significantly alter the mobilization of myeloid progenitors (LKS-) to the blood compared to vehicle control, in combination with G-CSF,
[00493] As shown in Figures SA-B, treatment with Compound A, Compound B> or Compound C in combination with G-CSF resulted in a significant increase of approximately 2-fbld in the number of phenotypic HSCs (LSK48- 150+) and HSPCs (LSK48+ and LSK) mobilized to the spleen compared to vehicle control in combination with G-CSF, Treatment ith Compound A, Compound B, or Compound C in combination with G-CSF also significantly increased by approximately 3-foid, the mobilization of myeloid progenitors (LKS-) to the spleen compared to vehicle control in combination with G-CSF.
[00494] As shown in Figures 9A~B, treatment with Compound A, Compound B, or
Compound C in combination with G-CSF resulted in a significant increase of approximately 2-fold in the total number of phenotypic HSCs (LSK48-150+) -.and HSPCs (LSK48÷ an LSK) mobilized from the bone marrow in comparison to vehicle control in combination with G-CSF. Additionally, treatment with Compound A, Compound B, or Compound C in combination with G-CSF resulted in a significant increase of approximately 6-fold in tine total number of myeloid progenitors (LKS-) mobilized from the bone marrow in comparison to vehicle control in combination with G-CSF.
[00495] As shown in Figure 10, treatment with Compound A or Compound C in combination with G-CSF -significantly increased -the number of colony forming units mobilized from the bone ma r w' to the blood in comparison. to vehicle control in combination with G-CSF. There was no. significant change in the number of CFUs mobilized to the biood following treatment with Compound B in combination with G-CSF. This finding is believed to reflect a more rapid induction of mobilization fay Compound B compared to the other compounds. Accordingly, the timepoint used in the experiment for blood and tissue collection misses the period of peak induction for Compound B. This hypothesis is supported by the statistically significant number of CFUs mobilized to the spleen for Compound B in combination with G-CSF compared to vehicle and G-CSF as it is known that mobilized stem and progenitor cells are taken up and retained over time by the spleen,
Further, the total number of CFUs mobilized by Compound B in combination with G-CSF is comparable to number of CFUs mobilized by Compound A or Compound C in combination with G-CSF.
[00496] As shown in Figure 1 1 , treatment with Compound A, Compound B or Compound C in combination with G-CSF resulted in a significant increase in the number of white blood cells per mL of blood compared to vehicle control in combination with G-CSF. Furthermore, treatment with. Compound A, Compound B, or Compound C in combination with G-CSF resulted in a significant increase in spleen weight of approximately 2-fold compared to vehicle control in combination with G-CSF.
[00497] In conclusion, the results show that the exemplary PHD inhibitors enhanced the mobilization of hematopoietic stem and/or progenitor ceils with G-CSF. Significantl elevated levels of phenotypic HSCs and HSPCs were mobilized using the combination of a PHD inhibitor and G-CSF compared to the use of G-CSF alone. Further, the mobilized HSCs and HSPCs were functional and retained the ability to proliferate and differentiate as demonstrated by the CFU assays. EXAMPLE 7
COMPOUNDS D, E AND F ENHANCE BSC MOBILIZATION
[00498] Further HlF-a potentiating agents in the form of PHD inhibitors were tested for their ability to enhance mobilization of hematopoietic stem and/or progenitor cells induced by G-CSF treatment. Compounds D. E and F were tested in combination with rhuG- CSF in m le, 7 weeks-old C57BL/6 as described in Example 6, above. Compound D was administered at 60 g kg, while Compounds E and F were administered at 20 mg/kg, Blood and tissue samples were collected, processed and analyzed by flow cytometry and colony assays as described in Example 6.
[00499] Treatment with Compound D, Compound E or Compound F in combination with G-CSF did not significantly alter the total number ofHSCs and progenitors in the BM compared to .vehicle control in combination with G-CSF.
}00500I The total mobilization to blood and spleen of phenotypic myeloid progenitors (L S-) and hematopoietic stem and progenitor cells (LKS+) following treatment with Compound D, Compound E or Compound F in combination with G-CSF is shown in Figures 12A-B. Treatment with Compound E or Compound F in combination with G-CSF resulted in a significant increase in the total nunlber of liSCs (LSK48-I5Q+) HSPCs (LSK48+ and LSK) and myeloid progenitors (LKS-) mobilized from the bone marrow in comparison to vehicle control in combination with G-CSF. Compound D in combination with G -CSF significantly inc reased the total number of mobilized myeloid progenitors (LKS in comparison to vehicle control in combination with G-CSF.
