WO2015089495A2 - Compositions and methods for treating, preventing and diagnosing cancer and other proliferative disorders - Google Patents

Compositions and methods for treating, preventing and diagnosing cancer and other proliferative disorders Download PDF

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WO2015089495A2
WO2015089495A2 PCT/US2014/070221 US2014070221W WO2015089495A2 WO 2015089495 A2 WO2015089495 A2 WO 2015089495A2 US 2014070221 W US2014070221 W US 2014070221W WO 2015089495 A2 WO2015089495 A2 WO 2015089495A2
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cancer
agx5
compound
treatment
subjects
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PCT/US2014/070221
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French (fr)
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WO2015089495A3 (en
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William A. Garland
Jaideep Chaudhary
Glen STOLLER
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Angiogenex, Inc.
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Priority to EP14870415.8A priority Critical patent/EP3079680A4/en
Priority to AU2014361814A priority patent/AU2014361814A1/en
Priority to CN201480075495.2A priority patent/CN107847470A/en
Priority to JP2016558543A priority patent/JP2017506257A/en
Priority to MX2016007748A priority patent/MX2016007748A/en
Publication of WO2015089495A2 publication Critical patent/WO2015089495A2/en
Publication of WO2015089495A3 publication Critical patent/WO2015089495A3/en

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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A61K31/36—Compounds containing methylenedioxyphenyl groups, e.g. sesamin
    • 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
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • 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
    • A61P35/04—Antineoplastic agents specific for metastasis
    • 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

  • the present invention relates to compositions and methods for preventing, diagnosing and treating cancer and other proliferative disorders, including pathogenic angiogenesis. in mammalian subjects.
  • the Id family of helix-loop-helix proteins is implicated in the regulation of virtually all underlying cellular mechanisms and determinative events in cancer: cellular differentiation, cell cycle progression, senescence, lineage commitment and apoptosis (Perk et al., 2005; Morse, 2006; Ling et al., 2006, Nair ct al, 2013).
  • the term d itself is a characterization of the ability of these proteins to inhibit both cell differentiation and binding of important regulatory proteins to DNA (deoxyribonucleic acid).
  • Id 1 -4 the role of Id 1 in tumor invasiveness, metastasis and angiogenesis is best characterized, and findings concerning the role of Idl in cancer provide a foundation for the discussion that follows.
  • Idl is highly conserved in vertebrates and invertebrates as well as among species (Deed et al., 1994) as illustrated in the sequence alignment chart provided below that shows remarkable "homology" (sequence identity) of >90% for human Idl (SEQ ID NO. 1) (UniProKB P41134) and mouse Idl (SEQ ID NO.2) (UniProKB P20067) Idl proteins.
  • Human Id 2 (Uni ProKB Q02363) and mouse Id2 (UniProKB P4 1 1 36) proteins share all but two out of 1 34 identical residues, whi le human Id4 (UniProKB P47928) and mouse ld4 (Uni ProKB P4 1 1 39) are 1 00% sequence identical .
  • Id I The most studied of the Ids is Id I , a regulator of transcription found in extremely low (typically non-measu rable or iindedtcctable concentrations) in d ifferentiated tissue of healthy adults (in contrast to elevated Id levels routinely delectable in cancer tissue or developmental (fetal) tissue).
  • Id3 is considered a paralog of Id 1 , whi le ld2 and Id4 are d istinctly di fferent from each other and from Id l and Id3.
  • I I LI I al l Id helix-loop-hel ix proteins
  • bH LH basic hel ix-loop-hel ix
  • E proteins E proteins
  • E proteins are bHLH proteins that are ubiquitously expressed, bind to the E-box element of DNA and are sequestered by Id proteins.
  • E47 is a basic hel ix-loop-hel ix protei n that is a spl ice product of the E2A gene.
  • E-twenty six "ETS" is another fam i ly of regulatory transcription factors (approximately 20) also proposed to be inhibited by Id proteins. Both E and ETS proteins have been shown to drive cel l differentiation and growth arrest in a variety of cel lular contexts.
  • Id H LH proteins include retinoblastoma (RB, a protein that functions as a tumor suppressor) for Id2 (Desprez et al habit 2003) and for Id l , non-bH LH proteins E-twenty-six (ETS) (a fam i ly of regulatory transcription factors implicated in the development of different tissues as wel l as cancer progression through, eg, pl6Ink4a, a tumor suppressor gene): Paired box (Pax); Mouse Id assocciated-1 (MIDA-1); and Sterol regulatory binding protein-lc, SREBP-lc, among others.
  • ETS E-twenty-six
  • HLH family proteins are comparable to functions of Id proteins. Generally, all of these proteins are fundamental mediators of stasis or change in cellular differentiation, cell cycle progression, senescence, cellular commitment (to a determined lineage/fate) and/or apoptosis. However, the activities of Id proteins are typically apposite in effect to activities of HLH family proteins. This is attributed in part to a deactivating result from binding between the HLH and bHLH proteins, as described below.
  • bHLH proteins generally mediate a restrained or no growth environment in mammalian cellular systems. Consistent with this role, a reduction of E proteins in B and T- cells correlates with development of B and T-cell leukemias (Kee, 2009). In contrast, increased Id protein expression is associated with a pro-growth environment, attributable to Id " s potential for neutralizing bHLH proteins.
  • Id proteins also possess an HLH domain, but without the adjacent basic region thai mediates DNA binding of bHLH proteins. As a result of this construction, Id proteins are capable of binding other HLH transcription factors and altering their activity in gene transcription. In this manner, Idl has been reported to inhibit the activity of bHLH transcription factors like the E protein, E47, by binding and restraining the ability of the bHLH binding partner to bind DNA and mediate transcriptional changes (proposed to promote pro-differentiation and pro-apoptotic cellular changes— believed to be correlated with anti-neoplastic and anti-angiogenic activity in different cellular developmental contexts).
  • Idl is present in both cytosol and nuclear compartments of cells, with shuttling from the nucleus possibly regulated by protein kinase-A (PKA) (Nishiyama et al., 2007).
  • PKA protein kinase-A
  • Intracellular levels of Idl are also proposed to be regulated through an ubiquitin-proteasome degradation pathway (Sun et al..2005), resulting in half-lives for Id proteins of approximately one hour or less. This degradation process is possibly linked to TNF-a- induced apoptosis in prostate cancer cells (Ling et al., 2006). Heterodimerization with bULH proteins reportedly affects rates of Id degradation and may be protective to extend Id protein half-life (Bounpheng et al, 1999).
  • Idl and Id3 have been proposed to play critical roles in the production of new blood vessels, including "pathogenic neovascularization” associated with growth and spread of tumors (“tumor-associated angiogenesis”). Transfer of the Idl gene into HUVEC cells elevates angiopoetin- 1 and confers a pro-angiogenic phenotype (Nishiyama ct al., 2005).
  • Idl reportedly acts '"downstream " of pro-angiogenic factors vascular endothelial growth factor-A (VEGF-A) Lee et al., 2006), bFGF (Ruzinova ct al., 2003), HIF-1 (Kim et al., 2007), and EGF-R (Ling et al., 2004), so loss of Idl activity may impair multiple angiogenic pathways.
  • VEGF-A vascular endothelial growth factor-A
  • Idl is also proposed to interact negatively with p2I (a cyclin kinase inhibitor, proposed to function via inactivation of cyclins.
  • P2I is reported to negatively control endothelial progenitor cell formation in bone marrow neovascularization (Ciarrocchi et al... 2007), which progenitor cells are contemplated to play a pivotal role in tumor-associated angiogenesis (Seandel et al., 2008), and cancer metastasis (Gao et al., 2008; Gao et al..2009).
  • Id 1 proteins have been reported to be linked to a diverse array of signaling and control elements involved in the initiation and progression of cancer (Fong et al., 2004).
  • a particularly interesting link has been proposed between Id l and cancer metastasis.
  • Id l has been reported to be associated in a mechanistic way with metastatic change in human breast cancer (M inn et al., 2005). Over-expression of Id l reportedly contributes to metastasis in breast cancer cel ls transplanted into animals (Fong et al., 2003).
  • over- expression of Id l was reported to immortal ize myeloid progenitor cel ls and lead to myeloprol iferative d isease in mice (Suh et al., 2008).
  • Id l is highly expressed in a large number of cancers including sol id tumors of the bladder (Perk et al., 2006), breast (for example, Schoppmann et al., 2003), cervix/uterus (Li et al., 2009; Schindl, et al ., 2001 ; Maw et al., 2008), colorectal (Zhao ct al ., 2008), endometrium (Takai et al., 2004), gastric (Han et al., 2004; Iwatsuki et al.. 2009), gl ia (Vandeputte et al..
  • Id l gene expression is reported to be stimulated by growth factors including bone morphogenetic protein-2 (BM P-2) (Le Page et al., 2009), BMP-6 (Darby et al ., 2008), growth/differentiation factor-5 (GDF5) (Chen et al., 2006), and insul in-like growth factor- 1 (IGF- 1 ) (Prisco et al., 200 1 ; Bel letti et al., 2002).
  • VEGF-A Vascular-endothelial-growth-factor-A
  • VEGF-A is reported to be both an upstream gene inducer ( Benezra et al..
  • SMAD proteins are proposed to modulate activity of transforming growth factor beta l igands by form i ng complexes with other SMADs that function as transcription factors (Liang et al.. 2009).
  • Early growth response protein 1 (Egr l ) (Subbaramaiah et al., 2008), Sp l (Jorga et al.,
  • FX03a a transcription factor reported to reverse leukem ic phcnotypes, may promote differentiation by transcriptional down-regu lation of Id l (Birkenkamp et al.,
  • Id l Complexity of Downstream Id Targets Id l is also proposed to target various downstream oncogenic tyrosine kinases (Tarn et al ., 2008 ), such as Bcr-Abl, Tel-A BL
  • PDG-FBR a hybrid fusion protein found in patients with myeloid neoplasms associated with eosinophi l ia
  • FLT3 a tyrosine kinase 1 associated with one of the receptors for V EG F-1TD
  • Id l is postulated to cooperate with oncogen ic rat RAS (a GTPase in the mitogen-activated protein kinase (MAPK) pathway), to induce metastatic mammary carcinoma by subverting cellular senescence responses
  • Id l has been reported to stimulate a phosphatidyl inositol -3- kinase (PI3 K)/protein kinases B (Akt)/nuclear factor kappa beta (N FkB) signal ing pathway important to cancer cel l proliferation, survival and invasion (Li et al ., 2007).
  • PI3 K phosphatidyl inositol -3- kinase
  • Akt protein kinases B
  • N FkB nuclear factor kappa beta
  • Id l may restrain expression of p i 6, which in turn positively regulates cell senescence
  • Id l may also regulate B-cell lymphoma cell-2 (Bcl-2) and BCL-2-associated X protein (Bax), through p53 and N FkB (K im et al., 2008), and chromosomal instability through anaphase-promoting complex-C
  • Id l is proposed to activate Akt-mediated wingless type (Wnt) signal ing and p27 phosphorylation through phosphatase and tensin homolog (PTEN) inhibition (Lee et al., 2009).
  • Metastasis is a multi-step, multi-factorial process, broadly described as having two major phases: ( 1 ) physical dissemination of cancer cel ls from a "primary" site (i.e., initial tissue/organ where the cancer originated); and (2 ) colon ization by cancer cel ls from a primary site to distant tissues/organs (Chaffer and Weinberg, 201 1 ).
  • EMT epidermal to mesenchymal transition
  • EMT-inducing transcription factors such as twist-related protein 1 (Twist 1 , or class A bHLH protein 38) has suggests molecular linkage of EMT-driven metastatic d issem ination, and the generation of TICs (Man i et al., 2008; Morel et al., 2008). How ever, less is known about the biology of TICs during the second phase of metastasis (colonization of distant tissues organs). The proposal that EMT induces breast cancer TICs has been challenged based on cl inical observations that most metastases present a differentiated epithelial morphology (Tarin et al., 2005).
  • EMT is a transient process
  • MET meenchymal to epithel ial transition
  • EMT may affect cancer cell dissemination (including invasion of the local parenchyma (Yook et al., 2006), intravasation into the circulatory system (Drake et al., 2009). survival during migration (Gal et al.. 2008), and extravasation into the secondary site (Labellc et al., 201 1 ; Vuori luoto et al., 201 1 )), loss of mesenchymal phenotype may enhance formation of macro-rnetastatic colonies. This may be l inked to a reversal of growth arrest associated with EMT (Brabletz et al., 2001 ; Mej lvang et al., 2007; Vega et al.. 2004).
  • M ET breast cancer metastasis
  • studies showing that after dissem ination, engineered loss of the EMT transcription factor Twist 1 , (Tsai et al., 20 1 2) and expression of microRNAs inhibiting the EMT transcription factor "zinc finger E-box-bind ing homeobox" (Zeb) (Korpal et al., 20 1 1 ) enhance lung colon ization by metastatic breast cancer cells.
  • the transcription factor "paired related homeobox ⁇ * (Prrx l ) (w h ich induces EMT during dissemination but suppresses sternness traits necessary for lung colonization), must be lost prior to colonization (uncoupling in this instance EMT from the TIC phenotype) (Ocana et al.. 20 1 2).
  • the present invention fulfi l ls the foregoing needs and satisfies add itional objects and advantages by providing novel tools and methods to modulate Id function in mammal ian subjects suffering from a neoplasm or other cellular prol iferative disorder.
  • the invention further provides novel technical discoveries to elucidate the role and effects of Id proteins and their biochemical and molecular targets in mediating cancer and metastasis, yielding tools and methods to manage and treat cancer and metastasis and other proliferative disorders in humans and other ma mmals.
  • compositions and methods for treating a cellular proliferative disorder for example inhibiting or reducing metastasis of a tumor or primary cancer cel l.
  • These methods comprise administering to a mammal ian subject an effective amount of an "anti-Id compound", sufficient to reduce or prevent pathogenic cel lular proliferation, angiogenesis, cancer, and/or metastatic disease in the subject.
  • an anti-Id compound of formu la I, 11. I l l, or I V as described below, or an active salt. enantiomer, polymorph, solvate, hydrate, or prodrug thereof.
  • the methods and compositions herein employ an exemplary anti-Id compound or composition comprising an anti-metastatic (or anti-prol iterative, or anti-angiogen ic) effective amount of racem ic N-(3-(benzo[d]
  • the anti-Id compound is an isolated, anti-metastatical ly active (-)-enantiomcr of N-(3-(benzord] [ l ,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzylpropionamide.
  • the (-) or "m inus” enantiomer of AGX5 1 exh ibits an extraordinary and unexpected dom inance of anti-Id potency compared to the (+) or "plus” enantiomer.
  • novel, enantomerically-enriched preparations of (-)-AGX5 1 enantiomer (substantia l ly purified to yield an increased amount or concentration of (-)-AGX5 1 , relative to an amount or concentration of the (+)-AGX5 I enantiomer (compared to conventional ly-prepared racem ic N-(3-(benzo[d] [ l ,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzyl propionam ide), provide surprising advantages and cl inical benefits within the compositions and methods of the invention.
  • the invention provides "anti-metastatic" compositions and therapeutic methods, which are effective to treat or prevent metastatic disease in mammal ian subjects. These methods may employ monotherapy or coordinate or combinatorial therapy.
  • the compounds and methods of the invention are "anti-metastatical ly effective " , for exam ple to reduce an incidence, size, tissue or organ distribution, or number of metastases in a subject presenting with cancer.
  • anti-metastatic activity corresponds to an observed reduction in one or more histopathological indices of metastasis, for example quantitative reduction in occurrence, size, number or distribution of metastasized cel ls or "foci " of primary tumor character observed at a secondary tissue or anatom ic site.
  • anti -metastatic efficacy is evinced by prevention and/or treatment of metastatic cancer, e.g., as demonstrated by an increase in a time period of disease free survival for subjects receiving anti-Id treatment according to the invention.
  • compositions and methods of the invention target distinct cel lular proli ferative disorders characterized by aberrant blood vessel growth, or "pathogenic angiogenesis" .
  • Examples of these disease targets include ocular disease mediated by aberrant vascular growth (e.g., macular degeneration), and tumor-associated angiogenesis.
  • Anti-Id com pounds of the invention function also as "anti-angiogenic" agents, as described below, making them useful to treat or prevent pathogenic angiogenesis, including quite powerful ly tumor- associated angiogenesis (to mediate a multi-pronged, anti-metastatic and anti-angiogen ic assault on secondary tumor initiation and growth.
  • novel diagnostic assays measuri ng Id protei ns in blood or tissues are demonstrated to provide useful diagnostic tools to monitor occurrence and progression of metastatic disease, and/or evaluate effectiveness of anti- Id treatment.
  • compositions and methods of the invention may employ an effective amount of the anti-Id compound combined in a kit or formulation, or coordinately administered, along with a secondary treatment agent, treatment modal ity or treatment method.
  • a secondary treatment agent for example selected from : radiation, chemotherapy, surgery, or combinations thereof.
  • Figure 1 provides a Western blot gel showing that the Id l and Id3 levels are potently reduced in leukem ic cel ls following treatment with an exemplary anti-Id compound, N-(3- (benzo[d][ l ,3]dioxo l-5-yI)-3-(2-methoxyphenyl)propyl)-N-benzylpropionamide (AGX5 1 ).
  • a purified (-)-AGX5 1 enantiomer exhibits surprising stereospeci flc effects compared to the (+)- AGX5 1 enantiomer.
  • Figure 2 provides a Western blot gel showing that the Idl and Id3 levels are variably affected in breast cancer cells following treatment with AGX51, revealing additional surprising stereospecific effects of this anti-Id compound.
  • Figure 3 is an immunoblot showing rescue ofpl6 levels in a leukemic cell line derived from a mouse overexpressing the MML-AF9 fusion protein following treatment with AGX51.
  • Figure 4 is a Western blot showing rescue of p21 levels in a human bladder carcinoma line following treatment with AGX5I.
  • Figure 5 graphically compares effects of racemic-AGX51, (+)-AGX51 ("El") and (- )-AGX51 ("E2) on restoring cell cycle control in DU-145 human prostate cancer cells.
  • the (- )- enantiomer of AGX51 shows pronounced stereospecific efficacy for restoring cell cycle control, whereas the (+)- enantiomer of AGX51, surprisingly, mediates no detectable effect.
  • Figure 6 illustrates potent anti-migration effects of an exemplary anti-Id compound of the invention, AGX51, in a model of migration-dependent metastatic disease potential.
  • This cellular motility "Scratch” assay demonstrates that AGX51 is a potent inhibitor of metastatic activity involving migration of cancer cells.
  • Figure 7 is a chart illustrating strong anti-angiogenic effects of AGX 1 to reduce formation of blood vessels in VEGF-165 and FGF-2 treated Matrigel plugs implanted into flanks of C57/BL mice.
  • DMSO vehicle
  • Boxplots are tumor volumes 53 days post implantation (last day of study).
  • Figure 9 provides ion chromatograms of control (A) and low calibrant of AGX5 I (B).
  • Figure 10 is a log-linear plot of plasma concentration data showing that AGX51 is rapidly absorbed with C ma x estimated here at 15 minutes.
  • Figure 11 is a simulation of plasma concentrations following 60mg/kg. bid of AGX51.
  • Figure 12 shows the design and results of studies demonstrating potent anti- metastatic efficacy of AGX51, directly reducing lung metastasis of Lewis Lung Carcinoma
  • FIG. 13 graphically demonstrates the effects of AGX5 1 on lung metastasis of breast cancer (4T 1 ) cells directly injected via tail vein into Balb/c m ice.
  • AGX5 1 potently reduces metastasis of injected breast cancer cel ls.
  • Figure 14 shows biolum inescence visual ization of AGX5 1 suppression of lung metastasis in Balb/c mice following direct injection of breast cancer (4T I ) cel ls via tai l vein.
  • the (-)-enantiomer of AGX5 1 potently, stereospecifically protects against metastasis in l iving model subjects predictive of cancer drug efficacy in humans.
  • Figure 15 is a graph demonstrating that a combination of pacl itaxel and AGX5 1 , even at a m.l dose, significantly decreased tumor growth in mice implanted with M DA-M B- 23 1 tumors.
  • Figure 16 is a chart showing the change in final-initial tumor volume on day 1 9 in m ice implanted with MDA-M B-23 1 tumors and treated with pacl itaxel and varying amounts of AGX5 1 .
  • Figu re 17 is a graph showing that 60mg/kg. bid, AGX5 1 sign i ficantly increased the e ffectiveness of pacl itaxel on reducing tumor growth.
  • Figure 18 is a chart showing the change in final-in itial tumor volume on day 4 1 in m ice implanted with M DA-M B-23 1 tumors and treated with pacl itaxel and AGX5 1 .
  • Figure 19 is a graph showing that the addition of AGX5 1 to a treatment with paclitaxel signi ficant ly increased the effectiveness of pacl itaxel on decreasing tumor growth.
  • Figure 20 is a graph showing tumor growth in Id l knockout m ice treated with pacl itaxel and AGX5 1 .
  • Figure 21 is a chart showing mean tumor volume on day 20 i n Id I knockout mice treated with paclitax l and AGX5 1 .
  • Figure 22 is a chart showing a comparison of selected chemistry and hematology values in mice treated with pacl itaxel and/or AGX5 1 .
  • Figure 23 is a graph illustrating effects of ld3 genetic knockout on pathogenic retinal neovascularization in a mouse model.
  • Figure 24 is a graph demonstrating that (-)-AGX5 1 administered intravitreal ly (ivt) protects against pathogenic retinal neovascularization in a murine model of human age- related macular degeneration (AMD)
  • Figure 25 is a graph demonstrating that (-)-AGX5 1 administered intraperitoneally (ip) protects against pathogenic retinal neovascularization in a murine model of human AM D,
  • Figure 26 is a schematic depiction of a modified sandwich immunoassay for detection of Id protein levels/activity in biological samples for use in implementing reflexive diagnostic-treatment methods of the invention.
  • Figure 27 is a graph demonstrating the use of Id diagnostic tools and methods of the invention for predicti ng and managing cancer.
  • Figure 28 is a graph demonstrating the use of Id diagnostic tools and methods of the invention for predicting and managing breast cancer in human subjects.
  • Figure 29 is a graphic depiction of AGX5 1 (+)- and (-)-enantiomer elution profi les using nantiomer Separation Method A of the invention.
  • Figure 30 is a graphic depiction of AGX5 1 (+)- and (-)-enantiomer elution profi les using Enantiomer Separation Method B of the invention.
  • Metastasis refers to the spread of a cancer from one organ or part to another non- adjacent organ or pa rt. Metastases are new occurrences of cancer at secondary sites generated by metastasis.
  • chemotherapeutic drug or agent typically refers to approved anti-cancer and other anti-hyperproliferative drugs or chem ical agents.
  • “Chemotherapeutic” wil l general ly apply to a drug or chemical active to destroy cells and tissues, typically cancer cel ls and alternatively or conj unctively cells of blood vessels newly formed as part of a pathologic condition attending a hyperproli ferative disorder, as typical ly attends a neoplasm or a cancer.
  • Chemotherapeutic agents for adjunctive use with in the invention include, but are not l im ited to: ( I ) tubul in depolymerizing agents such as taxanes l ike paclitaxel, docetaxel, BAY 59-
  • 8862 albumin bound paclitaxel (2) DNA damaging agents and agents that inhibit DNA synthesis, (3) anti-metabolites, (4) anti-angiogenics and vascular disrupting agents, (5) antibodies, (6) endocrine therapy, (7) immuno-modulators, ( 8) histone deacetylase inhibitors.
  • inhibitors of signal transduction ( 1 0) inhibitors of heat shock proteins, ( 1 1 ) retinoids such as al l-trans retinoic acid, ( 1 2) inhibitors of growth factor receptors or the growth factors themselves, ( 1 3) anti-mitotic compounds, ( 1 4) anti-inflammatory agents such as COX inhibitors, and ( 1 5) cell cycle regulators, eg, check point regulators and telomerase inhibitors.
  • combination therapy refers to a therapeutic regimen that involves the provision of at least two distinct therapies to achieve an indicated therapeutic effect.
  • a combination therapy may involve the adm inistration of two or more chemical ly distinct active ingred ients, for example, an anti-Id compound as we l l as a chemotherapeutie agent.
  • a combination therapy may involve the adm inistration of an anti-Id treatment and/or one or more chemotherapeutie agents, alone or together w ith the del ivery o f another treatment, such as radiation therapy and/or surgery.
  • the active ingredients may be administered as part of the same composition or as d i fferent compositions.
  • the com positions comprising the di fferent active ingredients may be adm inistered at the same or di fferent times, by the same or different routes, using the same of different dosing regimens, al l as the particular context requires and as determ ined by the attending physician.
  • the drug(s) may be delivered before or al ter surgery or radiation treatment.
  • “Monotherapy” refers to a treatment regimen based on del ivery of one therapeutical ly effective compound, whether adm inistered as a single dose or in several doses over time.
  • Neoplasia refers to abnormal and uncontrol led cell growth.
  • a “neoplasm”, or tumor is an abnorm al, unregulated, and disorganized proliferation of cell grow th and is sometimes referred to as a cancer.
  • a neoplasm may be benign or mal ignant.
  • a neoplasm is mal ignant, or cancerous, if it has properties of destructive growth, invasiveness, and metastasis.
  • Invasiveness refers to the local spread of a neoplasm by infiltration or destruction of surrounding tissue, typically including penetrating basal lam inae that define boundaries of tissues (thereby often entering the body's circulatory system).
  • Metastasis typical ly refers to dissemination of tumor cells to distant sites, often via lymphatics or blood vessels.
  • Metastasis also refers to migration of tumor cells to adjacent sites through serous cavities or subarachnoid or othe r spaces. Through the process of metastasis, tumor cell migration and dissemination to other compartments, tissues and areas of the body establ ishes "secondary " neoplasms in areas away from the "primary" site of initial cancer appearance.
  • a “subject” or “patient” refers to an animal in need of treatment that can be affected by methods and compositions of the invention.
  • Subjects and patients amenable to treatment using anti-Id compounds and methods of the invention include vertebrates, particularly mammals such as bovine, canine, equine, fel ine, ovine, porcine, and primate (including humans and non-humans primates) mammals, presenting with, or at elevated risk of developing, cancer, metastatic disease, or any proliferative disease inc luding pathogen ic angiogenesis.
  • treatment or therapy "system” as employed herein refers to a plurality of treatment agents or modalities, such as a combined formulation or protocol employing an anti-Id active agent, a chemotherapeutic and possibly a toxicity reducing agent— used in a coord inate treatment regimen (formulated alone or together, and adm inistered simultaneously or sequential ly).
  • Treatment systems may also combine drug (e.g., anti-Id and/or conventional chemotherapy) treatment with another intervention or treatment modal ity, such as rad iation therapy or surgery.
  • intervention or treatment modal ity such as rad iation therapy or surgery.
  • Optional treatment systems employing anti-Id agents and methods of the invention can be integrated in any combinatorial "therapeutic regimen" combining complementary tools or methods, such as chemotherapeutic agents, radiation therapy, surgery, gene therapy, DNA vaccines and therapy, siRNA therapy, anti-angiogen ic therapy, immunotherapy, bone marrow transplants, aptamers and other biologies such as antibodies and antibody variants, receptor decoys and other protein-based therapeutics.
  • treatment means any treatment of a disease or disorder, including preventing or protecting against the disease or disorder (that is, causing the clinical symptoms not to develop); inhibiting the disease or disorder (i.e., arresting or suppressing the development of cl ini cal symptoms; and/or rel ieving the disease or disorder (i .e., causing regression of clinical symptoms).
  • the methods and compositions of the invention wi l l often serve to prevent one or more symptoms of disease, or delay onset or recurrence/relapse of disease ("prophylaxis”), as wel l as to slow, inhibit or prevent disease progression (e.g., as marked by increased severity of disease symptoms, or onset of more advanced disease symptoms).
  • novel methods and compositions of the invention flow from a surprising course of discovery.
  • Early comparisons of gene expression data between cell lines with variable metastatic potential prompted reports that certain candidate genes are required during different steps of the metastatic disease cascade (Kang et al., 2003b; Minn et al., 2005 ; Yang et al., 2004).
  • Some of these reports suggested that expression of Id proteins, including products of Id l and l d3 genes, are involved in lung colonization of breast cancer cel ls (Gupta et al., 2007).
  • Id proteins have long been reported to be dominant negative regulators of basic hel ix-loop-helix (bHLH) transcription factors (Perk et al., 2005).
  • Id proteins have further been reported to play key roles in maintaining embryonic stem cel l self-renewal (Romero-Lanman et al. ; Ying et al., 2003), and to continue th is function in adult tissue stem cells (Nam and Benezra, 2009) and hematopoietic stem cells (Jankovic et al., 2007).
  • Id genes have also been impl icated as regulators of TIC phenotypes in certain cancers, such as gl ioblastoma. (Anido et al., 20 1 0; Barrett et al., 20 1 2) and colon cancer (O'Brien et al., 20 1 2).
  • Id 1 is predom inantly expressed in the more aggressive triple negative [negative for estrogen receptor, progesterone and negative for the neu type human epidermal growth factor-2 (H ER2 ) and metaplastic subtypes, with high Id l expression correlating with poor cl inical outcomes (Gupta et al., 2007).
  • the inventio n targets Id functional ity in promoting cancer and metastasis, by employing novel suppressors of Id proteins, including suppressors of Id l and Id3.
  • anti-Id novel suppressors of Id proteins, including suppressors of Id l and Id3.
  • the anti-Id compounds of the invention are general ly in an "anti-Id effective amount" by direct appl ication or injection to a cell population, physiological compartment, tumor, or individual.
  • These compounds and methods exhibit "anti-cancer", “anti-metastatic”, “anti-proliferative”, and/or "anti-angiogenic " effective activities, as described herein for different aspects and embod iments of the invention.
  • compositions and methods for treating a cellular proliferative disorder for example inhibiting or reducing metastasis of a tumor or primary cancer cel l.
  • These methods comprise adm inistering to a mammal ian subject an effective amount of an "anti-Id compound", sufficient to reduce or prevent pathogenic cellular proliferation, angiogenesis, cancer, and/or metastatic disease in the subject.
  • Ri may be a substituted or unsubstituted lower hydrocarbon selected from the group consisting of alkyl, alkenyl. alkanoyl, alkynyl, aryl, aroyl, aralkyl, alkylam ino.
  • R2 and R3 may independently, collectively, or in any combination that yields an active anti-Id (apoptosis inducing, cel lular prol iferation inhibiting, chemotherapeutic enhancing, transcrip tion regulating, anti-inflammatory, cellular differentiation promoting, cellular transformati on modulating) composition be a hydrogen, hydroxyl, sulfyhydryl, fluorine, methyl, ethyl, propyl, benzyl, 2-bromovinyl amino, hydroxymethyl, methoxy, halogen, pseudohalogen, cyano, carboxyl, nitro, thioal
  • R4 and R5 may be may independently, collectively, or in any combination that yields an active anti-Id (apoptosis inducing, cellular proliferation inhibiting, chemotherapeutic enhancing, transcription regulating, anti-inflammatory, cellular differentiation promoting, cel lular transformation modulating) composition be an acyl or a substituted or unsubstituted lower hydrocarbon selected from the group consisting of alkyl, alkenyl, alkanoyl, aryl, aroyl, aralkyl or alkylamino; R6 may be a heteroatom such as oxygen, sulfur or nitrogen; R7 may be a heteroatom such as sulfur, nitrogen or oxygen as well as a carbon; R8, 9, 10, 1 1 and 12 may independently, collectively, or in any combination that yields an active anti-Id (apoptosis inducing, cellular proliferation inhibiting, chemotherapeutic enhancing, transcription regulating, anti-inflammatory
  • pseudohalogen cyano and a substituted or unsubstituted lower hydrocarbon containing 1 to 20 carbons.
  • R group When more than one R group is present, the R group may be selected from any of the stated groups so as to be the same or different. In additional embodiments, two or more R groups may be joined together.
  • R2 and R3 may be members of a 5, or 6, member exocycl ic ring structure.
  • R3 and R4 may be members of a 5, or 6, member exocyclic ring structure.
  • R5 and R6 may be members of a 5 or 6 member exocycl ic ring structure.
  • R l 1 and R 1 2 may be members of a 5 or 6 member exocycl ic ring structure.
  • i f R7 is nitrogen
  • R6 and R7 may be members of a 5 or 6 member exocycl ic ring structure.
  • I n other embodiments, R6 and R I 2 may be members of a 5 or 6 member exocycl ic ring structure.
  • rational ly designed anti-Id compounds of Formula I can be selected from additional candidates, wherein R i , R4, R5.
  • Rf>, RH, Ry, R io are independently selected from the group consisting of hydrogen, hydroxyl. sul fyhydryl, benzyl. 2-bromovinyl am ino, hydroxymethyl, methoxy, halogen, pseudohalogen, cyano, carboxyl.
  • thioalkyl, thioaryl, thiol substituted or unsubstituted hydrocarbons containing 1 to 20 carbons, alkoxycarbonyl, alkoxycarbonylam ino, am ino, am ino acid, am inocarbonyl, aminocarbonyloxy, aryloxy, carboxyl, cycloalkenyl, substituted or unsubstituted cycloalkyl. substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, peptidyl.
  • R 7 cannot be methoxy if Rn is propionyl while R 7 and Re can be ortho, as shown in Formula I, or para to one another;
  • the anti-Id compound selected from Formula I is N-(3- (benzo[d][ l,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzylpropionamidc
  • Novel and surprisingly effective methods and compositions that inhibit Id proteins may additionally comprise compounds of Formula III, below, and their active salts, enantiomers, polymorphs, metabolites, solvates, hydrates, and prodrugs.
  • R i , R2, R3, 3 ⁇ 4, Rs, Re, Rx, R9, and Rio may independently, collectively, or in any combination that yields an active anti-Id compound be hydrogen, hydroxyl, sulfyhydryl. fluorine, methyl, ethyl, propyl, benzyl, 2-bromovinyl am ino, hydroxymethyl, mcthoxy.
  • halogen cyano, carboxyl, nitro, thioalkoxy, thioaryloxy, thiol, substituted or unsubstituted lower hydrocarbon containing 1 to 20 carbons; alkoxycarbonyl,
  • R7 may be selected from hydrogen, hydroxyl, benzoyl; substituted benzoyl or hydroxyl substituted with unsubstituted lower hydrocarbon containing 1 to 20 carbons;
  • Rn may be a hetcroatom such as oxygen, sul fur or nil rogen;
  • R i2 inay be a lower hydrocarbon independently selected from the group consisting of alkyl, alkenyi, alkanoyi, aikynyl, aryl, aroyl, aralkyi, alkylam ino, aryloxy.
  • R group when more than one R group is present, the R group may be selected from any of the stated groups so as to be the same or different. In additional embodiments, two or more R groups may be joined together. In some embodiments, R4 may become a member of a 5 or 6 member ring structure with neighboring rings.
  • An exemplary compounds of Formula III for use within the invention is N-[3-(l,3-benzodioxol-5-yl)-3-(2- methoxyphenyl) propyl]-N-benzylpropanamide, as shown in Formula IV.
  • An exemplary anti-Id compound for use within the compositions and methods of the invention is N-(3-(benzo[d][l,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N- benzylpropionamide ("AGX51 " ).
  • AGX51 refers to N-(3- (benzo[d][ 1 ,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzylpropionamide in isolated or substantially purified form, as well all functional equivalent salts, prodrugs, metabolites, derivatives, analogs, and conjugates of AGX51.
  • AGX51 encompasses desired prodrug forms and biotransformation products, and readily designed and tested analogs, derivatives and complexed or conjugated forms of the drug.
  • the invention encompasses compositions and methods employing demethylated forms of AGX15, for example N-(3-(benzo[d][l,3]dioxol-5-yl)-3-(2-hydroxyphenyl)propyl)- N-benzylpropionamide; deamidated forms, for example (3-(benzo[dj[ 1.3
  • demethylated forms of AGX15 for example N-(3-(benzo[d][l,3]dioxol-5-yl)-3-(2-hydroxyphenyl)propyl)- N-benzylpropionamide
  • the anti-Id compound is a selected salt form of a racemate or purified enantiomer of a biotransformation product of N-(3-(benzo[d][l,3]dioxol-5-yl)-3-(2- methoxyphenyl)propyl)-N-benzylpropionamide, for example a salt of (3- (benzo[d][l,3]dioxol-5-yl)-N-benzyl-3-(2-methoxyphenyl)propan-l-amine or 2-(l- (benzo[ ⁇ /J [ l ,3]dioxol-5-yl)-3-(benzylamino)propyl)phenol.
  • Exemplary salt forms of these and other anti-Id compounds herein include, but are not limited to, hydrochloride, hydrobrom ide, hydroiodide, phosphate, sulfate, oxalate, malate, maleate and succinate salts.
  • the anti-Id compound is an isolated, anti-metastatically active enantiomer of N-(3-(benzo[d][ l ,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N- benzylpropionam ide.
  • the discoveries herein demonstrate extraordinary anti-Id potency residing in the (-)-enantiomer form of AGX5 1 .
  • the invention employs novel, enantomerically-enriched preparations of (-)-AGX5 1 , substantial ly purified to yield an i ncreased relative amount or concentration of (-)-AGX5 1 (relative to an amount or concentration of the other, (+)-AGX5 1 enantiomer, or to an amount found in conventional, racem ic preparations of N-(3-(benzo[d][ l ,3]dioxol-5-yl)-3-(2- mcthoxyphenyl)propyl)-N-benzylpropionamide).
  • the methods and compositions of the invention employ a "substantially pure” or "essential ly pure” anti-metastatic effective (-)-enantiomer of N-(3- (benzo d ] [ l ,31dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzylpropionam ide.
  • the purified (-)-AGX5 1 enantiomer is at least initial ly (i.e., pre- formulation) provided in a form that exhibits at least 80-90%, 90-95%, greater than 95%, or 98% or greater "enantiomeric enrichment” (ce) or "enantiomeric purity " .
  • initial ly i.e., pre- formulation
  • enantiomeric enrichment ce
  • enantiomeric purity .
  • the identity and quantity of a selected AGX5 1 enantiomer within the compositions and methods of the invention may be determined by a variety of means. For example, these determinations and values may be demonstrated using conventional chiral chromatography and/or polarimitry.
  • the methods and compositions of the invention employ highly puri fied or isolated (-)-AGX5 1 (at least as a starting material prior to formu lation, storage or administration) in an enantiomeric excess of greater than 98%> (e.g.. as determined by chiral chromatography and/or optical purity assay).
  • Preparations of (-)-AGX 1 in enantiomeric excess greater than 85% or 90%> are considered substantial ly free of the corresponding (+)- AGX5 1 enantiomer and are highly desired drug preparations for cl in ical use.
  • compositions of the invention for treating cancer, preventing or treating metastatic disease, or treating an angiogenic or other prol iferative disorder, wi l l contain no more than about 5% w/w, and in some embodiments no more than about 2% or 1 % or lower w/w, of the (+)-AGX5 1 enantiomer (i .e., measured as a percent of total N-(3- (benzo[d ] [ l ,3 ]dioxo l-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzylpropionamide mass or molar content present in the composition).
  • enantiomerical ly enriched (-)-AGX5 1 compositions provides enhanced therapeutic efficacy for treating cancer, preventing or treating metastatic disease, and treating angiogen ic and other prol iferative disorders, with reduced side effects at eq uivalent dosages, compared to racem ic AGX 1 or the (+)-AGX5 1 enantiomer.
  • enriched (-)- AGX5 1 yields equal or greater therapeutic efficacy with fewer side effects at lower or less frequent dosages, compared to racem ic AGX5 1 or the (+)-AGX5 1 enantiomer.
  • the (-)-AGX5 1 - enriched compositions and methods of the invention exhibit increased activity or therapeutic efficacy of at least 1 0%, 20%, 30%, or 50%, up to 75-95%, 100% or even 200% greater than an observed activity or therapeutic efficacy determined for the racemate (or for the substantially pure (+)-AGX5 1 enantiomer)— using any one or combination of activity measures, therapeutic indices and clinical assays employed or referenced herein.
  • Anti-Cancer and nti-Metastasis Compositions and Methods exhibit increased activity or therapeutic efficacy of at least 1 0%, 20%, 30%, or 50%, up to 75-95%, 100% or even 200% greater than an observed activity or therapeutic efficacy determined for the racemate (or for the substantially pure (+)-AGX5 1 enantiomer)— using any one or combination of activity measures, therapeutic indices and clinical assays employed or referenced herein.
  • anti-Id compositions and methods of the invention effectively block Id binding to bHLH proteins, promote functionally significant Id degradation (i.e., therapeutical ly decrease Id levels in cells), and suppress Id activity when administered to cells, tissues and living subjects. These ant-Id activities in turn effectively suppress tumor metastasis, as well as tumor-associated angiogenesis and other prol iferative disease states and symptoms.
  • the compositions and methods of the invention afford effective tools to m itigate the most lethal and refractory neoplasms.
  • the instant invention has immense cl inical promise in view that so many Americans are critically affected by cancer.
  • the National Cancer Institute of the N I H estimates that approximately 1 3.7 m i ll ion Americans with a history of cancer were al ive in January, 20 1 3. Some of these indiv iduals were cancer-free, whi le others sti ll had evidence of cancer and may have been undergoing treatment. In 201 3, about 58,350 Americans were expected to die of cancer, or approximately 1 ,600 people a day. In the US, cancer accounts for 1 of every 4 deaths.
  • Id proteins are potent inducers of these tumor initiation events, which are in turn disabled or blocked by the potent anti-Id compositions and methods of the invention.
  • Anti-Id compounds and treatment methods provided here focus on multiple complementary anti-metastasis strategies, including: 1 ) prevention of cancer cell dissem ination; and 2) suppression of existing metastases.
  • compositions of the invention find use in therapeutic applications in which anti-Id administration is indicated, as described in a cl inically effective context for the first time here.
  • Rep resentative therapeutic applications of the invention include treatment of cellular proliferative diseases, including cancer and other diseases characterized by adverse cellular prol iferation (hyperplasias).
  • the anti-Id c ompositions and treatment methods of the invention are effective to reduce or prevent ce l lular prol iferative disorders in mammal ian subjects, including cl inical ly effective treatment of cancer in veterinary (e.g., dog, cat, large animal) and human patients.
  • anti-Id effectiveness of the compositions and methods of the invention may be correlated with a by a decrease in one or more symptoms of a cel lular prol i ferative disorders (e.g., cancer, metastatic disease, tumor-associated angiogenesis). This may be detected or quanti fied based on an observed decrease in cel lular prol iferation, vascular growth, cel lular m igration, secondary tumor appearance or growth, inflammation, or any other symptom associated with the targeted cel lular prol iferative disorder.
  • Various assays and model systems can be readily employed to illustrate therapeutic effectiveness of the anti-Id compounds of Formula I, I I, I I I and IV described herein.
  • assays are wel l known and include widely accepted cl inical correlates for each of the subject prol i ferative disorders contemplated herein.
  • Exemplary assays for ready use in this context include assays to detect or quantify cellular proliferation markers, circulating endothel ial cel ls, circulating endothel ial progenitor cells (EPCs), circulating tumor cells, various cancer/tumor markers (e.g., PSA), and histological, histochemical, and immunohistochem ical markers to detect, local ize, anatomically map, and/or quantify primary and metastatic cancer cells, among others.
  • EPCs circulating endothel ial progenitor cells
  • PSA cancer/tumor markers
  • histological, histochemical, and immunohistochem ical markers to detect, local ize, anatomically map, and/or quantify primary and metastatic cancer cells, among others.
  • markers for tagging , visual izing, quantifying and/or separating cells within various assays and diagnostic or cl inical embodiments of the invention include: for metastatic cel ls bearing markers of increased metastatic potential (e.g., markers associated with loss of epithelial character, increased migration potential, secondary site colonization potential)-Vimentin+, N-Cadherin+, E-Cadherin-; for EPCs-Lin-, GFP+, VE-Cadherin+, CD 1 1 -; for cancer stem cells (breast cancer example) ⁇ CD44(h igh) Cd24 (low).
  • markers of increased metastatic potential e.g., markers associated with loss of epithelial character, increased migration potential, secondary site colonization potential
  • the anti-Id compositions and methods of the invention are effective to reduce occurrence, relapse or growth of a neoplasm.
  • Administering an anti- neoplasm effective amount of the anti-Id compound wil l reduce incidence, number or growth of neoplasms in treated versus control subjects by 5%, 1 0%, 25%, 30%, 50%, 75%, 90% or more.
  • the invention provides "anti-metastatic" compositions and methods, which are effective to reduce an incidence or severity of tumor metastasis in mammalian subjects. These methods may employ monotherapy or coordinate or
  • anti-metastatical ly e ffective as demonstrated by a signi ficant reduction in incidence, number or size of metastases in treated versus control subjects presenting with, or at elevated risk for developing, cancer.
  • anti-metastatic efficacy correlates with a reduction in one or more histopathological indices of metastasis, for example quantitative reduction in occurrence, size, number or distribution of metastasized cells or ⁇ 'foc i'" of primary tumor character observed at a secondary tissue or anatom ic site.
  • anti-metastatic efficacy is demonstrated by a significant positive increase in one or more patient therapeutic indices correlating with effective prevention and/or treatment of cancer or metastas is— for example by an increase in a time period of disease-free survival for subjects receiving the anti-Id compound compared to control subjects not receiving the anti-Id compound.
  • Anti-metastatic efficacy of the compounds, compositions and methods of the invention yield substantial therapeutic benefits and improved treatment outcomes in patients treated for cellular prol iferative disorders and/or neoplasms.
  • cancer patients treated with the anti-Id methods and compositions of the invention exh ibit improved treatment outcomes with no increase, and often a significant decrease in adverse side effects that attend conventional cancer treatments (e.g., chemotherapy and radiation therapy, which may be reduced or el iminated in patients treated with the compositions and methods herein).
  • methods of the invention will yield at least a 20% increase in one or more positive therapeutic indices of metastatic disease prevention or arrest, for example a reduc tion in occurrence, size, number or distribution of metastasized cel ls or "foci " of primary tu mor character observed at a secondary tissue or anatomic site.
  • anti-rnetastatic efficacy of the anti-Id compounds and methods wi l l yield at least a 20% increase in an accepted metastatic index, including gross indices such as disease- free survival of Id-treated patients compared to quali fied control patients not treated with the anti-Id compound.
  • anti-Id compounds, formulations and methods of the invention wi l l yield even more significant anti-metastatic cl inical benefits, for example a 20-50% increase, 50-70% increase, up to a 75%- 1 00% reduction in a recogn ized metastasis indicator or marker (e.g., cancer cel l m igration or dissemination observed at or near a primary tumor site, metastasis observed in blood or lymphatic biological samples, or secondary tumor formation detected in a distant tissue or organ (e.g., by radiological or other imaging, biopsy, or post-surgical or post-mortum histolopathology).
  • a recogn ized metastasis indicator or marker e.g., cancer cel l m igration or dissemination observed at or near a primary tumor site, metastasis observed in blood or lymphatic biological samples, or secondary tumor formation detected in a distant tissue or organ (e.g., by radiological or other imaging, biopsy, or post-surgical or post-mor
  • the anti-Id methods and compositions of the invention wi l l be anti-metasta ically effective to yield at least a 20% decrease in metastasis, a 20%- 50%, a 50%-75%, up to a 90% or greater decrease in metastasis (e.g., as demonstrated by conventional, comparative histopathology, computerized tomography, positron em ission tomography and/or magnetic resonance imaging to detect, locate and/or quantify metastatic cells) in anti-Id-treated versus non-treated or placebo-treated subjects.
  • anti-metastatic efficacy will typica l ly correlate with no increase, or even a decrease, in observed adverse side effects (e.g., nausea, weight loss, hair loss, immunological damage, etc.) between anti-Id treated patients and a taxane, or an alternate cancer therapy, such as radiation therapy).
  • adverse side effects e.g., nausea, weight loss, hair loss, immunological damage, etc.
  • anti-ld-treated subjects including subjects treated with anti-Id compound monotherapy, and subjects treated with combinatorial methods, such as anti-Id plus chemotherapy, or anti-Id plus radiation therapy
  • wi ll exhibit no increase in one or more general ly-seen adverse cancer treatment side effects (e.g., general chemo- or radiation- therapy side effects), and will often exh ibit at least a 20% reduction, a 20-50% reduction, up to a 50-90% or greater reduction in occurrence or severity of one or more adverse cancer treatment side effects (e.g., compared to positive control subjects treated with conventional chemotherapy or radiation therapy alone).
  • Side effects that may be associated with anti-Id therapy may include side effects associated with anti -angiogenic effects of the novel, anti-Id compounds described here.
  • various management tools can effectively l imit or prevent such adverse sequelae.
  • potential impairment of wound healing due O anti-angiogenic activity of the claimed compositions and methods can be avoided by staging anti-Id treatment prior to cancer surgery when indicated, and/or post- surgery al lowing for an effective heal ing period prior to initiation of the anti-Id therapy.
  • Additional pro-heal ing agents and methods can be coordinately adm inistered, such as coordinate administration of pro-heal ing cytokines or growth factors (e.g., platelet-derived growth factor (PDGF).
  • PDGF platelet-derived growth factor
  • the anti-Id compounds and methods are also effective to reduce a metastasis-associated increase in circu lating endothel ial cel ls.
