WO2007059099A2 - Boroproline combination therapy for cancer - Google Patents

Boroproline combination therapy for cancer Download PDF

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Publication number
WO2007059099A2
WO2007059099A2 PCT/US2006/044130 US2006044130W WO2007059099A2 WO 2007059099 A2 WO2007059099 A2 WO 2007059099A2 US 2006044130 W US2006044130 W US 2006044130W WO 2007059099 A2 WO2007059099 A2 WO 2007059099A2
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Prior art keywords
compound
formula
cancer
administered
day
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PCT/US2006/044130
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French (fr)
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WO2007059099A3 (en
Inventor
Sharlene Adams
Barry Jones
Margaret J. Uprichard
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Point Therapeutics, Inc.
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Publication of WO2007059099A2 publication Critical patent/WO2007059099A2/en
Publication of WO2007059099A3 publication Critical patent/WO2007059099A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/69—Boron compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • This invention relates to methods and compositions for the treatment and prevention of cancer.
  • Cancer is the second leading cause of death, resulting in one out of every four deaths, in the United States.
  • the estimated total number of new diagnoses for lung, breast, prostate, colorectal and ovarian cancer was approximately two million. Due to the ever increasing aging population in the United States, it is reasonable to expect that rates of cancer incidence will continue to grow.
  • Cancer is currently treated using a variety of modalities including surgery, radiation therapy and chemotherapy.
  • the choice of treatment modality will depend upon the type, location and dissemination of the cancer.
  • One of the advantages of surgery and radiation therapy is the ability to control to some extent the impact of the therapy, and thus to limit the toxicity to normal tissues in the body.
  • Chemotherapy is arguably the most appropriate treatment for disseminated cancers such as leukemia and lymphoma as well as metastases.
  • Chemotherapy is generally administered systemically and thus toxicity to normal tissues is a major concern. Not all tumors, however, respond to chemotherapeutic agents and others, although initially responsive to chemotherapeutic agents, may develop resistance. As a result, the search for anti-cancer treatments has intensified in an effort to find more effective treatments with less non-specific toxicity.
  • the invention provides compositions and methods of use in the prevention and treatment of cancer.
  • the invention is based, in part, on the finding that Val-boroPro interacts and thereby enhances the activity of certain anti-cancer agents that have different mechanisms of action in the treatment of cancer.
  • the surprising enhancement is associated with the administration of certain doses of either agent and/or the use of certain administration regimens.
  • the combination of Val-boroPro with pemetrexed ALTA ®
  • NSCLC non-small cell lung cancer
  • Val-boroPro is a dipeptidyl peptidase IV (DPP-IV or DP-IV) inhibitor while pemetrexed is an anti-folate antimetabolite.
  • DPP-IV dipeptidyl peptidase IV
  • DP-IV dipeptidyl peptidase IV
  • pemetrexed is an anti-folate antimetabolite.
  • Val-boroPro acts, in part, by stimulating a variety of cytokines which can stimulate the immune system to combat cancer as well as having a direct anti-cancer effect.
  • Val-boroPro to stimulate cytokine and chemokine production endogenously is beneficial since exogenous administration of some of these factors, such as for example IL-I, has sometimes been associated with toxicity. Production of IL-I endogenously thereby overcomes these toxicity problems.
  • Val-boroPro also exerts a direct anti-tumor effect by inhibiting the enzymatic activity of fibroblast activation protein (FAP), a dipeptidyl peptidase (DPP) that has the potential to promote tumor growth.
  • FAP fibroblast activation protein
  • DPP dipeptidyl peptidase
  • pemetrexed an anti-folate antimetabolite, disrupts folate-dependent metabolism which is required for cell replication.
  • the invention relates, in part, to methods of treating cancer in a subject comprising administering a DPP-IV inhibitor represented by a compound (or an agent, as used interchangeably herein) of Formula I and an anti-metabolite preferably using particular dosing and timing regimens that are unexpectedly better than other regimens.
  • the invention also relates to compositions and kits comprising compounds of Formula I and an antimetabolite and uses thereof in the treatment of cancer.
  • a method for treating a subject having cancer comprises administering to a subject in need thereof an effective amount of pemetrexed (ALIMTA ® ) and a compound of Formula I:
  • P is a targeting group which binds to the reactive site of a post proline- cleaving enzyme
  • R is a reactive group which reacts with the reactive site of a post proline cleaving enzyme
  • the cancer is a refractory cancer.
  • the cancer is refractory to platinum-based therapy, such as but not limited to cisplatin or carboplatin.
  • the cancer is refractory to a platinum-based therapy and a taxane (e.g., paclitaxel or docetaxel) or a nucleoside analogue (e.g., gemcitabine).
  • the subject is further treated with surgery and radiation.
  • the compound of Formula I and pemetrexed may be administered before, during, and/or after treating the subject with surgery or radiation.
  • the compound of Formula I and pemetrexed may be administered before and after, before and during, or during and after treating the subject with surgery or radiation.
  • the compound of Formula I is administered via a route of administration different from that of pemetrexed.
  • the compound of Formula I may be administered orally.
  • Pemetrexed may be administered parenterally including intravenously.
  • the compound of Formula I may be administered to the subject before, during and/or after pemetrexed administration.
  • the compound of Formula I is administered to the subject about 24 hours after the administration of pemetrexed.
  • pemetrexed is administered on one or more days of a multi-day or multi-week cycle (e.g., a 3 week cycle).
  • pemetrexed is administered at the beginning of the cycle (e.g., on days 1, 2, and/or 3 of a three week cycle).
  • the compound of Formula I is administered on one multiple days of the same multi-day or multi-week cycle (e.g., at least 7 days, at least 10 days, or at least 14 days).
  • pemetrexed is administered on day 1 of a 21 day cycle, followed by administration of the compound of Formula I on days 2-15 of the same cycle, followed by a rest period on days 16-21 of the same cycle.
  • the rest period is a "treatment-free period" during which time the subject may be administered placebo or nothing at all.
  • the cycle may be repeated one, two, three, four, five, six, or more times.
  • pemetrexed is administered on days 1, 2 and 3 of a multi- day cycle (e.g., a 24 day cycle).
  • the compound of Formula I may be administered on days 4- 17 of the same 24 day cycle. Days 18-24 of the 24 day cycle may be a rest period (treatment- free period).
  • pemetrexed is administered intravenously over a period of about 10 minutes.
  • Pemetrexed and/or the compound of Formula I may be administered at a therapeutic dose or a sub-therapeutic dose.
  • a therapeutic dose of pemetrexed is about 500 mg/m 2 per day.
  • a sub-therapeutic dose is a dose that is less than 500 mg/m 2 per day, including 50 mg/m 2 per day to less than 500 mg/m 2 per day, 100 mg/m 2 per day to 450 mg/m 2 per day, 200 to 400 mg/m 2 per day, and 300 mg/m 2 per day to 400 mg/m 2 per day.
  • a therapeutic dose of the compound of Formula I such as for example Val-boroPro is 200-600 ⁇ g per day, In some instances the compound is administered in doses of 200 or 300 ⁇ g twice a day, in which case the sub-therapeutic dose is less than 400 ⁇ g per day. In other instances the compound is administered in the same doses but only once a day in which case the sub-therapeutic dose is less than 200 ⁇ g per day.
  • the effective amount of pemetrexed and/or the compound of Formula I is less than 90%, less than 80%, less than 75%, less than 60%, less than 50%, about 50%, less than 40%, less than 30%, less than 20% or less than 10% of the effective amount of the compound when administered as a single agent.
  • the cancer may be a tumor.
  • the size or volume of the tumor is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% from pretreatment size or volume (i.e., size prior to treatment according to the invention with pemetrexed and the compound of Formula
  • the method may further comprise administering a platinum-containing compound such as cisplatin or carboplatin, or gemcitabine, or erlotinib, or a taxane such as docetaxel to the subject, possibly before or during administration of the compound of Formula I and/or pemetrexed.
  • a platinum-containing compound such as cisplatin or carboplatin, or gemcitabine, or erlotinib, or a taxane such as docetaxel
  • a composition comprising a compound of Formula I and pemetrexed in an effective amount to reduce tumor size or volume by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% from pretreatment size or volume.
  • the composition may further comprise a pharmaceutically acceptable carrier.
  • the invention is further premised, in part, on the unexpected finding that Val-boroPro enhances the activity of erlotinib (T ARCEV ATM) when particular dosing and timing regimens are used.
  • a method for treating a subject having cancer comprising administering to a subject in need thereof erlotinib and a compound of Formula I:
  • PR wherein P is a targeting group which binds to the reactive site of a post proline- cleaving enzyme, and wherein R is a reactive group which reacts with the reactive site of a post proline cleaving enzyme, in an effective amount to reduce the cancer load by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% from pretreatment size or volume compared to pretreatment load.
  • a method for treating a subject having cancer comprising administering to a subject in need thereof an effective amount of erlotinib and a compound of Formula I:
  • a method for treating a subject having cancer comprising administering to a subject in need thereof an effective amount of erlotinib and a compound of Formula I:
  • P is a targeting group which binds to the reactive site of a post proline- cleaving enzyme
  • R is a reactive group which reacts with the reactive site of a post proline cleaving enzyme
  • the erlotinib is administered once daily and the compound of Formula I is administered twice daily.
  • Erlotinib and the compound may be administered on the same days of a multi-day cycle (e.g., a 21 day cycle). Administration of erlotinib and the compound of Formula I may be followed by a rest period of 1-7 days, after which the cycle may be repeated one, two, three, four, five, six, or more times.
  • the cancer may be a refractory cancer.
  • the cancer is refractory to a platinum-containing agent such as cisplatin or carboplatin, and/or to gemcitabine.
  • the compound of Formula I and erlotinib may both be administered orally.
  • Erlotinib and/or the compound of Formula I may be administered at a therapeutic dose or a sub-therapeutic dose.
  • a therapeutic dose of erlotinib is about 150 mg per day.
  • a subtherapeutic dose is a dose that is less than 150 mg per day, including 25 mg per day to less than 150 mg per day, 50 mg per day to 125 mg per day, and 75 mg per day to 100 mg per day.
  • Therapeutic doses and sub-therapeutic doses of the compound of Formula I such as for example Val-boroPro are as described above.
  • the effective amount of erlotinib and/or the compound of Formula I is less than 90%, less than 80%, less than 75%, less than 60%, less than 50%, about 50%, less than 40%, less than 30%, less than 20% or less than 10% of the effective amount of the compound when administered as a single agent.
  • the method further comprises administering pemetrexed, a platinum-containing compound such as cisplatin or carboplatin, or gemcitabine, or a taxane such as docetaxel to the subject.
  • a platinum-containing compound such as cisplatin or carboplatin, or gemcitabine
  • a taxane such as docetaxel
  • a composition comprising a compound of Formula I and erlotinib in an effective amount to reduce the cancer load by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% from pretreatment cancer load.
  • the composition may further comprise a pharmaceutically acceptable carrier.
  • the invention is further premised, in part, on the finding that particular treatment regimens for the combination of Formula I agents and a taxane such as docetaxel
  • TAXOTERE ® are particularly and unexpectedly effective in the treatment of certain cancers.
  • a method for treating cancer in a subject comprising administering a compound of Formula I and docetaxel in an effective amount to reduce the cancer load by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% from pretreatment cancer load.
  • a composition comprising a compound of Formula I and docetaxel in an effective amount to reduce cancer load by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% from pretreatment cancer load.
  • the composition may further comprise a pharmaceutically acceptable carrier.
  • a method is provided for treating a subject having cancer. The method comprises administering to a subject in need thereof an effective amount of docetaxel and a compound of Formula I:
  • P is a targeting group which binds to the reactive site of a post proline- cleaving enzyme
  • R is a reactive group which reacts with the reactive site of a post proline cleaving enzyme and wherein docetaxel is administered on day 1 of a multi-day or multi-week cycle (e.g., a 21 or 22-day cycle), and the compound of Formula I is administered on multiple days (e.g., at least 7, at least 10, at least 14 days) of the same cycle.
  • the compound of Formula I is administered on days 2-15 of a 21 or 22 day cycle. The cycle may be repeated one, two, three, four, five, six or more times.
  • Docetaxel and/or the compound of Formula I may be administered at a therapeutic dose or a sub-therapeutic dose.
  • a therapeutic dose of docetaxel is about 75 mg/m 2 per day.
  • a sub-therapeutic dose is a dose that is less than 75.mg/m 2 per day, including 25 mg/m 2 per day to less than 75 mg/m 2 per day, 25 mg/m 2 per day to 70 mg/m 2 per day, and 40 mg/m 2 per day to 50 mg/m 2 per day.
  • Therapeutic doses and sub-therapeutic doses of the compound of Formula I such as for example Val-boroPro are as described above.
  • the effective amount of docetaxel and/or the compound of Formula I is less than 90%, less than 80%, less than 75%, less than 60%, less than 50%, about 50%, less than 40%, less than 30%, less than 20% or less than 10% of the effective amount of the compound when administered as a single agent.
  • the subject has been previously treated with a regimen comprising a platinum-containing compound such as cisplatin or carboplatin, and optionally an antimetabolite or a taxane.
  • a platinum-containing compound such as cisplatin or carboplatin
  • an antimetabolite or a taxane is gemcitabine.
  • the taxane is docetaxel.
  • the subject may have been previously treated with a prior regimen for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 13 months, at least 14 months, at least 15 months, at least 16 months, at least 17 months, at least 18 months, at least 19 months, at least 20 months, at least 21 months, at least 22 months, at least 23 months, at least 24 months, at least 25 months, or more prior to treatment with docetaxel and the compound of Formula I.
  • the subject is treated with carboplatin and docetaxel about 5 months prior to treatment according to the invention.
  • the subject is treated with carboplatin and gemcitabine 1-4 months prior to treatment according to the invention.
  • the subject has adenocarcinoma while in others the subject has squamous cell carcinoma.
  • a method for treating a subject having cancer comprises administering to a subject in need thereof an effective amount of docetaxel and a compound of Formula I:
  • P is a targeting group which binds to the reactive site of a post proline- cleaving enzyme
  • R is a reactive group which reacts with the reactive site of a post proline cleaving enzyme
  • the antimetabolite is gemcitabine.
  • the taxane is docetaxel.
  • the platinum-containing compound is carboplatin or cisplatin. The subject preferably has been administered the platinum-containing compound and the anti-metabolite or the taxane within 5, 4, 3, 2, or 1 month of treatment according to the invention.
  • a kit comprising a first container comprising a compound of Formula I and a second container comprising pemetrexed.
  • the compound of Formula I may be formulated for oral administration.
  • the compound of Formula I may be formulated as a tablet, pill, capsule or caplet.
  • Pemetrexed may be formulated for parenteral administration such as intravenous or intramuscular administration.
  • Pemetrexed may be provided in a vial suitable for liquid reconstitution.
  • Pemetrexed and/or the compound of Formula I may be provided in therapeutic doses or sub-therapeutic doses, as described above.
  • the compound of Formula I may be provided in a unit dosage ranging from 100 ⁇ g to 500 ⁇ g, preferably 200 ⁇ g to 400 ⁇ g, and more preferably 200 ⁇ g to 300 ⁇ g.
  • the compound of Formula I is in a unit dosage of about 200 or 300 micrograms.
  • Pemetrexed may be provided in a unit dosage of 200 mg to 1000 mg, 300 mg to 900 mg, 400 mg to 800 mg, 500 mg to 600 mg, or about 700 mg. In some embodiments, pemetrexed is provided in a unit dosage of about 850 mg.
  • the kit may further comprise a daily dispenser.
  • the day of the month may be printed on the daily dispenser.
  • the first container may be a blister package having day indicia and time indicia. There may be two time indicia for every day indicium.
  • the kit includes a 1, 2 or 3 day supply of pemetrexed, a 7, 10 or 14 day supply of a compound of Formula I, and optionally a 1-7 day supply of pills containing no medication (i.e., placebo).
  • the kit includes a 3-day supply of pemetrexed, a 14-day supply of a compound of Formula I, and a 7-day supply of pills containing no medication.
  • the kit may further comprise instructions of use.
  • the instructions of use comprise instructing a subject or a health care provider to administer pemetrexed on day 1, 2 and/or 3 of a multi-day cycle (e.g., a 21 or 22 day cycle), the compound of Formula I on at least 7, at least 10, or at least 14 days of the same cycle (e.g., days 2-15 of a 21 or 22 day cycle), and the pills containing no medication on 1-7 days of the same cycle (e.g., days 16-21 or days 16-22 of the 21 or 22 day cycle respectively).
  • the instructions of use comprise instructing a subject or a health care provider to administer pemetrexed on days 1-3 of a 24 day cycle, the compound of Formula I on days 4-17 of the 24 day cycle, and the pills containing no medication on days 18-24 of the 24 day cycle.
  • the instructions of use instruct the subject or health care provider to administer pemetrexed parenterally (e.g., intravenously) over a period of about 10 minutes.
  • a kit is provided that comprises a first container comprising a compound of Formula I and a second container comprising erlotinib, or alternatively comprises a multiple physically separate and divided regions which each comprise the compound of Formula I and erlotinib.
  • the compound of Formula I and erlotinib may be formulated for oral administration, and thus both may be provided as tablets, pills, capsules or caplets.
  • the compound of Formula I and/or erlotinib may be provided in therapeutic or subtherapeutic unit doses, as described above.
  • the compound of Formula I may be provided in unit dosages as recited above for the pemetrexed kit.
  • the compound of Formula I is in a unit dosage of about 100, 200, 300, 400 or 500 micrograms.
  • Erlotinib may be provided in a unit dosage of 10-150 mg, 25-125 mg, 50-100 mg, and 75-100 mg, including preferably about 25 mg, 50 mg or 150 mg.
  • the kit may further comprise a daily dispenser.
  • the day of the month may be printed on the daily dispenser.
  • the first container may be a blister package having day indicia and time indicia and optionally two time indicia for every day indicium.
  • the second container may be a blister package having day indicia and optionally time indicia.
  • the kit includes a multi-day (e.g., at least 7, at least 10, at least 14, at least 21, at least 28 days, or at least 31 days) supply of erlotinib, a multi-day (e.g., at least 7, at least 10, at least 14, at least 21, or at least 28 days, or at least 31 days) supply of a compound of Formula I, and a multi-day (e.g., 1-7 day) supply of pills containing no medication.
  • a multi-day e.g., at least 7, at least 10, at least 14, at least 21, at least 28 days, or at least 31 days
  • a multi-day e.g., 1-7 day
  • the kit may further comprise instructions for use.
  • the instructions of use comprise instructing a subject or a health care provider to administer erlotinib on multiple days of a multi-day cycle, preferably once a day, the compound of Formula I on the same days as erlotinib but preferably twice a day, and the pills containing no medication on the remaining days of the cycle.
  • the instructions may describe administration of erlotinib on days 1-24 of a 31 day cycle, the compound of Formula I on days 1 -24 of the 31 day cycle, and the pills containing no medication on days 25-31 of the 31 day cycle.
  • the invention provides yet another kit that comprises a compound of Formula I and docetaxel.
  • the kit may comprise one container comprising docetaxel, preferably in a vial suitable for liquid reconstitution, and the compound of Formula I in an oral formulation (e.g., a tablet).
  • Each kit may proyide only one dose of docetaxel and multiple days (e.g., at least 7, at least 10, at least 14, or more days) dose of the compound of Formula I, and optionally multiple days (e.g., 1-7 days) dose of placebo.
  • the kits contain doses sufficient for one, two, three, four, five, six or more cycles.
  • the kit may further comprise one or more unit dosages for a carboplatin-containing compound such as cisplatin or carboplatin.
  • m is an integer between 0 and 100, inclusive;
  • a and A 1 are L- or D- naturally or non- naturally occurring amino acid or a peptide or peptidomimetic such that when A is an amino acid residue and m > 1 each A in A m may be 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 L- or R-conf ⁇ guration; and each X 1 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 compound of Formula I is a compound of Formula III:
  • m is an integer between 0 and 100, inclusive;
  • a and A 1 are L- or D- naturally occurring or non-naturally occurring amino acid residues such that when m>l, A in each repeating bracketed unit is independently selected; the C bonded to B is in the L- or Reconfiguration; and each X 1 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 compound of Formula I is Val-boroPro. In some embodiments the compound of Formula I is Ile-boroPro.
  • the compound of Formula I may be provided as a mixture of linear and cyclic forms.
  • the cancer is a carcinoma.
  • the cancer is non-small cell lung cancer (NSCLC), and in related embodiments, the cancer is adenocarcinoma or squamous cell carcinoma.
  • NSCLC non-small cell lung cancer
  • the cancer is a tumor.
  • the cancer may be a metastasis.
  • the subject may or may not have undergone an anti-cancer therapy with surgery, and/or radiation and/or chemotherapy.
  • the compound of Formula I is administered at a dose of about 50 - 1000 micrograms per day or 0.001 - 0.01 mg/kg/day.
  • the total daily dose may be administered in one, two or more administrations per day.
  • the method further comprises administering an antigen to the subject.
  • the antigen may be targeted to a tissue or a cell.
  • the antigen is a cancer antigen.
  • Some important cancer antigens include but are not limited to PGP9.5, H/Ley/Leb, B7-DC, and B7-H1.
  • the method further comprises administering an antibody or an antigen-binding antibody fragment to the subject.
  • Some important antibodies or antibody fragments include but are not limited to anti-a5 ⁇ l integrin antibody, anti-EGFR antibody, or alpha IR-3.
  • anti-a5 ⁇ l integrin antibody is Volocimixab.
  • anti-EGFR antibody Nimotuzumab (TheraCIM).
  • the method further comprises administering an adjuvant to the subject.
  • FIG. 1 is a graph of the dose response of A549 NSCLC (non-small cell lung cancer) xenografts to single administrations of pemetrexed (ALIMT A ® ).
  • BALB/c Ragl 1' mice were inoculated s.c. with A549 cells on day 0 and administered the indicated doses of ALIMT A ® or saline on day 7. Groups of 5 mice were treated with each dose of ALIMT A ® .
  • FIG. 2 is a graph of anti-tumor effect of pemetrexed (ALIMTA ® ) administered on days 7 and 29 after tumor inoculation in combination with multiply administered 5 ⁇ g doses of talabostat.
  • BALB/c Ragl' ' mice were inoculated with A549 tumor cells on day 0 and administered ALIMTA ® on days 7 and 29.
  • Talabostat was administered twice daily from days 8-21 and days 30-43.
  • Experimental treatments were saline (o), 5 ⁇ g dose of talabostat (•), 750-mg/kg dose of ALIMTA ® ( ⁇ ), 600 mg/kg dose of ALIMTA ® (A), 5 ⁇ g talabostat + 750 mg/kg ALIMTA ® ( ⁇ ), 5 ⁇ g talabostat + 600 mg/kg ALIMTA ® ( ⁇ ).
  • FIG. 3 is a graph of the anti-tumor effect of pemetrexed (ALIMTA ® ) administered on days 7 and 29 after tumor inoculation in combination with multiply administered 10 ⁇ g doses of talabostat.
  • BALB/c Rag2 ⁇ / ⁇ mice were inoculated with A549 tumor cells and administered ALIMTA ® and talabostat as described in FIG. 2.
  • FIG. 4 is a graph of the anti-tumor effect of pemetrexed (ALIMTA ® ) administered on days 7 and 29 after tumor inoculation in combination with multiply administered 20 ⁇ g doses of talabostat.
  • BALB/c Rag2 'A mice were inoculated with A549 tumor cells and administered ALIMTA ® and talabostat as described in FIGs. 2 and 3.
  • Experimental treatments were saline (o), 20 ⁇ g dose of talabostat (•), 750 mg/kg dose of ALIMTA ® ( ⁇ ), 600 mg/kg dose of ALIMTA ® (A), 20 ⁇ g talabostat + 750 mg/kg ALIMTA ® ( ⁇ ), 20 ⁇ g talabostat + 600 mg/kg ALIMTA ® ( ⁇ ).
  • FIG. 5 is a graph of the dose response of A549 NSCLC xenografts to multiple administrations of pemetrexed (ALIMTA ® ).
  • BALB/c Rag2 'A mice were inoculated s.c. with A549 cells on day 0 and administered the indicated doses of ALIMTA ® on days 7, 8 and 9.
  • FIG. 6 is a protocol of the schedule and frequencies of administration of talabostat and pemetrexed (ALIMTA ® ) as referenced in a part of Example 1.
  • FIG. 7 is a graph of the anti-tumor effect of pemetrexed (ALIMTA ® ) administered on days 7, 8, 9, 31, 32, and 33 after tumor inoculation in combination with multiply administered 5 ⁇ g doses of talabostat.
  • BALB/c Rag2 'A mice were inoculated with A549 tumor cells on day 0, administered ALIMTA ® on days 7, 8, 9, 31, 32 and 33.
  • Talabostat was administered twice daily on days 10-23 and days 34-47.
  • Experimental treatments were: saline (o), 5 ⁇ g dose of talabostat (•), 500 mg/kg dose of ALIMTA ® (A), 5 ⁇ g talabostat + 500 mg/kg ALIMTA ®
  • FIG. 8 is a graph of the anti-tumor effect of pemetrexed (ALIMTA ® ) administered on days 7, 8, 9, 31, 32, and 33 after tumor inoculation in combination with multiply administered 10 ⁇ g doses of Talabostat.
  • BALB/c Rag2 'A mice were inoculated with A549 tumor cells on day 0 and administered ALIMTA ® and talabostat as described in FIG. 6.
  • Experimental treatments were saline (o), 10 ⁇ g dose of talabostat (•), 500 mg/kg dose of ALIMTA ® (A) 5 10 ⁇ g talabostat + 500 mg/kg ALIMTA ® ( ⁇ ).
  • FIG. 9 is a protocol of the schedule and frequencies of administration of talabostat and erlotinib (TARCEVATM) in Example 2.
  • FIG. 10 shows two graphs showing the cooperative anti-tumor effect of talabostat and erlotinib (TARCEVATM) (25 mg/kg) in A549 NSCLC xenograft model.
  • FIG. 11 shows two graphs showing the cooperative anti-tumor effect of talabostat and erlotinib (TARCEVATM) (50 mg/kg) in A549 NSCLC xenograft model.
  • TARCEVATM talabostat and erlotinib
  • FIG. 12 is a protocol of the schedule and frequencies of administration of talabostat and docetaxel in Example 3.
  • FIG. 13 shows two graphs showing the anti-tumor effect of talabostat and docetaxel in A549 NSCLC xenograft model.
  • FIG. 14 shows two graphs showing the Kaplan-Meier estimate of (A) median progression-free survival (PFS) and (B) median survival for all patients enrolled. It is to be understood that the Figures are not required for enablement of the invention.
  • the invention provides compositions and methods for preventing and treating cancer.
  • the invention is based, in part, on the finding that the DPP-IV inhibitor Val-boroPro (talabostat, PT-100) interacts with certain anti-cancer agents that have different mechanisms of action in the treatment of cancer.
  • Val-boroPro with pemetrexed (ALIMTA ® ), erlotinib (TARCEVATM), or docetaxel (TAXOTERE®) in some aspects of the invention acts unexpectedly synergistically to treat non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • the findings were surprising at least because Val-boroPro exerts its anti-tumor activity via a different mechanism of action than pemetrexed, erlotinib, and docetaxel.
  • a synergistic response is one that is greater than that induced by either of the two or more agents when used alone.
  • the invention relates, in part, to methods of treating and/or preventing cancer in a subject comprising administering a compound (or agent, as used interchangeably herein) of Formula I and an antimetabolite, optionally with a human epidermal growth factor receptor type I/epide ⁇ nal growth factor receptor (HERl /EGFR) tyrosine kinase inhibitor, a taxane, or a platinum-containing compound.
  • a compound or agent, as used interchangeably herein
  • an antimetabolite optionally with a human epidermal growth factor receptor type I/epide ⁇ nal growth factor receptor (HERl /EGFR) tyrosine kinase inhibitor, a taxane, or a platinum-containing compound.
  • HERl /EGFR human epidermal growth factor receptor type I/epide ⁇ nal growth factor receptor
  • the invention also relates to compositions and kits comprising compounds of Formula I and an antimetabolite, and optionally an HERl /EGFR ty
  • the compound of Formula I and an antimetabolite are administered as a synergistic combination in an effective amount to treat a cancer.
  • the effect resulting from the combination of the at least two agents may be greater than the additive effect of the individual agents when used alone. Accordingly, when used together either or both agents may be used at lower doses than would be used if either agent was administered alone.
  • either or both agents may be administered in a "sub-therapeutic" dose and the combination would be therapeutic. In other words, the agents may be administered in reduced doses relative to therapeutically appropriate doses when agents are administered alone.
  • the unit dosage of one or both agents may be reduced by a factor of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100 or more relative to the unit dosage required when a single agent is administered.
  • one agent may be used at a dose which shows no response when used alone, and the compound of Formula I may enhance that effect when used in combination, resulting in a response that is greater than the response of either the agent or the compound of Formula I alone.
  • Treatment or therapy after a disorder has started aims to reduce, ameliorate or altogether eliminate the disorder and/or its associated symptoms, or prevent it from becoming worse.
  • Treatment of subjects before a disorder has started i.e., prophylactic treatment
  • the term "prevent” refers to the prophylactic treatment of patients who are at risk of developing a disorder (resulting in a decrease in the probability that the subject will develop the disorder) and to the inhibition of further development of an already established disorder.
  • Val-boroPro and other compounds of Formula I stimulate a variety of cytokines and chemokines which can stimulate the immune system.
  • the resultant immune stimulation can be exploited to enhance the anti-cancer efficacy of other agents.
  • the invention provides methods that exploit the effect that may be achieved when the compounds of Formula I are used together with an antimetabolite, a HERl /EGFR tyrosine kinase inhibitor, or a taxane.
  • the effect of particular combinations is unexpected.
  • the combination provides a synergistic response, resulting for example in greater than the additive effect expected from using the agents singly.
  • Compounds of Formula I are effective against a number of different cancers and tumors. It has been previously demonstrated that these compounds, when administered to tumor-bearing mice, rapidly stimulate the production of growth factors, cytokines and chemokines (e.g., IL-I, G-CSF, and IL-8 (KC in mice)). These mediators collectively stimulate the proliferation, activation and chemoattraction to the tumor microenvironment of effector cells involved in both adaptive and non-adaptive (innate) immunity and in immune lysis and growth inhibition of tumor cells.
  • cytokines and chemokines e.g., IL-I, G-CSF, and IL-8 (KC in mice).
  • the immune and non-immune effector cell populations mobilized and/or activated by compounds of Formula I enhance the anti-cancer effects of antimetabolites, HER1/EGFR tyrosine kinase inhibitors, and taxanes.
  • the anti-metabolite is pemetrexed
  • the HERl /EGFR tyrosine kinase inhibitor is erlotinib
  • the taxane (or taxoid) is docetaxel.
  • these particular combinations are administered in according to particular dosing, timing and administration regimens.
  • the invention therefore provides therapeutic and prophylactic methods that involve the administration of compounds of Formula I (or mixtures thereof).
  • the compounds of Formula I are combined, preferably in pharmaceutical form, with anti-metabolites (e.g., pemetrexed and gefitinib (IRESSA ® )), HER1/EGFR tyrosine kinase inhibitors (e.g., erlotinib), taxanes (e.g., docetaxel or paclitaxel), platinum-containing compounds (e.g., carboplatin or cisplatin), nucleoside analogues (e.g., gemcitabine (GEMZAR ® ) antibodies (or fragments thereof), antigens, or adjuvants.
  • anti-metabolites e.g., pemetrexed and gefitinib (IRESSA ® )
  • HER1/EGFR tyrosine kinase inhibitors e.g., erlotinib
  • taxanes e.
  • P is a targeting group which binds to the reactive site of a post proline-cleaving enzyme
  • R is a reactive group that reacts with a functional group in the reactive site of a post proline-cleaving enzyme.
  • Post proline-cleaving enzymes are enzymes which have a specificity for cleaving Xaa-Pro or Xaa-Ala dipeptides (where Xaa represents any amino acid) at the carboxy end of the dipeptide.
  • Dipeptidyl peptidases are exopeptidases that cleave Xaa-Pro or Xaa-Ala dipeptides from the amino terminus of a peptide or protein.
  • post-proline cleaving enzymes include, but are not limited to, dipeptidyl peptidase IV (DP IV or DPP IV) and fibroblast activation protein (FAP) both of which show DPP activity.
  • DP IV or DPP IV dipeptidyl peptidase IV
  • FAP fibroblast activation protein
  • POP Prolyl endopeptidase
  • POP POP
  • FAP also possesses endopeptidase activity.
  • the targeting group (P) can be composed of single or multiple amino acid residues, whether naturally occurring or not, or of a peptide or a peptidomimetic. In certain If
  • the portion of P that is involved in binding to the reactive site of a post proline- cleaving enzyme is formed of amino acid residues and the remaining portion of P is formed of non-amino acid components. Therefore, P can be composed wholly of amino acid residues, wholly of non-amino acid substituents, or a combination of both. P may be 100 or more residues in length including 30, 20, 10 or less than 10 residues in length. P or a portion(s) thereof may mimic a substrate of the protease.
  • Targeting groups can be synthesized from other biomolecules including but not limited to saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like. Also envisioned in the invention is the use of targeting groups made from peptoids, random bio-oligomers (U.S. Patent 5650489), benzodiazepines, diversomeres such as dydantoins, benzodiazepines and dipeptides, nonpeptidyl peptidomimetics with a beta-D-glucose scaffolding, oligocarbamates or peptidyl phosphonates.
  • a vast array of candidate targeting groups can be generated from libraries of synthetic or natural compounds.
  • the methods of the invention utilize this library technology to identify small molecules which bind to protease reactive sites.
  • One advantage of using libraries for inhibitor identification is the facile manipulation of millions of different putative candidates of small size in small reaction volumes (i.e., in synthesis and screening reactions).
  • Another advantage of libraries is the ability to synthesize targeting groups which might not otherwise be attainable using naturally occurring sources.
  • Libraries can be screened to identify naturally or non-naturally occurring putative targeting groups by assaying protease binding (and optionally cleavage activity) in the presence of the library molecule or member.
  • the cleavage assay includes determining whether the library molecule inhibits cleavage by the protease of a known substrate or of a substrate analog (e.g., a chromophoric substrate analog which is easily detectable in a spectrophotometric assay).
  • Those library molecules which exhibit binding and optionally inhibition of a post-prolyl cleaving enzyme then can be covalently coupled to the reactive groups disclosed herein and again tested for binding and inhibition of the enzyme. In this manner, a simple, high-through-put screening assay is provided for identifying inhibitors.
  • P is covalently coupled to R.
  • the covalent coupling occurs via a carboxyl group at the carboxyl terminus in P.
  • reactive groups useful in the invention include 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-acylhydroxylamine, an azapeptide, a fluoroolefin, a peptidyl (alpha-aminoalkyl) phosphonate ester, or a nitrile.
  • m is an integer between 0 and 100 or more, inclusive;
  • a and A 1 are L- or D- naturally or non-naturally occurring amino acid residues or a peptide or a peptidomimetic such that when m >1 and A is an amino acid each A in A n , may be an amino acid residue different from another or all other A in A m , and when A is a peptide or a peptidomimetic m is preferably 1 ;
  • the C bonded to B is in the L- or R- configuration; optionally the bonds between A 1 and N and between A and A 1 are peptide bonds;
  • each X 1 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 C bonded to B is in the L-configuration
  • the absolute configuration of the C is like that of an L-amino acid.
  • a 1 is alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, aspartate, glutamate, asparagine, glutamine, lysine, arginine, histidine, cysteine, methionine, or proline.
  • the agent is L-Ala-L-boroPro, L-Asp-L-boroPro, L-GIu- L-boroPro, L-Asn-L-boroPro, L-Gln-L-boroPro, L-Lys-L-boroPro, L-Arg-L-boroPro, L-His- L-boroPro, L-Pro-L-boroPro, L-Thr-L-boroPro, L-Ser-L-boroPro, L-Cys-L-boroPro, L-GIy- L-boroPro, L-Tyr-L-boroPro, L-Trp-L-boroPro, L-Phe-L-boroPro, L-Leu-L-boroPro, L-IIe-L- boroPro, L-Met-L-boroPro, or L-Val-L-boroPro.
