US20090264382A1 - Methods for Treating Hematopoietic Neoplasms - Google Patents

Methods for Treating Hematopoietic Neoplasms Download PDF

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US20090264382A1
US20090264382A1 US12/276,064 US27606408A US2009264382A1 US 20090264382 A1 US20090264382 A1 US 20090264382A1 US 27606408 A US27606408 A US 27606408A US 2009264382 A1 US2009264382 A1 US 2009264382A1
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David Chaplin
Shahin Rafii
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    • 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
    • A61K31/66—Phosphorus compounds
    • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
    • A61K31/6615—Compounds having two or more esterified phosphorus acid groups, e.g. inositol triphosphate, phytic acid
    • 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
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • 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
    • A61K31/66—Phosphorus compounds
    • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
    • 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
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • 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
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • Adhesion of leukemic cells to stromal cells has been shown to confer increased resistance to chemotherapeutic agents and diminish the rate of apoptosis of the leukemic cells.
  • This process named cell adhesion-mediated drug resistance (CAM-DR)
  • CAM-DR cell adhesion-mediated drug resistance
  • VLA4 very late antigen 4, ⁇ 4 ⁇ 1 integrin-positive myeloid cells
  • VCAM-1+ stromal cells is an important mediator of CAM-DR.
  • expression of VLA4 by leukemic cells portends a poor prognosis and a decreased five-year survival rate. Therefore, identification of novel anti-leukemic agents that inhibit interaction of leukemic cells with vascular cells provides novel strategies to target organ-infiltrating, angiogenesis-dependent leukemias.
  • hematopoietic neoplasms are closely associated with endothelium, supporting establishment of neo-vessels by elaboration of angiogenic factors.
  • leukemic cells may activate endothelial cells by releasing pro-inflammatory factors, including interleukin-1 (IL-1), facilitating invasion into tissues and formation of infiltrative organ disease or subcutaneous tumors, namely chloromas, thereby establishing chemotherapy-refractory leukemic minimal residual disease.
  • IL-1 interleukin-1
  • Combretastatin-A4 a novel tubulin-destabilizing agent, was isolated from the South African tree Combreturn caffrum .
  • Combretastatin-A4 binds to tubulin at the same site as colchicine does, but with even higher affinity.
  • Its pro-drug, combretastatin-A4 phosphate (CA4P) induces rapid microtubule depolymerization and vascular shutdown in subcutaneous solid tumors causing tumor necrosis at concentrations well below the maximum tolerated dose.
  • CA4P also can induce apoptosis of the endothelial cells by disengaging VE-cadherin interaction.
  • CA4P may not only target rapidly proliferating leukemic cells directly, but also diminish interaction of the leukemic cells with activated endothelial cells, thereby preventing establishment of a perivascular nidus for leukemic chloromas.
  • CA4P at low, non-toxic doses, surprisingly induces rapid cell death of non-adherent leukemic cells through caspase activation, mitochondria destabilization and accumulation of reactive oxygen species (ROS), accompanied by the release of pro-apoptotic mitochondrial membrane proteins.
  • ROS reactive oxygen species
  • single-agent CA4P treatment is effective in eradicating both circulating, and vascular-adherent leukemic cells in subcutaneous and systemic mouse models of AML, without affecting normal hematopoiesis.
  • CA4P-treated mice had significantly prolonged survival and showed a drastic reduction of detectable leukemic cells in the marrow and peripheral circulation, and significantly decreased leukemic organ infiltration.
  • CA4P decreases expression of VCAM-1 on endothelial cells both in vitro and in vivo, thereby decreasing leukemic cell adhesion to the vascular cells, thereby reversing drug resistance. Therefore, CA4P delivered in combination with chemotherapeutic agents represents a promising novel therapeutic approach to treat hematopoietic neoplasms.
  • One aspect of the invention provides methods of treating a hematopoietic neoplasm comprising administering a therapeutically effective amount of combretastatin A-4 phosphate (CA4P), or a pharmaceutically acceptable salt thereof, to a subject having a hematological malignancy.
  • CA4P combretastatin A-4 phosphate
  • Another aspect of the invention provides the use of combretastatin A-4 phosphate, or a pharmaceutically acceptable salt thereof, for the treatment of a hematopoietic neoplasm.
  • the invention also contemplates use of combretastatin A-4 phosphate and salts thereof in the preparation of a medicament for use in treating a hematopoietic neoplasm.
  • Yet another aspect of the invention provides methods of treating a non-solid tumor comprising administering a therapeutically effective amount of combretastatin A-4 phosphate or a pharmaceutically acceptable salt thereof, to a subject suffering from non-solid tumor.
  • FIG. 1 illustrates combretastatin A-4 phosphate (CA4P) blocking leukemic cell growth of a panel of leukemic cell lines.
  • CA4P combretastatin A-4 phosphate
  • FIG. 2 illustrates CA4P induction of G2/M arrest.
  • FIG. 3 illustrate CA4P induction of DNA fragmentation in leukemic cells.
  • FIG. 4 demonstrates CA4P inducing cell death without evidence of necrosis.
  • FIG. 5 demonstrates that CA4P mediated apoptosis of leukemic cells is partially reversed by the caspase inhibitor Q-VD.
  • FIG. 6 illustrates the decrease in mitochondrial transmembrane potential of leukemic cells in response to exposure to CA4P.
  • FIG. 7 demonstrates that Combretastatin A-4 phosphate (CA4P) induced cell death can be partially prevented by co-incubation with ascorbic acid, an ROS scavenger, in a concentration-dependent manner.
  • CA4P Combretastatin A-4 phosphate
  • FIG. 8 demonstrates early ROS accumulation during CA4P treatment.