  • Circulating endothelial cells are general ly absent in the blood of healthy ind ividuals, but significantly elevated in individuals suffering from diseases marked by pathogenic angiogenesis, including cancer.
  • the number (titer or hematocrit count) of c irculating endothel ial cel ls may be determined by any means applicable, such as through flow cytometry, immunobead capture, fl uorescence m icroscopy, standard and density
  • An anti-metastatic or anti-angiogenic effective amount of the anti-Id compound of the invention will decrease the number of circulating endothel ial cells by 5%. 10%, 25%o, 30%, 50%, 75%, 90% or more compared to levels observed in placebo-treated subjects presenting with similar pathology (e.g., an equivalent state of metastatic or angiogenic disease prior to treatment, normal ly attended by tumor-associated elevation of EPCs).
  • the anti-Id compounds and methods of the invention are effective to block or reduce a metastasis-associated increase in circulating endothel ial progenitor cel ls (EPCs) implicated in metastatic disease progression.
  • EPCs endothel ial progenitor cel ls
  • Endothel ial cell production is generally responsible for repair of damaged vasculature (including associated with tumors, and in other cases of pathogenic angiogenesis) through mobil ization of EPCs from the bone marrow. This is followed by hom ing of the EPCs to target sites for repair (including sites of damaged vasculature associated with tumors) (Shaked et al., 2006; Shaked et al., 2008).
  • This process of vascular repair often follows treatment with certa in, cytotoxic "chemotherapeutic * ' drugs, such a pacl itaxel, and vascular disrupting agents (VDAs, for example ZD6 1 26 or AVE8062), which have attendant adverse cytoxic side effects on blood vessels.
  • chemotherapeutic * ' drugs such as pacl itaxel
  • VDAs vascular disrupting agents
  • This angiogen ic repair process fol lowing paclitaxel treatment is observed in normal mice, but reportedly absent in mice lack ing the Id l gene (Shaked et al, 2008).
  • Id 1 is required for the previously described ability of phenotypic HSCs to give rise to endothel ial progeny to highl ight the opposi ng effects of Id l and its target gene p2 l on endothel ial and myeloid l ineage differentiation in the HSC subset.
  • Id l Ablation of p2 1 i n the Id l -/- animals restores a functional endothel ial population, rescues the angiogenic defect observed in the Id l -/- mice, and reverses the premature myeloid commitment of Id l nul l HSCs.
  • Id l appears to play a therapeutical ly negative, protective role for cancer cells undergoing threat with cytotoxics through a general anti-apoptotic effect (Zhang et al., 2006; Wong et al., 2004). This was reported based on s tudies of paclitaxel/docetaxel-induced apoptosis in prostate cancer cells.
  • nasopharyngeal carcinoma cells HeLa (cervical) cancer cells and MCF7 (breast) cancer cells.
  • Up-regulation by Id l of Raf/ME (Zhang et al., 2006), and/or MAPK signaling pathway (Cheung et al., 2004; Lin et al., 2005) has also been proposed to account for resistance to cytotoxics acquired by cancer cells post-treatment.
  • anti-Id compounds and methods of the invention fundamentally disable Id proteins at critical intersections of these pathways. I n this manner, anti -metastatic and anti-angiogenic effective anti-Id compounds of the invention also target EPC production and secondari ly disable tumor-associated angiogenesis and related tumor growth.
  • the anti-Id compositions and methods of the invention wi l l effectively decrease the number of EPCs in a circulating blood sample of treated subjects by at least 5%, 1 0%, 25%.
  • sim i lar pathology e.g., an equivalent state of metastatic or angiogen ic disease prior to treatment, normal ly attended by tumor-associated elevation of EPCs.
  • Anti-Id compositions and methods of the invention further exert anti-cancer and anti- metastatic effects by sh ifting a cellular determination or fate of tumor cel ls toward senescence or apoptosis.
  • anti-Id compositions and methods of the invention wi ll effectively increase the number of apoptotic or senescent cel ls in a primary or secondary tumor of anti-Id-treated patients.
  • the anti-Id compositions and methods of the invention wi l l increase the number of apoptotic or senescent cel ls in tumors (e.g., as observed through biopsy or necropsy of existing tumors) of treated subjects by at least 20%, 30%.
  • sim ilar pathology e.g., an equ ivalent state of metastatic or angiogenic disease prior to treatment, normal ly attended by tumor growth and a low incidence of apoptotic or senescent cells.
  • Cancer stem cells are a subopulation among bulk of tumor cel ls capable of in itiating new tumors (with a capacity to recapitulate the l ineage heterogeneity of the parental tumor). Cancer stem cells share characteristics of tissue stem cel ls, including sel f-renewal and multipotency.
  • Id proteins on cancer stem cel ls have been reported in colon cancer and malignant glioma, although other types of cancer stem cel ls also appear to depend on Id proteins (Hua et al., 2006; James et al, 20 1 0; Jankovic et al., 2007; Perry et al., 2007; Rawlins et al, 2009; Suh et al., 2009; Lyden et al.; 1999; Anido et al., 2010; Jeon et al., 2011).
  • colon cancer stem cells combined expression of Idl and ld3 reportedly increases both self-renewal and tumor initiation (O'Brien et al, 2012).
  • Cancer stem cells exhibit resistance to chemotherapeutic agents, and silencing of Idl and Id3 in culture-based assays reportedly sensitizes cells to oxaliplatin (0 * Brien et al, 2012).
  • Id proteins are co-expressed in diverse tumor cell populations, including glioma stem cells.
  • deletion of conditional Id 1 , Id2 and Id3 alleles in the tumor cells reportedly decrease the glioma stem cell population (nestin-positive and stage-specific embryonic antigen 1
  • SSEA l Embryonic neural stem cells
  • Id4 has also been reported to de-repress miR-9*-mediated suppression of SRY-box 2 (SOX2), increasing glioma stem cell potential and chemorcsistance (Jeon et al., 2011).
  • somatic stem cells cancer stem cells are anchored to a niche and derive supportive signals through cell-cell contacts with endothelial cells in blood vessels. The ability to adhere to the niche is a crucial feature of normal stem cells and cancer stem cells (Calabrcse C et al., 2007; Chen S et al., 2013; Lewallen M et al.. 2013; Fietzetal., 2011; Lathia JD et al., 2010; Park DM et al., 2009).
  • Id proteins reportedly disrupts stem cell adhesion to endothelial cells in the niche in NSCs and in glioma stem cells (Niola et al., 2012; Niola et al., 2013).
  • Id-mediated repression of bl 1LH transcription limits expression of RAS-related protein 1 (RAPL) GTPase-activating protein (RAP 1 GAP), a bHLH target gene that encodes an inhibitor of the RAP1 GTPase, which controls cell adhesion via integrin signaling (Boettner et al, 2009).
  • RAPL RAS-related protein 1
  • RAP 1 GAP RAS-related protein 1 GTPase-activating protein
  • both stem-l ike cel ls and cel ls with features of committed progenitors may have capacity to propagate tumors efficiently.
  • Id proteins have been postulated to play key roles as regulators of stem cell identity in both colorectal cancer and mal ignant glioma, essential for both self-renewal and tumor-initiating capacity of cancer stem cel ls. Again despite the mechanistic complexity and indefin ite pathways involved in cancer stem cel l deve lopment. anti-Id compounds and methods of the invention potently disable Id proteins at a critical foundation to disru pt stem cel l identity and impair stem cell tumor initiation.
  • Anti-metastatic and anti-angiogenic effective compounds of the invention specifically target tumor stem cell viabi l ity, prol iferat ion capacity, tumor-initiation potential, and/or cel l fate determination- w ith the result of substantial ly decreasing populations of new tumor induction-competent stem cel ls present i n new or established tumors.
  • the anti-Id compositions and methods of the invention will effectively decrease the number of cells bearing one or more selected stem cell markers in tumors (e.g., as observed through biopsy or necropsy of existing tumors) of treated subjects, by at least 5%, 1 0%, 25%, 30%, 50%. 75%.
  • sim i lar pathology e.g., an equivalent state of neoplastic or metastatic disease prior to treatment, normal ly attended by tumor growth, new tumor formation, and a high i ncidence of tumor- associated cancer stem cells.
  • sim i lar pathology e.g., an equivalent state of neoplastic or metastatic disease prior to treatment, normal ly attended by tumor growth, new tumor formation, and a high i ncidence of tumor- associated cancer stem cells.
  • assays are readi ly designed and implemented to identify and quantify cancer stem cells, for example based on detection of positive stem cel l markers, such as nestin and SSEA 1 , using conventional assay technologies such as cytometry, imm unobead capture, and immunocytochemistry. More discrete assays wil l determ ine d i fferences in tumor stem cel l viabil ity, prol iferation capacity, tumor-initiation potential, and/or cel l fate. speci fically correlated in anti-Id-treated subjects with reduction of cancer disease risk, accord ing to the teachings here and following reports of others above (al l of which are incorporated herein by reference for economy of description).
  • Effectiveness of anti-Id treatment methods of the invention directed against cancer and metastatic disease may be monitored in terms of cl in ical success by any of a variety of methods, for example by tumor imaging with x-rays or M l s (e.g., to determ ine i f tumors have decreased in size or number in treated patients).
  • Effectiveness wil l often be determ ined by radiographic or M RI observation of a decrease in tumor size.
  • Effective anti-Id compositions and methods of the invention for treating cancer wi l l routinely yield at least a 10%, 25%, 50%, 75%o, 90% or greater reduction of tumor size in treated patients, or average tumor size among a group of treated patients, compared to qual i fied, comparable control subjects.
  • Effectiveness of anti-Id treatment methods of the invention directed against cancer and metastatic disease may further be determ ined by measuring the number of circu lating tumor cel ls in blood samples between suitable test and control subjects. This may be accompl ished by any means applicable including, but not limited to immunomagnetic selection, flow cytometry, immunobead capture, fluorescence m icroscopy, cytomorphologic analysis, or cel l separation technology.
  • Effective anti-Id compositions and methods of the invention for treati ng cancer will routinely yield at least a 1 0%, 25%, 50%, 75%, 90% or greater reduction of circulating tumor cells in blood samples of treated patients, or among a group of treated pa tients, compared to qualified, comparable control subjects.
  • Effectiveness of anti-Id treatment methods of the invention directed against cancer and metastatic disease may further may also be determ ined by detecting or measuring primary tumor cel l occurrence or number in secondary tissues or organs, including but not l im ited to bone, lymph nodes and lung.
  • Effective anti-Id compositions and methods of the invention for treating cancer wi l l routinely yield at least a 1 0%, 25%, 50%. 75%, 90% or greater reduction in the occurrence or number of primary tumor cel ls metastasized to secondary tissues or organs among treated patients compared to qua l ified, comparable control subjects.
  • anti-Id compositions and methods for prevention or treatment of cancer involve coordinate adm inistration of an effective amount of the anti-Id compound, along with a secondary treatment agent, treatment modality or treatment method.
  • a secondary treatment agent or method selected from : a chemotherapeutic drug (i.e., using a second anti-cancer or anti-metastatic drug, compound or chem ical agent), radiation, chemotherapy, surgery, or any combination of these agents/methods.
  • the invention employs the anti-Id com pound administered simultaneously (at the same time, optionally in a combined formulation) with a secondary drug, compound or chemical agent possessing combinatorial anti-cancer or anti-metastatic activity.
  • Secondary chemotherapy drugs in this context are contemplated to broadly include agents classified as conventional chemotherapy drugs (for example taxanes); vascular disrupting agents ( V DAs); or HS P-90 inh ibitors.
  • the anti-Id compound and the secondary drug or treatment wil l be "combinatorial ly effective", meaning biological activity (e.g., anticancer or anti-metastatic activity as defined herein), side effects, patient outcomes, or other positive therapeutic indicia wi ll be improved over results observed in relevant control subjects treated with the anti-Id compound alone, or secondary drug alone.
  • biological activity e.g., anticancer or anti-metastatic activity as defined herein
  • side effects e.g., side effects, patient outcomes, or other positive therapeutic indicia wi ll be improved over results observed in relevant control subjects treated with the anti-Id compound alone, or secondary drug alone.
  • Anti-Id compounds and methods of the invention can be coordinately employed with any of a range of secondary anti-cancer drugs, agents or interventions, in combinatorial formulations or coordinate treatment protocols (with anti-Id therapy admin istered
  • an anti-Id compound such as AGX5 1 is adm inistered coordinately with a chemotherapeutic drug or therapy.
  • Chemotherapeutic drugs and therapies for use within these aspects of the invention include anti-cancer and anti-hypcrprol iterative agents, agents that destroy or "reprogram' ' cancer cells, agents that destroy blood vessels associated with neoplasms or hyperproli ferative conditions, and other classes of drugs harmful to neoplastic cel lu lar targets.
  • useful chemotherapeutics with in the invention include, but are not l im ited to:
  • V DAs anti-angiogenics agents and vascular disrupting agents
  • anti-inflammatory agents such as COX inhibitors, and
  • cel l cycle regu lators eg, check point regulators and telomerase inhibitors.
  • combinatorial formulations of the invention include an anti-Id compound as described in a combined formulation with one or more conventional chemotherapeutic drugs or other anti-metastatic compound or agent, optional ly includ ing a side-effect reducing agent as known in the art (which will depend on what combinatorial therapy is being employed, e.g.. chemotherapy versus radiation therapy, or both).
  • Anti-Id compounds of the invention may be provided in the form of a pharmaceutical ly acceptable salt. These compounds can be routinely formulated for oral, topical, parenteral, transdermal or intravenous (iv) adm inistration.
  • multiple pharmaceutical compositions may be provided (each containing a different active agent.
  • a single formu lation is provided that comprises an anti-Id active agent, a secondary chemotherapeutic agent, and optional ly a side effect reducing agent.
  • Combinatorial efficacy observed for coordinate therapies can occur for a variety of reasons but in general is due to combined inhibition of two or more independent pathways.
  • Individual pathways may provide "bypass" routes for targeted cel ls (e.g.. metastatic cancer cells), requiring that multiple pathways be targeted to prevent the escape.
  • cel ls at the periphery of tumors may "bypass" the disruption of normal tumor-associated angiogenesis coopting local vessels.
  • This stress in turn may be al leviated by endogenous activity of heat shock factor 90 (Hsp90).
  • an Hsp90 inh ibitor is coord inately administered with an anti-Id compound to yield combinatorial lv effective anti-Id and anti-HSP90 activity, attended by enhanced cl inical results.
  • Anti-Id compounds and methods of the invention can be particularly e ffectively employed in combination with vascular disrupting agents (VDAs), often with the attendant benefit of al lowing for a lowering of VDA effective dosage and/or reduction of V DA- associated adverse side effects.
  • VDAs arc mostly chemical agents that d isrupt the cytoskeletal vascular network causing cel l-shape and permeabi l ity changes resulting in vascular resistance, vasoconstriction, increased vascular permeabi l ity, platelet thrombi and vascular shutdown .
  • Vascular disruption represents a val idated therapeutic strategy to deprive tumors (and, in the case of eye disease, pathologic neovascular lesions) of blood supply.
  • vascular disrupting agents rapidly disrupt the vascu lature with in the tumor, reduce blood flow, and deprive the tumor of oxygen and nutrients, resu lting in tumor cell death.
  • This disruption of the newly formed blood vessels contrasts with the action of anti- angiogenic therapies, which are designed to prevent new blood vessel formation.
  • anti-angiogenic therapies which are designed to prevent new blood vessel formation.
  • VDAs resl rict tumor blood supply and drive them to hypoxic stress (ameliorated by heat shock protein (HSPs)
  • HSPs heat shock protein
  • Vascular disrupting agents target the tumor-associated, fragi le and relatively newly- constructed vasculature in tumors (Tozer et al., 2005, Mita et al., 20 1 3).
  • the prototype VDAs are combrestatins, natural antim itotic agents isolated from the root bark of the South A frican tree. Com oretum caffrum (C irca and Mann, 2003; Tozer et al., 2001 ). The most potent of these compounds is combretastatin A-4 (CA4), an antitumor drug.
  • CA4 combretastatin A-4
  • CA4 whose prodrug is CA4-phosphate (CA4P) binds to tubul in in endothelial cells at the same site as colchicine leading to strong inhibition of tubulin polymerization.
  • CA4P causes shape changes, cytotoxic ity, changes in cel l permeability and apoptosis of prol i ferating endothel ial cells, but not of qu iescent cel ls.
  • the cytoskeleton of mature cells is not sensitive to CA4P as opposed to newly formed cel ls, w hich are particularly sensitive.
  • There is a preferential sensitivity of endothel ial cells in tumor vessels to CA4P which unl ike those in normal vessels, become thrombogenic, resulting in hemorrhagic necrosis of tumors.
  • CA4P is currently being evaluated in multiple clinical trials as a treatment for various sol id tumors as wel l as a treatment for age-related macular degeneration (AM D), a vision l im iting condition (Nanbu et al., 2003 : Eichler et al., 2006).
  • AM D features an overgrowth of blood vessels as part of the underlying pathology (Campochiaro and 1 lackctt. 2003).
  • anti-angiogenic agents such as bevacizumab (Avastin 1 M ), that lim it tumor growth by preventing the formation of new blood vessels, have been approved for some cancer indications and are widely used to treat sol id tumors.
  • An anti-angiogenic agent such as bevacizumab (Avastin 1 M ), that lim it tumor growth by preventing the formation of new blood vessels, have been approved for some cancer indications and are widely used to treat sol id tumors.
  • An anti-angiogenic agent such as bevacizumab (
  • ranibizumab (Lucentis I M ) mechanistically related to bevacizumab, ranibizumab (Lucentis I M ), is used to treat AMD.
  • VDAs and anti-angiogenic agents both work in di fferent, yet complementary ways.
  • a nti-angiogcnesis drugs attempt to keep new blood vessels from forming.
  • anti-angiogen ic agents do not act on blood vessels that already feed existing tumors.
  • V DAs degrade blood vessels within the tumor and cause widespread cel l death in central parts of the tumor that historically have been resistant to conventional treatments, such as cytotoxic chemotherapy, rad iation, and biologies.
  • V DAs thus have demonstrated cl inical efficacy (Hasani and Leighl, 20 1 I ; H innen and Eskens, 2007; Mc Keage and Baguley, 20 1 0), preliminary evidence for the induction of EPCs after V DA treatment has been reported recently in Phase 1 clinical trials using the VDAs ZD6 126, AVE8062 or CA4P (Beerepoot et al., 2006; Farace et al., 2007). A robust elevation in EPC levels has also been observed within hours of treatment with microtubule-inhibiting cytotoxic-l ike vascular disrupting agents (VDAs) in mice (Shaked et a!., 2006). This induction of EPCs likely dim inishes effectiveness of the VDA treatment (Daenen et al., 201 0).
  • VDAs microtubule-inhibiting cytotoxic-l ike vascular disrupting agents
  • anti-Id compositions and methods of the invention are highly complementary to VDA treatment for reducing cancer incidence, metastasis, disease progression and tumor growth/invasiveness.
  • anti-Id compounds of the invention such as ANGX5 1 effectively block Id-mediated EPC responses to VDA treatments.
  • ANGX5 1 provides for reduction of VDA dosing or treatment duration with comparable anti-angiogenic effects, and less angiogenic rebound involving increase of EPCs responsive to tumor vascular destruction by VDAs.
  • Treatment of tumor-bearing m ice with VDAs leads to an acute mobi lization of EPCs, w hich home to the viable tumor rim that characteristical ly remains after VDA therapy.
  • Other coordinate treatment compositions and methods herein targets cel lular division as a secondary pathway for cancer and metastasis intervention.
  • Taxancs inhibit cancer cel l growth by antagon izing the machinery in cells that distribute chromosomes between mother and daughter cel ls. Taxane resistance has been reported to involve activation of the Notch signaling pathway, which in turn activates ld l .
  • Combining an anti-I d 1 compound with a taxane coordinately prevents this bypass.
  • a coordinate diagnosis and management protocol is contem plated to treat or prevent breast or ovarian cancer.
  • Women with fami ly histories of breast cancer or ovarian cancer may be selected for treatment, for example using a BrCA l genetic test to establ ish elevated risk of breast or ovarian cancer.
  • H igh risk subjects wil l be provided prophylactic anti-Id treatment (e.g., using (-)-AGX5 1 ) for several months, up to 1 -2 years or longer, post-surgery to prevent recurrence of the disease.
  • prophylactic anti-Id treatment e.g., using (-)-AGX5 1
  • Periodical ly, system ic Id levels are determ ined during this time.
  • I f Id is detected at a concentration above trace or non-measiireable, the anti-id treatment is continued or increased, optional ly supplemented by chemotherapy.
  • Other patients amenable to treatment according to the invention wi ll be identified by routine mammography of human females positive for a breast tumor, fol lowed by tumor excision and in some cases radiation.
  • anti-Id compound is administered chronically daily for up to a year or more to provide protection against recurrence of the disease. Recurrence is prevented through one or more of the anti-tumor, ant i-metastasis, pro-apoptosis and pro-cell cycle control effects of the drug.
  • systemic Id levels are determ ined during th is time.
  • I f Id is detected at a concentration above trace or non-measureable, the anti-Id treatment is continued or increased.
  • Additional coord inate diagnostic and management protocols are provided to treat breast cancer or prevent metastatic progression of an exist ing breast tumor.
  • Patients are identi fied by routine mammography of human females as positive for a breast tumor, fol lowed by tumor excision and in some cases radiation.
  • Cancer cel ls are identified in the lymph nodes of the subject.
  • the subject is adm inistered a course of conventional pacl itaxel treatment (e.g., every three weeks for 12 weeks). Over this period, anti-Id compound is adm inistered chronically daily day to maxim ize the effect of the pac l itaxel.
  • the anti-Id treatment is continued for an extended period (e.g., 9- 1 2 months) post pac l itaxel treatment to provide additional protection against recurrence of the disease.
  • Periodical ly, system ic Id levels and optional ly other markers are determ ined during this time.
  • I f Id is detected at a concentration above trace or non-measureable, the anti-Id treatment is continued or increased.
  • patients previously identified by routine mammography as positive for a breast tumor and treated by tumor excision (no cancer found in the lymph nodes), are exam ined two years post-surgery f r tumors (e.g., by computerized tomography (CT) scanning followed by Positron Emission Tomography (PET scanning).
  • CT computerized tomography
  • PET Positron Emission Tomography
  • the subject wi l l be administered conventional taxane therapy (e.g., paclitaxel every three weeks for 1 2 weeks), fol lowed by anti-Id compound dai ly for 9- 1 2 months to prevent rem ission of the disease, coupled with monitoring of Id levels and optionally other markers as described.
  • conventional taxane therapy e.g., paclitaxel every three weeks for 1 2 weeks
  • subjects presenting with Stage I I I are selected for cancer treatment and management.
  • Subjects thus identified are treated using an aggressive, combinatorial treatment regimen employing high doses of radiation and multiple doses of paclitaxel.
  • histopathology and/or bioscans eg, computerized tomography (CT), positron em ission tomography (PET) and/or magnetic resonance imaging] are used to identify patients presenting with no detectable cancer after the first l ine treatment above.
  • the anti-Id treatment would continue for a year post-treatment and longer i f the Id blood levels fai l to decrease to a value consistent with an acceptable (basel ine or low-risk) for metastatic potential or the presence of microtumors.
  • wil l employ an array of combinatorial therapies.
  • Patients identified with recurring or metastatic breast cancer may be treated fi rst with an aggressive course of taxane therapy (e.g., 5 single doses treatment of taxane (e.g., pacl itaxel. docetaxel or album in-bound paclitaxel), every three weeks for 12 weeks), to wh ich may be added a a vascular disrupting agent (VDA), such as combretastatin-A4 phosphate.
  • VDA vascular disrupting agent
  • anti-Id compound is also administered chronically dai ly to maximize the effect of taxane/VDA treatment.
  • the anti-Id treatment is continued for 9- 1 2 months or longer to prevent recurrence or metastatic progression of the d isease, with Id and other marker monitoring as described.
  • these patients may be adm inistered taxane therapy in combination with bimonthly or month ly treatment with an anli-VEGF agent (e.g.. bevacizumab), or anti-VEGF receptor antagonist (e.g., sunitinib, sorafenib) rein forced by extended, prophylactic admin istration of anti-Id compound of the invention.
  • anli-VEGF agent e.g. bevacizumab
  • anti-VEGF receptor antagonist e.g., sunitinib, sorafenib
  • coordinate diagnosis and management focuses on patients testing positive for the presence of the H ER2/neu receptor.
  • These subjects may be administered conventional taxane therapy along with bimonth ly or monthly treatment with trastuzumab (Herceptin), supplemented or followed by daily anti-Id compound treatment as described.
  • the anti-ld treatment is continued for 6- 12 months post the taxane treatment to provide additional protection against recurrence of the disease.
  • Clinical management methods of the invention can also be adapted to treat more specific cancers, for example estrogen and progesterone receptor-negative breast tumors.
  • Exemplary protocols here may employ tumor excision and in some cases radiation fol lowed by chemotherapy (e.g. with pacl itaxel and doxorubicin every three weeks for 1 2 weeks), accompanied or fo llowed by daily anti-Id therapy (continuing for an extended post- chemotherapy period for prevention of d isease recurrence).
  • cancer patients wi l l be treated coordinately with taxane chemotherapy (e.g., with paclitaxel, docetaxel or albumin-bound paclitaxel) supplemented with concurrent or sequential cisplatin treatment, and anti-Id therapy wi l l fol low the taxane therapy and extend 6- 12 months or more to protect against recurrence of the disease.
  • taxane chemotherapy e.g., with paclitaxel, docetaxel or albumin-bound paclitaxel
  • anti-Id therapy wi l l fol low the taxane therapy and extend 6- 12 months or more to protect against recurrence of the disease.
  • Comparable coordinate diagnosis and management protocols are provided for treating other forms of cancer.
  • prostate cancer patients wi l l be se lected based on PSA screening and/or biopsy to produce a Glecson score reflecting the stage and metastatic risk of the cancer.
  • Subjects at elevated risk are treated with radiation and chemotherapy, coupled with or followed by extended anti-Id treatment and mon itoring as above.
  • Th is treatment may be coordinated with conventional anti-androgen therapy.
  • Coordinate diagnosis and management protocols for treating melanoma in situ may include topical taxane treatment coupled with concurrent or subsequent, therapeutic or prophylactic anti-l d treatment according to the invention.
  • Coordinate diagnosis and management protocols for treating Kaposi sarcoma may include intralesional or topical taxane treatment coupled with concurrent or subsequent, therapeutic or prophylactic anti-Id treatment according to the invention.
  • This and other coordinate treatment methods may optionally include concurrent or sequential interferon- alpha treatment.
  • inc luding for al l types and stages of cancer and other proliferative disorders including but not limited to bladder cancer, colon cancer, pancreatic cancer, lung cancer, brain cancer, esophageal cancer and leukemias.
  • the entire armamentarium of known effective ch emotherapeutic agents may be combined w ith anti-Id therapy, l ikewise the ful l range of vascular disrupting agents (VDAs).
  • VDAs vascular disrupting agents
  • HSP 90 inhibitors can be combined with anti-Id drugs such as AGX5 1 in combinatorial formulations, and any configuration of coordinate treatment regimens, optional ly with other interventions such as rad iation and surgery.
  • compositions and methods of the invention target distinct cel lular prol i ferative disorders characterized by aberrant blood vessel growth, or "pathogen ic angiogenesis " .
  • pathogen ic angiogenesis characterized by aberrant blood vessel growth, or "pathogen ic angiogenesis " .
  • samples of these disease targets include ocular disease mediated by aberrant vascular growth (e.g., macu lar degeneration), and tumor-associated angiogenesis.
  • Anti-Id compounds of the invention function also as "anti-angiogenic” agents, as described below, making them useful to treat or prevent pathogenic angiogenesis, including quite powerful ly tumor- associated angiogenesis to mediate a multi-pronged assault on tumor development (i.e., both anti-metastatic and anti-angiogenic).
  • Anti-Id compositions and methods of the invention target both tumors and their supporting blood vessels simultaneously.
  • Anti-Id compositions and methods of the invention mediate both anti-tumor (including anti-metastatic) and anti-angiogenic effects.
  • Other anti-angiogenic agents alone have marginal efficacy for treating cancer, often requ iring coordinate use of cytotoxic chemotherapies to achieve a therapeutic response.
  • Additional embodiments of the invention employ anti-Id compounds such as AGX5 1 to effectively treat or prevent any pathogenic angiogenic or ncovascu lar cond ition or disease.
  • anti-Id compounds such as AGX5 1 to effectively treat or prevent any pathogenic angiogenic or ncovascu lar cond ition or disease.
  • Exemplary pathologic neovascularization phenomena are associated with the ocular d isease, age related macular degeneration (AM D).
  • AMD AMD is the most common cause of irreversible vision loss in the elderly (Jager et al., 2008). This condition is mediated in large part by angiogenic changes typified by a neovascular lesion complex which can be differentiated by ocular angiography into choroidal neovascular membrane (CNV) and non-CNV components. AMD is characterized by a spectrum of clinical and pathologic findings, including drusen formation, disruption of the RPE, CNV, disciform scar formation and sub-retinal fibrosis.
  • CNV choroidal neovascular membrane
  • the sustaining event for AM D is believed to be chronic ischem ia-reperfusion (I-R) inj ury of ocular tissue damage caused when blood supply returns to the tissue after a period of ischemia or lack of oxygen.
  • I-R ischem ia-reperfusion
  • the absence of oxygen and nutrients from blood during the ischem ic period creates a cond ition in which the restoration of circulation results in inflammation and oxidative damage through the induction of oxidative stress rather than restoration of normal function.
  • Tissue damage caused by cumulat ive insu lt by excess sunlight, pol lution, dust and d irt is bel ieved to be the in itiating event for the ischemia that precedes the l-R.
  • A has two forms: non-exudative (dry) and exudative (neovascular or wet).
  • anti-VEGF treatments represent a major advance in treating AM D (Rosenfeld et al.. 2006; Brown et al., 2009.
  • Useful drugs include ran ibizumab, an anti-V EG F fab fragment for injection, reported to stabi l ize or mediate reversal of vision loss in 95% and 40% of patients, respectively.
  • a majority of patients treated with th is drug d id not experience vision gain, and patients who had positive responses to treatment did not regain abi l ity to drive or read normal ly.
  • Results with bevacizumab, an anti-VEGF monoclonal antibody, for injection appear comparable (Rosenfeld et al., 2005).
  • Anti-VEGF therapies appear to exert most of their beneficial effect via an anti- permeability action resulting in resolution of intra and sub-retinal edema, as the actual C N V lesion does not markedly involute (Eichler et al., 2006).
  • Flovvever, exudative AM D- related vision loss is not due to solely choroid neovascularization (CNV) induced sub-retinal and intra-retinal edema.
  • CNV choroid neovascularization
  • pan-VEGF inhibition in the eye has yet to be established.
  • VEGF produced by a number of ce l ls in the neurosensory retina, is neuroprotective in nature and chronic inhibition could be detrimental to neuronal health (Greenberg et al.. 2005).
  • VEGF is also constitutively expressed by RPE cells and is a survival factor for quiescent choriocapil laris endothel ium (Witmer et al.. 2003), suggesting that long term VEGF inhibition could be detrimental to this vital structure and the cel ls wh ich rely upon it for metabolic support.
  • chronic anti-VEG F treatment a recent re-evaluation of patients treated for seven years with anti-VEGF treatment reported that macu lar atrophy was detected by fluorescein angiography in 98% of eyes, and that the area of atrophy correlated significantly with poor visual outcome ( Rofagha et al. 20 1 3).
  • the three anti-VEGF agents most commonly used to treat ocular conditions are ranibizumab for i njection), afl ibercept for injection, and bevacizumab for injection.
  • these agents are also used also used to treat macular edema that results from central retinal vein occlusion (CRVO) or branch retinal vein occlusion ( B VO) as wel l as for the treatment of macular edema due to diabetic retinopathy.
  • CRVO central retinal vein occlusion
  • B VO branch retinal vein occlusion
  • V DA vascu lar disrupting agent
  • CA-4 phosphate is also being evaluated in multiple cl in ical trials as a prospective treatment for AM D (Eichler et al., 2006).
  • Two surgical procedures are also occasional ly used to remove the ocu lar lesions that arc the essential pathology of A MD. particularly wet AMD: Laser photocoagulation ( and photodynam ic therapy (Cook et al., 2008).
  • exudative AM D The exact etiology and pathogenesis of exudative AM D is sti l l not wel l understood but is thought to consist of vascular and extravascular components orchestrated by mu ltiple factors (Tezel et al ., 2004; Ambati et al., 2003). However, AMD features an overgrowth o f blood vessels as p .rt of the underlying pathology (Campchiaro et al., 2003 ).
  • the vascu lar component of exudative AM D is comprised of vascular endothel ial cells, endothelial cel l precursors and per cytes. VEGF appears to be an important mediator in the pathogenesis of vascular component.
  • Tissue damage can result from either the vascular or extravascular component of the disease process.
  • the extravascular component often appears to be the largest component volumetrical ly, and appears, by histopathology, to be the source of the angiogenic stimuli. This extravascular component is composed primari ly of inflammatory cells and less so fibroblasts and gl ial or RPE.
  • Macrophages and the complement system are now known to play an important rol e in affecting CNV and propagating the pathogenesis of exudative A MD (Bushin i et al, 20 1 1 ; Gold et al., 2006; Klein et al., 2005 ; Hageman et al.. 2005 ; Tsutsum i et al .. 2003 ; Espinosa- Heidmann et al., 2005 ; Oh et al., 1 999; Grossniklaus et al., 2002; Forrester et al.. 2003).
  • the invention provides methods for inhibiting pathologic ocular neovascularization comprising adm in istering to a subject presenting with a neoplasm an e ffective amount of an anti-Id compound in a monotherapy protocol employing a single drug or method of treatment.
  • treatment methods of the invention inc ludes adm inistering to the subject an effective amount of the anti-Id compound along with a secondary treatment agent, treatment modality or treatment method (for example by treating the subject with the anti-Id compound simultaneously or sequential ly with a secondary treatment modality or agent selected from for example: An anti-VEGF agent, a V DA, interferon- ⁇ (Naldini et al., 2005), a potent anti-angiogenic cytokine, or an agent that ind uces interferon- ⁇ for example IL- 1 2 (Del Vecchio et al., 2007 and Kleinman et al., 2008).
  • a secondary treatment modality or agent selected from for example: An anti-VEGF agent, a V DA, interferon- ⁇ (Naldini et al., 2005), a potent anti-angiogenic cytokine, or an agent that ind uces interferon- ⁇ for example IL- 1 2 (Del Vec
  • the invention may employ the anti-Id compound administered simultaneously (for example adm inistered at the same time or in a combined formulation) with a secondary drug, compound or chemical agent possessing combinatorial anti-growth activity).
  • secondary chemotherapy drugs are chosen, for example an anti-VEGF agent or, a VDA, interferon- ⁇ .
  • the anti-Id compound and the secondary drug wi ll be "combinatorially effective" , meaning biological activity, eg, anti - growth or anti-angiogenesis activity as defined herein, side effects, patient outcomes, or other positive therapeutic indicia wi ll be improved over results observed in relevant control subjects treated with the anti-Id compound or secondary drug alone.
  • anti-Id compounds wi l l be employed in conj uction with an anti-VEGF agent, a V DA, and/or , interferon- ⁇ , with an attendant benefit of lowering dosage and/or side effects of these complementary treatment agents whi le retaining coordinate clinical benefit from their conjunctive use.
  • anti-Id therapy attended by coordinate use of an anti-VEGF agent and/or a VDA wi ll employ lower than conventional dosages of the anti-VEGF agent and/or a VDA drug for the indicated treatment, while combinatorial efficacy wi l l be greater than ful l dosage conventional anti-V EGF agent and/or a V DA, and side e ffects such as long term vision loss wil l be reduced.
  • anti-angiogenic compositions and methods of the invention are effective to reduce pathologic ocular neovascularization in a mammal ian subject. These methods may empl oy monotherapy or coordinate therapy, as above.
  • the methods (and related compounds and compositions) of the invention are "anti-angiogen ic effective " , for example to reduce incidence, size, or number of vascular lesions in an ocular tissue of a subject presenting with AMD.
  • "reducing neovascularization" wi l l correspond to an observed reduction in a histopathologic or ocular angiography index o f AMD lesion size, for example a reduced occurrence, size, number or distribution of lesions or "foci" of lesions observed at a secondary ocular site.
  • anti-angiogenic efficacy w i l l be determ ined by a positive change in one or more patient therapeutic indices correlating with effective prevention and/or treatment of AM Ds, e.g.. by an increase in a time period of disease free or disease stable conditions for subjects receiving the anti-Id compound compared to suitable control subjects not receiving the anti-Id compound.
  • Anti-AMD lesion efficacy i.e., efficacy diminishing or stabi lizing growth of the neovascular lesion complex
  • wil l routinely yield substantial therapeutic benefits and improved treatment outcomes in patients treated for an ocular condition (or any other pathogenic condition) w ith harmful angiogenesis as pa rt of its underlying pathology.
  • patients treated with the anti-ld methods and compositions of the invention wi ll exhibit improved treatment outcomes with no increase or an observed decrease in adverse side effects.
  • methods of the invention will yield at least a 20% increase in one or more positive clinical therapeutic indices for example a beneficial change in AMD lesion index (eg.
  • anti-AM D lesion efficacy of the anti-Id compounds and methods wi l l be demonstrable indirectly by at least a 20% increase in a disease-free or d isease stable condition for Id-treated patients compared to survival determined in suitable control patients (not treated with the anti-Id compound). In other embodiments.
  • anti-Id compounds, formulations and methods of the invention will resu lt in even greater anti-AM D clinical benefit, for example yielding a 20- 50% increase in a positive therapeutic index, 50-90% increase, up to a 75%- 1 00% increase, including total reir ission of observed primary AMD lesion enduri ng for 6 months to a year. 1 -2 years, 2-5 years, 5 years or greater, including 1 0 year and longer rem ission.
  • the anti-Id methods and compositions of the invention wi l l be anti-AM D effective to yield at least a 20% decrease in lesion size, a 20%-50%, a 50%-75%.
  • lesion size up to a 90% or greater decrease in lesion size, e.g., as demonstrated by comparative h istopathology, ocu lar angiography, optical coherence tomography (OCT) or another ocu lar imaging technique in anti-Id-treated versus non-treated or placebo-treated subjects.
  • OCT optical coherence tomography
  • anti-AM D efficacy w i l l typical ly correlate with no increase or even a decrease in observed symptoms of AM D, e.g., loss of visual acuity between anti-Id treated patients and positive control -treated subjects.
  • anti-Id-treated subjects included ing subjects treated with anti-Id compound monotherapy, and subjects treated with combinatorial methods such as anti-ld plus anti-VEGF therapy wi ll exhibit no increase in Snellen chart score and wi l l often exhibit at least a 20% increase, a 20-50% increase, up to a 50-90% or greater increase in Snel len chart score compared to positive control subjects treated with conventional (e.g., anti-VEGF) therapy.
  • cancer patients and other subjects identified for treatment according to the compositions and methods of the invention wi ll be evaluated and selected for refined clinical management using novel diagnostic and managed tools discovered here.
  • subjects are selected for treatment with anti-Id compositions and methods using novel Id diagnostic methods and kits provided here.
  • these methods and materials prov ide for detection and tracking of elevated Id levels (e.g.. ld l measured in blood or a biopsied tumor sample), wh ich wi l l correlate with a decision to initiated, continue, lower or increase anti-Id treatment.
  • these diagnostic methods may include diagnosing the subject for the presence of other disease indices (e.g., biochem ical or h istologic markers of cancer, or of metastasis, or of an angiogenic pathology) to yield coordinate diagnostic values to enhance disease assessment and management.
  • other disease indices e.g., biochem ical or h istologic markers of cancer, or of metastasis, or of an angiogenic pathology
  • Treatments that m itigate ld l and Id3 i.e., impair or inhibit their expression, activity or function are demonstrated here as potent tools for e ffectuating reduct ion of Id levels and attendant blockade of metastatic pathways and cel lular activities, along with other disease impacts in cancer patients.
  • Dosage, duration of treatment and efficacy of the novel anti-Id compounds employed within these methods wi l l best be determined rcflexively by monitoring changing Id levels (which in these methods w i l l often respond directly to changes in anti-Id treatment).
  • the novel treatment methods of the invention create a need for corresponding diagnostic and reflexive (cl inical managerial) monitoring of endogenous Id levels, which can be focused on any relevant test sample, such as cel ls (e.g., tumor cel ls, EPCs, cancer stem cells), tissues or organs (e.g., neoplasms, metastases, exploratory biopsy spec imens, lymph nodes) or physiological flu ids (e.g., blood, CNS fluid, lymphatic fluid).
  • cel ls e.g., tumor cel ls, EPCs, cancer stem cells
  • tissues or organs e.g., neoplasms, metastases, exploratory biopsy spec imens, lymph nodes
  • physiological flu ids e.g., blood, CNS fluid, lymphatic fluid.
  • Selection o f subjects amenable to treatment using the anti-Id compounds of the invention is useful ly guided by detection of elevated levels of ld l and/or Id3 in a biological sample (e.g., blood, urine or saliva) taken from a patient at risk of cancer, particularly of metastatic disease.
  • a biological sample e.g., blood, urine or saliva
  • Employment of assays to detect Id levels in th is manner in adj unct application with the invention yields observations of a bio-environment in certain patients with elevated Id levels, ind icating a high metastatic potential and thus a critical need for effective treatment using the anti-Id compounds and methods of the invention. Dosage and Formulation.
  • treatment and prophylaxis methods of the invention em ploy an "effective amount" of an anti-Id compound or composition as described.
  • Th is may refer to an amount or dosage of AGX 5 1 or another anti-Id compound that is effective to detectably, significantly reduce a level or concentration of a targeted Id protein (e.g., Id l or Id3) in a cel l, tissue, neoplasm, or subject.
  • a targeted Id protein e.g., Id l or Id3
  • Such demonstration of efficacy is readily using standard Id protein assays, for example using labeled id-specific antibodies or other quantitat ive Id detection reagents.
  • an anti-Id compound of the invention will be demonstrated as an amount or dosage effective to measurably inhibit or Id protein binding to a cognate binding partner (for example, Id dimerization to a bHLl I protei n, such as E-47.
  • a cognate binding partner for example, Id dimerization to a bHLl I protei n, such as E-47.
  • a lternate measures or assays to determ ine what is an effective amount or dosage of an anti-Id compound in this context include effective amounts and dosages of the compound that mediate a reduction in any detectable and/or quanti fiable Id activity or correlated biological index (e.g., a histopathological or clinical index of metastasis or tumor- associated angiogenesis), whether direct or indirect (as long as the activity is subject to modulation by reduction of Id activity or Id levels.
  • correlated biological index e.g., a histopathological or clinical index of metastasis or tumor- associated angiogenesis
  • Anti-Id compositions and methods in th is context may thus be alternatively demonstrated as '"effecti ve" through a demonstrated increase in apoptosis or cellular differentiation between test and control samples, by a decrease in cellu lar prol iferation, decrease in cel l m igration, decrease i n secondary site colonization by pri mary tumor cel ls, decrease in tumor associated ang iogenesis, decrease in EMT or MET progression, etc.
  • compositions of the invention wi l l often be referred to as comprising an "anti-cellular prol iferative effective amount " or unit dosage of an anti-Id compound, for example AGX5 1 .
  • compositions wi l l contain an "anti-metastatic dosage or amount" of the active anti-Id compound.
  • the active compositions may comprise an "anti-angiogenic effective amount" or dosage of the anti-Id drug.
  • Anti-Id compounds of the invention may be formulated with one or more
  • Suitable effective unit dosage amounts of the active anti-Id compounds for adm inistration to mammal ian subjects, including humans, may range from 1 0 to 1 500 mg, 20 to 1 000 mg, 25 to 750 mg. 50 to 500 mg, or 1 50 to 500 mg.
  • the anti-Id effective dosage may be selected within narrower ranges of, for example, 1 0 to 25 mg, 30-50 mg, 75 to 1 00 mg, 1 00 to 250 mg, or 250 to 500 mg.
  • These and other effective unit dosage amounts may be adm inistered in a single dose, or in the form of mul tiple dai ly, weekly or monthly doses, for example in a dosing regimen comprising from I to 5. or 2-3, doses administered per day. per week, or per month.
  • dosages of 1 0 to 25 mg, 30-50 mg, 75 to 1 00 mg, 1 00 to 250 mg, or 250 to 500 mg. are adm inistered one.
  • dosages of 50-75 mg, 1 00-200 mg. 250-400 mg. or 400-600 mg are adm inistered once or twice dai ly.
  • dosages are calculated based on body weight, and may be adm inistered, for example, in amounts from about 0.5mg/kg to about l OOmg/kg per day, l mg/kg to about 75mg/kg per day, I mg/kg to about 50mg/kg per day, 2mg/kg to about 50mg/kg per day, 2mg/kg to about 30mg/kg per day or 3mg/kg to about 30mg/kg per day.
  • compositions of the invention comprising an anti -Id effective amount of a compound of Formula I and/or I I I wi l l be routinely adj usted on an ind ividual basis, depending on such factors as weight, age. gender, and condition of the individual, the acuteness of the cel lular prol i ferative disorder and/or related symptoms, whether the adm inistration is prophylactic or therapeutic, and on the basis of other factors known to effect drug del ivery, absorption, pharmacokinet ics, inc luding hal f- l ife, and efficacy.
  • An effective dose or multi-dose treatment regimen for the instant anti-Id formulations wil l ordinari ly be selected to approximate a mrangel dosing regimen that is necessary and sufficient to substantial ly prevent or al leviate a targeted cel lular prol i ferati ve d isease (e.g., cancer, metastatic cancer, tumor-associated angiogenesis) in the subject, and/or to substantially prevent or alleviate one or more symptoms associated with the cel lular prol i ferative d isease in the subject.
  • a dosage and administration protocol w i l l often include repeated dosing therapy over a course of several days or even one or more weeks or years.
  • an effective treatment regime may also involve prophylactic dosage adm inistered on a day or multi-dose per day basis lasting over the course of days, weeks, months or even years.
  • the anti-Id active agent can be admixed with conventional pharmaceutical ly acceptable carriers and excipients (ie, vehicles) and used in the form of aqueous solutions, tablets, capsules, elixirs, suspensions, syrups, wafers, and the l ike.
  • Such pharmaceutical compositions contain, in certain embod iments, from about 0. 1 to about 90% by weight of the active compound, and more general ly from about 1 to about 30% by weight of the active compound.
  • compositions may contain common carriers and excipients, such as corn starch or gelatin, lactose, dextrose, sucrose, microcrystal l ine cellulose, kaolin, mannitol, di-calci um phosphate, sodium ch loride, and algin ic acid.
  • common carriers and excipients such as corn starch or gelatin, lactose, dextrose, sucrose, microcrystal l ine cellulose, kaolin, mannitol, di-calci um phosphate, sodium ch loride, and algin ic acid.
  • Disintegrators commonly used in the form ulations of this invention include croscarmcl lose, m icrocrystal l ine ce l lulose, corn starch, sodium starch glycolate and alginic acid.
  • a l iquid composition wi l l general ly consist of a suspension or solution of the compound or pharmaceutically acceptable salt in a suitable liquid carrier(s).
  • a suitable liquid carrier(s) for example, ethanol, glycerine, sorbitol, non-aqueous solvent such as polyethylene glycol, oi ls or water, w ith a suspending agent, preservative, surfactant, wetting agent, flavoring or coloring agent.
  • a l iq .lid formu lation can be prepared from a reconstitutablc powder.
  • a powder containing active compound, suspending agent, sucrose and a sweetener can be reconstituted with water to form a suspension and a syrup can be prepared from a powder containing active ingredient, sucrose and a sweetener.
  • composition in the form of a tablet can be prepared using any suitable
  • compositions routinely used for preparing sol id compositions.
  • examples of such carriers include magnesium stearate, starch, lactose, sucrose, microcrystal l i ne cel lulose and binders, for example, polyvinylpyrrolidone.
  • the tablet can also be provided with a color fi lm coating, or color included as part of the carrier(s).
  • active compound can be formulated in a control led release dosage form as a tablet comprising a hydrophi lic or hydrophobic matrix.
  • a compositi on in the form of a capsule can be prepared using routi ne encapsulation procedures, for example, by incorporation of active compound and excipients into a hard gelatin capsule.
  • a semi-solid matrix of active compound and high molecu lar weight polyethylene glycol can be prepared and filled into a hard gelatin capsule, or a solution of active compound in polyethylene glycol or a suspension in edible oil, eg, liquid paraffin or fractionated coconut oil, can be prepared and filled into a soft gelatin capsule.
  • Tablet binders that can be included are acacia, methylcellulose, sodium
  • Lubricants that can be used include magnesium stearate or other metallic stearates, stearic acid, silicone fluid, talc, waxes, oi ls and col loidal silica.
  • Flavoring agents such as peppermint, oil of wintergreen, cherry flavoring or the l ike can also be used. Additionally, it may be desirable to add a coloring agent to make the dosage form more attracti e in appearance or to help identify the product.
  • the compounds of the invention and their pharmaceutical ly acceptable salts that are active when given parenterally can be formulated for intramuscular, intrathecal or intravenous adm inistration.