  • m is 0 and/or X 1 and X 2 are hydroxyl groups. In some important embodiments, X 1 and X 2 are hydroxyl groups, m is 0, and A 1 is valine, and the resulting compound is referred to as Val-boroPro (or PT-100 or talabostat).
  • n is an integer from 0-30, or an integer from 0-20, or an integer from 0-10. In some important embodiments m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or it is a multiple of two (e.g., 2, 4, 6, 8, 10, etc.), such as a repeating dipeptide having a proline residue at the C terminal (e.g., A - Pro).
  • the proline residue attached to the reactive group is referred to as a pyrrolidine ring.
  • the pyrrolidine may be replaced with an azetidine or a thiazolidine.
  • the compound may comprise an isostere.
  • the compound may be a 4-cyanothiazolidine rather than an organo boronate containing compound.
  • m is an integer between 0 and 100 or more, inclusive;
  • a and A 1 are L- or D- naturally or non-naturally occurring amino acid residues or a peptide or a peptidomimetic such that when m >1 A in each repeating bracketed unit is independently selected; the C bonded to B is in the L-conf ⁇ guration; optionally the bonds between A and N, A 1 and C, and A 1 and N are peptide bonds; and each X 1 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 proline residue attached to the boronyl group is referred to as a pyrrolidine ring. It may be bonded, at its amino terminus, to an amino acid optionally in an R- or an S- configuration (and likewise in an L- or D- configuration).
  • inhibitors have an analogous structure to the agents of Formulae II or III but with the boronyl group replaced by, for example, 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-acylhydroxylamine, an azapeptide, a fluoroolefin, a peptidyl (alpha-aminoalkyl) phosphonate ester, or a nitrile.
  • organo phosphonate for example, an organo phosphonate, a fluoroalkylketone, an halomethyl ketone, a diazomethyl ketone, a dimethyls
  • 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 (Tip), cysteine (Cys), methionine (Met), serine (Ser), threonine (Thr), lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp), glutamic acid (GIu) 5 asparagine (Asn), glutamine (GIn) and proline (Pro).
  • Non-naturally occurring amino acids include the D-forms of Ala, VaI, Leu, He, Phe, Tyr, Trp, 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, cyclohexylglycine (Chg), N- Methylglycine (N-MeGIy), 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-aminopentanoi
  • Non-naturally occurring amino acids also include beta-amino acids and alpha-amino acids with side chains replaced with synthetic derivatives. Representative side chains of naturally occurring and non-naturally occurring ⁇ -amino acids are shown below in Table 1.
  • Non-naturally occurring amino acids also include D, L, and racemic configurations of hydrophobic amino acids.
  • Hydrophobic amino acids include amino acid analogs having the formula -NH-CHR-CO-, wherein R is an aliphatic group, a substituted aliphatic group, a benzyl group, a substituted benzyl group, an aromatic group or a substituted aromatic group and wherein R does not correspond to the side chain of a naturally-occurring amino acid.
  • aliphatic groups include straight chained, branched or cyclic C1-C8 hydrocarbons which are completely saturated, which contain one or two heteroatoms such as nitrogen, oxygen or sulfur and/or which contain one or more units of desaturation.
  • Aromatic groups include carbocyclic aromatic groups such as phenyl and naphthyl and heterocyclic aromatic groups such as imidazolyl, indolyl, thienyl, furanyl, pyridyl, pyranyl, oxazolyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl and acridintyl.
  • Suitable substituents on an aliphatic, aromatic or benzyl group include -OH, halogen (- Br, -Cl, -I and -F) -O (aliphatic, substituted aliphatic, benzyl, substituted benzyl, aryl or substituted aryl group), -CN, -NO 2 , -COOH, -NH 2 , -NH (aliphatic group, substituted aliphatic, benzyl, substituted benzyl, aryl or substituted aryl group), -N (aliphatic group, substituted aliphatic, benzyl, substituted benzyl, aryl or substituted aryl group) 2 , -COO (aliphatic group, substituted aliphatic, benzyl, substituted benzyl, aryl or substituted aryl group), -CONH 2 , -CONH (aliphatic, substituted aliphatic group, benzyl, substitute
  • a substituted benzylic or aromatic group can also have an aliphatic or substituted aliphatic group as a substituent.
  • a substituted aliphatic group can also have a benzyl, substituted benzyl, aryl or substituted aryl group as a substituent.
  • a substituted aliphatic, substituted aromatic or substituted benzyl group can have one or more substituents. Modifying an amino acid substituent can increase, for example, the lypophilicity or hydrophobicity of natural amino acids which are hydrophilic.
  • amino acids amino acids analogs and salts thereof can be obtained commercially. Others can be synthesized by methods known in the art. Synthetic techniques are described, for example, in Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, Chapters 5 and 7, 1991.
  • linear or cyclic forms can be provided in linear or cyclic form or as mixtures thereof, as described in U.S. Patent No. 6355614, issued March 12, 2002.
  • the proportion of linear (versus cyclic) forms in these mixtures may vary (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of cyclic relative to the total amount of the Formula I compound) depending on the formulation.
  • the agents may be provided as A-boroPro containing compounds that are converted (via enzymatic, chemical, metabolic, or any other means, in vivo or ex vivo) to A-boroPro.
  • A- boroPro containing compounds include prodrugs.
  • 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.
  • A-boroPro examples include alcohol precursors of A-boroPro that are oxidized in vivo (e.g., in the liver) and a boroxine 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, as discussed above.
  • 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. 5462928 issued October 31, 1995; and 6100234 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 may be 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.
  • All amino acids contain an asymmetric or chiral carbon and may contain more than one chiral carbon atom.
  • the asymmetric ⁇ carbon atom of the amino acid is referred to as a chiral center and can occur in two different isomeric forms. These forms are identical in all chemical and physical properties with one exception, the direction in which they can cause the rotation of plane-polarized light.
  • These amino acids are referred to as being "optically active," i.e., the amino acids can rotate the plane-polarized light in one direction or the other.
  • the four different substituent groups attached to the ⁇ carbon can occupy two different arrangements in space. These arrangements are not superimposable mirror images of each other and are referred to as optical isomers, enantiomers, or stereo isomers.
  • a solution of one stereo isomer of a given amino acid will rotate plane polarized light to the left and is called the levorotatory isomer [designated (-)]; the other stereo isomer for the amino acid will rotate plane polarized light to the same extent but to the right and is called dextrorotatory isomer [designated (+)].
  • a more systematic method for classifying and naming stereo isomers is the absolute configuration of the four different substituents in the tetrahedron around the asymmetric carbon atom (e.g., the ⁇ carbon atom).
  • a reference compound was selected (glyceraldehyde), which is the smallest sugar to have an asymmetric carbon atom.
  • L and D the two stereo isomers of glyceraldehyde.
  • Their absolute configurations have been established by x-ray analysis.
  • the designations, L and D also have been assigned to the amino acids by reference to the absolute configuration of glyceraldehyde.
  • the stereo isomers of chiral compounds having a configuration related to that of L-glyceraldehyde are designed L, and the stereo isomers having a configuration related to D-glyceraldehyde are designated D, regardless of the direction in which they rotate the plane-polarized light.
  • L and D refer to the absolute configuration of the four substituents around the chiral carbon.
  • naturally occurring compounds which contain a chiral center are only in one stereo isomeric form, either D or L.
  • the naturally occurring amino acids are the L stereo isomers.
  • the invention embraces inhibitors comprising amino acid residues which can be in the D stereo isomer configuration also.
  • the RS system was invented to avoid ambiguities when a compound contains two or more chiral centers.
  • the system is designed to rank the four different substituent atoms around an asymmetric carbon atom in order of decreasing atomic number or in order of decreasing valance density when the smallest or lowest-rank group is pointing directly away from the viewer.
  • the different rankings are well known in the art and are described on page i
  • the agents of the invention may be substantially optically pure. That is, at least 90%,
  • the agents may comprise the carbon of the pyrrolidine ring bonded to the boron in the R-configuration.
  • the agent may be provided as 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%, at least 96%, at least 97%, at least 98%, or at least 99% of the R-enantiomer of boron substituted pyrrolidine.
  • compositions of the invention comprise a compound of Formula I and an anti-metabolite used in particular dosing and/or administration and/or timing regimens.
  • Anti-metabolites include folic acid analogs (anti-folates), pyrimidine analogs, and purine analogs.
  • folic acid analogs (anti-folates) include but are not limited to methrotrexate and pemetrexed (ALIMTA ® ).
  • pyrimidine analogs include but are not limited to fluorouracil, (5-fluorouracil; 5-FU), fioxuridine (fluorodeoxyuridine; FUdR), cytarabine (cytosine arabinoside) and gemcitabine.
  • purine analogs include but are not limited to mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG), and pentostatin (2'-deoxycoformycin).
  • antimetabolites include fludarabme, cladribine, capecitabine, L- alanosine, 5-azacytidine, acivicin, aminopterin derivatives, antifols, Baker's soluble antifol, dichlorallyl lawsone, brequinar, ftorafur, 5,6-dihydro-5-azacytidine, methotrexate derivatives, N-(phosphonoacetyl)-L-aspartate (PALA), pyrazofurin, trimetrexate, 3-HP 5 2'-deoxy-5- fluorouridine, 5-HP 5 alpha-TGDR, aphidicolin glycinate, ara-C, 5-aza-2'-deoxycytidine, beta- TGDR, cyclocytidine, guanazole, hydroxyurea, inosine glycodialdehyde, macbecin II, I
  • the antimetabolite is pemetrexed (ALIMTA ® ).
  • compositions and kits of the invention comprise a compound of Formula I and a HERl /EGFR tyrosine kinase inhibitor used in particular dosing and/or administration and/or timing regimens.
  • HER1/EGFR tyrosine kinase inhibitor include but are not limited to gefitinib (IRESSA ® ), erlotinib (TARCEV ATM), Cetuximab (Erbitux), and quinazoline and pyrido-[3,4-d]-pyrimidine molecules (e.g., GW2974, GW0277, and WHI-P97), LFM-A12(leflunomide metabolite analog), and AG 1458.
  • compositions and kits comprise a compound of Formula I and a taxane/taxoid used in particular dosing and/or administration and/or timing regimens.
  • taxanes include but are not limited to paclitaxel (TAXOL), docetaxel (TAXOTERE ® ), cephalomannine, Baccatin, xylotaxol, 10 Deacetyltaxol, and taxinine.
  • the taxane is docetaxel.
  • Certain methods and compositions of the invention may further comprise a cancer antigen.
  • a cancer antigen as used herein is a compound differentially associated with a tumor or cancer, preferably at the cell surface of a tumor or cancer cell that is capable of invoking an immune response.
  • the cancer antigen may be peptide in nature but it is not so limited.
  • the antigen may be a lipid antigen, as described in U.S. Patents US 5679347, issued on October 21, 1997 and US 6238676 Bl, issued on May 29, 2001. If the antigen is a peptide, then it invokes an immune response when it is presented (in a digested form) on the surface of an antigen presenting cell in the context of an MHC molecule.
  • Cancer antigens can be prepared from cancer cells either by preparing crude extracts of cancer cells, for example, as described in Cohen, et al., 1994, Cancer Research, 54: 1055, by partially purifying the antigens, by recombinant technology, or by de novo synthesis of known antigens.
  • Cancer antigens include but are not limited to antigens that are recombinantly expressed, an immunogenic portion of, or a whole tumor or cancer. Such antigens can be isolated or prepared recombinantly or by any other means known in the art.
  • a cancer antigen encompasses antigens that are differentially expressed between cancer and normal cells.
  • Cancer antigens may be expressed in a regulated manner in normal cells. For example, they may be expressed only at certain stages of differentiation or at certain points in development of the organism or cell. Some are temporally expressed as embryonic and fetal antigens. Still others are never expressed in normal cells, or their expression in such cells is so low as to be undetectable.
  • cancer antigens are encoded by mutant cellular genes, such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes such as those carried on RNA and DNA tumor viruses.
  • cancer antigens examples include HER 2 (pl85), CD20, CD33, GD3 ganglioside, GD2 ganglioside, carcinoembryonic antigen (CEA), CD22, milk mucin core protein, TAG-72, Lewis A antigen, ovarian associated antigens such as OV-TL3 and MOvI 8, high Mr melanoma antigens recognized by antibody 9.2.27, HMFG-2, SM-3, B72.3, PR5C5, PR4D2, and the like.
  • Other cancer antigens are described in U.S. Pat. No. 5776427. Still other cancer antigens are listed in Table 2.
  • MAGE MART-1/Melan-A, gplOO, Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (AD Abp), FAP, cyclophilin b, Colorectal associated antigen (CRC)-CO 17- 1A/GA733, Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-I and CAP-2, etv6, amll, Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-I, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-Al, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8,
  • DPPIV Dipeptidyl peptidase IV
  • AD Abp
  • E2A and PBX or HLF B-cell acute lymphoblastic leukemia E2A and PBX or HLF B-cell acute lymphoblastic leukemia
  • NPM MLF-I Myelodysplastic syndrome/acute myeloid leukemia
  • CD20 CD22 Non-Hodgkin's lymphoma, B-cell lymphoma, Chronic lymphocytic leukemia
  • AML Acute myelogenous leukemia
  • CDlO gplOO
  • Common pre-B
  • TCR T-cell receptor
  • BCR B-cell receptor
  • CD26 Epithelial and lymphoid malignancies
  • HLA Human leukocyte antigen
  • HLA-DP HLA-DP
  • HLA-DQ HLA-DQ
  • EGFR HERl or erbBl
  • Tyrosinase Melan-A/MART-1, tyrosinase Malignant melanoma related protein (TRP)-l/gp75 Polymorphic epithelial mucin (PEM) Breast tumors Human epithelial mucin (MUCl) Breast, ovarian, colon and lung cancers
  • antigens include MAGE-Al, -A3, -A6, -Al 2, BAGE, GAGE, HAGE, LAGE-I 5 NY- ESO-I, RAGE, SSX-I, -2, -3, -4, -5, -6, -7, -8, -9, HOM-TES-14/SCP-1, HOM-TES-85 and PRAME.
  • antigens are expressed in some normal tissues such as testis and in some cases placenta. Their expression is common in tumors of diverse lineages and as a group the antigens form targets for immunotherapy. Examples of tumor expression of CT antigens are as follows.
  • SSX member Head and neck cancer ovarian cancer, lymphoid tumors, colorectal cancer and breast cancer
  • HOM-TES14/SCP-1, SSX-I, PRAME and Non-Hodgkin's lymphoma CT-7 PRAME Acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML) and chronic lymphocytic leukemia (CLL)
  • Carcinoembryonic antigen (CEA) family CD66a, CD66b, CD66c, CD66d and CD66e.
  • antigens can be expressed in many different malignant tumors and can be targeted by immunotherapy.
  • EBV-encoded nuclear antigen (EBNA)-I (lymphomas of neck and oral cancer)
  • Cancer or tumor antigens can also be classified according to the cancer or tumor they are associated with (i.e., expressed by). Cancers or tumors associated with tumor antigens include acute lymphoblastic leukemia (etv6; amll ; cyclophilin b), B cell lymphoma (Ig- idiotype); Burkitt's (Non-Hodgkin's) lymphoma (CD20); glioma (E-cadherin; ⁇ -catenin; ⁇ - catenin; ⁇ -catenin; pl20ctn), bladder cancer (p21ras), biliary cancer (p21ras), breast cancer (MUC family; HER2/neu; c-erbB-2), cervical carcinoma (p53; p21ras), colon carcinoma (p21ras; HER2/neu; c-erbB-2; MUC family), colorectal cancer (Colorectal associated antigen (CRC) ⁇ CO17-1A/GA733; APC),
  • the cancer antigen is VEGF 5 Anti-idiotypic mAb (GD3 ganglioside mimic), CD20, CD52, Anti-idiotypic mAb (CEA mimic), ERBB2, EGFR 5 CD22, ERBB2 X CD65 (fc ⁇ RI), EpCam, PEM and CD33.
  • VEGF 5 Anti-idiotypic mAb GD3 ganglioside mimic
  • CD20 CD52
  • Anti-idiotypic mAb CEA mimic
  • ERBB2 EGFR 5 CD22
  • ERBB2 X CD65 fc ⁇ RI
  • EpCam EpCam
  • PEM EpCam
  • the antigens are administered in a substantially purified form.
  • substantially purified refers to a compound which is substantially free of other compounds such as proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • One skilled in the art can purify antigens using standard techniques such as for example protein purification.
  • the substantially pure polypeptide will often yield a single major band on a non-reducing polyacrylamide gel. In the case of partially glycosylated polypeptides or those that have several start codons, there may be several bands on a non- reducing polyacrylamide gel, but these will form a distinctive pattern for that polypeptide.
  • the purity of the viral or bacterial polypeptide can also be determined by amino-terminal amino acid sequence analysis.
  • Some important cancer antigens include but are not limited to PGP9.5, H/Ley/Leb, B7-DC, and B7-Hl.
  • compositions comprise, in addition to the compounds of Formula I, an antibody or an antigen-binding fragment thereof.
  • the invention embraces the use of antibodies of all isotypes including IgM, IgAl, IgA2, slgA, IgD, IgE, IgGl, IgG2 5 IgG3, and IgG4, having light chains that are either kappa or lambda chains.
  • the invention encompasses the use of both antibodies and antibody fragments.
  • the antibodies may be monoclonal or polyclonal, and can be prepared by conventional methodology. Such antibodies can be further manipulated to create chimeric or humanized antibodies as will be discussed in greater detail below.
  • the antibodies or fragments thereof useful in the invention can be specific for any component of a particular target. Accordingly, the antibody can recognize and bind to proteins, lipids, carbohydrates, DlSTA, RNA, and any combination of these in molecular or supra-molecular structures (e.g., cell organelles such as mitochondria or ribosomes).
  • the antibody or fragment thereof can also recognize a modification of the tumor cell, such as e.g., chemical modifications, or genetic modifications made by transfection ex vivo or in vivo with DNA or RNA.
  • the terms "antibody” and "immunoglobulin" are used interchangeably.
  • Bispecif ⁇ c antibodies can also be used in the invention.
  • a bispecific antibody is one having one variable region that specifically recognizes a tumor antigen and the other variable region that specifically recognizes an antigenic epitope of a host immune effector cell that has lytic or growth inhibitory activity against the tumor.
  • Bispecific and multispecific antibody complexes can be created by linkage of two or more immunoglobulins of different specificity for tumor antigens and/or effector cell antigens, either at the peptide or nucleic acid level.
  • Immunoglobulin can be produced in vivo in human or non-human species, or in vitro from immunoglobulin encoding DNA or cDNA isolated from libraries of DNA (e.g., phage display libraries). Immunoglobulin can also be modified genetically or chemically to incorporate human polypeptide sequences into non-human coding sequences (commonly referred to as humanization). Additionally, immunoglobulins can be modified chemically or genetically to incorporate protein, lipid, or carbohydrate moieties. Potential modifications could also include naturally occurring or synthetic molecular entities that are either directly toxic for tumor cells or serve as ligands or receptors for biologically active molecules that could suppress tumor growth.
  • growth factors for example, growth factors, cytokines, chemokines and their respective receptors, immunologically active ligands or receptors, hormones or naturally occurring or synthetic toxins all represent biologically active molecules that could interact with suitably modified immunoglobulins and their targets.
  • an antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc 1 region designated an F(ab') 2 fragment
  • an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an Fab fragment
  • Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd.
  • the Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
  • CDRs complementarity determining regions
  • FRs framework regions
  • CDRl through CDR3 complementarity determining regions
  • the present invention also provides for F(ab') 2 , Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab') 2 fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDRl and/or CDR2 regions have been replaced by homologous human or non-human sequences.
  • the present invention also includes so-called single chain antibodies.
  • Some commercially available anti-cancer antibodies along with their commercial source are as follows: anti-CD20 mAb (monoclonal antibody), rituximab, RituxanTM, Non- Hodgkin's lymphoma, B cell lymphoma (IDEC/Genentech); anti-CD20 mAb, tositumomab Bexxar, Non-Hodgkin's lymphoma (Corixa/GlaxoSmithKline); anti-HER2, trastuzumab, HerceptinTM, breast and ovarian cancer (Genentech); anti-HER2, MDX-210, prostate, non- small cell lung cancer, breast, pancreatic, ovarian, renal and colon cancer (Medarex/Novartis); anti-CA125 mAb, oregovomab, B43.13, OvarexTM, ovarian cancer (Altarex); Breva-Rex, multiple myeloma, breast, lung,
  • anti-EGF receptor mAb EMD-72000, cancer (Merck KgaA); anti-CD22 humanized Ab, LymphoCide, Non-Hodgkin's lymphoma (Immunomedics); anti-CD33 mAb conjugate with calicheamicin (CMA 676), gemtuzumab ozogamicin, MylotargTM, acute myelogenous leukemia (Wyeth); Monopharm-C, colon, lung and pancreatic cancer (Viventia Biotech); anti- idiotypic human mAb to GD2 ganglioside, 4B5, melanoma, small-cell lung cancer, neuroblastoma (Viventia Biotech); anti-EGF receptor humanized Ab, ior egf/r3, cancers of epithelial origin (Center of Molecular Immunology); anti-ior c2 glycoprotein mAb, ior c5, colorectal and ovarian cancer (Center of Molecular Immunology);
  • antibodies that can be used according to the invention include anti-TNF ⁇ antibody such as infliximab (Remicade) and etanercept (Enbrel) and palivizumab; bevacizumab, breast cancer; alemtuzumab, Campath-IH, breast and renal cancer, melanoma, B cell chronic lymphocytic leukemia (Millennium and ILEX); BLyS -mAb, fSLE; anti- VEGF2, melanoma, breast cancer; anti-Trail receptor; B3 mAb, breast cancer; ml 70 mAb, breast cancer; niAB BR96, breast cancer; Abx-Cbl mAb, graft versus host disease.
  • the invention embraces a number of classes of antibodies and fragments thereof including but not limited to antibodies directed to cancer antigens (as described above), cell surface molecule, stromal cell molecules, extracellular matrix molecules,, and tumor vasculature associated molecules.
  • a cell surface molecule is a molecule that is expressed at the surface of a cell. In addition to an extracellular domain, it may further comprise a transmembrane domain and a cytoplasmic domain. Examples include HER 2, CD20, CD33, EGF receptor, HLA markers such as HLA-DR, CD52, CDl 5 CEA, CD22, GD2 ganglioside, FLK2/FLT3, VEGF 5 VEGFR, and the like.
  • a stromal cell molecule is a molecule expressed by a stromal cell. Examples include but are not limited to FAP and CD26.
  • An extracellular matrix molecule is a molecule found in the extracellular matrix. Examples include but are not limited to collagen, glycosaminoglycans (GAGs), proteoglycans, elastin, fibronectin and laminin.
  • GAGs glycosaminoglycans
  • proteoglycans elastin
  • fibronectin fibronectin
  • laminin laminin
  • a tumor vasculature associated molecule is a molecule expressed by vasculature of a tumor (i.e., a solid cancer rather than a systemic cancer such as leukemia).
  • a tumor vasculature associated molecule may be expressed by normal vasculature however its presence on vasculature of a tumor makes it a suitable target for anti-cancer therapy.
  • the tumor vasculature associated molecule is expressed at a higher level in tumor vasculature than it is in normal vasculature. Examples include but are not limited to endoglin (see U.S. Pat. No.
  • Antibodies to aminophospholipids are described in U.S. Pat. No. 6,312,694. Antibodies that inhibit VEGF are described in U.S. Pat. No. 6,342,219 and include 2C3 (ATCC PTA 1595). Other antibodies that are specific for tumor vasculature include antibodies that react to a complex of a growth factor and its receptor such as a complex of FGF and the FGFR or a complex of TGF ⁇ and the TGF ⁇ R. Antibodies of this latter class are described in U.S. Pat. No. 5,965,132, and include GV39 and GV97.
  • antibodies such as the following, all of which are commercially available: Apoptosis Antibodies:
  • BAX Antibodies Anti-Human Bax Antibodies (Monoclonal), Anti-Human Bax Antibodies (Polyclonal), Anti-Murine Bax Antibodies (Monoclonal), Anti-Murine Bax Antibodies (Polyclonal);
  • Fas / Fas Ligand Antibodies Anti-Human Fas / Fas Ligand Antibodies, Anti-Murine Fas / Fas Ligand Antibodies Granzyme Antibodies Granzyme B Antibodies;
  • BCL Antibodies Anti Cytochrome C Antibodies, Anti-Human BCL Antibodies (Monoclonal), Anti-Human bcl Antibodies (Polyclonal), Anti-Murine bcl Antibodies (Monoclonal), Anti-Murine bcl Antibodies (Polyclonal);
  • Miscellaneous Apoptosis Antibodies Anti TRADD, TRAIL, TRAFF, DR3 Antibodies Anti-Human Fas / Fas Ligand Antibodies Anti-Murine Fas / Fas Ligand Antibodies; Miscellaneous Apoptosis Related Antibodies: BIM Antibodies: Anti Human, Murine bim Antibodies (Polyclonal), Anti-Human, Murine bim Antibodies (Monoclonal);
  • PARP Antibodies Anti-Human PARP Antibodies (Monoclonal), Anti-Human PARP Antibodies (Polyclonal) Anti-Murine PARP Antibodies;
  • Caspase Antibodies Anti-Human Caspase Antibodies (Monoclonal), Anti-Murine Caspase Antibodies;
  • Anti-CD Antibodies Anti-CD29, PL 18-5 PanVera, Anti-CD29, PL4-3 PanVera, Anti- CD41a, PT25-2 PanVera, Anti-CD42b, PL52-4 PanVera, Anti-CD42b, GUR20-5 PanVera, Anti-CD42b, WGA-3 PanVeraAnti-CD43, 1D4 PanVera, Anti-CD46, MCP75-6 PanVera, Anti-CD61, PLl 1-7 PanVera, Anti-CD61, PL8-5 PanVera, Anti-CD62/P-slctn, PL7-6 PanVera, Anti-CD62/P-slctn, WGA-I PanVera, Anti-CD154, 5F3 PanVera; and anti-CDl, anti-CD2, anti-CD3, anti-CD4, anti-CD5, anti-CD6, anti-CD7, anti-CD8, anti-CD9, anti- CDlO, anti-CDl 1, anti-CD12, anti-CD13, anti
  • Human Chemokine Antibodies Human CNTF Antibodies, Human Eotaxin Antibodies, Human Epitherlial Neutrophil Activating Peptide-78, Human Exodus Antibodies, Human GRO Antibodies, Human HCC-I Antibodies, Human 1-309 Antibodies, Human IP-IO Antibodies, Human I- TAC Antibodies, Human LIF Antibodies, Human Liver-Expressed Chemokine Antibodies, Human lymphotoxin Antibodies, Human MCP Antibodies, Human MIP Antibodies, Human Monokine Induced by IFN-gamma Antibodies, Human NAP-2 Antibodies, Human NP-I Antibodies, Human Platelet Factor-4 Antibodies, Human RANTES Antibodies, Human SDF Antibodies, Human TECK Antibodies;
  • Murine Chemokine Antibodies Human B-CeIl Attracting Murine Chemokine Antibodies, Chemokine- 1 Antibodies, Murine Eotaxin Antibodies, Murine Exodus Antibodies, Murine GCP-2 Antibodies, Murine KC Antibodies, Murine MCP Antibodies, Murine MIP Antibodies, Murine RANTES Antibodies, Rat Chemokine Antibodies, Rat Chemokine Antibodies, Rat CNTF Antibodies, Rat GRO Antibodies, Rat MCP Antibodies, Rat MIP Antibodies, Rat RANTES Antibodies;
  • Cytokine / Cytokine Receptor Antibodies Human Biotinylated Cytokine / Cytokine Receptor Antibodies, Human IFN Antibodies, Human IL Antibodies, Human Leptin Antibodies, Human Oncostatin Antibodies, Human TNF Antibodies, Human TNF Receptor Family Antibodies, Murine Biotinylated Cytokine / Cytokine Receptor Antibodies, Murine IFN Antibodies, Murine IL Antibodies, Murine TNF Antibodies, Murine TNF Receptor Antibodies; anti-CCR4 antibody;
  • Rat Cytokine / Cytokine Receptor Antibodies Rat Biotinylated Cytokine / Cytokine Receptor Antibodies, Rat IFN Antibodies, Rat IL Antibodies, Rat TNF Antibodies;
  • ECM Antibodies Collagen / Procollagen, Laminin, Collagen (Human), Laminin (Human), Procollagen (Human), Vitronectin / Vitronectin Receptor, Vitronectin (Human), Vitronectin Receptor (Human), Fibronectin / Fibronectin Receptor, Fibronectin (Human), Fibronectin Receptor (Human);
  • Growth Factor Antibodies Human Growth Factor Antibodies, Murine Growth Factor Antibodies, Porcine Growth Factor Antibodies; Miscellaneous Antibodies: Baculovirus Antibodies, Cadherin Antibodies,
  • Complement Antibodies CIq Antibodies, VonWillebrand Factor Antibodies, Cre Antibodies, HIV Antibodies, Influenza Antibodies, Human Leptin Antibodies , Murine Leptin Antibodies, Murine CTLA-4 Antibodies, Human CTLA-4 Antibodies, P450 Antibodies, RNA Polymerase Antibodies; Neurobio Antibodies: Amyloid Antibodies, GFAP Antibodies, Human NGF
  • antibodies can be used in the invention and these include antibodies listed in references such as the MSRS Catalog of Primary Antibodies, and Linscott's Directory.
  • the antibody is rituximab (RITUXAN), bevacizumab (AVASTIN), cetuximab (ERBITUX), trastuzumab (HERCEPTIN), tositumomab (BEXXAR), or alemtuzumab (CAMPATH).
  • mitumomab (BEC2), CeaVac, centuximab (IMC-C225), epratuzumab (L YMPHOCIDE), MDX-210, gemtuzumab ozogamicin (MYLOTARG), edrecolomab (PANOREX), pemtumomab (THERAGYN), Zamyl, and ibritumomab tituxetan (ZEVALIN).
  • the invention also covers antibody fragments thereof.
  • the antibody may be an anti-a5 ⁇ l integrin antibody (e.g., Volocimixab), an anti-EGFR antibody (e.g., Nimotuzumab (TheraCIM)) , or alpha IR-3.
  • an anti-a5 ⁇ l integrin antibody e.g., Volocimixab
  • an anti-EGFR antibody e.g., Nimotuzumab (TheraCIM)
  • alpha IR-3 e.g., alpha IR-3.
  • the antibodies or antigen-binding fragments thereof provided herein can be used additionally for delivery of toxic substances to cancer cells.
  • Antibodies are commonly 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 such as, for example, antimetabolites, anthracyclines, vinca alkaloids, antibiotics, alkylating agents, and epipodophyllotoxins.
  • the antibodies or antigen-binding fragments thereof may also be conjugated 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 are specific for cancer antigens 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 thus most tumors are capable of recruiting and stimulating the growth of new blood vessels. As a result, one strategy of many cancer medicaments is to attack the blood vessels feeding a tumor and/or the connective tissues (or stroma) supporting such blood vessels.
  • the invention also embraces the use of adjuvants.
  • adjuvants derived from microorganisms, such as Bacillus Calmette-Guerin, heighten the immune response and enhance resistance to tumors in animals.
  • Adjuvants that may be combined with the compounds of Formula I include alum, immunostimulatory oligonucleotides such as CpG oligonucleotides, QS-21, MLP, MPD, and the like. These and other adjuvants are listed herein.
  • Adjuvants include but are not limited to nucleic acid adjuvants and non-nucleic acid adjuvants.
  • a "nucleic acid adjuvant” is an adjuvant that is a nucleic acid. Examples include immunostimulatory nucleic acid molecules such as those containing CpG dinucleotides, as described in U.S. Patents US 6,194,388Bl, issued February 27, 2001, US 6,207,646 Bl, issued March 27, 2001, and US 6,239,116 Bl, issued May 29, 2001.
  • non-nucleic acid adjuvant is any molecule or compound except for the immunostimulatory nucleic acids described herein which can stimulate the humoral and/or cellular immune response.
  • Non-nucleic acid adjuvants include, for instance, adjuvants that create a depo effect, immune-stimulating adjuvants, adjuvants that create a depo effect and stimulate the immune system and mucosal adjuvants.
  • an "adjuvant that creates a depo effect” as used herein is an adjuvant that causes an antigen, such as a cancer antigen present in a cancer vaccine, to be slowly released in the body, thus prolonging the exposure of immune cells to the antigen.
  • This class of adjuvants includes but is not limited to alum (e.g., aluminum hydroxide, aluminum phosphate); or emulsion-based formulations including mineral oil, non-mineral oil, water-in-oil or oil-in- water-in oil emulsion, oil-in- water emulsions such as Seppic ISA series of Montanide adjuvants (e.g., Montanide ISA 720, AirLiquide, Paris, France); MF-59 (a squalene-in-water emulsion stabilized with Span 85 and Tween 80; Chiron Corporation, Emeryville, CA; and PROVAX (an oil-in- water emulsion containing a stabilizing detergent and a micelle-forming agent; IDEC Pharmaceuticals Corporation, San Diego, CA).
  • alum e.g., aluminum hydroxide, aluminum phosphate
  • emulsion-based formulations including mineral oil, non-mineral oil, water-in-oil or oil-in-
  • an “immune stimulating adjuvant” is an adjuvant that causes activation of a cell of the immune system. It may, for instance, cause an immune cell to produce and secrete cytokines.
  • This class of adjuvants includes but is not limited to saponins purified from the bark of the Q.
  • saponaria tree such as QS21 (a glycolipid that elutes in the 21 st peak with HPLC fractionation; Antigenics, Inc., Waltham, MA); poly [di (carboxylatophenoxy) phosphazene (PCPP polymer; Virus Research Institute, USA); derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, MT), muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, WA).
  • QS21 a glycolipid that elutes in the 21 st peak with HPLC fractionation; Antigenics, Inc., Waltham, MA
  • adjuvants that create a depo effect and stimulate the immune system are those compounds which have both of the above- identified functions.
  • This class of adjuvants includes but is not limited to ISCOMS (Immunostimulating complexes which contain mixed saponins, lipids and form virus-sized particles with pores that can hold antigen; CSL,
  • SB-AS2 SmithKline Beecham adjuvant system #2 which is an oil-in- water emulsion containing MPL and QS21 : SmithKline Beecham Biologicals [SBB], Rixensart, Belgium
  • SB-AS4 SmithKline Beecham adjuvant system #4 which contains alum and MPL; SBB, Belgium); non-ionic block copolymers that form micelles such as CRL 1005 (these contain a linear chain of hydrophobic polyoxpropylene flanked by chains of polyoxyethylene; Vaxcel, Inc., Norcross, GA); and Syntex Adjuvant Formulation (SAF, an oil-in-water emulsion containing Tween 80 and a nonionic block copolymer; Syntex Chemicals, Inc., Boulder, CO).
  • non-nucleic acid mucosal adjuvant is an adjuvant other than an immunostimulatory nucleic acid that is capable of inducing a mucosal immune response in a subject when administered to a mucosal surface in conjunction with an antigen.
  • Mucosal adjuvants include but are not limited to Bacterial toxins: e.g., Cholera toxin (CT), CT derivatives including but not limited to CT B subunit (CTB) (Wu et al, 1998, Tochikubo et al, 1998); CTD53 (VaI to Asp) (Fontana et al., 1995); CTK97 (VaI to Lys) (Fontana et al., 1995); CTKl 04 (Tyr to Lys) (Fontana et al., 1995); CTD53/K63 (VaI to Asp, Ser to Lys) , (Fontana et al., 1995); CTH54 (Arg to His) (Fontana et al., 1995); CTNl 07 (His to Asn) (Fontana et al., 1995); CTEl 14 (Ser to GIu) (Fontana et al., 1995); CTEl 12K
  • LT Labile Toxin
  • LT derivatives including but not limited to LT B subunit (LTB) (Verweij et al., 1998); LT7K (Arg to Lys) (Komase et al., 1998, Douce et al., 1995); LT61 F (Ser to Phe) (Komase et al., 1998); LTl 12K (GIu to Lys) (Komase et al..