  • FIG. 9 illustrates that CA4P mediated apoptosis of leukemic cells is reversed by inhibiting ROS and caspase pathways.
  • FIG. 10 provides quantification of the microvessel density in HL60 tumor sections after CA4P treatment.
  • FIG. 11 demonstrates that combretastatin A-4 phosphate (CA4P) improves survival of xenotransplanted mice with human leukemia cells.
  • CA4P combretastatin A-4 phosphate
  • FIG. 12 illustrates the CA4P-mediated decrease in leukemic cell circulation in the peripheral blood and engraftment in the bone marrow, spleen, liver and lung.
  • FIG. 13 demonstrates that CA4P inhibits IL-1-mediated upregulation of VCAM-1 in HUVECs.
  • FIGS. 14A and 14B demonstrate that CA4P reduces leukemic cell adhesion to HUVECs.
  • FIGS. 15A and 15B illustrate that leukemic cells adherent to HUVECs are more resistant to CA4P.
  • a “therapeutically effective amount” of combretastatin A-4 phosphate (CA4P), or a therapeutically acceptable salt thereof, according to the present invention is intended to mean that amount of the CA4P that will inhibit the growth of, or retard cancer, or kill malignant cells, and cause the regression and palliation of cancer, i.e., reduce the proliferation rate and/or the number of malignant cells within the body.
  • CA4P combretastatin A-4 phosphate
  • Other desired anti-tumor effects include, without limitation, the modulation of neoplasm growth rates, the enhancement of necrosis or hypoxia in malignant cells, reduced retention of CEPs and other pro-angiogenic cells, amelioration or minimization of the clinical impairment or symptoms of hematopoietic neoplasms, extending the survival of the subject beyond that which would otherwise be expected in the absence of such treatment, and the prevention of neoplastic growth in an animal lacking any neoplasm formation prior to administration, i.e., prophylactic administration.
  • the terms “modulate”, “modulating” or “modulation” refer to changing the rate at which a particular process occurs, inhibiting a particular process, reversing a particular process, and/or preventing the initiation of a particular process. Accordingly, if the particular process is neoplastic growth or metastasis, the term “modulation” includes, without limitation, decreasing the rate at which neoplastic growth and/or metastasis occurs; inhibiting neoplastic growth and/or metastasis, including tumor re-growth following treatment with an anticancer agent; reversing neoplastic growth and/or metastasis (including tumor shrinkage and/or eradication) and/or preventing neoplastic growth and/or metastasis.
  • Hematopoietic neoplasm refers to a cell proliferative disorder arising from cells of the hematopoietic lineage.
  • hematopoiesis is the physiological process whereby undifferentiated cells or stem cells develop into various cells found in the peripheral blood.
  • hematopoietic stem cells typically found in the bone marrow, undergo a series of cell divisions to form multipotent progenitor cells that commit to two main developmental pathways: the lymphoid lineage and the myeloid lineage.
  • the committed progenitor cells of the myeloid lineage differentiate into three major sub-branches comprised of the erythroid, megakaryocyte, and granulocyte/monocyte developmental pathways.
  • Neoplasms of hematopoietic cells can involve cells of any phase of hematopoiesis, including hematopoietic stem cells, multipotent progenitor cells, oligopotent committed progenitor cells, precursor cells, and mature differentiated cells.
  • the categories of hematopoietic neoplasms can generally follow the descriptions and diagnostic criteria employed by those of skill in the art (see, e.g., International Classification of Disease and Related Health Problems (ICD 10), World Health Organization (2003)).
  • Hematopoietic neoplasms can also be characterized based on the molecular features, such as cell surface markers and gene expression profiles, cell phenotype exhibited by the aberrant cells, and/or chromosomal aberrations (e.g., deletions, translocations, insertions, etc.) characteristic of certain hematopoietic neoplasms, such as the Philadelphia chromosome found in chronic myelogenous leukemia. Other classifications include National Cancer Institute Working Formulation (Cancer, 1982, 49:2112-2135) and Revised European-American Lymphoma Classification (REAL).
  • hematopoietic neoplasm includes, but is not limited to, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, and myeloplastic syndrome.
  • ALL acute lymphoblastic leukemia
  • AML acute myelogenous leukemia
  • CML chronic myelogenous leukemia
  • CLL chronic lymphocytic leukemia
  • hairy cell leukemia Hodgkin's disease
  • non-Hodgkin's lymphoma multiple myeloma
  • myeloplastic syndrome myeloplastic syndrome
  • Myeloid neoplasm refers to proliferative disorder of cells of the myeloid lineage of hematopoiesis. Neoplasms can arise from hematopoietic stem cells, myeloid committed progenitor cells, precursor cells, and terminally differentiated cells. Myeloid neoplasms can be subdivided based on the phenotypic attributes of the aberrant cells or the differentiated state from which the abnormal cells arise. Subdivisions include, among others, myeloproliferative diseases, myelodysplastic/myeloproliferative diseases, myelodysplastic syndromes, acute myeloid leukemia, and acute biphenotypic leukemia.
  • Lymphoid neoplasm refers a proliferative disorder involving cells of the lymphoid lineage of hematopoiesis. Lymphoid neoplasms can arise from hematopoietic stem cells as well as lymphoid committed progenitor cells, precursor cells, and terminally differentiated cells. These neoplasms can be subdivided based on the phenotypic attributes of the aberrant cells or the differentiated state from which the abnormal cells arise. Subdivisions include, among others, B cell neoplasms, T cell neoplasms, NK cell neoplasms, and Hodgkin's lymphoma.
