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WO2007117419A2 - Procédés et compositions associés à des inhibiteurs d'une enzyme de clivage post-prolyle - Google Patents

Procédés et compositions associés à des inhibiteurs d'une enzyme de clivage post-prolyle Download PDF

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Publication number
WO2007117419A2
WO2007117419A2 PCT/US2007/008242 US2007008242W WO2007117419A2 WO 2007117419 A2 WO2007117419 A2 WO 2007117419A2 US 2007008242 W US2007008242 W US 2007008242W WO 2007117419 A2 WO2007117419 A2 WO 2007117419A2
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WIPO (PCT)
Prior art keywords
class
post
cancer
cleaving enzyme
enzyme inhibitor
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PCT/US2007/008242
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WO2007117419A8 (fr
WO2007117419A3 (fr
Inventor
Sharlene Adams
Michael I. Jesson
Glenn T. Miller
Paul A. Mclean
Barry Jones
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Point Therapeutics Inc
Dara Biosciences Inc
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Point Therapeutics Inc
Dara Biosciences Inc
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Publication of WO2007117419A2 publication Critical patent/WO2007117419A2/fr
Publication of WO2007117419A8 publication Critical patent/WO2007117419A8/fr
Publication of WO2007117419A3 publication Critical patent/WO2007117419A3/fr
Anticipated expiration legal-status Critical
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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
    • A61K31/69—Boron compounds

Definitions

  • the invention relates to post-prolyl cleaving enzyme inhibitors in the treatment of proliferative disorders such as cancer.
  • Abnormal cell proliferation is usually characterized by an increased rate of division and in some cases uncontrolled growth.
  • a proliferative cell disorder is a tumor.
  • primary malignant tumors are particularly problematic given their tendency to invade surrounding tissues and metastasize to distant organs in the body.
  • the most frequently used methods for treating neoplasia, especially solid tumor forms of neoplasia include surgical procedures, radiation therapy, drug therapies, and combinations of the foregoing. These methods involve significant risk (e.g., of infection, death) to the patient. More importantly, the probability of eliminating all malignant cells is small particularly if the zone of malignant growth is not well defined or if the primary tumor has metastasized by the time of surgery. Achieving therapeutic doses effective for treating the cancer is often limited by the toxic side effects of the anti-cancer agent on normal, healthy tissue. An ideal anti-cancer agent or therapy has tissue specificity, thereby reducing side-effects on normal (dividing) cells.
  • the invention provides novel cancer therapies.
  • these therapies are associated with reduced side effects in a subject.
  • these therapies modulate the timing and magnitude of an anti-cancer immune response.
  • the invention provides, in one aspect, a method for treating a subject having a condition characterized by abnormal cell proliferation comprising administering to a subject in need thereof a Class I post-prolyl cleaving enzyme inhibitor and a Class II post-prolyl cleaving enzyme inhibitor, wherein the inhibitors are administered in an amount effective to inhibit the condition.
  • the Class I post-prolyl cleaving enzyme inhibitor is Val-boroPro, Ile-boroPro, Leu-boroPro, or Met-boroPro. In one embodiment, the Class I post-prolyl cleaving enzyme inhibitor has a structure of Formula TVa, IVb, IVc, IVd, IVe, Va, Vb, Vc, Vd, Ve, Via, VIb, VIc 5 VId, VIe, Vila, VIIb, VIIc, VIId, VIIe, Villa, VIIIb, VIIIc, VIIId and VIIIe.
  • the Class II post-prolyl cleaving enzyme inhibitor is Glu-boroPro, GIn-boroPro, Arg-boroPro, Phe-boroPro, Lys-boroPro, or acetyl-Gly-boroPro.
  • the Class I inhibitor is Val-boroPro and the Class II inhibitor is Glu-boroPro.
  • the Class I post-prolyl cleaving enzyme inhibitor and the Class II post-prolyl cleaving enzyme inhibitor are administered substantially simultaneously. In one embodiment, the Class I post-prolyl cleaving enzyme inhibitor is administered prior to the Class ⁇ post-prolyl cleaving enzyme inhibitor. In one embodiment, the Class I post-prolyl cleaving enzyme inhibitor is administered after (and optionally before) the Class II post-prolyl cleaving enzyme inhibitor. In one embodiment, the inhibitors are administered in an alternating manner.
  • the Class I post-prolyl cleaving enzyme inhibitor and the Class II post-prolyl cleaving enzyme inhibitor are formulated together. In one embodiment, the inhibitors are formulated separately. In one embodiment, the inhibitors are administered orally.
  • the Class I post-prolyl cleaving enzyme inhibitor and the Class II post-prolyl cleaving enzyme inhibitor are administered in a molar ratio of 1 : 10, 1 :20, 1 :30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90 or 1:100.
  • the Class II post-prolyl cleaving enzyme inhibitor is administered in a greater than 100-fold molar excess as compared to the Class I post-prolyl cleaving enzyme inhibitor.
  • the amount effective to inhibit the condition is less than 1 mg/kg body weight per day. In one embodiment, the amount effective to inhibit the condition is in the range of 500 ⁇ g to 10 mg per day.
  • the condition is cancer.
  • the cancer may be carcinoma, sarcoma or melanoma.
  • the sarcoma may be osteosarcoma or fibrosarcoma.
  • the cancer may be non- small cell lung cancer, pancreatic cancer, colorectal cancer, leukemia or lymphoma.
  • the condition is chronic lymphocytic leukemia.
  • the method further comprises administration of a second therapeutic agent that is an anti-cancer agent.
  • the second therapeutic agent is a chemotherapeutic agent.
  • the chemotherapeutic agent may be docetaxel, cisplatin, gemcitabine, pemetrexed (ALIMTA), erlotinib (TARCEVA), gefitinib (IRESSA), temozolomide (TEMODAR), carboplatin, cyclophosphamide or doxorubicin.
  • the second therapeutic agent is an antibody.
  • the antibody may be rituximab (RITUXAN), bevacizumab (AVASTIN), cetuximab (ERBITUX), trastuzumab (HERCEPTIN), tositumomab (BEXXAR), or alemtuzumab (CAMPATH).
  • RITUXAN rituximab
  • AVASTIN bevacizumab
  • ERBITUX cetuximab
  • trastuzumab HERCEPTIN
  • tositumomab BEXXAR
  • alemtuzumab CAMPATH
  • the Class I post-prolyl cleaving enzyme inhibitor is administered before the second therapeutic agent.
  • the condition is refractory to a prior treatment.
  • the condition is a primary tumor.
  • the invention provides a method for improving treatment with a Class I post-prolyl cleaving enzyme inhibitor of a subject having a condition characterized by abnormal cell proliferation comprising administering to a subject in need thereof, who has been treated with a Class I post-prolyl cleaving enzyme inhibitor and who is experiencing one or more dose-limiting side effects, a Class II post-prolyl cleaving enzyme inhibitor in an amount effective to inhibit the condition.
  • the Class II post-prolyl cleaving enzyme inhibitor is administered until the dose-limiting side effects are reduced. In one embodiment, the Class I post-prolyl cleaving enzyme inhibitor is administered to the subject after the dose-limiting side effects are reduced. In one embodiment, the dose-limiting side effects are hypotension, edema, pyrexia, rigors, and/or dehydration.
  • the invention provides a method for treating a subject having myelodysplasia comprising administering to a subject in need thereof a Class II post-prolyl cleaving enzyme inhibitor in an amount effective to inhibit the myelodysplasia.
  • the invention provides a method for treating a subject having a condition characterized by abnormal cell proliferation comprising administering to a subject in need thereof a Class II post-prolyl cleaving enzyme inhibitor in an amount effective to inhibit the condition, wherein the subject is not administered an anti-side effect agent.
  • the anti-side effect agent is an anti-hypotension agent, an anti- edema agent, an anti-rigors agent, an anti-pyrexia agent, or an anti-dehydration agent.
  • the invention provides a method for treating a subject having a condition characterized by abnormal cell proliferation comprising administering to a subject in need thereof an immunostimulatory agent and a Class II post-prolyl cleaving enzyme inhibitor in an amount effective to inhibit the condition, wherein the immunostimulatory agent is administered prior to the Class II post-prolyl cleaving enzyme inhibitor, and the subject is - A -
  • the immunostimulatory agent may be alum, cholera toxin, CpG immunostimulatory nucleic acids, MPL, MPD, or QS-21.
  • the invention provides a method for treating a subject having a condition characterized by abnormal cell proliferation comprising administering to a subject in need thereof a Class I post-prolyl cleaving enzyme inhibitor in an amount effective to inhibit the condition and an IL-1/IL-lR antagonist in an amount to reduce side effects of the Class I post-prolyl cleaving enzyme inhibitor.
  • the invention provides a method for treating a subject having a condition characterized by abnormal cell proliferation comprising administering to a subject in need thereof a Class I post-prolyl cleaving enzyme inhibitor in an amount effective to inhibit the condition and an IL-6/IL-6R antagonist in an amount to reduce side effects of the Class I post-prolyl cleaving enzyme inhibitor.
  • the side effects are reduced by 50%.
  • the invention provides a method for treating a subject having a condition characterized by abnormal cell proliferation comprising administering to a subject in need thereof a Class I post-prolyl cleaving enzyme inhibitor in an amount effective to inhibit the condition and a granulocyte stimulating factor.
  • the subject is not administered an antibody or an antigen.
  • the granulocyte stimulating factor may be G-CSF or GM-CSF. In one embodiment, the granulocyte stimulating factor is administered prior to the Class I post-prolyl cleaving enzyme inhibitor. In one embodiment, the condition is sarcoma. In one embodiment, the method further comprises administration of a neutrophil chemoattractant to the subject.
  • the invention provides a method for treating a subject having a condition characterized by abnormal cell proliferation comprising administering to a subject in need thereof a Class I post-prolyl cleaving enzyme inhibitor in an amount effective to inhibit the condition and a macrophage stimulating factor.
  • the macrophage stimulating factor is M-CSF. In one embodiment, the macrophage stimulating factor is administered prior to the Class I post- prolyl cleaving enzyme inhibitor. In one embodiment, the condition is sarcoma.
  • a method for treating a subject who is in remission from cancer.
  • the method comprises administering to a subject in need thereof a Class II post-prolyl cleaving enzyme inhibitor in an effective amount to maintain remission in the subject.
  • the Class II post-prolyl cleaving enzyme inhibitor is selected from the group consisting of Glu-boroPro, Gln-boroPro, Leu-boroPro, Arg-boroPro, Phe- boroPro, and Lys-boroPro.
  • the Class II post-prolyl cleaving enzyme inhibitor is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoeth
  • each Xi and X 2 is, independently, a hydroxyl group or a group capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH.
  • the Class II post-prolyl cleaving enzyme inhibitor is a cyclic version of Glu-boroPro.
  • the Class II post-prolyl cleaving enzyme inhibitor is an ester of Glu-boroPro, a boroxine derivative of Glu-boroPro, or an alcohol precursor of Glu-boroPro.
  • the Class II post-prolyl cleaving enzyme inhibitor is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoeth
  • A is a peptide or a peptidomimetic
  • each X 1 and X ⁇ is, independently, a hydroxyl group or a group capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH, optionally wherein the C bonded to the B is in the R configuration, and further optionally wherein the bond between the glutamic acid residue and pyrrolidine is in the S configuration.
  • the Class II post-prolyl cleaving enzyme inhibitor is
  • A is any naturally or non-naturally occurring amino acid bonded in either an S- or an R-configuration
  • m is an integer from 0 - 100, such that when m is greater than one, each A in A m may be a different amino acid residue from every other A in A m
  • each Xi and X 2 is, independently, a hydroxyl group or a group capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH, optionally wherein the C bonded to the B is in the R configuration, and further optionally wherein the bond between the glutamic acid residue and pyrrolidine is in the S configuration.
  • the Class II post-prolyl cleaving enzyme inhibitor is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoeth
  • A is any naturally or non-naturally occurring amino acid in an S- or an R- configuration
  • m is an integer from 0 - 100, provided that when m is greater than one, A in each repeating bracketed unit can be a different amino acid residue
  • each Xi and X 2 is, independently, a hydroxyl group or a group capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH, optionally wherein the C bonded to the B is in the R configuration, and further optionally wherein the bond between the glutamic acid residue and pyrrolidine is in the S configuration.
  • the Class II post-prolyl cleaving enzyme inhibitor may comprise a glutamic acid bonded to a pyrrolidine bond in an S-configuration.