  • LTl 18E GIy to GIu
  • LTl 46E Arg to GIu
  • LTl 92G Arg to GIy
  • LTK63 Ser to Lys
  • LTR72 Al to Arg
  • Lipid A derivatives e.g., monophosphoryl lipid A, MPL
  • Lipid A derivatives e.g., monophosphoryl lipid A, MPL
  • MPL Muramyl Dipeptide
  • MDP Muramyl Dipeptide
  • Bacterial outer membrane proteins e.g., outer surface protein A (OspA) lipoprotein of Borrelia burgdorferi, outer membrane protine of Neisseria meningiti ⁇ is
  • Oil-in-water emulsions e.g., MF59
  • chemotherapeutic agents such as but not limited to those currently recited herein.
  • chemotherapeutic agents can be categorized as DNA damaging agents and these include topoisomerase inhibitors (e.g., etoposide, ramptothecin, topotecan, teniposide, mitoxantrone), antimicrotubule agents (e.g., vincristine, vinblastine), antimetabolites (e.g., cytarabine, methotrexate, hydroxyurea, 5-fluorouracil, floxuridine, 6-thioguanine, 6-mercaptopurine, fludarabine, pentostatin, chlorodeoxyadenosine), DNA alkylating agents (e.g., cisplatin, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chorambucil, busulfan, thiotepa, carmustine
  • topoisomerase inhibitors
  • chemotherapeutic agents are those selected from the group consisting of: Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Adriamycin; Aldesleukin; Alitretinoin; Allopurinol Sodium; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Annonaceous Acetogenins; Anthramycin; Asimicin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bexarotene; Bicalutamide; Bisantrene Hydrochloride; Bisnaf ⁇ de Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Bullatacin; Busulfan; Cabergoline; Cactinomycin; Calusterone; Car
  • DACA N-[2-(Dimethyl-amino)ethyl]acridine-4-carboxamide); Dactinomycin; Daunorubicin Hydrochloride; Daunomycin; Decitabine; Denileukin Diftitox; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflornithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Ethiodized Oil 1 131; Etoposide; E
  • Pipobroman Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin
  • Tiazofurin Tirapazamine; Tomudex; TOP-53; Topotecan Hydrochloride; Toremifene Citrate;
  • Glucuronate Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Valrubicin; Vapreotide; Verteporfin; Vinblastine; Vinblastine Sulfate; Vincristine; Vincristine Sulfate;
  • Vindesine Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate;
  • Vinorelbine Tartrate Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin;
  • Zinostatin Zinostatin; Zorubicin Hydrochloride; 2-Chlorodeoxyadenosine; 2'-Deoxyformycin; 9- aminocamptothecin; raltitrexed; N-propargyl-5,8-dideazafolic acid; 2-chloro-2'-arabino- fluoro-2'-deoxyadenosine; 2-chloro-2'-deoxyadenosine; anisomycin; trichostatin A; hPRL-
  • G129R G129R; CEP-751; linomide; sulfur mustard; nitrogen mustard (mechlor ethamine); cyclophosphamide; melphalan; chlorambucil; ifosfamide; busulfan; N-methyl-N-nitrosourea
  • MNU N, N'-Bis(2-chloroethyl)-N-nitrosourea
  • BCNU N-(2-chloroethyl)-N'-cyclohexyl-
  • CCNU N-nitrosourea
  • MeCCNU N-(2-chloroethyl)-N'-(trans-4-methylcyclohexyl-N-nitrosourea
  • MeCCNU N-(2-chloroethyl)-N'-(diethyl)ethylphosphonate-N-nitrosourea
  • streptozotocin diacarbazine (DTIC); mitozolomide; temozolomide; thiotepa; mitomycin C;
  • AZQ adozelesin
  • Cisplatin Carboplatin; Ormaplatin
  • Oxaliplatin C 1-973; DWA 2114R;
  • chemotherapeutic agents include: 20-epi-l,25 dihydroxyvitamin D3;
  • DCF 2'deoxycoformycin
  • chemotherapeutic agents include: Antiproliferative agents (e.g., Piritrexim Isothionate), Antiprostatic hypertrophy agent (e.g., Sitogluside), Benign prostatic hyperplasia therapy agents (e.g., Tamsulosin Hydrochloride), Prostate growth inhibitor agents (e.g., Pentomone), and Radioactive agents: Fibrinogen 1 125; Fludeoxyglucose F 18; Fluorodopa F 18; Insulin 1 125; Insulin 1 131; Iobenguane 1 123; Iodipamide Sodium 1 131; Iodoantipyrine I 131; Iodocholesterol 1 131; Iodohippurate Sodium 1 123; Iodohippurate Sodium 1 125; Iodohippurate Sodium 1 131; Iodohippurate Sodium 1 131; Iodohippurate Sodium 1 131; Iodopyracet 1 125
  • chemotherapeutic agents are anti-cancer Supplementary Potentiating Agents, including: Tricyclic anti-depressant drugs (e.g., imipramine, desipramine, amitryptyline, clomipramine, trimipramine, doxepin, nortriptyline, protriptyline, amoxapine and maprotiline); non-tricyclic anti-depressant drugs (e.g., sertraline, trazodone and citalopram); Ca “ " " " " antagonists (e.g., verapamil, nifedipine, nitrendipine and caroverine); Calmodulin inhibitors (e.g., prenylamine, trifluoroperazine and clomipramine); Amphotericin B; Triparanol analogues (e.g., tamoxifen); antiarrhythmic drugs (e.g., quinidine); antihypertensive drugs (e.g., re
  • chemotherapeutic agents are those selected from the group consisting of: annonaceous acetogenins; asimicin; rolliniastatin; guanacone, squamocin, bullatacin; squamotacin; taxanes; paclitaxel; gemcitabine; methotrexate FR-900482; FK-973; FR-66979; FK-317; 5-FU; FUDR; FdUMP; Hydroxyurea; Docetaxel; discodermolide; epothilones; vincristine; vinblastine; vinorelbine; meta-pac; irinotecan; SN-38; 10-OH campto; topotecan; etoposide; adriamycin; flavopiridol; platinum-containing compounds (e.g., cisplatin(Cis-Pt) and carboplatin (carbo-Pt); bleomycin; mitomycin C; mithramycin
  • the agents are administered together with chemotherapeutic agents selected from the group consisting of aldesleukin, asparaginase, bleomycin sulfate, carboplatin, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, docetaxel, doxorubicin, doxorubicin hydrochloride, epirubicin hydrochloride, etoposide, etoposide phosphate, floxuridine, fludarabine, fluorouracil, gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, interferons, interferon- ⁇ 2a, interferon- ⁇ 2b, interferon- ⁇ n3, interferon- ⁇ lb, interleukins, irinote
  • cancer therapies include hormonal manipulation.
  • Compounds of Formula I and pemetrexed are also useful in combination with tamoxifen or aromatase inhibitor arimidex (i.e., anastrozole).
  • the methods and compositions of the invention may be used with enzyme inhibitor agents such as CDK inhibitors, tyrosine kinase inhibitors and MAP kinase inhibitors.
  • enzyme inhibitor agents such as CDK inhibitors, tyrosine kinase inhibitors and MAP kinase inhibitors.
  • the tyrosine kinase inhibitor is selected from the group consisting of Genistein (4',5,7-trihydroxyisoflavone), Tyrphostin 25 (3,4,5-trihydroxyphenyl), methylene]- propanedinitrile, Herbimycin A, Daidzein (4',7-dihydroxyisoflavone), AG-126, trans-l-(3'- carboxy-4'-hydroxyphenyl)-2-(2" 5 5"-dihydroxy-phenyl)ethane, and HDBA (2-Hydroxy5- (2,5-Dihydroxybenzylamino)-2-hydroxybenzoic acid.
  • the CDK inhibitor is selected from the group consisting of p21, p27, p57, pl5, pl6, pl8, and pl9.
  • the MAP kinase inhibitor is selected from the group consisting of KY12420 (C 23 H 24 O 8 ), CNI-1493, PO98059, 4-(4-Fluorophenyl)-2-(4-methylsulfmyl phenyl)- 5-(4-pyridyl) lH-imidazole.
  • the methods and compositions of the invention may be used in subjects having or at risk of developing cancer.
  • a subject having a cancer is a subject that has detectable cancerous cells.
  • a subject at risk of developing a cancer is one who has a higher than normal probability of developing cancer. These subjects include, for instance, subjects having a genetic abnormality that has been demonstrated to be associated with a higher likelihood of developing a cancer, subjects having a familial disposition to cancer, subjects exposed to cancer causing agents (i.e., carcinogens) such as tobacco, asbestos, or other chemical toxins, and subjects previously treated for cancer and in apparent remission.
  • cancer refers to an uncontrolled growth of cells which interferes with the normal functioning of the bodily organs and systems.
  • Hemopoietic cancers such as leukemia, are able to outcompete the normal hemopoietic compartments in a subject, thereby leading to hemopoietic failure (in the form of anemia, thrombocytopenia and neutropenia) ultimately causing death.
  • a metastasis is a region of cancer cells, distinct from the primary tumor location resulting from the dissemination of cancer cells from the primary tumor to other parts of the body.
  • the subject may be monitored for the presence of metastases. Metastases are most often detected through the sole or combined use of magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, blood and platelet counts, liver function studies, chest X-rays and bone scans in addition to the monitoring of specific symptoms.
  • MRI magnetic resonance imaging
  • CT computed tomography
  • a cancer cell is a cell that divides and reproduces abnormally due to a loss of normal growth control. Cancer cells almost always arise from at least one genetic mutation. In some instances, it is possible to distinguish cancer cells from their normal counterparts based on profiles of expressed genes and proteins, as well as to the level of their expression. Genes commonly affected in cancer cells include oncogenes, such as ras, neu/HER2/erbB, myb, myc and abl, as well as tumor suppressor genes such as p53, Rb, DCC, RET and WT. Cancer- related mutations in some of these genes leads to a decrease in their expression or a complete deletion. In others, mutations cause an increase in expression or the expression of an activated variant of the normal counterpart.
  • neoplasm is usually equated with neoplasm, which literally means “new growth” and is used interchangeably with “cancer.”
  • a "neoplastic disorder” is any disorder associated with cell proliferation, specifically with a neoplasm.
  • a “neoplasm” is an abnormal mass of tissue that persists and proliferates after withdrawal of the carcinogenic factor that initiated its appearance.
  • the method of the invention can be used to treat neoplastic disorders in humans, including but not limited to: sarcoma, carcinoma, fibroma, leukemia, lymphoma, melanoma, myeloma, neuroblastoma, rhabdomyosarcoma, retinoblastoma, and glioma as well as each of the other tumors described herein.
  • Cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cervical cancer; choriocarcinoma; 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 including acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia; liver cancer; lung cancer (e.g., small cell lung cancer and non-small cell lung cancer); lymphoma including Hodgkin's andNon-Hodgkin's lymphoma; melanoma; mesothelioma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx);
  • the cancer is non-small cell lung cancer and more particularly adenocarincoma or squamous cell carcinoma.
  • Carcinomas are cancers of epithelial origin.
  • Carcinomas intended for treatment with the methods of the invention include, but are not limited to, acinar carcinoma, acinous carcinoma, alveolar adenocarcinoma (also called adenocystic carcinoma, adenomyoepithelioma, cribriform carcinoma and cylindroma), carcinoma adenomatosum, adenocarcinoma, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma (also called bronchiolar carcinoma, alveolar cell tumor and pulmonary adenomatosis), basal cell carcinoma, carcinoma basocellulare (also called basaloma, or basiloma, and hair matrix carcinoma), basaloid carcinoma, basosquamous cell carcinoma, breast carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma (also called cholangioma and cholangiocarcinoma), chorionic carcinoma, col
  • sarcomas are rare mesenchymal neoplasms that arise in bone and soft tissues. Different types of sarcomas are recognized and these include: liposarcomas (including myxoid liposarcomas and pleomorphic 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., non-bone) Ewing's sarcoma, and primitive neuroectodermal tumor [PNET]), synovial sarcoma, angiosarcomas, hemangiosarcomas, lymphangiosarcomas, Kaposi's sarcoma, hemangioendot
  • liposarcomas
  • 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. These cancers may appear initially responsive to a treatment (and then they may recur), or they may be completely non-responsive to the treatment from the outset.
  • 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. Subjects being treated according to the invention for a refractory cancer therefore may have already been exposed to another treatment for their cancer.
  • the subject may not have already been exposed to another treatment.
  • the subject is administered a combination therapy wherein one of the agents was previously administered to the subject and to which the cancer was previously considered refractory.
  • the compound of Formula I acts to enhance the effect of the other therapy and thus the response of the cancer to the other therapy.
  • refractory cancers include but are not limited to leukemias, melanomas, renal cell carcinomas, colon cancer, liver (hepatic) cancers, pancreatic cancer, Non-Hodgkin's lymphoma, and lung cancer including non-small cell lung cancer such as adenocarcinoma and squamous cell carcinoma of the lung.
  • refractory cancers are cancers that are refractory to treatment with platinum-containing compounds, such as carboplatin and cisplatin.
  • 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.
  • immunogenic cancers include malignant melanoma and renal cell cancer.
  • Subjects at risk of developing a cancer include subjects that are known or are suspected of being exposed to a carcinogen.
  • a carcinogen is an agent capable of initiating development of malignant cancers. Exposure to carcinogens generally increases the risk of neoplasms in subjects, usually by affecting DNA directly.
  • Carcinogens may take one of several forms such as chemical, electromagnetic radiation, or may be an inert solid body. Examples of chemical carcinogens include tobacco, asbestos, and the like.
  • a subject shall mean a human or animal including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent e.g., rats and mice, primate, e.g., monkey, and fish or aquaculture species such as fin fish (e.g., salmon) and shellfish (e.g., shrimp and scallops).
  • Subjects suitable for therapeutic or prophylactic methods include vertebrate and invertebrate species.
  • Subjects can be house pets (e.g., dogs, cats, fish, etc.), agricultural stock animals (e.g., cows, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), zoo animals (e.g., lions, giraffes, etc.), but are not so limited. Although many of the embodiments described herein relate to human disorders, the invention is also useful for treating other nonhuman vertebrates.
  • pets e.g., dogs, cats, fish, etc.
  • agricultural stock animals e.g., cows, horses, pigs, chickens, etc.
  • laboratory animals e.g., mice, rats, rabbits, etc.
  • zoo animals e.g., lions, giraffes, etc.
  • an effective amount refers to the amount necessary or sufficient to realize a desired biologic effect.
  • an effective amount is that amount that provides a biologically beneficial effect.
  • the biologically beneficial effect may be the amelioration and or absolute elimination of symptoms resulting from the disorder being treated in the short term (e.g., weeks or months) or the long term (e.g., years).
  • the biologically beneficial effect is the complete abrogation of the disorder as evidenced for example, by the absence of a tumor or a biopsy or blood smear which is free of cancer cells.
  • the effective amount may vary depending upon the particular compound and the particular antibody used.
  • the effective amount for any particular application can also vary depending on such factors as the cancer being treated, the size of the subject, or the severity of the disease or condition.
  • One of ordinary skill in the art can empirically determine the effective amount of a particular compound of Formula I and anti-cancer agent(s) combination without necessitating undue experimentation.
  • an effective therapeutic or prophylactic treatment regimen can be planned which does not cause substantial toxicity and yet is entirely effective to treat the particular subject.
  • a sub-therapeutic dosage of either the compound of Formula I or the anti-cancer agent(s), or a sub-therapeutic dosage of both is used in the treatment of a subject having, or at risk of developing, cancer.
  • erlotinib can be administered in a sub-therapeutic dose and still produce a desirable therapeutic result.
  • a "sub-therapeutic dose” as used herein refers to a dosage which is less than the dosage that would produce a therapeutic result in the subject if administered in the absence of the other agent.
  • the sub-therapeutic dose of an anticancer agent is one which would not produce the desired therapeutic result in the subject in the absence of the administration of the compound of Formula I.
  • Therapeutic doses of anticancer agents are well known in the field of medicine for the treatment of cancer. These dosages have been extensively described in references such as Remington's Pharmaceutical Sciences, 18th ed., 1990, or the Physician Desktop Reference; as well as many other medical references relied upon by the medical profession as guidance for the treatment of cancer.
  • a therapeutically effective amount can be initially determined from cell culture assays.
  • the effective amount of a compound of Formula I can be determined using in vitro stimulation assays.
  • the stimulation index of immune cells can be used to determine an effective amount of the particular compound for the particular subject, and the dosage can be adjusted upwards or downwards to achieve the desired levels in the subject.
  • Therapeutically effective amounts can also be determined in animal studies. For instance, the effective amount of a compound of Formula I and pemetrexed, erlotinib, or docetaxel to induce a response, including a synergistic response, can be assessed using in vivo assays of tumor regression and/or prevention of tumor formation. Relevant animal models include assays in which malignant cells are injected into the animal subjects, usually in a defined site. Generally, a range of compound of Formula I doses are administered into the animal along with a range of pemetrexed, erlotinib, or docetaxel doses. Inhibition of the growth of a tumor following the injection of the malignant cells is indicative of the ability to reduce the risk of developing a cancer.
  • Inhibition of further growth (or reduction in size) of a pre-existing tumor is indicative of the ability to treat the cancer.
  • Mice which have been modified to have human immune system elements can be used as recipients of human cancer cell lines to determine the effective amount of the synergistic combination.
  • an effective amount of a combination of a compound of Formula I and pemetrexed is an amount that reduces tumor size or volume or cancer load (e.g., as measured by number of lesions or extent of metastasis) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of pretreatment tumor size or volume or cancer load.
  • the effective amount is one that reduces the tumor size, vol ⁇ me or cancer load by at least 50% or at lest 60% of the pretreatment tumor size or volume or cancer load.
  • the applied dose of both agents can be adjusted based on the relative bioavailability and potency of the administered compounds, including the adjuvants used. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods are well within the capabilities of the ordinarily skilled artisan.
  • Subject doses of the compounds described herein will depend on the particular compound although they typically range from about 0.1 ⁇ g to 10,000 mg per day, more typically from about 50 ⁇ g to 5000 mg per day, even more typically from about 100 ⁇ g to 2000 mg per day, and most typically from about 200 ⁇ g to 1000 mg per day.
  • the compound of Formula I is administered in amounts of less than or equal to 1.0 mg/kg per day. This includes amounts equal to or less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 and 0.1 mg/kg per day.
  • the agents may also be administered in amounts of equal to or less than 0.1 mg/kg per day. This includes amounts equal to or less than 0.09, 0.08, 0.07, 0.06, 0.5, 0.04, 0.03, 0.02 or 0.01 mg/kg/day.
  • the agents are administered in a range of about 0.005 mg/kg per day to less than 1.0 mg/kg per day (or about 0.005 mg/kg per day to equal to or less than 0.1 mg/kg per day).
  • timing of the administration of the compound of Formula I and pemetrexed, erlotinib or docetaxel may be particularly important.
  • the agents may be administered to the subject on a routine schedule.
  • routine schedule refers to a predetermined designated period of time.
  • the routine schedule may encompass periods of time which are identical or which differ in length, as long as the schedule is predetermined.
  • the routine schedule may involve administration on a daily basis, every two days, every three days, every four days, every five days, every six days, a weekly basis, a monthly basis or any set number of days or weeks there-between, every two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, etc.
  • the predetermined routine schedule may involve administration on a daily basis for the first week, followed by a monthly basis for several months, and then every three months after that. Any particular combination would be covered by the routine schedule as long as it is determined ahead of time that the appropriate schedule involves administration on a certain day.
  • the compound of Formula I is administered orally and pemetrexed is administered parenterally. In some embodiments the compound of Formula I is administered prior to pemetrexed. In other embodiments the compound of Formula I is administered after pemetrexed.
  • pemetrexed is administered about 10 minutes to 24 hours prior to the compound of Formula I.
  • pemetrexed is administered 1-10 days prior to the compound of Formula I.
  • the pemetrexed may be administered on a first day of multi-day cycle, with the compound of Formula I administered on the remaining days of the cycle.
  • the cycle may be a 2, 3, 4, 5, 6, 7, or more day cycle.
  • the compound of Formula I may be administered once, twice, or more times per day.
  • pemetrexed is administered on the first day of a seven day cycle, followed by a twice daily administration of the compound of Formula I on each of the remaining days of the seven day cycle.
  • the multi-day cycle may be repeated twice, thrice, four times, or more. It may also be repeated for various lengths of time, including but not limited to a week, a month, two months, or more.
  • the multi-day cycle may be at least 7, at least 14, at least 21, at least 28, at least 35 days or more in length.
  • the agents such as pemetrexed and docetaxel and any platinum-containing compound may be administered at the beginning of such cycles for one or more days (e.g., days 1, 2 and 3), followed by the administration of the compound of Formula I (preferably Val-boroPro) for at least 7, at least 14, or more days, followed by a rest period in which the subject is administered no anti-cancer therapy but may be administered a placebo.
  • the anti-cancer agent is administered substantially simultaneously with the compound of Formula I.
  • substantially simultaneously means that the compounds are administered within minutes of each other (e.g., within 10 minutes of each other) and intends to embrace joint administration as well as consecutive administration, but 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.
  • single or multiple administrations of pemetrexed followed by 7-14 day courses of twice a day (BID) administration of varying doses of a compound of Formula I e.g., Val-boroPro (talabostat)
  • BID twice a day
  • pemetrexed is administered on day 1 of a multi-day cycle (e.g., a 21 day cycle).
  • the compound of Formula I is administered on days 2-15 of the same cycle.
  • pemetrexed is administered on days 1-3 of a multi-day cycle (e.g., a 21, 24, 28 or 31 day cycle).
  • the compound of Formula I is administered on days 4-17 of the same cycle.
  • the remaining days of the cycle may be a rest period (treatment-free period).
  • the cycles described herein may be repeated one, two, three, four, five, six or more times with or without an additional rest period (treatment-free period) in between each cycle.
  • the intervening rest periods may last days or weeks.
  • pemetrexed is administered at a dose of 500 mg/m 2 15-25 days apart. Each administration of pemetrexed is followed by administration of 100-500 ⁇ g of a compound of Formula I (e.g., Val-boroPro (talabostat)) BID orally the following day and for 7-14 consecutive days. This may be followed by a 7-day rest period (treatment-free period), then followed by administration of 100 -500 ⁇ g of a compound of Formula I (e.g., Val- boroPro (talabostat)) BID orally for another 7-14 consecutive days. This cycle may be repeated as necessary. Pemetrexed may be administered at doses ranging from 100 to 800 mg/m 2 .
  • a compound of Formula I e.g., Val-boroPro (talabostat)
  • pemetrexed 400-700 mg/kg is administered for 3 consecutive days followed by 100-500 ⁇ g of a compound of Formula I (e.g., Val-boroPro (talabostat)) for 10-20 consecutive days.
  • 100-500 ⁇ g of a compound of Formula I e.g., Val-boroPro
  • T ARCEV ATM 150 mg erlotinib
  • docetaxel is administered at a dose of 75 mg/m 2 on day 1 of a 22 day cycle and 100-500 ⁇ g of a compound of Formula I (e.g., Val-boroPro (talabostat)) is administered twice daily (BID) on day 2-15 of the 22 day cycle followed by a 7-day rest period (treatment-free period).
  • a compound of Formula I e.g., Val-boroPro (talabostat)
  • BID twice daily
  • the combination therapies may also be followed by repeated multi-day cycles of compound of Formula I optionally with rest periods.
  • the subject may be treated with one, two or more multi-day cycles of compound of Formula I.
  • the latter cycles may include administration of the agent on days 2-15 of 22 day cycle (with a rest period on days 16-22).
  • Administration of the agent may be once or twice a day but it is not so limited.
  • the compounds of the invention may be administered neat, or in the context of a vector or delivery system.
  • An example of a chemical/physical vector of the invention is a colloidal dispersion system.
  • Colloidal dispersion systems include lipid-based systems including oil-in- water emulsions, micelles, mixed micelles, and liposomes.
  • a preferred colloidal system of the invention is a liposome. Liposomes are artificial membrane vessels which are useful as a delivery vector in vivo or in vitro. It has been shown that large unilamellar vessels (LUV), which range in size from 0.2 - 4.0 ⁇ m can encapsulate large macromolecules.
  • LUV large unilamellar vessels
  • Liposomes may be targeted to a particular tissue by coupling the liposome to a specific ligand such as a sugar, glycolipid, or protein.
  • Ligands which may be useful for targeting a liposome to an immune cell include, but are not limited to intact or fragments of molecules which interact with immune cell specific receptors and molecules, such as antibodies, which interact with the cell surface markers of immune cells. Such ligands may easily be identified by binding assays well known to those of skill in the art.
  • the liposome may be targeted to the cancer by coupling it to a one of the immunotherapeutic antibodies discussed earlier.
  • the vector may be coupled to a nuclear targeting peptide, which will direct the vector to the nucleus of the host cell.
  • Lipid formulations for transfection are commercially available from QIAGEN, for example, as EFFECTENETM (a non-liposomal lipid with a special DNA condensing enhancer) and SUPERFECTTM (a novel acting dendrimeric technology).
  • EFFECTENETM a non-liposomal lipid with a special DNA condensing enhancer
  • SUPERFECTTM a novel acting dendrimeric technology
  • Liposomes are commercially available from Gibco BRL, for example, as LIPOFECTINTM and LIPOFECTACETM, which are formed of cationic lipids such as N-[I -(2, 3 dioleyloxy)-propyl]-N, N, N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB).
  • LIPOFECTINTM and LIPOFECTACETM are formed of cationic lipids such as N-[I -(2, 3 dioleyloxy)-propyl]-N, N, N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB).
  • DOTMA N-[I -(2, 3 dioleyloxy)-propyl]-N, N, N-trimethylammonium chloride
  • DDAB dimethyl dioctadecylammonium bromide
  • Non-biodegradable and biodegradable polymeric matrices can be used to deliver the compound of Formula I and/or the anti-cancer agent(s) to the subject.
  • Biodegradable matrices are preferred.
  • Such polymers may be natural or synthetic polymers. 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 multi-valent ions or other polymers.
  • the polymeric matrix preferably is in the form of a microparticle such as a microsphere (wherein the agents are dispersed throughout a solid polymeric matrix) or a microcapsule (wherein the agents are stored in the core of a polymeric shell).
  • Other forms of the polymeric matrix for containing the agents 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 is introduced.
  • the size of the polymeric matrix further is 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 and the compound of Formula I and the pemetrexed, erlotinib, or docetaxel are encompassed in a surfactant vehicle.
  • 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 matrix is administered to a nasal and/or pulmonary surface that has sustained an injury.
  • the matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time.
  • the compounds of Formula I are administered to the subject via an implant while the anti-cancer agent(s) is administered acutely.
  • Bioadhesive polymers of particular interest include bioerodible hydrogels described by
  • polyhyaluronic acids casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), polyQaurel methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).
  • Other delivery vehicles can be used and these include cochleates (Gould-Fogerite et al., 1994, 1996); Emulsomes (Vancott et al., 1998, Lowell et al., 1997); ISCOMs (Mowat et al., 1993, Carlsson et al., 1991, Hu et., 1998, Morein et al., 1999); liposomes (Childers et al., 1999, Michalek et al., 1989, 1992, de Haan 1995a, 1995b); live bacterial vectors (e.g., Salmonella, Escherichia coli, Bacillus calmatte-guerin, Shigella, Lactobacillus) (Hone et al., 1996, Pouwels et al., 1998, Chatfield et al., 1993, Stover et al., 1991, Nugent et al., 1998); live viral vectors (e.g., Vaccinia, adeno
  • compositions and methods of the invention in certain instances may be useful for replacing existing surgical procedures or drug therapies. In some instances the present invention is useful in improving the efficacy of existing therapies for treating such conditions. Accordingly, compositions and methods of the invention may be used to treat the subjects that are undergoing or that will undergo a treatment for cancer.
  • the agents may be administered to a subject in combination with other anti-cancer therapies.
  • Suitable anticancer therapies include surgical procedures to remove the tumor mass, chemotherapy or localized radiation.
  • the other antiproliferative therapy may be administered before, concurrent with, or after treatment with the compositions and methods of the invention.
  • compositions and methods of the invention may be administered before or after the other treatment.
  • the administration of compounds of Formula I induce memory within the immune cell compartment, for example, by the induction of memory T cells, and B cells. This is believed to occur via the cytokine cocktail that is induced by compounds of Formula I, particularly the induction of IL-I .
  • the ability to generate memory T cells can enhance immune responses to, for example, cancerous cells that remain following a surgical procedure, or following chemotherapy or radiation.
  • the invention further contemplates the use of compositions and methods of the invention in cancer subjects prior to and following surgery, radiation or chemotherapy in order to create memory immune cells to the cancer antigen.
  • memory cells of the immune system can be primed with cancer antigens and thereby provide immune surveillance in the long term.
  • This is particularly suited to radiotherapy of subjects where immune cells so primed can invade a tumor site and effectively clear any remaining tumor debris.
  • This in turn promotes further immunity to the cancer, particularly to antigens that might not have been exposed in the context of a tumor mass pre-treatment.
  • the subjects can be treated with compositions and methods of the invention without any other therapy, as well.
  • the methods are particularly directed to subjects at high risk of cancer, such as those predisposed for familial (e.g., familial colon polyposis, BRCAl- or BRCA2- associated breast cancer, Wilms tumor, colorectal cancer, Li-Fraumeni Syndrome, ovarian cancer, and prostate cancer), or non-familial genetic reasons.
  • Subjects at high risk are also those that manifest pre-cancerous symptoms such as pre-cancerous polyps (e.g., in colon cancer), or pre-cancerous lesions (e.g., in HPV-induced cervical cancer).
  • compositions and methods of the invention can also be used in combination with non-surgical antiproliferative (e.g., anti-cancer) drug therapy.
  • the compositions and methods of the invention may be used in combination with an anti-cancer compound such as a cytostatic compound.
  • a cytostatic compound is a compound (e.g., a nucleic acid, a protein) that suppresses cell growth and/or proliferation.
  • the cytostatic compound is directed towards the malignant cells of a tumor.
  • the cytostatic compound is one which inhibits the growth and/or proliferation of vascular smooth muscle cells or fibroblasts.
  • compounds of Formula I and pemetrexed, erlotinib, or docetaxel may be administered prior to, concurrent with, or following other anti- cancer compounds.
  • the administration schedule may involve administering the different agents in an alternating fashion.
  • the combination therapy of the invention may be delivered before and during, or during and after, or before and after treatment with other therapies.
  • the agent is administered more than 24 hours before the administration of the other anti-proliferative treatment.
  • more than one anti-proliferative therapy may be administered to a subject.
  • the subject may receive the agents of the invention, in combination with both surgery and at least one other anti-proliferative compound.
  • the agent may be administered in combination with more than one anti-cancer drug.
  • Cytokines and chemokines can potentially be cleaved and thereby inactivated by post proline cleaving enzymes.
  • Administration of compounds of Formula I with cytokines and/or chemokines can enhance the efficacy of these latter agents by protecting them from degradation.
  • Immune responses can also be induced or augmented by the co-administration or co- linear expression of cytokines or chemokines (Bueler & Mulligan, 1996; Chow et at, 1997; Geissler et «/., 1997; Iwasaki et at, 1997; Kim et at, 1997) or B-7 co-stimulatory molecules (Iwasaki et at, 1997; Tsuji et at, 1997) with the compounds of Formula I.
  • the cytokines and/or chemokines can be administered directly or may be administered in the form of a nucleic acid vector that encodes the cytokine, such that the cytokine can be expressed in vivo.
  • the cytokine or chemokine is administered in the form of a plasmid expression vector.
  • cytokine is used as a generic name for a diverse group of soluble proteins and peptides which act as humoral regulators at nano- to picomolar concentrations and which, either under normal or pathological conditions, modulate the functional activities of individual cells and tissues. These proteins also mediate interactions between cells directly and regulate processes taking place in the extracellular environment. Cytokines also are central in directing the T cell response.
  • cytokines examples include, but are not limited to IL-I, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL- 12, IL- 15, IL- 18, granulocyte- macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G- CSF), interferon- ⁇ (IFN- ⁇ ), IFN- ⁇ , tumor necrosis factor (TNF), TGF- ⁇ , FLT-3 ligand, and CD40 ligand.
  • the cytokine is a ThI cytokine.
  • the cytokine is a Th2 cytokine.
  • chemokine is used as a generic name for peptides or polypeptides that act principally to chemoattract effector cells of both innate and adaptive immunity. Chemokines are thought to coordinate immunological defenses against tumors and infectious agents by concentrating neutrophils, macrophages, eosinophils and T and B lymphocytes at the anatomical site in which the tumor or infectious agent is present. In addition, many chemokines are known to activate the effector cells so that their immune functions (e.g., cytolysis of tumor cells) are enhanced on a per cell basis. Two groups of chemokines are distinguished according to the positions of the first two cysteine residues that are conserved in the amino-terminal portions of the polypeptides.
  • the residues can either be adjacent or separated by one amino acid, thereby defining the CC and CXC cytokines respectively.
  • the activity of each chemokine is restricted to particular effector cells, and this specificity results from a cognate interaction between the chemokine and a specific cell membrane receptor expressed by the effector cells.
  • the CXC chemokines IL-8, Gro ⁇ / ⁇ and ENA 78 act specifically on neutrophils
  • the CC chemokines RANTES, MIP- l ⁇ and MCP-3 act on monocytes and activated T cells.
  • the CXC chemokine IP-IO appears to have anti-angiogenic activity against tumors as well as being a chemoattractant for activated T cells.
  • MIP- l ⁇ also reportedly has effects on hemopoietic precursor cells.
  • the invention relates to kits that are useful in the treatment of cancer.
  • kits of the invention includes a sustained release vehicle containing a compound of Formula I and a container housing pemetrexed, erlotinib, or docetaxel and instructions for timing of administration of the various agents contained therein.
  • a sustained release vehicle is used herein in accordance with its prior art meaning of any device which slowly releases the compound of Formula I.
  • Such systems can avoid repeated administrations of the compounds, 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 polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and poly anhydrides.
  • 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; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di- and tri-glycerides
  • hydrogel release systems such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di- and tri-glycerides
  • sylastic systems such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di- and tri-glycerides
  • peptide based systems such as fatty acids
  • wax coatings such as those described in U.S. Patent Nos.
  • compositions of the invention contain an effective amount of a compound of Formula I and pemetrexed, erlotinib, or docetaxel and/or other therapeutic agents, optionally included 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 vertebrate animal.
  • 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 also are capable of being commingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.
  • the agents may be administered per se (neat) or in the form of a pharmaceutically acceptable salt.
  • the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof.
  • Such 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-sul ⁇ honic, and benzene sulphonic.
  • such salts can be prepared as 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 benzalkoniurn 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).
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection 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. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, 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. Another suitable compound for sustained release delivery is GELFOAM 5 a commercially available product consisting of modified collagen fibers.
  • the active compounds may be 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 pharmaceutical 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 agents can be administered by any ordinary route for administering medications, depending upon the type of cancer to be treated, the compounds of Formula I 5 pemetrexed, erlotinib, or docetaxel may be inhaled, ingested or administered by systemic routes. Systemic routes include oral and parenteral. Inhaled medications are preferred in some embodiments because of the direct delivery to the lung, particularly in lung cancer patients.
  • metered dose inhalers are regularly used for administration by inhalation. These types of devices include metered dose inhalers (MDI), breath-actuated MDI, dry powder inhaler (DPI), spacer/holding chambers in combination with MDI, and nebulizers.
  • MDI metered dose inhalers
  • an effective amount of the compound of Formula I can be administered to a subject by any mode that delivers the compound to the affected organ or tissue, or alternatively to the immune system.
  • administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan. Preferred routes of administration include but are not limited to oral, parenteral, intramuscular, intranasal, intratracheal, inhalation, ocular, vaginal, and rectal.
  • the administration route of the compound of Formula I and the other agents described herein is not limiting on the administration route of the pemetrexed, erlotinib, or docetaxel.
  • the compound of Formula I may be administered in the same route, and in the same formulation as the pemetrexed, erlotinib, or docetaxel, or it may be administered in a different route, different formulation, and even on a different schedule.
  • the compound of Formula I is administered orally, and the pemetrexed, erlotinib, or docetaxel is administered parenterally, preferably by intramuscular or subcutaneous injection, although it is not so limited.