  • a typical composition for intramuscular or intrathecal adm inistration wi l l be of a suspension or solution of active ingredient in an oi l. for example, arachis oil or sesame oil.
  • a typical composition for intravenous or intrathecal administration wi l l be a steri le isotonic aqueous solution containing, for example, active ingredient and dextrose or sodium chloride, or a m ixture of dextrose and sodium chloride.
  • lactated Ringer's injection lactated Ringer's plus dextrose injection, Normosol-M and dextrose, Isolyte B, acylated Ringer's injection, and the like.
  • a co-solvent for example, polyethylene glycol
  • a chelating agent for example, ethylened iam ine tetracetic acid
  • an anti-oxidant for example, sodium metabisulphite
  • the sol ution can be freeze dried and then reconstituted with a su itable solvent just prior to admin istration.
  • the compounds of th i s invention are active on topical adm inistration, and can be formulated as transdermal compositions or in tegrated in a transdermal delivery device (e.g., a transdermal patch).
  • a transdermal delivery device e.g., a transdermal patch
  • Such compositions include, for example, a backing, active compound reservoir, a control membrane, l iner and contact adhesive.
  • Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts.
  • Formulations suitable for oral adm inistration can consist of (a) l iqu id solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules; (c) suspensions in an appropriate liquid; and (d) suitable emulsions.
  • Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients. colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients.
  • Lozenge forms can comprise the active ingredient in a flavor, usual ly sucrose and acacia or tragacanth, as well as pastil les comprising the active ingred ient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the l ike containing, in addi tion to the active ingredient, such excipients as are known in the art.
  • an inert base such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the l ike containing, in addi tion to the active ingredient, such excipients as are known in the art.
  • the anti-Id compositions of the present invention can also include aerosol formulations to be administered via pulmonary inhalation or intranasal spray. These aerosol formulations can be placed into pressurized acceptable propel lants. such as
  • dich lorodi fluoromethane propane, n itrogen, and the l ike. They may also be formulated as pharmaceuticals for non-prcssurcd preparations such as for use in a nebul izer or an atom izer.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic steri le injection solutions, which can contain anti-oxidants, bu ffers, baeteriostats, and solutes that render the formulation isoton ic with the blood of the intended rec ipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers. th icken ing agents, stabilizers, and preservatives.
  • the formu lations can be presented in un it- dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the steri le liquid excipient. for example, water, for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions can be prepared from steri le powders, granules, and tablets of the kind previously described.
  • Formulations suitable for topical administration may be presented as creams, gels, pastes, or foams, contain ing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.
  • Suppository formulations are also provided by m ixing with a variety of bases such as emulsifying bases or water-soluble bases.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams.
  • Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors.
  • unit dosage forms for injection or intravenous administration may comprise the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and an imal subjects, each unit contain ing a pre- determined quanti :y of compounds of the present invention calcu lated in an amount sufficient to produce the desired effect in association with a pharmaceutical ly acceptable di luent, carrier or vehicle.
  • the specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be ach ieved, and the
  • Suitable dosages for a given compound are readi ly determ inable by those of skil l in the art by a variety of means.
  • the dose administered to an animal, particularly a human, in the context of the present invention should be suffic ient to cause a prophylactic or therapeutic response in the animal over a reasonable t ime frame.
  • the pharmaceutical composition may contain other pharmaceutical ly acceptable components such as bu ffers, surfactants, antioxidants, viscosity mod i fying agents, preservatives and the l ike.
  • these components are wel l-known in the art. See, eg, U . S. Patent No. 5,985,3 10, the disclosure of which is herein incorporated by reference.
  • Other components su itable for use in the formulations of the present invention can be found in Rem ington, 1 985.
  • the aqueous cyclodextrin solution also incl udes dextrose, e.g., about 5% dextrose. Kits and Systems
  • kits and systems that correlate with the foregoing description regarding combinatorial formulations, coordinate methods, diagnostic and treatment tools and systems, etc. All combinations of these primary tools disclosed for coordinate or combinatorial use are contemplated to also be provided herein in combined "kit" form.
  • kits and systems for practicing the subject methods may include one or more pharmaceutical formulations, which include for example an anti-Id compound packaged or referenced for use with a secondary cancer treatment agent, such as a chemotherapeutic drug, VDA, or HSP90 inhibitor.
  • kits may include a single pharmaceutical composition, present as one or more unit dosages, where the composition includes both an anti-Id compound (e.g., (-)-AGX5 1 and a chemotherapeutic agent or toxicity reducing agent.
  • the kits may include three or more separate pharmaceutical compositions, each containi ng an anti-Id compound, a chemotherapeutic or possibly a toxicity reducing agent, or a combination of these elements.
  • the subject kits may further incl ude instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, e.g., as printed information or reference on a su itable med ium or substrate on the packaging of the kit or on a package insert, etc.
  • Metastasis is the greatest therapeutic challenge for prolonging survival in most cancer patients, perhaps most significantly among the most populous cancer sufferers, breast cancer patients. Identification of genes and regulatory factors involved in metastasis has proven exceedingly complex and intractable from a clinical standpoint.
  • the inhibitor of DNA- binding (Id) proteins have been variously implicated to play diverse roles in cancer metastasis, including breast cancer metastasis.
  • H igh Id l expression is observed at metastatic sites. Id l reportedly functions in regulating critical mesenchymal-to-epithelial transition (MET) changes which al low circulating ("disseminating") cancer cel ls to seed ("colonize”) distant metastatic sites.
  • MET critical mesenchymal-to-epithelial transition
  • Artificial genetic knockdown of Id l is reported to disrupt M ET by tumor cells in the lung, and to block development of lung metastases in murine models of breast cancer.
  • the invention provides the first clinically practicable showing that a smal l molecule drug can effectively target and block Id function in mammal ian subjects in vivo, to disrupt metastasis, slow or prevent cancer progression, and ultimately reduce or prevent mortality in cancer patients.
  • Anti-Id compounds of the invention provide a novel and profoundly effective approach for treating and/or preventing metastases in cancer patients.
  • the Examples below i llustrate developm ent, characterization and anti-metastatic use of an exemplary smal l molecule anti-Id drug.
  • N-(3-(benzo[d][ l ,3]dioxol-5-yl)-3-(2-methoxyphenyl )propyl )-N- benzylpropionamicle (“AGX5 I " ).
  • This exemplary lead com pound provides a foundation for rational design of equivalent drugs possessing anti-Id activity, based on the AGX5 1 platform (e.g., as described above in relation to alternate drug designs set forth in Formulae 1.
  • Th is mundane discovery was surprising in many ways, not the least being the nature and complexity of HLH-bH LH dimerization rendering prior efforts to effectively d isrupt these molecular interactions, using a host of cand idate blocking agents, profoundly uncertai n and persistently unsuccessful.
  • AGX5 1 and other Anti-Id compounds of the invention not on ly bind and impair Id dimerization function with bHLH proteins, but by this mechanism also interfere functionally, in cl inically practicable ways, with downstream Id activities in critical metastatic pathways.
  • AGX-5 1 enhances E47 binding to DNA in a dose-dependent man ner (correlated w ith AGX-5 1 ? s effective antagonism of Id 1 -E47 dimerization).
  • anti-Id compounds of the invention effectively mediate reduction of Id levels in cel ls and living subjects (likely through
  • AGX5 1 decreases cell viabil ity (as measured by alamar bl ue assays), and inhibits formation of mammospheres.
  • Anti-Id compositions and methods of the invention also effect therapeutic changes in regulatory proteins p i 6 and p2 1 , reducing ci rculating levels of Id in blood of AGX5 1 -treated subjects, down regulate circulating endothelial cells and EPCs, directly down-regulate pathogenic neovascularization associated with metastasis and tumor growth, among many additional, distinct activities demonstrated here.
  • AGX5 1 the lead candidate anti-Id drug AGX5 1 has now been shown to profoundly reduce actual tumor metastasis in in vivo murine mode ls of multiple cancer types widely accepted as predictive of cl inical drug uti l ity in other mammal ian subjects, including h umans.
  • AGX5 1 When administered at 50mg/kg via intraperionteal injection. 24- hours after tai l vein injection of 4T I cel ls, AGX5 1 sharply reduces development of lung metastases. Significantly greater inhibition of lung metastases was observed when AG X- 1 adm in istration was increased from once to twice daily. Comparable studies and results are provided from murine breast cancer models.
  • Examples use an exemplary small molecule inhibitor AGX57 to block and d isable Id protein levels and metasta:is-mediating functions, while other like compounds described herein wil l be constructed based on these teachings to provide additional agents effective for neutral izing Id binding and function.
  • the experiments disclosed here summarize the identification of 364 compounds, and concurrent design of twelve peptides useful for inhibiting Id. These experiments further provide means for identifying additional anti-Id compounds, as wel l as for analyzing effectiveness of candidate compounds capable of inhibiting Id.
  • the assays described belo include practicable means for testing effectiveness of cand idate compounds, for example to confirm Id binding, Id dimerization blockade, and/or abi lity to mediate destabi l ization and degradation of Id in cel ls and l iving subjects.
  • Cognate Id l binding structures were conceptualized and virtual screening employed a Monte Carlo simulation for a complex of Id l and a smal l compound screen (hel ical fragments fixed) was run, incl udi ng 1 ,000,000 steps and 1 00 conformations collected and analyzed. ⁇ complex conformation with the best score and total energy was selected for further analysis.
  • Table 1 Chemical compounds identified by E47-W 1 interaction mapping as potentially inhibiting E47-ldl interaction.
  • the E47 m olecule from an X-ray of the structure of an E47-Id l heterodimer was used as a template for peptide design.
  • the peptides were designed to form leucine-zipper type dimers with Id l (b t not with E47), which could be stabilized by introduction of polar or charged side chain s forming inter-molecular H-bonds and salt bridges with Id l .
  • the designed peptides were deemed to have significant probabil ity of retaining an a-hel ical con formation in solution.
  • the hel ical propensity of peptides was enhanced by introd uction of polar or charged side chains to form intra-molecular H-bonds and salt bridges at positions that d id not interact with Id I . Only peptides containing natural am ino acids only were considered.
  • Root mean square deviations between peptide positions in the init ial model and in the final multiple dynamics structure was 0.50 A for C" atoms and 0.74 A for al l heavy atoms.
  • the inter-molecular H-bonds anchoring the peptide to Id l were calculated to remain stable during the multiple dynamics simulations.
  • Table 2 Twelve synthetic peptides created using the E47 molecule from an X-ray of the structure of E47- Idl heterodimer as a template for peptide design.
  • AGX51 to disrupt Id dimerization with bl lLH proteins and cause rapid degradation of unbound Id protein in cellular systems.
  • TA leukemic cells expressing ETO chimeric protein were treated with either vehicle control (DMSO) or 20 ⁇ , concentrations of either, racemic AGX5 1 , puri fied (-)-enantiomer of AGX5 1 (peak “E- l “ in “Enantiomer Separation A “ , described below), or purified (-)-enantiomer of AGX5 1 (peak “E-2 " in Enantiomer
  • Figure 1 provides a Western blot gel demonstrating that Id l and Id3 levels are potently reduced following treatment of TA leukem ic cells with racemic AGX5 1 .
  • anti-Id activity of the (-)-AGX5 1 enantiomer (second eluting peak " ⁇ -2'” from “ Enantiomer Separation A” (see below)) is surprisingly much greater than the anti-Id activity of racemic AGX5 1 , while the (+)-AGX5 1 (denoted as the first cluting peak "E- I " from "Enantiomer Separation A”) has mducel effect on Id l ( Figure 1 ).
  • Th is stcreos pecific (-)-ABX5 1 -mediated destruction of Id l protein levels in both leukem ic and breast cancer cells profoundly implicates this enantiomer as a potent tool for intervention in preventing and treating metastatic disease. 70221
  • AGX5 1 enantiome r mediates potent destabi lization and destruction of both Id l - and Id3- bl 1 LI I complexes (corresponding to total ablation of Id 1 and Id3 protein at the levels of detection afforded here) in leukem ic cel ls, with comparable anti-Id 1 stereospecific activity residing in the (-)-AGX5 1 enantiomer in breast cancer cells, but l ittle or no anti-ld3 activity observed for any fc rm of ANGX5 1 in breast cancer cel ls (whi le al l three preparations exerted strong anti-Id3 protein ablation effects in the leukemic cells— possibly pointing to minor contamination and 2xtraordinary anti-ld3 potency of (-)-AGX5 I , but exh ibited only in this cel l type between s:udies, or alternatively to an incomplete stereospecific effect (wherein (-)- AGX-5 I is profoundly anti- I
  • AGX51 is a pioneer anti-Id drug in a nascent field of discovery, first explored and charted here.
  • the cells were cultured in flam ' s 1 2 (Gibco, Carlsbad, CA) medium containing 10% BCS (Hyclone, Logan, UT) and appropriate antibiotics (pen/strep, fungizaone, and gentamycin (Invitrogn Inc.. Carlsbad. CA). All cells were cultured at 37°C in a fully humidified atmosphere containing 5% C() 2 .
  • the cells were treated with either 100 ⁇ D SO, lOOOmOsmol of urea +NaCL, I ⁇ N-(3-(bcnzo[d][l,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N- bcnzylpropionamide(AGX51), 10 ⁇ N-(3-(benzo[d][l,3]dioxol-5-yl)-3-(2- methoxyphenyl)propyl)-N-benzylpropionamide, and 100 ⁇ N-(3-(benzo
  • the cell morphology and growth was monitored daily for I week by microscopy for changes in morphology or cell death.
  • Apoptosis was determined by measuring caspase 3 and caspase 7 activities using the Caspase-Glo 3/7 Assay system from Promega (Madison. WI).
  • N-(3-(benzo[d][ 1 ,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N- benzylpropionamide led to cell death.
  • N-(3-(benzo[d][l,3]dioxol-5-yl)-3-(2- methoxyphenyl)propyl)-N-benzylpropionamide was also able to induce apoptosis in DLi 145 at concentrations as low as ⁇ ⁇ (Fig.3 (C)).
  • N ' -(4- isopropylphcnyl)- 1 -benzothiophene-2-carbohydrazide N ' -(4-isopropylphcnyl)- 1 -benzothiophene-2-carbohydrazide, at a concentration of 1 urn was able to induce cell death in DLI 145 cells.
  • two small molecule inhibitors of E47-ldl interaction were identified that induce massive cell death in prostate cancer cell lines.
  • a leukemic cell line derived from a mouse overexpressing the MML-AF9 lusion protein was treated with increasing concentrations of N-(3-(benzo[d][l,3]dioxol-5-yl)-3-(2- methoxyphenyl)propyl)-N-benzylpropionamide (AGX51).50% growth inhibition was observed at 10 ⁇ of AGX51 relative to a DMSO control.
  • Total cell lysates were collected using buffer containing 50 mM Tris -HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholic acid and 0.02% sodium azide along with freshly added complete protease inhibitors.
  • the protein lysates (20 ⁇ g) were separated by SDS-PAGE and transferred to nitrocellulose membranes. Immunoblots were analyzed by Western blotting and visualized using a Western lightening chemiluminescence detection kit. As shown in Figure 3, AGX51 at the ICso concentration restored pi 6 compared to the DMSO vehicle.
  • the primary antibodies used in this study were purchased from Epitomics, Burlingame, CA. USA.
  • a human bladder carcinoma cell line was treated with increasing concentrations of N- (3-(benzo[d][l,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzylpropionamide (AGX51 ) and processed for p2l concentration impacts of AGX51, as described above in Example VII for AGX51-media1ed rescue of 16.
  • Total cell lysates were collected, processed, separated and transferred to a Western blot substrate as described. The Western blots were visualized using as shown in Figure 3.
  • AGX51 mediated a pro ounced dose-dependent restoration of p21.
  • the cells were starved of serum for 24 h and treated 24 h with luM N " -(4- isopropylphenyI)-l -benzothiophene-2-carbohydrazide (AGX8) or N-(3-(benzo[d][l,3]dioxol- 5-yl)-3-(2-methoxyphenyl)propyl)-N-benzylpropionamide (AGX51). After treatment, the cells were washed, fixed in ethanol and stained with propidium iodide before being analyzed by fluorescence-activated cell sorting (FACS) analysis (Accuri Cytometers, Inc., Ann Arbor, MI).
  • FACS fluorescence-activated cell sorting
  • ANGX5 I also functions to stabilize cell cycle control and dim inish production of endothelial progenitor cells (EPCs) and EPC-dependent tumor-associated angiogenesis.
  • EPCs endothelial progenitor cells
  • Cel l migration reflects the sum of many individual biologic processes that, overal l. promote the capabi l ity of a particular cel l to move from one place to another.
  • Cel l m igration is an integral mechanism essential tor tumor metastasis, the most lethal process associated with neoplasms (C iiang and Massague. 2008).
  • the instant Example demonstrates how exemplary anti-Id compounds of the invention such as AG X5 1 can reduce or prevent metastatic disease by blocking Id-dependent metastatic cel l m igration.
  • the basic steps of a Scratch Assay involve creating a "scratch" in a cel l monolayer, capturi ng the images at the beginning and at regular intervals duri ng cell m igration to c lose t he scratch, and comparing the images to quantify m igration rates of the cells.
  • the in vitro Scratch Assay is particularly suitable for studies concern ing cel l-matrix and cell-cell interactions in cel l m igration.
  • Results are provided in the photographs presented in Figure 6.
  • a fter 4 days the scratches or channels in plated cel l samples treated w ith media containing dosing vehicle (DMSO) have disappeared while the channels in plated cel l samples treated with AGX-5 1 remain open.
  • Th is assay, consistently repeated with comparable results, unambiguously shows that anti-Id active AGX5 1 potently inhibits Id-dependent cel l migration.
  • the invention provides yet additional tools and methods to directly block cancer development and metastasis by reducing or disabl ing Id-dependent cell migration.
  • VEGF- 1 65 and FGF-2 treated Matrigel plugs were implanted on Day 0 into C57B E/6 m ice. Mice were t reated with either vehicle or AGX5 1 .
  • the anti-Id compound was provided either in the plugs (25 ⁇ / ⁇ 3 ⁇ 4) or by daily ip treatment (30 or l OOmg/kg) for 10 days. Plugs were harvested on Day 1 0, fixed and paraffin embedded. Three sections (5 ⁇ thickness) of each plug were stai ned with an anti-CD3 1 antibody and counterstained with hematoxylin and eosin stain. CD3 1 - ositive m icrovcssels were counted for one entire cross-section per plug and the average m icrovessel density ⁇ SD was determined. Student ' s t-test was used for statistical analysis.
  • MB-23 1 cells were trypsinized, centrifuged and re-suspended in a 50% DPBS/50% Matrigel solution to a concentration of 5 x 1 0 6 cells/50 ⁇ , and frozen unti l use.
  • the frozen Matrigel was thawed at 4°C for 24 hours.
  • Syringe, needle and cells were kept on ice unti l used.
  • cel ls Prior to the injections, cel ls were gently mixed by inversion and the needed vol ume drawn up into a cold syringe equipped with a 25 x 5/8" gauge needle. The syringe w as gently inverted to m ix taking care to avoid bubbles.
  • the m ice were injected with 50 ⁇ ⁇ of Matrigel containing 1 0 x 1 0 6 MDA-MB-23 1 cells into the orthotopic site.
  • the cel ls were injected orthotopically into the right, caudal mammary fat pad of the animals anesthetized by ketamine ( 1 20mg/kg)/xylazine (6mg/kg) adm inistered im using a 26 x 3/8" gauge needle.
  • Tunors were then al lowed to establish to a volume of 1 00 mm 3 , after which animals, (typically 5 animals/treatment group) were randomized and treated ip with vehicle (DMSO) or 60mg/kg AGX5 1 bid.
  • DMSO vehicle
  • the m ice were sacrificed by cervical dislocation, and tumors excised and fixed in 1 0% buffered (neutral) formalin for histopathologic analyses. Tissue was removed from the formalin and washed with 3xPBS. soaked in 30% sucrose in PBS for 24-48 hours at 4°C to cryoprotect, and then embedded in paraffin. The para ffin blocks were stored at -20°C or -80°C unti l cryo-section ing into 5 ⁇ th ick sections for mounting on slides. The slides were treated with anti-CD3 l and washed with PBS, and endothelial cells were detected using an avidin-biotin com plex (ABC ) system. The sl ides were counter stained with hematoxylin and eosin stain to visual ize nuclei.
  • ABS avidin-biotin com plex
  • Additional compositions and methods of the invention discretely impai r tumor- associated angiogenesis by inhibiting production and reducing survival of endothelial progenitor cel ls (EPCs).
  • EPCs are responsible for promoting tumor growth and metastasis by contributing to neovascularization of new tumors (and by rescuing established tumors from vascular destruction caused by tumor growth).
  • EPC production and survival is Id-dependent in cancerous and metastatic systems, and therefore the anti-Id compositions and methods of the invention effectively reduce or prevent metastasis through reduction or prevention of EPCs and/or by im pairment of EPC function and developmental potential.
  • the instant Example utilizes the same test subjects as presented throughout the other Examples here, comparing anti-ld-treated animals with control animals under conditions that lead to cancer progression and metastasis in the non-treated control an imals.
  • the further object of this Example is demonstrating Id-dependent variation of EPC levels in animals treated or untreated using the compositions and methods of the invention.
  • each class of treated and untreated model animal described in the foregoing Examples I V-VI 1 will be evaluated for EPC levels (e.g., using multivariate flow sorting with antibodies directed against EPC markers (such as GFP+ and VE-cadherin+) to measure EPCs in samples from bone marrow or circulating blood).
  • compositions and methods of the invention particularly (-)-ANGX5 l wi ll exh ibit anti-Id compound-mediated, dose-dependent reduction in EPC levels (compared to control animals, and animals treated with lesser active anti-Id compounds, such as racem ic ANGX5 1 and particularly the anti-metaslatical ly inactive (+)-ANGX5 1 enantiomer.
  • Anti-Id Compounds Inhibit Tumor Growth In Vivo
  • CD 1 male mice (3/timepoint) were treated ip with 30mg/kg AGX5 1 in DMSO.
  • One ⁇ g of the internal standard for the assay (S I 09037 from Aldrich, St. Louis, MO) and 1 00 of pH 7.4 PBS were added to the plasma harvested from the blood. The mixture was vortexed for one minute with I m L methyl t-butyl ether (MTBE), and the MTBE removed after centrifugation with dry N2 (g).
  • I m L methyl t-butyl ether MTBE
  • 5M/5F per group were treated daily intraperitoneally ( ip) for 25 days with either dosing veh icle (DMSO), or 50 mg/kg racem ic N-(3-(benzo[ d][ 1 ,3]d ioxol-5-yl)-3-(2- methoxyphenyl)propyl)-N-benzylpropionamide (AGX5 1 ).
  • DMSO dosing veh icle
  • AGX5 1 racem ic N-(3-(benzo[ d][ 1 ,3]d ioxol-5-yl)-3-(2- methoxyphenyl)propyl)-N-benzylpropionamide
  • Example I I I above demonstrated therapeutic efficacy of AGX5 1 against metastasis of implanted lung tumors in m ice, the instant study measures anti-metastatic effects of AGX5 1 against breast cancer cells directly injected into the bloodstream of test subjects.
  • DPBS/50% atrigel solution to a concentration of 5 x 1 0 6 cel ls/50 ⁇ and frozen.
  • the frozen Matrigel was thawed at 4°C for 24 hours.
  • Syringe, needle and cel ls are kept on ice until used.
  • cells Prior to injections, cells are gently mixed by inversion and the needed volume drawn up into a co ld syringe equipped with a 25 x 5/8" gauge needle. The syringe is gently inverted to mix taking care to avoid bubbles.
  • the m ice were treated // with DM SO (vehicle), DMSO solutions of I 5mg/kg paclitaxel, I 5mg/kg, paclitaxel with either 6.7mg/kg, 20mg/kg. or 60mg/kg of AGX5 I or 60mg/kg of AGX5 1 alone for five days.
  • the m ice were sacrificed by cervical dislocat ion and final tumor volumes and weights recorded and compared.
  • mice 22.5mg/kg dose although the mice experienced significant hypotherm ia and lethargy compared to the 1 5mg/kg group.
  • D SO vehicle
  • I5mg/kg paclitaxel or 15mg/kg
  • paclitaxel with 60mg/kg of AGX51.
  • MDA-MB-231 cells were trypsinized, centrifuged and re-suspended in a 50% DPBS/50% Matrigel solution to a concentration of 5 x 10 6 cells/50 ⁇ , and frozen. Prior to use, the frozen Matrigel was thawed at 4°C for 24 hours. Syringe, needle and cells were kept on ice until used. Prior to injections, cells are gently mixed by inversion and the needed volume drawn up into a cold syringe equipped with a 25 x 5/8" gauge needle. The syringe is gently inverted to mix taking care to avoid bubbles.
  • mice 50 ⁇ ⁇ Matrigel containing 10 x 10 6 MDA-MB-231 cells were injected orthotopically into the right, caudal mammary fat pad of four to six week old female nude mice anesthetized by ketamine (120mg/kg)/xylazine (6mg/kg) administered im using a 26 x 3/8" gauge needle. Tumors are then allowed to establish to a volume of 100 mm 3 , after which animals are randomized and treeted ip with the test articles or vehicle. Fourteen days after implantation, the mice were treated ip with DMSO (vehicle), 15mg/kg of docetaxel q5d.
  • DMSO vehicle
  • Anti-Id compounds of the invention appear to specifical ly target Id proteins and mediate their potent anti-cancer and anti-metastatic effects without disrupting fundamental cell functions or conferring substantial toxicity, contrary to observations for most conventional chemotherapeutic drugs.
  • CD 1 mice 5/treatment group
  • Heparinized blood samples were obtained by retro-orbital puncture on Day 6, 12 hours after the last N-(3-(benzo
  • Tie plasma harvested from the blood was analyzed for standard clinical chemistry and hematology analytes and intact paclitaxel and AGX5 1 .
  • AGX5 1 No adverse effects were observed of AGX5 1 on weight or cl inical chem istry (ALB .
  • WBC Lym, Mon. Gra, RBC, Hg, Hematocrit & Platelet
  • AGX5 1 The anti-Id and anti-angiogenic activity of AGX5 1 is demonstrated in the instant example to powerful ly protect against pathogenic vascularization in the retina of m ice, using a model system wicely accepted as predictive of disease mechanisms and drug efficacy for treating human age related macular degeneration (AMD).
  • the retinas of 14 mice with the Id3 deleted were dilated with phenylephrine/atropine for 1 0 minutes and then anesthetized with ketam ine/xylene (5 : 1 ) for 5 minutes prior 1o affecting the laser burns.
  • a cover slip was placed on the surface of the eye (lower side) with a clear ophthalmologic med ia to act as a lens for the laser.
  • a l ight was shone into the eye to visual ize the optical nerve and the neural retina.
  • a fine laser was then focused unto the back of the retina, set perpendicular to the back of the eye.
  • Id3 deletion impairs neovacularization fol low ing laser induced disruption of Bruch's mem brane, indicating Id3 is involved at least in part in ful l development of a pathogenic angiogenic response in this model system of AM D.
  • This model is widei y accepted and has been used successful ly to develop numerous anti- AMD drugs.
  • the mean CNV area for the Id3 deleted animals was approximately hal f of the area of the wild type anima ls, and the result was highly signi ficant statistical ly (p ⁇ 0.00 l ).
  • a comparab le protective anti-AMD effect (as seen in the above Id genetic knockout study) has now surprisingly been achieved using a small molecule anti-Id drug of the invention, ANGX5 ' , .
  • the anti-A MD (anti-angiogenic) effects of ANGX5 I were observed in subjects treated intravitrcally (directly into the affected eye), as well as intraperitoneal ly (requiring the drug to transit to the retinal tissue in a stable, effective state).
  • the data here convincingly show that the anti-angiogen ic and anti-AMD effects of AGX5 1 are stereospecific, with activity residing principal ly or exclusively with the novel (-)-AGX5 1 enantiomer.

Abstract

The present invention provides methods for impairing function or reducing or ablating levels of Id (inhibitors of differentiation) proteins to suppress metastasis of cancer cells in mammalian subjects presenting with cancer or elevated risk of metastatic disease. Exemplary anti-Id compounds include N-(3-(benzo[d] [1,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N- benzylpropionamide and isolated enantiomers of N-(3-(benzo[d] [ 1,3]dioxol-5-yl)-3-(2- methoxyphenyl)propyl)-N-benzylpropionamide. Anti-Id compositions and methods of the invention can be combined with standard anticancer treatment modalities, such as surgery, radiation and chemotherapy. Also provided are compositions and methods to treat other hyperproliferative disorders, including pathogenic angiogenic conditions, including tumor-associated neoangiogenesis and ocular vascular pathologies including age-related macular degeneration.

Description

COMPOSITIONS AND METHODS FOR TREATING, PREVENTING AND
DIAGNOSING CANCER AND OTHER PROLIFERATIVE DISORDERS
TECHNICAL FIELD
The present invention relates to compositions and methods for preventing, diagnosing and treating cancer and other proliferative disorders, including pathogenic angiogenesis. in mammalian subjects.
CROSS REFERENCES TO RELATED APPLICATIONS
This application claims benefit of U. S. Provisional Application No.60/916, 116, filed
13 December 2013, and U.S. Provisional Application No.61/965,776, filed 6 February 2014. each of which is incorporated herein by reference its entirety for all purposes.
BACKGROUND
Inhibitor of Differentiation (Id) Proteins
The Id family of helix-loop-helix proteins (HLH) is implicated in the regulation of virtually all underlying cellular mechanisms and determinative events in cancer: cellular differentiation, cell cycle progression, senescence, lineage commitment and apoptosis (Perk et al., 2005; Morse, 2006; Ling et al., 2006, Nair ct al, 2013). The term d" itself is a characterization of the ability of these proteins to inhibit both cell differentiation and binding of important regulatory proteins to DNA (deoxyribonucleic acid). Of the four Id proteins (Id 1 -4), the role of Id 1 in tumor invasiveness, metastasis and angiogenesis is best characterized, and findings concerning the role of Idl in cancer provide a foundation for the discussion that follows.
Idl is highly conserved in vertebrates and invertebrates as well as among species (Deed et al., 1994) as illustrated in the sequence alignment chart provided below that shows remarkable "homology" (sequence identity) of >90% for human Idl (SEQ ID NO. 1) (UniProKB P41134) and mouse Idl (SEQ ID NO.2) (UniProKB P20067) Idl proteins.
Human MKVASGSTATAAAGPSCALKAGKTASGAGEVVRCLSEQSVAl SRCAGGAGARLPALLDEQ 060
Mouse MKVASGS-AAAAAGPSCSLKAGRTA GKVVLGLSEQSVAI SRCAG TRLPALLDEQ 053 Human QVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEI LQHVI DYIRDLQLEL SESEVGTPGG 120
Mouse QV VLLYDM GCYSRLKELVPTLPQ RKVSKVEI LQHVI DY 1 DLQLELNSESEVGTTGG 11
Human RGLPVRAPLSTLNGEI SALTAEAACVPADDRILCR 15h Mouse RGLPVRAPLSTLNGEISALAAEAACVPADDRILCR 148
A comparison between human ld3 (SEQ ID NO. 3) (UniProKB Q02535) and mouse Id3 (SEQ ID NO. 4) (UniProKB P41 1 33) Id3 proteins yields even closer sequence identity .
Human MKALS PVRGCYEAVCCLSERSLAI RGRGKGPAAEEPLSLLDDM HCYSRLRELVPGVPR 060 Mouse MKALSPVRGCYEAVCCLSERSLAIARGRGKSPSTEEPLSLLDDMNHCYSRLRELVPGVPR 060
Human GTQLSQVEILQRVIDYILDLQVVLAEPAPGPPDGPHLPIQTAELAPELVISNDKRSFCH 119
Mouse GTQLSQVEILQRVIDYILDLQVVLAEPAPGPPDGPHLPIQTAELTPELVI SKDKRSFCH 119
Human Id 2 (Uni ProKB Q02363) and mouse Id2 (UniProKB P4 1 1 36) proteins share all but two out of 1 34 identical residues, whi le human Id4 (UniProKB P47928) and mouse ld4 (Uni ProKB P4 1 1 39) are 1 00% sequence identical .
This unusual ly high degree of homology or sequence identity between human and murine Ids provides r heightened confidence in extrapolating experimental findings from m ice, as accepted model subjects, to humans, in relation to Id structure, function and biology .
The most studied of the Ids is Id I , a regulator of transcription found in extremely low (typically non-measu rable or iindedtcctable concentrations) in d ifferentiated tissue of healthy adults (in contrast to elevated Id levels routinely delectable in cancer tissue or developmental (fetal) tissue). Id3 is considered a paralog of Id 1 , whi le ld2 and Id4 are d istinctly di fferent from each other and from Id l and Id3.
The principal binding partners for Id 1 and al l Id helix-loop-hel ix (I I LI I) proteins are the basic hel ix-loop-hel ix (bH LH) fami ly of transcription factors that contai ns over 200 members and functions broadly as regulators of developmental processes (Powe l l and Jarman, 2008; Massari, 2000). Particularly important members of the bH LH fam i ly are the so-called E proteins.
E proteins are bHLH proteins that are ubiquitously expressed, bind to the E-box element of DNA and are sequestered by Id proteins. E47 is a basic hel ix-loop-hel ix protei n that is a spl ice product of the E2A gene. E-twenty six "ETS" is another fam i ly of regulatory transcription factors (approximately 20) also proposed to be inhibited by Id proteins. Both E and ETS proteins have been shown to drive cel l differentiation and growth arrest in a variety of cel lular contexts. Less frequent bind ing partners of the Id H LH proteins include retinoblastoma (RB, a protein that functions as a tumor suppressor) for Id2 (Desprez et al„ 2003) and for Id l , non-bH LH proteins E-twenty-six (ETS) (a fam i ly of regulatory transcription factors implicated in the development of different tissues as wel l as cancer progression through, eg, pl6Ink4a, a tumor suppressor gene): Paired box (Pax); Mouse Id assocciated-1 (MIDA-1); and Sterol regulatory binding protein-lc, SREBP-lc, among others.
The functions of HLH family proteins are comparable to functions of Id proteins. Generally, all of these proteins are fundamental mediators of stasis or change in cellular differentiation, cell cycle progression, senescence, cellular commitment (to a determined lineage/fate) and/or apoptosis. However, the activities of Id proteins are typically apposite in effect to activities of HLH family proteins. This is attributed in part to a deactivating result from binding between the HLH and bHLH proteins, as described below.
bHLH proteins generally mediate a restrained or no growth environment in mammalian cellular systems. Consistent with this role, a reduction of E proteins in B and T- cells correlates with development of B and T-cell leukemias (Kee, 2009). In contrast, increased Id protein expression is associated with a pro-growth environment, attributable to Id"s potential for neutralizing bHLH proteins.
Various repotts have been published concerning the nature of interactions between bHLI I proteins and other bHLH proteins (Slattery et al, 2008) and between bl ILH proteins and HLH proteins such as Id proteins (Norton, 2000). All members of the bl ILH family possess a highly conserved I ILH region containing two amphipathic a helices separated by a shorter intervening loop. This "'HLH domain" mediates homo- or hetero-dimerization that is essential for bHLH protein activity. Adjacent this HLH domain is a basic region capable of binding deoxyribonucleic acid (DNA) elements containing a canonical "Έ-box" sequence.
Id proteins also possess an HLH domain, but without the adjacent basic region thai mediates DNA binding of bHLH proteins. As a result of this construction, Id proteins are capable of binding other HLH transcription factors and altering their activity in gene transcription. In this manner, Idl has been reported to inhibit the activity of bHLH transcription factors like the E protein, E47, by binding and restraining the ability of the bHLH binding partner to bind DNA and mediate transcriptional changes (proposed to promote pro-differentiation and pro-apoptotic cellular changes— believed to be correlated with anti-neoplastic and anti-angiogenic activity in different cellular developmental contexts).
Idl is present in both cytosol and nuclear compartments of cells, with shuttling from the nucleus possibly regulated by protein kinase-A (PKA) (Nishiyama et al., 2007).
Intracellular levels of Idl are also proposed to be regulated through an ubiquitin-proteasome degradation pathway (Sun et al..2005), resulting in half-lives for Id proteins of approximately one hour or less. This degradation process is possibly linked to TNF-a- induced apoptosis in prostate cancer cells (Ling et al., 2006). Heterodimerization with bULH proteins reportedly affects rates of Id degradation and may be protective to extend Id protein half-life (Bounpheng et al, 1999).
Id Proteins and Angiogenesis
Idl and Id3 have been proposed to play critical roles in the production of new blood vessels, including "pathogenic neovascularization" associated with growth and spread of tumors ("tumor-associated angiogenesis"). Transfer of the Idl gene into HUVEC cells elevates angiopoetin- 1 and confers a pro-angiogenic phenotype (Nishiyama ct al., 2005). Idl reportedly acts '"downstream" of pro-angiogenic factors vascular endothelial growth factor-A (VEGF-A) Lee et al., 2006), bFGF (Ruzinova ct al., 2003), HIF-1 (Kim et al., 2007), and EGF-R (Ling et al., 2004), so loss of Idl activity may impair multiple angiogenic pathways. Such plural targeting of angiogenic pathways would be an important objective, as tumors have proven refractory to mono-directed antiangiogenic therapy (including by responding through up-regulation of alternative pro-angiogenic growth factors).
Idl is also proposed to interact negatively with p2I (a cyclin kinase inhibitor, proposed to function via inactivation of cyclins. P2I is reported to negatively control endothelial progenitor cell formation in bone marrow neovascularization (Ciarrocchi et al... 2007), which progenitor cells are contemplated to play a pivotal role in tumor-associated angiogenesis (Seandel et al., 2008), and cancer metastasis (Gao et al., 2008; Gao et al..2009). A direct role for endothelial Id protein expression in tumor-associated angiogenesis has been proposed based on xenograft studies in Idl "7"; ld3tA mice. Xenograft tumors in Idl Id3' mice reportedly fail to grow completely, or show slower growth with extensive hemorrhage and necrosis, compared to Id wild type controls, while transplantation of wild-type bone marrow-derived encothelial precursor cells reportedly restores tumor angiogenesis and growth in Idl ; Id3 +/" subjects (Lyden et al., 1999; Lyden et al., 2001).
Id Proteins and Cancer
Id 1 proteins have been reported to be linked to a diverse array of signaling and control elements involved in the initiation and progression of cancer (Fong et al., 2004). A particularly intriguing link has been proposed between Id l and cancer metastasis. Id l has been reported to be associated in a mechanistic way with metastatic change in human breast cancer (M inn et al., 2005). Over-expression of Id l reportedly contributes to metastasis in breast cancer cel ls transplanted into animals (Fong et al., 2003). In another study, over- expression of Id l was reported to immortal ize myeloid progenitor cel ls and lead to myeloprol iferative d isease in mice (Suh et al., 2008).
Id l is highly expressed in a large number of cancers including sol id tumors of the bladder (Perk et al., 2006), breast (for example, Schoppmann et al., 2003), cervix/uterus (Li et al., 2009; Schindl, et al ., 2001 ; Maw et al., 2008), colorectal (Zhao ct al ., 2008), endometrium (Takai et al., 2004), gastric (Han et al., 2004; Iwatsuki et al.. 2009), gl ia (Vandeputte et al.. 2002), hepatic (Matsuda et al., 2005), ovarian (Schindl et al., 2003 and Maw et al., 2009). prostate (Forootan et al., 2007; Yu ct al., 2009; Coppe et al., 2004; Ouyang et al., 2002a ). renal (Li et al., 2007), squamous cel l cancer head and neck (SCCHN) (Kamal ian et al., 2008). thyroid (Kebebew et al., 2004; Ciarrocchi et al., 20 1 0) and non-smal l cel l lung canccr (NSCLC) (Bhattach irya et al., 20 1 0) as wel l as l iqu id tumors such as acute myelogenous leukem ia (AM L) (Ta ng et al., 2009). In virtually al l these cancer types, over-expression of Id l has been reported to be assoc iated with an aggressive phenotype and poor cl in ical outcome. Fundamental questions remain, however, concern ing the mechanisms and pathways through which Id proteins interact and mediate negative impacts i n complex developmental systems involved in cancer and other prol iferative d iseases and cel lular pathogenic events.
The fol lowing additional observations and reports have contributed to an emerging but incomplete understanding of Id l expression, function and biology in cancer, cancer metastasis, and pathogenic angiogenic development in mammal ian subjects.
· Complex Factors Involved in Id Expression and Function Id l gene expression is reported to be stimulated by growth factors including bone morphogenetic protein-2 (BM P-2) (Le Page et al., 2009), BMP-6 (Darby et al ., 2008), growth/differentiation factor-5 (GDF5) (Chen et al., 2006), and insul in-like growth factor- 1 (IGF- 1 ) (Prisco et al., 200 1 ; Bel letti et al., 2002). Vascular-endothelial-growth-factor-A (VEGF-A) is reported to be both an upstream gene inducer ( Benezra et al.. 200 1 ) and downstream mediator of I d l (Lee et al., 2006; Lin et al., 2005). SMAD proteins are proposed to modulate activity of transforming growth factor beta l igands by form i ng complexes with other SMADs that function as transcription factors (Liang et al.. 2009). Early growth response protein 1 (Egr l ) (Subbaramaiah et al., 2008), Sp l (Jorga et al.,
2007), a member of the Sp/KLF (specificity protein/ ruppel-like factor) fami ly of human transc ription factors involved in gene expression in the early development of an organism and activating transcription-3 (ATF3) (Li et al., 2009), are reported to positively affect promoter activity, while the tumor suppressor ATF5 is reported to suppress Id l gene expression (Gho et al., 2008). Src, a proto-oncogene tyrosine protein kinase mediator of growth factor activity, is also reported to play a role in regulating Id l expression (Gautschi et al., 2008). Sim i larly, forkhead box 0-3a
(FOX03a), a transcription factor reported to reverse leukem ic phcnotypes, may promote differentiation by transcriptional down-regu lation of Id l (Birkenkamp et al.,
2007). Dow -regulation of the Id l gene has also been proposed as a mechan ism for a mechanism for anti-cancer activity of non-steroidal anti-inflammatory drugs
(NSA I Ds) in gastric cancer (Jang et al., 2006). A reduction in pro-in flammatory prostaglandin PGE2 in breast cancer cells mediated by the NSA I D celecoxib has also been correlated with reduced Id l levels (Subbaramaiah ct al.. 2008)
Complexity of Downstream Id Targets Id l is also proposed to target various downstream oncogenic tyrosine kinases (Tarn et al ., 2008 ), such as Bcr-Abl, Tel-A BL
(an aberrant fusion protein associated with chronic myelogenous leukem ia), TF.L-
PDG-FBR (a hybrid fusion protein found in patients with myeloid neoplasms associated with eosinophi l ia), FLT3 (a tyrosine kinase 1 associated with one of the receptors for V EG F)-1TD, and others. Id l is postulated to cooperate with oncogen ic rat RAS (a GTPase in the mitogen-activated protein kinase (MAPK) pathway), to induce metastatic mammary carcinoma by subverting cellular senescence responses
(Svvarbrick et al., 2008). Id l has been reported to stimulate a phosphatidyl inositol -3- kinase (PI3 K)/protein kinases B (Akt)/nuclear factor kappa beta (N FkB) signal ing pathway important to cancer cel l proliferation, survival and invasion (Li et al ., 2007).
Id l may restrain expression of p i 6, which in turn positively regulates cell senescence
(Zheng et al. , 2004; Cummings et al., 2008). Id l may also regulate B-cell lymphoma cell-2 (Bcl-2) and BCL-2-associated X protein (Bax), through p53 and N FkB (K im et al., 2008), and chromosomal instability through anaphase-promoting complex-C
(APC/C) during m itosis (Wang et al., 2008) to enhance survival of cancer cel ls. Additionally, Id l is proposed to activate Akt-mediated wingless type (Wnt) signal ing and p27 phosphorylation through phosphatase and tensin homolog (PTEN) inhibition (Lee et al., 2009). Id Proteins and Metastasis
Current know ledge fal ls even further short of practical ly comprehend ing the roles of Id proteins and other regulatory factors in cancer metastasis. Metastatic disease accounts for 90% of carcinoma-related deaths in humans. The complexity and refractory nature of metastasis present the greatest obstac les to developing new methods and tools to reduce cancer morbidity and mortality. Metastasis is a multi-step, multi-factorial process, broadly described as having two major phases: ( 1 ) physical dissemination of cancer cel ls from a "primary" site (i.e., initial tissue/organ where the cancer originated); and (2 ) colon ization by cancer cel ls from a primary site to distant tissues/organs (Chaffer and Weinberg, 201 1 ). Numerous studies ha ve shown that the first phase is attended by a de-differentiation or re- activation of development, switch ing determ ined cells to a more plastic fate, common ly referred to as the "epithelial to mesenchymal transition" (EMT). EMT conversion endow s primary cancer cel ls with a much greater metastatic potential, associated with a highly invasive phenotype ( Th iery et al., 2009).
During the course of EMT, immobile epithelial cells lose their epithel ial traits and acquire mesenchymal properties, coupled with an abil ity and propensity to m igrate to other locations (secondary tumor sites) to seed new tumors. Cancer cel ls that have undergone EMT reportedly share key characteristics with tumor initiating cel ls (TICs) (Mani et al., 2008) (functionally defined by their ability to seed new tumors and restore heterogeneity of primary tumors) (Valent et al., 20 12). The generation of breast cancer TICs by
overexpression of EMT-inducing transcription factors, such as twist-related protein 1 (Twist 1 , or class A bHLH protein 38) has suggests molecular linkage of EMT-driven metastatic d issem ination, and the generation of TICs (Man i et al., 2008; Morel et al., 2008). How ever, less is known about the biology of TICs during the second phase of metastasis (colonization of distant tissues organs). The proposal that EMT induces breast cancer TICs has been challenged based on cl inical observations that most metastases present a differentiated epithelial morphology (Tarin et al., 2005). This may indicate that EMT is a transient process, and that the re-differentiation of carcinoma cells by a "mesenchymal to epithel ial transition" (MET) is a driving force in metastatic colonization, at least in some cancers (Brabletz, 2012).
While EMT may affect cancer cell dissemination (including invasion of the local parenchyma (Yook et al., 2006), intravasation into the circulatory system (Drake et al., 2009). survival during migration (Gal et al.. 2008), and extravasation into the secondary site (Labellc et al., 201 1 ; Vuori luoto et al., 201 1 )), loss of mesenchymal phenotype may enhance formation of macro-rnetastatic colonies. This may be l inked to a reversal of growth arrest associated with EMT (Brabletz et al., 2001 ; Mej lvang et al., 2007; Vega et al.. 2004). The importance of M ET in breast cancer metastasis is suggested by stud ies showing that after dissem ination, engineered loss of the EMT transcription factor Twist 1 , (Tsai et al., 20 1 2) and expression of microRNAs inhibiting the EMT transcription factor "zinc finger E-box-bind ing homeobox" (Zeb) (Korpal et al., 20 1 1 ) enhance lung colon ization by metastatic breast cancer cells. Further, the transcription factor "paired related homeobox Γ* (Prrx l ) (w h ich induces EMT during dissemination but suppresses sternness traits necessary for lung colonization), must be lost prior to colonization (uncoupling in this instance EMT from the TIC phenotype) (Ocana et al.. 20 1 2). Detai ls of how a colon izing cancer cel l sheds its mesenchymal phenotype required for dissem ination, whi le retaining TIC properties essential for found ing a metastatic colony, remain unclear.
In view o f the foregoing, many critical questions remain regarding the mechanisms and pathways through which Id proteins interact and mediate negative impacts in cancer and other prol i ferative disorders (such as tumor-associated angiogenesis). There are there fore many obstacles and hurdles to be overcome before these agents and processes can be exploited to provide new tools and methods for treating cancers in mammal ian subjects. Additional needs remain unfulfi lled for tools and methods to evaluate, manage and treat metastatic cancer in mammal ian subjects.
SUMMARY OF EXEMPLARY EMBODIMENTS OF THE INVENTION
The present invention fulfi l ls the foregoing needs and satisfies add itional objects and advantages by providing novel tools and methods to modulate Id function in mammal ian subjects suffering from a neoplasm or other cellular prol iferative disorder. The invention further provides novel technical discoveries to elucidate the role and effects of Id proteins and their biochemical and molecular targets in mediating cancer and metastasis, yielding tools and methods to manage and treat cancer and metastasis and other proliferative disorders in humans and other ma mmals.
In exemplary aspects the invention provides compositions and methods for treating a cellular proliferative disorder, for example inhibiting or reducing metastasis of a tumor or primary cancer cel l. These methods comprise administering to a mammal ian subject an effective amount of an "anti-Id compound", sufficient to reduce or prevent pathogenic cel lular proliferation, angiogenesis, cancer, and/or metastatic disease in the subject. I n exemplary embodiments, the compositions and methods of the invention employ an exemplary anti-Id compound of formu la I, 11. I l l, or I V as described below, or an active salt. enantiomer, polymorph, solvate, hydrate, or prodrug thereof.