  • Committed progenitor cells of the lymphoid lineage develop into the B cell pathway, T cell pathway, or the non-T/B cell pathway. Similar to the myeloid lineage, an additional lymphoid pathway appears to give rise to dendritic cells involved in antigen presentation.
  • the B cell progenitor cell develops into a precursor B cell (pre-B), which differentiates into B cells responsible for producing immunoglobulins.
  • Progenitor cells of the T cell lineage differentiate into precursor T cells (pre-T) that, based on the influence of certain cytokines, develop into cytotoxic or helper/suppressor T cells involved in cell mediated immunity.
  • Non-T/B cell pathway leads to generation of natural killer (NK) cells.
  • hematopoietic neoplasm includes, but is not limited to, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, and myeloplastic syndrome.
  • ALL acute lymphoblastic leukemia
  • AML acute myelogenous leukemia
  • CML chronic myelogenous leukemia
  • CLL chronic lymphocytic leukemia
  • hairy cell leukemia Hodgkin's disease
  • non-Hodgkin's lymphoma multiple myeloma
  • myeloplastic syndrome myeloplastic syndrome
  • salts that are physiologically tolerated by a subject. Such salts are typically prepared from an inorganic and/or organic acid. Examples of suitable inorganic acids include, but are not limited to, hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, and phosphoric acid. Organic acids may be aliphatic, aromatic, carboxylic, and/or sulfonic acids.
  • Suitable organic acids include, but are not limited to, formic, acetic, propionic, succinic, camphorsulfonic, citric, fumaric, gluconic, lactic, malic, mucic, tartaric, para-toluenesulfonic, glycolic, glucuronic, maleic, furoic, glutamic, benzoic, anthranilic, salicylic, phenylacetic, mandelic, pamoic, methanesulfonic, ethanesulfonic, pantothenic, benzenesulfonic (besylate), stearic, sulfanilic, alginic, galacturonic, and the like.
  • salts include alkali metal cations (such as Na, K, Li), alkali earth metal salts (such as Mg or Ca), or organic amine salts (such as those disclosed in PCT International Application Nos. WO 02/22626 or WO 00/48606 and U.S. Pat. Nos. 6,855,702 and 6,670,344, which are incorporated herein by reference in their entireties).
  • Particularly preferred salts include organic amine salts such tromethamine (TRIS) and amino acid salts such as histidine.
  • TMS tromethamine
  • Other exemplary salts that can be synthesized using the methods of the invention include those described in U.S. Pat. No. 7,018,987, which is incorporated by reference herein.
  • Adhesion of leukemic cells to vascular cells may confer resistance to chemotherapeutic agents. Therefore, disruption of leukemic cell cytoskeletal stability and interference with vascular cell interactions should promote leukemic cell death. Indeed, as disclosed in greater detail below, low and non-toxic doses of combretastatin-A4 phosphate (CA4P) inhibit leukemic cell proliferation in vitro and induce mitotic arrest and cell death. Treatment of acute myeloid leukemias (AMLs) with CA4P leads to disruption of mitochondrial membrane potential, release of pro-apoptotic mitochondrial membrane proteins (MMPs) and DNA fragmentation, resulting in cell death in part through a caspase-dependent manner.
  • AMLs acute myeloid leukemias
  • MMPs pro-apoptotic mitochondrial membrane proteins
  • CA4P rapidly increases intracellular reactive oxygen species (ROS), and antioxidant treatment imparts partial protection from cell death, suggesting that ROS accumulation contributes to CA4P-induced cytotoxicity in AML.
  • ROS reactive oxygen species
  • CA4P inhibits proliferation and circulation of leukemic cells and diminishes the extent of peri-vascular leukemic infiltrates, thereby prolonging the survival of xenotransplanted mice, without inducing hematological toxicity.
  • CA4P decreases the interaction of leukemic cells with neo-vessels by down-regulating the expression of adhesion molecule, VCAM-1, thereby augmenting leukemic cell death.
  • combretastatin A-4 phosphate provides for an effective means to treat refractory organ-infiltrating leukemias.
  • one aspect of the present invention provides a method of treating a hematopoietic neoplasm, the method comprising administering a therapeutically effective amount of combretastatin A-4 phosphate, or a pharmaceutically acceptable salt thereof, to a mammal suffering from a hematopoietic neoplasm.
  • the pharmaceutically acceptable salt is a tromethamine salt of combretastatin A-4 phosphate.
  • combretastatins were initially identified in the 1980's as potent inhibitors of tubulin polymerization.
  • Combretastatin A-4 has been shown to bind a site at or near the colchicine binding site on tubulin with high affinity.
  • combretastatins are potent cytotoxic agents against a diverse spectrum of tumor cell types in culture.
  • Phosphate prodrugs of combretastatin A-4 were subsequently developed to combat problems with aqueous insolubility.
  • CA4P has also been shown to cause a rapid and acute shutdown of the blood flow to tumor tissue that is separate and distinct from the anti-proliferative effects of the agents on tumor cells themselves.
  • a number of studies have shown that combretastatins cause extensive shut-down of blood flow within the tumor microvasculature, leading to secondary tumor cell death (Dark et al., Cancer Res., 57: 1829-34, (1997); Chaplin et al., Anticancer Res., 19: 189-96, (1999); Hill et al., Anticancer Res., 22(3):1453-8 (2002); Holwell et al., Anticancer Res., 22(2A):707-11, (2002).
  • Blood flow to normal tissues is generally far less affected by CA4P than blood flow to tumors, although blood flow to some organs, such as spleen, skin, skeletal muscle and brain, can be inhibited (Tozer et al., Cancer Res., 59: 1626-34 (1999)).