  • the compounds of Formula I are administered orally, preferably by ingestible tablets that enter the gastrointestinal tract.
  • the antigens or antibodies are also administered via the same route.
  • the compounds of Formula I are administered locally, and optionally the pemetrexed, erlotinib, or docetaxel are administered locally as well.
  • the agents can be formulated readily by combining the active compound(s) 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 by a subject to be treated.
  • Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum 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.
  • 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.
  • stabilizers may be added.
  • Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention 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 pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.
  • gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • a powder mix of the compound such as lactose or starch.
  • suitable powder base such as lactose or starch.
  • 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.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • 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.
  • 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.
  • suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin.
  • the pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above.
  • the pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer, Science 249:1527-1533, 1990, which is incorporated herein by reference.
  • kits that comprise the agents of the invention and optionally instructions of use thereof.
  • the agents may be present in oral forms such as tablets, pills, capsules, caplets and the like.
  • the agents may be provided in a one a day dispensing unit such as a blister pack or dial pack type dispenser, preferably with days of the week or day of the month (e.g., 1, 2, 3, 4, etc.) (and doses per day, where applicable) printed on the dispenser. For example, if the agents are to be administered every other day or twice (or more) a day, the dispensing unit can be modified accordingly, with no more than routine reconfiguration, known in the art.
  • the kit may further contain a second agent such as a second anti-cancer agent.
  • kits may optionally comprise a housing such as a box or bag. Instructions for use may be supplied separately from the dispensing unit or housing or they may be imprinted on one or both.
  • This study investigated the interaction between talabostat and pemetrexed (ALIMTA ® ) in the A549 NSCLC xenograft model.
  • Single or multiple administrations of pemetrexed to mice inoculated subcutaneously (s.c.) with the A549 cell line were followed by 14-day courses of twice a day (BID) administration of varying doses of talabostat.
  • BID twice a day
  • the effects of pemetrexed and talabostat combination treatment were compared with vehicle treatment and monotherapy with each agent.
  • Talabostat mesylate is the methane sulphonate salt of boronic acid, [(2R)-l-[(2S)-2-amino-3-methyl-l-oxobutyl]-2-pyrrolidinyl]-.
  • the material used in this study was synthesized by Evotec OAI (Abingdon, Oxon, UK; batch #51218-06).
  • Pemetrexed pemetrexed disodium heptahydrate
  • ALTA ® has the chemical name L- Glutamic acid, N-[4-[2-(2-amino-4,7-dihydro-4-oxo-lH-pyrrolo[2,3-d]pyrimidin-5 ⁇ yl)ethyl]benzoyl]-, disodium salt, heptahydrate.
  • the molecular formula is C 20 H 19 N 5 Na 2 O 6 7H 2 O.
  • Pemetrexed an antifolate anti-cancer agent, has a mechanism of action involving the disruption of folate-dependent metabolic processes required for cell replication. Pemetrexed for injection was purchased from Eli Lilly and Co. (Indianapolis, IN). Normal saline, referred to as saline, was 0.9% (weight/volume) sodium chloride (Sigma, St Louis, MO) in water.
  • C.129S6 (B6)-Rag2 tmFwa mice referred to as BALB/c Rag2 ⁇ / ⁇ mice, were purchased from Taconic (Germantown, NY).
  • the A549 NSCLC cell line, CCL- 185,TM American Tissue Type Culture Collection [ATCC], Manassas, VA) was propagated in vitro at 37°C in an atmosphere of 95% air and 5% carbon dioxide by serial culture in Ham's F12K medium with 2mM L-glutamine, adjusted to contain 1.5g/L sodium bicarbonate and 10% fetal bovine serum. The subculture procedure was that described by ATCC.
  • Assays of Talabostat and Pemetrexed Antitumor Activity in Vivo :
  • mice were injected s.c. with 5 x 10 6 A549 tumor cells in a shaved flank as follows.
  • Pemetrexed was administered (doses are described below) by intraperitoneal injection of saline-diluted solutions.
  • Talabostat 5, 10, or 20 ⁇ g saline solutions, or saline alone, were administered by gavage BID at approximately 8h intervals.
  • the time of tumor inoculation was defined as Day 0, and pemetrexed and talabostat were administered according to the regimens described below.
  • mice contained 5 replicate mice for dose- ranging studies of pemetrexed, and 10 replicate mice in studies comparing the antitumor effects of pemetrexed and talabostat as monotherapies as well as in combination.
  • Tumor volumes (c.c.) are reported as values rounded to 2 decimal places. P-values are rounded to a sufficient number of decimal place to depict significance as values greater or smaller than 0.05, representing the 95% confidence limit.
  • A549 NSCLC xenografts to pemetrexed was investigated in vivo to select doses of pemetrexed that could be studied in combination with talabostat.
  • BALB/c Rag 2 ⁇ ' mice were inoculated s.c. with A549 cells and 7 days later administered a single injection of pemetrexed at doses ranging from 25 to 750 mg/kg.
  • pemetrexed 600 and 750 mg/kg appeared to slow tumor growth (FIG. 1), but the effect was not consistently significant (P ⁇ 0.05) at all timepoints.
  • BALB/c Rag2 'A mice were inoculated s.c. with A549 cells and on Days 7 and 29 were administered pemetrexed at either 750 or 600 mg/kg. The administration of pemetrexed on each day was followed by administration of talabostat or saline BID by gavage from Days 8 through 21 and Days 30 through 43. Pemetrexed, talabostat, and saline were also administered as single agents according to the same regimens.
  • the antitumor effect of the 500 mg/kg dose was also significant (P ⁇ 0.05) at all the later timepoints; therefore this dose was selected for study in combination with talabostat using a regimen of pemetrexed administration on 3 consecutive days followed by talabostat for 14 consecutive days.
  • BALB/c Rag2 ' ⁇ mice were inoculated s.c. with A549 cells and administered pemetrexed 500 mg/kg on each of Days 7, 8, 9, 31, 32, and 33 (see FIG. 6 for schedule and frequencies of administration).
  • Saline or talabostat (5 ⁇ g or 10 ⁇ g) was administered BID on Days 10 to 23 and on Days 34 to 47.
  • Pemetrexed, talabostat, and saline were also administered as single agents according to the same regimens.
  • A549 tumors grew to a mean volume of 2.5 c.c. on Day 50.
  • Tumors in mice receiving pemetrexed monotherapy FIG.
  • mice In BALB/c Rag 2 ' ⁇ mice, the antitumor effect of pemetrexed was much less than that previously observed with docetaxel. Pemetrexed is a multitargeted antifolate. Inhibition of de novo nucleotide biosynthesis can be circumvented by the salvage of extracellular preformed nucleosides. Compared with humans, mice have relatively high levels of circulating thymidine, and nucleoside salvage could have been responsible for the relatively weak activity of pemetrexed observed in the A549 NSCLC xenograft model.
  • talabostat As previously described, monotherapy with talabostat is highly effective in inhibiting growth of A549 tumor xenografts. It is important to note that in all pemetrexed/talabostat regimens investigated in this study, talabostat' s activity did not appear to be diminished by prior pemetrexed treatment.
  • Protocol A A549 Human NSCLC; Pemetrexed 1, Chemotherapy Alone, Dose- Titration.
  • Rodent Strain Mice (BALB/c Rag2 '/" ), Taconic Farms, females, 6-8 weeks of age,
  • Day 0 Inject 5xlO 6 A549 cells s.c. into shaven flank of each mouse, in 0.2 ml/mouse of D- PBS.
  • Day 7 Administer pemetrexed as a single i.p. injection in a 0.3 to 0.5 ml/mouse volume. Measure tumors: Days 7, 1 1, 15, 20, 25, 30, 35 etc. Pemetrexed Administration: Inject @ 0.6 ml/mouse, i.p.
  • Stock solution Resuspend 1 vial of 500 mg in 20 ml of saline to give a stock of 25 mg/ml, as per package instructions.
  • Protocol B A549 Human NSCLC; Actual Combination Therapy Assay Combining Talabostat (Day 8 Start) with Sub-Maximal Doses of Pemetrexed [i.p. injection, 1 dose/cycle].
  • Day 0 Inject A549 s.c. @ 5x10 6 / mouse in 0.2 ml saline/ mouse.
  • Day 7 Inject ALIMTA ® i.p. in 0.6 ml or less per mouse, as a single injection per cycle.
  • Day 8 Begin talabostat treatment, dosing @ 0.2 ml/ dose/ mouse; p.o., BID in saline. Measure tumors: days 7, 11, 15, 20, 25, 30, 35, 40, etc.
  • Pemetrexed Administration Stock solution Resuspend 2 vials of 500 mg in 20 ml of saline each to give stocks of 25 mg/ml (as per package inserts).
  • Day 0 Inject 5x10 6 A549 cells s.c. into the shaven flank of each mouse, in 0.2 ml/ mouse of D-PBS.
  • Day 7 Administer the first dose of ALIMTA ® as an i.p. injection @ 0.3 to 0.5 ml/ mouse.
  • Protocol D A549 Human NSCLC; Pemetrexed 3: Actual Combination Therapy Assay Combining Talabostat with an Effective Dose of Pemetrexed [i.p. injection, 3 consecutive doses per cycle].
  • the dose of pemetrexed was chosen based on results from the second titration assay, in which various doses were given as i.p. injections on 3 consecutive days: Days 7, 8, and 9.
  • Day 10 Begin talabostat treatment, dosing @ 0.2 ml/dose/mouse; p.o., BID in saline. Measure tumors: Days 7, 11, 15, 20, 25, 30, 35, 40, 45, 50, 54 END.
  • Stock solution Resuspend 1 vial of 500 mg in 20 ml saline to give a stock of 25 mg/ml (as per package inserts).
  • Example 2 BALB/c Rag2 'A mice were injected subcutaneously on one flank with 5 x 10 6 A549 human non-small cell lung cancer (NSCLC) cells.
  • NSCLC human non-small cell lung cancer
  • oral administration of a dose of 5 ⁇ g talabostat twice daily (b.i.d.) and 25 or 50 mg/kg erlotinib (TARCEVATM) once per day was started.
  • Oral administration of both agents was continued on consecutive days from day 7 to day 30.
  • Mice received no treatment from days 31 to 37 and then both agents were again administered on days 38 and 39 (see FIG. 9 for schedule and frequencies of administration). Tumor growth in experimental groups of 10 replicate mice was monitored by measurement with vernier calipers.
  • mice BALB/c Rag2 '/" mice were injected subcutaneously on one flank with 5 x 10 6 A549 human non-small cell lung cancer (NSCLC) cells.
  • NSCLC human non-small cell lung cancer
  • Docetaxel 20 mg/kg was administered on each of Days 7, 29, and 51.
  • Saline or talabostat 5 ⁇ g was administered BID on Days 8 to 21, Days 30 to 43, and Days 52 to 65.
  • the mice received no treatments on Days 22 to 28, Days 44 to 50, and Days 66 to 72 (rest days). See FIG. 12 for schedule and frequencies of administration.
  • Tumor growth in experimental groups of 10 or 20 replicate mice was monitored by measurement with vernier calipers. Data are presented as mean tumor volumes + SE, and significance was determined by Student's 2 -tailed t test (FIG 13).
  • talabostat can promote a T- cell independent anti-tumor response that interacts cooperatively with the antitumor agents that have very different mechanisms of action such as pemetrexed that disrupts folate- dependent metabolism required for cell replication, erlotinib which is a HERl /EGFR tyrosine kinase inhibitor, and docetaxel which disrupts the microtubular network essential for mitosis.
  • Talabostat is an orally administered inhibitor of dipeptidyl peptidases such as DPP-IV (also known as CD26) and fibroblast activation protein (FAP) found on the stroma of epithelial tumors such as non-small cell lung cancer (NSCLC).
  • DPP-IV also known as CD26
  • FAP fibroblast activation protein
  • NSCLC non-small cell lung cancer
  • Administration of talabostat stimulates cytokine and chemokine production in humans and animals resulting in specific T- cell immunity and T-cell independent activity.
  • talabostat significantly enhanced the activity of docetaxel in the A549 model of NSCLC .
  • Stage IIIB/IV NSCLC Stage IIIB with cytologically documented pleural effusion
  • Serum cytokines (IL- l ⁇ , IL-I ⁇ , IL-2, IL-6, and IL-8) were obtained prior to talabostat administration on Study Day 3 of Cycle 1 and at 1, 2, 4, and 6 hours after the morning dose of talabostat.
  • FIGs.14A and 14B The Kaplan-Meier estimates for median PFS and survival for all patients enrolled are shown in FIGs.14A and 14B respectively. In cases where patients were lost to follow-up, data were censored at the last observation. If a new cancer treatment was initiated due to PD, data were censored at the point the new treatment started.
  • the Kaplan-Meier estimate of median PFS for all patients enrolled is 4.2 months.
  • the Kaplan-Meier estimate of median survival is 8.4 months.
  • an agent that binds IL-6 or inhibits the activity of IL-6 may help reduce or prevent the adverse events of edema/peripheral swelling, hypotension or dehydration/hypovolemia.

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Abstract

The present invention relates to methods and compositions for treating a subject having cancer comprising administering an effective amount of pemetrexed and a compound of formula (I): PR, wherein P is a targeting group which binds to the reactive site of a post proline-cleaving enzyme, and wherein R is a reactive group which reacts with thre reactive site of a post proline-cleaving enzyme. Particularly preferred compound of Formula (I) are boroproline compounds, such as Ile-boroPro and Val-boroPro.

Description

BQROPROLINE COMBINATIONTHERAPYFORCANCER
Related Applications
This application claims priority to U.S. Provisional Application Serial No. 60/736918, filed November 14, 2005 entitled "BOROPROLINE COMBINATION THERAPY FOR CANCER", the entire contents of which are incorporated by reference herein.
Field of the Invention
This invention relates to methods and compositions for the treatment and prevention of cancer.
Background of the Invention
Cancer is the second leading cause of death, resulting in one out of every four deaths, in the United States. In 1997, the estimated total number of new diagnoses for lung, breast, prostate, colorectal and ovarian cancer was approximately two million. Due to the ever increasing aging population in the United States, it is reasonable to expect that rates of cancer incidence will continue to grow.
Cancer is currently treated using a variety of modalities including surgery, radiation therapy and chemotherapy. The choice of treatment modality will depend upon the type, location and dissemination of the cancer. One of the advantages of surgery and radiation therapy is the ability to control to some extent the impact of the therapy, and thus to limit the toxicity to normal tissues in the body. Chemotherapy is arguably the most appropriate treatment for disseminated cancers such as leukemia and lymphoma as well as metastases. Chemotherapy is generally administered systemically and thus toxicity to normal tissues is a major concern. Not all tumors, however, respond to chemotherapeutic agents and others, although initially responsive to chemotherapeutic agents, may develop resistance. As a result, the search for anti-cancer treatments has intensified in an effort to find more effective treatments with less non-specific toxicity.
Summary of the Invention The invention provides compositions and methods of use in the prevention and treatment of cancer. The invention is based, in part, on the finding that Val-boroPro interacts and thereby enhances the activity of certain anti-cancer agents that have different mechanisms of action in the treatment of cancer. In some instances, the surprising enhancement is associated with the administration of certain doses of either agent and/or the use of certain administration regimens. For example, the combination of Val-boroPro with pemetrexed (ALIMTA®) acted synergistically to treat non-small cell lung cancer (NSCLC). The anticancer effect of the combination of Val-boroPro with pemetrexed was greater than expected based on the response observed when the agents were used alone. The finding was surprising because of the magnitude of the response and also because Val-boroPro exerts its anti-cancer activity via a different mechanism of action than pemetrexed. Val-boroPro is a dipeptidyl peptidase IV (DPP-IV or DP-IV) inhibitor while pemetrexed is an anti-folate antimetabolite. Although not intending to be bound by any specific theory or mechanism, it is believed that the DPP-IV inhibitor Val-boroPro (also referred to as talabostat or PT- 100) acts, in part, by stimulating a variety of cytokines which can stimulate the immune system to combat cancer as well as having a direct anti-cancer effect. The ability of Val-boroPro to stimulate cytokine and chemokine production endogenously is beneficial since exogenous administration of some of these factors, such as for example IL-I, has sometimes been associated with toxicity. Production of IL-I endogenously thereby overcomes these toxicity problems. Val-boroPro also exerts a direct anti-tumor effect by inhibiting the enzymatic activity of fibroblast activation protein (FAP), a dipeptidyl peptidase (DPP) that has the potential to promote tumor growth.
Also without intending to be bound by any specific theory or mechanism, it is believed that pemetrexed, an anti-folate antimetabolite, disrupts folate-dependent metabolism which is required for cell replication.
Thus, the invention relates, in part, to methods of treating cancer in a subject comprising administering a DPP-IV inhibitor represented by a compound (or an agent, as used interchangeably herein) of Formula I and an anti-metabolite preferably using particular dosing and timing regimens that are unexpectedly better than other regimens. The invention also relates to compositions and kits comprising compounds of Formula I and an antimetabolite and uses thereof in the treatment of cancer.
According to one aspect of the invention, a method is provided for treating a subject having cancer. The method comprises administering to a subject in need thereof an effective amount of pemetrexed (ALIMTA®) and a compound of Formula I:
PR wherein P is a targeting group which binds to the reactive site of a post proline- cleaving enzyme, and wherein R is a reactive group which reacts with the reactive site of a post proline cleaving enzyme.
In some embodiments the cancer is a refractory cancer. In some important embodiments, the cancer is refractory to platinum-based therapy, such as but not limited to cisplatin or carboplatin. In other embodiments, the cancer is refractory to a platinum-based therapy and a taxane (e.g., paclitaxel or docetaxel) or a nucleoside analogue (e.g., gemcitabine).
In some embodiments the subject is further treated with surgery and radiation. The compound of Formula I and pemetrexed may be administered before, during, and/or after treating the subject with surgery or radiation. The compound of Formula I and pemetrexed may be administered before and after, before and during, or during and after treating the subject with surgery or radiation.
In some embodiments, the compound of Formula I is administered via a route of administration different from that of pemetrexed. The compound of Formula I may be administered orally. Pemetrexed may be administered parenterally including intravenously.
The compound of Formula I may be administered to the subject before, during and/or after pemetrexed administration. In some embodiments the compound of Formula I is administered to the subject about 24 hours after the administration of pemetrexed. In some embodiments pemetrexed is administered on one or more days of a multi-day or multi-week cycle (e.g., a 3 week cycle). Preferably, pemetrexed is administered at the beginning of the cycle (e.g., on days 1, 2, and/or 3 of a three week cycle). In some embodiments, the compound of Formula I is administered on one multiple days of the same multi-day or multi-week cycle (e.g., at least 7 days, at least 10 days, or at least 14 days). In one embodiment, pemetrexed is administered on day 1 of a 21 day cycle, followed by administration of the compound of Formula I on days 2-15 of the same cycle, followed by a rest period on days 16-21 of the same cycle. The rest period is a "treatment-free period" during which time the subject may be administered placebo or nothing at all. The cycle may be repeated one, two, three, four, five, six, or more times. In some other embodiments pemetrexed is administered on days 1, 2 and 3 of a multi- day cycle (e.g., a 24 day cycle). The compound of Formula I may be administered on days 4- 17 of the same 24 day cycle. Days 18-24 of the 24 day cycle may be a rest period (treatment- free period). In some important embodiments pemetrexed is administered intravenously over a period of about 10 minutes.
Pemetrexed and/or the compound of Formula I may be administered at a therapeutic dose or a sub-therapeutic dose. A therapeutic dose of pemetrexed is about 500 mg/m2 per day. A sub-therapeutic dose is a dose that is less than 500 mg/m2 per day, including 50 mg/m2 per day to less than 500 mg/m2 per day, 100 mg/m2 per day to 450 mg/m2 per day, 200 to 400 mg/m2 per day, and 300 mg/m2 per day to 400 mg/m2 per day.
A therapeutic dose of the compound of Formula I such as for example Val-boroPro is 200-600 μg per day, In some instances the compound is administered in doses of 200 or 300 μg twice a day, in which case the sub-therapeutic dose is less than 400 μg per day. In other instances the compound is administered in the same doses but only once a day in which case the sub-therapeutic dose is less than 200 μg per day.
In some embodiments, the effective amount of pemetrexed and/or the compound of Formula I is less than 90%, less than 80%, less than 75%, less than 60%, less than 50%, about 50%, less than 40%, less than 30%, less than 20% or less than 10% of the effective amount of the compound when administered as a single agent.
The cancer may be a tumor. In some embodiments, the size or volume of the tumor is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% from pretreatment size or volume (i.e., size prior to treatment according to the invention with pemetrexed and the compound of Formula
I)-
The method may further comprise administering a platinum-containing compound such as cisplatin or carboplatin, or gemcitabine, or erlotinib, or a taxane such as docetaxel to the subject, possibly before or during administration of the compound of Formula I and/or pemetrexed.
According to another aspect of the invention, a composition is provided. The composition comprises a compound of Formula I and pemetrexed in an effective amount to reduce tumor size or volume by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% from pretreatment size or volume. The composition may further comprise a pharmaceutically acceptable carrier.
The invention is further premised, in part, on the unexpected finding that Val-boroPro enhances the activity of erlotinib (T ARCEV A™) when particular dosing and timing regimens are used. Thus, according to another aspect of the invention, a method is provided for treating a subject having cancer comprising administering to a subject in need thereof erlotinib and a compound of Formula I:
PR wherein P is a targeting group which binds to the reactive site of a post proline- cleaving enzyme, and wherein R is a reactive group which reacts with the reactive site of a post proline cleaving enzyme, in an effective amount to reduce the cancer load by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% from pretreatment size or volume compared to pretreatment load.
According to yet another aspect of the invention, a method is provided for treating a subject having cancer comprising administering to a subject in need thereof an effective amount of erlotinib and a compound of Formula I:
PR wherein P is a targeting group which binds to the reactive site of a post proline- cleaving enzyme, and wherein R is a reactive group which reacts with the reactive site of a post proline cleaving enzyme, and wherein the effective amount of erlotinib and/or the compound of Formula I is less than 90%, less than 80%, less than 75%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20% or less than 10% of the effective amount of erlotinib and/or the compound of Formula I when either is administered as a single agent. According to still another aspect of the invention, a method is provided for treating a subject having cancer comprising administering to a subject in need thereof an effective amount of erlotinib and a compound of Formula I:
PR wherein P is a targeting group which binds to the reactive site of a post proline- cleaving enzyme, and wherein R is a reactive group which reacts with the reactive site of a post proline cleaving enzyme, and wherein the erlotinib is administered once daily and the compound of Formula I is administered twice daily. Erlotinib and the compound may be administered on the same days of a multi-day cycle (e.g., a 21 day cycle). Administration of erlotinib and the compound of Formula I may be followed by a rest period of 1-7 days, after which the cycle may be repeated one, two, three, four, five, six, or more times.
The cancer may be a refractory cancer. In some embodiments the cancer is refractory to a platinum-containing agent such as cisplatin or carboplatin, and/or to gemcitabine. The compound of Formula I and erlotinib may both be administered orally. Erlotinib and/or the compound of Formula I may be administered at a therapeutic dose or a sub-therapeutic dose. A therapeutic dose of erlotinib is about 150 mg per day. A subtherapeutic dose is a dose that is less than 150 mg per day, including 25 mg per day to less than 150 mg per day, 50 mg per day to 125 mg per day, and 75 mg per day to 100 mg per day. Therapeutic doses and sub-therapeutic doses of the compound of Formula I such as for example Val-boroPro are as described above.
In some embodiments, the effective amount of erlotinib and/or the compound of Formula I is less than 90%, less than 80%, less than 75%, less than 60%, less than 50%, about 50%, less than 40%, less than 30%, less than 20% or less than 10% of the effective amount of the compound when administered as a single agent.
In some embodiments the method further comprises administering pemetrexed, a platinum-containing compound such as cisplatin or carboplatin, or gemcitabine, or a taxane such as docetaxel to the subject.
According to another aspect of the invention, a composition is provided. The composition comprises a compound of Formula I and erlotinib in an effective amount to reduce the cancer load by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% from pretreatment cancer load. The composition may further comprise a pharmaceutically acceptable carrier.
The invention is further premised, in part, on the finding that particular treatment regimens for the combination of Formula I agents and a taxane such as docetaxel
(TAXOTERE®) are particularly and unexpectedly effective in the treatment of certain cancers. Thus, according to another aspect of the invention, a method is provided for treating cancer in a subject comprising administering a compound of Formula I and docetaxel in an effective amount to reduce the cancer load by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% from pretreatment cancer load.
According to another aspect of the invention, a composition is provided. The composition comprises a compound of Formula I and docetaxel in an effective amount to reduce cancer load by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% from pretreatment cancer load. The composition may further comprise a pharmaceutically acceptable carrier. According to another aspect of the invention, a method is provided for treating a subject having cancer. The method comprises administering to a subject in need thereof an effective amount of docetaxel and a compound of Formula I:
PR wherein P is a targeting group which binds to the reactive site of a post proline- cleaving enzyme, and wherein R is a reactive group which reacts with the reactive site of a post proline cleaving enzyme and wherein docetaxel is administered on day 1 of a multi-day or multi-week cycle (e.g., a 21 or 22-day cycle), and the compound of Formula I is administered on multiple days (e.g., at least 7, at least 10, at least 14 days) of the same cycle. In one embodiment, the compound of Formula I is administered on days 2-15 of a 21 or 22 day cycle. The cycle may be repeated one, two, three, four, five, six or more times.
Docetaxel and/or the compound of Formula I may be administered at a therapeutic dose or a sub-therapeutic dose. A therapeutic dose of docetaxel is about 75 mg/m2per day. A sub-therapeutic dose is a dose that is less than 75.mg/m2 per day, including 25 mg/m2 per day to less than 75 mg/m2 per day, 25 mg/m2 per day to 70 mg/m2 per day, and 40 mg/m2 per day to 50 mg/m2 per day.
Therapeutic doses and sub-therapeutic doses of the compound of Formula I such as for example Val-boroPro are as described above.
In some embodiments, the effective amount of docetaxel and/or the compound of Formula I is less than 90%, less than 80%, less than 75%, less than 60%, less than 50%, about 50%, less than 40%, less than 30%, less than 20% or less than 10% of the effective amount of the compound when administered as a single agent.
In some embodiments the subject has been previously treated with a regimen comprising a platinum-containing compound such as cisplatin or carboplatin, and optionally an antimetabolite or a taxane. In some important embodiments the antimetabolite is gemcitabine. In some important embodiments the taxane is docetaxel. Thus, these aspects of the invention are further premised on the ability of the compound of Formula I to enhance a subject's response to docetaxel since the method embraces the treatment of subjects that were previously refractory to docetaxel. The subject may have been previously treated with a prior regimen for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 13 months, at least 14 months, at least 15 months, at least 16 months, at least 17 months, at least 18 months, at least 19 months, at least 20 months, at least 21 months, at least 22 months, at least 23 months, at least 24 months, at least 25 months, or more prior to treatment with docetaxel and the compound of Formula I. In some embodiments, the subject is treated with carboplatin and docetaxel about 5 months prior to treatment according to the invention. In some embodiments, the subject is treated with carboplatin and gemcitabine 1-4 months prior to treatment according to the invention.
In some embodiments, the subject has adenocarcinoma while in others the subject has squamous cell carcinoma.
According to another aspect of the invention, a method is provided for treating a subject having cancer. The method comprises administering to a subject in need thereof an effective amount of docetaxel and a compound of Formula I:
PR wherein P is a targeting group which binds to the reactive site of a post proline- cleaving enzyme, and wherein R is a reactive group which reacts with the reactive site of a post proline cleaving enzyme, and wherein the subject has been previously administered a regimen comprising a platinum-containing compound and optionally an antimetabolite or a taxane.
In some important embodiments the antimetabolite is gemcitabine. In some important embodiments the taxane is docetaxel. The platinum-containing compound is carboplatin or cisplatin. The subject preferably has been administered the platinum-containing compound and the anti-metabolite or the taxane within 5, 4, 3, 2, or 1 month of treatment according to the invention.
According to yet another aspect of the invention, a kit is provided that comprises a first container comprising a compound of Formula I and a second container comprising pemetrexed.
The compound of Formula I may be formulated for oral administration. The compound of Formula I may be formulated as a tablet, pill, capsule or caplet. Pemetrexed may be formulated for parenteral administration such as intravenous or intramuscular administration. Pemetrexed may be provided in a vial suitable for liquid reconstitution. Pemetrexed and/or the compound of Formula I may be provided in therapeutic doses or sub-therapeutic doses, as described above. For example, the compound of Formula I may be provided in a unit dosage ranging from 100 μg to 500 μg, preferably 200 μg to 400 μg, and more preferably 200 μg to 300 μg. In some embodiments the compound of Formula I is in a unit dosage of about 200 or 300 micrograms. Pemetrexed may be provided in a unit dosage of 200 mg to 1000 mg, 300 mg to 900 mg, 400 mg to 800 mg, 500 mg to 600 mg, or about 700 mg. In some embodiments, pemetrexed is provided in a unit dosage of about 850 mg.
In some embodiments, the kit may further comprise a daily dispenser. The day of the month may be printed on the daily dispenser.
The first container may be a blister package having day indicia and time indicia. There may be two time indicia for every day indicium.
In some embodiments the kit includes a 1, 2 or 3 day supply of pemetrexed, a 7, 10 or 14 day supply of a compound of Formula I, and optionally a 1-7 day supply of pills containing no medication (i.e., placebo). In other embodiments the kit includes a 3-day supply of pemetrexed, a 14-day supply of a compound of Formula I, and a 7-day supply of pills containing no medication.
The kit may further comprise instructions of use. In some embodiments the instructions of use comprise instructing a subject or a health care provider to administer pemetrexed on day 1, 2 and/or 3 of a multi-day cycle (e.g., a 21 or 22 day cycle), the compound of Formula I on at least 7, at least 10, or at least 14 days of the same cycle (e.g., days 2-15 of a 21 or 22 day cycle), and the pills containing no medication on 1-7 days of the same cycle (e.g., days 16-21 or days 16-22 of the 21 or 22 day cycle respectively). In other embodiments, the instructions of use comprise instructing a subject or a health care provider to administer pemetrexed on days 1-3 of a 24 day cycle, the compound of Formula I on days 4-17 of the 24 day cycle, and the pills containing no medication on days 18-24 of the 24 day cycle. In some important embodiments the instructions of use instruct the subject or health care provider to administer pemetrexed parenterally (e.g., intravenously) over a period of about 10 minutes. According to still another aspect of the invention, a kit is provided that comprises a first container comprising a compound of Formula I and a second container comprising erlotinib, or alternatively comprises a multiple physically separate and divided regions which each comprise the compound of Formula I and erlotinib.
The compound of Formula I and erlotinib may be formulated for oral administration, and thus both may be provided as tablets, pills, capsules or caplets.
The compound of Formula I and/or erlotinib may be provided in therapeutic or subtherapeutic unit doses, as described above. The compound of Formula I may be provided in unit dosages as recited above for the pemetrexed kit. Thus, in some embodiments the compound of Formula I is in a unit dosage of about 100, 200, 300, 400 or 500 micrograms. Erlotinib may be provided in a unit dosage of 10-150 mg, 25-125 mg, 50-100 mg, and 75-100 mg, including preferably about 25 mg, 50 mg or 150 mg.
In some embodiments, the kit may further comprise a daily dispenser. The day of the month may be printed on the daily dispenser.
The first container may be a blister package having day indicia and time indicia and optionally two time indicia for every day indicium. The second container may be a blister package having day indicia and optionally time indicia.
In some embodiments the kit includes a multi-day (e.g., at least 7, at least 10, at least 14, at least 21, at least 28 days, or at least 31 days) supply of erlotinib, a multi-day (e.g., at least 7, at least 10, at least 14, at least 21, or at least 28 days, or at least 31 days) supply of a compound of Formula I, and a multi-day (e.g., 1-7 day) supply of pills containing no medication.
The kit may further comprise instructions for use. In some embodiments the instructions of use comprise instructing a subject or a health care provider to administer erlotinib on multiple days of a multi-day cycle, preferably once a day, the compound of Formula I on the same days as erlotinib but preferably twice a day, and the pills containing no medication on the remaining days of the cycle. As an example, the instructions may describe administration of erlotinib on days 1-24 of a 31 day cycle, the compound of Formula I on days 1 -24 of the 31 day cycle, and the pills containing no medication on days 25-31 of the 31 day cycle.
The invention provides yet another kit that comprises a compound of Formula I and docetaxel. The kit may comprise one container comprising docetaxel, preferably in a vial suitable for liquid reconstitution, and the compound of Formula I in an oral formulation (e.g., a tablet). Each kit may proyide only one dose of docetaxel and multiple days (e.g., at least 7, at least 10, at least 14, or more days) dose of the compound of Formula I, and optionally multiple days (e.g., 1-7 days) dose of placebo. In some embodiments, the kits contain doses sufficient for one, two, three, four, five, six or more cycles. The kit may further comprise one or more unit dosages for a carboplatin-containing compound such as cisplatin or carboplatin. The following embodiments apply equally to the various aspects of the invention set forth herein unless indicated otherwise.
In some embodiments the compound of Formula I is a compound of Formula II:
Figure imgf000013_0001
wherein m is an integer between 0 and 100, inclusive; A and A1 are L- or D- naturally or non- naturally occurring amino acid or a peptide or peptidomimetic such that when A is an amino acid residue and m > 1 each A in Am may be a different amino acid residue from every other A in Am and when A is a peptide or peptidomimetic m is 1; the C bonded to B is preferably in the L- or R-confϊguration; and each X1 and X2 is, independently, a hydroxyl group or a group capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH. In some embodiments the compound of Formula I is a compound of Formula III:
Figure imgf000013_0002
wherein m is an integer between 0 and 100, inclusive; A and A1 are L- or D- naturally occurring or non-naturally occurring amino acid residues such that when m>l, A in each repeating bracketed unit is independently selected; the C bonded to B is in the L- or Reconfiguration; and each X1 and X2 is, independently, a hydroxyl group or a group capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH.
In one important embodiment the compound of Formula I is Val-boroPro. In some embodiments the compound of Formula I is Ile-boroPro.
The compound of Formula I may be provided as a mixture of linear and cyclic forms.
In some embodiments the cancer is a carcinoma. In one important embodiment the cancer is non-small cell lung cancer (NSCLC), and in related embodiments, the cancer is adenocarcinoma or squamous cell carcinoma.
In some embodiments the cancer is a tumor. The cancer may be a metastasis.
The subject may or may not have undergone an anti-cancer therapy with surgery, and/or radiation and/or chemotherapy. In some embodiments the compound of Formula I is administered at a dose of about 50 - 1000 micrograms per day or 0.001 - 0.01 mg/kg/day. The total daily dose may be administered in one, two or more administrations per day.
In some embodiments the method further comprises administering an antigen to the subject. The antigen may be targeted to a tissue or a cell. In some embodiments the antigen is a cancer antigen. Some important cancer antigens include but are not limited to PGP9.5, H/Ley/Leb, B7-DC, and B7-H1.
In some embodiments the method further comprises administering an antibody or an antigen-binding antibody fragment to the subject. Some important antibodies or antibody fragments include but are not limited to anti-a5βl integrin antibody, anti-EGFR antibody, or alpha IR-3. One example of an anti-a5βl integrin antibody is Volocimixab. On example of an anti-EGFR antibody is Nimotuzumab (TheraCIM).
In some embodiments the method further comprises administering an adjuvant to the subject. These and other aspects of the invention will be described in greater detail below.
Throughout this disclosure, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains unless defined otherwise.
Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention.
Brief Description of the Figures
FIG. 1 is a graph of the dose response of A549 NSCLC (non-small cell lung cancer) xenografts to single administrations of pemetrexed (ALIMT A®). BALB/c Ragl1' mice were inoculated s.c. with A549 cells on day 0 and administered the indicated doses of ALIMT A® or saline on day 7. Groups of 5 mice were treated with each dose of ALIMT A®.