I n certain detailed aspects of the invention, the methods and compositions herein employ an exemplary anti-Id compound or composition comprising an anti-metastatic (or anti-prol iterative, or anti-angiogen ic) effective amount of racem ic N-(3-(benzo[d]| l
Figure imgf000011_0001
5-yl)-3-(2-mcthoxyphenyl)propyl)-N-benzylpropionam ide ("AGX51 "). I n distinct embodiments, the anti-Id compound is an isolated, anti-metastatical ly active (-)-enantiomcr of N-(3-(benzord] [ l ,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzylpropionamide. The (-) or "m inus" enantiomer of AGX5 1 exh ibits an extraordinary and unexpected dom inance of anti-Id potency compared to the (+) or "plus" enantiomer. On this basis, novel, enantomerically-enriched preparations of (-)-AGX5 1 enantiomer (substantia l ly purified to yield an increased amount or concentration of (-)-AGX5 1 , relative to an amount or concentration of the (+)-AGX5 I enantiomer (compared to conventional ly-prepared racem ic N-(3-(benzo[d] [ l ,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzyl propionam ide), provide surprising advantages and cl inical benefits within the compositions and methods of the invention.
I n certain em bodiments, the invention provides "anti-metastatic" compositions and therapeutic methods, which are effective to treat or prevent metastatic disease in mammal ian subjects. These methods may employ monotherapy or coordinate or combinatorial therapy. The compounds and methods of the invention are "anti-metastatical ly effective", for exam ple to reduce an incidence, size, tissue or organ distribution, or number of metastases in a subject presenting with cancer. In certain embodiments, anti-metastatic activity corresponds to an observed reduction in one or more histopathological indices of metastasis, for example quantitative reduction in occurrence, size, number or distribution of metastasized cel ls or "foci" of primary tumor character observed at a secondary tissue or anatom ic site. In other aspects, anti -metastatic efficacy is evinced by prevention and/or treatment of metastatic cancer, e.g., as demonstrated by an increase in a time period of disease free survival for subjects receiving anti-Id treatment according to the invention.
Other compositions and methods of the invention target distinct cel lular proli ferative disorders characterized by aberrant blood vessel growth, or "pathogenic angiogenesis".
Examples of these disease targets include ocular disease mediated by aberrant vascular growth (e.g., macular degeneration), and tumor-associated angiogenesis. Anti-Id com pounds of the invention function also as "anti-angiogenic" agents, as described below, making them useful to treat or prevent pathogenic angiogenesis, including quite powerful ly tumor- associated angiogenesis (to mediate a multi-pronged, anti-metastatic and anti-angiogen ic assault on secondary tumor initiation and growth.
I n additional aspects of the invention, novel diagnostic assays measuri ng Id protei ns in blood or tissues (particu larly Id l and/or Id3) are demonstrated to provide useful diagnostic tools to monitor occurrence and progression of metastatic disease, and/or evaluate effectiveness of anti- Id treatment.
In more detai led embodiments, compositions and methods of the invention may employ an effective amount of the anti-Id compound combined in a kit or formulation, or coordinately administered, along with a secondary treatment agent, treatment modal ity or treatment method. I n exemplary embod iments, subjects are treated with the anti-Id compound sim ultaneously or sequential ly with a secondary treatment modal ity or agent, for example selected from : radiation, chemotherapy, surgery, or combinations thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
This patent appl ication contains at least one figure executed in color. Copies of th is patent application w ith color drawing(s) wil l be provided upon request and payment of the necessary fee.
Figure 1 provides a Western blot gel showing that the Id l and Id3 levels are potently reduced in leukem ic cel ls following treatment with an exemplary anti-Id compound, N-(3- (benzo[d][ l ,3]dioxo l-5-yI)-3-(2-methoxyphenyl)propyl)-N-benzylpropionamide (AGX5 1 ). A purified (-)-AGX5 1 enantiomer exhibits surprising stereospeci flc effects compared to the (+)- AGX5 1 enantiomer. Figure 2 provides a Western blot gel showing that the Idl and Id3 levels are variably affected in breast cancer cells following treatment with AGX51, revealing additional surprising stereospecific effects of this anti-Id compound.
Figure 3 is an immunoblot showing rescue ofpl6 levels in a leukemic cell line derived from a mouse overexpressing the MML-AF9 fusion protein following treatment with AGX51.
Figure 4 is a Western blot showing rescue of p21 levels in a human bladder carcinoma line following treatment with AGX5I.
Figure 5 graphically compares effects of racemic-AGX51, (+)-AGX51 ("El") and (- )-AGX51 ("E2) on restoring cell cycle control in DU-145 human prostate cancer cells. The (- )- enantiomer of AGX51 shows pronounced stereospecific efficacy for restoring cell cycle control, whereas the (+)- enantiomer of AGX51, surprisingly, mediates no detectable effect.
Figure 6 illustrates potent anti-migration effects of an exemplary anti-Id compound of the invention, AGX51, in a model of migration-dependent metastatic disease potential. This cellular motility "Scratch" assay demonstrates that AGX51 is a potent inhibitor of metastatic activity involving migration of cancer cells.
Figure 7 is a chart illustrating strong anti-angiogenic effects of AGX 1 to reduce formation of blood vessels in VEGF-165 and FGF-2 treated Matrigel plugs implanted into flanks of C57/BL mice.
Figure 8 is a chart providing data from the xenograft study performed in nude mice (n=8/treatment group) implanted with DA 231 human breast tumors and treated iv 14 days after implantation with either vehicle (DMSO) or 50mg/kg per day of AGX51 for five days and 7.5mg/kg paclitaxel starting on days 8 and 22. Boxplots are tumor volumes 53 days post implantation (last day of study).
Figure 9 provides ion chromatograms of control (A) and low calibrant of AGX5 I (B).
Figure 10 is a log-linear plot of plasma concentration data showing that AGX51 is rapidly absorbed with Cmax estimated here at 15 minutes.
Figure 11 is a simulation of plasma concentrations following 60mg/kg. bid of AGX51.
Figure 12 shows the design and results of studies demonstrating potent anti- metastatic efficacy of AGX51, directly reducing lung metastasis of Lewis Lung Carcinoma
(LLC) tumors implanted into C 57/BL mice (excised 14 days after implantation). In this widely accepted model for predicting cancer drug efficacy in humans, AGX5 1 provides an unprecedented decrease in metastasis of implanted cancer cells.
Figure 13 graphically demonstrates the effects of AGX5 1 on lung metastasis of breast cancer (4T 1 ) cells directly injected via tail vein into Balb/c m ice. AGX5 1 potently reduces metastasis of injected breast cancer cel ls.
Figure 14 shows biolum inescence visual ization of AGX5 1 suppression of lung metastasis in Balb/c mice following direct injection of breast cancer (4T I ) cel ls via tai l vein. The (-)-enantiomer of AGX5 1 potently, stereospecifically protects against metastasis in l iving model subjects predictive of cancer drug efficacy in humans.
Figure 15 is a graph demonstrating that a combination of pacl itaxel and AGX5 1 , even at a m inimal dose, significantly decreased tumor growth in mice implanted with M DA-M B- 23 1 tumors.
Figure 16 is a chart showing the change in final-initial tumor volume on day 1 9 in m ice implanted with MDA-M B-23 1 tumors and treated with pacl itaxel and varying amounts of AGX5 1 .
Figu re 17 is a graph showing that 60mg/kg. bid, AGX5 1 sign i ficantly increased the e ffectiveness of pacl itaxel on reducing tumor growth.
Figure 18 is a chart showing the change in final-in itial tumor volume on day 4 1 in m ice implanted with M DA-M B-23 1 tumors and treated with pacl itaxel and AGX5 1 .
Figure 19 is a graph showing that the addition of AGX5 1 to a treatment with paclitaxel signi ficant ly increased the effectiveness of pacl itaxel on decreasing tumor growth.
Figure 20 is a graph showing tumor growth in Id l knockout m ice treated with pacl itaxel and AGX5 1 .
Figure 21 is a chart showing mean tumor volume on day 20 i n Id I knockout mice treated with paclitax l and AGX5 1 .
Figure 22 is a chart showing a comparison of selected chemistry and hematology values in mice treated with pacl itaxel and/or AGX5 1 .
Figure 23 is a graph illustrating effects of ld3 genetic knockout on pathogenic retinal neovascularization in a mouse model.
Figure 24 is a graph demonstrating that (-)-AGX5 1 administered intravitreal ly (ivt) protects against pathogenic retinal neovascularization in a murine model of human age- related macular degeneration (AMD) Figure 25 is a graph demonstrating that (-)-AGX5 1 administered intraperitoneally (ip) protects against pathogenic retinal neovascularization in a murine model of human AM D,
Figure 26 is a schematic depiction of a modified sandwich immunoassay for detection of Id protein levels/activity in biological samples for use in implementing reflexive diagnostic-treatment methods of the invention.
Figure 27 is a graph demonstrating the use of Id diagnostic tools and methods of the invention for predicti ng and managing cancer.
Figure 28 is a graph demonstrating the use of Id diagnostic tools and methods of the invention for predicting and managing breast cancer in human subjects.
Figure 29 is a graphic depiction of AGX5 1 (+)- and (-)-enantiomer elution profi les using nantiomer Separation Method A of the invention.
Figure 30 is a graphic depiction of AGX5 1 (+)- and (-)-enantiomer elution profi les using Enantiomer Separation Method B of the invention.
DESCRIPTION OF EXEMPLARY EMBODIMENTS OF TH E INVENTION
Before describing the present invention in detai l, sev eral terms used in the context of the present invention wi ll be defined. In addition to these terms, others arc defined elsew here in the specification, as necessary. Unless otherwise expressly defined herein, terms of art used in this specification wil l have their art-recogn ized meanings. In addition, the fol low ing terms have the following meanings unless otherwise indicated.
Metastasis refers to the spread of a cancer from one organ or part to another non- adjacent organ or pa rt. Metastases are new occurrences of cancer at secondary sites generated by metastasis.
The term "chemotherapeutic" drug or agent typically refers to approved anti-cancer and other anti-hyperproliferative drugs or chem ical agents. "Chemotherapeutic" wil l general ly apply to a drug or chemical active to destroy cells and tissues, typically cancer cel ls and alternatively or conj unctively cells of blood vessels newly formed as part of a pathologic condition attending a hyperproli ferative disorder, as typical ly attends a neoplasm or a cancer. Chemotherapeutic agents for adjunctive use with in the invention include, but are not l im ited to: ( I ) tubul in depolymerizing agents such as taxanes l ike paclitaxel, docetaxel, BAY 59-
8862 albumin bound paclitaxel, (2) DNA damaging agents and agents that inhibit DNA synthesis, (3) anti-metabolites, (4) anti-angiogenics and vascular disrupting agents, (5) antibodies, (6) endocrine therapy, (7) immuno-modulators, ( 8) histone deacetylase inhibitors. (9) inhibitors of signal transduction, ( 1 0) inhibitors of heat shock proteins, ( 1 1 ) retinoids such as al l-trans retinoic acid, ( 1 2) inhibitors of growth factor receptors or the growth factors themselves, ( 1 3) anti-mitotic compounds, ( 1 4) anti-inflammatory agents such as COX inhibitors, and ( 1 5) cell cycle regulators, eg, check point regulators and telomerase inhibitors.
The term "combination therapy" refers to a therapeutic regimen that involves the provision of at least two distinct therapies to achieve an indicated therapeutic effect. For example, a combination therapy may involve the adm inistration of two or more chemical ly distinct active ingred ients, for example, an anti-Id compound as we l l as a chemotherapeutie agent. Alternatively , a combination therapy may involve the adm inistration of an anti-Id treatment and/or one or more chemotherapeutie agents, alone or together w ith the del ivery o f another treatment, such as radiation therapy and/or surgery. In the context of the
administration of two or more chem ical ly distinct active ingredients, it is understood that the active ingredients may be administered as part of the same composition or as d i fferent compositions. When adm inistered as separate compositions, the com positions comprising the di fferent active ingredients may be adm inistered at the same or di fferent times, by the same or different routes, using the same of different dosing regimens, al l as the particular context requires and as determ ined by the attending physician. Similarly, when one or more anti-Id treatment alone, or in conj unction, with one or more chemotherapeutie agents combined w ith, for example, radiation and/or surgery, the drug(s) may be delivered before or al ter surgery or radiation treatment. "Monotherapy" refers to a treatment regimen based on del ivery of one therapeutical ly effective compound, whether adm inistered as a single dose or in several doses over time.
"Neoplasia" refers to abnormal and uncontrol led cell growth. A "neoplasm", or tumor, is an abnorm al, unregulated, and disorganized proliferation of cell grow th and is sometimes referred to as a cancer. A neoplasm may be benign or mal ignant. A neoplasm is mal ignant, or cancerous, if it has properties of destructive growth, invasiveness, and metastasis. Invasiveness refers to the local spread of a neoplasm by infiltration or destruction of surrounding tissue, typically including penetrating basal lam inae that define boundaries of tissues (thereby often entering the body's circulatory system). Metastasis typical ly refers to dissemination of tumor cells to distant sites, often via lymphatics or blood vessels. Metastasis also refers to migration of tumor cells to adjacent sites through serous cavities or subarachnoid or othe r spaces. Through the process of metastasis, tumor cell migration and dissemination to other compartments, tissues and areas of the body establ ishes "secondary" neoplasms in areas away from the "primary" site of initial cancer appearance.
A "subject" or "patient" refers to an animal in need of treatment that can be affected by methods and compositions of the invention. Subjects and patients amenable to treatment using anti-Id compounds and methods of the invention include vertebrates, particularly mammals such as bovine, canine, equine, fel ine, ovine, porcine, and primate (including humans and non-humans primates) mammals, presenting with, or at elevated risk of developing, cancer, metastatic disease, or any proliferative disease inc luding pathogen ic angiogenesis.The term treatment or therapy "system" as employed herein refers to a plurality of treatment agents or modalities, such as a combined formulation or protocol employing an anti-Id active agent, a chemotherapeutic and possibly a toxicity reducing agent— used in a coord inate treatment regimen (formulated alone or together, and adm inistered simultaneously or sequential ly). Treatment systems may also combine drug (e.g., anti-Id and/or conventional chemotherapy) treatment with another intervention or treatment modal ity, such as rad iation therapy or surgery. Optional treatment systems employing anti-Id agents and methods of the invention can be integrated in any combinatorial "therapeutic regimen" combining complementary tools or methods, such as chemotherapeutic agents, radiation therapy, surgery, gene therapy, DNA vaccines and therapy, siRNA therapy, anti-angiogen ic therapy, immunotherapy, bone marrow transplants, aptamers and other biologies such as antibodies and antibody variants, receptor decoys and other protein-based therapeutics.
The term "treatment" or "treating" means any treatment of a disease or disorder, including preventing or protecting against the disease or disorder (that is, causing the clinical symptoms not to develop); inhibiting the disease or disorder (i.e., arresting or suppressing the development of cl ini cal symptoms; and/or rel ieving the disease or disorder (i .e., causing regression of clinical symptoms). The methods and compositions of the invention wi l l often serve to prevent one or more symptoms of disease, or delay onset or recurrence/relapse of disease ("prophylaxis"), as wel l as to slow, inhibit or prevent disease progression (e.g., as marked by increased severity of disease symptoms, or onset of more advanced disease symptoms). The novel methods and compositions of the invention flow from a surprising course of discovery. Early comparisons of gene expression data between cell lines with variable metastatic potential prompted reports that certain candidate genes are required during different steps of the metastatic disease cascade (Kang et al., 2003b; Minn et al., 2005 ; Yang et al., 2004). Some of these reports suggested that expression of Id proteins, including products of Id l and l d3 genes, are involved in lung colonization of breast cancer cel ls (Gupta et al., 2007). Id proteins have long been reported to be dominant negative regulators of basic hel ix-loop-helix (bHLH) transcription factors (Perk et al., 2005).
Id proteins have further been reported to play key roles in maintaining embryonic stem cel l self-renewal (Romero-Lanman et al. ; Ying et al., 2003), and to continue th is function in adult tissue stem cells (Nam and Benezra, 2009) and hematopoietic stem cells (Jankovic et al., 2007).
Id genes have also been impl icated as regulators of TIC phenotypes in certain cancers, such as gl ioblastoma. (Anido et al., 20 1 0; Barrett et al., 20 1 2) and colon cancer (O'Brien et al., 20 1 2). In human breast cancer, Id 1 is predom inantly expressed in the more aggressive triple negative [negative for estrogen receptor, progesterone and negative for the neu type human epidermal growth factor-2 (H ER2 ) and metaplastic subtypes, with high Id l expression correlating with poor cl inical outcomes (Gupta et al., 2007).
The inventio n targets Id functional ity in promoting cancer and metastasis, by employing novel suppressors of Id proteins, including suppressors of Id l and Id3. These compounds are referred to herein as "anti-Id" compounds, and the attendant methods of the invention are collectively termed "anti-Id" methods. The anti-Id compounds of the invention are general ly in an "anti-Id effective amount" by direct appl ication or injection to a cell population, physiological compartment, tumor, or individual. These compounds and methods exhibit "anti-cancer", "anti-metastatic", "anti-proliferative", and/or "anti-angiogenic" effective activities, as described herein for different aspects and embod iments of the invention.
In exemplary aspects the invention provides compositions and methods for treating a cellular proliferative disorder, for example inhibiting or reducing metastasis of a tumor or primary cancer cel l. These methods comprise adm inistering to a mammal ian subject an effective amount of an "anti-Id compound", sufficient to reduce or prevent pathogenic cellular proliferation, angiogenesis, cancer, and/or metastatic disease in the subject. I n
N
Formula I R wherein Ri may be a substituted or unsubstituted lower hydrocarbon selected from the group consisting of alkyl, alkenyl. alkanoyl, alkynyl, aryl, aroyl, aralkyl, alkylam ino. aryloxy, hydrogen, carboxyl, nitro, thioalkoxy, thioaryloxy, thiol, cycloalkenyl cycloalkyl, heterocycloalkyl, he ieroaryl, aralkyl, am ino acid, peptide, dye, fluorophore, carbohydrate or polypeptide; R2 and R3 may independently, collectively, or in any combination that yields an active anti-Id (apoptosis inducing, cel lular prol iferation inhibiting, chemotherapeutic enhancing, transcrip tion regulating, anti-inflammatory, cellular differentiation promoting, cellular transformati on modulating) composition be a hydrogen, hydroxyl, sulfyhydryl, fluorine, methyl, ethyl, propyl, benzyl, 2-bromovinyl amino, hydroxymethyl, methoxy, halogen, pseudohalogen, cyano, carboxyl, nitro, thioalkoxy, thioaryloxy, th iol, substituted or unsubstituted lower hydrocarbon containing 1 to 20 carbons such as alkoxycarbonyl.
allkoxycarbonylamino, amino, am ino acid, aminocarbonyl, am inocarbonyloxy, aralkyl, aryloxy, carboxyl, cycloalkenyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl. amino acid, peptide, dye, fluorophore, carbohydrate or polypeptide; R4 and R5 may be may independently, collectively, or in any combination that yields an active anti-Id (apoptosis inducing, cellular proliferation inhibiting, chemotherapeutic enhancing, transcription regulating, anti-inflammatory, cellular differentiation promoting, cel lular transformation modulating) composition be an acyl or a substituted or unsubstituted lower hydrocarbon selected from the group consisting of alkyl, alkenyl, alkanoyl, aryl, aroyl, aralkyl or alkylamino; R6 may be a heteroatom such as oxygen, sulfur or nitrogen; R7 may be a heteroatom such as sulfur, nitrogen or oxygen as well as a carbon; R8, 9, 10, 1 1 and 12 may independently, collectively, or in any combination that yields an active anti-Id (apoptosis inducing, cellular proliferation inhibiting, chemotherapeutic enhancing, transcription regulating, anti-inflammatory, cellular differentiation promoting, cel lular transformation modulating) compos ition be selected from hydrogen, hydroxyl, sulfyhydryl, fluorine, methyl, ethyl, propyl, benzyl, 2-bromovinyl amino, hydroxymethyl, methoxy, halogen,
pseudohalogen, cyano and a substituted or unsubstituted lower hydrocarbon containing 1 to 20 carbons.
When more than one R group is present, the R group may be selected from any of the stated groups so as to be the same or different. In additional embodiments, two or more R groups may be joined together. In some embodiments, R2 and R3 may be members of a 5, or 6, member exocycl ic ring structure. In other embodiments, R3 and R4 may be members of a 5, or 6, member exocyclic ring structure. In further embodiments, R5 and R6 may be members of a 5 or 6 member exocycl ic ring structure. In additional embodiments, R l 1 and R 1 2 may be members of a 5 or 6 member exocycl ic ring structure. In some embodiments, i f R7 is nitrogen, R6 and R7 may be members of a 5 or 6 member exocycl ic ring structure. I n other embodiments, R6 and R I 2 may be members of a 5 or 6 member exocycl ic ring structure.
In more detai led embodiments, rational ly designed anti-Id compounds of Formula I can be selected from additional candidates, wherein R i , R4, R5. Rf>, RH, Ry, R io are independently selected from the group consisting of hydrogen, hydroxyl. sul fyhydryl, benzyl. 2-bromovinyl am ino, hydroxymethyl, methoxy, halogen, pseudohalogen, cyano, carboxyl. nitro, thioalkyl, thioaryl, thiol, substituted or unsubstituted hydrocarbons containing 1 to 20 carbons, alkoxycarbonyl, alkoxycarbonylam ino, am ino, am ino acid, am inocarbonyl, aminocarbonyloxy, aryloxy, carboxyl, cycloalkenyl, substituted or unsubstituted cycloalkyl. substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, peptidyl. dye, fluorophore, carbohydrate or polypeptidyl ; R» and R9 can also have a para configuration in addition to the ortho orientation shown in Formula I ; R7 is independently selected from hydrogen, hydroxyl. methoxy, azido, nitrile, sulfhydryl, halogen, benzoyl, substituted benzoyl or hydroxyl substituted with substituted or unsubstituted hydrocarbon containing 1 to 20 carbons, alkanoyl of a main chain of 1 to 20 carbon atoms, CF3(CH2)n CO where n= l to 1 0, CH3(C F2)n C=0 where n= l to 1 0, CF3(CF2)n where n=0 to 3, aryl, alkoxy, halogen, or n itro, aryloxy. esters of aryloxy, adamantoyl, substituted adamantoyl or aroyl of 1 to 20 carbons: R7 cannot be methoxy if Rn is propionyl while R7 and Re can be ortho, as shown in Formula I, or para to one another; Rn is independently hydrogen, propionyl, pivoyl, benzoyl, substituted benzoyl, alkanoyl of a main chain of 1 to 20 carbon atoms,
Figure imgf000021_0001
where n=l to 10. CH3(CF2)nC=0 where n=l to 10 or CF3(CF2)n where n=0 to 3 and Rl 1 cannot be propionyl if R7 is methoxy; R12 is independently hydrogen, halogen, 2H, CH3(CH2)n where n=0 to 5, CF3(CH2)nC=0 where n=l to 5; CH3(CF2)nC=0 where n=l to 5 or CF3(CF2)n where n=0 to 5.
Additional description pertaining to these and other aspects of the invention can be found, for example, in United States Patent No.8,138,356, issued 20 March 2012, and United States Patent Application Serial No. 14/341,756. filed 25 July 2014, each of which is incorporated herein by reference its entirety for all purposes.
In illustrative embodiments, the anti-Id compound selected from Formula I is N-(3- (benzo[d][ l,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzylpropionamidc
(alternatively, "AGX5 ), as shown in Figure II, below. It will be understood from the teachings herein that the various Formula I compounds, inclusive of AGX51. include pharmaceutically acceptable active salts of said compounds, as well as active enantiomers, polymorphs, metabolites, solvates, hydrates, and prodrugs of said compounds.
Figure imgf000021_0002
Formula II
Novel and surprisingly effective methods and compositions that inhibit Id proteins may additionally comprise compounds of Formula III, below, and their active salts, enantiomers, polymorphs, metabolites, solvates, hydrates, and prodrugs.
Figure imgf000022_0001
wherein R i , R2, R3, ¾, Rs, Re, Rx, R9, and Rio may independently, collectively, or in any combination that yields an active anti-Id compound be hydrogen, hydroxyl, sulfyhydryl. fluorine, methyl, ethyl, propyl, benzyl, 2-bromovinyl am ino, hydroxymethyl, mcthoxy.
halogen, pseudohalogen, cyano, carboxyl, nitro, thioalkoxy, thioaryloxy, thiol, substituted or unsubstituted lower hydrocarbon containing 1 to 20 carbons; alkoxycarbonyl,
allkoxycarbonylam i no, am ino, am ino acid, aminocarbonyl, aminocarbonyloxy, aralkyi, aryloxy, carboxyl, cycloalkenyl, substituted or unsubstituted alkyl, substituted or
unsubstituted cycloa ikyi, substituted or unsubstituted heterocycloaikyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyi, am ino acid, peptide, dye, fluorophore, carbohydrate or polypeptide; R7 may be selected from hydrogen, hydroxyl, benzoyl; substituted benzoyl or hydroxyl substituted with unsubstituted lower hydrocarbon containing 1 to 20 carbons; Rn may be a hetcroatom such as oxygen, sul fur or nil rogen; R i2 inay be a lower hydrocarbon independently selected from the group consisting of alkyl, alkenyi, alkanoyi, aikynyl, aryl, aroyl, aralkyi, alkylam ino, aryloxy. hydrogen, carboxyl, nitro, thioalkoxy, thioaryloxy, thiol, cycloalkenyl substituted or unsubstituted heteroatom, substituted or unsubstituted alkyl, substituted or unsubstituted cycloaikyi, substituted or unsubstituted heterocycloaikyl, substituted or unsubstitutcd aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyi, amino acid. peptide, dye, fluorophore, carbohydrate or polypeptide. With regard to Formula III, when more than one R group is present, the R group may be selected from any of the stated groups so as to be the same or different. In additional embodiments, two or more R groups may be joined together. In some embodiments, R4 may become a member of a 5 or 6 member ring structure with neighboring rings. An exemplary compounds of Formula III for use within the invention is N-[3-(l,3-benzodioxol-5-yl)-3-(2- methoxyphenyl) propyl]-N-benzylpropanamide, as shown in Formula IV.
Figure imgf000023_0001
Formula IV
An exemplary anti-Id compound for use within the compositions and methods of the invention is N-(3-(benzo[d][l,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N- benzylpropionamide ("AGX51"). As used herein, AGX51 refers to N-(3- (benzo[d][ 1 ,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzylpropionamide in isolated or substantially purified form, as well all functional equivalent salts, prodrugs, metabolites, derivatives, analogs, and conjugates of AGX51. For example, those skilled in the art will that AGX51 encompasses desired prodrug forms and biotransformation products, and readily designed and tested analogs, derivatives and complexed or conjugated forms of the drug.
Thus, the invention encompasses compositions and methods employing demethylated forms of AGX15, for example N-(3-(benzo[d][l,3]dioxol-5-yl)-3-(2-hydroxyphenyl)propyl)- N-benzylpropionamide; deamidated forms, for example (3-(benzo[dj[ 1.3|dioxol-5-yl)-N- benzyl-3-(2-methoxyphenyl)propan-l -amine, or multiply-modified (e.g., demethylated and deamidated) forms, such as 2-(l-(benzo[d][l,3]dioxol-5-yl)-3-(benzylamino)propyl)phenol. In related embodiments, the anti-Id compound is a selected salt form of a racemate or purified enantiomer of a biotransformation product of N-(3-(benzo[d][l,3]dioxol-5-yl)-3-(2- methoxyphenyl)propyl)-N-benzylpropionamide, for example a salt of (3- (benzo[d][l,3]dioxol-5-yl)-N-benzyl-3-(2-methoxyphenyl)propan-l-amine or 2-(l- (benzo[</J [ l ,3]dioxol-5-yl)-3-(benzylamino)propyl)phenol. Exemplary salt forms of these and other anti-Id compounds herein include, but are not limited to, hydrochloride, hydrobrom ide, hydroiodide, phosphate, sulfate, oxalate, malate, maleate and succinate salts.
In related embodiments, the anti-Id compound is an isolated, anti-metastatically active enantiomer of N-(3-(benzo[d][ l ,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N- benzylpropionam ide. Surprisingly, the discoveries herein demonstrate extraordinary anti-Id potency residing in the (-)-enantiomer form of AGX5 1 . Thus, in preferred aspects the invention employs novel, enantomerically-enriched preparations of (-)-AGX5 1 , substantial ly purified to yield an i ncreased relative amount or concentration of (-)-AGX5 1 (relative to an amount or concentration of the other, (+)-AGX5 1 enantiomer, or to an amount found in conventional, racem ic preparations of N-(3-(benzo[d][ l ,3]dioxol-5-yl)-3-(2- mcthoxyphenyl)propyl)-N-benzylpropionamide).
In more detai led aspects, the methods and compositions of the invention employ a "substantially pure" or "essential ly pure" anti-metastatic effective (-)-enantiomer of N-(3- (benzo d ] [ l ,31dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzylpropionam ide. In exemplary composit ions, the purified (-)-AGX5 1 enantiomer is at least initial ly (i.e., pre- formulation) provided in a form that exhibits at least 80-90%, 90-95%, greater than 95%, or 98% or greater "enantiomeric enrichment" (ce) or "enantiomeric purity". As used herein. (-)- AGX5 1 enantiomer preparations of 90-95% ee are "substantially pure", wh i le preparations of 98% ee or greater are or "essential ly pure." In alternative aspects, the methods and compositions of the invention employ a "substantially pure" (-)-N-(3-(benzo[d] [ l ,3]dioxol-5- yl)-3-(2-methoxyphenyl)propyl)-N-benzylpropionamide preparation is defined as being "essential ly free of the (+)-N-(3-(benzo[ d] [ l ,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N- benzylpropionamide enantiomer (i.e., having less than 5%, less than 2% or less than 1 % of the total N-(3-(benzo[d] [ l ,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N- benzylpropionam ide present being in the form of the (+)-enantiomer with the remainder exclusively the (-)-enantiomer.
The identity and quantity of a selected AGX5 1 enantiomer within the compositions and methods of the invention may be determined by a variety of means. For example, these determinations and values may be demonstrated using conventional chiral chromatography and/or polarimitry. As used herein the term "purified" or "enriched" (-)-AGX5 1 "\ or is intended to correspond to any composition artificially enriched to contain, in stable form, a substantially higher concentration of (-J-AGX5 1 than (+)-AGX5 1 (or than an amount of (-)- AGX5 1 present in conventional (non-chiral) racemic preparations of the compound)— wherein the (-)-AGX5 1 occurs in an enantiomeric excess (ee) of at least about 60%. 70%, 75%, 80%), up to approximately 90% or greater, up to 95-98%) or greater ee.
In certain aspects the methods and compositions of the invention employ highly puri fied or isolated (-)-AGX5 1 (at least as a starting material prior to formu lation, storage or administration) in an enantiomeric excess of greater than 98%> (e.g.. as determined by chiral chromatography and/or optical purity assay). Preparations of (-)-AGX 1 in enantiomeric excess greater than 85% or 90%> are considered substantial ly free of the corresponding (+)- AGX5 1 enantiomer and are highly desired drug preparations for cl in ical use. Typical ly, pharmaceutical compositions of the invention for treating cancer, preventing or treating metastatic disease, or treating an angiogenic or other prol iferative disorder, wi l l contain no more than about 5% w/w, and in some embodiments no more than about 2% or 1 % or lower w/w, of the (+)-AGX5 1 enantiomer (i .e., measured as a percent of total N-(3- (benzo[d ] [ l ,3 ]dioxo l-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzylpropionamide mass or molar content present in the composition).
The use of substantial ly pure (-)-N-(3-(benzo| d ] [ l ,3 ]dioxol-5-yl)-3-(2- methoxyphenyl)propyl)-N-bcnzylpropionam ide ((-)-AGX5 l ), or sign ificantly
enantiomerical ly enriched (-)-AGX5 1 compositions provides enhanced therapeutic efficacy for treating cancer, preventing or treating metastatic disease, and treating angiogen ic and other prol iferative disorders, with reduced side effects at eq uivalent dosages, compared to racem ic AGX 1 or the (+)-AGX5 1 enantiomer. In alternate embodiments, enriched (-)- AGX5 1 yields equal or greater therapeutic efficacy with fewer side effects at lower or less frequent dosages, compared to racem ic AGX5 1 or the (+)-AGX5 1 enantiomer. I n certain aspects, at equivalent (by weight or molarity) dosing with racem ic AGX5 1 , the (-)-AGX5 1 - enriched compositions and methods of the invention exhibit increased activity or therapeutic efficacy of at least 1 0%, 20%, 30%, or 50%, up to 75-95%, 100% or even 200% greater than an observed activity or therapeutic efficacy determined for the racemate (or for the substantially pure (+)-AGX5 1 enantiomer)— using any one or combination of activity measures, therapeutic indices and clinical assays employed or referenced herein. Anti-Cancer and nti-Metastasis Compositions and Methods
According to the discoveries and teachings herein, anti-Id compositions and methods of the invention effectively block Id binding to bHLH proteins, promote functionally significant Id degradation (i.e., therapeutical ly decrease Id levels in cells), and suppress Id activity when administered to cells, tissues and living subjects. These ant-Id activities in turn effectively suppress tumor metastasis, as well as tumor-associated angiogenesis and other prol iferative disease states and symptoms. In this surprising manner, the compositions and methods of the invention afford effective tools to m itigate the most lethal and refractory neoplasms.
The instant invention has immense cl inical promise in view that so many Americans are critically affected by cancer. The National Cancer Institute of the N I H estimates that approximately 1 3.7 m i ll ion Americans with a history of cancer were al ive in January, 20 1 3. Some of these indiv iduals were cancer-free, whi le others sti ll had evidence of cancer and may have been undergoing treatment. In 201 3, about 58,350 Americans were expected to die of cancer, or approximately 1 ,600 people a day. In the US, cancer accounts for 1 of every 4 deaths.
Patients typi cal ly present with cancer that is already wel l establ ished, with a tumor burden that is alread y an existential medical problem. These patients are treated with surgery, radiation, chemotherapy (optional ly including hormone therapy), sometimes with adjuvant, biologic or targeted therapy to reduce tumor burden. I f this first treatment or scries of treatments doesn' t yield complete success, the tumor often exhibits resistance to further treatments, wh ile in other cases the cancer metastasizes to distant organs with often fatal consequences. Death from cancer is almost universal ly preceded by cancer metastasis, which is therefore among the most critical, unresolved targets for cancer intervention.
Suppression of cancer metastasis is an urgent therapeutic need, in part because most existing cancer drugs inhibit only cell prol iferation. The molecular biology of metastasis has proven more complex and difficult to interpret than for primary cancer cell transformation. Because the molecules that drive dissemination and colonization by mal ignant cel ls are shared among different types of cancers, drugs that inhibit these processes wi ll be much more broadly beneficial than conventional chemotherapy drugs. Successful interventions to disrupt the metastati c cascade will focus on mechanisms and activities cruc ial for dissemination of cancer cel ls from the primary tumor, and for subsequent colonization of secondary tumor site. As elucidated with particularity herein, Id proteins are potent inducers of these tumor initiation events, which are in turn disabled or blocked by the potent anti-Id compositions and methods of the invention. Anti-Id compounds and treatment methods provided here focus on multiple complementary anti-metastasis strategies, including: 1 ) prevention of cancer cell dissem ination; and 2) suppression of existing metastases.
The methods and compositions of the invention find use in therapeutic applications in which anti-Id administration is indicated, as described in a cl inically effective context for the first time here. Rep resentative therapeutic applications of the invention include treatment of cellular proliferative diseases, including cancer and other diseases characterized by adverse cellular prol iferation (hyperplasias).
The anti-Id c ompositions and treatment methods of the invention are effective to reduce or prevent ce l lular prol iferative disorders in mammal ian subjects, including cl inical ly effective treatment of cancer in veterinary (e.g., dog, cat, large animal) and human patients.
In general terms, anti-Id effectiveness of the compositions and methods of the invention may be correlated with a by a decrease in one or more symptoms of a cel lular prol i ferative disorders (e.g., cancer, metastatic disease, tumor-associated angiogenesis). This may be detected or quanti fied based on an observed decrease in cel lular prol iferation, vascular growth, cel lular m igration, secondary tumor appearance or growth, inflammation, or any other symptom associated with the targeted cel lular prol iferative disorder. Various assays and model systems can be readily employed to illustrate therapeutic effectiveness of the anti-Id compounds of Formula I, I I, I I I and IV described herein. These assays are wel l known and include widely accepted cl inical correlates for each of the subject prol i ferative disorders contemplated herein. Exemplary assays for ready use in this context include assays to detect or quantify cellular proliferation markers, circulating endothel ial cel ls, circulating endothel ial progenitor cells (EPCs), circulating tumor cells, various cancer/tumor markers (e.g., PSA), and histological, histochemical, and immunohistochem ical markers to detect, local ize, anatomically map, and/or quantify primary and metastatic cancer cells, among others. Useful, cell-specific markers for each of the foregoing cell types and functional ities (i.e., cell commitments/fates, differentiation states, developmental potential ities), are wel l known in the art for use within these methods and compositions of the invention. Exemplary markers for tagging , visual izing, quantifying and/or separating cells within various assays and diagnostic or cl inical embodiments of the invention include: for metastatic cel ls bearing markers of increased metastatic potential (e.g., markers associated with loss of epithelial character, increased migration potential, secondary site colonization potential)-Vimentin+, N-Cadherin+, E-Cadherin-; for EPCs-Lin-, GFP+, VE-Cadherin+, CD 1 1 -; for cancer stem cells (breast cancer example)~CD44(h igh) Cd24 (low). Many additional markers, and combinations of markers, wil l be routinely employed in various assays and diagnostic and clinical methods, having l ike or additional specificities, to distinguish Anti-Id activity, function and cel lular and clinical effects, as discussed further herein.
In certain embodiments, the anti-Id compositions and methods of the invention are effective to reduce occurrence, relapse or growth of a neoplasm. Administering an anti- neoplasm effective amount of the anti-Id compound wil l reduce incidence, number or growth of neoplasms in treated versus control subjects by 5%, 1 0%, 25%, 30%, 50%, 75%, 90% or more.
In other embodiments, the invention provides "anti-metastatic" compositions and methods, which are effective to reduce an incidence or severity of tumor metastasis in mammalian subjects. These methods may employ monotherapy or coordinate or
combinatorial therapy. The subject compositions and methods "anti-metastatical ly e ffective", as demonstrated by a signi ficant reduction in incidence, number or size of metastases in treated versus control subjects presenting with, or at elevated risk for developing, cancer. In certain embodiments, anti-metastatic efficacy correlates with a reduction in one or more histopathological indices of metastasis, for example quantitative reduction in occurrence, size, number or distribution of metastasized cells or■'foc i'" of primary tumor character observed at a secondary tissue or anatom ic site. Alternatively there may be a substantia decrease in cancer cell m igration or dissemination mediated by the anti- Id compound. In other embodiments, anti-metastatic efficacy is demonstrated by a significant positive increase in one or more patient therapeutic indices correlating with effective prevention and/or treatment of cancer or metastas is— for example by an increase in a time period of disease-free survival for subjects receiving the anti-Id compound compared to control subjects not receiving the anti-Id compound.
Anti-metastatic efficacy of the compounds, compositions and methods of the invention yield substantial therapeutic benefits and improved treatment outcomes in patients treated for cellular prol iferative disorders and/or neoplasms. In exemplary embodiments, cancer patients treated with the anti-Id methods and compositions of the invention exh ibit improved treatment outcomes with no increase, and often a significant decrease in adverse side effects that attend conventional cancer treatments (e.g., chemotherapy and radiation therapy, which may be reduced or el iminated in patients treated with the compositions and methods herein).
I llustrative of these benefits, methods of the invention will yield at least a 20% increase in one or more positive therapeutic indices of metastatic disease prevention or arrest, for example a reduc tion in occurrence, size, number or distribution of metastasized cel ls or "foci" of primary tu mor character observed at a secondary tissue or anatomic site. In certain embodiments, anti-rnetastatic efficacy of the anti-Id compounds and methods wi l l yield at least a 20% increase in an accepted metastatic index, including gross indices such as disease- free survival of Id-treated patients compared to quali fied control patients not treated with the anti-Id compound.
In other embodiments, anti-Id compounds, formulations and methods of the invention wi l l yield even more significant anti-metastatic cl inical benefits, for example a 20-50% increase, 50-70% increase, up to a 75%- 1 00% reduction in a recogn ized metastasis indicator or marker (e.g., cancer cel l m igration or dissemination observed at or near a primary tumor site, metastasis observed in blood or lymphatic biological samples, or secondary tumor formation detected in a distant tissue or organ (e.g., by radiological or other imaging, biopsy, or post-surgical or post-mortum histolopathology). In many instances, effective cl inical management employing anti-Id compositions and methods of the invention wi l l yield total prevention, clearance or stable rem ission of primary and/or metastatic cancer symptoms, metastatic disease or other cancer symptoms enduring for 6 months to a year. I -2 years, 2-5 years, 5- 1 0 years or longer.
In exemplary embodiments, the anti-Id methods and compositions of the invention wi l l be anti-metasta ically effective to yield at least a 20% decrease in metastasis, a 20%- 50%, a 50%-75%, up to a 90% or greater decrease in metastasis (e.g., as demonstrated by conventional, comparative histopathology, computerized tomography, positron em ission tomography and/or magnetic resonance imaging to detect, locate and/or quantify metastatic cells) in anti-Id-treated versus non-treated or placebo-treated subjects.
Respective of al l embodiments presented here, anti-metastatic efficacy will typica l ly correlate with no increase, or even a decrease, in observed adverse side effects (e.g., nausea, weight loss, hair loss, immunological damage, etc.) between anti-Id treated patients and a taxane, or an alternate cancer therapy, such as radiation therapy). In exemplary embodiments, anti-ld-treated subjects (including subjects treated with anti-Id compound monotherapy, and subjects treated with combinatorial methods, such as anti-Id plus chemotherapy, or anti-Id plus radiation therapy) wi ll exhibit no increase in one or more general ly-seen adverse cancer treatment side effects (e.g., general chemo- or radiation- therapy side effects), and will often exh ibit at least a 20% reduction, a 20-50% reduction, up to a 50-90% or greater reduction in occurrence or severity of one or more adverse cancer treatment side effects (e.g., compared to positive control subjects treated with conventional chemotherapy or radiation therapy alone).
Side effects that may be associated with anti-Id therapy may include side effects associated with anti -angiogenic effects of the novel, anti-Id compounds described here. A lthough such prospective side effects have yet to be evaluated, various management tools can effectively l imit or prevent such adverse sequelae. For example, potential impairment of wound healing due O anti-angiogenic activity of the claimed compositions and methods can be avoided by staging anti-Id treatment prior to cancer surgery when indicated, and/or post- surgery al lowing for an effective heal ing period prior to initiation of the anti-Id therapy. Additional pro-heal ing agents and methods can be coordinately adm inistered, such as coordinate administration of pro-heal ing cytokines or growth factors (e.g., platelet-derived growth factor (PDGF).
In more deta i led aspects of the invention, the anti-Id compounds and methods are also effective to reduce a metastasis-associated increase in circu lating endothel ial cel ls.
Circulating endothelial cells are general ly absent in the blood of healthy ind ividuals, but significantly elevated in individuals suffering from diseases marked by pathogenic angiogenesis, including cancer. The number (titer or hematocrit count) of c irculating endothel ial cel ls may be determined by any means applicable, such as through flow cytometry, immunobead capture, fl uorescence m icroscopy, standard and density
centrifugation, or mononuclear cel l culturing on fibronectin-coated plates and
immunocytochemistry. An anti-metastatic or anti-angiogenic effective amount of the anti-Id compound of the invention will decrease the number of circulating endothel ial cells by 5%. 10%, 25%o, 30%, 50%, 75%, 90% or more compared to levels observed in placebo-treated subjects presenting with similar pathology (e.g., an equivalent state of metastatic or angiogenic disease prior to treatment, normal ly attended by tumor-associated elevation of EPCs).
In other deta iled embodiments, the anti-Id compounds and methods of the invention are effective to block or reduce a metastasis-associated increase in circulating endothel ial progenitor cel ls (EPCs) implicated in metastatic disease progression. Mal ignant
transformation and metastasis is associated with increased numbers of circulating EPCs. Endothel ial cell production is generally responsible for repair of damaged vasculature (including associated with tumors, and in other cases of pathogenic angiogenesis) through mobil ization of EPCs from the bone marrow. This is followed by hom ing of the EPCs to target sites for repair (including sites of damaged vasculature associated with tumors) (Shaked et al., 2006; Shaked et al., 2008). This process of vascular repair often follows treatment with certa in, cytotoxic "chemotherapeutic*' drugs, such a pacl itaxel, and vascular disrupting agents (VDAs, for example ZD6 1 26 or AVE8062), which have attendant adverse cytoxic side effects on blood vessels. This angiogen ic repair process fol lowing paclitaxel treatment is observed in normal mice, but reportedly absent in mice lack ing the Id l gene (Shaked et al, 2008).
The exact nature of the process in bone marrow responsible for tumor-associated EPC production has not been establ ished, but the source of the EPC* s is l ikely hematopoietic stem cells (HSCs) in the marrow. Eoss of Id l in bone marrow reportedly leads to a loss of EPCs in peripheral blood, correlated with impairment of tumor neovascularization and growth. The absence of Id l also comprom ises the sel f-renewing capacity of HSCs in bone marrow;
increasing their tendency to differentiate towards the myeloid l ineage. Th is functional defect is associated with transcriptional changes in Id l null HSCs, including increased expression of p2 1 , another target of Id I repression (Ciarrocchi et al., 2007). Id 1 is required for the previously described ability of phenotypic HSCs to give rise to endothel ial progeny to highl ight the opposi ng effects of Id l and its target gene p2 l on endothel ial and myeloid l ineage differentiation in the HSC subset. Ablation of p2 1 i n the Id l -/- animals restores a functional endothel ial population, rescues the angiogenic defect observed in the Id l -/- mice, and reverses the premature myeloid commitment of Id l nul l HSCs. Id l appears to play a therapeutical ly negative, protective role for cancer cells undergoing threat with cytotoxics through a general anti-apoptotic effect (Zhang et al., 2006; Wong et al., 2004). This was reported based on s tudies of paclitaxel/docetaxel-induced apoptosis in prostate cancer cells. nasopharyngeal carcinoma cells, HeLa (cervical) cancer cells and MCF7 (breast) cancer cells. Up-regulation by Id l of Raf/ME (Zhang et al., 2006), and/or MAPK signaling pathway (Cheung et al., 2004; Lin et al., 2005) has also been proposed to account for resistance to cytotoxics acquired by cancer cells post-treatment.
Despite the complex and indefinite pathways involved, anti-Id compounds and methods of the invention fundamentally disable Id proteins at critical intersections of these pathways. I n this manner, anti -metastatic and anti-angiogenic effective anti-Id compounds of the invention also target EPC production and secondari ly disable tumor-associated angiogenesis and related tumor growth. In certain embodiments, the anti-Id compositions and methods of the invention wi l l effectively decrease the number of EPCs in a circulating blood sample of treated subjects by at least 5%, 1 0%, 25%. 30%, 50%, 75%, 90% or more compared to levels observed in placebo-treated subjects presenting with sim i lar pathology (e.g., an equivalent state of metastatic or angiogen ic disease prior to treatment, normal ly attended by tumor-associated elevation of EPCs).
Anti-Id compositions and methods of the invention further exert anti-cancer and anti- metastatic effects by sh ifting a cellular determination or fate of tumor cel ls toward senescence or apoptosis. Accordingly, in yet additional aspects of the invention, anti-Id compositions and methods of the invention wi ll effectively increase the number of apoptotic or senescent cel ls in a primary or secondary tumor of anti-Id-treated patients. I n certain embodiments, the anti-Id compositions and methods of the invention wi l l increase the number of apoptotic or senescent cel ls in tumors (e.g., as observed through biopsy or necropsy of existing tumors) of treated subjects by at least 20%, 30%. 50%, 1 00%, up to 5- fold, ten-fold or higher, compared to numbers of apoptotic or senescent cel ls in control samples from place bo-treated subjects presenting with sim ilar pathology (e.g., an equ ivalent state of metastatic or angiogenic disease prior to treatment, normal ly attended by tumor growth and a low incidence of apoptotic or senescent cells).