[00501] Treatment with Compound D, Compound E or Compound F in combination with G-CSF significantl increased in the number of CFUs mobilized from the bone marrow to the blood and spleen in comparison to vehicle eoniroi in combination with G-CSF. See Figure 13.
[00502] Treatment with Compound D or Compound E in combination with G-CSF resulted in a significant increase in the number of white blood cells per h of blood compared to vehicle control in combination with G-CSF. Furthermore, treatment with Compound O, Cornpoimd E or Compound F in combination with G-CSF resulted in a significant increase in spleen weight of approximately 2-fold compared to vehicle control in combination with G-CSF.
[00503] in summary, the results obtained with the exemplary PHD inhibitors demonstrated that these PHD inhibitors enhanced the mobilizatio of phenotypic HSCs and HSPCs induced with G-CSF, Significantly elevated levels of phenotypie HSCs and HSPCs were. mobilized using the combination of a PHD inhibitor and G-CSF compared to the use of G-CSF alone. Further, the mobilized HSCs and HSPCs were functional and retained the ability to proliferate and differentiate as demonstrated by the CFU assays,
EXAMPLE 8
COMPOUNDS H, J AND ENHANCE HSC MOBILIZATION
[00504] Additional PHD inhibitors were tested for their ability to enhance mobilization induced with G-CST treatment. Compounds H, J and K were tested in combination with G-CSF in male, 7 weeks-old C57BL/6 as described in Example 6» above. Compounds H and J were administered at 60 mg/kg, while Compound was administered at 100 mg kg; Blood and tissue samples were collected, processed and analyzed by flow cytometry and colony assays as described in Example 6.
(00505] Treatment with Compound I I, Compound J or Compound in combination with G-CSF did not significantly alter the total number of HSCs and progenitors in the BM compared to vehicle control in combination with G-CSF.
[00506] Treatment with Compound H or Compound J in combination with G-CSF resulted in a significant increase in the total number of phenotypie HSCs (LSK48-15G+) and HSPCs -(L.SK48+ and LSK) mobilized blood and spleen in comparison to vehicle control in combination with G-CSF, See Figures 14A-B.
[00507] Treatment with Compound J in combination with G-CSF resulted in a significant increase in the number of colony forming units mobilized to the blood compared to vehicl control. See Figure 15, Treatment with Compound H or Compound J in combination with G-CSF resulted in significant increase in the number of colony forming units mobilized to the spleen and total number (blood and spleen) of mobilized CFUs in comparison to vehicle control in combination with G-CSF. There was no significant .change in the number of CFUs mobilized to the spleen or total number of CFUs following treatment with Compound K or vehicle control in combinat ion with G-CSF. The lack of significant mobilization with Compound K in combination with G-CSF may reflect poor bioavailability or the use of a sub-optima! dosing regimen,
[00508] The results demonstrate that the exemplar PHD inhibitors enhanced the mobilization of phenotypie HSCs arid FISPCs induced with rhuG-CSF treatment.
Significantly elevated levels of phenotypie- HSCs..and HSPCs were mobilized using the combination of PHD inhibitor and G-CSF compared to the use of vehicle control and G-CSF. Further, the mobilized: HSCs and HSPCs were functional and retained the ability to proliferat and differentiate as demonstrated by the CFU assays.
DISCUSSION OF THE EXAMPLES
[00509] The present study is the first to explore tine in vivo effects of
pharmacological stabilization of HIF- i protein on mobilization of HSC to the blood for transplantation. The present. inventors ound that pharmacological stabilization of HIF- l a using an inhibitor of HIF prolyl hydroxylase, Compound X, synergized with both G-GSF and Plerixafor to significantly increase the number of HSC mobilized from the bone marrow to the blood. Indeed administration of Compound X in combination vviih Ό-CSF and Plerixafor tripled the number of long-term repopulating HSC per mL of blood. Similar results were found wi th other inhibitors of HIF proly l hydroxylase (Compounds A, B, C, D, E, F, F J, and K). Finally, conditional deletion of the ifla g e demonstrates that HIF- 3 a expression in HSC is critical to efficient mobilization in response to G-CSF. interestingly, the presence of FUF-Ι is also required in niche cells such as oste progemtors and osteoblasts for optimal mobilization HSC.