FIG. 2 is a graph of anti-tumor effect of pemetrexed (ALIMTA®) administered on days 7 and 29 after tumor inoculation in combination with multiply administered 5 μg doses of talabostat. BALB/c Ragl'' mice were inoculated with A549 tumor cells on day 0 and administered ALIMTA® on days 7 and 29. Talabostat was administered twice daily from days 8-21 and days 30-43. Experimental treatments were saline (o), 5 μg dose of talabostat (•), 750-mg/kg dose of ALIMTA® (Δ), 600 mg/kg dose of ALIMTA® (A), 5 μg talabostat + 750 mg/kg ALIMTA® (□), 5 μg talabostat + 600 mg/kg ALIMTA® (■).
FIG. 3 is a graph of the anti-tumor effect of pemetrexed (ALIMTA®) administered on days 7 and 29 after tumor inoculation in combination with multiply administered 10 μg doses of talabostat. BALB/c Rag2~/~ mice were inoculated with A549 tumor cells and administered ALIMTA® and talabostat as described in FIG. 2. Experimental treatments were: saline (o), 10 μg dose of talabostat (•), 750 mg/kg dose of ALIMTA® (Δ), 600 mg/kg dose of ALIMTA® (A)3 10 μg talabostat + 750 mg/kg ALIMTA® (α), 10 μg talabostat + 600 mg/kg ALIMTA® (■). FIG. 4 is a graph of the anti-tumor effect of pemetrexed (ALIMTA®) administered on days 7 and 29 after tumor inoculation in combination with multiply administered 20 μg doses of talabostat. BALB/c Rag2'A mice were inoculated with A549 tumor cells and administered ALIMTA® and talabostat as described in FIGs. 2 and 3. Experimental treatments were saline (o), 20 μg dose of talabostat (•), 750 mg/kg dose of ALIMTA® (Δ), 600 mg/kg dose of ALIMTA® (A), 20 μg talabostat + 750 mg/kg ALIMTA® (□), 20 μg talabostat + 600 mg/kg ALIMTA® (■).
FIG. 5 is a graph of the dose response of A549 NSCLC xenografts to multiple administrations of pemetrexed (ALIMTA®). BALB/c Rag2'A mice were inoculated s.c. with A549 cells on day 0 and administered the indicated doses of ALIMTA® on days 7, 8 and 9. FIG. 6 is a protocol of the schedule and frequencies of administration of talabostat and pemetrexed (ALIMTA®) as referenced in a part of Example 1.
FIG. 7 is a graph of the anti-tumor effect of pemetrexed (ALIMTA®) administered on days 7, 8, 9, 31, 32, and 33 after tumor inoculation in combination with multiply administered 5 μg doses of talabostat. BALB/c Rag2'A mice were inoculated with A549 tumor cells on day 0, administered ALIMTA® on days 7, 8, 9, 31, 32 and 33. Talabostat was administered twice daily on days 10-23 and days 34-47. Experimental treatments were: saline (o), 5 μg dose of talabostat (•), 500 mg/kg dose of ALIMTA® (A), 5 μg talabostat + 500 mg/kg ALIMTA®
(■)•
FIG. 8 is a graph of the anti-tumor effect of pemetrexed (ALIMTA®) administered on days 7, 8, 9, 31, 32, and 33 after tumor inoculation in combination with multiply administered 10 μg doses of Talabostat. BALB/c Rag2'A mice were inoculated with A549 tumor cells on day 0 and administered ALIMTA® and talabostat as described in FIG. 6. Experimental treatments were saline (o), 10 μg dose of talabostat (•), 500 mg/kg dose of ALIMTA® (A)5 10 μg talabostat + 500 mg/kg ALIMTA® (■).
FIG. 9 is a protocol of the schedule and frequencies of administration of talabostat and erlotinib (TARCEVA™) in Example 2. FIG. 10 shows two graphs showing the cooperative anti-tumor effect of talabostat and erlotinib (TARCEVA™) (25 mg/kg) in A549 NSCLC xenograft model.
FIG. 11 shows two graphs showing the cooperative anti-tumor effect of talabostat and erlotinib (TARCEVA™) (50 mg/kg) in A549 NSCLC xenograft model.
FIG. 12 is a protocol of the schedule and frequencies of administration of talabostat and docetaxel in Example 3.
FIG. 13 shows two graphs showing the anti-tumor effect of talabostat and docetaxel in A549 NSCLC xenograft model.
FIG. 14 shows two graphs showing the Kaplan-Meier estimate of (A) median progression-free survival (PFS) and (B) median survival for all patients enrolled. It is to be understood that the Figures are not required for enablement of the invention.
Detailed Description of the Invention
The invention provides compositions and methods for preventing and treating cancer. The invention is based, in part, on the finding that the DPP-IV inhibitor Val-boroPro (talabostat, PT-100) interacts with certain anti-cancer agents that have different mechanisms of action in the treatment of cancer. In particular, the combination of Val-boroPro with pemetrexed (ALIMTA®), erlotinib (TARCEVA™), or docetaxel (TAXOTERE®) in some aspects of the invention acts unexpectedly synergistically to treat non-small cell lung cancer (NSCLC). The findings were surprising at least because Val-boroPro exerts its anti-tumor activity via a different mechanism of action than pemetrexed, erlotinib, and docetaxel. As used herein and in its broadest sense, a synergistic response is one that is greater than that induced by either of the two or more agents when used alone.
Thus, the invention relates, in part, to methods of treating and/or preventing cancer in a subject comprising administering a compound (or agent, as used interchangeably herein) of Formula I and an antimetabolite, optionally with a human epidermal growth factor receptor type I/epideπnal growth factor receptor (HERl /EGFR) tyrosine kinase inhibitor, a taxane, or a platinum-containing compound. The invention also relates to compositions and kits comprising compounds of Formula I and an antimetabolite, and optionally an HERl /EGFR tyrosine kinase inhibitor, a taxane, or a platinum-containing compound and uses thereof in the treatment of cancer.
In some embodiments, the compound of Formula I and an antimetabolite are administered as a synergistic combination in an effective amount to treat a cancer. As an example, the effect resulting from the combination of the at least two agents may be greater than the additive effect of the individual agents when used alone. Accordingly, when used together either or both agents may be used at lower doses than would be used if either agent was administered alone. In these embodiments, either or both agents may be administered in a "sub-therapeutic" dose and the combination would be therapeutic. In other words, the agents may be administered in reduced doses relative to therapeutically appropriate doses when agents are administered alone. For example, the unit dosage of one or both agents may be reduced by a factor of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100 or more relative to the unit dosage required when a single agent is administered. As shown in the Examples, one agent may be used at a dose which shows no response when used alone, and the compound of Formula I may enhance that effect when used in combination, resulting in a response that is greater than the response of either the agent or the compound of Formula I alone.
Treatment or therapy after a disorder has started aims to reduce, ameliorate or altogether eliminate the disorder and/or its associated symptoms, or prevent it from becoming worse. Treatment of subjects before a disorder has started (i.e., prophylactic treatment) aims to reduce the risk of developing the disorder. As used herein, the term "prevent" refers to the prophylactic treatment of patients who are at risk of developing a disorder (resulting in a decrease in the probability that the subject will develop the disorder) and to the inhibition of further development of an already established disorder.
Val-boroPro and other compounds of Formula I stimulate a variety of cytokines and chemokines which can stimulate the immune system. The resultant immune stimulation can be exploited to enhance the anti-cancer efficacy of other agents. Thus, in one aspect, the invention provides methods that exploit the effect that may be achieved when the compounds of Formula I are used together with an antimetabolite, a HERl /EGFR tyrosine kinase inhibitor, or a taxane. The effect of particular combinations is unexpected. In some instances, the combination provides a synergistic response, resulting for example in greater than the additive effect expected from using the agents singly.
Compounds of Formula I are effective against a number of different cancers and tumors. It has been previously demonstrated that these compounds, when administered to tumor-bearing mice, rapidly stimulate the production of growth factors, cytokines and chemokines (e.g., IL-I, G-CSF, and IL-8 (KC in mice)). These mediators collectively stimulate the proliferation, activation and chemoattraction to the tumor microenvironment of effector cells involved in both adaptive and non-adaptive (innate) immunity and in immune lysis and growth inhibition of tumor cells. The immune and non-immune effector cell populations mobilized and/or activated by compounds of Formula I enhance the anti-cancer effects of antimetabolites, HER1/EGFR tyrosine kinase inhibitors, and taxanes. In preferred embodiments, the anti-metabolite is pemetrexed, the HERl /EGFR tyrosine kinase inhibitor is erlotinib, and the taxane (or taxoid) is docetaxel. In still more preferred embodiments, these particular combinations are administered in according to particular dosing, timing and administration regimens.
The invention therefore provides therapeutic and prophylactic methods that involve the administration of compounds of Formula I (or mixtures thereof). Depending upon the indication being treated or prevented, the compounds of Formula I are combined, preferably in pharmaceutical form, with anti-metabolites (e.g., pemetrexed and gefitinib (IRESSA®)), HER1/EGFR tyrosine kinase inhibitors (e.g., erlotinib), taxanes (e.g., docetaxel or paclitaxel), platinum-containing compounds (e.g., carboplatin or cisplatin), nucleoside analogues (e.g., gemcitabine (GEMZAR®) antibodies (or fragments thereof), antigens, or adjuvants. Compounds of Formula I have the following structure: Formula I
PR wherein P is a targeting group which binds to the reactive site of a post proline-cleaving enzyme, and R is a reactive group that reacts with a functional group in the reactive site of a post proline-cleaving enzyme. Post proline-cleaving enzymes are enzymes which have a specificity for cleaving Xaa-Pro or Xaa-Ala dipeptides (where Xaa represents any amino acid) at the carboxy end of the dipeptide. Dipeptidyl peptidases (DPP) are exopeptidases that cleave Xaa-Pro or Xaa-Ala dipeptides from the amino terminus of a peptide or protein. Examples of post-proline cleaving enzymes include, but are not limited to, dipeptidyl peptidase IV (DP IV or DPP IV) and fibroblast activation protein (FAP) both of which show DPP activity. Prolyl endopeptidase (PEP or POP) are endopeptidases with the same specificity. FAP also possesses endopeptidase activity.
The targeting group (P) can be composed of single or multiple amino acid residues, whether naturally occurring or not, or of a peptide or a peptidomimetic. In certain If
-17- embodiments, the portion of P that is involved in binding to the reactive site of a post proline- cleaving enzyme is formed of amino acid residues and the remaining portion of P is formed of non-amino acid components. Therefore, P can be composed wholly of amino acid residues, wholly of non-amino acid substituents, or a combination of both. P may be 100 or more residues in length including 30, 20, 10 or less than 10 residues in length. P or a portion(s) thereof may mimic a substrate of the protease.
Targeting groups can be synthesized from other biomolecules including but not limited to saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like. Also envisioned in the invention is the use of targeting groups made from peptoids, random bio-oligomers (U.S. Patent 5650489), benzodiazepines, diversomeres such as dydantoins, benzodiazepines and dipeptides, nonpeptidyl peptidomimetics with a beta-D-glucose scaffolding, oligocarbamates or peptidyl phosphonates.
Many if not all of these compounds can be synthesized using recombinant or chemical library approaches. A vast array of candidate targeting groups can be generated from libraries of synthetic or natural compounds. The methods of the invention utilize this library technology to identify small molecules which bind to protease reactive sites. One advantage of using libraries for inhibitor identification is the facile manipulation of millions of different putative candidates of small size in small reaction volumes (i.e., in synthesis and screening reactions). Another advantage of libraries is the ability to synthesize targeting groups which might not otherwise be attainable using naturally occurring sources.
The use of library technology, such as phage display, and combinatorial chemistry, such as compound array methods, in the synthesis and screening of protease inhibitors has been previously described in U.S. Patent No. 6875737, the contents of which are incorporated in their entirety by reference. Examples of parallel synthesis mixtures and parallel synthesis methods are provided in published PCT patent application WO 95/18972, published July 13, 1995 and U.S. Patent No. 5712171 granted January 27, 1998 and its corresponding published PCT patent application WO 96/22529, which are hereby incorporated by reference.
Libraries can be screened to identify naturally or non-naturally occurring putative targeting groups by assaying protease binding (and optionally cleavage activity) in the presence of the library molecule or member. The cleavage assay includes determining whether the library molecule inhibits cleavage by the protease of a known substrate or of a substrate analog (e.g., a chromophoric substrate analog which is easily detectable in a spectrophotometric assay). Those library molecules which exhibit binding and optionally inhibition of a post-prolyl cleaving enzyme then can be covalently coupled to the reactive groups disclosed herein and again tested for binding and inhibition of the enzyme. In this manner, a simple, high-through-put screening assay is provided for identifying inhibitors. In general, P is covalently coupled to R. In some embodiments, the covalent coupling occurs via a carboxyl group at the carboxyl terminus in P.
Examples of reactive groups useful in the invention include 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-acylhydroxylamine, an azapeptide, a fluoroolefin, a peptidyl (alpha-aminoalkyl) phosphonate ester, or a nitrile.
Compounds of Formula I can be further defined by Formula II as follows:
Formula II
Figure imgf000020_0001
wherein m is an integer between 0 and 100 or more, inclusive; A and A1 are L- or D- naturally or non-naturally occurring amino acid residues or a peptide or a peptidomimetic such that when m >1 and A is an amino acid each A in An, may be an amino acid residue different from another or all other A in Am, and when A is a peptide or a peptidomimetic m is preferably 1 ; the C bonded to B is in the L- or R- configuration; optionally the bonds between A1 and N and between A and A1 are peptide bonds; and each X1 and X2 is independently a hydroxyl group or a group capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH.
By "the C bonded to B is in the L-configuration" is meant that the absolute configuration of the C is like that of an L-amino acid. Thus, the
Figure imgf000020_0002
group has the same relationship to the C as the -COOH group of an L-amino acid has to its α carbon.
In some embodiments A1 is alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, aspartate, glutamate, asparagine, glutamine, lysine, arginine, histidine, cysteine, methionine, or proline.
Thus, in some embodiments the agent is L-Ala-L-boroPro, L-Asp-L-boroPro, L-GIu- L-boroPro, L-Asn-L-boroPro, L-Gln-L-boroPro, L-Lys-L-boroPro, L-Arg-L-boroPro, L-His- L-boroPro, L-Pro-L-boroPro, L-Thr-L-boroPro, L-Ser-L-boroPro, L-Cys-L-boroPro, L-GIy- L-boroPro, L-Tyr-L-boroPro, L-Trp-L-boroPro, L-Phe-L-boroPro, L-Leu-L-boroPro, L-IIe-L- boroPro, L-Met-L-boroPro, or L-Val-L-boroPro.
In some embodiments m is 0 and/or X1 and X2 are hydroxyl groups. In some important embodiments, X1 and X2 are hydroxyl groups, m is 0, and A1 is valine, and the resulting compound is referred to as Val-boroPro (or PT-100 or talabostat).
Depending on the embodiment, m is an integer from 0-30, or an integer from 0-20, or an integer from 0-10. In some important embodiments m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or it is a multiple of two (e.g., 2, 4, 6, 8, 10, etc.), such as a repeating dipeptide having a proline residue at the C terminal (e.g., A - Pro).
The proline residue attached to the reactive group is referred to as a pyrrolidine ring. In some embodiments, the pyrrolidine may be replaced with an azetidine or a thiazolidine. In some embodiments, the compound may comprise an isostere. In still other embodiments, the compound may be a 4-cyanothiazolidine rather than an organo boronate containing compound.
The compounds of Formula I can be further defined by Formula III as follows:
Formula III
Figure imgf000021_0001
wherein m is an integer between 0 and 100 or more, inclusive; A and A1 are L- or D- naturally or non-naturally occurring amino acid residues or a peptide or a peptidomimetic such that when m >1 A in each repeating bracketed unit is independently selected; the C bonded to B is in the L-confϊguration; optionally the bonds between A and N, A1 and C, and A1 and N are peptide bonds; and each X1 and X2 is, independently, a hydroxyl group or a group capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH.
The proline residue attached to the boronyl group is referred to as a pyrrolidine ring. It may be bonded, at its amino terminus, to an amino acid optionally in an R- or an S- configuration (and likewise in an L- or D- configuration).
Many of the agents of the invention and methods for their manufacture have been previously disclosed in U.S. Patent 4935493, the contents of which are incorporated by reference herein. It is to be understood that each and every reactive group described herein can be substituted for the reactive group in Formulae II and III (e.g., boronyl group in the form of a boronic ester or a boronic acid). Thus, other inhibitors have an analogous structure to the agents of Formulae II or III but with the boronyl group replaced by, for example, 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-acylhydroxylamine, an azapeptide, a fluoroolefin, a peptidyl (alpha-aminoalkyl) phosphonate ester, or a nitrile.
The amino acid residues may be naturally and non-naturally occurring amino acids. Examples of 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 (Tip), cysteine (Cys), methionine (Met), serine (Ser), threonine (Thr), lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp), glutamic acid (GIu)5 asparagine (Asn), glutamine (GIn) and proline (Pro). Non-naturally occurring amino acids include the D-forms of Ala, VaI, Leu, He, Phe, Tyr, Trp, Cys, Met, Ser, Thr, Lys, Arg, His, Asp, GIu, Asn, GIn, and Pro.
Other examples of 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, cyclohexylglycine (Chg), N- Methylglycine (N-MeGIy), 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-aminopentanoic acid (N- MeNva), methylthreonine, nitroglutamine, norleucine (NIe), norvaline, ornithine, phosphoserine, pipecolic acid, sarcosine, taurine, tert-leucine, thiazolidine carboxylic acid, thyroxine, trans-4-hydroxyproline, and trans-3-methylproline.
Non-naturally occurring amino acids also include beta-amino acids and alpha-amino acids with side chains replaced with synthetic derivatives. Representative side chains of naturally occurring and non-naturally occurring α-amino acids are shown below in Table 1.
Figure imgf000024_0001
Non-naturally occurring amino acids also include D, L, and racemic configurations of hydrophobic amino acids. Hydrophobic amino acids include amino acid analogs having the formula -NH-CHR-CO-, wherein R is an aliphatic group, a substituted aliphatic group, a benzyl group, a substituted benzyl group, an aromatic group or a substituted aromatic group and wherein R does not correspond to the side chain of a naturally-occurring amino acid. As used herein, aliphatic groups include straight chained, branched or cyclic C1-C8 hydrocarbons which are completely saturated, which contain one or two heteroatoms such as nitrogen, oxygen or sulfur and/or which contain one or more units of desaturation. Aromatic groups include carbocyclic aromatic groups such as phenyl and naphthyl and heterocyclic aromatic groups such as imidazolyl, indolyl, thienyl, furanyl, pyridyl, pyranyl, oxazolyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl and acridintyl.
Suitable substituents on an aliphatic, aromatic or benzyl group include -OH, halogen (- Br, -Cl, -I and -F) -O (aliphatic, substituted aliphatic, benzyl, substituted benzyl, aryl or substituted aryl group), -CN, -NO2, -COOH, -NH2, -NH (aliphatic group, substituted aliphatic, benzyl, substituted benzyl, aryl or substituted aryl group), -N (aliphatic group, substituted aliphatic, benzyl, substituted benzyl, aryl or substituted aryl group)2, -COO (aliphatic group, substituted aliphatic, benzyl, substituted benzyl, aryl or substituted aryl group), -CONH2, -CONH (aliphatic, substituted aliphatic group, benzyl, substituted benzyl, aryl or substituted aryl group)), -SH, -S (aliphatic, substituted aliphatic, benzyl, substituted benzyl, aromatic or substituted aromatic group) and -NH-C(=NH)-NH2. A substituted benzylic or aromatic group can also have an aliphatic or substituted aliphatic group as a substituent. A substituted aliphatic group can also have a benzyl, substituted benzyl, aryl or substituted aryl group as a substituent. A substituted aliphatic, substituted aromatic or substituted benzyl group can have one or more substituents. Modifying an amino acid substituent can increase, for example, the lypophilicity or hydrophobicity of natural amino acids which are hydrophilic.
A number of the suitable amino acids, amino acids analogs and salts thereof can be obtained commercially. Others can be synthesized by methods known in the art. Synthetic techniques are described, for example, in Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, Chapters 5 and 7, 1991.
These compounds can be provided in linear or cyclic form or as mixtures thereof, as described in U.S. Patent No. 6355614, issued March 12, 2002. The proportion of linear (versus cyclic) forms in these mixtures may vary (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of cyclic relative to the total amount of the Formula I compound) depending on the formulation.
The agents may be provided as A-boroPro containing compounds that are converted (via enzymatic, chemical, metabolic, or any other means, in vivo or ex vivo) to A-boroPro. A- boroPro containing compounds include prodrugs. 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 a boroxine 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, as discussed above.
Another category of 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. 5462928 issued October 31, 1995; and 6100234 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 may be 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.
All amino acids, with the exception of glycine, contain an asymmetric or chiral carbon and may contain more than one chiral carbon atom. The asymmetric α carbon atom of the amino acid is referred to as a chiral center and can occur in two different isomeric forms. These forms are identical in all chemical and physical properties with one exception, the direction in which they can cause the rotation of plane-polarized light. These amino acids are referred to as being "optically active," i.e., the amino acids can rotate the plane-polarized light in one direction or the other.
The four different substituent groups attached to the α carbon can occupy two different arrangements in space. These arrangements are not superimposable mirror images of each other and are referred to as optical isomers, enantiomers, or stereo isomers. A solution of one stereo isomer of a given amino acid will rotate plane polarized light to the left and is called the levorotatory isomer [designated (-)]; the other stereo isomer for the amino acid will rotate plane polarized light to the same extent but to the right and is called dextrorotatory isomer [designated (+)].
A more systematic method for classifying and naming stereo isomers is the absolute configuration of the four different substituents in the tetrahedron around the asymmetric carbon atom (e.g., the α carbon atom). To establish this system, a reference compound was selected (glyceraldehyde), which is the smallest sugar to have an asymmetric carbon atom. By convention in the art, the two stereo isomers of glyceraldehyde are designated L and D. Their absolute configurations have been established by x-ray analysis. The designations, L and D, also have been assigned to the amino acids by reference to the absolute configuration of glyceraldehyde. Thus, the stereo isomers of chiral compounds having a configuration related to that of L-glyceraldehyde are designed L, and the stereo isomers having a configuration related to D-glyceraldehyde are designated D, regardless of the direction in which they rotate the plane-polarized light. Thus, the symbols, L and D, refer to the absolute configuration of the four substituents around the chiral carbon. In general, naturally occurring compounds which contain a chiral center are only in one stereo isomeric form, either D or L. The naturally occurring amino acids are the L stereo isomers. As discussed herein, the invention embraces inhibitors comprising amino acid residues which can be in the D stereo isomer configuration also.
Most amino acids that are found in proteins can be unambiguously named using the D L system. However, compounds which have two or more chiral centers may be in 2n possible stereo isomer configurations, where n is the number of chiral centers. These stereo isomers sometimes are designated using the RS system to more clearly specify the configurations of amino acids that contain two or more chiral centers. For example, compounds such as threonine and isoleucine contain two asymmetric carbon atoms and therefore have four stereo isomer configurations. The isomers of compounds having two chiral centers are known as diastereomers. A complete discussion of the RS system of designating optical isomers for amino acids is provided in Principles in Biochemistry, editor A.L. Lehninger, page 99-100. A brief summary of this system follows.
The RS system was invented to avoid ambiguities when a compound contains two or more chiral centers. In general, the system is designed to rank the four different substituent atoms around an asymmetric carbon atom in order of decreasing atomic number or in order of decreasing valance density when the smallest or lowest-rank group is pointing directly away from the viewer. The different rankings are well known in the art and are described on page i
-26-
99 of Lehninger (supra). If the decreasing rank order is seen to be clock- wise, the configuration around the chiral center is referred to as R; if the decreasing rank order is counter-clockwise, the configuration is referred to as S. Each chiral center is named accordingly using this system. Applying this system to threonine, one skilled in the art would determine that the designation, L-threonine, refers to (2S, 3R)-threonine in the RS system. The more traditional designations of L-, D-, L-allo, and D-allo, for threonine have been in common use for some time and continue to be used by those of skill in this art. However, the R S system increasingly is used to designate the amino acids, particularly those which contain more than one chiral center. The agents of the invention may be substantially optically pure. That is, at least 90%,
92%, 94%, 95%, 96%, 97%, 98% or 99% of the carbon atoms bearing boron are of the L- configuration in some embodiments. Methods for synthesizing optically pure isomers of compounds of Formula I are disclosed in published PCT application WO 93/08259.
The agents may comprise the carbon of the pyrrolidine ring bonded to the boron in the R-configuration. The agent may be provided as a mixture of R- and S-enantiomers of boron substituted pyrrolidine. In a related embodiment 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.
Certain methods and compositions of the invention comprise a compound of Formula I and an anti-metabolite used in particular dosing and/or administration and/or timing regimens. Anti-metabolites include folic acid analogs (anti-folates), pyrimidine analogs, and purine analogs. Examples of folic acid analogs (anti-folates) include but are not limited to methrotrexate and pemetrexed (ALIMTA®). Examples of pyrimidine analogs include but are not limited to fluorouracil, (5-fluorouracil; 5-FU), fioxuridine (fluorodeoxyuridine; FUdR), cytarabine (cytosine arabinoside) and gemcitabine. Examples of purine analogs include but are not limited to mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG), and pentostatin (2'-deoxycoformycin).
Other examples of antimetabolites include fludarabme, cladribine, capecitabine, L- alanosine, 5-azacytidine, acivicin, aminopterin derivatives, antifols, Baker's soluble antifol, dichlorallyl lawsone, brequinar, ftorafur, 5,6-dihydro-5-azacytidine, methotrexate derivatives, N-(phosphonoacetyl)-L-aspartate (PALA), pyrazofurin, trimetrexate, 3-HP5 2'-deoxy-5- fluorouridine, 5-HP5 alpha-TGDR, aphidicolin glycinate, ara-C, 5-aza-2'-deoxycytidine, beta- TGDR, cyclocytidine, guanazole, hydroxyurea, inosine glycodialdehyde, macbecin II, I
-27- pyrazoloimidazole, and thiopurine. In preferred embodiments the antimetabolite is pemetrexed (ALIMTA®).
Other methods, compositions and kits of the invention comprise a compound of Formula I and a HERl /EGFR tyrosine kinase inhibitor used in particular dosing and/or administration and/or timing regimens. Examples of HER1/EGFR tyrosine kinase inhibitor include but are not limited to gefitinib (IRESSA®), erlotinib (TARCEV A™), Cetuximab (Erbitux), and quinazoline and pyrido-[3,4-d]-pyrimidine molecules (e.g., GW2974, GW0277, and WHI-P97), LFM-A12(leflunomide metabolite analog), and AG 1458. In one important embodiment the HER1/EGFR tyrosine kinase inhibitor is erlotinib (T ARCEV A™). Still other methods, compositions and kits comprise a compound of Formula I and a taxane/taxoid used in particular dosing and/or administration and/or timing regimens. Examples of taxanes include but are not limited to paclitaxel (TAXOL), docetaxel (TAXOTERE®), cephalomannine, Baccatin, xylotaxol, 10 Deacetyltaxol, and taxinine. In one important embodiment the taxane is docetaxel. Certain methods and compositions of the invention may further comprise a cancer antigen. A cancer antigen as used herein is a compound differentially associated with a tumor or cancer, preferably at the cell surface of a tumor or cancer cell that is capable of invoking an immune response. The cancer antigen may be peptide in nature but it is not so limited. As an example, the antigen may be a lipid antigen, as described in U.S. Patents US 5679347, issued on October 21, 1997 and US 6238676 Bl, issued on May 29, 2001. If the antigen is a peptide, then it invokes an immune response when it is presented (in a digested form) on the surface of an antigen presenting cell in the context of an MHC molecule. If the antigen is a lipid, then it invokes an immune response when it is presented in the context of a CDl molecule. Cancer antigens can be prepared from cancer cells either by preparing crude extracts of cancer cells, for example, as described in Cohen, et al., 1994, Cancer Research, 54: 1055, by partially purifying the antigens, by recombinant technology, or by de novo synthesis of known antigens. Cancer antigens include but are not limited to antigens that are recombinantly expressed, an immunogenic portion of, or a whole tumor or cancer. Such antigens can be isolated or prepared recombinantly or by any other means known in the art. A cancer antigen encompasses antigens that are differentially expressed between cancer and normal cells. Due to this differential expression, these antigens can be targeted in anti-tumor therapies. Cancer antigens may be expressed in a regulated manner in normal cells. For example, they may be expressed only at certain stages of differentiation or at certain points in development of the organism or cell. Some are temporally expressed as embryonic and fetal antigens. Still others are never expressed in normal cells, or their expression in such cells is so low as to be undetectable.
Other cancer antigens are encoded by mutant cellular genes, such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes such as those carried on RNA and DNA tumor viruses.
Examples of cancer antigens include HER 2 (pl85), CD20, CD33, GD3 ganglioside, GD2 ganglioside, carcinoembryonic antigen (CEA), CD22, milk mucin core protein, TAG-72, Lewis A antigen, ovarian associated antigens such as OV-TL3 and MOvI 8, high Mr melanoma antigens recognized by antibody 9.2.27, HMFG-2, SM-3, B72.3, PR5C5, PR4D2, and the like. Other cancer antigens are described in U.S. Pat. No. 5776427. Still other cancer antigens are listed in Table 2.
Further examples include MAGE, MART-1/Melan-A, gplOO, Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (AD Abp), FAP, cyclophilin b, Colorectal associated antigen (CRC)-CO 17- 1A/GA733, Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-I and CAP-2, etv6, amll, Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-I, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-Al, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8,
MAGE-A9, MAGE-AlO, MAGE-AIl5 MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-Cl, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family of tumor antigens (e.g., GAGE-I, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-I, NAG, GnT-V, MUM- 1 , CDK4, tyrosinase, p53 , MUC family, HER2/neu, ρ21 ras, RCAS 1 , α-fetoprotein, E- cadherin, α-catenin, β-catenin and γ-catenin, pl20ctn, gpl00Pmel117, PRAME, NY-ESO-I, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, pi 5, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, Smad family of tumor antigens, lmp-1, PlA, EBV-encoded nuclear antigen (EBNA)-I, brain glycogen phosphorylase, SSX-I, SSX-2 (HOM-MEL-40), SSX-I, SSX-4, SSX-5, SCP-I and CT-7, CD20 and c-erbB-2.
These antigens can be classified as indicated in Table 2. Table 2. Classification of cancer antigens
Table 2a. Proteins encoded by genes that have undergone chromosomal alteration in lymphoma and leukemia
Genes Disease
Activation of quiescent genes
BCL-I and IgH Mantel cell lymphoma
BCL-2 andIgH Follicular lymphoma
BCL-6 Diffuse large B-cell lymphoma
TAL-I and TCRδ or SIL T-cell acute lymphoblastic leukemia c-MYC and IgH or IgL Burkitt lymphoma
MUN/IRF4 and IgH Myeloma
PAX-5 (BSAP) Immunocytoma
Creation of fusion genes
RARa, PML, PLZF, NPM or NuMA Acute promyelocytic leukemia BCR and ABL Chronic myeloid/acute lymphoblastic leukemia
MLL (HRX) Acute leukemia
E2A and PBX or HLF B-cell acute lymphoblastic leukemia
NPM, ALK Anaplastic large cell leukemia
NPM, MLF-I Myelodysplastic syndrome/acute myeloid leukemia
Adapted from Falini B. and Mason, D.Y. (2002) Blood 99: 409-426
Table 2b. Proteins specific to a tissue or cell lineage
Protein Disease
Cell-surface proteins
CD20, CD22 Non-Hodgkin's lymphoma, B-cell lymphoma, Chronic lymphocytic leukemia
(CLL)
CD52 B-cell CLL
CD33 Acute myelogenous leukemia (AML)
CDlO (gplOO) Common (pre-B) acute lymphocytic leukemia and malignant melanoma
CD3/T-cell receptor (TCR) T-cell lymphoma and leukemia
CD79/B-cell receptor (BCR) B-cell lymphoma and leukemia
CD26 Epithelial and lymphoid malignancies
Human leukocyte antigen (HLA)-DR, Lymphoid malignancies
HLA-DP, and HLA-DQ
RCASl Gvnecoloεical carcinomas, bilarv adenocarcinomas and ductal adenocarcinomas of the pancreas
Prostate specific membrane antigen Prostate cancer
Epidermal growth factor receptors (high expression
EGFR (HERl or erbBl) and EGFRvIII Brain, lung, breast, prostate and stomach cancer erbB2 (HER2 or HER2/neu) Breast cancer and gastric cancer erbB3 (HER3) Adenocarcinoma erbB4 (HER4) Breast cancer
Cell-associated proteins
Tyrosinase, Melan-A/MART-1, tyrosinase Malignant melanoma related protein (TRP)-l/gp75 Polymorphic epithelial mucin (PEM) Breast tumors Human epithelial mucin (MUCl) Breast, ovarian, colon and lung cancers
Secreted proteins
Monoclonal immunoglobulin Multiple myeloma and plasmacytoma Immunoglobulin light chains Multiple Myeloma α-fetoprotein Liver carcinoma Kallikreins 6 and 10 Ovarian cancer Gastrin-releasing peptide/bombesin Lung carcinoma Prostate specific antigen Prostate cancer Table 2c. Cancer testis (CT) antigens*
These antigens include MAGE-Al, -A3, -A6, -Al 2, BAGE, GAGE, HAGE, LAGE-I5 NY- ESO-I, RAGE, SSX-I, -2, -3, -4, -5, -6, -7, -8, -9, HOM-TES-14/SCP-1, HOM-TES-85 and PRAME.
* These antigens are expressed in some normal tissues such as testis and in some cases placenta. Their expression is common in tumors of diverse lineages and as a group the antigens form targets for immunotherapy. Examples of tumor expression of CT antigens are as follows.
Protein Disease
SSX-2, and -4 Neuroblastoma
SSX-2 (HOM-MEL-40), MAGE, GAGE, Malignant melanoma
BAGE and PRAME
HOM-TES-14/SCP-1 Meningioma
SSX-4 Oligodendrioglioma
HOM-TES-14/SCP-1, MAGE-3 and SSX-4 Astrocytoma
SSX member Head and neck cancer, ovarian cancer, lymphoid tumors, colorectal cancer and breast cancer
RAGE-I, -2, -4, GAGE-I, -2, -3, -4, -5, -6, -7 Head and neck squamous cell carcinoma and -8 (HNSCC)
HOM-TES14/SCP-1, SSX-I, PRAME and Non-Hodgkin's lymphoma CT-7 PRAME Acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML) and chronic lymphocytic leukemia (CLL)
Table 2d. Proteins not-specific to a tissue or cell lineage*
Carcinoembryonic antigen (CEA) family: CD66a, CD66b, CD66c, CD66d and CD66e.
* These antigens can be expressed in many different malignant tumors and can be targeted by immunotherapy.
Table 2e. Viral proteins
Human papilloma virus protein (cervical cancer)
EBV-encoded nuclear antigen (EBNA)-I (lymphomas of neck and oral cancer)
Table 2f. Mutated or aberrantly expressed molecules
CDK4 and beta-catenin in melanoma
Cancer or tumor antigens can also be classified according to the cancer or tumor they are associated with (i.e., expressed by). Cancers or tumors associated with tumor antigens include acute lymphoblastic leukemia (etv6; amll ; cyclophilin b), B cell lymphoma (Ig- idiotype); Burkitt's (Non-Hodgkin's) lymphoma (CD20); glioma (E-cadherin; α-catenin; β- catenin; γ-catenin; pl20ctn), bladder cancer (p21ras), biliary cancer (p21ras), breast cancer (MUC family; HER2/neu; c-erbB-2), cervical carcinoma (p53; p21ras), colon carcinoma (p21ras; HER2/neu; c-erbB-2; MUC family), colorectal cancer (Colorectal associated antigen (CRC)~CO17-1A/GA733; APC), choriocarcinoma (CEA), epithelial cell-cancer (cyclophilin b), gastric cancer (HER2/neu; c-erbB-2; ga733 glycoprotein), hepatocellular cancer (α- fetoprotein), Hodgkin' s lymphoma (Imp- 1 ; EBNA- 1), lung cancer (CEA; MAGE-3 ; NY- ESO-I)5 lymphoid cell-derived leukemia (cyclophilin b), melanoma (pi 5 protein, gp75, oncofetal antigen, GM2 and GD2 gangliosides), myeloma (MUC family; p21ras), non-small cell lung carcinoma (HER2/neu; c-erbB-2), nasopharyngeal cancer (lmp-1; EBNA-I)5 ovarian cancer (MUC family; HER2/neu; c-erbB-2), prostate cancer (Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA- 1 , PS A-2, and PS A-3 ; PSMA; HER2/neu; c-erbB-2), pancreatic cancer (p21ras; MUC family; HER2/neu; c-erbB-2; ga733 glycoprotein), renal (HER2/neu; c-erbB-2), squamous cell cancers of cervix and esophagus (viral products such as human papilloma virus proteins and non-infectious particles), testicular cancer (NY-ESO-I), T cell leukemia (HTLV-I epitopes), and melanoma (Melan-A/MART-1; cdc27; MAGE-3; p21ras; gpl00Pmel117).