  • the Class II post-prolyl cleaving enzyme inhibitor may comprise a carbon of pyrrolidine bonded to a boron in the R-configuration.
  • the Class II post-prolyl cleaving enzyme inhibitor may further comprise a mixture of R- and S- enantiomers of boron substituted pyrrolidine.
  • the mixture of R- and S-enantiomers of boron substituted pyrrolidine contains at least 95% of the R-enantiomer of boron substituted pyrrolidine.
  • the cancer is a solid cancer. In some embodiments, the cancer is of epithelial origin. In some important embodiments the cancer may be breast cancer, renal (kidney) cancer, lung cancer (such as non-small cell lung cancer), prostate cancer, pancreatic cancer, ovarian cancer or melanoma. In some embodiments the cancer is a leukemia. In some embodiments the cancer is a lymphoma. The lymphoma may be Hodgkin's lymphoma or non-Hodgkin's lymphoma. The cancer may be chronic lymphocytic leukemia. In some embodiments the cancer is a sarcoma (e.g., fibrosarcoma). In some embodiments, the cancer is a primary tumor. In other embodiments, it is a metastasis.
  • the cancer is a solid cancer. In some embodiments, the cancer is of epithelial origin. In some important embodiments the cancer may be breast cancer, renal (kidney) cancer, lung cancer (such as non-small cell
  • the remission induction therapy may comprise anti-cancer agents, radiation therapy, surgery, or a combination thereof.
  • Anti-cancer agents include chemotherapeutic agents, immunotherapeutic agents, and tumor vaccines.
  • chemotherapeutic agents examples include cisplatin, gemcitabine, 5-FU, taxol/paclitaxel, ortaxotere.
  • immunotherapy comprises administering an antibody such as but not limited to anti-neu/HER-2 antibody, anti-CD20 antibody, Rituximab (Rituxan), and trastuzumab (Herceptin).
  • an antibody such as but not limited to anti-neu/HER-2 antibody, anti-CD20 antibody, Rituximab (Rituxan), and trastuzumab (Herceptin).
  • the remission induction therapy comprises administering a DPP-TV inhibitor or a FAP inhibitor.
  • the Class II post-prolyl cleaving enzyme inhibitor is administered orally.
  • the effective amount may be less than 1 mg/kg/day. In some embodiments the effective amount is less than 1 mg/kg/day, less than 500 ⁇ g/kg/day, less than 250 ⁇ g/kg/day, less than 100 ⁇ g/kg/day, less than 50 ⁇ g/kg/day, less than 25 ⁇ g/kg/day or less than lO ⁇ g/kg/day.
  • the Class II post-prolyl cleaving enzyme inhibitor is administered at intervals. Examples of such intervals include but are not limited to every 12 hours, every 24 hours, every 36 hours, every 2, 3, 4, 5, 6, 7, 10, and 14 days. In some embodiments the Class ⁇ post-prolyl cleaving enzyme inhibitor is administered once every month. In some embodiments, the Class II post-prolyl cleaving enzyme inhibitor is administered every other day, every other 2 days, or every other week. In some important embodiments the Class II post-prolyl cleaving enzyme inhibitor is administered every 24 hours.
  • the method further comprises administering a second agent to the subject.
  • second agents include but are not limited to chemotherapeutic agents and immunotherapeutic agents.
  • FIG. IA is a graph showing the effects of Val-boroPro on WEHI 164 fibrosarcoma tumor growth as a function of time relative to a saline control in wild type BALB/c undepleted mice.
  • FIG. IB is a graph showing the effects of Val-boroPro on WEHI 164 fibrosarcoma tumor growth as a function of time relative to a saline control in BALB/c nu/nu mice.
  • FIG. 2 A is a graph showing the effects of Val-boroPro on A549 NSCLC xenograft growth as a function of time relative to a saline control in wild type undepleted mice.
  • FIG. 2B is a graph showing the effects of Val-boroPro on A549 NSCLC xenograft growth as a function of time relative to a saline control in neutrophil depleted mice (anti- Ly6G treated).
  • FIG. 2C is a graph showing the effects of Val-boroPro on A549 NSCLC xenograft growth as a function of time relative to a saline control in macrophage depleted mice (carrageenan treated).
  • FIG. 3 A is a graph showing the effects of Val-boroPro on Raji B-cell lymphoma xenograft growth as a function of time relative to a saline control in wild type undepleted mice.
  • FIG. 3B is a graph showing the effects of Val-boroPro on Raji B-cell lymphoma xenograft growth as a function of time relative to a saline control in neutrophil depleted mice (anti-Ly6G treated).
  • FIG. 3C is a graph showing the effects of Val-boroPro on Raji B-cell lymphoma xenograft growth as a function of time relative to a saline control in macrophage depleted mice (carrageenan treated).
  • FIG. 4A is a graph showing the effects of Val-boroPro on A549 and H460 lung carcinoma xenografts growth as a function of time relative to a saline control in BALB/c Rag2-/- mice.
  • FIG. 4B is a series of photographs of histological sections of explanted A549 xenografts from either saline or Val-boroPro treated mice.
  • FIG. 4C is a series of photographs of histological sections of explanted H460 xenografts from either saline or Val-boroPro treated mice.
  • FIG. 5 A is a series of photographs of histological sections of explanted A549 xenografts from either saline or Val-boroPro treated mice, showing neutrophil infiltration.
  • FIG. 5B is a series of photographs of histological sections of explanted H460 xenografts from either saline or Val-boroPro treated mice, showing lack of neutrophil infiltration.
  • FIG. 6 is a schematic showing a modeled FAP structure including Val-boroPro binding in the catalytic binding site.
  • FIG. 7A-D are photographs of HEK293-FAP and HT-29 xenografts stained by immunohistochernistry for FAP.
  • FIG. 8 is a graph showing the dose dependent effect of Glu-boroPro on HT-29 xenografts.
  • FIG. 9 is a graph showing the effect of Val-boroPro and Glu-boroPro on FAP enzymatic activity in HT-29 xenografts.
  • FIG. 10 is a graph showing the effect of Val-boroPro and Glu-boroPro on HEK-FAP xenograft growth as a function of time.
  • FIG. 11 is a graph showing the effect of Val-boroPro and Glu-boroPro on FAP enzymatic activity in HEK-FAP xenografts.
  • FIGs. 12A-B are graphs showing inhibition of human FAP (A) and human DPP-IV (B) by cyclopropyl-alanine-boroPro and cyclopentyl-glycine-boroPro.
  • FIGs. 13A-D are graphs showing inhibition of DPP-IV (A), DPP 8 (B), FAP (C), and DPP 2 (D) by Norleu-boroPro.
  • FIGs. 14A-B are histograms showing induction of IL-8 (A) and G-CSF (B) by human bone marrow stromal cells in vitro by cyclopropyl-alanine-boroPro and cyclopentyl-glycine- boroPro.
  • FIGs. 15A-D are graphs showing inhibition induction of human IL-I beta (A and D), human G-CSF (B), and mouse CXCL1/KC (C) by cyclopentyl-glycine-boroPro.
  • FIGs. 16A-B are graphs showing inhibition of serum DPP-IV activity (A) and induction of serum CXCL1/KC (B) by cyclopropyl-alanine-boroPro and cyclopentyl-glycine- boroPro.
  • FIGs. 17A-B are graphs showing inhibition of serum DPP-IV activity (A) and induction of serum CXCL1/KC (B) by Norleu-boroPro administered orally or subcutaneously.
  • FIGs. 18A-B are histograms showing induction of IFN-gamma from splenocytes by cyclopentyl-glycine-boroPro (A) and cyclohexyl-glycine-boroPro (B).
  • the invention provides novel anti-cancer therapies.
  • the invention provides combinations of therapeutic agents to be used in the treatment of cancer, hi various embodiments, these combinations surprisingly are able to regulate the timing and/or magnitude of an anti-cancer immune response.
  • the combinations comprise at least one post-prolyl cleaving enzyme inhibitor.
  • the combinations preferably are combinations of at least two different post-prolyl cleaving enzyme inhibitors.
  • Post-prolyl cleaving enzymes are enzymes that cleave peptides (including proteins) at the carboxy terminal of preferably a proline residue.
  • Post-prolyl cleaving enzymes can have endopeptidase and/or exopeptidase activity.
  • Post-prolyl cleaving enzymes with exopeptidase activity include dipeptidyl peptidases.
  • Dipeptidyl peptidases are enzymes that cleave dipeptides from the amino terminus of a peptide (e.g., a protein) provided that the penultimate residue is a proline (and sometimes an alanine). These enzymes cleave at the carboxy side of the penultimate residue.
  • Post-prolyl cleaving enzymes include but are not limited to DPP-II, DPP-IV, fibroblast activation protein (FAP) and prolylendopeptidase (PEP).
  • DPP-II and DPP-IV have exopeptidase activities.
  • PEP has endopeptidase activity.
  • FAP has both exo- and endopeptidase activity.
  • the invention in some aspects, distinguishes post-prolyl cleaving enzyme inhibitors according to the extent to which they induce cytokines and chemokines.
  • the invention is based in part on the surprising discovery that the use of post-prolyl cleaving enzyme inhibitors with different cytokine induction properties can be used beneficially in the treatment of abnormal cell proliferation.
  • Class I post-prolyl cleaving enzyme inhibitors induce cytokines to a greater extent than do Class II post-prolyl cleaving enzyme inhibitors.
  • Class I post-prolyl cleaving enzyme inhibitors are able to induce IL-I which in turn leads to the induction of a cytokine/chemokine cascade including IL-6, G-CSF, IL-8 (KC in mice), MCP-2, MAROMCP-3, MCP-5, JE, MIP-2, ENA78, LIX, lymphotactin, eotaxin, MIG, IP- 10, MDC, TARC, and thrombospondin, among others.
  • the invention therefore exploits these differences in cytokine/chemokine induction profiles between post-prolyl cleaving enzyme inhibitors and thereby achieves an unexpectedly improved anti-cancer therapy.
  • the particular Class I inhibitor induces at least 2-fold more of any given cytokine or chemokine than does the particular Class II inhibitor.
  • the specific Class I inhibitor induces at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 10-fold more, at least 20-fold more, at least 50-fold, at least 100-fold more, at least 500-fold more, or at least 1000-fold more of any given cytokine or chemokine than does the specific Class II inhibitor.
  • the Class I inhibitor induces at least 2, at least 3, at least 4, at least 5, or more cytokines or chemokines.
  • the Class II inhibitor is used in an amount that induces undetectable or negligible levels of any of the afore-mentioned cytokines and chemokines. Such an amount however is effective to inhibit a post-prolyl cleaving enzyme.
  • the Class II inhibitor may be used at an amount that inhibits one, two, three or more post-prolyl cleaving enzymes. The amount may inhibit post-prolyl cleaving enzyme activity by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more.
  • the Class II inhibitor inhibits DPP-IV and/or FAP.
  • the Class I inhibitor is used in an amount that induces detectable levels of any of the afore-mentioned cytokines and chemokines.
  • the level of any cytokine or chemokine may be increased at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 1000-fold over pre-treatment levels.
  • Such an amount however in some instances may not achieve maximal inhibition of any or all post-prolyl cleaving enzymes.
  • the Class I inhibitor may be used at an amount that inhibits, although not maximally, one, two, three or more post-prolyl cleaving enzymes. The amount may inhibit post-prolyl cleaving enzyme activity such as DPP-W or FAP activity less than 75%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less.
  • the Class I inhibitor is administered in a sub-therapeutic dose in the context of cancer therapy.
  • a sub-therapeutic dose in the context of cancer therapy is a dose that is less than the dose that produces the maximal, medically acceptable, therapeutic result in the subject if administered in the absence of another agent (e.g., in the absence of the Class II inhibitor).
  • a sub-therapeutic dose of a Class I inhibitor may be a dose that reduces tumor size to a lesser extent than does a therapeutic dose of the same inhibitor.
  • the sub-therapeutic dose may be equal to or less than 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or 5% of the therapeutic dose, or it may be at least 50-fold, 100-fold, 500-fold, or 1000-fold less than the therapeutic dose.
  • Therapeutic doses of Class I inhibitors for the treatment of cancer in human subjects are known in the art. Alternatively, therapeutic doses can be determined using murine model systems.
  • the sub-therapeutic dose of Class I inhibitors however is still a dose that induces one or more cytokines and/or chemokines to a level greater than a pre-treatment level.