Cancer stem cells are a subopulation among bulk of tumor cel ls capable of in itiating new tumors (with a capacity to recapitulate the l ineage heterogeneity of the parental tumor). Cancer stem cells share characteristics of tissue stem cel ls, including sel f-renewal and multipotency. A positive regulatory activity of Id proteins on cancer stem cel ls has been reported in colon cancer and malignant glioma, although other types of cancer stem cel ls also appear to depend on Id proteins (Hua et al., 2006; James et al, 20 1 0; Jankovic et al., 2007; Perry et al., 2007; Rawlins et al, 2009; Suh et al., 2009; Lyden et al.; 1999; Anido et al., 2010; Jeon et al., 2011). In colon cancer stem cells, combined expression of Idl and ld3 reportedly increases both self-renewal and tumor initiation (O'Brien et al, 2012). Cancer stem cells exhibit resistance to chemotherapeutic agents, and silencing of Idl and Id3 in culture-based assays reportedly sensitizes cells to oxaliplatin (0*Brien et al, 2012). In high- grade glioma. Id proteins are co-expressed in diverse tumor cell populations, including glioma stem cells. In an orthotopic model of brain cancer driven by the
Figure imgf000033_0001
oncogene, deletion of conditional Id 1 , Id2 and Id3 alleles in the tumor cells reportedly decrease the glioma stem cell population (nestin-positive and stage-specific embryonic antigen 1
(SSEA l)-positive cells), blocks tumor growth and extends survival. Cells selected in vitro for self-renewal potential reportedly lose tumorigenic capacity if Id genes are deleted after implantation of the cells into mouse brains, while they retain robust tumorigenic potential if Id genes are intact (Niola et al., 2013). Embryonic neural stem cells (NSCs) reportedly lose self-renewal and multi-potency in the absence of Idl, Id2 and Id3, but these properties are sustained near normal in cells that retain one Id2 allele (suggesting that Id proteins function redundantly in NSCs) (Niola et al., 2012). In the developing mouse brain, inaclivation of Idl and Id3 (but neither gene alone) can reportedly trigger premature differentiation of NSCs (Lyden etaL 1999). Short hairpin RNA-mediated silencing of Idl and Id3 reportedly abrogates glioma stem cell properties in vitro and in orthotopic transplantation experiments (Anido et al., 2010). In mouse astrocytes deiicient in cyclin-dependent kinase inhibitor 2A { dkn2a ), expression of Id4 induces glioma stem cell markers associated with cyclin H activation and Notch signaling (Jeon et al., 2008). Id4 has also been reported to de-repress miR-9*-mediated suppression of SRY-box 2 (SOX2), increasing glioma stem cell potential and chemorcsistance (Jeon et al., 2011). As with somatic stem cells, cancer stem cells are anchored to a niche and derive supportive signals through cell-cell contacts with endothelial cells in blood vessels. The ability to adhere to the niche is a crucial feature of normal stem cells and cancer stem cells (Calabrcse C et al., 2007; Chen S et al., 2013; Lewallen M et al.. 2013; Fietzetal., 2011; Lathia JD et al., 2010; Park DM et al., 2009). Loss of Id proteins reportedly disrupts stem cell adhesion to endothelial cells in the niche in NSCs and in glioma stem cells (Niola et al., 2012; Niola et al., 2013). Id-mediated repression of bl 1LH transcription limits expression of RAS-related protein 1 (RAPL) GTPase-activating protein (RAP 1 GAP), a bHLH target gene that encodes an inhibitor of the RAP1 GTPase, which controls cell adhesion via integrin signaling (Boettner et al, 2009). When Id protein levels decrease, for example, at the time of neural differentiation or in the absence of Id proteins in Id-knockout m ice, de-repression of Rap l gap inhibits RAP l and drives stem cell detachment from the niche. Thus, whereas in NSCs the Id-bHLH axis dynam ical ly regulates cel l intrinsic cues that direct stem cell interaction with the niche, continuous blockade of bHLH activity by increased Id activity in glioma stem cells may lock adhesion signals in an aberrant "on " state, correlati ng with reports that RAP l GAP, Id2 and Id3 are prognostic markers in patients with high-grade gl ioma. In other studies high-grade glioma cel ls with high Id l expression manifested high self-renewal potential in vitro and tumorigenic capacity when injected orthotopical ly, whereas cel ls with low Id l expression had impaired in vitro self- renewal but more robust tumorigen icity (Barrett et al., 20 12). These latter stem cells may be a stem cel l type analogous to "transit-ampl ifying progenitors" in neurogen ic areas of the brain. These progenitors, wh ich express lower levels of Id proteins than NSCs (Nam et al .. 2009) are capable of lim ited sel f-renewal but have high prol iferative potential (Diaz-Flores et al., 2006), possibly associated with origination of certain brain tumor subtypes (Liu et al.. 20 1 1 ). An inverse correlation between Id l expression and tumor progression m ight explain the sl ightly better prognosis for patients with gl ioblastomas having a pro-neural gene expression signatu re and h igh Id l expression, compared patients marked by lo Id l , although both subgroups fare poorly. In certain tumor types, both stem-l ike cel ls and cel ls with features of committed progenitors may have capacity to propagate tumors efficiently. These findings also ind icate that targeting both cell populations wi l l be important to e ffectively treat glioblastoma (Barrett et al., 20 12).
Based on the foregoing reports, Id proteins have been postulated to play key roles as regulators of stem cell identity in both colorectal cancer and mal ignant glioma, essential for both self-renewal and tumor-initiating capacity of cancer stem cel ls. Again despite the mechanistic complexity and indefin ite pathways involved in cancer stem cel l deve lopment. anti-Id compounds and methods of the invention potently disable Id proteins at a critical foundation to disru pt stem cel l identity and impair stem cell tumor initiation. Anti-metastatic and anti-angiogenic effective compounds of the invention specifically target tumor stem cell viabi l ity, prol iferat ion capacity, tumor-initiation potential, and/or cel l fate determination- w ith the result of substantial ly decreasing populations of new tumor induction-competent stem cel ls present i n new or established tumors. In certain embod iments, the anti-Id compositions and methods of the invention will effectively decrease the number of cells bearing one or more selected stem cell markers in tumors (e.g., as observed through biopsy or necropsy of existing tumors) of treated subjects, by at least 5%, 1 0%, 25%, 30%, 50%. 75%. 90% or more compared to levels observed in placebo-treated subjects presenting with sim i lar pathology (e.g., an equivalent state of neoplastic or metastatic disease prior to treatment, normal ly attended by tumor growth, new tumor formation, and a high i ncidence of tumor- associated cancer stem cells). As with the determination of epithel ial cel ls and EPCs, wel l known assays, markers and labeling reagents will be routinely employed to demonstrate anti- stem cell efficacy of the compositions and methods herein . Those ski lled in the art wi l l appreciate that such assays are readi ly designed and implemented to identify and quantify cancer stem cells, for example based on detection of positive stem cel l markers, such as nestin and SSEA 1 , using conventional assay technologies such as cytometry, imm unobead capture, and immunocytochemistry. More discrete assays wil l determ ine d i fferences in tumor stem cel l viabil ity, prol iferation capacity, tumor-initiation potential, and/or cel l fate. speci fically correlated in anti-Id-treated subjects with reduction of cancer disease risk, accord ing to the teachings here and following reports of others above (al l of which are incorporated herein by reference for economy of description).
Effectiveness of anti-Id treatment methods of the invention directed against cancer and metastatic disease may be monitored in terms of cl in ical success by any of a variety of methods, for example by tumor imaging with x-rays or M l s (e.g., to determ ine i f tumors have decreased in size or number in treated patients). Effectiveness wil l often be determ ined by radiographic or M RI observation of a decrease in tumor size. Effective anti-Id compositions and methods of the invention for treating cancer wi l l routinely yield at least a 10%, 25%, 50%, 75%o, 90% or greater reduction of tumor size in treated patients, or average tumor size among a group of treated patients, compared to qual i fied, comparable control subjects.
Effectiveness of anti-Id treatment methods of the invention directed against cancer and metastatic disease may further be determ ined by measuring the number of circu lating tumor cel ls in blood samples between suitable test and control subjects. This may be accompl ished by any means applicable including, but not limited to immunomagnetic selection, flow cytometry, immunobead capture, fluorescence m icroscopy, cytomorphologic analysis, or cel l separation technology. Effective anti-Id compositions and methods of the invention for treati ng cancer will routinely yield at least a 1 0%, 25%, 50%, 75%, 90% or greater reduction of circulating tumor cells in blood samples of treated patients, or among a group of treated pa tients, compared to qualified, comparable control subjects.
Effectiveness of anti-Id treatment methods of the invention directed against cancer and metastatic disease may further may also be determ ined by detecting or measuring primary tumor cel l occurrence or number in secondary tissues or organs, including but not l im ited to bone, lymph nodes and lung. Effective anti-Id compositions and methods of the invention for treating cancer wi l l routinely yield at least a 1 0%, 25%, 50%. 75%, 90% or greater reduction in the occurrence or number of primary tumor cel ls metastasized to secondary tissues or organs among treated patients compared to qua l ified, comparable control subjects.
In certain aspects of the invention, anti-Id compositions and methods for prevention or treatment of cancer, including for inh ibiting or reducing metastasis, involve coordinate adm inistration of an effective amount of the anti-Id compound, along with a secondary treatment agent, treatment modality or treatment method. In certain exemplary embod iments, subjects are treated with the anti-Id compound simultaneously or sequential ly with a secondary treatment agent or method, selected from : a chemotherapeutic drug ( i.e., using a second anti-cancer or anti-metastatic drug, compound or chem ical agent), radiation, chemotherapy, surgery, or any combination of these agents/methods.
In certain "coordinate therapy" or "combinatorial treatment" embodiments, the invention employs the anti-Id com pound administered simultaneously (at the same time, optionally in a combined formulation) with a secondary drug, compound or chemical agent possessing combinatorial anti-cancer or anti-metastatic activity. Secondary chemotherapy drugs in this context are contemplated to broadly include agents classified as conventional chemotherapy drugs (for example taxanes); vascular disrupting agents ( V DAs); or HS P-90 inh ibitors. Within these and related embodiments, the anti-Id compound and the secondary drug or treatment wil l be "combinatorial ly effective", meaning biological activity (e.g., anticancer or anti-metastatic activity as defined herein), side effects, patient outcomes, or other positive therapeutic indicia wi ll be improved over results observed in relevant control subjects treated with the anti-Id compound alone, or secondary drug alone.
Anti-Id compounds and methods of the invention can be coordinately employed with any of a range of secondary anti-cancer drugs, agents or interventions, in combinatorial formulations or coordinate treatment protocols (with anti-Id therapy admin istered
concurrently, prior or subsequent to the secondary treatment agent or method). In exemplary coordinate treatments, an anti-Id compound such as AGX5 1 is adm inistered coordinately with a chemotherapeutic drug or therapy. Chemotherapeutic drugs and therapies for use within these aspects of the invention include anti-cancer and anti-hypcrprol iterative agents, agents that destroy or "reprogram'' cancer cells, agents that destroy blood vessels associated with neoplasms or hyperproli ferative conditions, and other classes of drugs harmful to neoplastic cel lu lar targets. In this regard, useful chemotherapeutics with in the invention include, but are not l im ited to:
( 1 ) tubu l in depolymerizing agents l ike taxoids such a pacl itaxel. docetaxel, BA Y 59- 8862 album in bound a V DA,
(2) DNA damaging agents and agents that inhibit DNA synthesis
(3) anti-metabolites
(4) anti-angiogenics agents and vascular disrupting agents (V DAs),
(5) antibodies,
(6) endocri ne therapy,
(7) imm uno-modu lators,
(8) histone deacetylase inhibitors,
(9) inhibitors of signal transduction,
( 1 0) inh ibitors of heat shock proteins,
( 1 1 ) reti noids such as all-trans rctinoic acid,
( 1 2) inh ibitors of growth factor receptors or the growth factors themselves,
( 1 3) anti-mitotic compounds,
( 14) anti-inflammatory agents such as COX inhibitors, and
( 1 5) cel l cycle regu lators, eg, check point regulators and telomerase inhibitors.
In other aspects, combinatorial formulations of the invention include an anti-Id compound as described in a combined formulation with one or more conventional chemotherapeutic drugs or other anti-metastatic compound or agent, optional ly includ ing a side-effect reducing agent as known in the art (which will depend on what combinatorial therapy is being employed, e.g.. chemotherapy versus radiation therapy, or both). Anti-Id compounds of the invention may be provided in the form of a pharmaceutical ly acceptable salt. These compounds can be routinely formulated for oral, topical, parenteral, transdermal or intravenous (iv) adm inistration. In certain embodiments, e.g., where multiple compounds are administered separately, multiple pharmaceutical compositions may be provided (each containing a different active agent. In other embodiments, a single formu lation is provided that comprises an anti-Id active agent, a secondary chemotherapeutic agent, and optional ly a side effect reducing agent.
Combinatorial efficacy observed for coordinate therapies can occur for a variety of reasons but in general is due to combined inhibition of two or more independent pathways. Individual pathways may provide "bypass" routes for targeted cel ls (e.g.. metastatic cancer cells), requiring that multiple pathways be targeted to prevent the escape. When anti- angiogenic stress i s presented, for example using known anti-angiogen ic drugs, cel ls at the periphery of tumors may "bypass" the disruption of normal tumor-associated angiogenesis
Figure imgf000038_0001
coopting local vessels. However this is a poor escape mechanism, often subjecting the tumors to hypoxic stress. This stress in turn may be al leviated by endogenous activity of heat shock factor 90 (Hsp90). Thus, in certain embodiments of the invention an Hsp90 inh ibitor is coord inately administered with an anti-Id compound to yield combinatorial lv effective anti-Id and anti-HSP90 activity, attended by enhanced cl inical results.
Anti-Id compounds and methods of the invention can be particularly e ffectively employed in combination with vascular disrupting agents (VDAs), often with the attendant benefit of al lowing for a lowering of VDA effective dosage and/or reduction of V DA- associated adverse side effects. VDAs arc mostly chemical agents that d isrupt the cytoskeletal vascular network causing cel l-shape and permeabi l ity changes resulting in vascular resistance, vasoconstriction, increased vascular permeabi l ity, platelet thrombi and vascular shutdown . Vascular disruption represents a val idated therapeutic strategy to deprive tumors (and, in the case of eye disease, pathologic neovascular lesions) of blood supply. I n solid tumors, vascular disrupting agents rapidly disrupt the vascu lature with in the tumor, reduce blood flow, and deprive the tumor of oxygen and nutrients, resu lting in tumor cell death. This disruption of the newly formed blood vessels contrasts with the action of anti- angiogenic therapies, which are designed to prevent new blood vessel formation. Often, because VDAs resl rict tumor blood supply and drive them to hypoxic stress (ameliorated by heat shock protein (HSPs)), these combinatorial methods and formu lations wi l l often include anti-HSP 90 agents/methods to further enhance the coordinate therapeutic results. Vascular disrupting agents target the tumor-associated, fragi le and relatively newly- constructed vasculature in tumors (Tozer et al., 2005, Mita et al., 20 1 3). The prototype VDAs are combrestatins, natural antim itotic agents isolated from the root bark of the South A frican tree. Com oretum caffrum (C irca and Mann, 2003; Tozer et al., 2001 ). The most potent of these compounds is combretastatin A-4 (CA4), an antitumor drug. CA4 whose prodrug is CA4-phosphate (CA4P) binds to tubul in in endothelial cells at the same site as colchicine leading to strong inhibition of tubulin polymerization. CA4P causes shape changes, cytotoxic ity, changes in cel l permeability and apoptosis of prol i ferating endothel ial cells, but not of qu iescent cel ls. The cytoskeleton of mature cells is not sensitive to CA4P as opposed to newly formed cel ls, w hich are particularly sensitive. There is a preferential sensitivity of endothel ial cells in tumor vessels to CA4P, which unl ike those in normal vessels, become thrombogenic, resulting in hemorrhagic necrosis of tumors.
CA4P is currently being evaluated in multiple clinical trials as a treatment for various sol id tumors as wel l as a treatment for age-related macular degeneration (AM D), a vision l im iting condition (Nanbu et al., 2003 : Eichler et al., 2006). AM D features an overgrowth of blood vessels as part of the underlying pathology (Campochiaro and 1 lackctt. 2003). More recently, anti-angiogenic agents such as bevacizumab (Avastin 1 M), that lim it tumor growth by preventing the formation of new blood vessels, have been approved for some cancer indications and are widely used to treat sol id tumors. An anti-angiogenic agent
mechanistically related to bevacizumab, ranibizumab (Lucentis I M), is used to treat AMD.
VDAs and anti-angiogenic agents both work in di fferent, yet complementary ways. A nti-angiogcnesis drugs attempt to keep new blood vessels from forming. U n l ike V DA s. anti-angiogen ic agents do not act on blood vessels that already feed existing tumors. V DAs degrade blood vessels within the tumor and cause widespread cel l death in central parts of the tumor that historically have been resistant to conventional treatments, such as cytotoxic chemotherapy, rad iation, and biologies. A lthough V DAs thus have demonstrated cl inical efficacy (Hasani and Leighl, 20 1 I ; H innen and Eskens, 2007; Mc Keage and Baguley, 20 1 0), preliminary evidence for the induction of EPCs after V DA treatment has been reported recently in Phase 1 clinical trials using the VDAs ZD6 126, AVE8062 or CA4P (Beerepoot et al., 2006; Farace et al., 2007). A robust elevation in EPC levels has also been observed within hours of treatment with microtubule-inhibiting cytotoxic-l ike vascular disrupting agents (VDAs) in mice (Shaked et a!., 2006). This induction of EPCs likely dim inishes effectiveness of the VDA treatment (Daenen et al., 201 0).
The anti-Id compositions and methods of the invention are highly complementary to VDA treatment for reducing cancer incidence, metastasis, disease progression and tumor growth/invasiveness. As described herein, anti-Id compounds of the invention, such as ANGX5 1 effectively block Id-mediated EPC responses to VDA treatments. Thus, in combinatorial formulations and methods, ANGX5 1 provides for reduction of VDA dosing or treatment duration with comparable anti-angiogenic effects, and less angiogenic rebound involving increase of EPCs responsive to tumor vascular destruction by VDAs. Treatment of tumor-bearing m ice with VDAs leads to an acute mobi lization of EPCs, w hich home to the viable tumor rim that characteristical ly remains after VDA therapy. Because Id l and Id3 promote EPC prod uction, blockade of this EPC rebound spike by anti-Id compounds of the invention wi ll directly reduce or prevent further tumor-associated neovascularization, blocking tumor blood flow and preventing tumor growth.
Other coordinate treatment compositions and methods herein targets cel lular division as a secondary pathway for cancer and metastasis intervention. Taxancs inhibit cancer cel l growth by antagon izing the machinery in cells that distribute chromosomes between mother and daughter cel ls. Taxane resistance has been reported to involve activation of the Notch signaling pathway, which in turn activates ld l . Combining an anti-I d 1 compound with a taxane (e.g., laxol or paclitaxel) coordinately prevents this bypass. These combinatorial methods wi l l powerful ly reduce or prevent prol iferation w ithin tumors and other
hyperproliferative cell populations.
In exemplary embod iments of the invention, a coordinate diagnosis and management protocol is contem plated to treat or prevent breast or ovarian cancer. Women with fami ly histories of breast cancer or ovarian cancer may be selected for treatment, for example using a BrCA l genetic test to establ ish elevated risk of breast or ovarian cancer. H igh risk subjects wil l be provided prophylactic anti-Id treatment (e.g., using (-)-AGX5 1 ) for several months, up to 1 -2 years or longer, post-surgery to prevent recurrence of the disease. Periodical ly, system ic Id levels are determ ined during this time. I f Id is detected at a concentration above trace or non-measiireable, the anti-id treatment is continued or increased, optional ly supplemented by chemotherapy. Other patients amenable to treatment according to the invention wi ll be identified by routine mammography of human females positive for a breast tumor, fol lowed by tumor excision and in some cases radiation. When no cancer cells are identified, e.g., in the lymph nodes, anti-Id compound is administered chronically daily for up to a year or more to provide protection against recurrence of the disease. Recurrence is prevented through one or more of the anti-tumor, ant i-metastasis, pro-apoptosis and pro-cell cycle control effects of the drug. Periodical ly, systemic Id levels, optionally combined with other markers (e.g., metastatic, entothelial cell, EF'C and/or cancer stem cell markers), are determ ined during th is time. I f Id is detected at a concentration above trace or non-measureable, the anti-Id treatment is continued or increased.
Additional coord inate diagnostic and management protocols are provided to treat breast cancer or prevent metastatic progression of an exist ing breast tumor. Patients are identi fied by routine mammography of human females as positive for a breast tumor, fol lowed by tumor excision and in some cases radiation. Cancer cel ls are identified in the lymph nodes of the subject. The subject is adm inistered a course of conventional pacl itaxel treatment (e.g., every three weeks for 12 weeks). Over this period, anti-Id compound is adm inistered chronically daily day to maxim ize the effect of the pac l itaxel. The anti-Id treatment is continued for an extended period (e.g., 9- 1 2 months) post pac l itaxel treatment to provide additional protection against recurrence of the disease. Periodical ly, system ic Id levels and optional ly other markers are determ ined during this time. I f Id is detected at a concentration above trace or non-measureable, the anti-Id treatment is continued or increased.
In other coordinate diagnosis and management protocols, patients, previously identified by routine mammography as positive for a breast tumor and treated by tumor excision (no cancer found in the lymph nodes), are exam ined two years post-surgery f r tumors (e.g., by computerized tomography (CT) scanning followed by Positron Emission Tomography (PET scanning). When metastasized tumors are found, and/or a lymph node test is positive for cancer cel ls, the subject wi l l be administered conventional taxane therapy (e.g., paclitaxel every three weeks for 1 2 weeks), fol lowed by anti-Id compound dai ly for 9- 1 2 months to prevent rem ission of the disease, coupled with monitoring of Id levels and optionally other markers as described.
In further exemplary embod iments, subjects presenting with Stage I I I (metastatic ) breast cancer are selected for cancer treatment and management. Subjects thus identified are treated using an aggressive, combinatorial treatment regimen employing high doses of radiation and multiple doses of paclitaxel. After treatment, histopathology and/or bioscans [eg, computerized tomography (CT), positron em ission tomography (PET) and/or magnetic resonance imaging] are used to identify patients presenting with no detectable cancer after the first l ine treatment above. These subjects are then treated for a prolonged period (e.g., months, six-nine months, 1 -2 years or longer) with an anti-Id compound, in a prophylactic treatment regimen to block re-seeding of any remaining cancer cel ls to a d istant organ (e.g.. brain or liver). During this management phase, measurements of Id in the blood of the patient are performed periodical ly. The measurements continue after treatment. After another management period of several months to one year or more. Id blood levels may be observed to increase, indicating an increase in metastatic potential or even the presence of add itional disease. Treatment with the anti-Id compound would then be combined with another course of treatment with a non-taxane cytotoxic such as epirubicin. Fol lowing this treatment, the anti-Id treatment would continue for a year post-treatment and longer i f the Id blood levels fai l to decrease to a value consistent with an acceptable (basel ine or low-risk) for metastatic potential or the presence of microtumors.
Other exemplary embodiments wil l employ an array of combinatorial therapies. Patients identified with recurring or metastatic breast cancer may be treated fi rst with an aggressive course of taxane therapy (e.g., 5 single doses treatment of taxane (e.g., pacl itaxel. docetaxel or album in-bound paclitaxel), every three weeks for 12 weeks), to wh ich may be added a a vascular disrupting agent (VDA), such as combretastatin-A4 phosphate. In combination with these therapies. anti-Id compound is also administered chronically dai ly to maximize the effect of taxane/VDA treatment. The anti-Id treatment is continued for 9- 1 2 months or longer to prevent recurrence or metastatic progression of the d isease, with Id and other marker monitoring as described. A lternatively, these patients may be adm inistered taxane therapy in combination with bimonthly or month ly treatment with an anli-VEGF agent (e.g.. bevacizumab), or anti-VEGF receptor antagonist (e.g., sunitinib, sorafenib) rein forced by extended, prophylactic admin istration of anti-Id compound of the invention.
In other em bodiments of the invention, coordinate diagnosis and management focuses on patients testing positive for the presence of the H ER2/neu receptor. These subjects may be administered conventional taxane therapy along with bimonth ly or monthly treatment with trastuzumab (Herceptin), supplemented or followed by daily anti-Id compound treatment as described. The anti-ld treatment is continued for 6- 12 months post the taxane treatment to provide additional protection against recurrence of the disease.
Clinical management methods of the invention can also be adapted to treat more specific cancers, for example estrogen and progesterone receptor-negative breast tumors. Exemplary protocols here may employ tumor excision and in some cases radiation fol lowed by chemotherapy ( e.g. with pacl itaxel and doxorubicin every three weeks for 1 2 weeks), accompanied or fo llowed by daily anti-Id therapy (continuing for an extended post- chemotherapy period for prevention of d isease recurrence).
In other refractory cases, cancer patients wi l l be treated coordinately with taxane chemotherapy (e.g., with paclitaxel, docetaxel or albumin-bound paclitaxel) supplemented with concurrent or sequential cisplatin treatment, and anti-Id therapy wi l l fol low the taxane therapy and extend 6- 12 months or more to protect against recurrence of the disease.
Comparable coordinate diagnosis and management protocols are provided for treating other forms of cancer. For example prostate cancer patients wi l l be se lected based on PSA screening and/or biopsy to produce a Glecson score reflecting the stage and metastatic risk of the cancer. Subjects at elevated risk are treated with radiation and chemotherapy, coupled with or followed by extended anti-Id treatment and mon itoring as above. Th is treatment may be coordinated with conventional anti-androgen therapy.
Coordinate diagnosis and management protocols for treating melanoma in situ may include topical taxane treatment coupled with concurrent or subsequent, therapeutic or prophylactic anti-l d treatment according to the invention.
Coordinate diagnosis and management protocols for treating Kaposi sarcoma may include intralesional or topical taxane treatment coupled with concurrent or subsequent, therapeutic or prophylactic anti-Id treatment according to the invention. This and other coordinate treatment methods may optionally include concurrent or sequential interferon- alpha treatment.
Yet additional coord inate diagnosis and management methods are contemplated and routinely implemented according to the teachings herein, inc luding for al l types and stages of cancer and other proliferative disorders, including but not limited to bladder cancer, colon cancer, pancreatic cancer, lung cancer, brain cancer, esophageal cancer and leukemias.
In all coord inate treatment methods of the invention, the entire armamentarium of known effective ch emotherapeutic agents may be combined w ith anti-Id therapy, l ikewise the ful l range of vascular disrupting agents (VDAs). and HSP 90 inhibitors can be combined with anti-Id drugs such as AGX5 1 in combinatorial formulations, and any configuration of coordinate treatment regimens, optional ly with other interventions such as rad iation and surgery.
Fol lowing either surgery to excise a tumor and/or radiation treatment to shrink or destroy a tumor, metastasis from remaining tumor cells stil l is a risk to patients. In this regard, follow-up i:reatment with an anti-Id compound wi l l decrease this risk and, in th is way. increase the chance of patient survival. In addition, in brain cancer, radiation ki lls cells that express low levels of Id protein leaving behind cancer stem-l ike cel ls which can refuel tumor growth. Combinin g an anti-Id 1 agent with radiation therapy is pred icted to increase survival rates in this aggressive and lethal human cancer.
Other compositions and methods of the invention target distinct cel lular prol i ferative disorders characterized by aberrant blood vessel growth, or "pathogen ic angiogenesis". F.xamples of these disease targets include ocular disease mediated by aberrant vascular growth (e.g., macu lar degeneration), and tumor-associated angiogenesis. Anti-Id compounds of the invention function also as "anti-angiogenic" agents, as described below, making them useful to treat or prevent pathogenic angiogenesis, including quite powerful ly tumor- associated angiogenesis to mediate a multi-pronged assault on tumor development (i.e., both anti-metastatic and anti-angiogenic). Many cancer types rely on Id protein expression in both the primary and metastatic tumor cel l, as well as to support tumor-associated angiogenesis. Anti-Id compositions and methods of the invention target both tumors and their supporting blood vessels simultaneously. In these embodiments, Anti-Id compositions and methods of the invention mediate both anti-tumor ( including anti-metastatic) and anti-angiogenic effects. Other anti-angiogenic agents alone have marginal efficacy for treating cancer, often requ iring coordinate use of cytotoxic chemotherapies to achieve a therapeutic response.
Compositions and Methods for Treating Disease Involving Pathogenic Angiogenesis
Additional embodiments of the invention employ anti-Id compounds such as AGX5 1 to effectively treat or prevent any pathogenic angiogenic or ncovascu lar cond ition or disease. Exemplary pathologic neovascularization phenomena are associated with the ocular d isease, age related macular degeneration (AM D).
AMD is the most common cause of irreversible vision loss in the elderly (Jager et al., 2008). This condition is mediated in large part by angiogenic changes typified by a neovascular lesion complex which can be differentiated by ocular angiography into choroidal neovascular membrane (CNV) and non-CNV components. AMD is characterized by a spectrum of clinical and pathologic findings, including drusen formation, disruption of the RPE, CNV, disciform scar formation and sub-retinal fibrosis. The sustaining event for AM D is believed to be chronic ischem ia-reperfusion (I-R) inj ury of ocular tissue damage caused when blood supply returns to the tissue after a period of ischemia or lack of oxygen. The absence of oxygen and nutrients from blood during the ischem ic period creates a cond ition in which the restoration of circulation results in inflammation and oxidative damage through the induction of oxidative stress rather than restoration of normal function. Tissue damage caused by cumulat ive insu lt by excess sunlight, pol lution, dust and d irt is bel ieved to be the in itiating event for the ischemia that precedes the l-R.
A has two forms: non-exudative (dry) and exudative (neovascular or wet).
Currently, the prev alence of wet AMD in the US is estimated as 1 .2- 1 .5 m i l l ion cases with approximately 200,000 new cases occurring each year. Because A M D is a disease of aging, the number of people with AM D is expected to substantial ly increase as people l ive longer and the baby-boom generation grows older. By 2030, an estimated 500.000 cases of A M D wi l l be diagnosed each year (Seddon et al., 2004).
Recently approved anti-VEGF treatments represent a major advance in treating AM D (Rosenfeld et al.. 2006; Brown et al., 2009. Useful drugs include ran ibizumab, an anti-V EG F fab fragment for injection, reported to stabi l ize or mediate reversal of vision loss in 95% and 40% of patients, respectively. A majority of patients treated with th is drug d id not experience vision gain, and patients who had positive responses to treatment did not regain abi l ity to drive or read normal ly. Results with bevacizumab, an anti-VEGF monoclonal antibody, for injection, appear comparable (Rosenfeld et al., 2005).
Anti-VEGF therapies appear to exert most of their beneficial effect via an anti- permeability action resulting in resolution of intra and sub-retinal edema, as the actual C N V lesion does not markedly involute (Eichler et al., 2006). Flovvever, exudative AM D- related vision loss is not due to solely choroid neovascularization (CNV) induced sub-retinal and intra-retinal edema. In addition, the long term safety of pan-VEGF inhibition in the eye has yet to be established. VEGF. produced by a number of ce l ls in the neurosensory retina, is neuroprotective in nature and chronic inhibition could be detrimental to neuronal health (Greenberg et al.. 2005). VEGF is also constitutively expressed by RPE cells and is a survival factor for quiescent choriocapil laris endothel ium (Witmer et al.. 2003), suggesting that long term VEGF inhibition could be detrimental to this vital structure and the cel ls wh ich rely upon it for metabolic support. With respect to the safety of chronic anti-VEG F treatment, a recent re-evaluation of patients treated for seven years with anti-VEGF treatment reported that macu lar atrophy was detected by fluorescein angiography in 98% of eyes, and that the area of atrophy correlated significantly with poor visual outcome ( Rofagha et al. 20 1 3). Further em phasizing the need for better treatment tools against wet A M D, the follow- up evaluations of the patients after seven years of ant-VEGF treatment found that one th ird of the patients had poor outcomes with vision on the Snel len chart test decl in ing by 1 5 letters or more (Rofagha et al, 201 3).
The three anti-VEGF agents most commonly used to treat ocular conditions are ranibizumab for i njection), afl ibercept for injection, and bevacizumab for injection. Besides wet A MD, these agents are also used also used to treat macular edema that results from central retinal vein occlusion (CRVO) or branch retinal vein occlusion ( B VO) as wel l as for the treatment of macular edema due to diabetic retinopathy. Besides anti-V EG F agents, a vascu lar disrupting agent (V DA), CA-4 phosphate, is also being evaluated in multiple cl in ical trials as a prospective treatment for AM D (Eichler et al., 2006).
Two surgical procedures are also occasional ly used to remove the ocu lar lesions that arc the essential pathology of A MD. particularly wet AMD: Laser photocoagulation ( and photodynam ic therapy (Cook et al., 2008).
The exact etiology and pathogenesis of exudative AM D is sti l l not wel l understood but is thought to consist of vascular and extravascular components orchestrated by mu ltiple factors (Tezel et al ., 2004; Ambati et al., 2003). However, AMD features an overgrowth o f blood vessels as p .rt of the underlying pathology (Campchiaro et al., 2003 ). The vascu lar component of exudative AM D is comprised of vascular endothel ial cells, endothelial cel l precursors and per cytes. VEGF appears to be an important mediator in the pathogenesis of vascular component. However, the growth of choroidal blood vessels, involves a coordinated interaction among many other agents, not just VEGF. Targeting additional elements in the vascular process oilers the opportunity to modulate the entire process. Tissue damage can result from either the vascular or extravascular component of the disease process. The extravascular component often appears to be the largest component volumetrical ly, and appears, by histopathology, to be the source of the angiogenic stimuli. This extravascular component is composed primari ly of inflammatory cells and less so fibroblasts and gl ial or RPE. Macrophages and the complement system are now known to play an important rol e in affecting CNV and propagating the pathogenesis of exudative A MD (Bushin i et al, 20 1 1 ; Gold et al., 2006; Klein et al., 2005 ; Hageman et al.. 2005 ; Tsutsum i et al .. 2003 ; Espinosa- Heidmann et al., 2005 ; Oh et al., 1 999; Grossniklaus et al., 2002; Forrester et al.. 2003). I n addition, several biochem ical changes in the ocular macrophages with aging are apparent, i .e., the cytokine I L- 1 2 is lowered and the cytokine I L- 1 0 is increased (.lager et al., 2007) wh ich leads to an overal l less robust m itigation of trauma-induced angiogenesis [1 L- I 2 is strongly anti-angiogenic via its upregulation of I FN-γ (Dace et al., 2008) and I L- 1 0 is pro- angiogenic (Kelly et al., 2007)]. These changes provide a compel l ing explanation for the pronounced age dependency of AMD.
In l ight of I he foregoing d iscussion, an urgent unmet medical need exists for novel therapeutic modal ities to treat exudative AM D. Given the emerging l im itations of anti- VEGF therapy, the se novel therapeutics should target non-VEGF-dependent steps in the development of CNV that ultimately lead to exudative A M D. Targeting Id represents such a target because of the fundamental role the Ids, particularly Id l and Id3, in mediating the neovascularization process underlying the lesion associated with AM D.
In one aspect, the invention provides methods for inhibiting pathologic ocular neovascularization comprising adm in istering to a subject presenting with a neoplasm an e ffective amount of an anti-Id compound in a monotherapy protocol employing a single drug or method of treatment. In related embodiments, treatment methods of the invention inc ludes adm inistering to the subject an effective amount of the anti-Id compound along with a secondary treatment agent, treatment modality or treatment method (for example by treating the subject with the anti-Id compound simultaneously or sequential ly with a secondary treatment modality or agent selected from for example: An anti-VEGF agent, a V DA, interferon-γ (Naldini et al., 2005), a potent anti-angiogenic cytokine, or an agent that ind uces interferon-γ for example IL- 1 2 (Del Vecchio et al., 2007 and Kleinman et al., 2008).
In certain "coordinate therapy" or "combinatorial treatment" embod iments, the invention may employ the anti-Id compound administered simultaneously (for example adm inistered at the same time or in a combined formulation) with a secondary drug, compound or chemical agent possessing combinatorial anti-growth activity). I n exemplary embodiments, secondary chemotherapy drugs are chosen, for example an anti-VEGF agent or, a VDA, interferon-γ. Within these and related embodiments, the anti-Id compound and the secondary drug wi ll be "combinatorially effective", meaning biological activity, eg, anti - growth or anti-angiogenesis activity as defined herein, side effects, patient outcomes, or other positive therapeutic indicia wi ll be improved over results observed in relevant control subjects treated with the anti-Id compound or secondary drug alone.
All known treatment agents and methods for treating AMD and other pathogenic neovascular or angiogenic conditions wil l be useful in certain com binatorial formulations and coordinate treatment methods employing anti-Id compounds of the invention, such as AGX5 1 . In certain embodiments, anti-Id compounds wi l l be employed in conj uction with an anti-VEGF agent, a V DA, and/or , interferon-γ, with an attendant benefit of lowering dosage and/or side effects of these complementary treatment agents whi le retaining coordinate clinical benefit from their conjunctive use. For example, anti-Id therapy attended by coordinate use of an anti-VEGF agent and/or a VDA wi ll employ lower than conventional dosages of the anti-VEGF agent and/or a VDA drug for the indicated treatment, while combinatorial efficacy wi l l be greater than ful l dosage conventional anti-V EGF agent and/or a V DA, and side e ffects such as long term vision loss wil l be reduced.
I n certain embodiments, anti-angiogenic compositions and methods of the invention are effective to reduce pathologic ocular neovascularization in a mammal ian subject. These methods may empl oy monotherapy or coordinate therapy, as above. The methods (and related compounds and compositions) of the invention are "anti-angiogen ic effective", for example to reduce incidence, size, or number of vascular lesions in an ocular tissue of a subject presenting with AMD. I n certain embodiments, "reducing neovascularization" wi l l correspond to an observed reduction in a histopathologic or ocular angiography index o f AMD lesion size, for example a reduced occurrence, size, number or distribution of lesions or "foci" of lesions observed at a secondary ocular site. In other aspects, anti-angiogenic efficacy w i l l be determ ined by a positive change in one or more patient therapeutic indices correlating with effective prevention and/or treatment of AM Ds, e.g.. by an increase in a time period of disease free or disease stable conditions for subjects receiving the anti-Id compound compared to suitable control subjects not receiving the anti-Id compound.
Anti-AMD lesion efficacy, i.e., efficacy diminishing or stabi lizing growth of the neovascular lesion complex, of the compounds, compositions and methods of the invention wil l routinely yield substantial therapeutic benefits and improved treatment outcomes in patients treated for an ocular condition (or any other pathogenic condition) w ith harmful angiogenesis as pa rt of its underlying pathology. In exemplary embodiments, patients treated with the anti-ld methods and compositions of the invention wi ll exhibit improved treatment outcomes with no increase or an observed decrease in adverse side effects. I l lustrative of these benefits, methods of the invention will yield at least a 20% increase in one or more positive clinical therapeutic indices for example a beneficial change in AMD lesion index (eg. a reduction in occurrence, size, number or distribution of the lesion or "foci'* of the primary lesion observed at a secondary ocular site. In il lustrative embodiments, anti-AM D lesion efficacy of the anti-Id compounds and methods wi l l be demonstrable indirectly by at least a 20% increase in a disease-free or d isease stable condition for Id-treated patients compared to survival determined in suitable control patients (not treated with the anti-Id compound). In other embodiments. anti-Id compounds, formulations and methods of the invention will resu lt in even greater anti-AM D clinical benefit, for example yielding a 20- 50% increase in a positive therapeutic index, 50-90% increase, up to a 75%- 1 00% increase, including total reir ission of observed primary AMD lesion enduri ng for 6 months to a year. 1 -2 years, 2-5 years, 5 years or greater, including 1 0 year and longer rem ission. In exemplary embodiments, the anti-Id methods and compositions of the invention wi l l be anti-AM D effective to yield at least a 20% decrease in lesion size, a 20%-50%, a 50%-75%. up to a 90% or greater decrease in lesion size, e.g., as demonstrated by comparative h istopathology, ocu lar angiography, optical coherence tomography (OCT) or another ocu lar imaging technique in anti-Id-treated versus non-treated or placebo-treated subjects.
Respective of al l embodiments presented here, anti-AM D efficacy w i l l typical ly correlate with no increase or even a decrease in observed symptoms of AM D, e.g., loss of visual acuity between anti-Id treated patients and positive control -treated subjects. I n exemplary embodi ments, anti-Id-treated subjects includ ing subjects treated with anti-Id compound monotherapy, and subjects treated with combinatorial methods such as anti-ld plus anti-VEGF therapy wi ll exhibit no increase in Snellen chart score and wi l l often exhibit at least a 20% increase, a 20-50% increase, up to a 50-90% or greater increase in Snel len chart score compared to positive control subjects treated with conventional (e.g., anti-VEGF) therapy. Diagnostic Compositions and Methods, and Related Clinical [Management Tools
In certain embodiments, cancer patients and other subjects identified for treatment according to the compositions and methods of the invention wi ll be evaluated and selected for refined clinical management using novel diagnostic and managed tools discovered here.
In one embodiment, subjects are selected for treatment with anti-Id compositions and methods using novel Id diagnostic methods and kits provided here. Certain of these methods and materials prov ide for detection and tracking of elevated Id levels (e.g.. ld l measured in blood or a biopsied tumor sample), wh ich wi l l correlate with a decision to initiated, continue, lower or increase anti-Id treatment. In certain embodiments, these diagnostic methods may include diagnosing the subject for the presence of other disease indices (e.g., biochem ical or h istologic markers of cancer, or of metastasis, or of an angiogenic pathology) to yield coordinate diagnostic values to enhance disease assessment and management.
H igh ld l levels lead to reversion of EMT to an epithel ial phenotype, wh ich is requ i red for colonization of metastatic sites. Treatments that m itigate ld l and Id3 ( i.e., impair or inhibit their expression, activity or function) are demonstrated here as potent tools for e ffectuating reduct ion of Id levels and attendant blockade of metastatic pathways and cel lular activities, along with other disease impacts in cancer patients. Dosage, duration of treatment and efficacy of the novel anti-Id compounds employed within these methods wi l l best be determined rcflexively by monitoring changing Id levels (which in these methods w i l l often respond directly to changes in anti-Id treatment). Thus, the novel treatment methods of the invention create a need for corresponding diagnostic and reflexive (cl inical managerial) monitoring of endogenous Id levels, which can be focused on any relevant test sample, such as cel ls (e.g., tumor cel ls, EPCs, cancer stem cells), tissues or organs (e.g., neoplasms, metastases, exploratory biopsy spec imens, lymph nodes) or physiological flu ids (e.g., blood, CNS fluid, lymphatic fluid).
Selection o f subjects amenable to treatment using the anti-Id compounds of the invention is useful ly guided by detection of elevated levels of ld l and/or Id3 in a biological sample (e.g., blood, urine or saliva) taken from a patient at risk of cancer, particularly of metastatic disease. Employment of assays to detect Id levels in th is manner in adj unct application with the invention yields observations of a bio-environment in certain patients with elevated Id levels, ind icating a high metastatic potential and thus a critical need for effective treatment using the anti-Id compounds and methods of the invention. Dosage and Formulation.
Generally, treatment and prophylaxis methods of the invention em ploy an "effective amount" of an anti-Id compound or composition as described. Th is may refer to an amount or dosage of AGX 5 1 or another anti-Id compound that is effective to detectably, significantly reduce a level or concentration of a targeted Id protein (e.g., Id l or Id3) in a cel l, tissue, neoplasm, or subject. Such demonstration of efficacy is readily using standard Id protein assays, for example using labeled id-specific antibodies or other quantitat ive Id detection reagents. In other embodiments, effective amounts or dosages of an anti-Id compound of the invention will be demonstrated as an amount or dosage effective to measurably inhibit or Id protein binding to a cognate binding partner (for example, Id dimerization to a bHLl I protei n, such as E-47. A lternate measures or assays to determ ine what is an effective amount or dosage of an anti-Id compound in this context, include effective amounts and dosages of the compound that mediate a reduction in any detectable and/or quanti fiable Id activity or correlated biological index (e.g., a histopathological or clinical index of metastasis or tumor- associated angiogenesis), whether direct or indirect (as long as the activity is subject to modulation by reduction of Id activity or Id levels. Anti-Id compositions and methods in th is context may thus be alternatively demonstrated as '"effecti ve" through a demonstrated increase in apoptosis or cellular differentiation between test and control samples, by a decrease in cellu lar prol iferation, decrease in cel l m igration, decrease i n secondary site colonization by pri mary tumor cel ls, decrease in tumor associated ang iogenesis, decrease in EMT or MET progression, etc.
For conven ience, compositions of the invention wi l l often be referred to as comprising an "anti-cellular prol iferative effective amount" or unit dosage of an anti-Id compound, for example AGX5 1 . A lternatively, compositions wi l l contain an "anti-metastatic dosage or amount" of the active anti-Id compound. In other references, the active compositions may comprise an "anti-angiogenic effective amount" or dosage of the anti-Id drug.
Anti-Id compounds of the invention may be formulated with one or more
pharmaceutically acceptable carriers, excipients, vehicles, emulsifiers. stabi l izers, preservatives, buffers, and/or other additives that may enhance stabil ity, del ivery, absorption, half-life, efficacy, pharmacokinetics, and/or pharmacodynamics, reduce adverse side effects, or provide other advantages for pharmaceutical use. Suitable effective unit dosage amounts of the active anti-Id compounds for adm inistration to mammal ian subjects, including humans, may range from 1 0 to 1 500 mg, 20 to 1 000 mg, 25 to 750 mg. 50 to 500 mg, or 1 50 to 500 mg. In certain em bodiments, the anti-Id effective dosage may be selected within narrower ranges of, for example, 1 0 to 25 mg, 30-50 mg, 75 to 1 00 mg, 1 00 to 250 mg, or 250 to 500 mg. These and other effective unit dosage amounts may be adm inistered in a single dose, or in the form of mul tiple dai ly, weekly or monthly doses, for example in a dosing regimen comprising from I to 5. or 2-3, doses administered per day. per week, or per month. In one exemplary embodiment, dosages of 1 0 to 25 mg, 30-50 mg, 75 to 1 00 mg, 1 00 to 250 mg, or 250 to 500 mg. are adm inistered one. two, three, four, or five times per day. I n more detai led embod iments, dosages of 50-75 mg, 1 00-200 mg. 250-400 mg. or 400-600 mg are adm inistered once or twice dai ly. In alternate embodiments, dosages are calculated based on body weight, and may be adm inistered, for example, in amounts from about 0.5mg/kg to about l OOmg/kg per day, l mg/kg to about 75mg/kg per day, I mg/kg to about 50mg/kg per day, 2mg/kg to about 50mg/kg per day, 2mg/kg to about 30mg/kg per day or 3mg/kg to about 30mg/kg per day.
The amount, tim ing and mode of delivery of compositions of the invention comprising an anti -Id effective amount of a compound of Formula I and/or I I I wi l l be routinely adj usted on an ind ividual basis, depending on such factors as weight, age. gender, and condition of the individual, the acuteness of the cel lular prol i ferative disorder and/or related symptoms, whether the adm inistration is prophylactic or therapeutic, and on the basis of other factors known to effect drug del ivery, absorption, pharmacokinet ics, inc luding hal f- l ife, and efficacy.
An effective dose or multi-dose treatment regimen for the instant anti-Id formulations wil l ordinari ly be selected to approximate a m inimal dosing regimen that is necessary and sufficient to substantial ly prevent or al leviate a targeted cel lular prol i ferati ve d isease (e.g., cancer, metastatic cancer, tumor-associated angiogenesis) in the subject, and/or to substantially prevent or alleviate one or more symptoms associated with the cel lular prol i ferative d isease in the subject. A dosage and administration protocol w i l l often include repeated dosing therapy over a course of several days or even one or more weeks or years.
An effective treatment regime may also involve prophylactic dosage adm inistered on a day or multi-dose per day basis lasting over the course of days, weeks, months or even years. By way of illustration, the anti-Id active agent can be admixed with conventional pharmaceutical ly acceptable carriers and excipients (ie, vehicles) and used in the form of aqueous solutions, tablets, capsules, elixirs, suspensions, syrups, wafers, and the l ike. Such pharmaceutical compositions contain, in certain embod iments, from about 0. 1 to about 90% by weight of the active compound, and more general ly from about 1 to about 30% by weight of the active compound. The pharmaceutical compositions may contain common carriers and excipients, such as corn starch or gelatin, lactose, dextrose, sucrose, microcrystal l ine cellulose, kaolin, mannitol, di-calci um phosphate, sodium ch loride, and algin ic acid.
Disintegrators commonly used in the form ulations of this invention include croscarmcl lose, m icrocrystal l ine ce l lulose, corn starch, sodium starch glycolate and alginic acid.
A l iquid composition wi l l general ly consist of a suspension or solution of the compound or pharmaceutically acceptable salt in a suitable liquid carrier(s). for example, ethanol, glycerine, sorbitol, non-aqueous solvent such as polyethylene glycol, oi ls or water, w ith a suspending agent, preservative, surfactant, wetting agent, flavoring or coloring agent. Alternatively, a l iq .lid formu lation can be prepared from a reconstitutablc powder. For example, a powder containing active compound, suspending agent, sucrose and a sweetener can be reconstituted with water to form a suspension and a syrup can be prepared from a powder containing active ingredient, sucrose and a sweetener.
A composition in the form of a tablet can be prepared using any suitable
pharmaceutical carrier(s) routinely used for preparing sol id compositions. Examples of such carriers include magnesium stearate, starch, lactose, sucrose, microcrystal l i ne cel lulose and binders, for example, polyvinylpyrrolidone. The tablet can also be provided with a color fi lm coating, or color included as part of the carrier(s). In addition, active compound can be formulated in a control led release dosage form as a tablet comprising a hydrophi lic or hydrophobic matrix.