  • combretastatin A-4 phosphate denotes a compound of the Formula I:
  • One implementation comprises use of a compound of Formula II,
  • each OR 1 and OR 2 independently is selected from OH, —O ⁇ QH + and —O ⁇ M + , wherein M + is a monovalent or divalent metal cation, and Q is:
  • the combretastatin A-4 phosphate salt is a compound of Formula I, wherein one of OR 1 and OR 2 is hydroxyl and the other is —O ⁇ QH + and Q is tris(hydroxymethyl)amino methane (tromethamine or “TRIS”).
  • Another implementation comprises use of a compound of Formula II, wherein R 1 and R 2 are O ⁇ M + , wherein each M + independently is an aliphatic organic amine, an alkali metal, a transition metal, a heteroarylene, a heterocyclyl, a nucleoside, a nucleotide, an alkaloid, an amino sugar, an amino nitrile, or an nitrogenous antibiotic.
  • Yet another implementation comprises use of a compound of Formula II, wherein R 1 and R 2 are O ⁇ M + and each M + , independently, is sodium, TRIS, histidine, ethanolamine, diethanolamine, ethylenediamine, diethylamine, triethanolamine, glucamine, N-methylglucamine, ethylenediamine, 2-(4-imidazolyl)-ethylamine, choline, or hydrabamine.
  • each M + is sodium.
  • the method of the invention can further comprise co-administering a chemotherapeutic agent, such a cytosine arabinoside (Ara-C), to the subject.
  • a chemotherapeutic agent such as a cytosine arabinoside (Ara-C)
  • “Co-administration” or “co-administering” can be in the form of a single formulation (combining, for example, CA4P and a Ara-C with pharmaceutically acceptable excipients, optionally segregating the two active ingredients in different excipient mixtures designed to independently control their respective release rates and durations) or by independent administration of separate formulations containing the active agents.
  • Co-administration further includes concurrent administration (e.g.
  • CA4P and a Ara-C at the same time
  • time varied administration administration of CA4P at a time different from that of the Ara-C
  • the chemotherapeutic agent e.g., Ara-C
  • the chemotherapeutic agent is Ara-C, etoposide, thioguanine or cyclophosphamide.
  • chemotherapeutic agent is a chemical compound useful in the treatment of cancer.
  • examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® (cyclophosphamide); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan
  • dynemicin including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL®) and deoxydoxor
  • non-solid tumors are quite distinct from non-solid tumors, such as those found in hematopoietic-related cancers.
  • non-solid tumors include leukemias, such as myeloid leukemias and lymphoid leukemias, myelomas, and lymphomas.
  • the non-solid tumor cell is a hematopoietic neoplasm, which is aberrant growth of cells of the hematopoietic system.
  • Hematopoietic malignancies can have its origins in pluripotent stem cells, multipotent progenitor cells, oligopotent committed progenitor cells, precursor cells, and terminally differentiated cells involved in hematopoiesis.
  • hematopoietic stem cells which have the ability for self renewal. For instance, cells capable of developing specific subtypes of acute myeloid leukemia (AML) upon transplantation display the cell surface markers of hematopoietic stem cells, implicating hematopoietic stem cells as the source of leukemic cells.
  • AML acute myeloid leukemia
  • hematopoietic neoplasms often originate from stem cells, committed progenitor cells or more terminally differentiated cells of a developmental lineage can also be the source of some leukemias.
  • the hematopoietic malignancy treated by the method of the invention is acute lymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML).
  • Hematopoietic neoplasms differ from solid tumors in being capable of circulating and having access to various organs through interaction with activated vascular cells. Indeed, some hematopoietic neoplasms may adhere to vascular cells, establishing perivascular infiltrates, and as such may be endowed with a unique mechanism of resistance to chemotherapy. Both circulating and vascular-adherent hematopoietic neoplasms require cytoskeletal stability to maintain mitochondrial and cellular function and avoid cell death. Low and non-toxic doses of combretastatin A-4 phosphate can selectively induce apoptosis of circulating and vascular-bound leukemic cells, leading to cell death.
  • combretastatin A-4 phosphate is effective in treating hematopoietic neoplasms, as demonstrating by its ability to target hematopoietic neoplasms in vitro and in vivo and to eradicate circulating, marrow- and organ-resident vascular-adherent hematopoietic neoplasms.
  • the hematopoietic neoplasm treated is a lymphoid neoplasm, where the abnormal cells are derived from and/or display the characteristic phenotype of cells of the lymphoid lineage.
  • Lymphoid neoplasms can be subdivided into B-cell neoplasms, T and NK-cell neoplasms, and Hodgkin's lymphoma.
  • B-cell neoplasms can be further subdivided into precursor B-cell neoplasm and mature/peripheral B-cell neoplasm.
  • Exemplary B-cell neoplasms are precursor B-lymphoblastic leukemia/lymphoma (precursor B-cell acute lymphoblastic leukemia) while exemplary mature/peripheral B-cell neoplasms are B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone B-cell lymphoma, hairy cell leukemia, plasma cell myeloma/plasmacytoma, extranodal marginal zone B-cell lymphoma of MALT type, nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle-cell lymphoma, diffuse large B-cell lymphoma, mediastinal large B-cell lymphoma, primary effusion lymphoma, and Burkitt's lymphoma/Burkitt cell leukemia.
  • T-cell and NK-cell neoplasms are further subdivided into precursor T-cell neoplasm and mature (peripheral) T-cell neoplasms.