  • the Class I and Class II inhibitors are administered in synergistic amounts.
  • the term "synergistic” describes an effect resulting from the combination of at least two agents (e.g., the Class I and Class II inhibitors) which is greater than the sum of the individual effects observed when each agent is used alone.
  • either or both Class I and Class II inhibitors may be used at lower doses than would be used if either inhibitor was used alone.
  • either or both inhibitors may be administered in a sub-therapeutic dose with the combined effect however being therapeutic.
  • Class I post-prolyl cleaving enzyme inhibitors include but are not limited to VaI- boroPro, Ile-boroPro, Met-boroPro, Leu-boroPro, cyclopropylalanine boroPro (see Formula Ve), cyclopentylglycine boroPro (see Formula VIe), and norleucine boroPro (see Formula V ⁇ ie).
  • the Class I inhibitor is Val-boroPro.
  • Class I inhibitors include Vildagliptin ((2S- ⁇ [(3- hydroyxyadamantan-l-yl) amino]acetyl ⁇ -pyrrolidine-2-carbonitrile; Novartis, LAF237), Saxagliptin (BMS), Sitagliptin (Merck MK-0431), (2R)-4-oxo-4-[3-(trifluoromethyl)-5,6- dihydro[l,2,4]triazolo[4,3- ⁇ ]pyrazin-7(8H)-yl]-l-(2,5-difluorophenyl)butan-2-amine fumarate and hydrochloride salts (des-fluoro analog of sitagliptin; Lankas et al.
  • Class II post-prolyl cleaving enzyme inhibitors include but are not limited to GIu- boroPro, Gln-boroPro, Arg-boroPro, Phe-boroPro, Lys-boroPro, and acetyl-Gly-boroPro. In some embodiments, the Class II inhibitor is Glu-boroPro or Gln-boroPro.
  • the invention in some aspects relates to the use of a Class II post-prolyl cleaving enzyme inhibitor in the treatment of an abnormal cell proliferative condition, in the absence of a Class II inhibitor, and optionally in the absence of other therapeutic agents such as agents administered to control side effects associated with Class I post-prolyl cleaving enzyme inhibitors (e.g., pyrexia, edema, rigors, hypotension, dehydration, etc.), or immunotherapeutic agents such as antibodies or antigens.
  • the invention provides anti-cancer therapies that employ Class II inhibitors (e.g., Glu-boroPro) in the absence of Class I inhibitors.
  • the Class I inhibitor is Val-boroPro and/or the Class II inhibitor is Glu-boroPro.
  • post-prolyl cleaving enzyme inhibitors have been shown to have anti-tumor activity. (See U.S. Pat. No. 6,890,904, issued on May 10, 2005.) It has been discovered, according to the invention, that a combination of post-prolyl cleaving enzyme inhibitors that differ in their cytokine/chemokine induction profiles can be used to achieve an unexpectedly improved anti-tumor therapy. In particular, this new therapy is able to achieve the same level of ant ⁇ -tumor activity while reducing side effects.
  • hypotension i.e., low to below normal blood pressure
  • hypotension is defined as systolic blood pressure less than or equal to 90 mm Hg in adults. For children (i.e., less than 16 years of age), hypotension is defined as less than the fifth percentile by age.
  • the invention provides therapies with reduced incidence of side effects.
  • a reduced incidence of side effects embraces a lower frequency of one or more side effects in a given subject or in the subject population.
  • a reduced incidence of side effects may reflect that a lower percentage of subjects being treated experience side effect(s). It may also mean that a given subject experiences the side effect(s) less frequently. It also embraces a reduction in the severity of the side effect. As an example, the fevers experienced by the subject may be lower, on average, or the extent of edema experienced by the subject maybe lower, on average. Individual side effects or a group of side effects may be impacted.
  • the side effect(s) may be reduced by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.
  • Some embodiments of the invention relate to "dose-limiting side effects". As used herein, this term refers to side effects which limit the amount of post-prolyl cleaving enzyme inhibitor that can be administered to a subject.
  • the side effects observed are generally associated with Class I inhibitors. Accordingly, the invention embraces administration of Class I inhibitors with agents that modulate such side effects. The invention also embraces altered or interrupted administration of Class I inhibitors to achieve the same effect. The invention further embraces administration the use of Class II inhibitors alone or together with Class I inhibitors to reduce incidence of side effects. Anti-side effect agents may be administered to subjects.
  • anti-edema agents such as diuretics, ginkgo biloba, coumarin, and semi-synthetic fiavonoids (i.e., hydroxyethylrutosides); anti-pyrexia agents such as ibuprofen, aspirin and acetaminophen; anti-dehydration agents such as oral fluids and oral electrolytes; and the like.
  • anti-pyrexia agents are also anti-rigors, agents. Edema can also be treated using a salt- restricted diet.
  • an aspect of the invention relates to methods for reducing the side effects of Class I post-prolyl cleaving enzyme inhibitors through the use of additional immunomodulatory agents.
  • the immunomodulatory agent is an IL-I antagonist.
  • an IL-I antagonist is an agent that blocks or interferes with IL-I function.
  • An IL-I antagonist can (a) bind to IL-I and prevent its binding to an IL-I receptor, (b) bind to IL-I receptor in a manner that does not mimic IL-I and therefore does not stimulate signaling through the receptor, and/or (c) bind to and interfere with intracellular mediators of the IL-I signaling pathway.
  • IL-I antagonists examples include anakinra (KINERET), soluble type II IL-IR (IL-IRII) which binds to both IL- l ⁇ and IL- l ⁇ but does not induce intracellular signaling (McMahan et al., EMBO J. 10: 2821-2832, 1991), IL-I receptor antagonist (IL- Ira) which binds to IL-IRI with high affinity but does not activate the receptor or signaling from the receptor, thereby acting as a competitive antagonist to IL-I ⁇ and IL-l ⁇ (Arend, Prog. Growth Factor Res. 2(4): 193-205, 1990), anti-IL-lRI antibodies (Fredricks et al., Pro. Eng.
  • KINERET soluble type II IL-IR
  • IL-IRII soluble type II IL-IR
  • Ira IL-I receptor antagonist
  • the immunomodulatory agent is an IL-6 antagonist.
  • an IL-6 antagonist is an agent that blocks or interferes with IL-6 function.
  • An IL-6 antagonist can (a) bind to EL-6 and prevent its binding to an IL-6 receptor, (b) bind to IL-6 receptor in a manner that does not mimic IL-6 and therefore does not stimulate signaling through the receptor, and/or (c) bind to and interfere with intracellular mediators of the IL-6 signaling pathway.
  • Examples of IL-6 antagonists include antagonists described in U.S. Patent 6599875, 6838433, 6172042, 5888510, 5844099, 5527546, 5470952, and 5210075. The antagonist compounds disclosed in these references are incorporated herein by reference.
  • the invention provides methods of treating cancer by inducing neutrophil and/or macrophage production and/or chemotaxis systemically and/or locally to affected sites in the body.
  • Neutrophil production can be induced using granulocyte stimulating factors.
  • Granulocyte stimulating factors are factors that increase the number of granulocytes, and preferably neutrophils, in the subject.
  • Increased granulocyte numbers generally result from increased differentiation from precursors including granulocyte restricted precursors such as CFC-G or oligo- or multi-lineage precursors such as CFC-GM or CFC-GEMM.
  • Granulocyte stimulating factors are known in the art and include but are not limited to granulocyte colony stimulating factor (G-CSF) and granulocyte/macrophage colony stimulating factor (GM- CSF).
  • G-CSF granulocyte colony stimulating factor
  • GM- CSF granulocyte/macrophage colony stimulating factor
  • macrophage production can be induced using macrophage stimulating factors.
  • Macrophage stimulating factors are factors that increase the number of macrophages in the subject.
  • Increased macrophage numbers generally result from increased differentiation from precursors including macrophage restricted precursors such as CFC-M or oligo- or multi-lineage precursors such as CFC-GM or CFC-GEMM.
  • An example of a macrophage stimulating factor is macrophage colony stimulating factor (M-CSF) which is known in the art.
  • M-CSF macrophage colony stimulating factor
  • the invention contemplates the administration of a granulocyte and/or a macrophage stimulating factor prior to the administration of post-prolyl cleaving enzyme inhibitors.
  • the post-prolyl cleaving enzyme inhibitors are Class I inhibitors (e.g., Val-boroPro).
  • chemoattractants are agents that induce movement of cells along a concentration gradient (e.g., from a region of low chemoattractant concentration to a region of higher chemoattractant concentration). Examples of chemoattractants are provided in Table 1.
  • the chemoattractants are administered locally to an affected site (e.g., a melanoma lesion). The chemoattractants may also be administered locally in a sustained release formulation.
  • the invention further provides in part methods for treatment of cancers and precancerous conditions that are themselves responsive to one or more of the cytokines or chemokines induced by Class I inhibitors. Such conditions are treated according to the invention using one or more Class II inhibitors.
  • Class II inhibitors provides the anti-tumor response in the absence of the cytokine/chemokine cascade that might otherwise stimulate rather than inhibit growth of the cancerous or precancerous cells.
  • Conditions to be treated in this manner may be either known a priori to be cytokine and/or chemokine responsive or alternatively they may be assessed for such responsiveness prior to treatment. Accordingly, this aspect of the invention further contemplates testing cells to determine if they are cytokine or chemokine responsive.
  • This additional screening step avoids unnecessary treatment of subjects unlikely to respond to the intended therapy.
  • Testing of cells for cytokine or chemokine responsiveness can be accomplished using in vitro methods known in the art. Examples of conditions that may be treated using Class II inhibitors include but are not limited to multiple myeloma, plasmacytoma, and myelodysplasia.
  • Post-prolyl cleaving enzyme inhibitors are agents that inhibit the enzymatic activity of post-prolyl cleaving enzymes. These inhibitors are represented generally by Formula I
  • P is a targeting group that binds to the reactive site of post proline-cleaving enzyme
  • R is a reactive group that reacts with a functional group in a post proline cleaving enzyme, preferably in the reactive site of the post proline cleaving enzyme.
  • P may be a peptide or a peptidomimetic.
  • the reactive group may be a boronate, phosphonate, cyanopyrrolidine, thiazolide, fluoroalkylketone, alphaketo amide, alphaketo esters, alphaketo acids, N-peptidyl-O-acylhydroxylamine, azapeptide, azetidine, fluoroolefin, dipeptide isoestere, peptidyl (alpha-aminoalkyl) phosphonate ester, or aminoacyl pyrrolidine-2-nitrile.
  • Post-prolyl cleaving enzyme inhibitors may comprise thiazolidine or pyrrolidine groups.
  • Some post-prolyl cleaving enzyme inhibitors have the structure of
  • T is a reactive group such as those recited above (e.g., a boronate group of the formula
  • each D 1 and D 2 independently, is a hydroxyl group or a group which is capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH);
  • X comprises an amino acid or a peptide which mimics the site of a substrate recognized by a post-prolyl cleaving enzyme.
  • X further comprises an N-terminal blocking group.
  • Y may be I l I R 6 R 8
  • Each R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 may be a group which does not interfere significantly with site-specific recognition of the compound by the enzyme, while permitting a complex to be formed between the compound and the enzyme.
  • T is a boronate group
  • each D 1 and D 2 independently, is OH, or D 1 and D 2 together form a ring containing 1 to about 20 carbon atoms, and optionally heteroatoms such as N, S, or O
  • each R 1"8 is H
  • X comprises an amino acid and a blocking group, such as an acetyl group.
  • blocking groups can vary widely. In one blocking reaction, a hydrogen atom of the amino terminal amino group is replaced, generally in a dehydration reaction. Blocking groups can be
  • blocking groups may be employed not only to protect arnino-terrninal groups (and thereby act as N-terminal blocking groups), but also may be used to protect side chains of amino acid residues (e.g., side chains of Lys and Arg). Similarly, amino acid residues having acidic or hydroxy side chains can be protected using t-butyl, benzyl, or other suitable esters or ethers as blocking groups.
  • Additional inhibitors of this class may also be defined by Formula II:
  • a and Ai may be a naturally or non-naturally occurring amino acid residue, peptide or peptidomimetic such that when A is an amino acid residue each A in A n , (i.e., where m >1) maybe a different amino acid residue from every other A in A m and when A is a peptide or peptidomimetic m is 1 ; the C bonded to B is preferably in the R-configuration; and each Xi and X 2 is, independently, a hydroxyl group or a group capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH.
