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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English (en)
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 CN201480075495.2A priority Critical patent/CN107847470A/zh
Priority to JP2016558543A priority patent/JP2017506257A/ja
Priority to EP14870415.8A priority patent/EP3079680A4/en
Priority to MX2016007748A priority patent/MX2016007748A/es
Priority to AU2014361814A priority patent/AU2014361814A1/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.

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CN111246860A (zh) * 2015-02-18 2020-06-05 恩立夫克治疗有限责任公司 用于癌症治疗的组合免疫治疗和细胞因子控制治疗
CN113627763A (zh) * 2021-07-30 2021-11-09 厦门大学 一种风险量化评估模型建立方法
US11730761B2 (en) 2016-02-18 2023-08-22 Enlivex Therapeutics Rdo Ltd Combination immune therapy and cytokine control therapy for cancer treatment
EP3994123B1 (en) * 2019-07-02 2024-01-03 Universität Heidelberg Chemical inhibitors of id proteins for the treatment of cancer and other diseases
US11883429B2 (en) 2015-04-21 2024-01-30 Enlivex Therapeutics Rdo Ltd Therapeutic pooled blood apoptotic cell preparations and uses thereof
US12274714B2 (en) 2015-02-18 2025-04-15 Enlivex Therapeutics R&D Ltd Early apoptotic cells for use treating sepsis

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LU100900B1 (de) * 2018-08-10 2020-02-17 Thomas Melchior Homann Verbindungen zur modulation von a-ketoglutarsäure (2kg)-abhängigen oxygenasen
JP2022550589A (ja) * 2019-10-01 2022-12-02 メモリアル スローン ケタリング キャンサー センター Ｉｄタンパク質の小分子阻害剤
CN114480490A (zh) * 2021-12-31 2022-05-13 四川省医学科学院·四川省人民医院 一种构建视网膜新生血管性疾病动物模型的方法
CN115487358B (zh) * 2022-08-05 2023-05-30 核工业四一六医院 一种用于软骨组织修复的凝胶复合支架及制备方法

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US20130171158A1 (en) * 2010-06-30 2013-07-04 Garvan Institute Of Medical Research Treatment of abnormalities of glucose metabolism with an antagonist of inhibitor of differentiation 1
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CN111246860A (zh) * 2015-02-18 2020-06-05 恩立夫克治疗有限责任公司 用于癌症治疗的组合免疫治疗和细胞因子控制治疗
US11717539B2 (en) 2015-02-18 2023-08-08 Enlivex Therapeutics RDO Ltd. Combination immune therapy and cytokine control therapy for cancer treatment
US12274714B2 (en) 2015-02-18 2025-04-15 Enlivex Therapeutics R&D Ltd Early apoptotic cells for use treating sepsis
US11883429B2 (en) 2015-04-21 2024-01-30 Enlivex Therapeutics Rdo Ltd Therapeutic pooled blood apoptotic cell preparations and uses thereof
US11730761B2 (en) 2016-02-18 2023-08-22 Enlivex Therapeutics Rdo Ltd Combination immune therapy and cytokine control therapy for cancer treatment
EP3994123B1 (en) * 2019-07-02 2024-01-03 Universität Heidelberg Chemical inhibitors of id proteins for the treatment of cancer and other diseases
CN113627763A (zh) * 2021-07-30 2021-11-09 厦门大学 一种风险量化评估模型建立方法
CN113627763B (zh) * 2021-07-30 2023-12-01 厦门大学 一种风险量化评估模型建立方法

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