[Q05I0J The mechanisms by which HIF- l a stabilization synergistically enhances HSC mobilization in response to G-CSF and Plerixafor remain unclear. The results with conditional deletion of Hifla in HSCs and osteoprogenitors suggest that the dominant effect of HIF- l a protein stabilization on HSC mobilization is cell-autonomous with
microenvironrnent effects important for maximal mobilization.
MATERIALS AND METHODS
MOUSE STRAINS
[00511] C57BL 6 mice were purchased from the Australian Resource Centre. Perth Australia. All genetically modified mice had been backerossed at least 10 times into
C56BL 6 background. B6.129- Hifi atm3R¾f7j (HlfJa c* csx) m ice were purchased from the
Jackson Laboratory (Bar Harbor, Maine). SeiCreER transgenic mice expressing a tamoxifen- inducible Cre recombinase driven by a HSC specific Sd gene enhancer (1 1), B6- Gt( OSA)26Sortml (EYFP)Cos/J (Abbreviated as R26RYFP) with a ioxP-fianked STOP sequence followed by the enhanced yellow fluorescent protein reporter gene (EYFP) inserted into the genetrap ROSA26 locus, and osxYPFCre mice expressing a fusion protein of YFP and Cre recombinase under the control of the osteoprogenitor specific osterix (Sp7) gene promoter were backerossed a least 10 times into C57BL/6 background. [00512] SclCreER R26RYi;p/YFP HiflaMm mice with HSC specific tamoxifen- inducible deletion of the H if la gene and induction of the YPF reporter, and control SciCreER R26RV!'P YFI> mice with two Htf'l wild-type alleles were produced by
intercrossing the three parental strains. Likewise osxCre H;fJai)o !no mice were produced by intercrossing H7/7a °x/f,W£ mice with osxCre transgenic mice and R26RYt'iim'p strain.
Offspring were genctyped from ear clips by PGR using allele specific primers primers
MO BILIZATI ON TREATMENTS
[00513] All procedures were approved by the animal experimentation ethics committee of the University of Queensland. All experiments were performed on n ine to twelve-week old C57BL/6 male mice or genetically modified mice back-crossed at least 10 times into C57BL/6. Recombinant human G-CSF (Arogen, Thousand Oaks, CA) was injected twice daily subeutaneously at 125 ,ug/kg per injection for up to 4 consecutive days, Plerkafor (AMD3100 octohydrochlofide .Tocris Bioscience, Bristol, UK) was injected intraperitoneal!}' as a single 16 mg/kg dose corresponding to 10 mg/kg of AMD310Q base. Tissues were harvested 1 h after Pierixafor administration. Compounds X and A-F were injected daily intraperitoneal!}' at 20ing/'kg per injection with the exception of Compound D at 60 mg kg per injection. Compounds H and J were administered at 60 mg/kg and
Compound was administered at 100 mg kg. Contr l mice were injected with an equivalent volume of saline. At specified time-points, mice were anesthetized with isoflurane before cardiac puncture to harvest blood. Mice were then euthanized by cervical dislocation and BM and bones harvested.
Il UCTION OF THE SCLCREER TRANSGINE WITH TAMOXIFEN
[G05I4J SclCreER mice were gavaged daily with tamoxifen free base diluted in peanut oil containing 10% ethanol for three days t induce the SclCreER Iran sgene. G-CSF was administered for three days beginning on the final day of tamoxifen gavaging. Following Cj-CSF treatment, mice were then euthanized by cervical dislocation and BM and bones harvested.
CELL COUNTS AND COLONY ASSAYS
[00515] For myeloid colony assays. 10 pL whole blood or leukocyte suspension containing 50,000 BM or spleen cells were deposited in 35mm perri dishes and covered with I ml, 1MDM supplemented with 16% metb.yleeilulose (high viscosity Methoee.ll MC, Fluka Sigma-Aldfich, St Louis, MO) and 35% FCS, Optimal concentrations of mouse IL-3, ίί,-6 and soluble kit iigand were added as conditioned media from stably transfected BHK cell lines. Colonies were counted after ? days culture at 37°C in a humidified incubator containing 5% G(¾.