  • a 1 may be alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, aspartate, glutamate, asparagine, glutamine, lysine, arginine, histidine, cysteine, methionine, or proline.
  • Ai is an L- amino acid residue
  • Ai is an L- amino acid residue
  • optionally every A in A m is an L- amino acid residue.
  • m is 0.
  • Xi and X 2 are hydroxyl groups.
  • the proline residue in Formula II may be replaced with another amino acid residue such as, for example, alanine, lysine or glycine.
  • derivatives of Formula II in which the boronate group is replaced with a reactive group as described above are also embraced by the invention.
  • Some post-prolyl cleaving enzyme inhibitors have a structure of Formula III:
  • m is an integer between 0 and 10, inclusive;
  • a and Aj are naturally or non- naturally occurring amino acids, peptides or peptidomimetics; wherein if A is an amino acid residue, it can be a different amino acid residue in each repeating bracketed unit; the C bonded to B is in the R-configuration; and each Xi and X 2 is, independently, a hydroxyl group or a group capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH.
  • a and Ai maybe alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, aspartate, glutamate, asparagine, glutamine, lysine, arginine, histidine, cysteine, methionine, or proline.
  • Ai is an L- amino acid residue, and optionally every A is an L- amino acid residue.
  • the amino acid residues may be naturally and non-naturally occurring amino acids.
  • naturally occurring amino acids are glycine (GIy), and the L-forms of alanine (Ala), valine (VaI), leucine (Leu), isoleucine (He), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), cysteine (Cys), methionine (Met), serine (Ser), threonine (Thr), lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp), glutamic acid (GIu), asparagine (Asn), glutamine (GIn) and proline (Pro).
  • Non-naturally occurring amino acids include the D-forms of Ala, VaI, Leu, He, Phe, Tyr, Tip, Cys, Met, Ser, Thr, Lys, Arg, His, Asp, GIu, Asn, GIn, and Pro.
  • non-naturally occurring amino acids include 2-azetidinecarboxylic acid or pipecolic acid (which have 6-membered, and 4-membered ring structures respectively), 4-hydroxy-proline (Hyp), 5 -hydroxy-lysine, norleucine (NIe), 5- hydroxynorleucine (Hyn), 6-hydroxynorleucine, ornithine, cyclohexyl glycine (Chg), N- Methylglycine (N-MeGIy) 5 N-Methylalanine (N-MeAIa), N-Methylvaline (N-MeVaI), N- Methylleucine (N-MeLeu), N-Methylisoleucine (N-MeIIe), N-Methylnorleucine (N-MeNIe), N-Methyl-2-aminobutyric acid (N-MeAbu) and N-Methyl-2-aminopent
  • agents of Formulae IV-VIII (a through e). These agents may be provided in an isolated form. These agents may also be provided in compositions. As used herein, the terms agent and compound are used interchangeably.
  • T is a reactive group such as an organo boronate, an organo phosphonate, a fluoroalkylketone, an halomethyl ketone, a diazomethyl ketone, a dimethylsulphonium salt, an alphaketo carboxylic acid, an alphaketo ester, an alphaketo amide, an alpha-diketone, an acyloxymethyl ketone, an aldehyde, an epoxysuccinyl, an N-peptidyl-O-acylhydroxylarnine, an azapeptide, a fluoroolefin, a peptidyl (alpha-aminoalkyl) phosphonate ester, or a nitrile.
  • a reactive group such as an organo boronate, an organo phosphonate, a fluoroalkylketone, an halomethyl ketone, a diazomethyl ketone, a dimethylsulphonium salt, an alphaket
  • Rl in its broadest sense maybe a hydrophobic or branched side chain of an amino acid or amino acid analog (e.g., a non-naturally occurring amino acid), or it may be an amino acid or amino acid analog (e.g., a non-naturally occurring amino acid) that comprises at least 3 carbon atoms.
  • Rl comprises one or more ring structures or — R3 (i.e., an additional carbon bond from the peptide backbone), wherein R3 comprises one or more ring structures, and R2 is a hydrogen, peptide, peptidomimetic, or amino acid, whether naturally or non-naturally occurring.
  • the ring structures may be wholly carbon rings or they may be substituted at one or more positions with for example sulfur, nitrogen, and the like.
  • Rl comprises a five carbon ring structure.
  • R3 comprises a three carbon ring structure.
  • the proline residue attached to the reactive group is referred to as a pyrrolidine ring.
  • the dipeptide moiety can be an isostere.
  • the pyrrolidine may be replaced with an azetidine or a thiazolidine.
  • the pyrrolidine-T moiety is replaced with a A- cyanothiazolidine.
  • the bond between the carbon in the pyrrolidine ring and T may be in the L or D configuration.
  • the bond between C and Rl may be in the L or D configuration, although in some embodiments, it is preferably in the L configuration.
  • the agents of Formula IVa include agents of Formula IVb
  • Xl and X2 are independently selected from hydroxyl groups or groups capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH.
  • the bond between the C and B is preferably in the L configuration (otherwise referred to as the R configuration).
  • the agents of Formula IVb further include agents of Formula IVc
  • agents of Formula IVc even further include agents of Formula IVd
  • agents of Formula IVd even further include agents of Formula IVe
  • T is a reactive group as described above; and R2 is a peptide, peptidomimetic, or amino acid, whether naturally or non-naturally occurring.
  • the bond between the carbon in the pyrolidine ring and T may be in the L or D configuration.
  • the dipeptide moiety can be an isostere.
  • the pyrrolidine may be replaced with an azetidine or a thiazolidine. In other embodiments, the pyrrolidine-T moiety is replaced with a 4-cyanothiazolidine.
  • the agents of Formula Va include agents of Formula Vb
  • Xl and X2 are independently selected from hydroxyl groups or groups capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH.
  • the bond between the C and B is preferably in the L configuration (otherwise referred to as the R configuration).
  • the agents of Formula Vb further include agents of Formula Vc
  • agents of Formula Vc even further include agents of Formula Vd
  • agents of Formula Vd even further include agents of Formula Ve (referred to herein as cyclopropyl-alan ⁇ ne-boroPro)
  • T is a reactive group as described above; and R2 is a peptide, peptidomimetic, or amino acid, whether naturally or non-naturally occurring.
  • the bond between the carbon in the pyrolidine ring and T may be in the L or D configuration.
  • the dipeptide moiety can be an isostere.
  • the pyrrolidine may be replaced with an azetidine or a thiazolidine. In other embodiments, the pyrrolidine-T moiety is replaced with a 4-cyanothiazolidine.
  • the agents of Formula Via include agents of Formula VIb
  • Xl and X2 are independently selected from hydroxyl groups or groups capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH.
  • the bond between the C and B is preferably in the L configuration (otherwise referred to as the R configuration).
  • the agents of Formula VIb further include agents of Formula VIc
  • agents of Formula VIc even further include agents of Formula VId
  • agents of Formula VId even further include agents of Formula VIe (referred to herein as cyclopentyl-glycine-boroPro)
  • T is a reactive group as described above; and R2 is a peptide, peptidomimetic, or amino acid, whether naturally or non-naturally occurring.
  • the bond between the carbon in the pyrolidine ring and T may be in the L or D configuration.
  • the dipeptide moiety can be an isostere.
  • the pyrrolidine may be replaced with an azetidine or a thiazolidine. In other embodiments, the pyrrolidine-T moiety is replaced with a 4-cyanothiazolidine.
  • the agents of Formula Vila include agents of Formula VIIb
  • Xl and X2 are independently selected from hydroxyl groups or groups capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH.
  • the bond between the C and B is preferably in the L configuration (otherwise referred to as the R configuration).
  • the agents of Formula VIIb further include agents of Formula VIIc
  • agents of Formula VIIc even further include agents of Formula VIId
  • agents of Formula VIId even further include agents of Formula VIIe (referred to herein as cyclohexyl-glycine-boroPro)
  • Still other agents of the invention have the structure of Formula Villa
  • T is a reactive group as described above; and R2 is a peptide, peptidomimetic, or amino acid, whether naturally or non-naturally occurring.
  • the bond between the carbon in the pyrolidine ring and T may be in the L or D configuration.
  • the amino terminal amino acid residue may be in the L or D configuration, although in some embodiments, it is preferably in the L configuration.
  • the dipeptide moiety can be an isostere.
  • the pyrrolidine may be replaced with an azetidine or a thiazolidine. In other embodiments, the pyrrolidine-T moiety is replaced with a 4-cyanothiazolidine.
  • the agents of Formula Villa include agents of Formula VIIIb
  • Xl and X2 are independently selected from hydroxyl groups or groups capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH.
  • the bond between the C and B is preferably in the L configuration (otherwise referred to as the R configuration).
  • the agents of Formula VIIIb further include agents of Formula VIIIc
  • agents of Formula VIIIc even further include agents of Formula VIIId
  • agents of Formula VIIId even further include agents of Formula VIIIe (referred to herein as norleucine-boroPro)
  • inhibitors include L-threo-isoleucyl thiazolidide (Lankas et al. Diabetes 54:2988-2994, 2005), isoleucylthiazolidide, and cyclohexylglycylpyrrolidide.
  • the inhibitors can be provided in linear or cyclic form or as mixtures thereof, as described in U.S. Patent No. 6,355,614, issued March 12, 2002.
  • the proportion of linear (versus cyclic) forms in these mixtures may vary (e.g., less than 20% to more than 90%) depending on the formulation. In some embodiment, at least 30%, 40%, 50%, 60%, 70%, or 80% of the inhibitor is in the linear form.
  • the inhibitors may be provided as prodrugs that are converted (via enzymatic, chemical, metabolic, or any other means, in vivo or ex vivo) to the forms shown above.
  • a prodrug of for example "A-boroPro”, as used herein, is a compound that is metabolized in vivo to A-boroPro or that disintegrates (e.g., upon contact with stomach acid) to form A- boroPro.
  • Some prodrugs are converted into A-boroPro via hydrolysis or oxidation in vivo. These include alcohol precursors of A-boroPro that are oxidized in vivo (e.g.. in the liver) and aboroxine derivative of A-boroPro, as well as esters of Glu-boroPro and related compounds.
  • Prodrugs of A-boroPro also include cyclized versions of the molecule.
  • prodrugs includes compounds that are converted to A-boroPro by enzymes. These enzymes may be post-prolyl cleaving enzymes (e.g., DPP-IV) or non-post- prolyl cleaving enzymes. Examples of this class of prodrug moieties are disclosed in U.S. Patent Nos. 5,462,928 issued October 31, 1995; and 6,100,234 issued August 8, 2000; and published PCT applications WO 91/16339 published October 31, 1991; WO 93/08259 published April 29, 1993; and WO 03/092605, published November 13, 2003, among others.
  • the length of such prodrug compounds maybe 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 50, 100 or more residues in length (whereby the length includes A and proline residues). Multiples of 3 are also contemplated.
  • the inhibitors may be provided in a substantially optically pure form. That is, at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% of the inhibitors in the mixture possess boron- bearing carbon atoms that are in the L-configuration.
  • Methods for synthesizing substantially optically pure isomers of for example Formula I agents are disclosed in published PCT application WO 93/08259.
  • the mixture of R- and S-enantiomers of boron substituted pyrrolidine contains at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the R-enantiomer of boron substituted pyrrolidine.
  • the invention intends to treat subjects having or at risk of developing a condition characterized by abnormal mammalian cell proliferation.
  • subject means a mammal including humans, norihuman primates, dogs, cats, sheep, goats, horses, cows, pigs and rodents.
  • An abnormal mammalian cell proliferation disorder or condition refers to a cell population (e.g., a tumor) which exhibits an abnormal (e.g., increased) rate of division as compared to its normal cellular counterparts, or which grows in a factor independent manner.
  • Conditions characterized by an abnormal mammalian cell proliferation include but are not limited to solid and non-solid benign, pre-malignant or malignant conditions.
  • the condition therefore may be a cancer.
  • the cancer may be carcinoma, sarcoma or melanoma.
  • the cancer may be basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, CNS cancer, colon and rectum cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, cancer of the head and neck, gastric cancer, intra-epithelial neoplasm, kidney cancer, larynx cancer, leukemia, acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphoid (or lymphocytic) leukemia (CLL), T cell leukemia, B cell leukemia, liver cancer, small cell lung cancer, non-small cell lung cancer (NSCLC, including adenocarcinoma, giant (or oat) cell carcinoma, and squa
  • Sarcomas are rare mesenchymal neoplasms that arise in bone (osteosarcomas) and soft tissues (fibrosarcomas).