In some embodiments, the cancer antigen is VEGF5 Anti-idiotypic mAb (GD3 ganglioside mimic), CD20, CD52, Anti-idiotypic mAb (CEA mimic), ERBB2, EGFR5 CD22, ERBB2 X CD65 (fcγRI), EpCam, PEM and CD33.
For examples of cancer antigens which bind to either or both MHC class I and MHC class II molecules, see the following references: Coulie, Stem Cells 13:393-403, 1995; Traversari et al, J. Exp. Med. 176:1453-1457, 1992; Chaux et al., J Immunol. 163:2928- 2936, 1999; Fujie et al., Int. J. Cancer 80:169-172, 1999; Tanzarella et al., Cancer Res. 59:2668-26^ '4, 1999; van der Bruggen et al., Eur. J. Immunol. 24:2134-2140, 1994; Chaux et al., J Exp. Med. 189:767-778, 1999; Kawashima et al, Hum. Immunol. 59:1-14, 1998; Tahara et al., Clin. Cancer Res. 5:2236-2241, 1999; Gaugler et al., J Exp. Med. 179:921-930, 1994; van der Bruggen et al., Eur. J. Immunol. 24:3038-3043, 1994; Tanaka et al., Cancer Res. 57:4465-4468, 1997; Oiso et al., Int. J. Cancer 81:387-394, 1999; Herman et al., Immunogenetics 43:377-383, 1996; Manici et al., J Exp. Med. 189:871-876, 1999; Duffour et al., Eur. J. Immunol. 29:3329-3337, 1999; Zorn et al., Eur. J. Immunol. 29:602-607, 1999; Huang et al., J Immunol .162:6849-6854, 1999; Boel et al., Immunity 2:167-175, 1995; Van den Eynde et al., J Exp. Med. 182:689-698, 1995; De Backer et al., Cancer Res. 59:3157- 3165, 1999; Jager et al., J. Exp. Med. 187:265-270, 1998; Wang et al., J. Immunol 161:3596- 3606, 1998; Aarnoudse et al., Int. J. Cancer 82:442-448, 1999; Guilloux et al., J Exp. Med. 183:1173-1183, 1996; Lupetti et al., J Exp. Med. 188:1005-1016, 1998; Wδlfel et al., Eur. J. Immunol. 24:759-764, 1994; Skipper et al., J Exp. Med. 183:527-534, 1996; Kang et al., J. Immunol. 155:1343-1348, 1995; Morel et al., Int. J. Cancer 83:755-759, 1999; Brichard et al., Eur. J. Immunol. 26:224-230, 1996; Kittlesen et al., J Immunol. 160:2099-2106, 1998; Kawakami et al., J. Immunol. 161:6985-6992, 1998; Topalian et al., J Exp. Med. 183:1965- 1971, 1996; Kobayashi et al., Cancer Research 58:296-301, 1998; Kawakami et al., J. Immunol. 154:3961-3968, 1995; Tsai et al., J Immunol. 158:1796-1802, 1997; Cox et al., Science 264:716-719, 1994; Kawakami et al., Proc. Natl. Acad. Sci. USA 91 :6458-6462, 1994; Skipper et al., J Immunol. 157:5027-5033, 1996; Robbins et al., J Immunol. 159:303- 308, 1997; Castelli et al, J. Immunol. 162:1739-1748, 1999; Kawakami et al., J. Exp. Med. 180:347-352, 1994; Castelli et al., J. Exp. Med. 181:363-368, 1995; Schneider et al., Int. J. Cancer.75:451-458, 1998; Wang et al., J Exp. Med. 183:1131-1140, 1996; Wang et al., J Exp. Med. 184:2207-2216, 1996; Parkhurst et al., Cancer Research 58:4895-4901, 1998; Tsang et al., J Natl Cancer Inst 87:982-990, 1995; Correale et al., J Natl Cancer Inst 89:293- 300, 1997; Coulie et al., Proc. Natl. Acad. ScL USA 92:7976-7980, 1995; Wδlfel et al.,
Science 269:1281-1284, 1995; Robbins et al., J. Exp. Med. 183:1185-1192, 1996; Brandle et al., J Exp. Med. 183:2501-2508, 1996; ten Bosch et al., Blood 88:3522-3527, 1996; Mandruzzato et al., J Exp. Med. 186:785-793, 1997; Gueguen et al., J Immunol. 160:6188- 6194, 1998; Gjertsen et al., Int. J. Cancer 72:784-790, 1997; Gaudin et al., J. Immunol. 162:1730-1738, 1999; Chiari et al., Cancer Res, 59:5785-5792, 1999; Hogan et al., Cancer Res. 58:5144-5150, 1998; Pieper et al., J Exp. Med. 189:757-765, 1999; Wang et al., Science 284:1351-1354, 1999; Fisk et al., J Exp. Med. 181:2109-2117, 1995; Brossart et al., Cancer Res. 58:732-736, 1998; Ropke et al., Proc. Natl. Acad. Sci. USA 93:14704-14707, 1996; Ikeda et al., Immunity 6:199-208, 1997; Ronsin et al., J Immunol 163:483-490, 1999; Vonderheide et al., Immunity 10:673-679,1999. These antigens as well as others are disclosed in PCT Application PCT/US98/18601.
In some embodiments, the antigens are administered in a substantially purified form. The term "substantially purified" as used herein refers to a compound which is substantially free of other compounds such as proteins, lipids, carbohydrates or other materials with which it is naturally associated. One skilled in the art can purify antigens using standard techniques such as for example protein purification. The substantially pure polypeptide will often yield a single major band on a non-reducing polyacrylamide gel. In the case of partially glycosylated polypeptides or those that have several start codons, there may be several bands on a non- reducing polyacrylamide gel, but these will form a distinctive pattern for that polypeptide. The purity of the viral or bacterial polypeptide can also be determined by amino-terminal amino acid sequence analysis.
Some important cancer antigens include but are not limited to PGP9.5, H/Ley/Leb, B7-DC, and B7-Hl.
Certain methods and compositions comprise, in addition to the compounds of Formula I, an antibody or an antigen-binding fragment thereof. The invention embraces the use of antibodies of all isotypes including IgM, IgAl, IgA2, slgA, IgD, IgE, IgGl, IgG25 IgG3, and IgG4, having light chains that are either kappa or lambda chains. The invention encompasses the use of both antibodies and antibody fragments. The antibodies may be monoclonal or polyclonal, and can be prepared by conventional methodology. Such antibodies can be further manipulated to create chimeric or humanized antibodies as will be discussed in greater detail below.
The antibodies or fragments thereof useful in the invention can be specific for any component of a particular target. Accordingly, the antibody can recognize and bind to proteins, lipids, carbohydrates, DlSTA, RNA, and any combination of these in molecular or supra-molecular structures (e.g., cell organelles such as mitochondria or ribosomes). The antibody or fragment thereof can also recognize a modification of the tumor cell, such as e.g., chemical modifications, or genetic modifications made by transfection ex vivo or in vivo with DNA or RNA. As used herein, the terms "antibody" and "immunoglobulin" are used interchangeably.
Bispecifϊc antibodies can also be used in the invention. A bispecific antibody is one having one variable region that specifically recognizes a tumor antigen and the other variable region that specifically recognizes an antigenic epitope of a host immune effector cell that has lytic or growth inhibitory activity against the tumor. Bispecific and multispecific antibody complexes can be created by linkage of two or more immunoglobulins of different specificity for tumor antigens and/or effector cell antigens, either at the peptide or nucleic acid level.
Immunoglobulin can be produced in vivo in human or non-human species, or in vitro from immunoglobulin encoding DNA or cDNA isolated from libraries of DNA (e.g., phage display libraries). Immunoglobulin can also be modified genetically or chemically to incorporate human polypeptide sequences into non-human coding sequences (commonly referred to as humanization). Additionally, immunoglobulins can be modified chemically or genetically to incorporate protein, lipid, or carbohydrate moieties. Potential modifications could also include naturally occurring or synthetic molecular entities that are either directly toxic for tumor cells or serve as ligands or receptors for biologically active molecules that could suppress tumor growth. For example, growth factors, cytokines, chemokines and their respective receptors, immunologically active ligands or receptors, hormones or naturally occurring or synthetic toxins all represent biologically active molecules that could interact with suitably modified immunoglobulins and their targets.
Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W.R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The pFc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc1 region, designated an F(ab')2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FRl through FR4) separated respectively by three complementarity determining regions (CDRl through CDR3). The CDRs, and in particular the CDR3 regions, and more particularly the heavy chain CDR3, are largely responsible for antibody specificity. It is now well-established in the art that the non-CDR regions of a mammalian antibody may be replaced with similar regions of co-specific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody. Thus, for example, PCT International Publication Number WO 92/04381 teaches the production and use of humanized murine RSV antibodies in which at least a portion of the murine FR regions has been replaced by FR regions of human origin. Such antibodies, including fragments of intact antibodies with antigen-binding ability, are often referred to as "chimeric" antibodies. Commercial sources of humanized or chimeric antibodies include GenPharm, Xenotech, AbGenix and CellGeneSys.
Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab')2, Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDRl and/or CDR2 regions have been replaced by homologous human or non- human sequences. The present invention also includes so-called single chain antibodies. Some commercially available anti-cancer antibodies along with their commercial source are as follows: anti-CD20 mAb (monoclonal antibody), rituximab, Rituxan™, Non- Hodgkin's lymphoma, B cell lymphoma (IDEC/Genentech); anti-CD20 mAb, tositumomab Bexxar, Non-Hodgkin's lymphoma (Corixa/GlaxoSmithKline); anti-HER2, trastuzumab, Herceptin™, breast and ovarian cancer (Genentech); anti-HER2, MDX-210, prostate, non- small cell lung cancer, breast, pancreatic, ovarian, renal and colon cancer (Medarex/Novartis); anti-CA125 mAb, oregovomab, B43.13, Ovarex™, ovarian cancer (Altarex); Breva-Rex, multiple myeloma, breast, lung, ovarian (Altarex); AR54, ovarian, breast, lung (Altarex); GivaRex, pancreas, stomach, colorectal (Altarex); ProstaRex, prostate (Altarex); anti-EGF receptor mAb, IMC-C225, Erbitux™, breast, head and neck, non-small cell lung, renal, prostate, colorectal and pancreatic cancer (ImClone Systems); anti-EGF receptor mAb, MDX-447, head and neck, prostate, lung, bladder, cervical, ovarian cancer (Medarex/Merck); gemtuzumab ozogamicin, Mylotarg, CMA-676, anti-CD33 (Wyeth Pharmaceuticals); anti- tissue factor protein (TF), (Sunol); ior-c5, colorectal cancer; ceal, colorectal cancer; c5, colorectal cancer; anti-EGF receptor mAb, MDX-447, head and neck, prostate, lung, bladder, cervical and ovarian cancer (Medarex/Merck); anti-17-lA mAb, edrecolomab, Panorex, colorectal, pancreatic, lung, breast and ovarian cancer (Centocor/Glaxo/Ajinomoto); anti- CD20 mAb (Y-90 labeled), ibritumomab tiuxetan (IDEC- Y2B8), Zevalin, Non-Hodgkin's lymphoma (IDEC); anti-idiotypic mAb mimic of ganglioside GD3 epitope, BEC2, small cell lung carcinoma, melanoma (ImClone Systems); anti-HLA-DrlO mAb (131 1 LYM-I), Oncolym™, Non-Hodgkin's lymphoma (Peregrine Pharmaceuticals); anti-CD33 humanized mAb (SMART Ml 95), Zamyl™, acute myeloid leukemia, acute promyelocytic leukemia (Protein Design Labs); anti-CD52 humAb (LDP-03), CAMPATH, chronic lymphocytic leukemia (Millenium Pharmaceuticals/Ilex Oncology); anti-CD 1 mAb, ior t6, cancer (Center of Molecular Immunology); anti-CAR (complement activating receptor) mAb, MDX-11, myeloid leukemia (Medarex); humanized bispecific mAb conjugates (complement cascade activators), MDX-22, myeloid leukemia (Medarex); OVl 03 (Y-90 labeled antibody), celogovab, OncoScint™, ovarian and prostate cancer (Cytogen); anti-17-lA mAb, 3622W94, non-small cell lung carcinoma, prostate cancer (Glaxo Wellcome pic); anti-VEGF (RhumAb- VEGF), bevacizumab, Avastin™, lung, breast, prostate, renal and colorectal cancer (Genentech); anti-TAC (IL-2 receptor) humanized Ab (SMART), daclizumab, Zenapax, leukemia, lymphoma (Protein Design Labs); anti-TAG-72 partially humanized bispecific Ab, MDX-220, lung, colon, prostate, ovarian, endometrial, pancreatic and gastric cancer (Medarex); anti-idiotypic mAb mimic of high molecular weight proteoglycan (I-Mel-1), MELIMMUNE-I, melanoma (IDEC); anti-idiotypic mAb mimic of high molecular weight proteoglycan (I-Mel-2), MELIMMUNE-2, melanoma (IDEC); anti-CEA Ab (hMNl 4), CEACide™, colorectal cancer and other cancers (Immunomedics); Pretarget™ radioactive targeting agents, cancer (NeoRx); hmAbHl 1 scFv fragment (NovomAb-G2), Hl 1 scFv, cancer (Viventia Biotech); anti-DNA or DNA-associated proteins (histones) mAb and conjugates, TNT (e.g., Cotara™), cancer (Peregrine Pharmaceuticals); Gliomab-H mAb, brain cancer, melanoma, neuroblastoma (Viventia Biotech); GNI-250 mAb, colorectal cancer
(Wyeth); anti-EGF receptor mAb, EMD-72000, cancer (Merck KgaA); anti-CD22 humanized Ab, LymphoCide, Non-Hodgkin's lymphoma (Immunomedics); anti-CD33 mAb conjugate with calicheamicin (CMA 676), gemtuzumab ozogamicin, Mylotarg™, acute myelogenous leukemia (Wyeth); Monopharm-C, colon, lung and pancreatic cancer (Viventia Biotech); anti- idiotypic human mAb to GD2 ganglioside, 4B5, melanoma, small-cell lung cancer, neuroblastoma (Viventia Biotech); anti-EGF receptor humanized Ab, ior egf/r3, cancers of epithelial origin (Center of Molecular Immunology); anti-ior c2 glycoprotein mAb, ior c5, colorectal and ovarian cancer (Center of Molecular Immunology); BABS (biosynthetic antibody binding site) proteins, breast cancer (Chiron); anti-FLK-2/FLT-3 mAb, cancer (tumor-associated angiogenesis) (ImClone Systems); mAb/small-molecule conjugate, TAP (tumor-activated prodrug), cancer (ImmunoGen); anti-GD-2 bispecific mAb, MDX-260, melanoma, glioma, neuroblastoma (Medarex); antinuclear autoantibodies (binds nucleosomes), ANA Ab5 cancer (Procyon Biopharma); anti-HLA-DR Ab (SMART IDlO Ab), Remitogen™, Non-Hodgkin's B-cell lymphoma (Protein Design Labs); SMART ABL 364 Ab5 epithelial cell cancers, breast, lung and colon cancer (Protein Design Labs/Novartis); anti-CEA 1131-labeled mAb, ImmuRAIT-CEA, colorectal cancer (Immunomedics).
Other antibodies that can be used according to the invention include anti-TNFα antibody such as infliximab (Remicade) and etanercept (Enbrel) and palivizumab; bevacizumab, breast cancer; alemtuzumab, Campath-IH, breast and renal cancer, melanoma, B cell chronic lymphocytic leukemia (Millennium and ILEX); BLyS -mAb, fSLE; anti- VEGF2, melanoma, breast cancer; anti-Trail receptor; B3 mAb, breast cancer; ml 70 mAb, breast cancer; niAB BR96, breast cancer; Abx-Cbl mAb, graft versus host disease. The invention embraces a number of classes of antibodies and fragments thereof including but not limited to antibodies directed to cancer antigens (as described above), cell surface molecule, stromal cell molecules, extracellular matrix molecules,, and tumor vasculature associated molecules.
A cell surface molecule is a molecule that is expressed at the surface of a cell. In addition to an extracellular domain, it may further comprise a transmembrane domain and a cytoplasmic domain. Examples include HER 2, CD20, CD33, EGF receptor, HLA markers such as HLA-DR, CD52, CDl5 CEA, CD22, GD2 ganglioside, FLK2/FLT3, VEGF5 VEGFR, and the like.
A stromal cell molecule is a molecule expressed by a stromal cell. Examples include but are not limited to FAP and CD26.
An extracellular matrix molecule is a molecule found in the extracellular matrix. Examples include but are not limited to collagen, glycosaminoglycans (GAGs), proteoglycans, elastin, fibronectin and laminin.
A tumor vasculature associated molecule is a molecule expressed by vasculature of a tumor (i.e., a solid cancer rather than a systemic cancer such as leukemia). As with a cancer antigen, a tumor vasculature associated molecule may be expressed by normal vasculature however its presence on vasculature of a tumor makes it a suitable target for anti-cancer therapy. In some instances, the tumor vasculature associated molecule is expressed at a higher level in tumor vasculature than it is in normal vasculature. Examples include but are not limited to endoglin (see U.S. Pat. No. 5,660,827), ELAM-I, VCAM-I, ICAM-I5 ligand reactive with LAM-I, MHC class II antigens, aminophospholipids such as phosphatidylserine and phosphatidylethanolamine (as described in U.S. Pat. No. 6,312,694), VEGFRl (FIt-I) and VEGFR2 (KDR/Flk-1), and other tumor vasculature associated antigens such as those described in U.S. Pat. No. 5,776,427. Antibodies to endoglin are described in U.S. Pat. No. 5,660,827 and include TEC-4 and TEC-1 1, and antibodies that recognize identical epitopes to these antibodies. Antibodies to aminophospholipids are described in U.S. Pat. No. 6,312,694. Antibodies that inhibit VEGF are described in U.S. Pat. No. 6,342,219 and include 2C3 (ATCC PTA 1595). Other antibodies that are specific for tumor vasculature include antibodies that react to a complex of a growth factor and its receptor such as a complex of FGF and the FGFR or a complex of TGFβ and the TGFβR. Antibodies of this latter class are described in U.S. Pat. No. 5,965,132, and include GV39 and GV97.
It is to be understood that the antibodies embraced by the invention include those recited explicitly herein and also those that bind to the same epitope as those recited herein.
Also useful in the invention are antibodies such as the following, all of which are commercially available: Apoptosis Antibodies:
BAX Antibodies: Anti-Human Bax Antibodies (Monoclonal), Anti-Human Bax Antibodies (Polyclonal), Anti-Murine Bax Antibodies (Monoclonal), Anti-Murine Bax Antibodies (Polyclonal);
Fas / Fas Ligand Antibodies: Anti-Human Fas / Fas Ligand Antibodies, Anti-Murine Fas / Fas Ligand Antibodies Granzyme Antibodies Granzyme B Antibodies;
BCL Antibodies: Anti Cytochrome C Antibodies, Anti-Human BCL Antibodies (Monoclonal), Anti-Human bcl Antibodies (Polyclonal), Anti-Murine bcl Antibodies (Monoclonal), Anti-Murine bcl Antibodies (Polyclonal);
Miscellaneous Apoptosis Antibodies: Anti TRADD, TRAIL, TRAFF, DR3 Antibodies Anti-Human Fas / Fas Ligand Antibodies Anti-Murine Fas / Fas Ligand Antibodies; Miscellaneous Apoptosis Related Antibodies: BIM Antibodies: Anti Human, Murine bim Antibodies (Polyclonal), Anti-Human, Murine bim Antibodies (Monoclonal);
PARP Antibodies: Anti-Human PARP Antibodies (Monoclonal), Anti-Human PARP Antibodies (Polyclonal) Anti-Murine PARP Antibodies; Caspase Antibodies : Anti-Human Caspase Antibodies (Monoclonal), Anti-Murine Caspase Antibodies;
Anti-CD Antibodies: Anti-CD29, PL 18-5 PanVera, Anti-CD29, PL4-3 PanVera, Anti- CD41a, PT25-2 PanVera, Anti-CD42b, PL52-4 PanVera, Anti-CD42b, GUR20-5 PanVera, Anti-CD42b, WGA-3 PanVeraAnti-CD43, 1D4 PanVera, Anti-CD46, MCP75-6 PanVera, Anti-CD61, PLl 1-7 PanVera, Anti-CD61, PL8-5 PanVera, Anti-CD62/P-slctn, PL7-6 PanVera, Anti-CD62/P-slctn, WGA-I PanVera, Anti-CD154, 5F3 PanVera; and anti-CDl, anti-CD2, anti-CD3, anti-CD4, anti-CD5, anti-CD6, anti-CD7, anti-CD8, anti-CD9, anti- CDlO, anti-CDl 1, anti-CD12, anti-CD13, anti-CD14, anti-CD15, anti-CD16, anti-CD17, anti- CD18, anti-CD19, anti-CD20, anti-CD21, anti-CD22, anti-CD23, anti-CD24, anti-CD25, anti- CD26, anti-CD27, anti-CD28, anti-CD29, anti-CD30, anti-CD31, anti-CD32, anti-CD33, anti- CD34, anti-CD35, anti-CD36, anti-CD37, anti-CD38, anti-CD39, anti-CD40 anti-CD41, anti- CD42, anti-CD43, anti-CD44, anti-CD45, anti-CD46, anti-CD47, anti-CD48, anti-CD49, anti- CD50, anti-CD51, anti-CD52, anti-CD53, anti-CD54, anti-CD55, anti-CD56, anti-CD57, anti- CD58, anti-CD59, anti-CD60, anti-CD61, anti-CD62, anti-CD63, anti-CD64, anti-CD65, anti- CD66, anti-CD67, anti-CD68, anti-CD69, anti-CD70, anti-CD71, anti-CD72, anti-CD73, anti-CD74, anti-CD75, anti-CD76, anti-CD77, anti-CD78, anti-CD79, anti-CD80, anti-CD81, anti-CD82, anti-CD83, anti-CD84, anti-CD85, anti-CD86, anti-CD87, anti-CD88, anti-CD89, anti-CD90, anti-CD91, anti-CD92, anti-CD93, anti-CD94, anti-CD95, anti-CD96, anti-CD97, anti-CD98, anti-CD99, anti-CD 100, anti-CD 101, anti-CD 102, anti-CD 103 , anti-CD 104, anti- CD105, anti-CD106, anti-CD107, anti-CD108, anti-CD109, anti-CDllO, anti-CDl 11, anti- CDl 12, anti-CDl 13, anti-CDl 14, anti-CDl 15, anti-CDl 16, anti-CDl 17, anti-CDl 18, anti- CDl 19, anti-CD120, anti-CD121, anti-CD122, anti-CD123, anti-CD124, anti-CD125, anti- CD126, anti-CD127, anti-CD128, anti-CD129, anti-CD130, anti-CD131, anti-CD132, anti- CD133, anti-CD134, anti-CD135, anti-CD136, anti-CD137, anti-CD138, anti-CD139, anti- CD140, anti-CD141, anti-CD142, anti-CD143, anti-CD144, anti-CD145, anti-CD146, anti- CD147, anti-CD148, anti-CD149, anti-CD150, anti-CD151, anti-CD152, anti-CD153, anti- CD154, anti-CD155, anti-CD156, anti-CD157, anti-CD158, anti-CD159, anti-CD160, anti- CD161, anti-CD162, anti-CD163, anti-CD164, anti-CD165, anti-CD166, anti-CD167, anti- CD168, anti-CD169, anti-CD170, anti-CD171, anti-CD172, anti-CD173, anti-CD174, anti- CD175, anti-CD176, anti-CD177, anti-CD178, anti-CD179, anti-CD180, anti-CD18l, anti- CD 182, anti-CD 183, anti-CD 184, anti-CDl 85, anti-CDl 86, anti-CD 187, anti-CDl 88, anti- CD189, anti-CD190, anti-CD191, anti-CD192, anti-CD193, anti-CD194, anti-CD195, anti- CD196, anti-CD197, anti-CD198, anti-CD199, anti-CD200, anti-CD201, anti-CD202, anti- CD203, anti-CD204, anti-CD205, anti-CD206, anti-CD207, anti-CD208, anti-CD209, anti- CD210, anti-CD2115 anti-CD212, anti-CD213, anti-CD214, anti-CD215, anti-CD216, anti- CD217, anti-CD218, anti-CD219, anti-CD220, anti-CD221, anti-CD222, anti-CD223, anti- CD224, anti-CD225, anti-CD226, anti-CD227, anti-CD228, anti-CD229, anti-CD230, anti- CD231, anti-CD232, anti-CD233, anti-CD234, anti-CD235, anti-CD236, anti-CD237, anti- CD238, anti-CD239, anti-CD240 anti-CD241, anti-CD242, anti-CD243, anti-CD244, anti- CD245, anti-CD246, anti-CD247, anti-CD248, anti-CD249, anti-CD250, and the like. Human Chemokine Antibodies: Human CNTF Antibodies, Human Eotaxin Antibodies, Human Epitherlial Neutrophil Activating Peptide-78, Human Exodus Antibodies, Human GRO Antibodies, Human HCC-I Antibodies, Human 1-309 Antibodies, Human IP-IO Antibodies, Human I- TAC Antibodies, Human LIF Antibodies, Human Liver-Expressed Chemokine Antibodies, Human lymphotoxin Antibodies, Human MCP Antibodies, Human MIP Antibodies, Human Monokine Induced by IFN-gamma Antibodies, Human NAP-2 Antibodies, Human NP-I Antibodies, Human Platelet Factor-4 Antibodies, Human RANTES Antibodies, Human SDF Antibodies, Human TECK Antibodies;
Murine Chemokine Antibodies: Human B-CeIl Attracting Murine Chemokine Antibodies, Chemokine- 1 Antibodies, Murine Eotaxin Antibodies, Murine Exodus Antibodies, Murine GCP-2 Antibodies, Murine KC Antibodies, Murine MCP Antibodies, Murine MIP Antibodies, Murine RANTES Antibodies, Rat Chemokine Antibodies, Rat Chemokine Antibodies, Rat CNTF Antibodies, Rat GRO Antibodies, Rat MCP Antibodies, Rat MIP Antibodies, Rat RANTES Antibodies;
Cytokine / Cytokine Receptor Antibodies: Human Biotinylated Cytokine / Cytokine Receptor Antibodies, Human IFN Antibodies, Human IL Antibodies, Human Leptin Antibodies, Human Oncostatin Antibodies, Human TNF Antibodies, Human TNF Receptor Family Antibodies, Murine Biotinylated Cytokine / Cytokine Receptor Antibodies, Murine IFN Antibodies, Murine IL Antibodies, Murine TNF Antibodies, Murine TNF Receptor Antibodies; anti-CCR4 antibody;
Rat Cytokine / Cytokine Receptor Antibodies: Rat Biotinylated Cytokine / Cytokine Receptor Antibodies, Rat IFN Antibodies, Rat IL Antibodies, Rat TNF Antibodies;
ECM Antibodies: Collagen / Procollagen, Laminin, Collagen (Human), Laminin (Human), Procollagen (Human), Vitronectin / Vitronectin Receptor, Vitronectin (Human), Vitronectin Receptor (Human), Fibronectin / Fibronectin Receptor, Fibronectin (Human), Fibronectin Receptor (Human);
Growth Factor Antibodies: Human Growth Factor Antibodies, Murine Growth Factor Antibodies, Porcine Growth Factor Antibodies; Miscellaneous Antibodies: Baculovirus Antibodies, Cadherin Antibodies,
Complement Antibodies, CIq Antibodies, VonWillebrand Factor Antibodies, Cre Antibodies, HIV Antibodies, Influenza Antibodies, Human Leptin Antibodies , Murine Leptin Antibodies, Murine CTLA-4 Antibodies, Human CTLA-4 Antibodies, P450 Antibodies, RNA Polymerase Antibodies; Neurobio Antibodies: Amyloid Antibodies, GFAP Antibodies, Human NGF
Antibodies , Human NT-3 Antibodies , Human NT-4 Antibodies.
Still other antibodies can be used in the invention and these include antibodies listed in references such as the MSRS Catalog of Primary Antibodies, and Linscott's Directory. In some preferred embodiments of the invention, the antibody is rituximab (RITUXAN), bevacizumab (AVASTIN), cetuximab (ERBITUX), trastuzumab (HERCEPTIN), tositumomab (BEXXAR), or alemtuzumab (CAMPATH). mitumomab (BEC2), CeaVac, centuximab (IMC-C225), epratuzumab (L YMPHOCIDE), MDX-210, gemtuzumab ozogamicin (MYLOTARG), edrecolomab (PANOREX), pemtumomab (THERAGYN), Zamyl, and ibritumomab tituxetan (ZEVALIN). The invention also covers antibody fragments thereof.
In some important embodiments of the invention, the antibody may be an anti-a5βl integrin antibody (e.g., Volocimixab), an anti-EGFR antibody (e.g., Nimotuzumab (TheraCIM)) , or alpha IR-3.
The antibodies or antigen-binding fragments thereof provided herein can be used additionally for delivery of toxic substances to cancer cells. Antibodies are commonly 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 such as, for example, antimetabolites, anthracyclines, vinca alkaloids, antibiotics, alkylating agents, and epipodophyllotoxins. The antibodies or antigen-binding fragments thereof may also be conjugated 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. In addition to the use of antibodies which are specific for cancer antigens, 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 thus most tumors are capable of recruiting and stimulating the growth of new blood vessels. As a result, one strategy of many cancer medicaments is to attack the blood vessels feeding a tumor and/or the connective tissues (or stroma) supporting such blood vessels.
The invention also embraces the use of adjuvants. Adjuvant substances derived from microorganisms, such as Bacillus Calmette-Guerin, heighten the immune response and enhance resistance to tumors in animals. Adjuvants that may be combined with the compounds of Formula I include alum, immunostimulatory oligonucleotides such as CpG oligonucleotides, QS-21, MLP, MPD, and the like. These and other adjuvants are listed herein.
Adjuvants include but are not limited to nucleic acid adjuvants and non-nucleic acid adjuvants. A "nucleic acid adjuvant" is an adjuvant that is a nucleic acid. Examples include immunostimulatory nucleic acid molecules such as those containing CpG dinucleotides, as described in U.S. Patents US 6,194,388Bl, issued February 27, 2001, US 6,207,646 Bl, issued March 27, 2001, and US 6,239,116 Bl, issued May 29, 2001.
A "non-nucleic acid adjuvant" is any molecule or compound except for the immunostimulatory nucleic acids described herein which can stimulate the humoral and/or cellular immune response. Non-nucleic acid adjuvants include, for instance, adjuvants that create a depo effect, immune-stimulating adjuvants, adjuvants that create a depo effect and stimulate the immune system and mucosal adjuvants.
An "adjuvant that creates a depo effect" as used herein is an adjuvant that causes an antigen, such as a cancer antigen present in a cancer vaccine, to be slowly released in the body, thus prolonging the exposure of immune cells to the antigen. This class of adjuvants includes but is not limited to alum (e.g., aluminum hydroxide, aluminum phosphate); or emulsion-based formulations including mineral oil, non-mineral oil, water-in-oil or oil-in- water-in oil emulsion, oil-in- water emulsions such as Seppic ISA series of Montanide adjuvants (e.g., Montanide ISA 720, AirLiquide, Paris, France); MF-59 (a squalene-in-water emulsion stabilized with Span 85 and Tween 80; Chiron Corporation, Emeryville, CA; and PROVAX (an oil-in- water emulsion containing a stabilizing detergent and a micelle-forming agent; IDEC Pharmaceuticals Corporation, San Diego, CA). An "immune stimulating adjuvant" is an adjuvant that causes activation of a cell of the immune system. It may, for instance, cause an immune cell to produce and secrete cytokines. This class of adjuvants includes but is not limited to saponins purified from the bark of the Q. saponaria tree, such as QS21 (a glycolipid that elutes in the 21st peak with HPLC fractionation; Antigenics, Inc., Waltham, MA); poly [di (carboxylatophenoxy) phosphazene (PCPP polymer; Virus Research Institute, USA); derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, MT), muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, WA).
"Adjuvants that create a depo effect and stimulate the immune system" are those compounds which have both of the above- identified functions. This class of adjuvants includes but is not limited to ISCOMS (Immunostimulating complexes which contain mixed saponins, lipids and form virus-sized particles with pores that can hold antigen; CSL,
Melbourne, Australia); SB-AS2 (SmithKline Beecham adjuvant system #2 which is an oil-in- water emulsion containing MPL and QS21 : SmithKline Beecham Biologicals [SBB], Rixensart, Belgium); SB-AS4 (SmithKline Beecham adjuvant system #4 which contains alum and MPL; SBB, Belgium); non-ionic block copolymers that form micelles such as CRL 1005 (these contain a linear chain of hydrophobic polyoxpropylene flanked by chains of polyoxyethylene; Vaxcel, Inc., Norcross, GA); and Syntex Adjuvant Formulation (SAF, an oil-in-water emulsion containing Tween 80 and a nonionic block copolymer; Syntex Chemicals, Inc., Boulder, CO).
A "non-nucleic acid mucosal adjuvant" as used herein is an adjuvant other than an immunostimulatory nucleic acid that is capable of inducing a mucosal immune response in a subject when administered to a mucosal surface in conjunction with an antigen. Mucosal adjuvants include but are not limited to Bacterial toxins: e.g., Cholera toxin (CT), CT derivatives including but not limited to CT B subunit (CTB) (Wu et al, 1998, Tochikubo et al, 1998); CTD53 (VaI to Asp) (Fontana et al., 1995); CTK97 (VaI to Lys) (Fontana et al., 1995); CTKl 04 (Tyr to Lys) (Fontana et al., 1995); CTD53/K63 (VaI to Asp, Ser to Lys) , (Fontana et al., 1995); CTH54 (Arg to His) (Fontana et al., 1995); CTNl 07 (His to Asn) (Fontana et al., 1995); CTEl 14 (Ser to GIu) (Fontana et al., 1995); CTEl 12K (GIu to Lys) (Yamamoto et al., 1997a); CTS61F (Ser to Phe) (Yamamoto et al., 1997a, 1997b); CTS 106 (Pro to Lys) (Douce et al., 1997, Fontana et al., 1995); andCTK63 (Ser to Lys) (Douce et al.,
1997, Fontana et al., 1995), Zonula occludens toxin, zot, Escherichia coli heat-labile enterotoxin, Labile Toxin (LT), LT derivatives including but not limited to LT B subunit (LTB) (Verweij et al., 1998); LT7K (Arg to Lys) (Komase et al., 1998, Douce et al., 1995); LT61 F (Ser to Phe) (Komase et al., 1998); LTl 12K (GIu to Lys) (Komase et al.. 1998); LTl 18E (GIy to GIu) (Komase et al., 1998); LTl 46E (Arg to GIu) (Komase et al., 1998); LTl 92G (Arg to GIy) (Komase et al., 1998); LTK63 (Ser to Lys) (Marchetti et al., 1998, Douce et al., 1997, 1998, Di Tommaso et al., 1996); and LTR72 (Ala to Arg) (Giuliani et al., 1998), Pertussis toxin, PT. (Lycke et al., 1992, Spangler BD, 1992, Freytag and Clemments, 1999, Roberts et al., 1995, Wilson et al., 1995) including PT-9K/129G (Roberts et al., 1995, Cropley et al., 1995); Toxin derivatives (see below) (Holmgren et al., 1993, Verweij et al.,
1998, Rappuoli et al., 1995, Freytag and Clements, 1999); Lipid A derivatives (e.g., monophosphoryl lipid A, MPL) (Sasaki et al., 1998, Vancott et al., 1998; Muramyl Dipeptide (MDP) derivatives (Fukushima et al., 1996, Ogawa et al., 1989, Michalek et al., 1983, Morisaki et al., 1983); Bacterial outer membrane proteins (e.g., outer surface protein A (OspA) lipoprotein of Borrelia burgdorferi, outer membrane protine of Neisseria meningitiάis)(MeLrm' axo et al., 1999, Van de Verg et al., 1996); Oil-in-water emulsions (e.g., MF59) (Barchfield et al., 1999, Verschoor et al., 1999, O'Hagan, 1998); Aluminum salts (Isaka et al., 1998, 1999); and Saponins (e.g., QS21) Aquila Biopharmaceuticals, Inc., Worcester, MA) (Sasaki et al., 1998, MacNeal et al., 1998), ISCOMS, MF-59 (a squalene-in- water emulsion stabilized with Span 85 and Tween 80; Chiron Corporation, Emeryville, CA); the Seppic ISA series of Montanide adjuvants (e.g., Montanide ISA 720; AirLiquide, Paris, France); PROVAX (an oil-in-water emulsion containing a stabilizing detergent and a micell- forming agent; IDEC Pharmaceuticals Corporation, San Diego, CA); Syntext Adjuvant Formulation (SAF; Syntex Chemicals, Inc., Boulder, CO); poly[di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA) and Leishniania elongation factor (Corixa Corporation, Seattle, WA).