FLOW CYTOMETRY
[00516] Following flushing with PBS plus 2% CS, enriche central BM cells were pelleted at 37G g for 5 minutes at 4° C and resuspended in CDi6/CD32 hybridoma 2.4G2 supernatant to block lgGFc receptors. HSCs were stained with the biotinyiate lineage antibody cocktail (CD3, CDS, B220, CDl lb, Gt- K Terl l9) and biotiaylated CD41 together with streptavidin (SAV)-Pacifie Blue, antf-Sca-l-PECY7, anti-KlT-APC, CD48-FITC and. GDI 50-PE as previously described (17). Data were acquired on a C An (Dako Cytomation) flow cytometer and analyzed following compensation with single color controls using
FlowJo software (Tree Star, Ashland, OR).
COMPEWTIVE REIOPW.ATl.ON' ASSAYS
[00517] The content of mobilized blood samples in competiti ve repopulating BSC was determined in competitive re-population assays as previously described (12s17), The day prior to blood harvest, recipient congenic B6.SJL 0045, 1"*' female mice were lethally irradiated with 1 l.OGy in two split doses 4 hours apart, 50fiL blood samples from 6 mobilized GD45.2+ C57BL/6 donor mice were pooled within each treatment group and -'20μΙ, Mood aiiquots were taken from this pool and mixed with 200,000 competitive whole BM cells from untreated B6,SJL CD45.1 + in a total volume of 200 μΐ, saline, and injected retro- orbitally into each lethally irradiated recipient. Recipients were maintained with antibiotics for the first 3 weeks post-transplant and tail bled 8, 12. and 16 weeks post-transplant to determine CD45.2 (test donor blood) versus CD45.1. (competitive whole BM ceils)
©himerisra in myeloid, B and T lineages by flow cytometry using CD45.I-PE, CD45.2- altephycGcyanin (APC), CD3-FITC, B220-altophyeocyanin (A C)-cyanin (CY) 7, GDI lb- PBCY7, Ly6G-Paciile Blue, Content in repopulating units (RU) was calculated as previously described. (18-1 ),
STATISTICAL ANALYSES
[00518] Differences between treatment groups were analyzed using a two-tailed t- test or non-parametric Mann- Whitney depending o distribution normality. A value of p<0.05 was considered significant Dat are presented as mean ± standard deviation.
(005191 The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety. [00520] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.
[0052.1] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will theretbre apprec iate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present Invention. All such modifications and changes are intended to be included within the scope of the appended claims.
BIBLIOGRAPHY
1. To, L.B., Levesque, J.-P. & Herbert, KJE. How I treat patients who mobilize
hematopoietic stem cells poorly. -.Blood US, 4330-4540 (201 1).
2. Sanc o, 3.M., et al. Predictive factors for poor peripheral blood stem/cell mobilization and peak CD34(- ) ceil count to guide pre-emptive or immediate rescue mobilization.
Cyteth rapy 14, 823-829 (2012).
3. Foudi, A., et al Reduced retention of radioprotective hematopoietic cells within the bone marrow microenvirohment in CXCR4 - chimeric mice. Blood 107, 2243-225 i (2006).
4. Sugiyama, T,, Kohara. FL, Noda, . & Nagasawa, T, Maintenance of the
hematopoietic stem ceil pool by C.X.CL12~CXCR4 chemokine signaling in bone marrow stromal eel! niches. Immunity 25, 977-988 (2006).
5. Levesque. i.P., Hendy, j., Takamaisu, Y.« Simmons, PJ. & Bendail, L.J. Disruption of the CXCR4/CXCL12 chemotactic interaction during hematopoietic stem ceil mobilization induced by GCSF or cyclophosphamide. Clin Invest 111, 187-196
(2003).
6. Semerad, C.L., et al G-CSF potently inhibits osteoblast activity and CXCLi 2 m NA expression in the bone marrow. Blood 106, 3020-3027 (2005).
7. Broxmeyer, H.E., et al Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD310O, a CXCR4 antagonist. J Exp Med 201 , 1307-1318
(2005).
8. DiPersio, 'S.F.. et al. Phase ill prospective randomized double-blind placebo-controlled trial of plerixafor plus granulocyte colony-stimulating factor compared with placebo plus granulocyte -colony-stimulating factor for autologous stem-cell mobilization and transplantation for patients with non-Hodgkin's lymphoma. J Clin Oncol 27, 47 7-
4773 (2009).
9. DiPersio, J.F., et al. Plerixafor and G-CSF versus placebo and G-CSF to mobilize hematopoietic stem ceils for autologous stem cell transplantation in patients with multiple myeloma, Biood 113., 5720-5726 (2009).