  • Sarcomas include liposarcomas (including myxoid liposarcomas and pleiomorphic liposarcomas), leiomyosarcomas, rhabdomyosarcomas, malignant peripheral nerve sheath tumors (also called malignant schwannomas, neurofibrosarcomas, or neurogenic sarcomas), Ewing's tumors (including Ewing's sarcoma of bone, extraskeletal (i.e., not bone) Ewing's sarcoma, and primitive neuroectodermal tumor), synovial sarcoma, angiosarcomas, hemangiosarcomas, lymphangiosarcomas, Kaposi's sarcoma, hemangioendothelioma, des
  • Melanomas are tumors arising from the melanocyte system of the skin and other organs.
  • Examples of melanoma include lentigo maligna melanoma, superficial spreading melanoma, nodular melanoma, and acral lentiginous melanoma.
  • the cancer is breast cancer, colorectal cancer, chronic lymphocytic leukemia, non-small cell lung cancer, Non-Hodgkin's lymphoma, melanoma or prostate cancer.
  • the condition may be a primary tumor with undetectable metastatic lesions, or it may be a tumor that has metastasized.
  • the compositions and methods provided herein therefore are intended to treat primary tumors and/or metastases. In some instances, treatment may be of a primary tumor in the absence of detectable metastatic lesions in the subject.
  • the cancers to be treated may be refractory cancers.
  • a refractory cancer as used herein, is a cancer that is resistant to the ordinary standard of care prescribed. Refractory cancers may appear initially responsive to a treatment (and then recur), or they may be completely non-responsive to the treatment.
  • the ordinary standard of care will vary depending upon the cancer type, and the degree of progression in the subject. It may be a chemotherapy, or surgery, or radiation, or a combination thereof. Those of ordinary skill in the art are aware of such standards of care or are able to readily discern the ordinary standard of care based on FDA guidelines. Subjects being treated according to the invention for a refractory cancer therefore may have already been exposed to another treatment for their cancer.
  • refractory cancers include but are not limited to leukemia, melanoma, lung cancer including non-small cell lung cancer, pancreatic cancer and Non- Hodgkin's lymphoma.
  • the ordinary standard of care may comprise one or more chemotherapeutic or immunotherapeutic agents including but not limited to platinum- containing regimens such as cisplatin and carboplatin, docetaxel, pemetrexed, 5-fluorouracil, gemcitabine, cyclophosphamide, doxorubicin, rituximab, HERCEPTIN, and the like, either alone or in combination. Accordingly, the methods provided herein may be used as first line therapy or as subsequent line therapy.
  • the invention can also be used to treat cancers that are immunogenic.
  • Cancers that are immunogenic are cancers that are known to (or likely to) express immunogens on their surface or upon cell death. These immunogens are in vivo endogenous sources of cancer antigens and their release can be exploited by the methods of the invention in order to treat the cancer. These cancers therefore are also known to respond to immunotherapy such as vaccine or antibody therapy.
  • cancers examples include melanoma, renal cell cancer, colorectal cancer, breast cancer, ovarian cancer, prostate cancer, leukemia such as T cell leukemia, B cell leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, common (pre-B) acute lymphocytic leukemia, chronic lymphocytic leukemia (CLL), and lymphoma such as Non-Hodgkin's lymphoma, T-cell lymphoma, and B-cell lymphoma.
  • leukemia such as T cell leukemia, B cell leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, common (pre-B) acute lymphocytic leukemia, chronic lymphocytic leukemia (CLL), and lymphoma such as Non-Hodgkin's lymphoma, T-cell lymphoma, and B-cell lymphoma.
  • One category of benign or precancerous conditions characterized by abnormal cell proliferation is proliferative dermatologic disorders. These include conditions such as keloids, actinic keratosis, bowenoid actinic keratosis seborrheic keratosis, hemangiomas, papilloma virus infection (e.g.
  • verruca vulbaris verruca plantaris, verruca plana, condylomata, etc.
  • eczema hypertrophic actinic keratosis, arsenical keratosis, hydrocarbon keratosis, thermal keratosis, radiation keratosis, viral keratosis, Bowen's disease, erythroplaquia of queyrat, oral erythroplaquia, leukoplakia, and intraepidermal epithelialoma.
  • An precancerous lesion is a lesion that has a propensity to develop into a cancerous condition.
  • the epithelial lesions may develop into an invasive form of squamous cell carcinoma and may pose a significant threat of metastasis.
  • compositions and methods of the invention are useful in some instances for improving the efficacy of or replacing existing cancer therapies including but not limited to surgical procedures, radiation therapies, chemotherapies, immunotherapies and/or hormone therapies (e.g., the ordinary standard of care therapies).
  • the post-pro IyI cleaving enzyme inhibitors can be used in combination with surgery, radiation, chemotherapy, immunotherapy and/or hormone therapy to treat subjects according to the invention.
  • the second therapy i.e., the non-post prolyl cleaving enzyme inhibitor therapy
  • the post-prolyl cleaving enzyme inhibitors may be administered, according to some embodiments, with a second therapeutic agent.
  • the second therapeutic agent may be an anti-cancer agent.
  • An anti-cancer agent is an agent that at least partially inhibits the development or progression of a cancer, including inhibiting in whole or in part symptoms associated with the cancer even if only for the short term.
  • Anti-cancer agents function in a variety of ways. For example, some anti-cancer agents work by targeting physiological mechanisms that are specific to cancer cells. Examples include the targeting of specific genes and their gene products (i.e., proteins primarily) which are mutated in cancer.
  • Such genes include but are not limited to oncogenes (e.g., Ras, Her2, bcl-2), tumor suppressor genes (e.g., EGF, p53, Rb), and cell cycle targets (e.g., CDK4, p21, telomerase).
  • oncogenes e.g., Ras, Her2, bcl-2
  • tumor suppressor genes e.g., EGF, p53, Rb
  • cell cycle targets e.g., CDK4, p21, telomerase.
  • Cancer chemotherapy is any treatment involving the use of chemotherapeutic agents to treat cancer.
  • Cancer chemotherapy may consist of a single chemotherapeutic agent or a combination of chemotherapeutic agents.
  • chemotherapeutic agents include Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin; Bleomycin Sulfate; Bortezomib (VELCADE); Brequ ⁇ nar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambuci
  • Additional chemotherapeutic agents include but are not limited to actimomycin D; AD 32/Valrubicin; adrenocortical suppressant; Adrenocorticosteroids antagonists; adriamycin; AG3340; AG3433; alkylating agents; Alkyl sulfonates; 5-Azacitidine; 5- azacytidine; Alfa 2b; Amsacrine (m-AMSA); Anthracenedione; antiandrogens; antibiotics; antiestrogens; antimetabolites; antimitotic drugs; AraC; azathioprine; bacteriochlorophyll-a; BAY 12-9566; BB2516/Marmistat; BCH-4556; benzoporphyrin derivatives; Biological response modifiers; BMS-182751/oral platinum; bromodeozyuridine; 5-bromodeozyuridine; 2-CdA; Caelyx/liposomal doxorubicin; Campto/Levamisole;
  • Combinations of two, three, four or more chemotherapeutic agents may be used.
  • the invention also contemplates the use of a second agent that is a post-prolyl cleaving enzyme inhibitor such as a DPP-IV inhibitor, or a FAP inhibitor.
  • a second agent that is a post-prolyl cleaving enzyme inhibitor such as a DPP-IV inhibitor, or a FAP inhibitor.
  • DPP-IV inhibitors and FAP inhibitors alone or in combination with other agents may also be used.
  • the anti-cancer agent may be an immunotherapeutic agent.
  • Immunotherapeutic agents are medicaments which derive from antibodies or antibody fragments which specifically bind or recognize a cancer antigen.
  • a cancer antigen is broadly defined as an antigen expressed by a cancer cell.
  • the antigen is expressed at the cell surface of the cancer cell.
  • the antigen is one which is not expressed by normal cells, or at least not expressed to the same level as in cancer cells.
  • Antibody-based immunotherapies may function by binding to the cell surface of a cancer cell and thereby stimulating the endogenous immune system to attack the cancer cell. Another way in which antibody-based therapy functions is as a delivery system for the specific targeting of toxic substances to cancer cells.
  • Antibodies are usually conjugated to toxins such as ricin (e.g., from castor beans), calicheamicin and maytansinoids, to radioactive isotopes such as Iodine-131 and Yttrium-90, to chemotherapeutic agents (as described herein), or to biological response modifiers. In this way, the toxic substances can be concentrated in the region of the cancer and non-specific toxicity to normal cells can be minimized.
  • antibodies which bind to vasculature such as those which bind to endothelial cells, are also useful in the invention. This is because generally solid tumors are dependent upon newly formed blood vessels to . survive, and most tumors are capable of recruiting and stimulating the growth of new blood vessels. As a result, one strategy of some cancer medicaments is to attack the blood vessels feeding a tumor and/or the connective tissues (or stroma) supporting such blood vessels.
  • antibodies such as but not limited to bevacizumab (AVASTIN), trastuzumab (HERCEPTIN), alemtuzurnab (CAMPATH, indicated for B cell chronic lymphocytic leukemia,), gemtuzumab (MYLOTARG, hP67.6, indicated for leukemia), rituximab (RITUXAN), tositumomab (BEXXAR, anti-CD20, indicated for B cell malignancy), MDX-210 (bispecific antibody that binds simultaneously to HER-2/neu oncogene protein product and type I Fc receptors for immunoglobulin G (IgG) (Fc gamma RI)), oregovomab (OVAREX, indicated for ovarian cancer), edrecolomab (PANOREX), daclizumab (ZENAPAX), palivizumab (SYNAGIS, indicated for respiratory conditions such as RSV infection), ibritumomab (AV
  • immunotherapeutic agents include but are not limited to Rituximab (Rituxan), and trastuzumab (Herceptin), anti-neu/HER-2 antibodies, anti-CD20 antibodies, Quadramet, Panorex, IDEC- Y2B8, BEC2, C225, Oncolym, SMART M195, Anti-CTLA4 ATRAGEN, Ovarex, Bexxar, LDP-03, ior t6, MDX-210, MDX-11, MDX-22, OV103, 3622W94, anti-VEGF, Zenapax, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-I, CEACIDE, Pretarget, NovoMAb-G2, TNT, Gliomab-H, GNI-250, EMD-72000, LymphoCide, CMA 676, Monopharm-C, 4B5, ior egf.r3, ior c5, BABS, anti-FLK-2, MDX- 260,
  • Tumor vaccines are medicaments which are intended to stimulate an endogenous immune response against tumor cells.
  • Tumor vaccines generally enhance the presentation of tumor antigens to both antigen presenting cells (e.g., macrophages and dendritic cells) and/or to other immune cells such as T cells, B cells, and NK cells.
  • tumor vaccines may be used along with adjuvants, such as those described above.
  • Tumor antigens such as those present in tumor vaccines or those used to prepare tumor immunotherapies, can be prepared from crude tumor cell extracts, as described in Cohen PA et al. (1994) Cancer Res 54:1055-1058, or by partially purifying the antigens, using recombinant technology, or de novo synthesis of known antigens.
  • Tumor antigens can be used in the form of immunogenic portions of a particular antigen or in some instances a whole cell or a tumor mass can be used as the antigen.
  • Such antigens can be isolated or prepared recombinantly or by any other means known in the art.
  • dendritic cells which have been exposed to tumor antigens in vitro, have processed the antigens, and are able to express the tumor antigens at their cell surface in the context of MHC molecules for effective antigen presentation to other immune system cells.
  • Dendritic cells form the link between the innate and the acquired immune system by presenting antigens and through their expression of pattern recognition receptors which detect microbial molecules like LPS in their local environment.
  • tumor vaccines include but are not limited to EGF, Anti-idiotypic cancer vaccines, Gp75 antigen, GMK melanoma vaccine, MGV ganglioside conjugate vaccine, Her2/neu, Ovarex, M-Vax, O-Vax, L-Vax, STn-KHL theratope, BLP25 (MUC-I), liposomal idiotypic vaccine, Melacine, peptide antigen vaccines, toxin/antigen vaccines, MVA-based vaccine, PACIS, BCG vacine, TA-HPV, TA-CIN. DISC-virus and ImmuCyst/TheraCys.