The methods and compositions of the invention can further include chemotherapeutic agents such as but not limited to those currently recited herein. Several chemotherapeutic agents can be categorized as DNA damaging agents and these include topoisomerase inhibitors (e.g., etoposide, ramptothecin, topotecan, teniposide, mitoxantrone), antimicrotubule agents (e.g., vincristine, vinblastine), antimetabolites (e.g., cytarabine, methotrexate, hydroxyurea, 5-fluorouracil, floxuridine, 6-thioguanine, 6-mercaptopurine, fludarabine, pentostatin, chlorodeoxyadenosine), DNA alkylating agents (e.g., cisplatin, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chorambucil, busulfan, thiotepa, carmustine, lomustine, carboplatin, dacarbazine, procarbazine), DNA strand break inducing agents (e.g., bleomycin, doxorubicin, daunorubicin, idarubicin, mitomycin C), and radiation therapy.
Important chemotherapeutic agents are those selected from the group consisting of: Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Adriamycin; Aldesleukin; Alitretinoin; Allopurinol Sodium; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Annonaceous Acetogenins; Anthramycin; Asimicin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bexarotene; Bicalutamide; Bisantrene Hydrochloride; Bisnafϊde Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Bullatacin; Busulfan; Cabergoline; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Celecoxib; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine;
Dacarbazine; DACA (N-[2-(Dimethyl-amino)ethyl]acridine-4-carboxamide); Dactinomycin; Daunorubicin Hydrochloride; Daunomycin; Decitabine; Denileukin Diftitox; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflornithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Ethiodized Oil 1 131; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; 5-FdUMP; Flurocitabine; Fosquidone; Fostriecin Sodium; FK-317; FK-973; FR-66979; FR-900482; Gemcitabine; Gemcitabine Hydrochloride; Gemtuzumab Ozogamicin; Gold Au 198; Goserelin Acetate; Guanacone; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-nl; Interferon Alfa-n3; Interferon Beta- 1 a; Interferon Gamma- 1 b; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Methoxsalen; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mytomycin C; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic
Acid; Nocodazole; Nogalamycin; Oprelvekin; Ormaplatin; Oxisuran; Paclitaxel; Pamidronate
Disodium; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide;
Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin
Hydrochloride; Pyrazofurin; Riboprine; Rituximab; Rogletimide; Rolliniastatin; Safingol;
Safingol Hydrochloride; Samarium/Lexidronam; Semustine; Simtrazene; Sparfosate Sodium;
Sparsomycin; Spiro germanium Hydrochloride; Spiromustine; Spiroplatin; Squamocin;
Squamotacin; Streptonigrin; Streptozocin; Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane; Taxoid; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin;
Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Thymitaq;
Tiazofurin; Tirapazamine; Tomudex; TOP-53; Topotecan Hydrochloride; Toremifene Citrate;
Trastuzumab; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate
Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Valrubicin; Vapreotide; Verteporfin; Vinblastine; Vinblastine Sulfate; Vincristine; Vincristine Sulfate;
Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate;
Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin;
Zinostatin; Zorubicin Hydrochloride; 2-Chlorodeoxyadenosine; 2'-Deoxyformycin; 9- aminocamptothecin; raltitrexed; N-propargyl-5,8-dideazafolic acid; 2-chloro-2'-arabino- fluoro-2'-deoxyadenosine; 2-chloro-2'-deoxyadenosine; anisomycin; trichostatin A; hPRL-
G129R; CEP-751; linomide; sulfur mustard; nitrogen mustard (mechlor ethamine); cyclophosphamide; melphalan; chlorambucil; ifosfamide; busulfan; N-methyl-N-nitrosourea
(MNU); N, N'-Bis(2-chloroethyl)-N-nitrosourea (BCNU); N-(2-chloroethyl)-N'-cyclohexyl-
N-nitrosourea (CCNU); N-(2-chloroethyl)-N'-(trans-4-methylcyclohexyl-N-nitrosourea (MeCCNU); N-(2-chloroethyl)-N'-(diethyl)ethylphosphonate-N-nitrosourea (fotemustine); streptozotocin; diacarbazine (DTIC); mitozolomide; temozolomide; thiotepa; mitomycin C;
AZQ; adozelesin; Cisplatin; Carboplatin; Ormaplatin; Oxaliplatin; C 1-973; DWA 2114R;
JM216; JM335; Bis (platinum); tomudex; azacitidine; cytarabine; gemcitabine; 6-
Mercaptopurine; 6-Thioguanine; Hypoxanthine; teniposide; 9-amino camptothecin; Topotecan; CPT-11 ; Doxorubicin; Daunomycin; Epirubicin; darubicin; mitoxantrone; losoxantrone; Dactinomycin (Actinomycin D); amsacrine; pyrazoloacridine; all-trans retinol;
14-hydroxy-retro-retinol; all-trans retinoic acid; N-(4-Hydroxyphenyl) retinamide; 13-cis retinoic acid; 3-Methyl TTNEB; 9-cis retinoic acid; fludarabine (2-F-ara-AMP); 2- chlorodeoxyadenosine (2-Cda).
Other chemotherapeutic agents include: 20-epi-l,25 dihydroxyvitamin D3;
5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukii ALL-TK antagonists; altretamine; ambamustine; atnidox; amifostine; aminolevulinic acid amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein- 1; antiandro prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1 axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balan< batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaui'osporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bleomycin A2; bleomycin B2; bropiriminβ; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives (e.g., 10-hydroxy- camptothecin); canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castano spermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;
2'deoxycoformycin (DCF); deslorelin; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; discodermolide; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epothilones (A, R = H; B, R = Me); epithilones; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide; etoposide 4'-phosphate (etopofos); exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; homoharringtonine (HHT); hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor- 1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; irinotecan; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide + estrogen + progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum or platinum containig compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirirnostim; mismatched double stranded RNA; mithracin; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A + myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1 -based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone + pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; 06-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum or platinum-containing compounds; platinum-triamine complex; podophyllotoxin; porfϊmer sodium; porfiromycin; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohituldne; romurtide; roquinimex; rubiginone Bl; ruboxyl; safϊngol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1 ; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfmosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thalidomide; thiocoraline; thrombopoietin; tlirombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene ' dichloride; topotecan; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfm; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; zinostatin stimalamer.
Other chemotherapeutic agents include: Antiproliferative agents (e.g., Piritrexim Isothionate), Antiprostatic hypertrophy agent (e.g., Sitogluside), Benign prostatic hyperplasia therapy agents (e.g., Tamsulosin Hydrochloride), Prostate growth inhibitor agents (e.g., Pentomone), and Radioactive agents: Fibrinogen 1 125; Fludeoxyglucose F 18; Fluorodopa F 18; Insulin 1 125; Insulin 1 131; Iobenguane 1 123; Iodipamide Sodium 1 131; Iodoantipyrine I 131; Iodocholesterol 1 131; Iodohippurate Sodium 1 123; Iodohippurate Sodium 1 125; Iodohippurate Sodium 1 131; Iodopyracet 1 125; Iodopyracet 1 131; Iofetamine Hydrochloride 1 123; Iomethin 1 125; Iomethin 1 131; Iothalamate Sodium 1 125; Iothalamate Sodium 1 131; Iotyrosihe 1 131; Liothyronine 1 125; Liothyronine 1 131; Merisoprol Acetate Hg 197; Merisoprol Acetate Hg 203; Merisoprol Hg 197; Selenomethionine Se 75; Technetium Tc 99m Antimony Trisulfide Colloid; Technetium Tc 99m Bicisate; Technetium Tc 99m Disofenin; Technetium Tc 99m Etidronate; Technetium Tc 99m Exametazime; Technetium Tc 99m Furifosmin; Technetium Tc 99m Gluceptate; Technetium Tc 99m Lidofenin; Technetium Tc 99m Mebrofenin; Technetium Tc 99m Medronate; Technetium Tc 99m Medronate Disodium; Technetium Tc 99m Mertiatide; Technetium Tc 99m Oxidronate; Technetium Tc 99m Pentetate; Technetium Tc 99m Pentetate Calcium Trisodium; Technetium Tc 99m Sestamibi; Technetium Tc 99m Siboroxime; Technetium Tc 99m Succimer; Technetium Tc 99m Sulfur Colloid; Technetium Tc 99m Teboroxime; Technetium Tc 99m Tetrofosmin; Technetium Tc 99m Tiatide; Thyroxine 1 125; Thyroxine 1 131; Tolpovidone 1 131; Triolein 1 125; Triolein 1 131.
Another category of chemotherapeutic agents is anti-cancer Supplementary Potentiating Agents, including: Tricyclic anti-depressant drugs (e.g., imipramine, desipramine, amitryptyline, clomipramine, trimipramine, doxepin, nortriptyline, protriptyline, amoxapine and maprotiline); non-tricyclic anti-depressant drugs (e.g., sertraline, trazodone and citalopram); Ca""" antagonists (e.g., verapamil, nifedipine, nitrendipine and caroverine); Calmodulin inhibitors (e.g., prenylamine, trifluoroperazine and clomipramine); Amphotericin B; Triparanol analogues (e.g., tamoxifen); antiarrhythmic drugs (e.g., quinidine); antihypertensive drugs (e.g., reserpine); Thiol depleters (e.g., buthionine and sulfoximine) and Multiple Drug Resistance reducing agents such as Cremaphor EL. Some important chemotherapeutic agents are those selected from the group consisting of: annonaceous acetogenins; asimicin; rolliniastatin; guanacone, squamocin, bullatacin; squamotacin; taxanes; paclitaxel; gemcitabine; methotrexate FR-900482; FK-973; FR-66979; FK-317; 5-FU; FUDR; FdUMP; Hydroxyurea; Docetaxel; discodermolide; epothilones; vincristine; vinblastine; vinorelbine; meta-pac; irinotecan; SN-38; 10-OH campto; topotecan; etoposide; adriamycin; flavopiridol; platinum-containing compounds (e.g., cisplatin(Cis-Pt) and carboplatin (carbo-Pt); bleomycin; mitomycin C; mithramycin; capecitabine; cytarabine; 2-Cl-2'deoxy adenosine; Fludarabine-PO4; mitoxantrone; mitozolomide; Pentostatin; Tomudex.
In important embodiments, the agents are administered together with chemotherapeutic agents selected from the group consisting of aldesleukin, asparaginase, bleomycin sulfate, carboplatin, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, docetaxel, doxorubicin, doxorubicin hydrochloride, epirubicin hydrochloride, etoposide, etoposide phosphate, floxuridine, fludarabine, fluorouracil, gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, interferons, interferon-α2a, interferon-α2b, interferon- αn3, interferon-αlb, interleukins, irinotecan, mechlorethamine hydrochloride, melphalan, mercatopurine, methotrexate, methotrexate sodium, mitomycin, mitoxantrone, paclitaxel, pegaspargase, pentostatin, prednisone, profϊmer sodium, procabazine hydrochloride, taxol, docetaxel (TAXOTERE®), teniposide, topotecan hydrochloride, vinblastine sulfate, vincristine sulfate and vinorelbine tartrate.
Other cancer therapies include hormonal manipulation. Compounds of Formula I and pemetrexed are also useful in combination with tamoxifen or aromatase inhibitor arimidex (i.e., anastrozole).
The methods and compositions of the invention may be used with enzyme inhibitor agents such as CDK inhibitors, tyrosine kinase inhibitors and MAP kinase inhibitors.
The tyrosine kinase inhibitor is selected from the group consisting of Genistein (4',5,7-trihydroxyisoflavone), Tyrphostin 25 (3,4,5-trihydroxyphenyl), methylene]- propanedinitrile, Herbimycin A, Daidzein (4',7-dihydroxyisoflavone), AG-126, trans-l-(3'- carboxy-4'-hydroxyphenyl)-2-(2"55"-dihydroxy-phenyl)ethane, and HDBA (2-Hydroxy5- (2,5-Dihydroxybenzylamino)-2-hydroxybenzoic acid. In another embodiment, the CDK inhibitor is selected from the group consisting of p21, p27, p57, pl5, pl6, pl8, and pl9. In another embodiment, the MAP kinase inhibitor is selected from the group consisting of KY12420 (C23H24O8), CNI-1493, PO98059, 4-(4-Fluorophenyl)-2-(4-methylsulfmyl phenyl)- 5-(4-pyridyl) lH-imidazole.
The methods and compositions of the invention may be used in subjects having or at risk of developing cancer. A subject having a cancer is a subject that has detectable cancerous cells. A subject at risk of developing a cancer is one who has a higher than normal probability of developing cancer. These subjects include, for instance, subjects having a genetic abnormality that has been demonstrated to be associated with a higher likelihood of developing a cancer, subjects having a familial disposition to cancer, subjects exposed to cancer causing agents (i.e., carcinogens) such as tobacco, asbestos, or other chemical toxins, and subjects previously treated for cancer and in apparent remission. "Cancer" as used herein refers to an uncontrolled growth of cells which interferes with the normal functioning of the bodily organs and systems. Cancers which migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs. Hemopoietic cancers, such as leukemia, are able to outcompete the normal hemopoietic compartments in a subject, thereby leading to hemopoietic failure (in the form of anemia, thrombocytopenia and neutropenia) ultimately causing death.
A metastasis is a region of cancer cells, distinct from the primary tumor location resulting from the dissemination of cancer cells from the primary tumor to other parts of the body. At the time of diagnosis of the primary tumor mass, the subject may be monitored for the presence of metastases. Metastases are most often detected through the sole or combined use of magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, blood and platelet counts, liver function studies, chest X-rays and bone scans in addition to the monitoring of specific symptoms.
A cancer cell is a cell that divides and reproduces abnormally due to a loss of normal growth control. Cancer cells almost always arise from at least one genetic mutation. In some instances, it is possible to distinguish cancer cells from their normal counterparts based on profiles of expressed genes and proteins, as well as to the level of their expression. Genes commonly affected in cancer cells include oncogenes, such as ras, neu/HER2/erbB, myb, myc and abl, as well as tumor suppressor genes such as p53, Rb, DCC, RET and WT. Cancer- related mutations in some of these genes leads to a decrease in their expression or a complete deletion. In others, mutations cause an increase in expression or the expression of an activated variant of the normal counterpart. The term "tumor" is usually equated with neoplasm, which literally means "new growth" and is used interchangeably with "cancer." A "neoplastic disorder" is any disorder associated with cell proliferation, specifically with a neoplasm. A "neoplasm" is an abnormal mass of tissue that persists and proliferates after withdrawal of the carcinogenic factor that initiated its appearance. There are two types of neoplasms, benign and malignant. Nearly all benign tumors are encapsulated and are noninvasive; in contrast, malignant tumors are almost never encapsulated but invade adjacent tissue by infiltrative destructive growth. This infiltrative growth can be followed by tumor cells implanting at sites discontinuous with the original tumor. The method of the invention can be used to treat neoplastic disorders in humans, including but not limited to: sarcoma, carcinoma, fibroma, leukemia, lymphoma, melanoma, myeloma, neuroblastoma, rhabdomyosarcoma, retinoblastoma, and glioma as well as each of the other tumors described herein.
Cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cervical cancer; choriocarcinoma; 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 including acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia; liver cancer; lung cancer (e.g., small cell lung cancer and non-small cell lung cancer); lymphoma including Hodgkin's andNon-Hodgkin's lymphoma; melanoma; mesothelioma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; renal cancer; cancer of the respiratory system; sarcoma; skin cancer; stomach cancer; testicular cancer; thyroid cancer; uterine cancer; cancer of the urinary system, as well as other carcinomas and sarcomas.
In some important embodiments, the cancer is non-small cell lung cancer and more particularly adenocarincoma or squamous cell carcinoma.
Carcinomas are cancers of epithelial origin. Carcinomas intended for treatment with the methods of the invention include, but are not limited to, acinar carcinoma, acinous carcinoma, alveolar adenocarcinoma (also called adenocystic carcinoma, adenomyoepithelioma, cribriform carcinoma and cylindroma), carcinoma adenomatosum, adenocarcinoma, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma (also called bronchiolar carcinoma, alveolar cell tumor and pulmonary adenomatosis), basal cell carcinoma, carcinoma basocellulare (also called basaloma, or basiloma, and hair matrix carcinoma), basaloid carcinoma, basosquamous cell carcinoma, breast carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma (also called cholangioma and cholangiocarcinoma), chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epibulbar carcinoma, epidermoid carcinoma, carcinoma epitheliale adenoides, carcinoma exulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma (also called hepatoma, malignant hepatoma and hepatocarcinoma), Hϋrthle cell carcinoma, hyaline carcinoma, hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma mastitoides, carcinoma medullare, medullary carcinoma, carcinoma melanodes, melanotic carcinoma, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, carcinoma nigrum, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, ovarian carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prostate carcinoma, renal cell carcinoma of kidney (also called adenocarcinoma of kidney and hypernephoroid carcinoma), reserve cell carcinoma, carcinoma sarcomatodes, scheinderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, carcinoma vilosum. In preferred embodiments, the methods of the invention are used to treat subjects having cancer of the breast, cervix, ovary, prostate, lung, colon and rectum, pancreas, stomach or kidney.
Another important cancer type is sarcomas. Sarcomas are rare mesenchymal neoplasms that arise in bone and soft tissues. Different types of sarcomas are recognized and these include: liposarcomas (including myxoid liposarcomas and pleomorphic 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., non-bone) Ewing's sarcoma, and primitive neuroectodermal tumor [PNET]), synovial sarcoma, angiosarcomas, hemangiosarcomas, lymphangiosarcomas, Kaposi's sarcoma, hemangioendothelioma, fibrosarcoma, desmoid tumor (also called aggressive fibromatosis), dermatofibrosarcoma protuberans (DFSP), malignant fibrous histiocytoma (MFH), hemangiopericytoma, malignant mesenchymoma, alveolar soft-part sarcoma, epithelioid sarcoma, clear cell sarcoma, desmoplastic small cell tumor, gastrointestinal stromal tumor (GIST) (also known as GI stromal sarcoma), osteosarcoma (also known as osteogenic sarcoma)-skeletal and extraskeletal, and chondrosarcoma.
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. These cancers may appear initially responsive to a treatment (and then they may recur), or they may be completely non-responsive to the treatment from the outset. 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. Subjects being treated according to the invention for a refractory cancer therefore may have already been exposed to another treatment for their cancer. Alternatively, if the cancer is likely to be refractory (e.g., given an analysis of the cancer cells or history of the subject), then the subject may not have already been exposed to another treatment. In some embodiments, the subject is administered a combination therapy wherein one of the agents was previously administered to the subject and to which the cancer was previously considered refractory. In these latter embodiments as well as others contemplated by the invention, the compound of Formula I acts to enhance the effect of the other therapy and thus the response of the cancer to the other therapy.
Examples of refractory cancers include but are not limited to leukemias, melanomas, renal cell carcinomas, colon cancer, liver (hepatic) cancers, pancreatic cancer, Non-Hodgkin's lymphoma, and lung cancer including non-small cell lung cancer such as adenocarcinoma and squamous cell carcinoma of the lung.
In some important embodiments, refractory cancers are cancers that are refractory to treatment with platinum-containing compounds, such as carboplatin and cisplatin.
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. Examples of immunogenic cancers include malignant melanoma and renal cell cancer. Subjects at risk of developing a cancer include subjects that are known or are suspected of being exposed to a carcinogen. A carcinogen is an agent capable of initiating development of malignant cancers. Exposure to carcinogens generally increases the risk of neoplasms in subjects, usually by affecting DNA directly. Carcinogens may take one of several forms such as chemical, electromagnetic radiation, or may be an inert solid body. Examples of chemical carcinogens include tobacco, asbestos, and the like.
A subject shall mean a human or animal including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent e.g., rats and mice, primate, e.g., monkey, and fish or aquaculture species such as fin fish (e.g., salmon) and shellfish (e.g., shrimp and scallops). Subjects suitable for therapeutic or prophylactic methods include vertebrate and invertebrate species. Subjects can be house pets (e.g., dogs, cats, fish, etc.), agricultural stock animals (e.g., cows, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), zoo animals (e.g., lions, giraffes, etc.), but are not so limited. Although many of the embodiments described herein relate to human disorders, the invention is also useful for treating other nonhuman vertebrates.
The term "effective amount" of either or the combination of compounds refers to the amount necessary or sufficient to realize a desired biologic effect. Generally, an effective amount is that amount that provides a biologically beneficial effect. The biologically beneficial effect may be the amelioration and or absolute elimination of symptoms resulting from the disorder being treated in the short term (e.g., weeks or months) or the long term (e.g., years). In another embodiment, the biologically beneficial effect is the complete abrogation of the disorder as evidenced for example, by the absence of a tumor or a biopsy or blood smear which is free of cancer cells. The effective amount may vary depending upon the particular compound and the particular antibody used. The effective amount for any particular application can also vary depending on such factors as the cancer being treated, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular compound of Formula I and anti-cancer agent(s) combination without necessitating undue experimentation. Combined with the teachings provided herein, by choosing among the various compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and preferred mode of administration, an effective therapeutic or prophylactic treatment regimen can be planned which does not cause substantial toxicity and yet is entirely effective to treat the particular subject.
In some instances, a sub-therapeutic dosage of either the compound of Formula I or the anti-cancer agent(s), or a sub-therapeutic dosage of both, is used in the treatment of a subject having, or at risk of developing, cancer. For example, when the combination of VaI- boroPro and erlotinib is used, erlotinib can be administered in a sub-therapeutic dose and still produce a desirable therapeutic result. A "sub-therapeutic dose" as used herein refers to a dosage which is less than the dosage that would produce a therapeutic result in the subject if administered in the absence of the other agent. Thus, the sub-therapeutic dose of an anticancer agent is one which would not produce the desired therapeutic result in the subject in the absence of the administration of the compound of Formula I. Therapeutic doses of anticancer agents are well known in the field of medicine for the treatment of cancer. These dosages have been extensively described in references such as Remington's Pharmaceutical Sciences, 18th ed., 1990, or the Physician Desktop Reference; as well as many other medical references relied upon by the medical profession as guidance for the treatment of cancer.
For any compound described herein a therapeutically effective amount can be initially determined from cell culture assays. In particular, the effective amount of a compound of Formula I can be determined using in vitro stimulation assays. The stimulation index of immune cells can be used to determine an effective amount of the particular compound for the particular subject, and the dosage can be adjusted upwards or downwards to achieve the desired levels in the subject.
Therapeutically effective amounts can also be determined in animal studies. For instance, the effective amount of a compound of Formula I and pemetrexed, erlotinib, or docetaxel to induce a response, including a synergistic response, can be assessed using in vivo assays of tumor regression and/or prevention of tumor formation. Relevant animal models include assays in which malignant cells are injected into the animal subjects, usually in a defined site. Generally, a range of compound of Formula I doses are administered into the animal along with a range of pemetrexed, erlotinib, or docetaxel doses. Inhibition of the growth of a tumor following the injection of the malignant cells is indicative of the ability to reduce the risk of developing a cancer. Inhibition of further growth (or reduction in size) of a pre-existing tumor is indicative of the ability to treat the cancer. Mice which have been modified to have human immune system elements can be used as recipients of human cancer cell lines to determine the effective amount of the synergistic combination.
In some important embodiments, an effective amount of a combination of a compound of Formula I and pemetrexed is an amount that reduces tumor size or volume or cancer load (e.g., as measured by number of lesions or extent of metastasis) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of pretreatment tumor size or volume or cancer load. In preferred embodiments, the effective amount is one that reduces the tumor size, volμme or cancer load by at least 50% or at lest 60% of the pretreatment tumor size or volume or cancer load.
In some important embodiments, an effective amount of a combination of a compound of Formula I and erlotinib in an amount that reduces tumor size or volume or cancer load by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of pretreatment tumor size or volume or cancer load.
The applied dose of both agents can be adjusted based on the relative bioavailability and potency of the administered compounds, including the adjuvants used. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods are well within the capabilities of the ordinarily skilled artisan.
Subject doses of the compounds described herein will depend on the particular compound although they typically range from about 0.1 μg to 10,000 mg per day, more typically from about 50 μg to 5000 mg per day, even more typically from about 100 μg to 2000 mg per day, and most typically from about 200 μg to 1000 mg per day.
In some important embodiments, the compound of Formula I is administered in amounts of less than or equal to 1.0 mg/kg per day. This includes amounts equal to or less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 and 0.1 mg/kg per day. The agents may also be administered in amounts of equal to or less than 0.1 mg/kg per day. This includes amounts equal to or less than 0.09, 0.08, 0.07, 0.06, 0.5, 0.04, 0.03, 0.02 or 0.01 mg/kg/day. In some embodiments, the agents are administered in a range of about 0.005 mg/kg per day to less than 1.0 mg/kg per day (or about 0.005 mg/kg per day to equal to or less than 0.1 mg/kg per day).
In methods particularly directed at subjects at risk of developing a disorder, timing of the administration of the compound of Formula I and pemetrexed, erlotinib or docetaxel may be particularly important. For instance, in a subject with a genetic predisposition to cancer, the agents may be administered to the subject on a routine schedule.
A "routine schedule" as used herein, refers to a predetermined designated period of time. The routine schedule may encompass periods of time which are identical or which differ in length, as long as the schedule is predetermined. For instance, the routine schedule may involve administration on a daily basis, every two days, every three days, every four days, every five days, every six days, a weekly basis, a monthly basis or any set number of days or weeks there-between, every two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, etc. Alternatively, the predetermined routine schedule may involve administration on a daily basis for the first week, followed by a monthly basis for several months, and then every three months after that. Any particular combination would be covered by the routine schedule as long as it is determined ahead of time that the appropriate schedule involves administration on a certain day.
In some embodiments the compound of Formula I is administered orally and pemetrexed is administered parenterally. In some embodiments the compound of Formula I is administered prior to pemetrexed. In other embodiments the compound of Formula I is administered after pemetrexed.
In some embodiments pemetrexed is administered about 10 minutes to 24 hours prior to the compound of Formula I. In another embodiment, pemetrexed is administered 1-10 days prior to the compound of Formula I. The pemetrexed may be administered on a first day of multi-day cycle, with the compound of Formula I administered on the remaining days of the cycle. The cycle may be a 2, 3, 4, 5, 6, 7, or more day cycle. The compound of Formula I may be administered once, twice, or more times per day. In one embodiment, pemetrexed is administered on the first day of a seven day cycle, followed by a twice daily administration of the compound of Formula I on each of the remaining days of the seven day cycle.
The multi-day cycle may be repeated twice, thrice, four times, or more. It may also be repeated for various lengths of time, including but not limited to a week, a month, two months, or more. The multi-day cycle may be at least 7, at least 14, at least 21, at least 28, at least 35 days or more in length. The agents such as pemetrexed and docetaxel and any platinum-containing compound may be administered at the beginning of such cycles for one or more days (e.g., days 1, 2 and 3), followed by the administration of the compound of Formula I (preferably Val-boroPro) for at least 7, at least 14, or more days, followed by a rest period in which the subject is administered no anti-cancer therapy but may be administered a placebo. In yet another embodiment, the anti-cancer agent is administered substantially simultaneously with the compound of Formula I. As used herein, the term "substantially simultaneously" means that the compounds are administered within minutes of each other (e.g., within 10 minutes of each other) and intends to embrace joint administration as well as consecutive administration, but 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). As used herein, concurrent administration and substantially simultaneous administration are used interchangeably. In some embodiments single or multiple administrations of pemetrexed followed by 7-14 day courses of twice a day (BID) administration of varying doses of a compound of Formula I (e.g., Val-boroPro (talabostat)) are contemplated.
In some important embodiments pemetrexed is administered on day 1 of a multi-day cycle (e.g., a 21 day cycle). In some important embodiments, the compound of Formula I is administered on days 2-15 of the same cycle.
In some other embodiments pemetrexed is administered on days 1-3 of a multi-day cycle (e.g., a 21, 24, 28 or 31 day cycle). In some important embodiments the compound of Formula I is administered on days 4-17 of the same cycle. The remaining days of the cycle may be a rest period (treatment-free period).
The cycles described herein may be repeated one, two, three, four, five, six or more times with or without an additional rest period (treatment-free period) in between each cycle. The intervening rest periods may last days or weeks.
In one embodiment, pemetrexed is administered at a dose of 500 mg/m2 15-25 days apart. Each administration of pemetrexed is followed by administration of 100-500 μg of a compound of Formula I (e.g., Val-boroPro (talabostat)) BID orally the following day and for 7-14 consecutive days. This may be followed by a 7-day rest period (treatment-free period), then followed by administration of 100 -500 μg of a compound of Formula I (e.g., Val- boroPro (talabostat)) BID orally for another 7-14 consecutive days. This cycle may be repeated as necessary. Pemetrexed may be administered at doses ranging from 100 to 800 mg/m2.
In another embodiment, pemetrexed 400-700 mg/kg is administered for 3 consecutive days followed by 100-500 μg of a compound of Formula I (e.g., Val-boroPro (talabostat)) for 10-20 consecutive days. In one embodiment, 100-500 μg of a compound of Formula I (e.g., Val-boroPro
(talabostat)) is administered twice daily (BID) and 150 mg erlotinib (T ARCEV A™) is administered once per day for at least 7, at least 10, at least 21, at least 28 or more days in a cycle followed by a 1-7 day rest period (treatment-free period).
In one embodiment, docetaxel is administered at a dose of 75 mg/m2 on day 1 of a 22 day cycle and 100-500 μg of a compound of Formula I (e.g., Val-boroPro (talabostat)) is administered twice daily (BID) on day 2-15 of the 22 day cycle followed by a 7-day rest period (treatment-free period). The combination therapies may also be followed by repeated multi-day cycles of compound of Formula I optionally with rest periods. For example, following any number of combination multi-day cycles, the subject may be treated with one, two or more multi-day cycles of compound of Formula I. The latter cycles may include administration of the agent on days 2-15 of 22 day cycle (with a rest period on days 16-22). Administration of the agent may be once or twice a day but it is not so limited.
The compounds of the invention may be administered neat, or in the context of a vector or delivery system. An example of a chemical/physical vector of the invention is a colloidal dispersion system. Colloidal dispersion systems include lipid-based systems including oil-in- water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system of the invention is a liposome. Liposomes are artificial membrane vessels which are useful as a delivery vector in vivo or in vitro. It has been shown that large unilamellar vessels (LUV), which range in size from 0.2 - 4.0 μm can encapsulate large macromolecules. Liposomes may be targeted to a particular tissue by coupling the liposome to a specific ligand such as a sugar, glycolipid, or protein. Ligands which may be useful for targeting a liposome to an immune cell include, but are not limited to intact or fragments of molecules which interact with immune cell specific receptors and molecules, such as antibodies, which interact with the cell surface markers of immune cells. Such ligands may easily be identified by binding assays well known to those of skill in the art. In still other embodiments, the liposome may be targeted to the cancer by coupling it to a one of the immunotherapeutic antibodies discussed earlier. Additionally, the vector may be coupled to a nuclear targeting peptide, which will direct the vector to the nucleus of the host cell.
Lipid formulations for transfection are commercially available from QIAGEN, for example, as EFFECTENE™ (a non-liposomal lipid with a special DNA condensing enhancer) and SUPERFECT™ (a novel acting dendrimeric technology).
Liposomes are commercially available from Gibco BRL, for example, as LIPOFECTIN™ and LIPOFECTACE™, which are formed of cationic lipids such as N-[I -(2, 3 dioleyloxy)-propyl]-N, N, N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB). Methods for making liposomes are well known in the art and have been described in many publications. Liposomes also have been reviewed by Gregoriadis, G. in Trends in Biotechnology, (1985) 3:235-241. In another embodiment the chemical/physical vector is a biocompatible microsphere that is suitable for delivery, such as oral or mucosal delivery. Such microspheres are disclosed in Chickering et al, Biotech. And Bioeng. , (1996) 52:96-101 and Mathiowitz et al., Nature, (1997) 386-..410-414 and PCT Patent Application WO97/03702. Both non-biodegradable and biodegradable polymeric matrices can be used to deliver the compound of Formula I and/or the anti-cancer agent(s) to the subject. Biodegradable matrices are preferred. Such polymers may be natural or synthetic polymers. 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 multi-valent ions or other polymers.
The polymeric matrix preferably is in the form of a microparticle such as a microsphere (wherein the agents are dispersed throughout a solid polymeric matrix) or a microcapsule (wherein the agents are stored in the core of a polymeric shell). Other forms of the polymeric matrix for containing the agents 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 is introduced. The size of the polymeric matrix further is 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. Preferably when an aerosol route is used the polymeric matrix and the compound of Formula I and the pemetrexed, erlotinib, or docetaxel are encompassed in a surfactant vehicle. 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 matrix is administered to a nasal and/or pulmonary surface that has sustained an injury. The matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time. In some preferred embodiments, the compounds of Formula I are administered to the subject via an implant while the anti-cancer agent(s) is administered acutely. Bioadhesive polymers of particular interest include bioerodible hydrogels described by
H.S. Sawhney, C.P. Pathak and J.A. Hubell in Macromolecules, (1993) 26:581-587, the teachings of which are incorporated herein, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), polyQaurel methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate). Other delivery vehicles can be used and these include cochleates (Gould-Fogerite et al., 1994, 1996); Emulsomes (Vancott et al., 1998, Lowell et al., 1997); ISCOMs (Mowat et al., 1993, Carlsson et al., 1991, Hu et., 1998, Morein et al., 1999); liposomes (Childers et al., 1999, Michalek et al., 1989, 1992, de Haan 1995a, 1995b); live bacterial vectors (e.g., Salmonella, Escherichia coli, Bacillus calmatte-guerin, Shigella, Lactobacillus) (Hone et al., 1996, Pouwels et al., 1998, Chatfield et al., 1993, Stover et al., 1991, Nugent et al., 1998); live viral vectors (e.g., Vaccinia, adenovirus, Herpes Simplex) (Gallichan et al., 1993, 1995, Moss et al., 1996, Nugent et al., 1998, Flexner et al., 1988, Morrow et al., 1999); microspheres (Gupta et al., 1998, Jones et al., 1996, Maloy et al., 1994, Moore et al., 1995, O'Hagan et al., 1994, Eldridge et al., 1989); nucleic acid vaccines (Fynan et al., 1993, Kuklin et al., 1997, Sasaki et al., 1998, Okada et al., 1997, Ishii et al., 1997); polymers (e.g. carboxymethylcellulose, chitosan) (Hamajima et al., 1998, Jabbal-Gill et al., 1998); polymer rings (Wyatt et al., 1998); proteosomes (Vancott et al., 1998, Lowell et al., 1988, 1996, 1997); sodium fluoride (Hashi et al., 1998); transgenic plants (Tacket et al., 1998, Mason et al., 1998, Haq et al., 1995); virosomes (Gluck et al., 1992, Mengiardi et al., 1995, Cryz et al., 1998); and, virus-like particles (Jiang et al., 1999, Leibl et al., 1998).