10. Soriano, P. Generalized lacZ expressio with the RGSA26 Cre reporter strain. Nat Genet 21, 70-71 (.1 99). 11. Gothert, J.R., et al In vivo fate racing studies using the Sci stem cell enhancer: embryonic hematopoietic stem cells significantly contribute to aduit hematopoiesis. Blood 105, 2724-2732 (2005).
12. Winkler, 1G,» et al Bone marrow macrophages maintain hematopoietic stem eel! (HSC) niches and their depletion mobilizes HS s. Blood Ϊ 16, 481 5-4828 (2010).
13. Christopher. M.J, & Link, D.C. Granulocyte colony-stimulating factor induces osteoblast apopiosis and inhibits osteoblast differentiation. Journal Bone Miner Res 23, 1765-1774 (2008).
14. Katayaraa, Y., etaL Signals from the sympathetic nervous system regulate
hematopoietic stem eeO egress from bone marrow. Cell 124, 407-421 (2006).
15. Levesque, J.P., Heiwani, P.M. & Winkler, I.G. The endosteal 'osteoblastic' niche and its role in hematopoietic stem cell homing and mobilization. Leukemia 24, 1979-1992 (2030),
16. Raaijmak rs, M.PLG.P,, et aL Bone progenitor dysfunction induces myelodysplasia and secondary leukaemia. Nature 464, 852-857 (2010).
17. Barbier, V,, Winkler, I.G. & Levesque, J.P. Mobilization of hematopoietic stem cells by depleting bone marrow macrophages. Methods Mol Biol 904, 1 17-138 (2012),
18. Putton, L.E, & Scadden, D.T. Limiting factors in murine hematopoietic stem cell assays Cell Stem Cell I, 262-270 (2007),
19. Winkler, I.G., ei al Hematopoietic stem cell mobilizing agents G-CSF,
cyclophosphamide or AMD3100 have distinct mechanisms of action on bone marrow HSC niches and bone formation. Leukemia 26. 1594-1601 (2012).

Claims

WHAT IS CLAIMED IS
1. A use of a Ff!F-α potentiating agent and at least one mobilizer of
hematopoietic stein cells and/or progenitor cells for stimulating or enhancing mobilization of hematopoietic stem ceils and/or progenitor cells.
2. A use of a HIF-a potentiating agent and at least one mobilizer of
hematopoietic stem cells and qr progenitor cells for stem cell transplantation.
3. The use of claim 1 or claim 2, wherein the HIF-a potentiating agent is an inhibitor of a HIF prolyl hydroxylase (PHD).
4. The use of clai m 1 or claim 2S wherein the at least one mobilizer is selected from a colony stimulatin factor, a CXCR4 antagonist, or combination thereof.
5. The use of claim 1 or claim 2, wherein the at least one mobilizer is G-CSF.
6. The use of claim 1 or claim 2, wherein the at least one mobilizer is Plerixafor
7. The use of claim: 1 or claim 2, wherein the HI F-a potentiating agent is an inhibitor of a HIF prolyl hydroxylase, and the at least one mobilizer comprises a colony- stimulating factor.
8. The use of claim 7S wherein the colony-stimulating factor is G-CSF.
9. The use of claim 7, wherein the at least one mobilizer former comprises Plerixafor.
10. The use of claim 3, wherein the inhibitor of HIF prolyl hydroxylase is a heterocyclic carhoxarnide compound,
1 1. The use of claim 10, wherein the heterocyclic carboxamide compound is a heterocyclic carbonyl glycine.
12. The use of claim 3, wherein the Inhibitor of HIF prolyl hydroxy lase is selected from the group consisting of [(4-i-iydroxy~7-phenylsuSfan l-isoqiiinoiine-3- earbon l)~amino] -acetic acid, { [5 -( -C hi oro~phenoxy}~ 1 ~cy ano-4-hydroxy -isoqui no! i ne~ 3-carbonyl] -amino} -acetic acid, [(l -Cyano-4-hydroxy~5-phenoxy-isoqumoline-3- carbony1)-amino]-aeetic acid, [7-CyaBG~l~(2-fluoro~benzyl)-4-hyd5Oxy-iH-pyrrok>[2!>3 c]pyridine-5-carbonyl]-amino}~acetic acid, [(],3-Dicyclohexyi-6-hydroxy-2,4-dioxo-
Figure imgf000152_0001
biphenyI-4-yraiethy!)~5-hyd^
amino} -acetic acid, 2»(6-Moq3holin-4-}d-pynmidin^-yi)-4~[l52,33tria2:oi-l-yl-l}2- dihydro-pyrazol~3~one, [(4~Hydroxy-7-phe.noxy~isoqinno!m^
acid, {[4-Bydroxy~7~(4~meto acid, and { [5-{3-F!uoro-phen i)-3 iydtOxy-pyridiTie-2-carbonyl]-an"! mof-acetic acid.