  • angiogenesis inhibitors function by attacking the blood supply of solid tumors.
  • Angiogenic mediators include basic FGF, VEGF, angiopoietins, angiostatin, endostatin, TNF- ⁇ , TNP-470, thrombospondin-1, platelet factor 4, CAI, and certain members of the integrin family of proteins. Since the most malignant tumors are able to metastasize (i.e., exit the primary tumor site and seed a distal tissue, thereby forming a secondary tumor), therapies that impede this metastasis are also useful in the treatment of the cancer.
  • Anti-cancer agents may also be classified according to their targets.
  • One such target is the EGF receptor.
  • EGFR inhibitors include erlotinib (TARCEVA), gefitinib (IRESSA), WHI-P97 (quinazoline derivative), LFM-A12 (leflunomide metabolite analog), ABX-EGF, lapatinib, canertinib, ZD-6474 (ZACTIMA), AEE788, and AG1458.
  • Another such target is VEGF.
  • VEGF inhibitors include bevacizumab (AVASTIN), ranibizumab (LUCENTIS). pegaptanib (MACUGEN), sorafenib, sunitinib (SUTENT), vatalanib, ZD-6474 (ZACTIMA), anecortave (RETAANE), squalamine lactate, and semaphorin.
  • Immunotherapeutic agents may also be classified according to their targets.
  • One such target is CD20.
  • Antibodies that target CD20 include rituximab (RITUXAN), ibritumomab tiuxetan (ZEVALIN), AND tositumomab (BEXXAR).
  • Other targets include CD22, CD54, HER2. and EGFR.
  • Radiation therapy uses high-energy rays to damage or kill cancer cells by preventing them from growing and dividing. Radiation therapy may be externally or internally delivered. External radiation delivers high-energy rays directly to the tumor site from a machine outside the body. Internal radiation, or brachytherapy, involves the implantation of a small amount of radioactive material in or near the cancer.
  • Surgery is used to diagnose a cancer, determine its stage, and to induce remission of a cancer.
  • information gained during surgery is useful in predicting the likelihood of cancer recurrence and whether other treatment modalities will be necessary.
  • Cancer therapy is divided into two phases: remission induction and maintaining cancer remission (maintenance therapy).
  • the invention also encompasses compositions and methods for maintaining cancer remission (maintenance therapy) in a subject who had cancer and who has responded to cancer remission induction therapy.
  • Remission induction therapy consists of administering treatment (e.g., anti-cancer agents, and/or radiation therapy and/or surgery) over a period of time that varies from hours to weeks to months and even years depending on the type of cancer, stage of cancer, and overall condition of the subject.
  • Remission induction may take place over a period of time that varies from hours to weeks to months and even years.
  • more than one remission induction course of treatment may be given. For example, in subjects who have leukemia, bone marrow blood cell production requires about 2-3 weeks to recover. During this time, patients often require blood and platelet transfusions to maintain red blood cell and platelet levels.
  • the white blood cell growth factors Neupogen® and NeulastaTM may be used to reduce the severity of neutropenia, shorten hospital stays and to decrease the chance of dying from remission induction therapy.
  • the subject is examined for signs and/or symptoms and/or laboratory findings of the cancer in order to determine if a remission is achieved. If a remission is achieved and no further therapy given, a subject may have a recurrence of the cancer. To prevent recurrence of cancer, maintenance therapy may be initiated after induction of remission.
  • Maintenance maybe initiated 1, 2, 3, 4, 5, 6, 7, 8, 9 ,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 75, 90, 105, 120, 135, 150, 165 days, or more after induction of remission.
  • maintenance therapy is initiated as close to induction of remission as possible.
  • Maintenance therapy may be given for 1, 2, 3, 4, 5, 6, 7, 8, 9 ,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 months, or more.
  • the maintenance therapy is given for 8, 9, 10, 11, 12, 13, 14, 15, 16, .17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30 31, 32, 33, 34, 35 years, or more.
  • Maintenance therapy may be given at intervals. Examples of such intervals include but are not limited to every 12 hours, every 24 hours, every 36 hours, every 2, 3, 4, 5, 6, 7, 10, and 14 days.
  • the compound and/or agent is administered once every month. In some embodiments, the compound and/or agent is administered every other day, every other 2 days, or every other week.
  • maintenance therapy refers to treatment that is given to help keep cancer in remission and/or to prevent a relapse. Thus, maintenance therapy is given to prevent a cancer from recurring once that cancer has responded to the initial treatment.
  • treatment refers to the administration of one or more therapeutic agents to a subject for the purpose of achieving a medically desirable benefit.
  • treatment includes preventing, delaying, abating or arresting the clinical symptoms and or signs of a cancer.
  • remission refers to a complete or partial disappearance of the signs and/or symptoms and/or laboratory findings of a cancer in response to treatment. It is the period during which symptoms of disease of the cancer are reduced (partial remission) or disappear (complete remission). Remission does not necessarily mean cure because a medical professional cannot be sure that there are no cancer cells at all in the body.
  • Therapies to induce remission in a subject vary depending on the type of cancer, stage of cancer, and overall condition of the subject. Depending on these factors, remission may be induced in a subject. with one or more anti-cancer agents, radiation therapy, or surgery. There are various categories of anti-cancer agents. These include chemotherapeutic agents, immunotherapeutic agents, and tumor vaccines, as discussed herein.
  • a subject who had cancer is a subject who at least had one identifiable sign, symptom, or laboratory finding sufficient to make a diagnosis of a cancer in accordance with clinical standards known in the art for identifying such cancer. Examples of such clinical standards can be found, for example, in Harrison's Principles of Internal Medicine, 15th Ed. (Fauci AS et al., eds., McGraw-Hill, New York, 2001) and in other medical textbooks.
  • a diagnosis of a cancer will include identification of a particular cell type present in a sample of a body fluid or tissue obtained from the subject.
  • the agents of the invention are administered in effective amounts.
  • An effective amount is a dosage of the agent sufficient to provide a medically desirable result.
  • the effective amount will vary with the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent or combination therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. It is preferred generally that a maximum dose be used, that is 7 the highest safe dose according to sound medical judgment.
  • an effective amount to inhibit the condition would be an amount sufficient to reduce or halt altogether the abnormal mammalian cell proliferation so as to slow or halt the development of or the progression of a cell population such as, for example, a tumor, and/or to inhibit in whole or in part symptoms associated with the condition.
  • Inhibition can be assessed by the size of a cell mass (e.g., a tumor), or by the presence and/or frequency of cancer cells in the blood or other body fluid or tissue (e.g., a biopsy).
  • some inhibitors are administered in an amount effective to inhibit the condition while reducing in whole or in part side effects such as those associated with post-prolyl cleaving enzymes including Class I inhibitors.
  • Some embodiments of the invention relate to the administration of inhibitors substantially simultaneously.
  • the term “substantially simultaneously” means that the inhibitors (or other agents) are administered at the same time or within minutes of each other (e.g., within 10 minutes of each other).
  • the term embraces joint administration as well as consecutive administration. If the administration is consecutive, it is separated in time for only a short period (e.g., the time it would take a medical practitioner to administer two compounds separately).
  • concurrent administration and substantially simultaneous administration are used interchangeably.
  • the invention contemplates a variety of inhibitor administration dosings, schedules and regimens.
  • Class I and Class II inhibitors are administered alone or in combination so as to maximize the therapeutic benefit and minimize the side effect(s) of each or both inhibitor classes. In some embodiments, this is accomplished by providing a molar excess of Class II inhibitors relative to Class I inhibitors.
  • the Class LClass II molar ratio can be 1 :2, 1:3, 1:4, 1:5, 1:6, 1:7, 1 :8, 1:9, 1:10, 1:20, 1:30, 1:40, 1 :50, 1:60, 1:70, 1 :80, 1 :90 or 1 : 100.
  • the molar ratio as used herein refers to the total moles of the Class I inhibitortotal moles of Class II inhibitor.
  • the Class II inhibitor can also be administered in a greater than 100 molar excess (as compared to the Class I inhibitor) (e.g., the Class T.Class II molar ratio is 1:150, 1:200, etc.).
  • the combined amount of inhibitors administered may vary.
  • the combined amount of inhibitors refers to the sum mass of inhibitors administered.
  • the combined amounts typically range from about 0.1 ⁇ g to 10.000 mg per day, from about 1 ⁇ g to 5000 mg per day, from about 100 ⁇ g to 50 mg per day, from about 400 ⁇ g to 25 mg per day, from about 800 ⁇ g to 10 mg per day, from about 1500 ⁇ g to about 10 mg per day, from about 1500 ⁇ g to about 5 mg per day.
  • the combined amounts therefore maybe about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more micrograms per day.
  • the inhibitors may be administered in a single or multiple administrations per day. In some important embodiments, the inhibitors are administered in two administrations per day. Any administration per day may include both Class I and Class II inhibitors. Alternatively, one or all administrations per day may include one but not both inhibitor classes. For example, the first administration of a day may include the Class I inhibitor and the second administration of the day may include the Class II inhibitor (and optionally the Class II inhibitor).
  • the invention further embraces the administration of different inhibitors at different times.
  • the Class I inhibitor may be administered before, before and during, before and after, during and after, or before, during and after administration of the Class II inhibitor.
  • These embodiments seek to maximize immunostimulation against the condition.
  • the Class II inhibitor is administered before the Class I inhibitor.
  • the inhibitors are administered in an alternating manner.
  • the Class I inhibitor is administered first, followed by the Class II inhibitor, followed by the Class I inhibitor, followed by the Class II inhibitor, etc. More specifically, the Class I inhibitor may be administered on days 1, 3, 5, 7, etc. and the Class II inhibitor may be administered on days 2, 4, 6, 8, etc.
  • the Class I inhibitor is administered on a number of days (e.g., 2, 3, 4, 5, 6, 7, or more days), followed by administration of the Class II inhibitor for a number of days (e.g., 2, 3, 4, 5, 6, 7, or more days). The number of days in which the Class I inhibitor is administered may be equal to or different from the number of days in which the Class II inhibitor is administered.
  • the invention further contemplates administration of Class I inhibitors on alternating days or alternating administrations without intervening administration of a Class II inhibitor.
  • the Class I inhibitor may be administered on day 2, day 4 and day 6 of a weekly cycle, without administration of the Class II inhibitor on days 3, 5 or 7.
  • administration of Class I may be interrupted for a day or more depending on the presence and magnitude of side effects. The entire time course may therefore be shifted based on the length of the interruption.
  • the timing of administration may also depend upon whether a second therapy is used in combination with the inhibitor(s).
  • the inhibitor(s) are used in combination with a chemotherapeutic or an immunotherapeutic agent that is administered on day 1 of a treatment cycle.
  • the Class I/Class II inhibitor combination may be administered on days 2-7 of the same treatment cycle.
  • the cycle may be 7, 14, 21, 28, 35, 42, 49 days or longer.
  • the cycle may be performed once, twice, three times, four times, or more, optionally with rest periods (e.g., days or weeks in which no therapy is administered) in between.
  • the cycle may be a 21 day cycle in which the immunotherapeutic or chemotherapeutic agent is administered on days 1 and 8, the inhibitors are administered on days 2-7 and 9-14, and days 15-21 are rest days.
  • the cycle may be a 21 day cycle in which the immunotherapeutic or chemotherapeutic agent is administered on days 1, 8 and 15, and the inhibitors are administered on days 2-7, 9-14 and 15-21.
  • the Class I inhibitor may be administered on days 2-7 and 15-21 and the Class II inhibitor may be administered on days 9-14.
  • the Class I inhibitor may also be administered prior to the administration of the chemotherapeutic or immunotherapeutic agent, with administration of the Class II (and optionally Class I inhibitor) during and/or after administration of the chemotherapeutic or immunotherapeutic agent.
  • the inhibitors may be formulated separately or together. Inhibitors that are formulated together are present in the same composition prior to administration to the subject. Inhibitors that are formulated separately are present in separate and distinct compositions prior to administration to the subject. In this latter instance, however, they may be commingled immediately prior to administration to the subject.
  • the inhibitors can be administered to a subject by any route that delivers the inhibitor to the affected site, either directly or indirectly.
  • routes of administration include but are not limited to oral, topical (including intranasal, ocular, vaginal, rectal, transdermal, etc.), and parenteral (including intramuscular, intravenous, subcutaneous, etc.), by inhalation and intratracheal. Delivery may be local (e.g., mucosal) or systemic.