A compositi on in the form of a capsule can be prepared using routi ne encapsulation procedures, for example, by incorporation of active compound and excipients into a hard gelatin capsule. Alternatively, a semi-solid matrix of active compound and high molecu lar weight polyethylene glycol can be prepared and filled into a hard gelatin capsule, or a solution of active compound in polyethylene glycol or a suspension in edible oil, eg, liquid paraffin or fractionated coconut oil, can be prepared and filled into a soft gelatin capsule. Tablet binders that can be included are acacia, methylcellulose, sodium
carboxymethylceliulose, polyvinylpyrrolidone (Povidone), hydroxypropyl methylcellulose. sucrose, starch and ethylcellulose. Lubricants that can be used include magnesium stearate or other metallic stearates, stearic acid, silicone fluid, talc, waxes, oi ls and col loidal silica.
Flavoring agents such as peppermint, oil of wintergreen, cherry flavoring or the l ike can also be used. Additionally, it may be desirable to add a coloring agent to make the dosage form more attracti e in appearance or to help identify the product.
The compounds of the invention and their pharmaceutical ly acceptable salts that are active when given parenterally can be formulated for intramuscular, intrathecal or intravenous adm inistration. A typical composition for intramuscular or intrathecal adm inistration wi l l be of a suspension or solution of active ingredient in an oi l. for example, arachis oil or sesame oil. A typical composition for intravenous or intrathecal administration wi l l be a steri le isotonic aqueous solution containing, for example, active ingredient and dextrose or sodium chloride, or a m ixture of dextrose and sodium chloride. Other examples are lactated Ringer's injection, lactated Ringer's plus dextrose injection, Normosol-M and dextrose, Isolyte B, acylated Ringer's injection, and the like. Optionally, a co-solvent, for example, polyethylene glycol, a chelating agent, for example, ethylened iam ine tetracetic acid, and an anti-oxidant, for example, sodium metabisulphite may be included in the formulation. A lternatively, the sol ution can be freeze dried and then reconstituted with a su itable solvent just prior to admin istration.
For certain appl ications, such as treating skin cancer lesions, the compounds of th i s invention are active on topical adm inistration, and can be formulated as transdermal compositions or in tegrated in a transdermal delivery device (e.g., a transdermal patch). Such compositions include, for example, a backing, active compound reservoir, a control membrane, l iner and contact adhesive. Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts.
Formulations suitable for oral adm inistration can consist of (a) l iqu id solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients. colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usual ly sucrose and acacia or tragacanth, as well as pastil les comprising the active ingred ient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the l ike containing, in addi tion to the active ingredient, such excipients as are known in the art.
The anti-Id compositions of the present invention can also include aerosol formulations to be administered via pulmonary inhalation or intranasal spray. These aerosol formulations can be placed into pressurized acceptable propel lants. such as
dich lorodi fluoromethane, propane, n itrogen, and the l ike. They may also be formulated as pharmaceuticals for non-prcssurcd preparations such as for use in a nebul izer or an atom izer.
Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic steri le injection solutions, which can contain anti-oxidants, bu ffers, baeteriostats, and solutes that render the formulation isoton ic with the blood of the intended rec ipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers. th icken ing agents, stabilizers, and preservatives. The formu lations can be presented in un it- dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the steri le liquid excipient. for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from steri le powders, granules, and tablets of the kind previously described.
Formulations suitable for topical administration may be presented as creams, gels, pastes, or foams, contain ing, in addition to the active ingredient, such carriers as are known in the art to be appropriate. Suppository formulations are also provided by m ixing with a variety of bases such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams.
Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors. Simi larly, unit dosage forms for injection or intravenous administration may comprise the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and an imal subjects, each unit contain ing a pre- determined quanti :y of compounds of the present invention calcu lated in an amount sufficient to produce the desired effect in association with a pharmaceutical ly acceptable di luent, carrier or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be ach ieved, and the
pharmacodynam ics associated with each compound in the host.
Those of ski ll in the art wil l readily appreciate that dose levels can vary as a function of the specific compound, the nature of the delivery vehicle, and the like. Suitable dosages for a given compound are readi ly determ inable by those of skil l in the art by a variety of means. The dose administered to an animal, particularly a human, in the context of the present invention should be suffic ient to cause a prophylactic or therapeutic response in the animal over a reasonable t ime frame. One ski l led in the art wi l l recogn ize that dosage w i l l depend on a variety of factors includ ing the strength of the particu lar com pound employed, the cond ition of the animal, and the body weight of the animal, as well as the severity of the il lness and the stage of the disease. The size of the dose wi l l also be determ ined by the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular compound. Suitable doses and dosage regimens can be determined by comparisons to anticancer or immunosuppressive agents that arc known to cause the desired growth inhibitory or immunosuppressive response.
Optional ly, the pharmaceutical composition may contain other pharmaceutical ly acceptable components such as bu ffers, surfactants, antioxidants, viscosity mod i fying agents, preservatives and the l ike. Each of these components is wel l-known in the art. See, eg, U . S. Patent No. 5,985,3 10, the disclosure of which is herein incorporated by reference. Other components su itable for use in the formulations of the present invention can be found in Rem ington, 1 985. In an embodiment, the aqueous cyclodextrin solution also incl udes dextrose, e.g., about 5% dextrose. Kits and Systems
Also provided within the invention are kits and systems that correlate with the foregoing description regarding combinatorial formulations, coordinate methods, diagnostic and treatment tools and systems, etc. All combinations of these primary tools disclosed for coordinate or combinatorial use are contemplated to also be provided herein in combined "kit" form. Exemplary kits and systems for practicing the subject methods may include one or more pharmaceutical formulations, which include for example an anti-Id compound packaged or referenced for use with a secondary cancer treatment agent, such as a chemotherapeutic drug, VDA, or HSP90 inhibitor. I n certain embodiments the kits may include a single pharmaceutical composition, present as one or more unit dosages, where the composition includes both an anti-Id compound (e.g., (-)-AGX5 1 and a chemotherapeutic agent or toxicity reducing agent. In yet other embod iments, the kits may include three or more separate pharmaceutical compositions, each containi ng an anti-Id compound, a chemotherapeutic or possibly a toxicity reducing agent, or a combination of these elements. In addition to the above components, the subject kits may further incl ude instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, e.g., as printed information or reference on a su itable med ium or substrate on the packaging of the kit or on a package insert, etc. EXAMPLES
Anti-Cancer and Anti-Metastatic Compositions and Methods
Metastasis is the greatest therapeutic challenge for prolonging survival in most cancer patients, perhaps most significantly among the most populous cancer sufferers, breast cancer patients. Identification of genes and regulatory factors involved in metastasis has proven exceedingly complex and intractable from a clinical standpoint. The inhibitor of DNA- binding (Id) proteins have been variously implicated to play diverse roles in cancer metastasis, including breast cancer metastasis. H igh Id l expression is observed at metastatic sites. Id l reportedly functions in regulating critical mesenchymal-to-epithelial transition (MET) changes which al low circulating ("disseminating") cancer cel ls to seed ("colonize") distant metastatic sites. Artificial genetic knockdown of Id l is reported to disrupt M ET by tumor cells in the lung, and to block development of lung metastases in murine models of breast cancer.
The invention provides the first clinically practicable showing that a smal l molecule drug can effectively target and block Id function in mammal ian subjects in vivo, to disrupt metastasis, slow or prevent cancer progression, and ultimately reduce or prevent mortality in cancer patients.
Anti-Id compounds of the invention provide a novel and profoundly effective approach for treating and/or preventing metastases in cancer patients. The Examples below i llustrate developm ent, characterization and anti-metastatic use of an exemplary smal l molecule anti-Id drug. N-(3-(benzo[d][ l ,3]dioxol-5-yl)-3-(2-methoxyphenyl )propyl )-N- benzylpropionamicle ("AGX5 I "). This exemplary lead com pound provides a foundation for rational design of equivalent drugs possessing anti-Id activity, based on the AGX5 1 platform (e.g., as described above in relation to alternate drug designs set forth in Formulae 1. 11. I l l and I V). AGX5 1 was discovered through exhaustive molecu lar screening, inc luding a vast in silico small molecule screen (spanning 2 x 1 06 candidate compounds). AGX5 1 . as herein characterized, emerged from the screen as the first small molecular agent capable of binding a hydrophobic pock et adjacent to a dimerization domain of Id I , in a manner effective to disrupt dimerization of Id l and its cognate binding partners (including critical regulatory b! I LH proteins, such as E47). Th is monumental discovery was surprising in many ways, not the least being the nature and complexity of HLH-bH LH dimerization rendering prior efforts to effectively d isrupt these molecular interactions, using a host of cand idate blocking agents, profoundly uncertai n and persistently unsuccessful.
As described AGX5 1 and other Anti-Id compounds of the invention not on ly bind and impair Id dimerization function with bHLH proteins, but by this mechanism also interfere functionally, in cl inically practicable ways, with downstream Id activities in critical metastatic pathways. Among these activities, AGX-5 1 enhances E47 binding to DNA in a dose-dependent man ner (correlated w ith AGX-5 1 ?s effective antagonism of Id 1 -E47 dimerization). The Examples below further show that anti-Id compounds of the invention effectively mediate reduction of Id levels in cel ls and living subjects (likely through
"deprotection"' or ''u nmasking" Id by disrupting Id-BHLH protein dimers, exposing Id to ubiquitin-mediated degradation, and likely other Id-degradative mechanisms). This novel and unpredicted activity of anti-Id compounds herein, alone without other powerful anti-Id effects noted here, provides effective tools for reducing harmful Id levels in cl inical subjects, to reduce or preve nt cancer in these subjects by directly reducing risk of cancer development in precancerous cells, lowering cancer transformation in neoplastic cells, and reduc ing metastatic potentia l of existing neoplasms and cancers.
The Exam ples below demonstrate that, by treating murine breast cancer cel ls (4T 1 ) with AGX5 1 , a cli nically practicable decrease of harmful ld l protei n levels results.
Correlated with these surprising results, in cancer cel l culture assays AGX5 1 decreases cell viabil ity (as measured by alamar bl ue assays), and inhibits formation of mammospheres. Anti-Id compositions and methods of the invention also effect therapeutic changes in regulatory proteins p i 6 and p2 1 , reducing ci rculating levels of Id in blood of AGX5 1 -treated subjects, down regulate circulating endothelial cells and EPCs, directly down-regulate pathogenic neovascularization associated with metastasis and tumor growth, among many additional, distinct activities demonstrated here. These and other data, both in vitro and in vivo, are consistent with resu lts from genetic Id knockdown experiments, which can on ly be construed as surprising and unexpected for a first-in-class anti-I d small molecule drug.
These exem lary data further demonstrate that anti-Id compounds and methods of the invention potently disrupt several metastatic mechanisms and pathways (as demonstrated in leukem ic, lung cancer, bladder cancer, and breast cancer precl inical models, among others).
Culminating these studies, the lead candidate anti-Id drug AGX5 1 has now been shown to profoundly reduce actual tumor metastasis in in vivo murine mode ls of multiple cancer types widely accepted as predictive of cl inical drug uti l ity in other mammal ian subjects, including h umans. When administered at 50mg/kg via intraperionteal injection. 24- hours after tai l vein injection of 4T I cel ls, AGX5 1 sharply reduces development of lung metastases. Significantly greater inhibition of lung metastases was observed when AG X- 1 adm in istration was increased from once to twice daily. Comparable studies and results are provided from murine breast cancer models. Additional data are provided evincing that AGX5 1 will block Id-dependent M ET and thus prevent or curtail metastatic d isease in mammalian subjects. Further demonstration of these novel activities by anti-Id compounds of the invention are ongoing stud ies of AGX-5 1 effects on the 4T 1 transcriptome and EMT/MET pathway. The Examples provided here are presented for illustrative purposes on ly, and wi ll not be misconstrued by skilled artisans as limiting the scope, or practical appl ications, of the invention, including equivalent embodiments the teachings of this disclosure. These
Examples use an exemplary small molecule inhibitor AGX57 to block and d isable Id protein levels and metasta:is-mediating functions, while other like compounds described herein wil l be constructed based on these teachings to provide additional agents effective for neutral izing Id binding and function.
Example 1
Identification and Testing of Anti-Id Compounds
The experiments disclosed here summarize the identification of 364 compounds, and concurrent design of twelve peptides useful for inhibiting Id. These experiments further provide means for identifying additional anti-Id compounds, as wel l as for analyzing effectiveness of candidate compounds capable of inhibiting Id. The assays described belo include practicable means for testing effectiveness of cand idate compounds, for example to confirm Id binding, Id dimerization blockade, and/or abi lity to mediate destabi l ization and degradation of Id in cel ls and l iving subjects.
Several mil lion chem icals were screened using three dimensional E47-ld l interaction mapping to identify small molecules that could potential ly inhibit E47-Id l interaction.
Cognate Id l binding structures were conceptualized and virtual screening employed a Monte Carlo simulation for a complex of Id l and a smal l compound screen (hel ical fragments fixed) was run, incl udi ng 1 ,000,000 steps and 1 00 conformations collected and analyzed. Λ complex conformation with the best score and total energy was selected for further analysis.
The top 3,000 compounds from virtual screening were further analyzed for ClogP < 5. IPSA < 80, Molecular Weight < 600, and chem ical and biochemical stability. 364 compounds identified by the in silico screen are l isted in Table I .
Table 1 : Chemical compounds identified by E47-W 1 interaction mapping as potentially inhibiting E47-ldl interaction.
Compound Formula MW Chemical Name
1 C21H27CIN 603 446.94074 RCL Lll, 321-2
2 C19H2406 348.39953 2-methoxyethyl 5-(3,3-dimethyl-2-oxobutoxy)-2-methyl-l-benzofuran-3-carboxylate
3 C30H24N2O7 524.53498 di-me 3-(3,4-di-meo-p )7-(l-nap thoyl)pyrrolo(l,2-c)pyr'im'idine-5,6-d'icarboxylate
4 C25H30N4O6S 514.60505 RCL L18, 827-1
5 C27H25FN206S 524.5725 RCL L19.192-2
6 C19H25N503 371.4428 RCL L21, 483-3
7 C24H34N602 438.57758 RCL 134,410-9
8 C27H31Brf\205 543.46252 RCL L34,794-9
9 C26H29N307 495.53693 RCL L34.836-8
10 C28H33FN205 496.58401 RCL L34.930-5
11 C25H26BrFN205 533.39877 RCL L35.342-6
12 C19H21N502S 383.47552 N-(4-ethoxyphenyl)-2-((4-ET-5-(3-pyridinyl)-AH-l,2,4 -3-yl)t io)acetamide
13 C19H18N4C3S2 414.50831 ME 4-((((4-allyl-5-(2-thienyl)-4h-l,2,4-triazol-3-Yl)thio)acetYl)amino)benzoate
14 C25H27FN206 470.50214 RCL L42, 816-7
15 C32H27FN205 538.58079 RCL L42.856-6
16 C21H17N304S 407.45134 RCL L43, 208-3
17 C29H30N2C6 502.57225 RCL L43, 214-8
18 C21H29N5C5 431.49578 RCL R13, 767-7
19 C26H3808 478.58796 3-beta,5-diacetoxy-17-alpha-ethoxycarbonyl-5-beta, 14-beta-androstan-19-oic acid
20 C14H18CINi.O 307.78546 2-(4-amino-6-(pentylarriino)-l,3,5-triazin-2-yl)-4-chloro-phenol
21 C18H17N05S 359.40389 2,4-dimethoxYbenzYl (2-oxo-l,3-benzothiazol-3(2h)-yl)acetate
22 C18H29N04 323.43613 2-ethyl-4-(2-hydroxy-3-(isopropylarnino)propoxy)-3,6-di ethylphenyl acetate
23 C18H21N703S 415.47717 RCL R60,176-4
24 C25H22BrCIN203 513.82269 RCL R60.587-5
25 C18H2205 318.37304 ethyl 5-(3,3-dimethyl-2-oxobutoxy)-2-methyl-l-benzofuran-3-carboxYlate
26 C26H24N205 460.49098 ethyl 7-benzoyl-3-(3,4f5-trimethoxyphenyl)pyrrolo(l,2-c)pyrimidine-5-carboxylate
27 C17H21N70 ; 371.46722 RCL R77.075-2
28 C26H23CIN206 494.93601 et 7-(4-cl-benzoyl)-3-(3,4,5-tri-meo-ph)pyrrolo(l,2-c)pyrimidine-5-carboxylate 29 C24H23N50 !~ 429.48261 RCL R82.426-7
30 C21H16FN304S 425.44177 RCL R84, 320-2
31 C23H21N3045 435.50552 RCL R89.190-8
32 C23H29N05 399.49128 4-(3,5-Dimethoxy-phenyl)-2,7,7-trimethyl-5-oxo-l,4,5,6,7,8-hexahydro-quinoline-3
33 C24H31N06 429.51777 2,7,7-Trimethyl-5-oxo-4-(3,4,5-trimethoxy-phenyl)-l,4,5,6,7,8-hexahydro-quinolin
5-(acetyloxy)-3-(3,4-dihydro-2H-l,5-benzodioxepin-7-yl)-2-methyl-4-oxo-4H-
34 C23H20O8 424.41105
chromen-7-yl acetate
ethyl 7-(4-methoxyphenyl)-2-methyl-5-oxo-4-pyridin-2-yl-l,4,5,6,7,8-
35 1 C25H26N2CX 418.49697
hexahydro uinolirte-3-carboxylate
methYl (2E)-5-[4-(acetyloxy [phenyl]- 2-[4-(dimethylammo)benzylidene]-7-methyl-3-
35 C26H25N305S 491.57025
oxo-2, 3-dihydro-5H-[l,3]tbiazolo|3,2-a]pyrimidine-6-carboxylate
37 C22H3207 408.49614 Structure contains non-supported carbohydrate derivative!
ethyl (5-[(4-methoxyphenyl)aminoj [l,2,5]oxadiazolo[3,4-e][l,2,4]triazolo[4,3-
38 C16H15N704 369.34245
a]pyrazin-8-yl}acetate
39 C26H27N302 413.52399 N-{2-[(4-benzylpiperazin-l-yl)carbonyl]phenyl}-2-methylbenzamide
N-(2-ethoxyphenyl)-2-{((4-methoxyphenyl)acetyl]amino}-5,6-dihydro-4H-
40 C25H26N2045 450.56097
cyclopentaIb]thiophene-3-carboxamide
41 C27H27N05 445.51994 N-Benzo[l,3]dioxol-5-ylmethyl-N-(3-benzo[l,3]dioxol-5-yl-4-phenyl-butyl)-acetami
42 C20H28N2O3 344.45776 3-Cyclopentyl-l-[4-(3-methoxy-benzoyl)-piperazin-l-yl]-propan-l-one
43 C22H30N2O4 386.4954 l-(4-methoxybenzyl)-4-(2,4,5-trimethoxybenzyl)piperazine
N-((12)-(benzoylamino)[(6-methoxy-4-methylquinazolin-2-
44 C25H21N503 439.47782
yl)amino]methylene]benzamide
45 C17H19N502 325.37328 2-[(6-ethoxy-4-methylquinazolin-2-yl)amino]-5,6-dimethylpyrimidin-4(lH)-one
46 C22H24N206 412.44638 Acetic acid 4-(2-amino-3-cyano-7,7-dimethyl-5-oxo-5,6,7,8-tetrahydro-4H-chronien-
47 C15H13BrN2C 4 365.18586 5-Bromo-furan-2-carboxylic acid [l-carbamoyl-2 -|4-methoxy-phenyl)-vinyl]-amide
4-(2-amino-3-cyano-7 J-dimethyl-S-oxo-S.e .S-tetrahydro^H-chro en^-yll ^-
48 C24H27N306 453.49929
methoxyphenyl morpholine-4-carboxylate
ethyl 2-{[4-methoxy-3-(morpholin-4-ylsulfonyl)benzoyl]amino}-4,5,6,7-tetrahydro-l-
49 C23H28N207S2 508.61681
benzothiophene-3-carboxylate
N-{(12)-[(4,6-dimethylpYrimidin-2-yl)amino][(4-methoxYphenYl)amino]methylene}-4-
50 C21H23N503S 425.51316
methylbenzenesulfonamide Compound Formula MW Chemical Name
ethyl 5-i[(2-metboxypbenyl)amino]C3rbonyl}-4-methyl-2-[(2-
51 C24H24N205S 452.53328
rnethylbenzoyl)amino]thiophene-3-carboxylate
52 C18H2205 318.37304 ethyl 5-(3,3-dirnethyl-2-oxobutoxy)-2-methyl -benzofuran-3-carboxylate
53 C17H16N404S2 404.46947 2-[2-(4- ethyl-6-oxo ,6-dihydro-pyrimidin-2-ylsulfanyl)-acetylamino]-benzothiaz
N~l~-2,3-dihydro-lH-cyclopentaib]qu'inolin-9-yl-N~2~-[2-(3,4-
54 C25H29N303 419.52818
dimethaxyphenyl)ethyl]-N~2~-methylglycinamide
2-(l,3-dioxo ,3-dihydro-2H-isoindol-2-yl)-N-(4-{[(4-
55 C23H19N3D5S 449.48898
methylphenyl)sulfonyl]amino)phenyl)acetamide
ethyl 5-acetyl-2-{[(2,4-difluorophenoxy)acetyl]amino}-4-methylthiophene-3-
56 C18H17F2N05S 397.40069
carboxylate
ethyl 2,5-dimethyl-3-{2-[(3-methylphenyl)amino]-2-oxoethyl}-4-oxo-3,4-
57 C20H21N3O4S 399.47207
dihydrothieno[2,3-d]pyrimidine-6-carboxylate
ethyl 4-[(4-fluorophenyl)amino]-2-(2-methoxy-2-oxoethyl)-5-methylthieno[2,3-
58 C19H18FN:!04S 403.43541
d]pyrimidine-6-carboxylate
_59 C24H3607 436.55032 Structure contains non-supported carbohydrate derivative!
|9-(2-[(4-chlorobenzyl)oxy]phenyl}-l,8-dioxo-2, ,4,5,6,7, 8,9-octahydroacridin-10(lH)-
60 C28H26CIN05 491.97612
yljacetie acid
61 C19H28N205 364.44541 ethyl -(3-[(2,5-dimethoxyphenyl ]-3-oxopropyl)piperidine-4-carboxylate
62 C18H26N204 334.41892 ethyl l-{3-[(2-methoxyphenyl)amino]-3-oxopropylipiperidine-4-carboxylate
63 C20H19N5O 345.40733 N-(4-ethoxyphenyl)-l-(4-methylphenyl)-lH-pyrazolo[3,4-dJpyrimidin-4-amine
4-(2 "3-cyano-7,7-diinethyl-5-oxo-5,6,7,8-tetrahydro-4H-chromen-4-yl)-2,6-
64 C25H29N307 483.52578
dimethoxyphenyl morpholine-4-carboxylate
ethyl 2-[3-(4-fluorobenzoyl)-4-hydroxy-5-oxo-2-phenyl-2,5-dihydro-l H-pyrrol-l-yl]-4-
65 C24H19FN205S 466.49183
methyl-l,3-thiazole-5-carboxylate
66 C21H16FN304S 425.44177 5-(4-Fluoro-phenyl)-3-hydroxy-4-(4-methoxy-benzoyl)-l-(5-methyl-[l,3,4jthiadiazo
5 4-bromophenyl)-3-hydroxy-4-(4-rnethoxybenzoyl)-l-(5- ethyl-l,3,4-thiadiazol-2-
67 C21H16Br 304S 486.34737
yl)-l,5-dihydro-2H-pyrrol-2-one
5-(2-fluorophenyl)-3-hydroxy-4-(4-methoxybenzoyl)-l-(5-methyl-l,3,4-thiadiazol-2-
68 C21H16FN304S 425.44177
yl ,5-dihydro-2H-pyrrol-2-one
69 C23H24N205 408.45813 ethyl 6-methoxy-4-{[4-{propoxycarbanyl)phenyljamino)quinoline-3-C3rbox lafe
(4-{(Z -(4-chlorobenzyl)-2,5-dioxoimidazolidin-4-ylidene]methyl}-2-
70 C20H17CIN2O6 416.82129
methoxyphenoxyjacetic acid
5'(4-fluorophenyl)-3-hydroxy-4-(3-methyl-4-propoxybenzoyl)-l-|pyr'idin-3-ylmethyl)-
71 C27H25FN:04 460.5097
l,5-di ydro-2H-pyrrol-2-one
72 C20H20N2O5 368.3928 4-Acetyl-5-(2,4-dimethoxy-phenyl)-3-hydroxy-l-pyridin-3-ylmethyl-l,5-dihydro-pyr
73 C20H20N2O5 368.3928 4-Acetyl-5-(3,4-dimethoxy-phenyl)-3-hydroxy-l-pyridin-3-ylmethyl-l,5-dihydro-pyr
74 C24H19N304 Γ 413.43673 " ethyl 4-[(4-isonicotinoyl-l-oxo ,2-dihydroisoquinolin-3-yl)amino]benzoate
75 C19H23N07 377.39766 4-(3-Hydroxy-4-methoxy-pberiyl)-l-(2-methoxy-ethyl)-l,4-dihydro-pyridine-3,5-dica methyl N-([l-(4-chlorophenyl)-7,7-dimethyl-2,5-dioxo-l,2,5,6,7,8-hexahydroquinolin-
76 C28H27CIN 206 522.99019
3-yl]carbonyl}tyrosinate
77 C21H20N2O6 396.40335 4-[3-Hydroxy-4-(4-methoxy-benzoyl)-2-oxo-5-pyridin-3-yl-2,5-dihydro-pyrrol-l-yl]
78 C22H28N206 416.47826 4-{[4-(3,5-dimethoxybenzoyl)piperazin-l-yl]methyl}-2,6-dimethoxyphenol
79 C14H21N304S 327.40517 5-Butyl-6-hydroxy-2-(2-morpholin-4-yl-2-oxo-ethylsulfanyl)-3H-pyrimidin-4-one
80 C20H18N2O5 366.37686 3-([3-(ethoxycarbonyl)-8-methoxyquinolin-4-yl]amino}benzoic acid
N-(2-methoxyphenyl)-5-methyl-7-pyridin-2-yl-2-thien-2-yl-4,7-
81 C23H20N6O2S 444.51885
dihydro[l,2,4]triazolo|l,5-a]pyrirnidine-6-carboxamide
82 C15H21N50 287.36752 N~4~-(4-methoxyphenyl)-l,3,5-triazaspiro(5.5]undeca-l,3-diene-2,4-diarnine
83 C20H19F3N2O4 408.38063 methyl 4-[4-{acetylamino)phenoxyJ-2,3,5-tnfluoro-6-pyrrolidin-l-ylbenzoate
ethyl 3-bromo-4-([4-(ethoxycarbonyl)phenyl]amino)-2-methyl-5-oxo-l-phenyl-2,5
84 C23H23BrN205 487.35416
dihydro-lH-pyrrole-2-carboxylate
l-(2-([4,6-bis(ethylamino)-l,3,5-triazin-2-yllamino}-4-methyl-l,3-thiazol-5-
85 C13H19N7GS 321.40668
yl)ethanone
86 C20H18N4O5 394.39026 2-{5-|Bis-(5-hydroxy-3-methyl-]H-pyrazol-4-yl)-methyl]-furan-2-yl}-benzoic acid
87 C20H18N4CI5 394.39026 3-|5-[Bis-(5-hydroxy-3-methyl-lH-pyrazol-4-yl)-methyl]-furan-2-yl}-benzoic acid
88 C17H16N202 280.32927 2-Methoxy-6-(quinolin-3-ylarninomethyl)-phenol
(2Z)-3-(3,4-dimethoxyberizyl)-2-[(4-methoxyphenyl)imino]-N-rnethyl-4-oxo-l,3-
89 C22H25N30SS 443.52565
thiazinane-6-carboxamide
90 C18H21N502 339.40037 2-(6-Ethoxy-4-methyl-quinazolin-2-ylamino)-5-ethyl-6-methyl-lH-pyrimidin-4-one
91 C23H18CIN04 407.85721 2-((2-benzoyl-4-chlorophenyl)aminol-4-oxo-4-phenylbutanoic acid
92 C19H21N5Q2 351.41152 2-[(6-ethoxy-4-methytquinazolin-2-yl)amino]-5,6,7,8-tetrahydroquinazolin-4|lH)-one
8-butyl-3,3-dimethyl-6-[(tetrahydrofuran-2-ylmethyl)amino]-3,4-dihydro-l H-
93 C20H29N3O2 343.47303
pyrano[3,4-c] pyridine-5-carbonitrile
94 C21H23N06. 385.42056 2-Amino-4-(4-methoxycarbonyl-phenyl)-7,7-dimethyl-5-oxo-5,6,7,8-tetrahydro-4H-ch Compound Formula MW Chemical Name
95 C20H23NO 341.41061 isobutyl 2-benzyl-l-oxo-l,2,3,6,7,7a-hexahydro-3a,6-epoxyisoindole-7-carboxylate methyl 6-3cetyl-7-(3-ethoxy-4-hydroxyphenyl)-4,7-dihydrotetr3zolo[l,5-3]pyrimidine-
96 C16H17N505 359.34439
5-carboxylate
N-{2-[(6,7-din)etboxyi5oquinolin-l-yl)methyl)-4,5-dimethoxyphenyl}-l-methyi'4-
97 C25H25N5 7 507.5073
nitro-l H-pyrazole-5-carboxamide
4-([3-(aminocarbonyl)-6-tert-butyl-4,5,6,7-tetrahydro-l-benzothien-2-yl]aminD>-4-
98 C17H24N204S 352.45583
oxobutanoic acid
99 C26H31N303 433.55527 N-(3-morpholin-4-ylpropyl)-2-(4-propoxyphenyl)quinoline-4-carboxamide
100 C20H19NO4 337.37873 2-(2,4-diethoxyphenyl)quinoline-4-carboxylic acid
101 C20H22N4O2 350.42394 N-(2,4-diroethoxyphenyl)-2-pyrrolidin-l-ylquinazolin-4-amine
N-((12)-|(3-chloro-4-fluorophenyl)amino][(4,6-dimethylpyrimidin- 2-
102 C20H19CIFN5O2S 447.92213
yl)amino]methylene}-l-phenylm(;thanesulfonarnide
2-{2-[4-(l,3-benzodioxol-5-ylmethyl)piperazin-l-yl]-l-methyl-2-oxoetriyl}-l H-
103 C23H23N305 421.45686
isoindole-l,3(2H)-dione
N-(4-butoxyphenyl)-l-(4-methoxyphenyl)-7.7-dimethyl-2,5-dioxo-l,2,5,6,7,8-
104 C29H32N205 488.58879
hex3hydroquinoline-3-carboxamide
N-(4-butoxyphenyl)-l-(4-chloro henyl)-2,5-dioxo-l,2,5,6,7,8-hexahydroquinoline-3- 105 C26H25CIN204 464.95315
carboxamide
5-(4-chlorophenyl)-7-(4-ethoxyphenyl)-4,5,6,7-tetrahydro[l,2,41tnazolo(l,5-
106 C19H20CIN5O 369.85715
a]pynmidin-2-amine
5-(4-bromophenyl)-7-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydro(l,2,4}triazolo(l,5-
107 C19H20BrN5O2 430.30755
a]pyrimidin-2-amine
ethyl 2-amino-7,7-dimethyl-5-oxo-4-(3,4,5-trimethoxyphenyl)-5,6,7,8-tetrahydro-4H-
108 C23H29N07 431.49008
chromene-3-carboxylate
109 C23H22N206 422.44159 l',l"-propane-l,3-diylbisspiro[l,3-dioxolane-2,3'-indol]-2'(l'H)-one
2-Amino-7,7-dimethyl-4-(4-nitro-phenyl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-
110 C20H22N2O6 386.40814
carboxylate
111 C22H25N304S 427.52625 ethyl 10-(3-morpholin-4-ylpropanoyl)-10H-phenothiazin-3-ylcarbamate
4-(2-amino-3-cyano-7,7-dimethyl-5-oxo-5,6,7,8-tetrahydro-4H-chromen-4-yl)-2-
112 C25H29N3D6 467.52638
ethoxyphen l mor holine-4-carboxylate
N-l2-[3-(4-chlorobenzoyl)-4-hydroxy-5-oxo-2-phenyl-2,5-dihydro- lH-pyrrol-1 -
113 C21H19CIN204 398.84958
yllethyljacetamide
(3aR,7aS)-2-{[(4-acetylphenyl)amino]methyl}-3a,4,7,7a-tetrahydro-l H-isoindole
114 C17H18N203 298.34461
l,3(2H)-dione
_Π5 C21H25N3Q4 383.4511 2-Azepari-l-yl-N-(2-ethoxy-phenyl)-5-nitro-benzamide
4-(2-amino-3-cyano-7, 7-dimethyl-5-oxo-5,6,7,8-tetrahydro-4H-chromen-4-yl)-2 116 C24H22N205S 450.51734
methoxy phenyl thiophene-2-carboxylate
4-(2-3mino-3-cyano-7,7-dimethyl-5-oxo-5,6,7,8-tetrahydro-4H-chromen-4-yl)-2
117 C25H23N307S 509.54196
ethoxy-6-nitrophenyl thiophene-2-carboxylate _
118 C23H21CIN203 408.88842 N'-3cetyl-N'-(3-chloro-4-methylphenyl)-2-hydroxy-2,2-diphenylacet hydrazide
4-[2-amino-3-cyano-7-(2-furyl)-5-oxo-5,6,7,8-tetrahydro-4H-chromen-4-yl]-2-
119 C27H22N207 486.48559
ethoxyphenyl 2-furoate
dimethyl 2-({(Z)-[l-(4-fluomphenyl)-5-oxo-3-propyl-l,5-dihydro-4H-pyrazol-4-
120 C23H22FN305 439.44729
ylidenglmethyl}3mino)terephthalate
8-[(4-amino-6-morpholin-4-yl-l,3,5-triazin-2-yl)methoxy]-l,3-dimethyl-7-(4-
121 C23H27N904 493.52954
methylbenzyl)-3,7-dihydro-lH-purine-2,6-dione
122 C21H22N2CI5 382.41989 ethyl 4-[(3,4-dimethoxyphenyl)amino]-8-methoxyquinoline-3-c3f boxy late
ethyl 4-{[2,3-bis(4-methoxyphenyl)-l-oxo-l,2,3,4-tetrahydroisoquinolin-4-
123 C31H33N306 543.62516
yl]carbonyl}piperazine-l-carboxylate
N~2~-acetyl-N~l~-[2-(2,3-dihydro-l,4-benzodioxin-6-ylamino)-l-(4-methoxyphenyl)-
124 C21H23N3G6 413.43396
2-oxoethyl]glycinamide
ethyl l-[2-(cyclopentylamino)-l-(4-methoxyphenyl)-2-oxoethyl]piperidine-4-
125 C22H32N204 388.51134
carboxylate
126 C20H30N2CI2 330.4743 N-cyclohexyl-2-(4-methoxyphenyl)-2-piperidin-l-ylacetamide
N-cyclohexyl-2-[(2,2-dimethoxyethyl)(methyl)3minol-2-(6-nitro-l,3-benzodioxol-5-
127 C20H29IM3O7 423.47003
yl)acetamide
128 C18H23N305 361.40111 N-cyclopentyl-2-(6-nitro-l,3-benzodioxol-5-yl)-2-pyrrolidin-l-ylacetamide
129 C22H25N3G5 411.46165 2-[benzyl(methyl)amino]-N-cyclopentyl-2-|6-nitro-l)3-benzodioxol-5-yl)acetamide
N-cyclopentyl-2-I(2,2-dimethoxyethyl)(methyl)amino]-2-(6-nitro-l,3-benzodioxol-5-
130 C19H27N307 409.44294
yljacetamide
ethyl l-{[6-methyl-5-[(4-methylpiperidin-l-yl)sulfonyl]-4-oxothieno[2,3-d] pyrimidin-
131 C23H32N4 652 524.66269
3(4H)-yl]acetyl)piperidine-4-carboxyl3te
3-(4-methoxyphenyl)-N-(3-methoxypropyl)-2-rnethyl-l-oxo-l, 2,3,4-
132 C22H26N204 382.46352
tetrahydroisoquinoline-4-carboxamide
6 !
Figure imgf000064_0001
Compound Formula MW Chemical Name
(112)-ll-[(2E)-l-hydroxy-3-(2,3,4-tnmethoxypheriYl)prop'2-enYlidene]-5-methoxY-
167 C28H31NO ' 493.56177
1,2,3, ,9, 9a-hexahydro-4a,9-(epirninoethano)xanthen-12-one
168 C27H28N204 444.53521 2-(4-ethoxy-3-methoxybenzyl)-3-oxo-N-(2-phenylethyl)isoindoline-l-carboxamide
2-[3-(3,4-dimethylphenyl)-2,4-d'ioxo-3,4,6,7,8,9-hexa ydro-2H-
169 C24H29N3 4S 455.58043
cyclohepta[4,5]thieno[2,3-dlpyrimidin-l(5H)-yl]-N-(2'methoxyethyl)acetamide
3-[2,4-dioxo-l-[(4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)methyl)-l,4-dihydroquinazolin-
170 C25H21N505 471.47662
3(2H)-yl]-N-(2-furylmet yl)propanamide
N-(2-methoxybenzyl)-3-(l-(4-methylbenzyl)-2,4-dioxo-l,4-dihydroquinazo(in-3(2H)-
171 C27H27N304 457.53394
yljpropanamide
4-[2,4-dioxo-l-[(4-oxo-4H-pyndo[l,2-a]pyrimidin-2-yl)metriyl]-l,4-dihydroquinazolin-
172 C29H27N5 5 525.56904
3(2H)-ylJ-N-(2-metboxybenzyl)butanamide
N-(2-metrioxybenzyl)-4-[l-[(6-metriyl-4-oxo-4H-pyrido[l,2-a]pYnmidin-2-yl)met vl]-
173 C30H29N5O5 539.59613
2,4-dioxo-l,4-dihydroquinazolin-3(2H)-yllbutanamide
4-[l-(2-fluorobenzyl)-2,4-dioxo-l,4-dihydroquinazolin-3(2H)-yl]-N-(2-
174 C24H22FN304 435.45904
furylmethyl)butanamide
^(4-acetylphenyl)-2-13-(l,3-benzodioxol-5-ylrriethyl)-2,4-dioxo-3,4-
175 C26H21N306 471.47377
dihydroquinazolin-l(2H)-yl]acetamide
N-(4-bromophenyl)-2-(3-(2-(cyclopentylamino)-2-oxoethyl)-2,4-dioxo-3,4-
176 C23H23BrN404 499.36816
dihydroquinazolin-l(2H)-yl)acetamide
ethyl 6-(2,5-dimethoxyphenyl)-4-oxo-3-propyl-4,5,6,7-tetrahydro-lH-indole-2-
177 C22H27N05 385.46419
carboxylate
2-isopropoxyethyl 3-methyl-4-oxo-6-phenyl-4,5,6,7-tetrahydro-lH-indole-2-
178 C21H25N04 355.4377
carboxylate
N-(2,5-dimethoxybenzyl)-3-(lH-indol-3-yl)-2-(2-met oxyethyl)- 1-oxo-l, 2,3,4-
179 C30H31N3O5 513.59867
tetrahydroisoquinoline-4-carboxamide
2-(2-methoxyethyl)-3-(l-met yl-lH-indol-3-yi)-l-oxo-N-{thien-2-ylmethyl)-l, 2,3,4-
180 C27H27N303S 473.59854
tetrahydroisoquinoline-4-carboxamide
N-(2,5-dimethoxybenzyl)-2-(2-methoxyethyl)-3-(l-methyl-lH-indol-3-yl)-l-oxo-
181 C31H33N305 527.62576
l,2,3,4-tetrahydroisoquinoline-4-carboxamide
N-(4-ethoxybenzyl)-2-(2-methoxyethyl)-3-(l-methyl-lH-indol-3-yl)-l-oxo-l, 2,3,4-
182 C31H33N304 511.62636
tetrahydroisoquinoline-4-carboxamide
N-(4-ethoxyphenyl)-10,ll-dimethoxy-8-oxo-5,8,13,13a-tetrahydro-6H-isoquino[3,2-
183 C28H28N205 472.54576
a]isoqutnoline-13-carboxamide
184 C23H21N304 403.44152 2-f(3,4-dimethoxyphenyl)aminoJ-N-(2-furylmethyl)quinoline-4-carboxarri'ide
185 C23H27N303 393.48994 N-(3-isopropoxypropyl)-2-[(4-methoxyphenyl)amino]quinoline-4-carboxam'ide
2-[(2,4-dimethoxyphenyl)amino]-N-[3-(2-oxopyrrolidin-l-yl)propylSquinoline-4-
186 C25H28N404 448.52631
carboxamide
187 C24H27N303 405.50109 4-(azepan-l-ylcarbonyl)-N-(2,4-dimethoxyphenyl)quinolin-2-amine
ethyl l-({2-[(2,4-dimethoxyphenyl)amino)quinolin-4-yl}carbonyl)piperidinc-3-
188 C26H29N305 463.53813
carboxylate
189 C22H25N304 395.46225 2-[(2,5-dimethoxypheny(jamirio]"N-(3-methoxYpropyl)quinoline-4-carboxamide ethyl l-[(2-{[3-(methylthio)phenyl]amino)quinolin-4-yl)carbonyl]piperidine-3-
190 C25H27N303S 449.57624
carboxylate
191 C26H32N402 432.57054 2-[(4-isopropylphenyl)amino]-N-|3-morpho in-4-ylpropyl)quinoline-4-carboxamide
192 C24H29N302 391.51763 N-(3-ethoxYpropyl)-2-[(4-'isopropylphenyl)amino]quinoline-4-carboxam'ide
193 C24H30N2O4 410.5177 N-[2-(3,4-diethoxyphenyl)ethyl)-4-methyl-3-(2-oxopyrrolidin-l-yl)benzamide
194 C17H28N202 292.42491 4-acetyl-N,N-dibutyl-3,5-dimethyl-lH-pyrrole-2-carboxam'ide
195 C18H19N3Q2S 341.43503 N-(4-butylphenyl)-3-methyl-5-oxo-5H-[l,3]thiazoloi3,2-a]pyrimidine-6-carboxamide
7-(4-chlorobenzyl)-8-i(4-(2-hydroxyethyl)piperazin-l-yl]methyl)-l,3-dimethyl-3,7-
196 C21H27CIN 503 446.94074
dihydro-lH-purine-2,6-dione
8-[(3,5-dimethylpiperidin-l-yl)methyl]-7-(3-hydroxypropyl)-l,3-dimethyl-3,7-dihydro-
197 C18H29N503 363.46353
lH-purine-2,6-dione
N~2~-i(3,5-dimethyl-lH-pyrazol-4-yl)sulfonyl]-N~2~-(4-methoxYphenyl)-N~l~-(4-
198 C21H24N404S 428.51383 methylpheny glycinamide
4-(4-[4-(l,3-benzodioxol-5-ylmethyl)piperazin-l-yl]-4-oxobutoxy}-l-methylquinolin-
1 9 C26H29N30S 463.53813
2(lH)-one
methyl 2-{[2-(4-methoxyphenyl)-6,8-dimethyl-5,7-dioxo-5,6,7,8-
200 C20H22N4O5S 430.48614
tetrahydropyrimido[4,5-d)pyrimidin-4-yl]thio}butanoate
ethyl 4-{[7-(2-chlorobenzyl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-lH-purin-8-
201 C21H25CIN504 460.9242
yl]methyl}piperazine-l-carboxylate
8,9-dimethoxY-N-(3-methylbutyl)-2-pyridin-3-yl[l,2,4]triazolo[l,5-c]quinazolin-5-
202 C21H24N602 392.46443
amine
203 C25H19N303 409.44848 2,3-di-2-furyl-N-(2-phenylethyl)quinoxaline-6-carboxamide Compound Formula MW Chemical Name
204 C22H31N305 417.50947 N-cycloheptyl-3-(6,7-diethoxy-2,4-dioxo-l,4-dihYdroquinazolin-3(2H)-yl)propanamide
N-(4-fluorophenyl)-2-([3-(3-isopropoxypropyl)-4-oxo-3,4-dihydro[l]benzothieno[3,2-
205 C24H24FN.' 03S2 485.60358
d]pyrimidin-2-yl]thio}3cetamide
N-(4-methoxybenzyl)-3-(3-oxo-5-{[(4-oxo-4H-pyrido[l,2-a]pyrimidin-2-
206 C30H26N6O4S 566.64352
yl)methyl]thio}-2,3-dihydro'imidazo[l,2-c]quinazolin-2-yl)propanamide
2-{[3-(l,3-benzodioxol-5-ylmethyl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio}-N-(4-
207 C26H23N305S 489.55431
ethoxyphenyljacetamide
208 C21H19N503 389.41728 ethyl 4-{[l-(4-methoxyphenyl)-lH-pyrazolo[3,4-d]pynmidm-4-yl]amino}benzoate
N-(2,4'dimethoxyphenyl)-l-(2,4-dimethylphenyl)-lH-pYrazolo[3,4-dlpYrimidin-4 -
209 C21H21N502 375.43382
amine
210 C24H26N40 386.50102 N-(3-isopropoxypropyl)-5,7-diphenyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine
2-(2-{[7-(4-chlorophenyl)-5-phenyl-7H-pyrrolo[2i3-d)pyrimid'in-4-
211 C22H21CIN402 408.89127
yl]amino}ethoxy)ethanol
2-(2-{[7-(3-methylphenyl)-5-phenyl-7H-pyrroto[2,3-dlpyrimidin-4-
212 C23H24N402 388,47333
yl]amino}ethoxy)ethanol
l-(3-{[7-(3-methylphenyl)-5-phenyl-7H-pyrrolo[2,3-d]pyrirnidin-4-
213 C26H27N50 425.53799
yl]arnino}propyl)pyrrolidin-2-one
2-(3,4-dimethoxybenzoyi)-6,7-dimethoxy-l-[(2-methoxyphenoxy)methyl]-l, 2,3,4-
214 C28H31N07 493.56177
tetrahydroisoquinoline
6,7-dimethoxy-l-((4-nitrophenoxy)methyl)-2-(phenylacetyl)-l,2,3,4-
215 C26H26N206 462.50692
tetrahydroisoquinoline
methyl 4-{[6,7-dimethoxy-2-(phenylacetyl)-l,2,3,4-tetrahydroisoquinolin-l-
216 C28H29N06 475.54643
yl]methoxy}benzoate
6,7-dimethoxy-l-[(4-methoxyphenoxy)methyl)-2-(2-phenoxypropanoyl)-l,2,3,4- 217 C28H31N06 477.56237
tetrahydroisoquinoline
2-(!2-[5'(4-methoxyphenyl)-3-thien-2-yl-4,5-dihydro-lH'Pyrazol-l-yl]-2- 218 C22H21N503S3 499.63637
oxoethyl}thio)-6,7-dihydro[l,3,4]t iadiazolo[3,Z-a][l,3]diazepin-8(5H)-one
4-methoxy-N-[(Z)-l-[(8-oxo-l,5,6,8-tetrahydro-2H-l,5-methanopyrido[l,2-
219 C31H33N307 559.62456
a][l,5]dia20cin-3(4H)-yl)carbonyl)-2-{3,4,5-trimethoxyphenyl)vinyiIbenzamide 220 C19H22N206 374.39699 ethyl 4-{[2-(3,4-dimethoxyphenyl)ethyl]amino}-3-nitrobenzoate
221 C25H27N302S 433.57684 2-((2-[(4-i5opropylphenyl)amino]-2-oxoethyl)thio)-N-(4-methylbenzyl)nicotinamide
222 C24H34N204 414.54958 4-cyclopentyl-3-(3,4-dimethoxypbenyl)-l-(4-methylcyclohexyl)piperazine-2,5-dlone methyl 2-(4-butoxyphenyl)-3-(cydohexylamino)-lH-imidazoll,2-b!pyrazole-7-
223 C23H30N4O3 410.52055
carboxylate
224 C24H28N203 392.50236 4-cyclopentyl-3-(4-methoxyphenyl)-l-(2-phenylethyl)piperazlne-2,5-dione
225 C26H26N204 430.50812 2-benzyl-N'(2-(3,4-dimethoxyphenyl)ethyl]-3-oxoisoindoline-l-carboxamide
N-(5-bromo-2-methoxybenzyl)-2-(6-chloro--3-oxo-2,3-dihydiO-4H-l,4-benzoxazin-4-
226 C19H18BrCIN204 453.72331
yl)propanamide
ethyl l-[3-(3-oxo-2,3-dihydro-4H-l,4-benzoxazin-4-yl)propanoyl]piperidine-3-
227 C19H24N205 360.41353
carboxylate
N-(4-ethoxy-3-methoxybenzyl)-2-(3-oxo-2,3-dihydro-4H-l,4-benzoxazin-4-
228 C20H22N2O5 0.40874
yl)acetamide
N-[4-(dimethylamino)phenylJ-2-[ll-oxo-8-(pyrrolidin-l-
229 C28H28N4035 500.62436
ylcarbonyl)dibenzo[b,f][l,4]thiazepin-10(llH)-yl]acetamide
N-|4-ethoxyphenyl)-2-[ll -oxo-8-(pyrro)idin-l -ylcarbonyl)dibenzo|b,fJ|l,4]thiazepin-
230 C28H27N304S 501.60909
10(llH)-yl)acetamide
N-(4-ethylphenyl)-2-[ll-oxo-8-(pyrrolidin-l-ylcarbonyl)dibenzo[b,f][l,4]thiazepin-
231 C28H27N3035 485.60969
10(llH)-yl]acetamide
4-{[4-(l,3-benzodioxol-5-ylmethyl)piperazin-l-yl]carbonyl}-N-(2,4-
232 C30H30N4O5 526.5974
dimethoxyphenyl)quinolin-2-amine
233 C26H25N304 443.50685 2-[(2,4-dimethoxyphenyl)amino]- -(2-methoxybenzyl)quinollne-4-carboxamide
N-(2,4-dimethoxyphenyl)-2-[8-(morpholin-4-ylcarbonyl)-ll-
234 C28H27N306S 533.60789
oxodibenzo[b,f][l,4]t iazep'm-10(llH)-yl]acetamide
N-(4-isopropylphenyl)-2-[8-(morpholin-4-ylcarbonyl)-ll-
235 C29H29M304S 515.63618
oxodibenzo[b,f][l,4Jthiazepin-10(llH)-yljacetamide
ethyl 4-[(2-fluorciphenyl)amino]-2-(2-methoxy-2-oxoethyl)-5-methylth'ieno|2,3-
236 C19H18FN304S 403.43541
d)pyrim'idine-6-carboxylate
ethyl 4-(buty)amino)-2-(2-metboxy-2-oxoethy))-5-metbyltbienD|2,3-dJpynmidine'6-
237 C17H23N304S 365.45456
carboxylate
5-{4-methylphenyl)-7-(2,3,4-trimethoxypbenyl)-41516,7-tetrahydrotetrazolo[l,5-
238 C20H23N5O3 381.43801
ajpyrimidine
5-(4-bromophenyl)-7-(2,3,4-trimethoxyphenyl)-4,5,6,7-tetrahydrotetrazolo[l,5-
239 C19H20BrN5O3 446.30695
ajpyrimidine Compound Formula W Chemical Name
7-(2,5-dimethoxyphenyl)-5-(4-ethoxyphenvl)-4,7-dihY(iro[l,2,4]ti'iazolo[l,5-
240 C21H22N403 378.43449
ajpyrimidine
7-(2,S-dimethoxyphenyl)-5-(4-ethylphenyl)-4,5<6,7-tetrahydro[l,2,4]tria2olo[l,5-
241 C21H24N402 364.45103
a]pyrimidine
7-(2,5-dimethoxyphenyl)-5-(4-ethylphenyl)-4,5,6,7 etrahydrotetrazolo[l,5-
242 C20H23N5O2 365.43861
a]pyrimidine
diethyl 4-(3-chlorophenyl)-l-(4-methoxybenzyl)-l,4-dihydropyridine-3,5-
243 C25H26CIN05 455.94267
dicarboxylate
ethyl 2-amino-4-methyl-5-(([2-(trifluoroiTiethyl)phenyl]amino}carbonyl)thiophene-3-
244 C16H15F3i\ 203S 372.36875
carboxylate
l-(4-chlorophenyl)-2-{[4-hydroxy-5-(4-methoxyphenoxy)-6-methylpyrimidin-2-
245 C20H17CIN2O4S 416.88649
yl]thio}ethan-l-one
246 C19H21FN; 05S 408.45202 ethyl 3-l|(4-fluorophenyl)sulfonyl]amino}-4-morpho inobenzoate
2,5-di(tert-butyl)-4-({[4-(trifluoromethyl)phenyl]sulfonyl)oxy)phenyl 4-
247 C28H28F606S2 638.65016
(trifluoromethyl)benzene-l-sulfonate
2-{[4-hydroxy~5-(4-methoxyphenoxy)-6-methylpyrimidin-2-yl]thio}-l-(4-
248 C21H20N2O5S 412.46795
methoxyphenyl)etharvl-one
Ethyl 3- ydroxy-5-methyl-6-oxo-l-phenyl-l,6-dihydropyrano[2,3-c]pyrazole-4-
249 C16H14N205 314.30038
carboxylate
250 C19H21CIN204S 408.90722 l-(l,3-benzodioxol-5-ylmethyl)-4-[(4-chlorobenzyl)sulforiyl]piperazine
251 C22H23N02 333.43411 l-benzyl-3-butyl-4-hydroxy-6-phenylpyridin-2(lH)-one
252 C18H18N20S 310.42096 N'-(4-isopropylphenyl)'l-benzothiopheae-2-carbohydrazide
253 C16H21N3 4 319.36347 ethyl 5-[4-(2-methoxyethoxy)anilino]-3-methyl-lH-pyrazole-4-carboxylate
2-({5-[({[3-(2-chlorophenyl)-5-methyrisoxazol-4-yl]carbonyl}oxy)ethanimidoyl]-2-
254 C26H22CIN304S 507.99994
methoxybenzyl}thio)pyndine
(2-(2,3-dihydro-l,4-benzodioxi -6-ylamino)-4-phenYl-l,3-thiazol-5-
255 C24H18N203S 414.48666
yl](phenyl)methanone
2-(l,3-benzothiazol-2-ylsulfanyl)-l-[l-(trifluoromethyl)-l,3,4,9-tetrahydro-2H-beta-
256 C21H16F3N 30S2 447.50437
carbolin-2-yl]-l-ethanone
[2-(2,3^dihydro-l,4-benzodioxin-6-ylamino)-4-phenyl-l,3-thiazol-5-yl](4-
257 C24H17FN203S 432.47709
fluorophenyl)methanone
258 C17H18F3P> 302 353.34711 ethyl 2-(4-isopropylanilino)-4-(trifluoromethyl)-5-pyrlmidinecarboxylate
ethyl 6-methyl-3-(5-nitro-Z-thienyl)-lH-pyrazolo[5,l-c][l,2,4]triazole-7-
259 C12H11N504S 321.31657
carboxylate,80<90%
diethyl 2-({2-methyl-4-[5-(trifluorOmethyl)-l,3,4-oxadiazDl-2-
260 C18H18F3I\ 305 413.35646
yl]anilino}methylidene)malonate
261 C20H18F3 3O3 405.37996 2-furylmethyl 2-(4-isopropylanilino)-4-(trifluoromethyl)pyrimidine-5-carboxylate
4-(3,5-dimethoxyphenyl)-2,7,7-trimethyl-5-oxo-l,4,5,6,7,8-hexahydiO-3-
262 C23H29N05 399.49128
quinolinecarboxylate
263 C24H25N03 375,47175 N-[2-(3,4-dimethoxyphenyl)ethyl]-2,2-diphenylacetamide
264 C20H25NO5 359.42595 3-butyryl-4-{[2-(3,4-dimethoxyphenyl)ethyl]amino}-6-methyl-2H-pYran-2-one
265 C22H28N206 416.47826 4-{[4-(3,5-dimethoxybenzoyl)-l-piperazinYl]methyl}-2,6-dimethoxyphenol
266 C17H19N05 317.34468 l,3-benzodioxol-5-y 1(2,3, 4-trimethoxybenzyl)a mine
267 C15H19N04 277.32298 ethyl 6-[(dimethylamino)methyl] -5-hydroxy-2--methyl-l-benzofuran-3-carboxylate
268 C24H27N03 377.48769 (l,2-diphenylethyl)(2,3,4-trimethoxybenzyl)amine
269 C23H24N204S 424.52273 N-(2-methoxYethYl)-4-([phenyl(phenylsulfonYl)aminolmethYl)benzamide
270 C22H21CIN 204S 444.94067 N'-(4-chloro-2-methYlphenyl]-N2- (4-methoxYphenYl)-IM2-(phenYlsulfonyl)glYCinamide
2-[6-(2-chlorobenzoyl)-3,4-dimethoxy-2,4-cyclohexadien-l-yl]-l-(3,4-
271 C25H25CI05 456.9274
dimethoxyphenyl)ethanone
272 C21H24N204 368.43643 l-(4-ethoxyphenyl)-3-{i2-(4-methoxyphenyl)ethyl]amino)-2,5-pyrrolidinedione
273 C23H22N203 374.44339 (l-benzyl-lH-benzimid3zol-2-yl)(3,4-dimethoxyphenyl)methanol
274 C18H18CI2N203 381.26176 N-(4-([2-(2,4-dichlorophenoxy)acetyl]aminoJphenYl)butanamide
275 C20H24N2O3 340.42588 N-(4-{[2-(3,4-dimethylphenoxy)acetyl]amino}phenyl)butanamide
276 C20H28N2O3 344.45776 l-(3-cyclopentylpropanoyl)-4-(3-methoxybenzoyl)piperazine
277 Π9Η22Ν203 326.39879 N-(4-{[2-(2,4'dimethylphenoxy)acetyl]amino}phenyl)propanamide
278 C22H30N2O4 386.4954 l-(4-methoxybenzyl)'4-(2,4,5-trirnethoxybenzyl)piperazine
3-benzyl-5,5-diethyl-2-[(2-hydroxyethyl)amino]-5,6-dihydrobenzo[h]quinazolin-4(3H)-
279 C25H29N302 403.52878
one
280 C21H22BrF 204 465.32289 ethyl 4-[4-(3-bromo-4-methoxYbenzoyl)-2-fluorophenYl]-l-piperazinecarboxylate
281 C18H30N2O2 306.452 4-(2-[(4-methoxybenzyl)aminoJethyl}-l,2,5-trimethyl-4-piperidinol
4-{[(5 hloro-2-methylphenyl)(phenylsulfonyl)amino]methyl}-N-(2-
282 C27H24CIN 303S 506.02763
pyridinylmethyl)benzamide
4-([(5-chloro-2-methYlphenyl)(phenYlsulfonYl)aminolmethYl}-N-(tetrahydro-2-
283 C26H27CIN 204S 499.03309
furanylmethyl)benzamide
Figure imgf000068_0001
Figure imgf000069_0001
c]pyridin-6-yl]thio)butanoate
Example II
Design of Synthetic Anti-Id Peptides
The E47 m olecule from an X-ray of the structure of an E47-Id l heterodimer was used as a template for peptide design. The peptides were designed to form leucine-zipper type dimers with Id l (b t not with E47), which could be stabilized by introduction of polar or charged side chain s forming inter-molecular H-bonds and salt bridges with Id l . The designed peptides were deemed to have significant probabil ity of retaining an a-hel ical con formation in solution. For certain peptides, the hel ical propensity of peptides was enhanced by introd uction of polar or charged side chains to form intra-molecular H-bonds and salt bridges at positions that d id not interact with Id I . Only peptides containing natural am ino acids only were considered.