13. The use of claim 3, wherein the inhibitor of Ff IF prolyl hydroxylase is a compound of Formula (1), a compound of Formula (II), a compound of Formula (III), or a compound of Formula (I V).
14. A method for mobilizing hematopoietic stem cells and/or progenitor cells from bone marrow into peripheral blood of a donor subject, the method comprising, consisting or consisting essentially of:, administering; concurrently to the subject a HIF-ct potentiating agent and at least one mobilizer of hematopoietic stem cells and/or progenitor cells in effective amounts to mobilize hematopoietic stem cells ami/or progenitor cells from the bone marrow into the peripheral blood of the subject.
15. The method of claim 14, wherein the subject has an immimocompromised condition or is at risk of acquiring an immunocomproniised condition,
16. The method of claim 14, wherein the subject has a hyperproHferatiye cell disorder, and has been, is or will be subjected to a medical treatment that gives rise or is
'likely to give rise to an immunocompromised condition.
17. The method of claim 16, wherein the hyperprollferatlve ceil disorder is a cancer or a autoimmune disease.
18. The method of claim 16, wherein the hyperproliferative cell disorder is a cancer selected from leukemia, multiple myeloma, or lymphoma.
19. The method of claim 14, further comprising collecting- or harvesting mobilized hematopoietic stem cells and/or progenitor cells from the subject.
20. The method of claim 19, further comprising eulturing and/or storing the collected or harvested mobilized hematopoietic stem ceils and/or progenitor cells.
21. The method of claim 19 or claim 20, further comprising transplanting the collected or harvested mobilized hematopoietic stem cells and/or progenitor cells into a recipient subject.
22. The method of claim 21, wherei n the recipient subj ect is the donor of the collected or harvested mobilized hematopoietic ste cells and/or progenitor ceils and the transplantatio is an autologous transplantation.
23. The method of claim 2 L wherein the recipient subject is not the donor of the collected or harvested mobilized hematopoietic stem ceils and/or progenitor cells and the transplantation is a syngeneic, allogeneic or xenogeneic transplantation.
24. The method of any one of claims 21 to 23, wherein the recipient subject has an immunocompromised condition or has been exposed to a medical treatment that results in an immunocompromised condition.
25. The method of any one of claims 21 to 24, further comprising administering to the recipient subject prior to, simultaneously with, or after the stem ceil
transplantation a HIF-a potentiating agent and a mobilizer of hematopoietic stern cells and/or progenitor ceils in effective amounts to mobilize hematopoietic stem cells and/or progenitor cells from the bo e marrow into the peripheral blood of the subject