  • the administration route of the inhibitor(s) and other therapeutic agents may be the same or it may be different.
  • the inhibitors are administered orally, and the other therapeutic agent is administered by a non-oral route.
  • compositions are sterile compositions that comprise effective amounts of active agent, such as the inhibitors of the invention, preferably in a pharmaceutically-acceptable carrier.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other subject contemplated by the invention.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions are commingled in a manner that precludes interaction that would substantially impair their desired pharmaceutical efficiency.
  • the inhibitors may be administered pjer se (neat) or in the form of a salt.
  • the salts are preferably pharmaceutically acceptable. Salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Salts can also be alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
  • Suitable buffering agents include acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v).
  • Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).
  • the compounds when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • Pharmaceutical parenteral formulations include aqueous solutions of the active ingredients in water-soluble form.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of active ingredients may be prepared as oil-based suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • a suitable compound for sustained release delivery is GELFOAM, a commercially available product consisting of modified collagen fibers.
  • compositions also may comprise suitable solid or gel phase carriers or excipients.
  • suitable solid or gel phase carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • the active agents may be formulated in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • the agents can be formulated readily by combining with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion.
  • Pharmaceutical compositions for oral use can be obtained as solid excipients, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions or may be administered without any carriers.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • Pharmaceutical compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • suitable liquids such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. AU formulations for oral administration should be in dosages suitable for such administration.
  • compositions may take the form of tablets, lozenges, gum, dissolvable sheets or films, pastes (such as tooth pastes), washes (such as mouth washes), formulated in conventional manner.
  • the compounds may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • compositions may also be formulated for topical delivery (e.g., to the skin) by commingling the active ingredients with bases such as creams, ointments or lotions.
  • bases such as creams, ointments or lotions.
  • the compounds may also be formulated as a depot preparation.
  • Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • the preferred vehicle is a biocompatible microparticle or implant that is suitable for implantation into the mammalian recipient.
  • exemplary bioerodible implants that are useful in accordance with this method are described in PCT International Application No. PCT/US/03307 (Publication No. WO 95/24929, entitled “Polymeric Gene Delivery System", claiming priority to U.S. patent application serial no. 213,668, filed March 15, 1994).
  • PCT/US/0307 describes a biocompatible, preferably biodegradable polymeric matrix for containing a biological macromolecule. The polymeric matrix may be used to achieve sustained release of the agent in a subject.
  • the agent may be encapsulated or dispersed within the biocompatible, preferably biodegradable polymeric matrix disclosed in PCT/US/03307.
  • the polymeric matrix preferably is in the form of a microparticle such as a microsphere (wherein the agent is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein the agent is stored in the core of a polymeric shell).
  • Other forms of the polymeric matrix for containing the agent include films, coatings, gels, implants, and stents.
  • the size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix device is implanted.
  • the size of the polymeric matrix devise may be further selected according to the method of delivery which is to be used, typically injection into a tissue or administration of a suspension by aerosol into the nasal and/or pulmonary areas.
  • the polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when the devise is administered to a vascular or pulmonary surface.
  • the matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time.
  • Both non-biodegradable and biodegradable polymeric matrices can be used to deliver agents to the subject.
  • Biodegradable matrices are preferred.
  • Such polymers may be natural or synthetic polymers. Synthetic polymers are preferred.
  • the polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable.
  • the polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multivalent ions or other polymers.
  • agents may be delivered using the bioerodible implant by way of diffusion, or more preferably, by degradation of the polymeric matrix.
  • exemplary synthetic polymers which can be used to form the biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycol ⁇ des, polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionat
  • non-biodegradable polymers examples include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.
  • biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkyl ene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof.
  • synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone
  • natural polymers such as alginate and other
  • Bioadhesive polymers of particular interest include bioerodible hydrogels described by H.S. Sawhney, CP. Pathak and J.A.
  • the invention provides a composition of the inhibitors for use as a medicament, methods for preparing the medicament, and methods for the sustained release of the medicament in vivo.
  • Other delivery systems can include timed release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of agent, increasing convenience to the subject and the physician.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. They include the above-described polymeric systems, as well as polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109.
  • Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • Specific examples include, but are not limited to: (a) erosional systems in which the agent is contained in a form within a matrix such as those described in U.S. Patent Nos. 4,452,775, 4,675,189 and 5,736,152 and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Patent Nos. 3,854,480, 5,133,974 and 5,407,686.
  • pump-based hardware delivery systems can be used, some of which are adapted for implantation.
  • long-term sustained release implant may be particularly suitable for treatment of chronic conditions, such as a the suspected presence of dormant metastases.
  • Long-term release are used herein, means that the implant is constructed and arranged to delivery therapeutic levels of the active ingredient for at least 30 days, at least 60 days and more preferably for several months.
  • Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.
  • L-valinyl-L-boroproline (PT-100, Val-boroPro, VBP) is an orally available anti-tumor agent.
  • VBP can upregulate cytokine and chemokine expression in tumor and lymphoid tissue. Consistent with targeting of both adaptive and innate immune effector cells by the cytokines/chernokines involved, VBP can stimulate specific anti-tumor responses that cause tumor rejection in immunocompetent mice and T-cell independent activity that inhibits tumor growth by 50-90% in immunodeficient mice inoculated with mouse or human tumor cell lines. Natural killer cells appear to be non-essential for T-cell independent activity because anti-tumor activity of VBP was undiminished in SCID/beige mice.
  • Innate effector cells involved in anti-tumor activity of VBP have now been characterized utilizing immunocompetent and immunodeficient mice depleted of either neutrophils by treatment with anti-Ly6G mAb or macrophages by treatment with carrageenan. Both neutrophils and macrophages appear to contribute to the anti-tumor activity of VBP in immunocompetent BALB/c mice inoculated with WEHI 164 fibrosarcoma cells because depletion of either reduced the frequency of tumor rejection from 70% to ⁇ 2%. Depletion of macrophages in C.
  • B- 17 SCID mice has now been shown to significantly (P ⁇ 0.005) reduce the anti-tumor activity of VBP against A549 non-small cell lung cancer (NSCLC) and Raji B- cell lymphoma xenografts.
  • NSCLC non-small cell lung cancer
  • neutrophil depletion was previously shown to abolish anti-tumor activity of CB- 17 SCID mice xeno grafted with human tumor cell lines, including A549.
  • neutrophil infiltration of VBP sensitive A549 and insensitive H460 NSCLC tumors was compared in VBP treated BALB/c Rag2 'A mice.
  • VBP VBP treated A549 tumors were heavily infiltrated with neutrophils compared with vehicle treated tumors; but VBP caused little infiltration of refractory H460 tumors.
  • mice were injected (s.c.) in one flank with 4 x 10 6 WEHI 164, 5 x 10 6 A549 or 1 x 107 Raji cells on day 0.
  • Neutrophils were depleted in vivo by anti-Ly6G mAb (RB6-8C5) injection (i.p.) on days -2, 0, 2, 4, and 6 in WEHI 164 model, and on days 0, 3, 5, 7, 9, 12 and 15 in A549 and Raji models.
  • Macrophages were depleted in vivo by carrageenan injection (i.p.) on days -3, -1. 3 and 7 in WEHI 164 model and on days -3, -1, 1, 3, 6, 9, 12, 15 in A549 and Raji models. Tumor tissue sections were stained with hematoxylin and eosin.
  • Fibroblast activation protein is a serine protease that is selectively expressed on tumor stromal fibroblasts in many epithelial cancers. FAP overexpression has been shown to enhance tumor growth.
  • Val-boro-Pro PT-100, VBP
  • Glu-boro-Pro GBP
  • VBP treatment has been shown to decrease tumor size and cause tumor regression and rejection in a variety of animal tumor models (Cancer Res 64: 5471-5480, 2004).
  • the mechanism of action is complex, as it not only inhibits FAP enzymatic activity but also has been shown to have immunomodulatory effects including upregulation of cytokine and chemokine expression.
  • GBP inhibits FAP enzymatic activity to a similar degree as VBP, but without invoking a cytokine response. Therefore, the effects of these inhibitors in animal models that have strong FAP expression were analyzed.
  • HT-29 xenografts induce stromal FAP expression in the tumor fibroblasts without any detectable expression on the colorectal cancer cells themselves.
  • HEK293 cells transfected with FAP were also tested as an epithelial FAP overexpression animal model.
  • VBP and GBP when administered orally to mice inoculated subcutaneously with either HT-29 or HEK-FAP cells, significantly inhibited tumor growth when compared to saline treated controls. Both inhibitors decreased tumor volume in the HT-29 and HEK-FAP xenografts by approximately 50%.
  • mice Similar studies were performed using 3 x 106 HEK-293 cells transfected with FAP wild type. Three cohorts of 10 mice were inoculated subcutaneously with HEK-FAP and tumor growth assessed after treatment with VBP and GBP at 10 ⁇ g BID and 100 ⁇ g BID respectively.
  • Dipeptidyl Peptidase Assay of Xenografts The degree of FAP enzymatic activity in tumors was performed using an immunocapture assay with Ala-Pro-7-amido-4- trifluoromethylcoumarin (Ala-Pro-AFC) as a substrate. 96- well Fluoronunc MaxiSorb plates were coated overnight with anti-FAP rabbit polyclonal antibody at a 100 ⁇ g/ml dilution. HEK293 or HT-29 xenografts were excised and protein extracted using the detergent T-PER (tissue protein extraction reagent) (Pierce, Rockford, IL) according to manufacturer's instructions.
  • T-PER tissue protein extraction reagent
  • the total tumor protein extracts containing FAP were added to the wells and incubated for 1 hour, washed 10 times, and dipeptidyl peptidase activity assessed using 0.5 mM Ala- Pro- AFC. Release of the free AFC fluorescent substrate at 60 minutes was detected on a Cytofluor fluorimeter (Labsystems, Helsinki, Finland) with 396 nm excitation and 490 emission.
  • VBP and GBP inhibit tumor growth of HT-29 and HEK-FAP xenografts. Both inhibitors attenuate enzymatic activity in vivo in a dose dependent manner. Because GBP has minimal immunomodulatory effects, these experiments suggest that the anti-tumor activity seen in these animal models may be mediated by the inhibition of FAP at the tumor microenvironment.
  • This Example illustrates the inhibition of FAP and DPP-IV in vitro by cyclopropyl- alanine-boroPro and cyclopentyl-glycine-boroPro.
  • the dose response curves (FIG. 12) indicate that compound concentrations achieving 50% inhibition (ICso) of dipeptidyl peptidase activity were 20 nM and 70 nM for inhibition of FAP by cyclopropyl-alanine- boroPro and cyclopentyl-glycine-boroPro respectively, and less than 8 nM for inhibition of DPP-IV by cyclopropyl-alanine-boroPro or cyclopentyl-glycine-boroPro.
  • Assay reaction mixtures consisted of 120 ⁇ l 50 mM HEPES/Na buffer pH 7.6, 140 mM NaCl, 15 ⁇ l of FAP or DPP-IV enzyme preparations in a 96-well plate. Enzyme was pre-warmed at 32°C, and then 7.5 ⁇ l inhibitor added.
  • the assay was then started by addition to FAP of 7.5 ⁇ l of an 8 mM solution of tripeptidepeptide substrate, Ala-Gly-Pro-(7-amino-4-trifluoromethyl coumarin) (Ala-Gly-Pro-AFC; Enzyme System Products, Dublin, CA) diluted from a 0.1 M stock in dimethyl sulfoxide, or by addition to DPP-IV of 7.5 ⁇ l of a 8 mM solution of dipeptide substrate, Ala-Pro-(7-amino-4- trifluoromethyl coumarin) (Ala-Pro-AFC; Enzyme System Products, Dublin, CA) diluted from a 0.4 M stock in dimethyl formamide.
  • the inhibitor was incubated with the enzyme for 4 min before addition of substrate. Reaction mixtures were incubated at 32°C, and production of the fluorescent AFC product was measured continuously in a fluorometer (excitation, 400 nm; emission 505 nm). Fluorometric readings were made with a Molecular Dynamics Spectra Max GeminiXS capable of reading 96-well microtiter plates.
  • This Example illustrates the inhibition of DPP-IV, DPP 8, FAP and DPP 2 in vitro by Norleucine-boroPro.