Multiple dy nam ics simulations were performed for the model of Id I -peptide complex to assess stabil ity of proposed peptide-dimer structure. Simulations were carried out in vacuum at 300°K with the AM BER-94 force field. Positional constraints were appl ied to maintain backbone atoms of Id l close to the crystal structure; side chain atoms of Id l and all atoms of the peptide were not constrained. During the 40 picosecond equ i l ibration and 300 picosecond acquisition simulations, dimer structure remained close to the initial model with relatively small fluctuation in potential energy. The peptide maintained an a-hel ical structure during entire multiple dynamics trajectory. Root mean square deviations between peptide positions in the init ial model and in the final multiple dynamics structure was 0.50 A for C" atoms and 0.74 A for al l heavy atoms. The inter-molecular H-bonds anchoring the peptide to Id l were calculated to remain stable during the multiple dynamics simulations.
Twelve peptides that met the required criteria were identified as cand idate anti-Id compounds (Table 2).
Table 2: Twelve synthetic peptides created using the E47 molecule from an X-ray of the structure of E47- Idl heterodimer as a template for peptide design.
Figure imgf000071_0001
The identifi ed compounds and peptides were subjected to further testing and analysis using the gel shift and cel lular assays described in further detail in the examples that follow.
Example II I
Gel Shift Assays of Synthetic Anti-Id Compounds
Gel shift assays were performed using the 376 small molecules identified in the in silica drug screen a nd peptide synthesis. The identified compounds were dissolved in 5% DMSO to a concentration of Ι ΟΟμιη and reacted in binding mixtures that contained E47, Id l and Mck. As controls, recombinant human E47 was used in a standard binding reaction that contained 32P labeled MCK double stranded oligonucleotides containing consensus E-Box response element (MCK- 5 ' TTG ATC CCC CCA ACA CCT GCT GCC TGA AGC T (SHQ ID NO. 17)) and l OOng of Id l was added to a standard binding reaction using E47 and p32 labeled MCK. After 30 minutes of incubation, the reaction mixtures were resolved on a 5% non-denaturing polyacrylamide gel and autoradiographed. The bound E47-MCK resulted in a shifted band whe:*eas the unbound MCK migrated to the bottom of the gel. The reaction mixture containing Id l and E47 and MCK showed a failure of E47 to bind to MCK. If the identified molecules were capable of inhibiting Id l , the gel shift assay showed a shifted band of bound E47-MC . The presence of shifted band also suggests that the small molecules have no effect on the interaction between E47 and MCK, a critical observation that demonstrates the absence of any non-specific effect of small molecules on the normal b/ ILH mediated transcriptional pathway.
Of the 376 small molecules identified in the in silico drug screen, the 1 7 in Table 3 were identified as demonstrating the most pronounced anti-Id activity in the gel shi t assays. These molecules resulted in pronounced gel shifts in the presence of E47. Id l and MCK. Table 3: Compounds showing the most pronounced anti-id activity in gel shift assays.
Figure imgf000072_0001
The molecu les identified in Table 3 mediated gel shift in the presence of E47, Id l and E box. These 1 7 m olecules were subjected to additional gel shift assays to ensure repeatability and consistency of results. All these compounds resulted in a specific hit in the gel shift assays, indicating they are capable of disrupting Id 1 -E47 dimcrization interactions under conditions of the assay. Based on the intensity of the gel shift (considered equivalent to potency Id 1 -E47 dimerization blockade), two small molecules: N '-(4-isopropylphenyl)- l - benzothiophene-2-carbohydrazide and N-(3-(benzo[d][ l ,3]dioxoI-5-yl)-3-(2- methoxyphenyl)propyl)-N-benzylpropionamide (N-[3-( 1 ,3-benzodioxol-5-yl)-3-(2- methoxyphenyl)propyl]-N-benzylpropanamide) were selected for further testing. However, all 376 molecules in Tables 1 , 2 and specifically all 1 7 molecules listed in Table 3 are considered anti-Id candidate compounds for use within the methods and compositions of the invention.
N , -(4-isopropylphenyl)- l -benzothiophene-2-carbohydrazide (HTS 03876,
Maybridge) and N-[3-( 1 ,3-benzodioxol-5-yl)-3-(2-methoxyphenyl)propyl )-N- bcnzylpropanamide (N-(3-(benzo[d][ 1 ,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N- benzylpropionam ide) (6958667, Chembridge) were identified based on their abi l ity to restore the binding between E47-MC in the presence of Id 1 . The abi lit of these molecules to block E47-Id l inte 'action was confirmed at least twice in a sim i lar assay before analyzing their effects on cell based assays. In addition, the effect of both compounds demonstrated dose-proportionality in the gel shift assay.
Example IV
AGX5 1 Disruption of Id-bH LI I Binding
And AGX5 1 -Med iated Id Degradation i n Leukem ic Cel ls The instant Example demonstrates novel ability of an exemplary anti-Id compound. N-(3-(benzo[d] f l ,3]dioxol-5-yl)-3-(2-methoxyphenyI)propyl)-N-benzylpropionam ide
("AGX51 "), to disrupt Id dimerization with bl lLH proteins and cause rapid degradation of unbound Id protein in cellular systems. TA leukemic cells expressing ETO chimeric protein were treated with either vehicle control (DMSO) or 20 μΜ, concentrations of either, racemic AGX5 1 , puri fied (-)-enantiomer of AGX5 1 (peak "E- l " in "Enantiomer Separation A", described below), or purified (-)-enantiomer of AGX5 1 (peak "E-2" in Enantiomer
Separation A). Cell l sates (20 μg) were separated by SDS-PAG E and transferred to nitrocel lulose membranes. Immunoblots were analyzed by Western blotting and visual ized by chemiluminescence (Figure 1 ). The primary antibody for Id l and ld3 was obtained from B ioCheck (Foster City, CA). 1
Figure 1 provides a Western blot gel demonstrating that Id l and Id3 levels are potently reduced following treatment of TA leukem ic cells with racemic AGX5 1 . As in other investigations documented here, anti-Id activity of the (-)-AGX5 1 enantiomer (second eluting peak "Ε-2'" from " Enantiomer Separation A" (see below)) is surprisingly much greater than the anti-Id activity of racemic AGX5 1 , while the (+)-AGX5 1 (denoted as the first cluting peak "E- I " from "Enantiomer Separation A") has m inimal effect on Id l ( Figure 1 ).
Example V
AGX5 I -Mediated Disruption of Id-bl lLH B inding
And AGX5 1 -Mediated Id Degradation in Breast Cancer Cells
The ability of an AGX5 1 to bind Id and disrupt Id dimerization with bl l LH proteins and cause rapid degradation of unbound Id protein was also demonstrated in cellular models of breast cancer. 4T 1 murine breast cancer cells were treated as in Example I V above, with either vehicle control (DMSO) or 20 μΜ, concentrations of either, racem ic AGX5 I , puri fied (+)-enantiomer of AGX5 1 (peak 'Έ- Ι from Separation A"), or purified (-)-enantiomer of
AGX5 1 (peak "E-2 from Separation A"). Cel l lysates (20 μ ) were separated by SDS-PAG E and transferred to nitrocellulose membranes. Immunoblots were analyzed by Western blotting and visual ized by chem i luminescence. The same primary anti bod ies for Id l and Id3 (B ioCheck, Foster City, CA) were used.
Stcreospeci fic activity of anti-Id compound AGX5 1 was confirmed in part in this breast cancer mode l (4T I cells) (Figure 2). However, these resu lts render the molecular basis for this stereospeci ficity between AGX5 1 enantiomers even more complex. The comparable Western blot data for the breast cancer cells (Figure 2) again showed that the (-)-AGX5 1 enantiomer exhibited profound dominance over the (+)-ANG X5 1 enantiomer in terms of anti-Id 1 binding d isruption and degradation (though weak Id l signal was stil l detectable in all lanes, indicating, the ablation or knock-down may not have been complete in any sample).
Th is stcreos pecific (-)-ABX5 1 -mediated destruction of Id l protein levels in both leukem ic and breast cancer cells profoundly implicates this enantiomer as a potent tool for intervention in preventing and treating metastatic disease. 70221
In contrast to the results presented in Example IV for leukem ic cel ls, none of the AGX5 1 preparations (racemate "mix", or either (+)- or (-)-enriched enantiomer prep.) appeared to effecti vely impair Id3 binding to promote Id3 degradation in breast cancer cel ls.
The unexpected stereospecificity findings for AGX5 1 and its enantiomers traces a predominant if not exclusive anti-Id 1 activity to the (-)-AGX5 1 enantiomer. In comparison. anti-Id 1 activity is min imal or completely lacking in the (+)-AGX5 l enantiomer. The (-)- AGX5 1 enanliomer-specific activity is paradoxically strong for both Id l and Id3 in leukem ic cells, wh ile comparable levels of activity were observed for the (+)-AGX5 1 enantiomer preparation ("Ε-2'" ) in this model (Figure 1 ). This latter observation may be an artifact of the experiment, with possibly small amounts of the (-)-AGX5 1 enantiomer (5- 1 0%) in the (+)- AGX5 1 preparation (fraction "E- l " from Enantiomer Separation A) being capable of med iating potent reduction of Id3 levels (as observed for al l three preparations: racemate "m ix"; (+)-enantiomer "Έ- Γ"; and (-)-enantiomcr "E-2") (Figure 1 ).
The contrast of the Id- 1 and Id3 stereospecificity data here, however, calls this sim ple "contam inant" exp lanation into question. Alternatively, there may be a lower concentration of ld3 in certain cancer cel ls, or a greater activity of al l forms of AGX5 1 against Id3 than Id l . This latter interpretation is challenged, however, by the results in Figure 2, suggesting that neither the AGX5 1 racemate, nor either of the (-)-AGX5 1 or (+)-AGX5 1 enantiomers, appear to mediate significant Id3 degradation in breast cancer cells.
It must therefore be regarded as a surprising and confounding discovery that the ( -)-
AGX5 1 enantiome r mediates potent destabi lization and destruction of both Id l - and Id3- bl 1 LI I complexes (corresponding to total ablation of Id 1 and Id3 protein at the levels of detection afforded here) in leukem ic cel ls, with comparable anti-Id 1 stereospecific activity residing in the (-)-AGX5 1 enantiomer in breast cancer cells, but l ittle or no anti-ld3 activity observed for any fc rm of ANGX5 1 in breast cancer cel ls (whi le al l three preparations exerted strong anti-Id3 protein ablation effects in the leukemic cells— possibly pointing to minor contamination and 2xtraordinary anti-ld3 potency of (-)-AGX5 I , but exh ibited only in this cel l type between s:udies, or alternatively to an incomplete stereospecific effect (wherein (-)- AGX-5 I is profoundly anti- Id I dominant, with little or no andi-ld3 activity resides in the (+) enantiomer, yet both the (-)-AGX5 1 and (+)-AGX-5 1 enantiomer exhibit comparable anti-l d3 activity). The surprising anti-Id potency of the (-)-AGX51 enantiomer observed here could not have been predicted based on conventional understanding of stereochemistry in biological systems. Preconceptions of stereochemical interactions in simple biochemical systems might predict a disparate activity potential between opposite polarity enantiomers. But the art clearly fails to provide a similar foundation for predicting stereochemical functional disparit} among any compound such as ANGX 1. AGX51 is a first-in-class compound shown to interact with Id and disrupt Id-dimerization with bHLH proteins and other complex binding partners. The large helix-loop-helix structures that contain homo- and hetero-dimerization domains among these complex binding pairs, have no correlates among conventional stereo- biochemistry models (e.g.. simple enzyme-substrate interactions). There can be no scientifically well-founded predictions of stereospecific effects, of any kind, among any compound analogous to AGX51 , because AGX51 is a pioneer anti-Id drug in a nascent field of discovery, first explored and charted here.
Example VI
Lsthal Effects of Anti-Id Compounds on Cancer Cell Lines Prostate cancer cell lines DIJ 145 and PC3 (in 10% BCS) were obtained from
American Type Culture Collection (Rockville, MD). The cells were cultured in flam's 1 2 (Gibco, Carlsbad, CA) medium containing 10% BCS (Hyclone, Logan, UT) and appropriate antibiotics (pen/strep, fungizaone, and gentamycin (Invitrogn Inc.. Carlsbad. CA). All cells were cultured at 37°C in a fully humidified atmosphere containing 5% C()2.
At 50% confluence, the cells were treated with either 100 μΜ D SO, lOOOmOsmol of urea +NaCL, I μΜ N-(3-(bcnzo[d][l,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N- bcnzylpropionamide(AGX51), 10 μΜ N-(3-(benzo[d][l,3]dioxol-5-yl)-3-(2- methoxyphenyl)propyl)-N-benzylpropionamide, and 100 μΜ N-(3-(benzo|d][l,3]dioxol-5- y))-3-(2-methoxyphenyl)propyl)-N-benzylpropionamide, 1 μ N'-(4-isopropylphenyl)-l - benzothiophene-2-carbohydrazide, 10 μ N"-(4-isopropylphenyl)-l -benzothiophene-2- carbohydrazide, or 100 μΜ N'-(4-isopropylphenyl)-l-benzothiophene-2-carbohydrazide. The cell morphology and growth was monitored daily for I week by microscopy for changes in morphology or cell death. Apoptosis was determined by measuring caspase 3 and caspase 7 activities using the Caspase-Glo 3/7 Assay system from Promega (Madison. WI). N-(3-(benzo[d][l J]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzy]propionamide. at 1 ΟΟμιη concentration led to massive cell death of PC 3 cells within 3 days with no surviving cells observed after six days of treatment. While the effect of on DU145 cells was not as pronounced after three days, the cells appeared very unhealthy and were unable to proliferate as compared to the controls. Additionally, after six days, treatment of DU 145 cells with N-(3-(benzo[d][ 1 ,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N- benzylpropionamide led to cell death. N-(3-(benzo[d][l,3]dioxol-5-yl)-3-(2- methoxyphenyl)propyl)-N-benzylpropionamide was also able to induce apoptosis in DLi 145 at concentrations as low as Ι Μ (Fig.3 (C)). Cell survival was also sensitive to N'-(4- isopropylphcnyl)- 1 -benzothiophene-2-carbohydrazide. N"-(4-isopropylphenyl)- 1 - benzothiophene-2-carbohydrazide, at a concentration of 1 urn was able to induce cell death in DLI 145 cells. In short, two small molecule inhibitors of E47-ldl interaction were identified that induce massive cell death in prostate cancer cell lines.
The molecular mechanisms underlying the effects of N-(3-(bcnzo[d|[ l.3]dioxol-5-yl)- 3-(2-methoxyphenyl)propyl)-N-benzylpropionamide on prostate cancer cells was assessed by measuring activity of primary mediators of apoptosis, caspase 3/7. Treatment with N-(3- (benzofdJI 1 ,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzylpropionamide (1-1 ΟΟμιη) resulted in a significant increase in caspase 3/7 in both DU 145 and PC3 cells, which was higher than caspase activity in cells treated with the apoptosis inducing agent staurosporine (ΙΟμιη).
Example VII
AGX 1 Rescues pi 6 Levels in Leukemic Cells
A leukemic cell line derived from a mouse overexpressing the MML-AF9 lusion protein was treated with increasing concentrations of N-(3-(benzo[d][l,3]dioxol-5-yl)-3-(2- methoxyphenyl)propyl)-N-benzylpropionamide (AGX51).50% growth inhibition was observed at 10 μΜ of AGX51 relative to a DMSO control. Total cell lysates were collected using buffer containing 50 mM Tris -HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholic acid and 0.02% sodium azide along with freshly added complete protease inhibitors. The protein lysates (20 μg) were separated by SDS-PAGE and transferred to nitrocellulose membranes. Immunoblots were analyzed by Western blotting and visualized using a Western lightening chemiluminescence detection kit. As shown in Figure 3, AGX51 at the ICso concentration restored pi 6 compared to the DMSO vehicle. The primary antibodies used in this study were purchased from Epitomics, Burlingame, CA. USA.
Example VI II
AGX51 Rescues p2l Levels in Human Bladder Carcinoma Cells
A human bladder carcinoma cell line was treated with increasing concentrations of N- (3-(benzo[d][l,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzylpropionamide (AGX51 ) and processed for p2l concentration impacts of AGX51, as described above in Example VII for AGX51-media1ed rescue of 16. Total cell lysates were collected, processed, separated and transferred to a Western blot substrate as described. The Western blots were visualized using as shown in Figure 3. AGX51 mediated a pro ounced dose-dependent restoration of p21. This activity, as in the case of the p 16 rescue activity, indicates potent anti-Id efficacy in cellular systems modeling clinical disease. In the case of p21, Id natively impairs normal E protein regulation of p21 implicated in cell cycle control and anti-proliferative effects of p21. By virtue that the novel anti-Id compounds and methods of the invention functionally knock down or even ablate Id levels in metastatic environments, the knock down ofld here directly liberates E protein induction of p21 to restore healthy p21 levels to cancerous cells. These molecular rescue activities of AGX51 and other anti-Id compounds of the invention are shown here to mediate powerful anti-cancer and anti-metastatic impacts on cellular migration, cellular proliferation, apoptosis and other critical mechanisms for metastatic disease progrcssior and clinical arrest.
Example IX
Anti-Id Compounds Restore Critical Cell Cycle Control in Cancer Cell Lines
DU 145 prostate cancer cells were obtained from American Type Culture Collection
(Rockville, D). The cells were starved of serum for 24 h and treated 24 h with luM N"-(4- isopropylphenyI)-l -benzothiophene-2-carbohydrazide (AGX8) or N-(3-(benzo[d][l,3]dioxol- 5-yl)-3-(2-methoxyphenyl)propyl)-N-benzylpropionamide (AGX51). After treatment, the cells were washed, fixed in ethanol and stained with propidium iodide before being analyzed by fluorescence-activated cell sorting (FACS) analysis (Accuri Cytometers, Inc., Ann Arbor, MI). Cell pellets were suspended and numbers of cells in each identified phases of the cell cycle were measured by flow cytometry. AGX5 1 and AGX8 both exhibited distinct activity for positively restoring cell cycle control. AGX8 was less effective compared to AGX5 1 in this assay for rescue of cell cycle control. These data are consistent with reports that Id l mediates cancer-promoting and metastatic induction effects in part by impairing or disabling normal cell cycle controls.
In follow on studies here, the abil ity of AGX5 1 to mediate cel l cyc le control was further analyzed with regard to possible stereospecific effects. Figure 5 illustrates the surprising result that one specific enantiomer, (-)-AGX5 1 ("E2"), is signi licantly more active than racem ic AGX5 I ("E0") in restoring cell cycle control in DU- 145 prostate cancer cells, whereas the other enantiomer (+)-AGX5 1 ("E l ") appears virtually inactive. Data are mean values of the % cel ls in sub-GO (apoptotic), G 1 , S and G2/M phases. The error bars are SEs and an * designates p<0.05. The treatments concentrations were al l 10 iiM . I mproved cell cycle control is exemplified by fewer cells in G2/m phase of cel l cycle and more cel ls in the sub-GO cell dormancy phase.
The foregoing find ings demonstrate that anti-Id compounds of the invention med iate powerful disruption of ld-dependent cel l cycle deregulation. Thus the invention provides tools and methods :o directly disable cancer development through the curtai lment of Id- deregulated cel l cycl ing, correlated with uncontrolled, cancerous or metastatic cel lular proliferation. In a correlated array of studies, the inventors have demonstrated direct antiprol iferative effects of anti-Id compounds described here, shown to powerful ly reduce proliferation rates of many different types of cancer cells in both cel lular systems and in vivo models of human disease.
Direct anti-proliferation activity of AGX5 1 was demonstrated in a dose-proportional fashion to l im it cancer cel l prol i feration in human bladder cancer cel ls, among other models. U M UC-3 human b adder cancer cells were treated with D SO (control) or Ι ΟμΜ or 20μΜ AGX5 1 . After 48 hours, cel l viabil ity numbers were determ ined by trypan blue exclusion stain ing. In addition, total cell lysates were collected using a buffer containing 50 mM Tris- HCI (pH 7.5). 1 50 mM NaCI, 1 % Triton X- 1 00. 0. 1 % SDS, 0.5% deoxycholic acid and 0.02% sodium azide along with freshly added complete protease inhibitors. Three 20 μg protein lysates were separated by SDS-PAGE and transferred to nitrocel lulose membranes. The immunoblots were analyzed by Western blotting for Id l , p i 6 or p2 1 and the results on the blots visualized using a Western lightening chemiluminescence detection k it. The three primary antibodies were for Id 1 , p 16 or p2 1 . The primary antibody for Id 1 was obtained from BioCheck, Foster City, CA. p 16 and p21 antibodies were purchased from Pierce Antibodies, Roekford, IL. Actin staining was used for internal standardization.
Within these studies, ANGX 1 mediated approximately a fifty percent growth inhibition (observed at 10 μ AGX5 1 ) compared to DMSG* control. These studies also demonstrated that AGX5 1 -mediated anti-prol iferation activity was correlated with AGX-5 1 - mediated decrease in Id l protein and concomitant increase in p21 levels (with a less substantial effect on p i 6 levels). Acting thus as a direct modulator of p2 l
activity/expression, ANGX5 I also functions to stabilize cell cycle control and dim inish production of endothelial progenitor cells (EPCs) and EPC-dependent tumor-associated angiogenesis.
Example X
AGX I B locks Id-Dependent Cell M igration Essential for Metastasis Cel l migration reflects the sum of many individual biologic processes that, overal l. promote the capabi l ity of a particular cel l to move from one place to another. Cel l m igration is an integral mechanism essential tor tumor metastasis, the most lethal process associated with neoplasms (C iiang and Massague. 2008). The instant Example demonstrates how exemplary anti-Id compounds of the invention such as AG X5 1 can reduce or prevent metastatic disease by blocking Id-dependent metastatic cel l m igration.
Cel lular m i gration potential is readily determined employing conventional "Scratch
Assay" methods. The basic steps of a Scratch Assay involve creating a "scratch" in a cel l monolayer, capturi ng the images at the beginning and at regular intervals duri ng cell m igration to c lose t he scratch, and comparing the images to quantify m igration rates of the cells. The in vitro Scratch Assay is particularly suitable for studies concern ing cel l-matrix and cell-cell interactions in cel l m igration.
For this study, PC3 cells were plated and allowed to grow almost to confluence. Λ p200 pipet tip was used to scratch a channel in the cell layer, and the cel ls are w ashed to remove debris from the scratch area. Media contain ing AGX-5 1 dosing vehic le (DMSO) or media contain ing AGX-5 1 was then added to the cells and the cells were photographed daily.
Results are provided in the photographs presented in Figure 6. A fter 4 days, the scratches or channels in plated cel l samples treated w ith media containing dosing vehicle ( DMSO) have disappeared while the channels in plated cel l samples treated with AGX-5 1 remain open. Th is assay, consistently repeated with comparable results, unambiguously shows that anti-Id active AGX5 1 potently inhibits Id-dependent cel l migration. Thus the invention provides yet additional tools and methods to directly block cancer development and metastasis by reducing or disabl ing Id-dependent cell migration.
Example XI
AGX5 1 Blocks Id-Dependent Angiogenesis Essential for
Cancer Growth and Metastasis
VEGF- 1 65 and FGF-2 treated Matrigel plugs were implanted on Day 0 into C57B E/6 m ice. Mice were t reated with either vehicle or AGX5 1 . The anti-Id compound was provided either in the plugs (25 μ§/η¾) or by daily ip treatment (30 or l OOmg/kg) for 10 days. Plugs were harvested on Day 1 0, fixed and paraffin embedded. Three sections (5μΜ thickness) of each plug were stai ned with an anti-CD3 1 antibody and counterstained with hematoxylin and eosin stain. CD3 1 - ositive m icrovcssels were counted for one entire cross-section per plug and the average m icrovessel density ± SD was determined. Student' s t-test was used for statistical analysis.
M icrovessel data from this study is provided in Figure 7. Compared to vehicle control animals, all AGX5 1 subjects were significantly protected against new blood vesse l formation. Statistics for the study were: p>0.05 (n=7), 0.0 1 (n=6) and 0.01 (n=7) compared to vehicle (n=9) for 25 μg/plug, 30 and l OOmg/kg dose groups respectively. The maxim um protection was 44% at an ip dose of l OOmg/kg, qd. Slices of the Matrigel plugs were histologically analyzed at the end of the study, which revealed a major decrease in the presence of complete blood vessels and therefore endothelial cells in the AGX5 1 -treated subjects relative to control subjects.
In related studies, female nude m ice were orthotopical ly (mammary fat pad) implanted with ME>A-MB-23 1 human breast cancer cells. Prior to implantation, the MDA-
MB-23 1 cells were trypsinized, centrifuged and re-suspended in a 50% DPBS/50% Matrigel solution to a concentration of 5 x 1 06 cells/50 μΐ, and frozen unti l use. The frozen Matrigel was thawed at 4°C for 24 hours. Syringe, needle and cells were kept on ice unti l used. Prior to the injections, cel ls were gently mixed by inversion and the needed vol ume drawn up into a cold syringe equipped with a 25 x 5/8" gauge needle. The syringe w as gently inverted to m ix taking care to avoid bubbles. At four to six weeks of age, the m ice were injected with 50 μ ί of Matrigel containing 1 0 x 1 06 MDA-MB-23 1 cells into the orthotopic site. The cel ls were injected orthotopically into the right, caudal mammary fat pad of the animals anesthetized by ketamine ( 1 20mg/kg)/xylazine (6mg/kg) adm inistered im using a 26 x 3/8" gauge needle. Tunors were then al lowed to establish to a volume of 1 00 mm3, after which animals, (typically 5 animals/treatment group) were randomized and treated ip with vehicle (DMSO) or 60mg/kg AGX5 1 bid. After 4 1 days of treatment, the m ice were sacrificed by cervical dislocation, and tumors excised and fixed in 1 0% buffered (neutral) formalin for histopathologic analyses. Tissue was removed from the formalin and washed with 3xPBS. soaked in 30% sucrose in PBS for 24-48 hours at 4°C to cryoprotect, and then embedded in paraffin. The para ffin blocks were stored at -20°C or -80°C unti l cryo-section ing into 5 μιη th ick sections for mounting on slides. The slides were treated with anti-CD3 l and washed with PBS, and endothelial cells were detected using an avidin-biotin com plex (ABC ) system. The sl ides were counter stained with hematoxylin and eosin stain to visual ize nuclei.
As consistently observed among these study specimens, blood vessels with endothel ial cells were prom inent in vehicle-treated h istological samples, but were consistently absent in the AGX5 1 treated animals.
These findi ngs directly evince that anti-Jd function of AGX5 1 and other compounds of the invention ex :ends to specific blockade of Id-mediated pathogenic angiogenesis. This mechanism of intervention in the metastatic cascade will provide particularly powerful tools for managing cancer and preventing and reducing metastatic disease through blockade of tumor-associated angiogenesis.
Example XI I
Anti-Cancer Effects of AGX5 I Med iated by
Inhibition of Endothelial Progenitor Cel ls ( EPCs)
Additional compositions and methods of the invention discretely impai r tumor- associated angiogenesis by inhibiting production and reducing survival of endothelial progenitor cel ls (EPCs). EPCs are responsible for promoting tumor growth and metastasis by contributing to neovascularization of new tumors (and by rescuing established tumors from vascular destruction caused by tumor growth). EPC production and survival is Id-dependent in cancerous and metastatic systems, and therefore the anti-Id compositions and methods of the invention effectively reduce or prevent metastasis through reduction or prevention of EPCs and/or by im pairment of EPC function and developmental potential.
The instant Example utilizes the same test subjects as presented throughout the other Examples here, comparing anti-ld-treated animals with control animals under conditions that lead to cancer progression and metastasis in the non-treated control an imals. The further object of this Example is demonstrating Id-dependent variation of EPC levels in animals treated or untreated using the compositions and methods of the invention. According to th is Example, each class of treated and untreated model animal described in the foregoing Examples I V-VI 1 will be evaluated for EPC levels (e.g., using multivariate flow sorting with antibodies directed against EPC markers (such as GFP+ and VE-cadherin+) to measure EPCs in samples from bone marrow or circulating blood). Animals treated with anti-Id
compositions and methods of the invention, particularly (-)-ANGX5 l wi ll exh ibit anti-Id compound-mediated, dose-dependent reduction in EPC levels (compared to control animals, and animals treated with lesser active anti-Id compounds, such as racem ic ANGX5 1 and particularly the anti-metaslatical ly inactive (+)-ANGX5 1 enantiomer.
Example XI I I
Anti-Id Compounds Inhibit Tumor Growth In Vivo
Nude mice (n=8/treatment group) were implanted with MDA23 I human cancer cel ls. Fourteen days after implantation, the mice were treated ip with DMSO (vehic le) or 50mg/kg AGX5 1 in DM SO. Al l m ice were treated iv for 5 consecutive days with 7.5mg/kg Taxol starting on days 8 and 22. Bo plots are tumor volumes 53 days post implantation (last day of the study).
Two animal in the control group and one animal in the treated group died before day 53 (data not used). Tumor size findings for al l surviving an imals are provided in Figure 1 3. As shown in Figure 8, treatment with AGX 5 1 provides a significant p=0.05 compared to vehicle (DMSO) control] negative effect of approximately 50% on tumor growth.
Example XIV
Dose optim ization of AGX5 1
CD 1 male mice (3/timepoint) were treated ip with 30mg/kg AGX5 1 in DMSO. B lood was collected in he aarinized tubes by retro-orbital puncture pre-dose and at 0.25, 0.5, 1 .0, 2.0, 4.0, 8.0 & 24 hours post dose. One μg of the internal standard for the assay (S I 09037 from Aldrich, St. Louis, MO) and 1 00 of pH 7.4 PBS were added to the plasma harvested from the blood. The mixture was vortexed for one minute with I m L methyl t-butyl ether (MTBE), and the MTBE removed after centrifugation with dry N2 (g). The residue remaining after removal of the MTBE was reconstituted in 200 of ACN. Five of this solution was analyzed by LC/MS using a 2.0 x 250 mm C 1 8 column coupled to an electrospray LC- MS set to monitor m/z 432 (AGX5 1 ) and m/z 468 (S I 09037) in the HPLC effluent. The flow rate was 200 L/m inute of ACN (85)/H20 + 0. 1 % formic acid. Cal ibration curve samples were prepared in duplicate by fortifying 1 00 μL· control plasma w ith 0. 0.03. 0. 1 1 , 0.33, 1 or 3 μg of AGX 5 1 and analyzing the samples as described. The ratio of 432 to m/z 468 in the calibration samples (y) and the concentration of AGX5 1 in the sample (x) were analyzed by l inear regression and the resulting slope (m) and intercept (b) used to convert the ratio in an experimental samp le into a concentration of AGX5 1 . Typical ion chromatograms from the assays are provided in Figure 9.
As shown in Figure 1 0. AGX5 1 is rapidly absorbed with Cmax occurring at the first time point, 1 5 minutes. The observed profile is consistent with a two compartment model with tj/2 for elim ination from the first compartment of approximately 30 minutes and a terminal elimination ti/2 from the second compartment of approximately 6 hours. The drug is widely distributed, Vz/F of approximately 80 l iters/kg, and relatively rapid ly cleared, G/F of > 1 00 mL/kg/minute.
Various dosing simulations were performed using the two compartment model in WinNonl in (Pharsight, Sunnyvale, CA) and the PK parameter estimates from the lit of the plasma concentration data to the two compartment model in WinNonl i n. The target criteria was to maintain the plasma concentration of AGX5 1 above 2 μΜ (0.43 1 μg/mL) which is the lower end of the ran ge of IC50 values observed for AGX5 1 to inhibit prol iferation in various cancer cel l culture systems, e.g., DU 145 (as seen in Examples IV and V, above). As shown in Figure 1 1 , the simulations suggested that a dose of 60mg/kg, bid, would achieve the target criteria. Example XV
AGX51 Profoundly Reduces Lung Cancer Metastasis In Vivo
Using Accepted Murine Models Predictive of Cancer Dru¾ Efficacy In Human
Subjects
This Example demonstrates potent, in vivo anti-metastatic effects of the novel anti-Id compounds and methods herein. Tail vein injection of Lewis lung carci noma was fol lowed by measurement of lung metastases according to conventional methods. Thirty C57B L/6 m ice were implanted with 7.5 x l (P Lew is Lung Carcinoma (LLC) cells/animal. Seven days after implantation, 5M/5F per group were treated daily intraperitoneally ( ip) for 25 days with either dosing veh icle (DMSO), or 50 mg/kg racem ic N-(3-(benzo[ d][ 1 ,3]d ioxol-5-yl)-3-(2- methoxyphenyl)propyl)-N-benzylpropionamide (AGX5 1 ). Fourteen days after implantation, another group of 5M/5 F animals were treated daily ip for 1 8 days with 50 mg/kg N-(3- (benzo[d][ L3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzylpropionamide. Tumors were measured three times from Day 7 to 14. On Day 14, the tumors were excised. As i l lustrated in Figure 4, the animals were necropsied for the presence of lung metastases on day 32, 1 8 days post excision. Tumors at day 14 were approx imately the same size for each treatment group, provid ing assurance that any difference in lung metastases found at the end of the experiment was not the result of different tumor sizes in the compared groups.
The data presented in Figure 12 demonstrate unprecedented anti-metastatic acti vity mediated in vivo through treatment of animal models of human disease using an exem plar)' anti-Id drug of the i nvention. AGX5 1 . As the data evince, in this study racem ic AGX 1 mediated more than a 50% reduction in the number of treated animals presenting with greater than five metastatic tumor foci. The data here are further shown to be consistent among multiple test samples, and are further consistent with other evidence of anti-Id efficacy (AG X5 1 -mediated Id binding disruption and ablation of Id protein in cel ls, and d isruption of Id-dependent cellular migration crucial for metastasis). By virtue of these and other study results herein. anti-Id compounds of the invention provide potent tools for treating, reducing and preventing initiation and progression of metastatic cancer in mammalian subjects. Example XVI
AGX5 1 Profoundly Reduces Breast Cancer Metastasis In Vivo
Using a Clinically Predictive Model of Anti-Metastatic Drug Efficacy This Example focuses on breast cancer intervention using a widely accepted murine model of clinical breast cancer treatment efficacy. Breast cancer metastasis is a leading cause of cancer mortal ity in humans worldwide. A common site of breast cancer metastasis is the lung. Whereas Example I I I above demonstrated therapeutic efficacy of AGX5 1 against metastasis of implanted lung tumors in m ice, the instant study measures anti-metastatic effects of AGX5 1 against breast cancer cells directly injected into the bloodstream of test subjects.
Breast cancer cel ls (4T 1 ) tagged with a luciferase gene were injected via tail vein into Balb/c m ice (5.0 x 1 04 cells per mouse, two groups of 5 mice). Development of king metastases was monitored by biolum inescent imaging. One day after tumor cel l injection, mice were treated twice dai ly, i.p., for 4 weeks with either dosing vehicle ( DMSO) (5 m ice) or 50 mg/kg N-(3-(benzo[d] 1 ,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N- ben/.ylpropionamiC e (AGX 1 ) (5 m ice). B iolum inescent imaging was carried out once a week. At the end of week four, the lungs were collected and analyzed for metastatic burden (Figure 1 3).
As shown in Figure 1 3, treatment with AGX5 1 profoundly reduced breast cancer metastasis in this d rect injection model. An unprecedented 40% of the anti-Id treated animals remained completely metastasis free. In the AGX5 1 -treated versus DMSO control subjects, there was greater than a five-fold reduction in that average number of metastases observed.
Example X VI I
Anti-Metastatic E ffects of AGX5 1 Are Stereospecific, Dom inantly
or Exclusively Mediated By The (-)-ANGX5 I Enantiomer
Based on the pioneering studies above revealing stereospecific anti-Id binding and Id degradation (ablation) activities of AGX 1 enantiomers, characterized by a profound anti-Id 1 dom inance of the (-)-AGX5 1 enantiomer, further investigations were undertaken to elucidate stereospecific effec .s of AGX5 1 affecting cellular processes within the metastatic cascade. Following "he methodology of Example V, above, anti-metastatic effects of racem ic AGX5 1 and both the (-)-AGX5 1 and (+)-AGX51 enantiomers were compared side-by-side.
Metastasis was assessed according to the protocol described in Example V, above, with 5 subjects receiving DMSO or racemic AGX5 I , and two more groups of 5 mice each treated with either the (-)-AGX5 1 or (+)AGX5 1 enantiomer. For these studies, the AGX5 1 enantiomers were purified using a different separation method that yielded a h igher level of enantiomeric enrichment (approximately 98% enantiomeric purity). Interestingly, the second enrichment methoc ("Enantiomer Separation Method B") resu lted in a reversal of elution profi le compared to the profile seen in the first Separation Method A (in Method B the ( -)- AGX5 1 enantiome r eluted first ("peak 1 ), and the (+)-AGX5 1 enantiomer eluted second ("peak 2"). These distinct preparative designs and elution profiles serve to further identi fy and eluc idate the respective physicochem ical properties of the (-)-AGX5 1 and (+)-AGX 5 1 enantiomers.
The instant studies revealed yet another dom inant anti-Id activity exhibited by the (-)- AGX5 1 enantiomer; namely a profound dominance, or exc lusive stcreospeci flc ity, for mediating anti-metastatic effects in vivo. Upon bioluminescent imaging, positive metastatic signal was observed in the lung regions of al l an imals receiving veh icle or the (+)-ANGX enantiomer (two of the animals in the (+)-AGX5 1 enantiomer group died prior to the imaging stage). In contrast, only 3 out of 5 m ice receiving racemic AGX5 1 , or the active, (-)-AGX5 1 enantiomer, exhibited detectable metastatic signals in the lungs.
A confirming study identical in procedure to the foregoing study has been abbreviated at a two-week prel im inary visual ization for inclusion in this writing. Th is study involves four animals per group, treated with DMSO veh ic le, racem ic ANGX5 1 . (-)-ANGX5 1 ("peak I "). or (+)-ANGX5 1 ("peak 2''). As shown in Figure 14, two of the four animals in both of the DMSO and (+)-ANG X5 1 ("peak 2") groups have developed extensive lung metastasis
(marked by prom inent biolum inescing fields in thoracic areas), whereas all animals treated with racemic ANGX5 1 and (-)-ANGX5 l ("peak 1 ") remain completely clear of metastatic signal. Example XVII I
Coordinate Anti-Cancer Drug Therapy-AGX5 1 and Taxanes M DA-MB-23 1 cells were trypsinized, centrifuged and re-suspended in a 50%
DPBS/50% atrigel solution to a concentration of 5 x 1 06 cel ls/50 μΕ and frozen. Prior to use, the frozen Matrigel was thawed at 4°C for 24 hours. Syringe, needle and cel ls are kept on ice until used. Prior to injections, cells are gently mixed by inversion and the needed volume drawn up into a co ld syringe equipped with a 25 x 5/8" gauge needle. The syringe is gently inverted to mix taking care to avoid bubbles.
50 Matrigel containing 1 0 x 1 06 MDA-MB 23 1 cel ls were injected orthotopical ly i nto the right, caudal mammary fat pad of four to six week old female nude m ice anesthetized by ketamine ( I 20mg/kg)/xylazine (6mg/kg) adm in istered im using a 26 x 3/8" gauge needle. Tumors were al lowed to establish to a volume of 1 00 mm , after wh ich an imals were randomized and treated ip with test compound or veh icle.