  • Exemplary precursor T-cell neoplasm is precursor T-lymphoblastic lymphoma/leukemia (precursor T-cell acute lymphoblastic leukemia) while exemplary mature (peripheral) T-cell neoplasms are T-cell prolymphocytic leukemia T-cell granular lymphocytic leukemia, aggressive NK-cell leukemia, adult T-cell lymphoma/leukemia (HTLV-1), extranodal NK/T-cell lymphoma, nasal type, enteropathy-type T-cell lymphoma, hepatosplenic gamma-delta T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, Mycosis fungoides/Sezary syndrome, Anaplastic large-cell
  • the third member of lymphoid neoplasms is Hodgkin's lymphoma, also referred to as Hodgkin's disease.
  • Exemplary diagnosis of this class that can be treated with the compounds include, among others, nodular lymphocyte-predominant Hodgkin's lymphoma, and various classical forms of Hodgkin's disease, exemplary members of which are Nodular sclerosis Hodgkin's lymphoma (grades 1 and 2), Lymphocyte-rich classical Hodgkin's lymphoma, Mixed cellularity Hodgkin's lymphoma, and Lymphocyte depletion Hodgkin's lymphoma.
  • any of the lymphoid neoplasms can be treated with the combretastatin A-4 phosphate, or a pharmaceutically acceptable salt thereof.
  • the hematopoietic neoplasm treated is a myeloid neoplasm.
  • This group comprises a large class of cell proliferative disorders involving or displaying the characteristic phenotype of the cells of the myeloid lineage.
  • Myeloid neoplasms can be subdivided into myeloproliferative diseases, myelodysplastic/myeloproliferative diseases, myelodysplastic syndromes, and acute myeloid leukemias.
  • Exemplary myeloproliferative diseases are chronic myelogenous leukemia (e.g., Philadelphia chromosome positive (t(9;22)(qq34;q11)), chronic neutrophilic leukemia, chronic eosinophilic leukemia/hypereosinophilic syndrome, chronic idiopathic myelofibrosis, polycythemia vera, and essential thrombocythemia.
  • Exemplary myelodysplastic/myeloproliferative diseases are chronic myelomonocytic leukemia, atypical chronic myelogenous leukemia, and juvenile myelomonocytic leukemia.
  • Exemplary myelodysplastic syndromes are refractory anemia, with ringed sideroblasts and without ringed sideroblasts, refractory cytopenia (myelodysplastic syndrome) with multilineage dysplasia, refractory anemia (myelodysplastic syndrome) with excess blasts, 5q-syndrome, and myelodysplastic syndrome.
  • any of the myeloid neoplasms can be treated with combretastatin A-4 phosphate, or a pharmaceutically acceptable salt thereof.
  • combretastatin A-4 phosphate can be used to treat acute myeloid leukemias (AML), which represent a large class of myeloid neoplasms having its own subdivision of disorders. These subdivisions include, among others, AMLs with recurrent cytogenetic translocations, AML with multilineage dysplasia, and other AML not otherwise categorized.
  • AML acute myeloid leukemias
  • Exemplary AMLs with recurrent cytogenetic translocations include, among others, AML with t(8;21)(q22;q22), AML1 (CBF-alpha)/ETO, Acute promyelocytic leukemia (AML with t(15;17)(q22;q11-12) and variants, PML/RAR-alpha), AML with abnormal bone marrow eosinophils (inv(16)(p13q22) or t(16;16)(p13;q11), CBFb/MYH11X), and AML with 11q23 (MLL) abnormalities.
  • Exemplary AML with multilineage dysplasia are those that are associated with or without prior myelodysplastic syndrome.
  • AML minimally differentiated AML without maturation
  • AML with maturation AML with maturation
  • acute myelomonocytic leukemia acute monocytic leukemia
  • acute erythroid leukemia acute megakaryocytic leukemia
  • acute basophilic leukemia acute panmyelosis with myelofibrosis.
  • compositions useful for treating a hematopoietic neoplasm in a warm-blooded animal which composition comprises combretastatin A-4 phosphate, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable excipient.
  • the composition is prepared in accordance with known formulation techniques to provide a composition suitable for oral, topical, transdermal, rectal, by inhalation, parenteral (intravenous, intramuscular, or intraperitoneal) administration, and the like.
  • parenteral intravenous, intramuscular, or intraperitoneal
  • the pharmaceutical composition further comprises a chemotherapeutic agent, such as Ara-C, etoposide, thioguanine or cyclophosphamide.
  • Unit doses or multiple dose forms are contemplated, each offering advantages in certain clinical settings.
  • the unit dose would contain a predetermined quantity of active compound calculated to produce the desired effect(s) in the setting of treating cancer.
  • the multiple dose form may be particularly useful when multiples of single doses, or fractional doses, are required to achieve the desired ends. Either of these dosing forms may have specifications that are dictated by or directly dependent upon the unique characteristic of the particular compound, the particular therapeutic effect to be achieved, and any limitations inherent in the art of preparing the particular compound for treatment of cancer.
  • a unit dose will contain a therapeutically effective amount sufficient to treat a hematopoietic neoplasm in a subject and may contain from about 1.0 to 1000 mg of compound, for example about 50 to 500 mg.
  • the combretastatin A-4 phosphate, or a pharmaceutically acceptable salt thereof preferably is administered parenterally, e.g., intravenously, intramuscularly, intravenously, subcutaneously, or intraperitoneally.
  • the carrier or excipient or excipient mixture can be a solvent or a dispersive medium containing, for example, various polar or non-polar solvents, suitable mixtures thereof, or oils.
  • carrier or excipient means a pharmaceutically acceptable carrier or excipient and includes any and all solvents, dispersive agents or media, coating(s), antimicrobial agents, iso/hypo/hypertonic agents, absorption-modifying agents, and the like.