  • the dose response curves (FIG. 13) indicate that compound concentrations achieving 50% inhibition (IC 5 o) of dipeptidyl peptidase activity were: 0.6 nM for inhibition of DPP-P/, 1.8 to 2.2 nM for inhibition of DPP 8, 23.0 to 30.0 nM for inhibition of FAP and 0.8 to 1.0 nM for inhibition of DPP 2.
  • Assay reaction mixtures consisted of 120 ⁇ l 140 mM NaCl buffered with 50 mM HEPES/Na pH 7.6 for DPP-IV, DPP 8 and FAP, or 140 mM NaCl buffered with 120 ⁇ l 100 mM MES/Na pH 5.5 for DPP 2 mixed with 15 ⁇ l of each enzyme preparation in a 96-well plate. 15 ⁇ l of varying amounts of Norleucine- boroPro or buffer were added so as to obtain a concentration range of 0 to 100,000 nM in the final reaction mixture.
  • This Example illustrates that cyclopentyl-glycine-boroPro and cyclopropyl-alanine- boroPro stimulate cytokine and chemokine production by cultured human bone marrow stromal cells in vitro, as indicated by measurement of the levels of interleukin-8 (IL-8) and granulocyte colony stimulating factor (G-CSF) in culture supernatants (FIG. 14).
  • IL-8 interleukin-8
  • G-CSF granulocyte colony stimulating factor
  • Human bone marrow stromal cell cultures Samples of normal human bone marrow were purchased from Cambrex Bioproducts (Walkersville, MD) and mononuclear cells were purified over Ficoll-Hypaque (Nycomed, Oslo, Norway). Human stromal layers were established by seeding 4 x 10 7 mononuclear cells into T75 flasks (Corning) containing 20 ml MyeloCult medium (Stem Cell Technologies, Vancouver, BC) supplemented with 10 "6 M hydrocortisone (Sigma, St. Louis, MO) and incubation at 37°C in 100% humidified 5% CO 2 in air.
  • Assay ofIL-8 and G-CSF supernatant levels in stromal cell cultures After incubation of multi-well cultures for 24 hours, supernatant concentrations of human IL-8 and G-CSF were determined by Quantikine enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. ELISA was performed in duplicate for each sample. IL-8 and G-CSF concentrations were compared between cultures containing amino boronic dipeptides and control cultures.
  • This Example illustrates that cyclopentyl-glycine-boroPro stimulates cytokine and chemokine production in cultures of adherent monocytes derived from normal human blood incubated in the presence of the mouse MM46T fibroblast-derived cell line (FIGs. 15A to C) or in the presence of conditioned medium derived from cultures of MM46T cells (FIG. 15D), as indicated by measurement of the levels human IL- I ⁇ (FIG. 15A and D), human G-CSF (FIG. 15B), or mouse CXCL1/KC (FIG. 15C) in vitro.
  • Human monocyte cell cultures Previously frozen normal human peripheral blood mononuclear cells (Cambrex Bioproducts, Walkersville, MD) were incubated for one hour in 48-well plates at a concentration of 1.5 x 10 6 cells/ml. The adherent monocytes were washed three times and incubated for 18 hours with either no addition or cyclopentyl-glycine-boroPro at concentrations ranging from 0.1 to 100 nM in 10-fold increments. The 18 hour incubation of the monocyte was performed either in the presence of the mouse fibroblast-derived MM46T cells (ATCC) at a concentration of 5 x 10 4 cells/ml or in the presence of filtered conditioned medium obtained from cultures of the MM46T cell line as follows.
  • ATCC mouse fibroblast-derived MM46T cells
  • Conditioned medium was collected from MM46T cells incubated for 2 days in RPMI 1640 medium containing 10% fetal bovine serum and fractionated by Amicon Centriplus YM-3 (Millipore, Billerica, MA) filtration to produce a filtrate containing material of ⁇ 3 kDa in molecular size.
  • the filtrate was reconstituted with heat-inactivated fetal bovine serum (10%) and used as the culture medium for adherent monocytes incubated with or without cyclopentyl-glycine- boroPro.
  • TL-I ⁇ , G-CSF, and CXCLl /KC supernatant levels in human monocyte cultures After incubation of multi-well cultures for 18 hours, supernatant concentrations of human IL-I ⁇ and G-CSF and mouse CXClAfKC were determined by Quantikine enzyme- linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. ELISA was performed in duplicate for samples obtained from cultures performed in triplicate for each concentration of cyclopentyl-glycine-boroPro tested. The cytokine and chemokine concentrations were compared between cultures containing cyclopentyl-glycine-boroPro and control cultures.
  • ELISA Quantikine enzyme- linked immunosorbent assay
  • This Example illustrates that cyclopropyl-alanine-boroPro and cyclopentyl-glycine- boroPro can inhibit serum DPP-IV activity and stimulate increased levels of serum KC/CXCL1 in a dose dependent fashion in vivo when administered orally (by gavage) to BALB/c mice (FIG. 16).
  • Serum DPP-IV activity was assayed because its inhibition serves to indicate the oral bioavailability of small molecules that inhibit the enzyme.
  • Serum KC/CXCL1 was assayed because it was previously shown to be an indicator of increased levels of cytokines and chemokines in serum of mice administered active amino boronic dipeptide compounds.
  • Serum DPP-FV activity was assayed in reaction mixtures containing 10 ⁇ l serum and 90 ⁇ l 140 inM NaCl, 50 mM Hepes/Na, pH 7.6 containing 0.1 ImM of Gly-Pro-AFC. Reactions were incubated for 30 minutes at room temperature and stopped by addition of 100 ⁇ l 1 M sodium acetate. Fluorometric readings (excitation, 400 nm; emission, 505 nm) were made with a Molecular Dynamics Spectra Max Gemini XS. Serum concentrations of mouse KC/CXCL1 were determined by Quantikine enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN), according to the manufacturer's instructions. Assays were performed in duplicate for each sample. No detectable levels of KC/CXCL1 were measured in the serum of saline treated mice.
  • ELISA Quantikine enzyme-linked immunosorbent assay
  • This Example illustrates that Norleucine-boroPro can inhibit serum DPP-IV activity and stimulate increased levels of serum KC/CXCL1 in a dose dependent fashion in vivo when administered either orally (by gavage) or subcutaneously (by injection) to BALB/c mice (FIG. 17).
  • Serum DPP-IV activity was assayed because its inhibition serves to indicate the oral bioavailability of small molecules that inhibit the enzyme.
  • Serum KC/CXCL1 was assayed because it was previously shown to be an indicator of increased levels of cytokines and chemokines in serum of mice administered active amino boronic dipeptide compounds.
  • Example 9 This Example illustrates that oral administration of cyclopentyl-glycine-boroPro and cyclohexyl-glycine-boroPro can stimulate antigen-specific T-cell responses to subcutaneously injected synthetic peptides representing MHC class I- and class II-restricted antigenic epitopes.
  • mice co- immunized with a MHC class I (L d ) restricted peptide consisting of residues 118-126 of the nucleoprotein from lymphocytic choriomeningitis virus (LCMV NPm-ne) and a MHC class II (I-A d ) restricted peptide consisting of residues 323-336 of hen egg ovalbumin (OVA 323-336)-
  • L d MHC class I restricted peptide consisting of residues 118-126 of the nucleoprotein from lymphocytic choriomeningitis virus
  • I-A d MHC class II restricted peptide consisting of residues 323-336 of hen egg ovalbumin
  • mice Female BALB/c mice (6-8 week of age) were immunized on days 0 and 7 with equimolar amounts (50 nmol of each per mouse) of LCMV NP 118-126 and OVA 323-336 dissolved in saline and injected subcutaneously at the base of the tail. Cyclopentyl-glycine-boroPro and cyclohexyl-glycine- boroPro were co-administered individually, in separate treatment groups, with peptides on days 0 and 1, and were also given on each flanking day: for example, for priming the mice received PT-810 or PT-820 on days -1, 0, and +1.
  • PT-810 and PT-820 were administered by gavage in 0.2 ml saline at mid-day. On day 0 PT-810/PT-820 and peptides were administered within 60 minutes of each other. Specific T-cell responses from ex vivo splenocytes were quantitatively measured 5 weeks after last immunization by IFN- ⁇ ELISPOT.
  • ELISPOT assay of T cell responses in mice Spleen cells from immunized mice were assayed for their ability to secrete IFN- ⁇ during in vitro restimulation with antigenic peptides by an IFN- ⁇ specific ELISPOT assay (mouse IFN- ⁇ ELISPOT; R&D Systems, Minneapolis, MN). Pooled splenocytes from 2 mice per treatment group plated at 5xlO 5 cells/well were incubated with 1 ⁇ g/ml LCMV NP 118-126, 10 ⁇ g/ml OVA 323-336, 10 ⁇ g/ml Bag 28-39 (class I MHC matched irrelevant control peptide) or no peptide (medium alone).

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Abstract

L'invention concerne l'utilisation d'inhibiteurs d'une enzyme de clivage post-prolyle dans le traitement de troubles prolifératifs tels que le cancer.
PCT/US2007/008242 2006-03-31 2007-03-30 Procédés et compositions associés à des inhibiteurs d'une enzyme de clivage post-prolyle Ceased WO2007117419A2 (fr)

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CN105326841A (zh) * 2015-11-05 2016-02-17 中国科学院武汉病毒研究所 雷替曲塞或其药学上可接受的盐在制备治疗或预防疱疹病毒感染药物中的应用
CN105412113A (zh) * 2015-11-05 2016-03-23 中国科学院武汉病毒研究所 培美曲塞二钠或其药学上可接受的盐在制备治疗或预防疱疹病毒感染药物中的应用
WO2018056907A1 (fr) * 2016-09-21 2018-03-29 Agency For Science, Technology And Research Procédés et compositions pour inhiber l'inflammation de la peau et de déterminer la susceptibilité au cancer
CN107970442A (zh) * 2017-12-06 2018-05-01 成都康华生物制品有限公司 一种吸附无细胞百白破和b型嗜血流感四价联合疫苗的制备方法
JP2022121426A (ja) * 2016-09-07 2022-08-19 トラスティーズ オブ タフツ カレッジ イムノｄａｓｈ阻害剤及びｐｇｅ２アンタゴニストを用いた併用療法

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US5965532A (en) * 1996-06-28 1999-10-12 Trustees Of Tufts College Multivalent compounds for crosslinking receptors and uses thereof
US6890904B1 (en) * 1999-05-25 2005-05-10 Point Therapeutics, Inc. Anti-tumor agents
US20030130199A1 (en) * 2001-06-27 2003-07-10 Von Hoersten Stephan Dipeptidyl peptidase IV inhibitors and their uses as anti-cancer agents

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CN105326841A (zh) * 2015-11-05 2016-02-17 中国科学院武汉病毒研究所 雷替曲塞或其药学上可接受的盐在制备治疗或预防疱疹病毒感染药物中的应用
CN105412113A (zh) * 2015-11-05 2016-03-23 中国科学院武汉病毒研究所 培美曲塞二钠或其药学上可接受的盐在制备治疗或预防疱疹病毒感染药物中的应用
CN105412113B (zh) * 2015-11-05 2018-01-09 中国科学院武汉病毒研究所 培美曲塞二钠或其药学上可接受的盐在制备治疗或预防疱疹病毒感染药物中的应用
CN105326841B (zh) * 2015-11-05 2018-01-09 中国科学院武汉病毒研究所 雷替曲塞或其药学上可接受的盐在制备治疗或预防疱疹病毒感染药物中的应用
JP2022121426A (ja) * 2016-09-07 2022-08-19 トラスティーズ オブ タフツ カレッジ イムノｄａｓｈ阻害剤及びｐｇｅ２アンタゴニストを用いた併用療法
US11957657B2 (en) 2016-09-07 2024-04-16 Trustees Of Tufts College Combination therapies using immuno-dash inhibitors and PGE2 antagonists
US12478609B2 (en) 2016-09-07 2025-11-25 Trustees Of Tufts College Combination therapies using immuno-DASH inhibitors and PGE2 antagonists
WO2018056907A1 (fr) * 2016-09-21 2018-03-29 Agency For Science, Technology And Research Procédés et compositions pour inhiber l'inflammation de la peau et de déterminer la susceptibilité au cancer
CN107970442A (zh) * 2017-12-06 2018-05-01 成都康华生物制品有限公司 一种吸附无细胞百白破和b型嗜血流感四价联合疫苗的制备方法

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