Fourteen days after implantation, the m ice were treated // with DM SO (vehicle), DMSO solutions of I 5mg/kg paclitaxel, I 5mg/kg, paclitaxel with either 6.7mg/kg, 20mg/kg. or 60mg/kg of AGX5 I or 60mg/kg of AGX5 1 alone for five days. Tumor vol umes were determ ined throughout the study using digital cal iper and the formula: tumor volume = ½ ( length x width-) where the greatest longitudinal diameter is the length of the tumor and the greatest transverse diameter is the width. After 4 1 days of dosing, the m ice were sacrificed by cervical dislocat ion and final tumor volumes and weights recorded and compared.
As shown in Figures 1 5 and 1 6, at day 19, paclitaxel alone decreased tumor growth by approximately 40%. AGX5 1 alone even at 60mg/kg, bid, did not affect tumor growth.
However, al l three AGX5 I doses— 6.7, 20 & 60mg/kg, bid sign ificantly decreased tumor growth, p<0.0 l , with the 60mg/kg dose providing the greatest e ffect w ith many tumors smal ler than at the start of the study.
Additional groups of m ice were treated w ith two other doses of pacl itaxel— 7.5mg/kg and 22.5mg/kg, q5d with 60mg/kg, bid, AGX5 1— were compared with the 1 5mg/kg, q5d, dose. The 7.5mg/kg dose provided no additional efficacy while the efficacy at 22.5mg/kg appeared no better than that observed with the 1 5mg/kg dose. Compared to the 25mg/kg dose used in the prelim ir ary study, the animals appeared to somewhat better tolerate the
22.5mg/kg dose although the mice experienced significant hypotherm ia and lethargy compared to the 1 5mg/kg group. In a second study, nude mice (n=5/treatment group except for the 60mg/kg, bid, AGX51 & paclitaxel doses (n=8) were again implanted with MDA231 human cancer cells. Fourteen days after implantation, the mice were treated ip with D SO (vehicle), I5mg/kg paclitaxel, or 15mg/kg, paclitaxel with 60mg/kg of AGX51. The effect of the 15 mg/k, q5d, dose of paclitaxel on the mice in this study was considerably more pronounced than in previous studies: severe lethargy, hypothermia and poor grooming for many days. The effect of the paclitaxel treatment alone on tumor growth was slow to develop. However, ultimately paclitaxel alone did decrease tumor growth by approximately 50% and the 60mg/kg. bid in these studies. AGX51 significantly (p<0.01) increased this effect, as shown in Figures 17 and 18.
Additional studies examined time of administration of AGX51 to maximize tumor regression. Nude mice (n=4/treatment group) were implanted with MDA231 human cancer cells. Fourteen da s after implantation, the mice were treated ip with DMSO (vehicle). 40mg/kg of N-(3-(benzo[d][l,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N- benzylpropionamide in a single dose per day or two 20mg/kg doses per day. The mean observed increase (± SD) in tumor volumes on Day 41 (final minus initial) were 242.69 ± 133.58 (qd) and 47.98 ± 86.31 mm3 (bid) and the difference was statistically significant (p<0.03).
Additional studies here further evaluated combinatorial anti-cancer efficacy of AGX51 and docctaxel. MDA-MB-231 cells were trypsinized, centrifuged and re-suspended in a 50% DPBS/50% Matrigel solution to a concentration of 5 x 106 cells/50 μί, and frozen. Prior to use, the frozen Matrigel was thawed at 4°C for 24 hours. Syringe, needle and cells were kept on ice until used. Prior to injections, cells are gently mixed by inversion and the needed volume drawn up into a cold syringe equipped with a 25 x 5/8" gauge needle. The syringe is gently inverted to mix taking care to avoid bubbles.
50 μΐ^ Matrigel containing 10 x 106 MDA-MB-231 cells were injected orthotopically into the right, caudal mammary fat pad of four to six week old female nude mice anesthetized by ketamine (120mg/kg)/xylazine (6mg/kg) administered im using a 26 x 3/8" gauge needle. Tumors are then allowed to establish to a volume of 100 mm3, after which animals are randomized and treeted ip with the test articles or vehicle. Fourteen days after implantation, the mice were treated ip with DMSO (vehicle), 15mg/kg of docetaxel q5d. or 15mg/kg of docetaxel q5d and 20mg/kg of AGX5 1 bid. As shown in Figure 1 9, the mean (± SD) increase in tumor volumes on Day 4 1 (final-initial) were 497.47 ± 63.08 (n=5) & 57.55 ± 87.63 mm3 (n=4), for docetaxel and docetaxel + AGX5 1 , respectively. The difference was significant (p<0.00 1 ).
Example X IX
Comparison of the Effectiveness of Inhibiting Id l Genes vs Id l Protein Id l knockout m ice were implanted with 1 06 LLC cells in Matrigel. The tumors were allowed to grow to approximately 350 mm3 and then treated (5/treatment group) with DMSO vehicle control, 1 5mg/kg, q5d. paclitaxel or 1 5mg/kg, q5d. pacl itaxel + 30mg/kg AG X 5 1 . bid 1 9d. Included in the study, designated M S4, were two additional groups: C57 B L6 m ice implanted as described above and treated with DMSO vehicle control or 30mg/kg AGX5 1 , bid 1 9d. As shown in Figures 20 and 2 1 , AGX5 I had no effect on the LLC tumors implanted into the C57BL76 m ice, (mean volume ± SD of 5374. 1 8 ± 1 12.25 mm3 for the DMSO treated animals versus 5 143.44 ± I 122. 1 7 mm3 for the AGX5 1 treated animals). Contrary to reports in the literature of a modest anti-growth effect, the Id l deletion in MS4 provided no anti-growth effect in the very rapidly growing LLC cel ls. Even more surprising, the paclitaxel (PAX) treatment also had on ly a very modest effect over the first 1 8 days of the study and on day 20, no effect. However, the combination of AGX5 1 + pacl itaxel did provide a signi ficant (p<0.0 l ) anti-growth effect.
Example XX
Safety of AGX5 1
Anti-Id compounds of the invention appear to specifical ly target Id proteins and mediate their potent anti-cancer and anti-metastatic effects without disrupting fundamental cell functions or conferring substantial toxicity, contrary to observations for most conventional chemotherapeutic drugs. To evaluate the safety profi le of AGX5 1 , 8- 1 week old, CD 1 mice (5/treatment group) were dosed q5d with DMSO, I 5mg/kg, qd, PTX, 60mg/kg AGX5 1 , bid or 1 5mg/kg, qd, paclitaxel + 60mg/kg AGX5 1 , bid (designated MS3).
Heparinized blood samples were obtained by retro-orbital puncture on Day 6, 12 hours after the last N-(3-(benzo| d][ 1 ,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzylpropionamidc (AGX5 1 ) dose. Tie plasma harvested from the blood was analyzed for standard clinical chemistry and hematology analytes and intact paclitaxel and AGX5 1 .
No adverse effects were observed of AGX5 1 on weight or cl inical chem istry (ALB . ALP, ALT, AMY, TB1L, BUN, Ca, PHOS, CRE, GLU, Na, K, TP & Glob) or hematology (WBC, Lym, Mon. Gra, RBC, Hg, Hematocrit & Platelet) values, compared to pacl itaxel treatment alone. Sim i larly, no effect was observed for AGX5 1 on these same measures compared to DMSO vehicle control. The data were obtained using an Abaxis VetScan analyzer (Un ion City, CA). Data for a representative set of these laboratory tests are provided in Figure 22.
Example XXI
Anl i-ld Compounds Protect Against Pathogenic Anaiogenesis
In Age-Related Macular Degeneration
The anti-Id and anti-angiogenic activity of AGX5 1 is demonstrated in the instant example to powerful ly protect against pathogenic vascularization in the retina of m ice, using a model system wicely accepted as predictive of disease mechanisms and drug efficacy for treating human age related macular degeneration (AMD).
The retinas of 14 mice with the Id3 deleted (n=9 control and n=5 mice) were dilated with phenylephrine/atropine for 1 0 minutes and then anesthetized with ketam ine/xylene (5 : 1 ) for 5 minutes prior 1o affecting the laser burns. A cover slip was placed on the surface of the eye (lower side) with a clear ophthalmologic med ia to act as a lens for the laser. A l ight was shone into the eye to visual ize the optical nerve and the neural retina. A fine laser was then focused unto the back of the retina, set perpendicular to the back of the eye. Four burns were placed 1 optical disc (size of the optical nerve) away from the optical nerve between blood vessels (avoiding blood vessels). The settings for the laser were as follows: Duration of 1 00 ms, intensity of 250 m W and a diameter of 50 m icrons. The laser burns travel through the neural retina and foc us on the retinal pigment epithelial (RPE) layer causing a rupture of Bruch's membrane. Immediately after the burn, a pocket of fluid forms around the burn and marks the spot of the burn. The pocket results from fluid expanding from the heat of the burn. The pocket eventual ly diminishes but a small burn spot is sti l l observed.
Two weeks after rupture of Bruch's membrane, the animals were sacrificed and their eyes harvested and placed in paraformaldehyde overn ight. The eyes were then washed in PBS and the RPE-choroid-sclera complex isolated from the neural retina. The complex (~200microns in thickness) was then incubated overnight in PBS containing Triton X- 1 00 and an anti-collagen IV antibody, followed by an overnight wash, then overnight incubation with A lexaFl uor-conjugated secondary antibody. The complex was then washed overnight and flat mounted in anti-fade medium for evaluation. Using the Z-line imaging with confocal m icroscopy, 4 mic ron sections were imaged from the top to the bottom of the com plex (-50 images). The central scar/vascularized area (~ middle of the complex) was then manual ly outl ined and the images independently analyzed for background levels of fluorescence.
Background fluorescence was subtracted from the outl ined area of each image and then each area is analyzed for relative fluorescence. The total fluorescence was then calculated. Each animal, with the 4 burns, is an n of 1 . Data was expressed as the mean lesion volume ± standard error of the mean (SEM) and analyzed using S PSS software. Statistical sign i ficance of the differences between the experimental groups was calculated using one-tailed Student' s t-test of the volumes. As shown in Figure 23, Id3 deletion impairs neovacularization fol low ing laser induced disruption of Bruch's mem brane, indicating Id3 is involved at least in part in ful l development of a pathogenic angiogenic response in this model system of AM D. This model is widei y accepted and has been used successful ly to develop numerous anti- AMD drugs.
The mean CNV area for the Id3 deleted animals was approximately hal f of the area of the wild type anima ls, and the result was highly signi ficant statistical ly (p<0.00 l ).
A comparab le protective anti-AMD effect (as seen in the above Id genetic knockout study) has now surprisingly been achieved using a small molecule anti-Id drug of the invention, ANGX5 ', . Even more unexpected, the anti-A MD (anti-angiogenic) effects of ANGX5 I were observed in subjects treated intravitrcally (directly into the affected eye), as well as intraperitoneal ly (requiring the drug to transit to the retinal tissue in a stable, effective state). Yet more pioneering still, the data here convincingly show that the anti-angiogen ic and anti-AMD effects of AGX5 1 are stereospecific, with activity residing principal ly or exclusively with the novel (-)-AGX5 1 enantiomer.
In reference to Figure 24, (-)-AGX 1 was administered intravitreally (ivt) using the same methods as above. C57BL M ice (5/Treatinent Group) had CN V by laser treatment on day 1 . The test mice were administered the (-)-enantiomer of AGX5 1 ("E2") ( 10 μg), while controls were administered vehicle (70% DMSO/30% Water). Adm inistration was by ivt, one hour post laser and on day 7. Sacrifice and lesion size measurements (performed using fluorescein-labeled dextran) were conducted on day 14.
In reference to Figure 25, (-)-AGX5 1 was administered intraperitoneal ly (ip) using the same methods as above. C57BL Mice ( 1 0/Treatment Group) had CN V by laser treatment on day 1 . The test mice were administered the (-)-enantiomer of AGX5 1 ("Έ2") (20 mg/kg, qd) two hours post laser and then 20 mg/kg, bid, daily for 14 days, with control mice receiving vehicle (70% DMSO/30% Water). Sacrifice and lesion size measurements were completed on day 14.
The foregoi ng studies show powerful anti-angiogenic and anti-AMD mediated by an exemplary anti-ld c ompound of the invention, ANGX5 1 . Potent anti-Id activity is again demonstrated by th^ (-)-AGX5 1 enantiomer employed in these studies. Comparing Figures 23-25, it is apparent that (-)-AGX 1 mediates comparable anti-angiogenic and anti-A M D effects as is obtained by ld3 genetic knockdown. In both anti-ld drug tests (Figures 24 and 25 ), the extent of anti-angiogenic activity observed corresponded to a greater than 50% reduction of pathogenic angiogenic defects noted in the subjects, predicting a clinically very effective intervention provided by the invention for treating AM D and other pathogenic angiogenic diseases in humans. The nearly identical activity shown by (-)-AGX5 1 adm inistered by the remote route o f intraperitoneal (ip) adm in istration (Figure 25 ), com pared to the direct route c f intravitreal ( ivt) del ivery, portends an unprecedented efficacy of this drug for reaching remote clinical targets in in the body, such as dense tumors, CN S targets, and other compartments that are isolated or refractory to standard drug
dclivery/bioavailability.
Example XXI I
Cancer Treatment and Metastatic Disease Management
Aided by Novel Anti-Id Diagnostic Tools
The invention also provides novel compositions, methods and kits for detecting and quantifying Id proteins, such as ld l , in biological samples for diagnosis and reflexive treatment of proliferative diseases, including cancer. These methods can be effectively integrated as well into novel therapeutic methods coordinating Id detection in patients wi th modifications in treatment regiments to achieve sensitive and highly successful clinical management of cancer patients and other subjects amenable to anti-ld treatment as described herein. For clinical applications of the invention, it is important to determ ine anti-Id effects in treated patients on a continuing basis. Since Id proteins are degraded when Agx5 1 disrupts the interaction of Id with E proteins, measuring Id I loss in patients provides useful information to modulate treatment and manage status of these patients. Id levels are elevated in the serum of both experimental mice bearing tumors, as well as in breast cancer patients. High levels of Id in these circumstances are likely associated with circulating EPCs and/or circulating tumor cells that lyse during preparations of sam ples, or spontaneously. Therefore, a simple blood test to determ ine if Id I levels are reduced in serum of patients treated with anti-Id compounds of the invention will allow ongoing assessment of the efficacy of anti-Id compounds for reaching clinical trials. On the basis of these measurements, cl inical management can be tailored to individual patient's disease status and treatment response (e.g., by lowering anti-Id drug dosing upon determ ination of extensive lowering of Id levels upon initial, successful drug treatment, thereby reducing potential, unnecessary treatment costs and/or side effects).
Practical implementation of reflexive diagnostic methods herein provide coordinately integrated diagnostic/treatment cl inical management tools, i llustrated by the fol lowing Example. Assay methods are uniquely modified for detecting of Id targets in biological samples, lor example body fluids (e.g., urine, plasma, CNS fluid, expressed or extracted mammary fluid, etc.), tissues (e.g., bone marrow, blood, tumor biopsy specimens), and other diagnostically useful sample materials. A schematic representation of such a novel assay is provided in Figure 26. This assay employs modified "sandwich" techniques, whereby a highly specific monoclonal antibody against Id l is bound to microtiter we l ls and then exposed to a test sample (e.g., serum) from a known or at-risk cancer screen subject, to al low binding of Id l protein in the sample by the anti-Id monoclonal Ab. A fter washing the plates are exposed to E47 protein conj ugated to horse radish peroxidase ( E47-I I R P). Accord ing to this novel assay construction, Id l proteins from the sample bound to the monoclonal Abs bind and retain the MRP-labeled E47 in direct proportion to an amount of Id I original ly present in the samp le. E47-H RP bound to the plate is then exposed to 3.3',5,5'- Tetramethylbenzid ne (TMB), a chromogenic substrate of H RP for colorimetric quantitation.
The foregoing Id l -detection assay is demonstrated here ith human breast cancer patients and normal subject controls to faithfully discriminate between cancer bearing and cancer patients—thus affording sensitive and powerful clinical diagnostic, treatment and management tools.
According to the assay design il lustrated in Figure 26, EL 1 SA m icrotiter plates were coated with known amounts of anti-Id 1 -mouse monoclonal antibody (BD Pharm ingen San Diego, CA). 100 yiL Id l standards ( 1 0X, 20X, 40X, 50X, 1 60X, 320X and 640X (Id l fortified mouse sera)) were dispensed into wells. The solution was then thoroughly m ixed for 30 seconds and incubated at 4°C for 16 hours. After 16 hours, the incubation mi xture was removed by emptying the plate contents into a waste container. The microtiter plate was rinsed 5 times with distil led water and struck sharply onto absorbent paper to remove al l residual water droplets. 1 00 μί of E47 HRP Conjugate Reagents ( 1 :25 or 1 :50) was dispensed into each well and thoroughly mixed for 30 seconds. The plate was then incubated at room temperature for 2 hours after wh ich the incubation was dumped out an the m icrotiter plate was rinsed 5 times with distil led water before being struck sharply onto absorbent paper to remove all residual water droplets. 1 00 of TM B reagent was then dispensed into each wel l and gently mixed for 5 seconds. The microtiter plate was then incubated in the dark at room temperature for 20 minutes. After 20 minutes, the reaction was stopped by adding 1 00 μΐ , of Stop Solution to each wel l. The solution was gently mixed for 30 seconds and the absorbance was read at 450nm within 1 5 minutes. The resulting absorbance values were l t to a ichaelis- er ton hyperbolic equation using WinNonlin (Pharsight, Mountainvicvv, CA). The correlation coefficient for the fit of the concentration data to the absorbance values was >0.99.
A total of 42 p53 knockout mice were maintained according to standard cond itions. They were housed in plastic cages with hardwood chips in an air-cond itioned room with a 1 2 h light— 12 h dark cycle and were given basal diet (Oriental NMF; Oriental Yeast Co., Tokyo. Japan) and water ad libitum. Serum samples were obtained from the m ice. 1 00 μ ΐ , of serum from each mouse was then added to three wells on microtiter plates coated with known amounts of anti-Id 1 mouse monoclonal antibody (BD Pharmingen. San Diego, CA). The solution was then thoroughly m ixed for 30 seconds and incubated at 4°C for 1 6 hours. A fter 1 6 hours, the incubation m ixture was removed by emptying the plate contents into a waste container. The mic rotiter plate was rinsed 5 times with disti lled water and struck sharply onto absorbent paper to remove all residual water droplets. 100 μί of E47 HRP Conjugate Reagents ( 1 :25 or 1 :50) was dispensed into each wel l and thoroughly mixed for 30 seconds. The plate was then incubated at room temperature for 2 hours after which the incubation was dumped out an the microtiter plate was rinsed 5 times with distilled water before being struck sharply onto absorbent paper to remove all residual water droplets. 1 00 μΐ^ of TMB reagent was then dispensed into each well and gently mixed for 5 seconds. The microtiter plate w as then incubated in the dark at room temperature for 20 m inutes. After 20 minutes, the reaction was stopped by adding 1 00 \x of Stop Solution to each well. The solution was gently m ixed for 30 seconds and the absorbance was read at 450nm within 1 5 minutes.
After serum samples were obtained, the m ice were sacrificed and analyzed for tumors. As shown in Figure 27, the dividing line between tumor bearing and tumor free animals was calculated using an evaluation by Receiving Operation Characteristic ( ROC) curves. The ROC evaluation of the 42 concentration values predicts only one false positive and one f alse negative.
The powerful diagnostic utility of ld-detection assays within the invention was further demonstrated by applying the foregoing tools and methods for a breast cancer screen ing qual i ficat ion in to popu lations of human female patients presenting with, and without, breast cancer. 1 5 serum samples were obtained from women with either normal health or advanced breast cancer (BioCheck, Inc., Foster City, CA). In a blind analysis, 1 5 1 00 samples were added to three well s on microtiter plates coated with known amounts of anti-Id 1 mouse monoclonal antibody (BD Pharmingen, San Diego, Ca). The solution was thorough ly m ixed for 30 seconds and incubated at 4°C for 16 hours. After 1 hours, the incubation m ixture was removed by emptying the plate contents into a waste container. The m icrotiter plate was rinsed 5 times with distil led water and struck sharply onto absorbent paper to remove all residua! water droplets. 1 00 μL· of C47 I IRP Conjugate Reagents ( 1 :25 or 1 :50) w as d ispensed into each well and thoroughly mixed for 30 seconds. The plate was then incubated at room temperature for 2 hours after which the incubation was dumped out and the plate was rinsed 5 times with distil led water before being struck sharply onto absorbent paper to remove all residual water droplets. 1 00 of TMB reagent was then dispensed into each wel l and gently mixed for 5 seconds. The plate was then incubated in the dark at room temperature for 20 minutes. After 20 minutes, the reaction was stopped by add ing 1 00 μ ΐ. of Stop Solution to ea:h wel l. The solution was gently m ixed for 30 seconds and the absorbance was read at 450nm within 1 minutes. As shown in Figure 28, there was an extraordinary high accuracy of these assay results for distinguish ing between cancer-positive and normal samples.
In more detailed diagnostic and reflexive, integrated diagnostic-treatment methods of the invention, patients may be primarily or comp!ementariiy screened using any of a number of other specific cancer markers (e.g., using prostate-specific antigen (PSA) to screen for elevated prostate cancer risk). B iopsy, optionally coupled with histopathological and/or immimohistochem ical analysis may further refine primary or integrated diagnoses. In certain patients, add itional tests as described herein wil l further refine diagnostic assessments to focus on metastatic risk (e.g.. by measuring Id l in identified tumor samples, by measuring EPCs or cancer stem cells in tumors, blood, marrow (or their markers in other samples such as urine, sal iva, or CNS fluid), etc.
I l lustrating reflexi ve diagnostic-treatment methods of the invention, subjects with organ confined cancer may be selected for treatment employing an anti-Id compound administered in a prolonged, prophylactic regimen (e.g., daily for periods extending for multiple months, six-nine months, 1 -2 years, 2 years or longer), with routine follow-ups to assess maintained organ confinement of the prostate cancer. This regimen al lows long-term suppression of metastasis of the cancer to avoid unnecessary trauma and quality of l ife costs that attend traditional surgical and chemotherapy cancer treatments. In conj unction with this novel treatment re imen, regular blood testing is conducted in the patients to test for levels of Id, as described above, particularly elevated ld l and/or Id3, coupled with decisions based on these assay results. Reduced, static or elevated Id expression from an early compared to a successive treatment stage will often direct corresponding changes in treatment dosing, or treatment repertoire (including switch ing to more or fewer treatment agents or moda l ities, or more or less potenf'aggressive interventions, general ly balancing d isease risk, current and projected treatment efficacy, and adverse patient impacts).
Reduced or stable Id levels may be relied upon as a future treatment and/or prognostic indicator, and may be determinative of a continuing treatment decision to maintain, or even suspend, anti-Id treatment as successful . Further elevations of Id during a treatment history might indicate a treatment decision to increase anti-Id dosing using the methods and compositions of the invention, and/or to initiate combinatorial therapy using an anti-Id compound and a secondary anti-cancer or anti-metastatic drug or alternate treatment modality (e.g., chemotherapy and/or radiation). Combined with this management paradigm, additional methods of the invention wi l l employ other, conventional methods and tools to diagnose and manage patients in a coordinate management regime, for example using follow-up assays for blood cancer markers, radiologic and/or histopathologic screening to continue monitoring for metastatic disease development, etc., to further refine and manage monotherapy with anti-Id compounds and combinatorial therapies as described herein.
The foregoing coordinate diagnosis and management protocols can be modified and employed to treat p rostate cancer patients coordinately with both anti-Id methods and compositions and a secondary, anti-testosterone drug or therapy. Alternatively, prostate- speci fic radiation may be employed prior to, concurrent with, or subsequent to the anti-Id treatment.
The foregoing coordinate diagnosis and management protocols may be modi fied and employed to treat b reast cancer or prevent metastatic progression of existing breast cancer. Patients are identifi ed by routine mammography in human females positive for a breast tumor, followed by tumor excision where no cancer is found in the lymph nodes. f3ecause the cancer appears local ized, the subject is administered an anti-Id compound chronically to suppress metastasis of the cancer to avoid, perhaps, unnecessary trauma including greatly lowered qual ity of l ife associated with chemotherapy as well as the possibi lity of
development of a neoplasm later in life because of the chemotherapy (Vega-Stromberg, 2003). A blood test in the patient show ing elevated Id, particularly elevated Id I and/or ld3, will support a decision to treat with an anti-Id compound, because such indic ia suggest a positive environment in the body for metastasis. In related exemplary embodiments, patients within this management scheme may be coordinately treated with an anti-Id compound and chemotherapy, and 'or breast-speci fic rad iation, before, concurrent with, or subsequent to the anti-Id treatment.
Coordinate diagnosis and management protocols of the invention can also be employed to treat colon cancer or prevent metastatic progression of an existing colon cancer. Patients are selected by routine colonoscopy revealing a tumor in suitable treatment subjects, which are further selected fol lowing surgery (and/or histopathological testing) indicating that the colon cancer likely remains non-metastatic. These subjects are adm inistered an anti-Id compound chronical ly to suppress metastasis of the cancer to avoid, perhaps, unnecessary trauma including lowered quality of life associated with conventional chemotherapy. A blood test in the patient showing elevated Id, particularly elevated Id l and/or Id3, supports a a decision to treat with an anti-Id compound, because such a measurement suggests a positive environment in the body for metastasis. In related embodiments, patients within this management scheme may be coordinately treated with an anti-Id compound and
chemotherapy, and^or gastrointestinal (Gl)-specific radiation, before, concurrent with, or subsequent to the anti-Id treatment.
Other coordinate diagnostic and management protocols are employed to treat melanoma. Subjects are diagnosed with melanoma in situ (the tumor remains in the epiderm is, the outermost layer of skin.) The melanoma is excised by surgery and the subject is thereafter admini stered an anti-Id compound chronically to suppress metastasis of the cancer to avoid, pe rhaps, unnecessary trauma and lowered qual ity of li fe associated with chemotherapy or other conventional cancer treatments. A blood test in the patient showing elevated Id, particularly elevated Id I and/or Id3, supports a decision to treat with an anti-I d compound, as th is Ending is associated with a positive environment in the body for metastasis.
In yet another exemplary coordinate diagnosis and management protocol, female human are selected presenting with Stage I I I (metastatic), breast cancer. Subjects thus identified are treated using an aggressive, combinatorial treatment regimen employing high doses of rad iation and multiple doses of Taxol® (paclitaxel ). After th is primary treatment, histopathology and'Or bioscans , (e.g., computerized tomography, positron em ission tomography and/or magnetic resonance imaging) are used to identi fy patients presenting with no detectable residi ng cancer. These subjects are then treated for a prolonged period (e.g., six-nine months, 1 -2 years or longer) with an anti-Id compound, in a prophylactic treatment regimen to block re-seeding of any remaining cancer cel ls to a distant organ (e.g.. brain or l iver). During this management phase, measurements of Id in the blood of the patient are performed periodical ly. After another management period of several months to one year or more, Id blood levels may be observed to increase, indicating an increase in metastatic potential. Treatment with the anti-Id compound is then increased in dosage or periodicity ( or resumed). Thereaf ter when Id blood levels decrease to a value consistent w ith an acceptable (baseline or low-risk) metastatic potential the anti-Id treatment is reduced. Th is cycle of monitoring and reflexive intervention is maintained throughout the l ife of the woman. Example XXIII
Separation of N-(3-(benzo d [ I 1dioxol-5-yl)-3-(2-iriethoxyprienyl)propyl)-N- benzylpropionamide Enantiomers
Individual (+)- and (-)-enantiomers of AGX51 are obtained from a racemic (achiral) AGX51 composition using preparative HPLC with a chiral column. Separations on chiral stationary phases proved effective to obtaining enantiomers in acceptable purity.
Two different HPLC systems were designed and implemented here, referred to as "Enantiomer Separation Method A" and "Method B". Both methods were designed and adjusted to provide provide high mg or low gram amounts of the AGX5 1 enantiomers for biologic testing. Both methods employ supercritical CO2 with an organic alcohol as the eluting solvent. However, the methods use different columns: Method A uses a Pirkle- concept stationary phase [ l -(3. 5-dinitrobenzamido)- l , 2, 3, 4-tetrahydrophenanthreneJ and Method B uses a polysaccharide stationary phase (cellulose with the free hydroxy! groups derivatized with 3, 5-dimethylphenylcarbamate).
The two methods are described in further detail and compared in the Tables 4 and 5 below.
Figure imgf000100_0001
In both methods, product isolation post fraction collection is provided using a rotovap at reduced pressure at a water bath temperature of approximately 40°C. The resulting gum can be used as is, or the enantiomer gum following isolation can be crystalized with some loss of product from methanol-hexane or ethyl acetate-hexane. Purity of the isolated enantiomers is determined by analytical HPLC using similar HPLC conditions to those used to produce the enantiomers.
Figure imgf000101_0001
Intermediate from the Milligram to the Kilogram Scale. Journal of Chromatography A. 1999; 849: 309-317. Brittain HG, Applications of chiroptical spectroscopy for the characterization of pharmaceutical compounds, Journal of Pharmaceutical and Biomedical Analysis 1998;
17; 933-940.
The enantiomers prepared by either Method A or Method B show identical LC/MS
(electrospray ionization) profiles, i.e.. MH+=432, and 400 MHz proton NMR spectroscopy, i.e., 1 hydrogen triplet at 4. 18 ppm (benzyl ic hydrogen), 12 hydrogen signals in the 6.6 to 7.4 ppm aromatic region, 3 hydrogen triplet centered at 0.96 ppm (propionyl methyl), 3 hydrogen multiplet centered at 3.73 (methoxy group), 2 hydrogen unresolved multiplet centered at 5.9 ppm (methylene in the 1 ,3 dioxole moiety), 2 hydrogen multiplet centered at 4.50 ppm (benzyl methylene attached to the nitrogen), 2 hydrogen multiplet centered at 3.07 ppm (second methylene attached to the nitrogen), multiple peaks corresponding to 4 hydrogens (methylene adjacent to the asymmetric carbon and methylene adjacent to the carbonyl in the propionyl moiety).
The enantiomers produced by both Method A or Method B were evaluated by polarimetry to establish the optical rotation for each enantiomer. The ability of individual enantiomers among enantiomer pairs to rotate polarized light in opposite direction to the rotation observed with the other enantiomer in the pair is universally accepted as a discrete characterization to unambiguously differentiate and quantitate enantiomers. The polarimetry evaluation repeatedly and consistently conducted here used a Jasco P-2000 with a dichrom polarizer. The concentration of each enantiomer was approximately 1 % in methanol, the light source was a lungsten-halogen lamp and the wavelength of the emitted light was 589 nm. At 20°C, the enantiomers under the conditions described above rotated the polarized light either+28 degrees or -28 degrees.
HPLC prof les for analysis of racemic (achiral) AGX51 by Method A and Method B are provided in Figures 29 and and 30, respectively. The peaks in these chromatograms are labelled as to whether the individual peak rotated the polarized light in a (+) or (-) direction in the polarimetry identification. Interestingly, the order of elution for the enantiomers differs for Method A and Method B. In Method A, the first ekiting peak is the (+)-enantiomer and in Method B the first eluting peak is (-)-enantiomer.
Example XX IV
Biotransformation products of AGX5 1
Further i l lu stration of the invention is presented here through descri ption of in vivo biotransformation products of the exemplary anti-Id compound AGX5 1 . CD ! male mice (3/timepoint) were treated ip with 30mg/kg AGX5 1 in DMSO. B lood was col lected in heparinized tubes by retro-orbital puncture pre-dose and at 0.25, 0.5, 1 .0, 2.0. 4.0, 8.0 & 24 hours post dose. One hundred μΕ of pi I 7.4 PBS were added to the plasma harvested from the blood by centri ugation and the samples from all time points, except pre-dose, combined. The m ixture was vortexed for one minute with 1 mL methyl t-butyl ether (MTB E), and the MTBE removed after centrifugation with dry N2 (g). The residue remaining a ter removal of the MTBE was reconstituted in 200 of ACN. Five iL of this solution was analyzed by LC7M S using a 2.0 x 250 mm C 1 8 column coupled to an electrospray EC-MS set to monitor m/z 1 00-600 in the H PLC effluent. The flow rate was 200 μΕ/minute of ACN ( 85)/H20 + 0. 1 % form ic acid.
The mass spectral data was carefully screened for MH+ ions reflecting common biotransformation routes such as dealkylation, hydroxylation, etc. Ions corresponding to demethylation, M FI+ = m/z 41 8 (M l ), depropionylation, M H+ = m/z 376 ( M2) and demethylation + depropionylation, MH+ = m/z 348 (M3), were found and three structures. M l , M2 and M3 as shown below were identified.
Figure imgf000103_0001
-(3-Hbenzo[^[ -iV-benzyiproo(onamide
Figure imgf000103_0002
N-(3-(benzo(d] 1,3]dioxoi-5-y -3-(2-hydfoxyphenyi)propyl)-/V-b€nzyipropionaiinfde
Figure imgf000103_0003
-( 1 -(benzo[rf][1.3]dioxo]-5-yl)-3-(benzyiamino)propyl)pheno!
lOl All publications and patents cited within this specification and listed below are herein incorporated by reference for economy of description, for all purposes of disclosing and describing the methods and/or materials in connection with which the publ ications are cited. The citation of any publication herein should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publ ication dates which may need to be independently confirmed.
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Claims

We claim:
Claim 1 . A method for reducing metastasis in a mammalian subject suffering a neoplasm comprising administering an anti-metastatic effective amount of an anti-Id (inhibitor of differentiation) compound of Formula 1 to said subject;
Figure imgf000127_0001
wherein R i . R , Rs, Re, Rs, R9, Rio are independently selected from the group consisting of hydrogen, hydroxy!, sulfyhydryl, benzyl, 2-bromovinyl amino, hydroxymethyl. methoxy, halogen, pseudohalogcn, cyano. carboxyl, nitro, thioalkyl, tliioaryl, thiol, substituted or unsubstituted hydrocarbons contain ing 1 to 20 carbons, alkoxycarbonyl.
alkoxycarbonylamino. amino, amino acid, aminocarbonyl, aminocarbonyloxy. aryloxy. carboxyl, cvcloalkcnyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted hetcroaryl. substituted or unsubstituted aralkyl, peptidyl, dye, fluorophore. carbohydrate or polypeptidyl ; Rs and Ry can also have a para configuration in addition to the ortho orientation shown in Formula 1 ; R7 is independently selected from hydrogen, hydroxy!, methoxy. azido, nitri le, sul fhydryl, halogen , benzoyl, substituted benzoyl or hydroxyl substituted w ith substituted or unsubstituted hydrocarbon containing 1 to 20 carbons, alkanoyl of a main chain of 1 to 20 carbon atoms, CF3( H2)n CO where n= l to 10, CI-b<CF2)n C=0 where n= l to 1 0. CF3(CF2)n where n=0 to 3, aryl, alkoxy, halogen, or n itro, aryloxy, esters of aryloxy. adamantoy l.
substituted adamantoyl or aroyl of 1 to 20 carbons; R7 cannot be methoxy if n is propionyl while R? and Rd can be ortho, as shown in Formula 1, or para to one another; R i 1 is independently hydrogen, propionyl, pivoyl, benzoyl, substituted benzoyl, al kanoyl of a main chain of I to 20 carbon atoms, CF3(CH2)n C=0 where n= l to 1 0, CH3(CF2)n C=0 where n= l to 1 0 or CF3(CF2)n where n=0 to 3 and R l 1 cannot be propionyl if R7 is methoxy; R 12 is independently hydrogen, halogen, 2H, CH3(CH2)n vvhere n=0 to 5. CF3(CH2)nC=0 where n= l to 5; CH3(CF2)n C=0 where n= l to 5 or CF3(CF2)n where n=0 to 5.
Claim 2. The method of claim 1 , wherein the anti-ld compound is racem ic N-(3- (benzofd][ l ,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzylpropionam ide (AGX5 1 ).
Claim 3. The method of claim 1 , wherein reduction of metastasis mediated by the anti-ld compound yields a greater than 20% increase in cancer free survival for anti-Id treated subjects compared to non-treated control subjects.
Claim 4. The method of claim 1 . wherein reduction of metastasis mediated the anti-Id compound yields at least a 20-50% increase in cancer free survival for anti-Id treated subjects compared to non-treated control subjects.
Claim 5. The meth od of claim I . wherein reduction of metastasis mediated by the anti-ld compound yields at least a 20% average decrease in average size or number of organ metastases in anti-Id treated subjects compared to non-treated control subjects.
C laim 6. The meth od of claim 1 , wherein reduction of metastasis mediated by the anti-ld compound yields a 20-50% or larger decrease in average size or number of organ metastases in anti-Id treated subjects com pared to non-treated control subjects.
C laim 7. The method of claim 1 , wherein reduction of metastasis mediated by the anti-Id compound yields at least a 50% decrease in average size or number of organ metastases in anti-ld treated subjects compared to non-treated control subjects.
Claim 8. The method of claim 1 , wherein the anti-Id 1 compound is a metabol ite of N-(3- (benzo[d][ 1 ,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzylpropionamide (AGX5 1 ) selected from the fol lowing (Formula I I): N-(3-(benzo[c ][ K3]d ioxol-5-yl)-3-(2- hydroxyphenyl)propyl)-N-benzylpropionamide (Formula I la), (3-(benzo[i/] [ l ,3]dioxol-5-yl)- N-benzy!-3-(2-inethoxypheny!)propan-l -amine (Formula lib), 2-(l-(benzo[(/][],3]d yl)-3-(benzylamino)propyl)phenol (Formula 11c) or an enantiomer of one of the
aforementioned metabolites, or an isolated enantiomer of one of said metabolites.
Figure imgf000129_0002
Figure imgf000129_0001
Claim 9. The method of claim I, wherein the anti-Id compound is a salt of'N-(3- (benzo[d][ l,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzylpropionamide (AGX5 I ), (3-(benzo[d][ 1 ,3]dioxol-5-yl)-N-bcnzyl-3-(2-metlioxyphenyl)propan- 1 -amine, or 2-( 1 - (benzo[d][l,3]dioxol-5-yl)-3-(benzylamino)propyl)phenoI.
Claim 10. The method of claim 9. wherein the salt is a hydrochloride, hydrobromide.
hydroiodide, phosphate, sulfate, oxalate, malate, maleate or succinate salt.
Claim 11. The mefiod of claim 1, further comprising coordinately administering a secondary anti-metastatic agent selected from a chemothcrapeutic agent, a vascular disrupting agent (VDA), an anti-angiogenic agent, or an HSP90 inhibitor.
Claim 12. A pharmaceutical composition comprising an enantiomerically-cnriched, anti- metastatically active (-)-enantiomer of N-(3-(benzo[d][1.3]dioxol-5-yl)-3-(2- methoxyphenyl)propyl)-N-benzylpropionamide ((-)-AGX51), in a dosage form comprising the
(-)-AGX51) enantiomer in an effective amount to reduce or prevent one or more symptom(s) of a proliferative disorder or metastatic disease in a mammalian subject.
Claim 1 3. The pharmaceutical composition of claim 12, wherein the composition has an enantiomeric purity of the (-)-AGX5 1 enantiomer of 60% enantiomeric excess (ee) or greater.
Claim 14. The pharmaceutical composition of claim 1 2, wherein the composition has an enantiomeric purity of the (-)-AGX5 1 enantiomer of 90% enantiomeric excess (ee) or greater.
Claim 1 5. The pharmaceutical composition of claim 12, w herein the composition has an enantiomeric purity of the (-)-AGX5 1 enantiomer of 98% enantiomeric excess (ee) or greater.
C laim 1 6. The pharmaceutical composition of claim 1 2, wherei n the anti-metastatical ly active, substantially pure (-)-AGX5 1 ) enantiomer is formulated for oral, buccal, nasal, aerosol, topical, transdermal, mucosal, or injectable del ivery.
Claim 1 7. The pharmaceutical composition of claim 12, wherein the anti-metastatical ly active, substantial ly pure (-)-AGX5 1 ) enantiomer is formulated in a sustained release mucosal delivery formulation, or a sustained release topical delivery formulation or patch device.
Claim 1 8. The pharmaceutical composition of claim 1 2, wherein the anti-metastatical ly active, substantially pure (-)-AGX5 1 ) enantiomer is formulated in an effective delivery form to provide sustained anti-mctastatic effective plasma levels of (-)-AGX5 I ) in human subjects over a period of approximately 8 hours or longer, wherein the sustained anti-metastatic efficacy correlates with sustained reduction of circulating plasma levels of Id 1 or ld3 in treated subjects.
Claim 1 9. The pharmaceutical composition of claim 12, comprising an anti-metastatic effective daily dose of between about 1 00 mg to about 900 mg of the (-)-AGX5 I ) enantiomer.
C laim 20. The pharmaceutical composition of claim x, comprising an anfi-metastafic effective dose selected from a 50- 1 00 mg dose, a 1 00-200 mg dose, a 150-300 mg dose, a 300-400 mg dose, and a 400-600 mg dose, anti-metastatical ly effective on adm inistration 1 -3 times per day.
Claim 2 1 . A pharmaceutical composition comprising an cnantiomerically-cnriched, anti- metastatical ly active (-)-enantiomer of N-(3-(benzo[d][ l ,3 jdioxol-5-yI)-3-(2- methoxypheny l)propyl)-N-benzylpropionam ide ((-)-AGX5 1 ), and a secondary anti-metastatic agent selected from a chemotherapeutic agent, a vascular disrupting agent (VDA), an anti- angiogenic agent, or an HSP90 inhibitor.
Claim 22. A method for treating and pathogenic vascular prol iferative disease in a mammalian subject, comprising administering an anti-angiogenic effective amount of an anti- Id compound of Formula I to said subject;
Figure imgf000131_0001
wherein R i, R4, R5, Ri>. Rx, R , R io are independently selected from the group consisting of hydrogen, hydroxyl, su lfyhydryl, benzyl, 2-bromovinyl am ino, hydroxymethyl. methoxy. halogen, pseudohalogen, cyano, carboxyl, nitro, thioalkyl, thioaryl, thiol, substituted or unsubstituted hydrocarbons containing 1 to 20 carbons, alkoxycarbonyl,
alkoxycarbonylam ino, am ino, am ino acid, aminocarbonyl, aminocarbonyloxy, arylo.xy, carboxyl, cycloalkenyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, su bstituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, peptidyl, dye, (luorophore, carbohydrate or polypeptidyl ; Rx and R9 can also have a para configuration in add ition to the ortho orientation shown in Formula 1 ; R7 is independently selected from hydrogen, hydroxyl, methoxy, azido, nitri le. sulfhydryl, halogen, benzoyl, substituted benzoyl or hydroxyl substituted with substituted or unsubstituted hydrocarbon containing 1 to 20 carbons, alkanoyl of a main chain of 1 to 20 carbon atoms, CF3(CH2)nCO where n=l to 10, CH3(CF2)nC=0 where n=l to 10, CF3(CF2)n where n=0 to 3, aryl, alkoxy, halogen, or nitro, aryloxy, esters of ar loxy, adamantoyl.
substituted adamantoyl or aroyl of 1 to 20 carbons; R7 cannot be methoxy if RM is propiony! while R7 and e can be ortho, as shown in Formula I, or para to one another; Ri 1 is independently hydrogen, propionyl, pivoyl, benzoyl, substituted benzoyl, alkanoyl of a main chain of 1 to 20 carbon atoms, CF^CH^C ) where n=l to 10, CH3(CF2)nC=0 where n=I to 10 or CF.-)(CF2)i, where n=0 to 3 and RI 1 cannot be propionyl if R7 is methoxy; R|2 is independently hydrogen, halogen, 2H, CH3(CH2)n where n--0 to 5, CF3(CH2)nC=0 where n=l to 5; CH3(CF2)nC=0 where n=l to 5 or CF3(CF2)n where n=0 to 5.
Claim 23. The method of claim 22, wherein the anti-Id compound is racemic N-(3- (benzo[d][l,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzylpropionamide (AGX5 I ). or an isolated (-)-AGX51 enantiomer.
Claim 24. The method of claim 22, wherein the anti-Id compound is selected from racemic N-(3-(benzo[dj[l,3|dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzylpropionamidc, N-(3- (benzofilf l,3]dioxol-5-yl)-3-(2-hydroxyphcnyl)propyl)-V-benzylpropionamide (Formula Ila), (3-(benzo[f/J[[,3Jdioxol-5-y[)-N-benzyl-3-(2-methoxyphenyl)propan-I -amine (Formula lib), 2-(\-(benzo[i/]f l,3]dioxol-5-yl)-3-(bcnzylamino)propyl)phenol (Formula lie), or an isolated enantiomer thereof.
Figure imgf000132_0002
Figure imgf000132_0001
Claim 25. The method of claim 22, wherein the anti-Id compound is in a salt form selected from a hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, oxalate, malate, maleate or succinate salt.
Claim 26. The method of claim 22, wherein the pathogenic vascular proliferative disease is an ocular disease.
Claim 27. The method of claim 25, wherein the ocular disease is selected from the group consisting of Age-F elated Macular Degeneration (AMD), diabetic retinopathy, retinopathy of prematurity, sickle cel l retinopathy, retinal venous occlusive disease, central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), neovascular macular degeneration or an ocular cancer.
Claim 28. The method of claim 22, wherein the pathogen ic vascu lar proli ferative disease is Age-Related Macu lar Degeneration (AM D).
Claim 29. The method of claim 27, wherein the pathogenic vascular proliferative d isease is wet, exudative AMD.
Claim 30. The method of claim 22, wherein the anti-Id compound is administered coordiiiately with a secondary drug selected from an anti-VBGF agent, an anti-VnCF receptor antagonist, a vascular disrupting agent (VDA), an HSP90 inhibitor, and an antiinflammatory compound.
Claim 3 1 . The method of claim 22, further comprising photodynam ic therapy (PDT) or laser photocoagulation.
Claim 32. The meth od of claim 29, wherein the anti-Id compound yields a greater than 20% therapeutic reduction in occurrence (numbers of lesions among treatment groups) or size (total affected retinal surface area in a subject or among a group of subjects) of wet AMD lesions in anti-Id treated subjects compared to non-treated control subjects.
Claim 33. The method of claim 29, wherein the anti-Id compound yields a 30-50% therapeutic reduction in occurrence (numbers of lesions among treatment groups) or size (total affected retinal surface area in a subject or among a group of subjects) of wet AM D lesions in anti-Id treated subjects compared to non-treated control subjects.
Claim 34. The method of claim 29, wherein the anti-Id compound yields a 50% or greater therapeutic reduction in occurrence (numbers of lesions among treatment groups) or size (total a ffected retin al surface area in a subject or among a group of subjects) of the wet AM D lesion in anti-Id treated subjects compared to non-treated control subjects.
Claim 34. The method of claim 29, wherein suppression of wet AM D lesions by the anti-Id compound yields at least a 20% smaller decrease in Snellen line score over a selected treatment or monitoring period in anti-Id treated subjects compared to non-treated control subjects.
Claim 35. The method of claim 29, wherein suppression of the wet AM D lesion by the anti- Id compound yields a 20-50% smal ler decrease in Snel len line score over a selected treatment or monitoring period in anti-Id treated subjects compared to non-treated control subjects.
C laim 36. The method of claim 29, wherein suppression of the wet AM D lesion by the ant i- Id compound yields at least a 50% smaller decrease in Snel len l ine score over a selected treatment or monitoring period in anti-Id treated subjects compared to non-treated control subjects.
Claim 37. A composition or kit for use in treating subjects receiving drug treatment for a pathogen ic angiogenic condition comprising: (a) an anti-Id active compound and (b) a second drug compound selected from an anti-VEGF agent, an anti-VEGF receptor antagonist, a vascu lar disrupting agent (VDA), an HSP90 inhibitor, or an anti-inflammatory compound.
Claim 38. A inethod for diagnosing a hyperproli feratlve disorder in a mammalian subject by- determining a concentration in a biologic sample of an Id protein, comprising: reacting a b iological sample from a test subject with an Id binding protein that binds an HLH region of an Id protein;
contacting the sample with labeled antibody that binds specifical ly to the Id protein at an antibody binding site on the Id protein separate from the HLH region of the Id protein bound by the Id binding protein, wherein the labeled antibody binds to a di fferent location on the Id protein than the Id binding protein; and
quantifying an amount of labeled antibody complexed with the Id bind ing protein.
C laim 39. The method of claim 38, wherein the id binding protein or the anti-id antibody is bound to a solid-phase support.
Claim 40. The method of claim 38, wherein the Id binding protei n or the anti-Id antibody is labelled.
C laim 4 1 . The method of claim 38, wherein the both the anti-Id antibody and Id binding protein are labelled
Claim 42. The method of claim 38, wherein the Id binding protein is a bHLH protein selected from a group consisting of E l 2. E47, E2-2. F IBB. yoD and MRF.
Claim 43. The mct iod of claim 38, wherein the Id protein is Id ) or Id3.
Claim 44. The method of claim 38, wherein the biological sample is a body flu id, blood or tissue sample from a mammalian subject presenting with cancer, or with a diagnosed elevated risk of cancer or metastatic disease.
1
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