  • Solutions of the compound may be prepared in suitable diluents such as water, ethanol, glycerol, liquid polyethylene glycol(s), various oils, and/or mixtures thereof, and others known to those skilled in the art.
  • suitable diluents such as water, ethanol, glycerol, liquid polyethylene glycol(s), various oils, and/or mixtures thereof, and others known to those skilled in the art.
  • the pharmaceutical forms suitable for injectable use include sterile solutions, dispersions, emulsions, and sterile powders.
  • the final form must be stable under conditions of manufacture and storage. Furthermore, the final pharmaceutical form must be protected against contamination and must, therefore, be able to inhibit the growth of microorganisms such as bacteria or fungi.
  • a single intravenous or intraperitoneal dose can be administered. Alternatively, a slow long term infusion or multiple short term daily infusions may be utilized, typically lasting from 1 to 8 days. Alternate day or dosing once every several days may also be utilized.
  • Sterile, injectable solutions are prepared by incorporating a compound in the required amount into one or more appropriate solvents to which other ingredients, listed above or known to those skilled in the art, may be added as required.
  • Sterile injectable solutions are prepared by incorporating the compound in the required amount in the appropriate solvent with various other ingredients as required. Sterilizing procedures, such as filtration, then follow.
  • dispersions are made by incorporating the compound into a sterile vehicle which also contains the dispersion medium and the required other ingredients as indicated above. In the case of a sterile powder, the preferred methods include vacuum drying or freeze drying to which any required ingredients are added.
  • the final form must be sterile and must also be able to pass readily through an injection device such as a hollow needle.
  • the proper viscosity may be achieved and maintained by the proper choice of solvents or excipients.
  • the use of molecular or particulate coatings such as lecithin, the proper selection of particle size in dispersions, or the use of materials with surfactant properties may be utilized.
  • Prevention or inhibition of growth of microorganisms may be achieved through the addition of one or more antimicrobial agents such as chlorobutanol, ascorbic acid, parabens, thermerosal, or the like. It may also be preferable to include agents that alter the tonicity such as sugars or salts.
  • antimicrobial agents such as chlorobutanol, ascorbic acid, parabens, thermerosal, or the like. It may also be preferable to include agents that alter the tonicity such as sugars or salts.
  • Another aspect of this invention is a method for treating a hematopoietic neoplasm in a warm-blooded animal, which method comprises administering a therapeutically effective amount of combretastatin A-4 phosphate, or a pharmaceutically acceptable salt thereof.
  • the combretastatin A-4 phosphate, or a pharmaceutically acceptable salt thereof can be administered to an appropriate subject in a therapeutically effective dose by a medically acceptable route of administration such as orally, parentally (e.g., intramuscularly, intravenously, subcutaneously, interperitoneally), transdermally, rectally, by inhalation and the like.
  • body surface area may be approximately determined from the height and weight of an individual (see, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y. pp. 537-538 (1970)).
  • a suitable dose range is from 1 to 1000 mg of equivalent per m 2 body surface area of a compound of the invention, for instance from 50 to 500 mg/m 2 .
  • Another important feature of the method provided by the present invention relates to the relatively low apparent overall toxicity of the derivatives administered in accordance with the teachings herein.
  • Overall toxicity can be judged using various criteria. For example, loss of body weight in a subject over 10% of the initially recorded body weight (i.e., before treatment) can be considered as one sign of toxicity.
  • loss of overall mobility and activity and signs of diarrhea or cystitis in a subject can also be interpreted as evidence of toxicity.
  • CA4P Combretastatin A4 Phosphate
  • CA4P at concentrations as low as 1 nM initiated cell death of non-adherent, anchorage-independent AML cells in vitro, with the IC 50 ranging from 2.5 to 5 nM ( FIG. 1 ).
  • the majority of the AML cell lines tested was sensitive to CA4P at a concentration of 2.5 nM or less. All leukemic cell lines, as well as a recently established primary leukemic cell line (R81) 16 were sensitive at low doses of CA4P ( ⁇ 10 nM).
  • CA4P induces G2/M arrest and cell death, as evidenced by increase in the sub-G0/G1 peak ( FIG. 2 ).
  • Leukemic cells were seeded at 10 5 per ml in X-vivo supplemented with 5% FBS and then incubated with CA4P.
  • KG1a leukemic cells were treated with CA4P at 0, 5 and 10 nM concentrations. After incubation for 48 hours, apoptotic cells were quantified by ApoAlert Annexin-V-fluorescine isothiocyanate (FITC) propidium iodide (PI) Apoptosis Kit (BD) using a Coulter Elite flow cytometer.
  • FITC ApoAlert Annexin-V-fluorescine isothiocyanate
  • PI propidium iodide
  • BD Coulter Elite flow cytometer
  • DNA damage in CA4P-incubated leukemic cells was assessed by comet assay.
  • the concept behind this assay is based upon the ability of denatured, cleaved DNA fragments to migrate out of the cell under the influence of an electric field, whereas undamaged DNA migrates more slowly and remains within the confines of the nucleus.
  • Evaluation of the DNA “comet” tail shape and migration pattern allows for assessment of DNA damage.
  • Results were expressed as the percentage of cells with a comet tail in 100 randomly selected, non-overlapping cells visualized by standard light microscopy. Quantification of the number of leukemic cells displaying a comet tail strongly increased after CA4P treatment ( FIG. 3 ), consistent with CA4P-induced DNA damage.
  • CA4P-treated AML cells were subjected to Annexin-V/propidium iodide (PI) staining and quantification by flow cytometry.