26. The method of claim 14,, wherein the HI F-a potentiating agent is an inhibitor of H1F prolyl hydroxylase (PHD).
27. The method of claim 26, wherein the inhibitor of HIF prolyl hydroxylase is a heterocyclic carboxamide compound.
28. The method of claim 27, wherein the heterocyclic earhoxamide compound, is a heterocyclic carbohyl glycine
29. The method of claim 26, wherein the inhibitor of HIF prolyl hydroxylase is selected from the 'group consisting of [(4-Hydroxy-7~phenyisuifattyl-isoqu{ROline-3- carbo!iyi) arnino] -acetic acid, {[5-(4~Ch:loro-phenoxY)~l~cyano-4-hydroxy-isoquinoIine- 3-carbonyi]-amino}-acetic acid, [(l-Cyano-4-hydroxy-5-phenoxy-isoquinoiine-3- carbon !)-amin o] -acetic acid, { [7-Cyano- 1 -(2-fiuoiO-benz 'l)-4-hydroxy- 1 H-pyrrolo[2}3- c]pyridine-5-carbonyi]-amino}-aeetie ae.id5 [(i^-Dicyclohexyl-d-hydroxy-S^-dioxo- l ^J^-tetrahydro-pyflmidme-S-carbonylJ-aminoj-acetie acid, {[2-(3,4'-Difluoro- b i phenyl-4-y Irnet hy l)-5 -hydroxy-6-i5opropy 1-3 -oxo-2, 3 -d iliydro-p>T
amino} -acetic acid, .2-(6-Morpholin^-yl-pyrimidin .-y'I)^-(l-2 ]triaz I-i-y^iil2- dihydro-pyrazoi-3-one, [(4-Hydroxy-7-phenoxy-isoc]uinoiine-3-earbonyl)-ainino]-aceiic acid., {[4 iydroxy-7«(4-methoxy-phenoxy)~isoquinolm^ acid, and {[5-(3-Fluoro-pheny!}-3-hydroxy~pyridine--2-carbonyl]-amir!o}-acetic acid. 30, The method of claim 26? wherein the inhibitor of HIF prolyl hydroxylase is a compound of Formula (i), a compound of Formula (H)s a compound of Formula (Oi), or a compound of Formula (IV),
31 , The method of claim 1 , wherein the at least one mobilizer is selected from a colony stimulating factor, a CXCR4 antagonist, or a combination thereof.
32, The method of c!aim 14, wherein the at least one niobilizer is G-CSF.
33, The method of claim 14, wherein the at least one mobilizer is Plerixa&r.
34, The method of claim 14, wherein the HiF-a potentiating agent is an inhibitor of a HIF prolyl hydroxylase, and the at least one mobilizer comprises a colony- stimulating factor.
35, The method of claim 34, wherein the colony-stimulating factor is G-CSF.
36, The method of claim.35. wherein the at least one mobilizer further comprises Plerixafor.
37, The method of claim 14. wherein the rnobilizer(s) and the RlF- potentiating agent are administered simultaneousl to the subject.
38, The method of claim 14, wherein the HiF-a potentiating agent is administered to the subject prior to administration of the mobilizer.
39, The method of claim 14, wherein the HlF-a potentiating agent is administered after administration of the mobilizer to the subject.
40, A method for increasing the dose of a medicament in a subject, wherein the medicament results or increases the risk of developing an immunocompromised condition, the method comprising, consisting or consisting essentially of: administering coneurrently to the subject the medicament in a dose that ordinarily induces side effects, together with at least one mobilizer of hematopoietic stem cells and/or progenitor cells and a HIF- potentiating agent in amounts effective for inhibiting or preventing the induction of those side effects.
4.1. The method of claim 40, wherein the HIF-a potentiating agent is an inhibitor of a HIF prolyl hydroxylase (PHD).
42, The method of claim 40, wherein the at least one mobilizer is selected from a colony stimulating factor, a CXCR.4 antagonist, or a combinatio thereof.
43 , The method of claim 40, wherein the at least one mobilizer is G-CSF,
44. The method of claim 40. wherein the at least one mobilizer is Plerixafor.
45. The method of claim 40, wherein the HI F-a potentiating agent is an inhibitor of a HIF prolyl hydroxy lase, and the at least one mobilizer comprises a colony- stimulating factor.
46. The method of claim 45, wherein the colony-stimulating factor is G-CSF.
47. The method of claim 46, wherein the at least one mobil izer further comprises Plerixafor.
48. Use of a HIF- potentiating agent for stimulating or enhancing mobilization of hematopoietic stem ceils and/or progenitor cells.
49. Use of a HIF-α. potentiating agent for stem cell transplantation.
50. The use of claim 48 or claim 49, wherein the Hi F-oc potentiating agent is used for stimulating or enhancing mobilization of hematopoietic stem cells and/or progenitor ceils in donor subject that is concurrently administered at least one mobilizer of hematopoietic stem cells and/or progenitor cells.
51. A method for mobilizing hematopoietic stem cells and/or progenitor cells from bone marrow into peripheral blood of a donor subject, the method comprising, administering to the subject a .HIF-a potentiating agent in an effective amount to mobilize hematopoietic stem cells and/or progenitor cells from the bone marrow into the peripheral blood of th subj ect.
52. The method . of claim ! . wherein the donor subject is concurrently administered at least one mobilizer of hematopoietic stem cells and or progenitor cells,
53. The use of claim 48, claim 49, or claim 50, or the method of claim 51 or claim 52. wherein the HIF-a potentiating agent is. an inhibitor of HIF proly l hydroxylase and the. at least one mobilizer of hematopoietic stem cells and/or progenitor cells is a colon stimulating factor.
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