  • Combretastatin A-4 phosphate (CA4P) induces caspase-independent apoptosis in leukemic cells.
  • CA4P induces apoptosis of leukemic cells.
  • Leukemic cells were treated with or without CA4P for 48 hours and the percentage of apoptotic leukemic cells was determined by Annexin V and propidium iodide staining using flow cytometry. Results are representative of three independent experiments.
  • CA4P In contrast to endothelial cells, CA4P induced phosphatidylserine externalization in all leukemic cell lines tested, suggesting that CA4P promotes leukemic cell death through apoptosis ( FIG. 4 ).
  • CA4P In the majority of AML cell lines tested, CA4P at a concentration of 5 nM induced phosphatidylserine externalization (Annexin V+) in more than 50% of the leukemic cells. Only a small number of PI+, Annexin V( ⁇ ) cells were detected, suggesting that CA4P-mediated cell death is apoptotic rather than necrotic.
  • CA4P-Induced Cell Death is Partially Caspase-Dependent
  • leukemic cells incubated with CA4P for 48 hours were harvested, and incubated for 10 minutes at 37° C. in serum-free culture medium at a concentration of 2 ⁇ 105 cells/ml with 20 nM of 3,30-dihexyloxacarbocyanine (DiOC6(3), Molecular Probes), a cell-permeant, green-fluorescent, lipophilic dye that is selective for the mitochondria of viable cells.
  • Cells were collected by centrifugation and analyzed by flow cytometry.
  • the pattern of DiOC 6 (3) fluorescence taken up by control leukemic cells showed cell populations with bright fluorescence, representing cells with intact high MTP ( FIG. 6 ). Results are representative of three independent experiments. In contrast, the amount of DiOC 6 (3) dye taken up in CA4P-treated leukemic cells was strongly decreased. The percentage of cells with fluorescence below control ranged from 12 to 89% of total cells, and for each cell line tested, the results correlated well with the extent of Annexin-V positivity. These data indicated that CA4P-induced leukemic cell death is possibly mediated through alteration of mitochondrial permeability.
  • Mitochondrial damage may result in the release of pro-apoptotic mitochondrial membrane proteins (MMPs) such as cytochrome c, SMAC/diablo and ARTS.
  • MMPs pro-apoptotic mitochondrial membrane proteins
  • cytochrome c mitochondrial membrane proteins
  • SMAC/diablo mitochondrial membrane proteins
  • ARTS mitochondrial membrane proteins
  • Cytochrome c and ARTS were detected using mAb clone 6H2.B4 (1:100, BD Pharmingen) and polyclonal-antibody A3720 (1:50, Sigma), both followed by AlexaFluor 488 conjugated secondary antibody (1:200, Molecular Probes) and analyzed by confocal microscopy.
  • ROS Reactive Oxygen Species
  • CA4P induces cell death in part through a caspase-dependent as well as in part through a non-caspase dependent cell pathway, by accumulation of ROS as a result of tubulin-destabilization and disruption of the mitochondrial respiratory chain.
  • HL60 5 ⁇ 10 6 cells were injected subcutaneously into the dorsa of seven-week-old NOD-SCID mice (Jackson Laboratory). When mice bore a tumor (i.e. after 12 days), 4 experimental groups were randomized, each with 9 animals. Daily treatment was initiated at this time: the CA4P groups were subjected to intraperitoneal injection of CA4P at 10, 25 and 50 mg/kg body weight, and the control group received PBS. After a 3-day treatment, animals were sacrificed, tumors removed and then subjected to immunohistochemical analysis. Tumors were embedded in paraffin, serially sectioned, and stained with hematoxylin and eosin for histological analysis. Cell death was assessed by TUNEL assay.
  • TUNEL reaction (Roche Diagnostics). The detection of cell death in this assay relies on the detection of free 3′OH DNA ends. Positive signal was revealed by fast red and tumor sections analyzed by light microscopy after hematoxylin counterstain.
  • Tumors from mice treated with CA4P for 3 days were softer and hemorrhagic (particularly when treated with 50 mg/kg) than those observed in the control group.
  • Tumor sections of the control untreated mice showed large areas of viable HL60 cells without significant necrosis or fibrosis. In contrast, all tumors in CA4P treated mice were largely necrotic. The control tumor sections were negative for TUNEL reaction. In sharp contrast, most of the leukemic cells were non-viable following treatment with low to high doses of CA4P (data not shown).
  • the extent of intra-tumor vascularization within the different groups was assessed by immunostaining for the endothelial-specific antigen MECA-32.
  • NOD-SCID mice were intravenously inoculated with 1 ⁇ 10 7 GFP+HL60 cells.
  • the HL60 cells were labeled with green fluorescent protein (GFP) by a lentiviral construct, and inoculated systemically through tail vein injection.
  • GFP green fluorescent protein
  • mice were divided into 2 groups of five mice. One group was treated every other day with PBS (control) and the second group received 25 mg/kg CA4P every other day. Each experiment was done three times. At day 30 after the start of the experiment, two mice from each group were killed, and their organs (spleen, liver and lung), peripheral blood and marrow of surgically removed femurs were collected, and analyzed for the presence of human leukemic cells by flow cytometry.
  • organs spleen, liver and lung
  • peripheral blood and marrow of surgically removed femurs were collected, and analyzed for the presence of human leukemic cells by flow cytometry.
  • Single cell suspension was stained using phytoerythrin-labeled anti-human CD45 mAb (Pharmingen), and the percentage of double positive human CD45-PE and GFP cells was determined flow cytometer. The extent of GFP+HL60 cell infiltration was assessed by fluorescence microscopy. In a second set of experiments, GFP+U937 cells were used instead of HL60 cells, and the animals sacrificed after 30 days.
  • control mice had massive leukemic infiltrates in the spleen (5.2%), liver (2.6%) and lung (3.5%) ( FIG. 12 ).
  • Concurrent histological analysis showed the presence of leukemic infiltrates in spleen, liver and lung sections of control mice, but only minimal foci of leukemic cells in the liver of CA4P-treated mice, confirming a drastic decrease in the amount of residual disease.
  • CA4P can efficiently block systemic hematopoietic neoplasm growth in vivo and inhibit organ-specific spread of hematopoietic neoplasms, apparently through disruption of hematopoietic neoplasm growth, migration and possibly interfering with the activation of vascular stromal cells.
  • CA4P may modulate leukemia-vascular interactions, thereby disrupting chemo-protected niches for leukemic cells. Indeed, CA4P treatment of leukemia xenografts significantly reduced expression of VCAM-1, a VLA4 ligand and key molecule in leukemia-stroma adhesion. NOD-SCID mice with subcutaneous HL60 AML tumors were treated with CA4P or PBS (untreated control). Immunofluorescence for VCAM-1 showed significantly decreased VCAM-1 expression in CA4P-treated tumors (data not shown).
  • VCAM-1 Human umbilical vein endothelial cells (HUVECs) were activated with IL-1 ⁇ (5 ng/ml) for 24 hours with CA4P added at concentrations from 0 to 5 nM. VCAM-1 expression was determined by flow cytometry with phytoerythrin-conjugated anti-CD106 (VCAM-1) mAb. Treatment of HUVECs with low, non-toxic (1 to 5 nM range) doses of CA4P significantly reduced expression of VCAM-1 ( FIG. 13 ), without inducing apoptosis.
  • GFP+HL60 or U937 cells in X-vivo/5% FBS were added per well.
  • the percentage of GFP++adherent cells was quantified by fluorescent microscopy.
  • leukemic cells were seeded on either IL-1 ⁇ -activated or non-activated HUVECs, with addition of CA4P. After 48 hours, GFP++leukemic cells were removed from the wells by trypsinization and quantified by fluorescence microscopy and flow cytometry.
  • FIGS. 14A and 14B Treatment with CA4P led to a decreased attachment of HL60 and U937 AML cells to HUVECs in vitro ( FIGS. 14A and 14B ).
  • the number of adherent cells is expressed as a percentage of total leukemic cells and representative of three independent experiments performed in triplicate SEM (*p ⁇ 0.05 as compared to CA4P-untreated control).
  • FIGS. 15A and 15B survival of GFP+U937 (E) and HL60 (F) leukemic cells co-cultured with HUVECs is expressed as a percentage of total cells.
  • Adherent i.e. attached to IL-1 ⁇ activated HUVECs
  • non-adherent leukemic cells i.e. cultured with non-activated HUVECs
  • Results are representative of three independent experiments performed in triplicate ⁇ SEM (*p ⁇ 0.05).
  • CA4P reduces expression of VCAM-1 on vascular cells, thereby increasing the chemosensitivity of hematopoietic neoplasms. Most notably, low CA4P concentrations reduced the expression of VCAM-1 without inducing endothelial cell death, suggesting that CA4P exerts an anti-leukemic effect by modulating adhesive function of the endothelial cells prior to its anti-angiogenic effect.
  • CA4P has Minimal Bone Marrow Toxicity
  • mice Eight week-old sex matched CD1 mice (Jackson Laboratory, Bar Harbor, Me.) were treated with CA4P at 25 mg/kg subcutaneously every other day for 4 weeks. Serial complete blood counts were monitored using Bayer ADVIA 120 hematology analyzer. the mice so treated for 4 weeks displayed a slight decrease in WBC and absolute neutrophil count only, suggesting minimal marrow suppression.
  • human umbilical cord derived (CB) CD34+ cells cultured in vitro in the presence of kit-ligand (Stem Cell Factor) and CA4P for 48 h produced only minimal effect on cell viability as assayed by Annexin/PI staining.
  • Cord blood CD34+ cells were isolated by magnetic procedure and cultured with recombinant human SCF (50 ng/ml, Peprotech) and CA4P for 48 h. Annexin/PI staining was then performed.
  • CD34+ cells were also cultured in methylcellulose supplemented with cytokines and CA4P (Stem cell technologies, Vancouver, Canada) for 14 days. Colonies were scored.
  • CA4P did not impair colony-forming potential of CD34+ cells, demonstrating that CA4P at the concentrations used to target hematopoietic neoplasms has no major toxic effect on normal stem or progenitor cell function.
  • CA4P used as single agent can selectively target circulating and or tissue-resident hematopoietic neoplasms without incurring significant hematological toxicity.

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AU2008326251B2 (en) 2014-03-06
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US20150246064A1 (en) 2015-09-03
EP2219451A4 (en) 2011-01-19
CA2703283C (en) 2016-01-12
EP2219451A1 (en) 2010-08-25
US20090192098A1 (en) 2009-07-30
JP5302328B2 (ja) 2013-10-02
AU2008326251A1 (en) 2009-05-28
HRP20150040T1 (hr) 2015-02-27
EP2219451B1 (en) 2014-11-12
CA2703283A1 (en) 2009-05-28
US9040500B2 (en) 2015-05-26
DK2219451T3 (en) 2015-01-12
WO2009067706A1 (en) 2009-05-28
JP2011504510A (ja) 2011-02-10
US20120270831A1 (en) 2012-10-25
PL2219451T3 (pl) 2015-04-30
PT2219451E (pt) 2015-02-09

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