The compositions and methods of the invention in certain instances may be useful for replacing existing surgical procedures or drug therapies. In some instances the present invention is useful in improving the efficacy of existing therapies for treating such conditions. Accordingly, compositions and methods of the invention may be used to treat the subjects that are undergoing or that will undergo a treatment for cancer. For example, the agents may be administered to a subject in combination with other anti-cancer therapies. Suitable anticancer therapies include surgical procedures to remove the tumor mass, chemotherapy or localized radiation. The other antiproliferative therapy may be administered before, concurrent with, or after treatment with the compositions and methods of the invention. There may also be a delay of several hours, days and in some instances weeks between the administration of the different treatments, such that the compositions and methods of the invention may be administered before or after the other treatment. Although not intending to be bound by any particular mechanism, it is proposed that the administration of compounds of Formula I induce memory within the immune cell compartment, for example, by the induction of memory T cells, and B cells. This is believed to occur via the cytokine cocktail that is induced by compounds of Formula I, particularly the induction of IL-I . The ability to generate memory T cells can enhance immune responses to, for example, cancerous cells that remain following a surgical procedure, or following chemotherapy or radiation. The invention further contemplates the use of compositions and methods of the invention in cancer subjects prior to and following surgery, radiation or chemotherapy in order to create memory immune cells to the cancer antigen. In this way, memory cells of the immune system can be primed with cancer antigens and thereby provide immune surveillance in the long term. This is particularly suited to radiotherapy of subjects where immune cells so primed can invade a tumor site and effectively clear any remaining tumor debris. This in turn promotes further immunity to the cancer, particularly to antigens that might not have been exposed in the context of a tumor mass pre-treatment. It is to be understood that in other embodiments, the subjects can be treated with compositions and methods of the invention without any other therapy, as well. In some embodiments of the invention, the methods are particularly directed to subjects at high risk of cancer, such as those predisposed for familial (e.g., familial colon polyposis, BRCAl- or BRCA2- associated breast cancer, Wilms tumor, colorectal cancer, Li-Fraumeni Syndrome, ovarian cancer, and prostate cancer), or non-familial genetic reasons. Subjects at high risk are also those that manifest pre-cancerous symptoms such as pre-cancerous polyps (e.g., in colon cancer), or pre-cancerous lesions (e.g., in HPV-induced cervical cancer).
The compositions and methods of the invention can also be used in combination with non-surgical antiproliferative (e.g., anti-cancer) drug therapy. In some embodiments, the compositions and methods of the invention may be used in combination with an anti-cancer compound such as a cytostatic compound. A cytostatic compound is a compound (e.g., a nucleic acid, a protein) that suppresses cell growth and/or proliferation. In some embodiments, the cytostatic compound is directed towards the malignant cells of a tumor. In yet other embodiments, the cytostatic compound is one which inhibits the growth and/or proliferation of vascular smooth muscle cells or fibroblasts.
According to the methods of the invention, compounds of Formula I and pemetrexed, erlotinib, or docetaxel may be administered prior to, concurrent with, or following other anti- cancer compounds. The administration schedule may involve administering the different agents in an alternating fashion. In other embodiments, the combination therapy of the invention may be delivered before and during, or during and after, or before and after treatment with other therapies. In some cases, the agent is administered more than 24 hours before the administration of the other anti-proliferative treatment. In other embodiments, more than one anti-proliferative therapy may be administered to a subject. For example, the subject may receive the agents of the invention, in combination with both surgery and at least one other anti-proliferative compound. Alternatively, the agent may be administered in combination with more than one anti-cancer drug.
Cytokines and chemokines can potentially be cleaved and thereby inactivated by post proline cleaving enzymes. Administration of compounds of Formula I with cytokines and/or chemokines can enhance the efficacy of these latter agents by protecting them from degradation.
Immune responses can also be induced or augmented by the co-administration or co- linear expression of cytokines or chemokines (Bueler & Mulligan, 1996; Chow et at, 1997; Geissler et «/., 1997; Iwasaki et at, 1997; Kim et at, 1997) or B-7 co-stimulatory molecules (Iwasaki et at, 1997; Tsuji et at, 1997) with the compounds of Formula I. The cytokines and/or chemokines can be administered directly or may be administered in the form of a nucleic acid vector that encodes the cytokine, such that the cytokine can be expressed in vivo. In one embodiment, the cytokine or chemokine is administered in the form of a plasmid expression vector. The term "cytokine" is used as a generic name for a diverse group of soluble proteins and peptides which act as humoral regulators at nano- to picomolar concentrations and which, either under normal or pathological conditions, modulate the functional activities of individual cells and tissues. These proteins also mediate interactions between cells directly and regulate processes taking place in the extracellular environment. Cytokines also are central in directing the T cell response. Examples of cytokines include, but are not limited to IL-I, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL- 12, IL- 15, IL- 18, granulocyte- macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G- CSF), interferon-γ (IFN-γ), IFN-α, tumor necrosis factor (TNF), TGF-β, FLT-3 ligand, and CD40 ligand. In some embodiments, the cytokine is a ThI cytokine. In still other embodiments, the cytokine is a Th2 cytokine.
The term "chemokine" is used as a generic name for peptides or polypeptides that act principally to chemoattract effector cells of both innate and adaptive immunity. Chemokines are thought to coordinate immunological defenses against tumors and infectious agents by concentrating neutrophils, macrophages, eosinophils and T and B lymphocytes at the anatomical site in which the tumor or infectious agent is present. In addition, many chemokines are known to activate the effector cells so that their immune functions (e.g., cytolysis of tumor cells) are enhanced on a per cell basis. Two groups of chemokines are distinguished according to the positions of the first two cysteine residues that are conserved in the amino-terminal portions of the polypeptides. The residues can either be adjacent or separated by one amino acid, thereby defining the CC and CXC cytokines respectively. The activity of each chemokine is restricted to particular effector cells, and this specificity results from a cognate interaction between the chemokine and a specific cell membrane receptor expressed by the effector cells. For example, the CXC chemokines IL-8, Groα/β and ENA 78 act specifically on neutrophils, whereas the CC chemokines RANTES, MIP- lα and MCP-3 act on monocytes and activated T cells. In addition, the CXC chemokine IP-IO appears to have anti-angiogenic activity against tumors as well as being a chemoattractant for activated T cells. MIP- lα also reportedly has effects on hemopoietic precursor cells. In other aspects, the invention relates to kits that are useful in the treatment of cancer.
One kit of the invention includes a sustained release vehicle containing a compound of Formula I and a container housing pemetrexed, erlotinib, or docetaxel and instructions for timing of administration of the various agents contained therein.
A sustained release vehicle is used herein in accordance with its prior art meaning of any device which slowly releases the compound of Formula I. Such systems can avoid repeated administrations of the compounds, 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 polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and poly anhydrides. 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; sylastic 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 an agent or compound of the invention 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. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.
The pharmaceutical compositions of the invention contain an effective amount of a compound of Formula I and pemetrexed, erlotinib, or docetaxel and/or other therapeutic agents, optionally included in a pharmaceutically-acceptable carrier. The term "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 vertebrate animal. The term "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 also are capable of being commingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.
The agents may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. Such 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-sulρhonic, and benzene sulphonic. Also, such salts can be prepared as 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 benzalkoniurn 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).
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection 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. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, 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. Another suitable compound for sustained release delivery is GELFOAM5 a commercially available product consisting of modified collagen fibers.
Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle, e.g. , sterile pyrogen-free water, before use.
The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such 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 agents can be administered by any ordinary route for administering medications, depending upon the type of cancer to be treated, the compounds of Formula I5 pemetrexed, erlotinib, or docetaxel may be inhaled, ingested or administered by systemic routes. Systemic routes include oral and parenteral. Inhaled medications are preferred in some embodiments because of the direct delivery to the lung, particularly in lung cancer patients. Several types of metered dose inhalers are regularly used for administration by inhalation. These types of devices include metered dose inhalers (MDI), breath-actuated MDI, dry powder inhaler (DPI), spacer/holding chambers in combination with MDI, and nebulizers.
For use in therapy, an effective amount of the compound of Formula I can be administered to a subject by any mode that delivers the compound to the affected organ or tissue, or alternatively to the immune system. "Administering" the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan. Preferred routes of administration include but are not limited to oral, parenteral, intramuscular, intranasal, intratracheal, inhalation, ocular, vaginal, and rectal.
The administration route of the compound of Formula I and the other agents described herein is not limiting on the administration route of the pemetrexed, erlotinib, or docetaxel. The compound of Formula I may be administered in the same route, and in the same formulation as the pemetrexed, erlotinib, or docetaxel, or it may be administered in a different route, different formulation, and even on a different schedule. In an important embodiment, the compound of Formula I is administered orally, and the pemetrexed, erlotinib, or docetaxel is administered parenterally, preferably by intramuscular or subcutaneous injection, although it is not so limited. In some important embodiments, the compounds of Formula I are administered orally, preferably by ingestible tablets that enter the gastrointestinal tract. In some embodiments, the antigens or antibodies are also administered via the same route.
In still other embodiments, the compounds of Formula I are administered locally, and optionally the pemetrexed, erlotinib, or docetaxel are administered locally as well.
For oral administration, the agents can be formulated readily by combining the active compound(s) 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 by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally 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. 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 preparations 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. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use according to the present invention 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. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. Techniques for preparing aerosol delivery systems are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties of the therapeutic (see, for example, Sciarra and Cutie, "Aerosols," in Remington's Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712; incorporated by reference). Those of skill in the art can readily determine the various parameters and conditions for producing aerosols without resort to undue experimentation.
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. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. 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.
In addition to the formulations described previously, 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. Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer, Science 249:1527-1533, 1990, which is incorporated herein by reference.
The invention further provides kits that comprise the agents of the invention and optionally instructions of use thereof. The agents may be present in oral forms such as tablets, pills, capsules, caplets and the like. The agents may be provided in a one a day dispensing unit such as a blister pack or dial pack type dispenser, preferably with days of the week or day of the month (e.g., 1, 2, 3, 4, etc.) (and doses per day, where applicable) printed on the dispenser. For example, if the agents are to be administered every other day or twice (or more) a day, the dispensing unit can be modified accordingly, with no more than routine reconfiguration, known in the art. The kit may further contain a second agent such as a second anti-cancer agent. The unit dosages provided in each form (e.g., tablet, pill, capsule, etc.) will depend upon the particular therapy and desired result. The kit may optionally comprise a housing such as a box or bag. Instructions for use may be supplied separately from the dispensing unit or housing or they may be imprinted on one or both.
The following Examples are provided to illustrate specific instances of the practice of the present invention and are not intended to limit the scope of the invention. As will be apparent to one of ordinary skill in the art, the present invention will find application in a variety of compositions and methods. Examples
Example 1 Introduction
This study investigated the interaction between talabostat and pemetrexed (ALIMTA®) in the A549 NSCLC xenograft model. Single or multiple administrations of pemetrexed to mice inoculated subcutaneously (s.c.) with the A549 cell line were followed by 14-day courses of twice a day (BID) administration of varying doses of talabostat. The effects of pemetrexed and talabostat combination treatment were compared with vehicle treatment and monotherapy with each agent.
Materials and Methods Test Material:
Talabostat mesylate, referred to as talabostat, is the methane sulphonate salt of boronic acid, [(2R)-l-[(2S)-2-amino-3-methyl-l-oxobutyl]-2-pyrrolidinyl]-. The material used in this study was synthesized by Evotec OAI (Abingdon, Oxon, UK; batch #51218-06). Pemetrexed (pemetrexed disodium heptahydrate) (ALIMTA®) has the chemical name L- Glutamic acid, N-[4-[2-(2-amino-4,7-dihydro-4-oxo-lH-pyrrolo[2,3-d]pyrimidin-5~ yl)ethyl]benzoyl]-, disodium salt, heptahydrate. The molecular formula is C20H19N5Na2O67H2O. Pemetrexed, an antifolate anti-cancer agent, has a mechanism of action involving the disruption of folate-dependent metabolic processes required for cell replication. Pemetrexed for injection was purchased from Eli Lilly and Co. (Indianapolis, IN). Normal saline, referred to as saline, was 0.9% (weight/volume) sodium chloride (Sigma, St Louis, MO) in water.
Animals and Cell Lines: C.129S6 (B6)-Rag2tmFwa mice, referred to as BALB/c Rag2~/~ mice, were purchased from Taconic (Germantown, NY). The A549 NSCLC cell line, CCL- 185,™ (American Tissue Type Culture Collection [ATCC], Manassas, VA) was propagated in vitro at 37°C in an atmosphere of 95% air and 5% carbon dioxide by serial culture in Ham's F12K medium with 2mM L-glutamine, adjusted to contain 1.5g/L sodium bicarbonate and 10% fetal bovine serum. The subculture procedure was that described by ATCC. Assays of Talabostat and Pemetrexed Antitumor Activity in Vivo :
BALB/c Rag2~/~ mice were injected s.c. with 5 x 106 A549 tumor cells in a shaved flank as follows. Pemetrexed was administered (doses are described below) by intraperitoneal injection of saline-diluted solutions. Talabostat 5, 10, or 20 μg saline solutions, or saline alone, were administered by gavage BID at approximately 8h intervals. The time of tumor inoculation was defined as Day 0, and pemetrexed and talabostat were administered according to the regimens described below. Experimental groups contained 5 replicate mice for dose- ranging studies of pemetrexed, and 10 replicate mice in studies comparing the antitumor effects of pemetrexed and talabostat as monotherapies as well as in combination. Tumor growth was monitored by measurement of the length (L) and the width (W) with vernier calipers. Tumor volume was calculated by the formula: volume (c.c.) = W2 x 0.5 L.
Data Analysis:
Mean tumor volumes, standard errors, and significance by Student's Mest were calculated using Microsoft Excel software (Redmond, WA).
Tumor volumes (c.c.) are reported as values rounded to 2 decimal places. P-values are rounded to a sufficient number of decimal place to depict significance as values greater or smaller than 0.05, representing the 95% confidence limit.
Results and Discussion:
The dose response of A549 NSCLC xenografts to pemetrexed was investigated in vivo to select doses of pemetrexed that could be studied in combination with talabostat. BALB/c Rag 2^' mice were inoculated s.c. with A549 cells and 7 days later administered a single injection of pemetrexed at doses ranging from 25 to 750 mg/kg. Throughout the 45-day period of tumor growth, pemetrexed (600 and 750 mg/kg) appeared to slow tumor growth (FIG. 1), but the effect was not consistently significant (P<0.05) at all timepoints. Although the antitumor effect of these doses of pemetrexed on tumor growth was modest, their activity in combination with talabostat was investigated because higher doses of pemetrexed have been reported to be toxic in mice. To study talabostat' s antitumor effect in combination with pemetrexed,
BALB/c Rag2'A mice were inoculated s.c. with A549 cells and on Days 7 and 29 were administered pemetrexed at either 750 or 600 mg/kg. The administration of pemetrexed on each day was followed by administration of talabostat or saline BID by gavage from Days 8 through 21 and Days 30 through 43. Pemetrexed, talabostat, and saline were also administered as single agents according to the same regimens.
In the saline-treated control mice that received neither pemetrexed nor talabostat, A549 tumors grew to a mean volume of 2.4 c.c. on Day 50. Monotherapy with pemetrexed (750 or 600 mg/kg) again resulted in a small inhibition of tumor growth, but the tumors were not significantly smaller (P>Q.O5) than in the saline-treated control mice on Day 50. In contrast, monotherapy with talabostat 5, 10, and 20 μg resulted in highly significant suppression of tumor growth (P<0.0005 on Day 50; see FIG. 2-4). Treatment with pemetrexed (600 or 750 mg/kg) in combination with talabostat (5, 10, or 20 μg) also resulted in highly significant suppression of tumor growth (P≤O-0001 to 0.001). The antitumor effect of combination treatment was significantly greater than that of monotherapy with either dose of pemetrexed (P<0.00005 to 0.005).
The relatively high concentrations (~1 μM) of circulating thymidine in mice are thought to attenuate the activity of pemetrexed. Therefore, in order to attempt to improve the activity of pemetrexed in the A549 xenograft model, administration was repeated on 3 consecutive days. The dose response indicated that administration of pemetrexed (600 or 500 mg/kg) on Days 7, 8 and 9 post tumor inoculation resulted in a significant reduction (P<0.05) in tumor size on Days 11 and 15 (FIG. 5). The antitumor effect of the 500 mg/kg dose was also significant (P<0.05) at all the later timepoints; therefore this dose was selected for study in combination with talabostat using a regimen of pemetrexed administration on 3 consecutive days followed by talabostat for 14 consecutive days.
BALB/c Rag2'Λ mice were inoculated s.c. with A549 cells and administered pemetrexed 500 mg/kg on each of Days 7, 8, 9, 31, 32, and 33 (see FIG. 6 for schedule and frequencies of administration). Saline or talabostat (5 μg or 10 μg) was administered BID on Days 10 to 23 and on Days 34 to 47. Pemetrexed, talabostat, and saline were also administered as single agents according to the same regimens. In saline-treated control mice that received neither pemetrexed nor talabostat, A549 tumors grew to a mean volume of 2.5 c.c. on Day 50. Tumors in mice receiving pemetrexed monotherapy (FIG. 7) were significantly smaller than those in control mice receiving no anti-cancer agent on Days 11 (P≤O.0005) and 15 (P<0.05), but showed no significant effect thereafter (P>0.05). Pemetrexed 500 mg/kg was clearly less efficacious in this study than in the dose-response study (FIG. 5). In keeping with a previously reported study of the activity of pemetrexed in the H460 NSCLC xenograft model, the antitumor effect of pemetrexed in mice bearing A549 NSCLC xenografts appeared to occur early in tumor development. As expected, monotherapy with talabostat (5 or 10 μg) resulted in significant suppression of overall tumor growth (P≤O.01 on Day 50; see FIG. 7-8). The effect of talabostat monotherapy in this study appeared to be less than that previously observed (FIG. 2-3). This difference could have been due to the delayed initiation of talabostat treatment, to Day 10 as opposed to Day 8.
Treatment with pemetrexed 500 mg/kg in combination with either talabostat (5 or 10 μg) also resulted in significant suppression of overall tumor growth, as reflected by tumor sizes recorded in mice receiving combination treatment versus saline alone (P<5 x 10"8 to 0.003). The combination treatment achieved significantly greater reductions in tumor growth than with pemetrexed monotherapy as indicated by tumor sizes measured on Day 50 (see FIGS. 7 and 8; P≤O.001). The combination of talabostat (5 μg) with pemetrexed resulted in a significantly greater antitumor effect than did monotherapy with talabostat 5μg; this effect was apparent by comparison of tumor sizes at all timepoints studied (FIG. 7; P≤O.00001 to 0.05). The combination of pemetrexed and talabostat (10 μg) failed to achieve a significantly greater reduction in tumor size on Days 45 and 50 than did talabostat monotherapy at this dose (FIG. 8; P>0.1 for pemetrexed + talabostat (10 μg), vs. talabostat (10 μg) alone). Consequently, there appeared to be less of a cooperative interaction of pemetrexed with talabostat at the 10 μg than at the 5 μg dose.
Conclusions: In BALB/c Rag 2'^ mice, the antitumor effect of pemetrexed was much less than that previously observed with docetaxel. Pemetrexed is a multitargeted antifolate. Inhibition of de novo nucleotide biosynthesis can be circumvented by the salvage of extracellular preformed nucleosides. Compared with humans, mice have relatively high levels of circulating thymidine, and nucleoside salvage could have been responsible for the relatively weak activity of pemetrexed observed in the A549 NSCLC xenograft model. Nevertheless, the combination of two cycles of treatment incorporating 3 consecutive injections of pemetrexed followed by 14 days' of BID talabostat administration resulted in a significantly greater antitumor effect than that observed with either agent alone. The cooperative interaction of pemetrexed and talabostat was more apparent when pemetrexed 500mg/kg was combined with talabostat 5 μg as opposed to talabostat 10 μg. Talabostat and pemetrexed can be considered to interact synergistically because pemetrexed as a single agent had no significant effect on overall tumor growth and the combination of pemetrexed and talabostat had a significantly greater effect than talabostat alone.
As previously described, monotherapy with talabostat is highly effective in inhibiting growth of A549 tumor xenografts. It is important to note that in all pemetrexed/talabostat regimens investigated in this study, talabostat' s activity did not appear to be diminished by prior pemetrexed treatment.
References:
1. Adams S, Miller GT, Jesson MI, Watanabe T5 Jones B, Wallner BP. "Talabostat [talabostat], a small molecule dipeptidyl peptidase inhibitor, has potent antitumor effects and augments antibody-mediated cytotoxicity via a novel immune mechanism." Cancer Res. 2004; 64:5471-80.
2. Adams S, Miller G, Jones B. "T-cell independent activity of talabostat [TALABOSTAT] against human tumor xenografts in mice." J Immunother. 2004; 27:S54. 3. Worzall JF, Shih C, Schultz RM. "Role of folic acid in modulating the toxicity and efficacy of the multitargeted antifolate, LY231514." Anticancer Res. 1998; 18:3235- 9.
4. Teicher. BA, Chen V, Shih C, et al. "Treatment regimens including the multitargeted antifolate LY231514 in human tumor xenografts." Clin Cancer Res. 2000; 6:1016-23. 5. Smith PG, Thomas HD, Barlow HC, et al. "In vitro and in vivo properties of navel nucleoside transport inhibitors with improved pharmacological properties that potentiate antifolate activity." Clin Cancer Res. 2001; 7:2105-13.
Protocols:
Protocol A: A549 Human NSCLC; Pemetrexed 1, Chemotherapy Alone, Dose- Titration.
Rodent Strain: Mice (BALB/c Rag2'/"), Taconic Farms, females, 6-8 weeks of age,
5/group.
Objective: To determine effectiveness of various doses of chemotherapy alone at slowing tumor growth in this mouse model. Experimental Plan:
Table A.1. Group Inj. A549 s.c. @ Treatment ALIMTA® dose: 5x106/ mouse day 7, i.p.
1 Yes Saline (Saline)
2 Yes ALIMTA® 750 mg/kg
3 Yes ALIMTA® 600
4 Yes ALIMTA® 500
5 Yes ALIMTA® 400
6 Yes ALIMTA® 300
7 Yes ALIMTA® 200
8 Yes ALIMTA® 100
9 Yes ALIMTA® 50
10 Yes ALIMTA® 25
Day 0: Inject 5xlO6 A549 cells s.c. into shaven flank of each mouse, in 0.2 ml/mouse of D- PBS.
Day 7: Administer pemetrexed as a single i.p. injection in a 0.3 to 0.5 ml/mouse volume. Measure tumors: Days 7, 1 1, 15, 20, 25, 30, 35 etc. Pemetrexed Administration: Inject @ 0.6 ml/mouse, i.p.
Stock solution: Resuspend 1 vial of 500 mg in 20 ml of saline to give a stock of 25 mg/ml, as per package instructions.
Protocol B: A549 Human NSCLC; Actual Combination Therapy Assay Combining Talabostat (Day 8 Start) with Sub-Maximal Doses of Pemetrexed [i.p. injection, 1 dose/cycle].
Rodent Strain: Mice (BALB/c Rag2'/"), females, 6-8 weeks of age, 10/group = 120 total Objective: To determine effectiveness of the combination on slowing tumor growth in this model: in a multiple-cycle assay.
Experimental Plan:
Table B.I.
Group A549 @ 5xlO6 Talabostat: p.o., ALIMTA®:
BID, day 8 start i.p., day 7 Cycle 1
1 Yes Saline
2 Yes 5 μg 3 Yes 10 μg
4 Yes 20 μg
5 Yes 750 mg/kg
6 Yes 600 mg/kg
7 Yes 5 μg 750 mg/kg
8 Yes 5 μg 600 mg/kg
9 Yes 10 μg 750 mg/kg
10 Yes 10 μg 600 mg/kg
11 Yes 20 μg 750 mg/kg
12 Yes 20 μg 600 mg/kg
Day 0: Inject A549 s.c. @ 5x106/ mouse in 0.2 ml saline/ mouse. Day 7: Inject ALIMTA® i.p. in 0.6 ml or less per mouse, as a single injection per cycle. Day 8: Begin talabostat treatment, dosing @ 0.2 ml/ dose/ mouse; p.o., BID in saline. Measure tumors: days 7, 11, 15, 20, 25, 30, 35, 40, etc.
Table B.2. Treatment Cycles
Cycle ALIMTA® (i.p.) Talabostat (14 days) Rest (7 days) ϊ day 7 days 8-21 days 22-28
2 day 29 ; days 30-43 days 44-50
END day 50
Pemetrexed Administration Stock solution: Resuspend 2 vials of 500 mg in 20 ml of saline each to give stocks of 25 mg/ml (as per package inserts).
Inject @ 0.6 ml/mouse, i.p. for Cycle 1 Inject @ 0.7 ml/mouse, i.p. for Cycle 2 Protocol C: A549 Human NSCLC; Pemetrexed Alone Dose-Titration 2; Multiple pemetrexed Injections.
Rodent Strain: Mice (BALB/c Rag2'/~); Taconic Farms, females, 6-8 weeks of age, 5/group = 35 total. Objective: To determine effectiveness of various doses of chemotherapy alone at slowing tumor growth in this model.
Experimental Plan:
Table d.
Group Inj. A549 S.C. (ά ζ> Treatment ALIMTA® dose:
5xl06/mouse i.p., days 7, 8, 9
1 Yes Saline (Saline)
2 Yes ALIMTA® 600 mg/kg
3 Yes ALIMTA® 500
4 Yes ALIMTA® 400
5 Yes ALIMTA® 300
6 Yes ALIMTA® 200
7 Yes ALIMTA® 100
Day 0: Inject 5x106 A549 cells s.c. into the shaven flank of each mouse, in 0.2 ml/ mouse of D-PBS.
Day 7: Administer the first dose of ALIMTA® as an i.p. injection @ 0.3 to 0.5 ml/ mouse. ALIMTA® dosing days: 7, 8, 9; all doses q.i.d., all i.p. injection. ] Measure tumors: Days 7, 11, 15, 20, 25, 30, etc.
Pemetrexed Administration
Stock solution: Resuspend 1 vial of 500 mg in 20 ml of saline each to give a stock of 25 mg/ml (as per package inserts). Inject @ 0.6 ml/mouse, i.p. 5 mice per group
Protocol D: A549 Human NSCLC; Pemetrexed 3: Actual Combination Therapy Assay Combining Talabostat with an Effective Dose of Pemetrexed [i.p. injection, 3 consecutive doses per cycle]. Rodent Strain: Mice (BALB/c Rag2~/~); females, 6-8 weeks of age, 10/group = 60 total Objective: To determine effectiveness of the combination on slowing tumor growth in this model: in a multiple-cycle assay.
Experimental Plan:
The dose of pemetrexed was chosen based on results from the second titration assay, in which various doses were given as i.p. injections on 3 consecutive days: Days 7, 8, and 9.
Table P.1. Group A549 @ 5xlO6 Talabostat: p.o., AUMTA®:
BID, day 10 i.p., days 7,8,9 start Cycle 1
1 Yes Saline
2 Yes 5 μg
3 Yes 10 μg
4 Yes 500 mg/kg
5 Yes 5 μg 500 mg/kg
6 Yes 10 μg 500 mg/kg
Day 0: Inject A549 s.c. @ 5xl06/mouse in 0.2 ml saline/mouse. Days 7, 8 and 9: Inject ALIMT A® i.p. in 0.6 ml or less per mouse, as a single injection per day, for 3 consecutive days
Day 10: Begin talabostat treatment, dosing @ 0.2 ml/dose/mouse; p.o., BID in saline. Measure tumors: Days 7, 11, 15, 20, 25, 30, 35, 40, 45, 50, 54 END.
Table D-2. Treatment Cycles
Cycle ALIMTA® (i.p.) Talabostat (14 days) Rest (7 days) ϊ Days 7, 8, 9 Days 10-23 Days 24-30
2 Days 31, 32, 33 Days 34-47 Days 48-54
Pemetrexed Administration
Stock solution: Resuspend 1 vial of 500 mg in 20 ml saline to give a stock of 25 mg/ml (as per package inserts).
Average weight: 18.87 grams = 0.01887 kg Inject @ 0.6 ml/mouse, i.p. for Cycle 1. Abbreviations and definitions: BID or b.i.d.: twice daily; Gr: dosing group; h: hour; Lp.: intraperitoneal; Pem: pemetrexed, (ALIMTA®); P.O.: by mouth; QD: once daily; SAC: sacrifice(d); S.C. or s.c. : subcutaneous(ly); T: talabostat.
Example 2 BALB/c Rag2'A mice were injected subcutaneously on one flank with 5 x 106 A549 human non-small cell lung cancer (NSCLC) cells. On day 7, when the tumors were established and of 180 ± 10 m.m.3 mean volume, oral administration of a dose of 5 μg talabostat twice daily (b.i.d.) and 25 or 50 mg/kg erlotinib (TARCEVA™) once per day was started. Oral administration of both agents was continued on consecutive days from day 7 to day 30. Mice received no treatment from days 31 to 37 and then both agents were again administered on days 38 and 39 (see FIG. 9 for schedule and frequencies of administration). Tumor growth in experimental groups of 10 replicate mice was monitored by measurement with vernier calipers. Data are presented as mean tumor volumes + SE, and significance was determined by Student's 2-tailed t test. Administration of suboptimal doses of either 25 or 50 mg/kg doses of erlotinib together with talabostat for 26 days inhibited A549 tumor growth to a greater extent than administration of either agent alone (FIGS. 10 and 11). The anti-tumor effect of each combination treatment was significantly greater than that of either PT-100 or erlotinib alone for the doses of erlotinib and talabostat studied as determined by measurements of tumor volumes on day 40. The P values are tabulated below.
Figure imgf000084_0001
Example 3
BALB/c Rag2'/" mice were injected subcutaneously on one flank with 5 x 106 A549 human non-small cell lung cancer (NSCLC) cells. On day 7, when the tumors were established and of 180 ± 10 mm3 mean volume. Docetaxel 20 mg/kg was administered on each of Days 7, 29, and 51. Saline or talabostat 5 μg was administered BID on Days 8 to 21, Days 30 to 43, and Days 52 to 65. The mice received no treatments on Days 22 to 28, Days 44 to 50, and Days 66 to 72 (rest days). See FIG. 12 for schedule and frequencies of administration. Tumor growth in experimental groups of 10 or 20 replicate mice was monitored by measurement with vernier calipers. Data are presented as mean tumor volumes + SE, and significance was determined by Student's 2 -tailed t test (FIG 13).
It can be concluded that against A549 NSCLC xenografts, talabostat can promote a T- cell independent anti-tumor response that interacts cooperatively with the antitumor agents that have very different mechanisms of action such as pemetrexed that disrupts folate- dependent metabolism required for cell replication, erlotinib which is a HERl /EGFR tyrosine kinase inhibitor, and docetaxel which disrupts the microtubular network essential for mitosis.
Example 4 Phase 2 Trial of Talabostat and Docetaxel in Patients with Stage IIIB/IV NSCLC Introduction
Talabostat is an orally administered inhibitor of dipeptidyl peptidases such as DPP-IV (also known as CD26) and fibroblast activation protein (FAP) found on the stroma of epithelial tumors such as non-small cell lung cancer (NSCLC). Administration of talabostat stimulates cytokine and chemokine production in humans and animals resulting in specific T- cell immunity and T-cell independent activity. In a mouse xenograft model, talabostat significantly enhanced the activity of docetaxel in the A549 model of NSCLC .
Study Objectives This Phase 2 trial was conducted to evaluate the activity of talabostat and docetaxel in patients with Stage IIIB/IV NSCLC who failed a platinum-based therapy.
Study Design
Open-label, single-arm, multicenter study Treatment regimen (up to six 3 -week cycles) o Docetaxel 75 mg/m (Day 1 of each cycle) with appropriate pre-medication as needed o Talabostat 200 μg tablets administered orally BID Days 2-15. Dose-escalation of talabostat to 300 μg BID allowed in subsequent cycles depending on tolerability. o Single-agent talabostat continued past 6 cycles at the discretion of the investigator Key outcome measures: o Primary: Response rate (per WHO criteria) o Secondary: Duration of response, progression-free survival (PFS), and survival Patients were followed for 12 months for survival and disease progression (PD)
Methods
Key Eligibility Criteria
• Men or women age >18 years with measurable disease
• Histologically or cytologically confirmed Stage IIIB/IV NSCLC (Stage IIIB with cytologically documented pleural effusion) • Failed or relapsed following a platinum-based regimen
• No more than 2 prior chemotherapy regimens
• ECOG performance status 0 or 1 and life expectancy >12 weeks
• No CNS metastases (unless resected or irradiated and asymptomatic)
• Baseline laboratory results within the following parameters: ♦ Platelets >100,000/μL, ANC >1500/μL
♦ ALT or AST < 1.5 x upper limit of normal (ULN) and alk phos <2.5 x ULN
♦ Total bilirubin < ULN
♦ Serum creatinine <2.0mg/dL
• Significant cardiac history (MI within 1 year, CABG within 6 months, severe CHF, ventricular arrhythmia or other uncontrolled cardiac arrhythmia)
• No chemotherapy, radiation or immunotherapy within 30 days of Study Day 1
• Written informed consent
Patients were seen in clinic after each 3 week cycle. CT scans of the chest and upper abdomen were obtained at baseline and every 6 weeks for 18 weeks and then every 2 months thereafter until PD. Adverse events, clinical laboratories, and vital signs were monitored throughout the study. Serum cytokines (IL- lα, IL-I β, IL-2, IL-6, and IL-8) were obtained prior to talabostat administration on Study Day 3 of Cycle 1 and at 1, 2, 4, and 6 hours after the morning dose of talabostat.
Results
Patient Population
Fifty-five patients (26 men and 29 women) enrolled into the study. The median age was 61 years (range 34 to 87); 46 patients were Caucasian and 9 were African- American. The majority of patients had Stage IV adenocarcinoma. Thirty-five patients (64%) received only 1 prior regimen for advanced NSCLC, and 20 (36%) received 2 prior regimens; most patients (75%) had received a platinum-based combination with a taxane.
All patients have had the opportunity to complete the 6-cycle treatment period, and 15 patients received additional cycles of talabostat. The median number of cycles completed was 4 (range 1 to 18). The majority (65%) of patients increased their dose of talabostat to 300μg BID, including the five patients with a clinical response to treatment (see Table 3).
Clinical Disease Assessments
Of 55 patients enrolled, 43 met criteria for evaluability (defined as 2 cycles of treatment and a post-baseline CT or MRI scan).
Figure imgf000087_0001
a Lesion size decreased by 45% from baseline, and 52% from maximum observed at the end of Cycle 4
The Kaplan-Meier estimates for median PFS and survival for all patients enrolled are shown in FIGs.14A and 14B respectively. In cases where patients were lost to follow-up, data were censored at the last observation. If a new cancer treatment was initiated due to PD, data were censored at the point the new treatment started.
The Kaplan-Meier estimate of median PFS for all patients enrolled is 4.2 months. The Kaplan-Meier estimate of median survival is 8.4 months.
The addition/use of an agent that binds IL-6 or inhibits the activity of IL-6 (such as, for example, an IL-6 antibody or an IL-6 antisense molecule) may help reduce or prevent the adverse events of edema/peripheral swelling, hypotension or dehydration/hypovolemia.
Conclusions
• The addition of talabostat to docetaxel shows promising activity in patients with advanced NSCLC who have failed a platinum-based regimen.
• Five objective responses, including 2 CRs, were observed in 43 evaluable patients.
• Median PFS in is currently estimated at 4.2 months; median survival is estimated 8.4 months.
• The results of this study support a Phase 3 randomized study of talabostat/docetaxel versus docetaxel in patients with Stage IIIB/IV NSCLC.
Equivalents
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention. All references, patents and patent publications that are recited in this application are incorporated in their entirety herein by reference.
What is claimed is:

Claims

1. A method for treating a subject having cancer comprising administering to a subject in need thereof an effective amount of pemetrexed and a compound of Formula I:
PR wherein P is a targeting group which binds to the reactive site of a post proline- cleaving enzyme, and wherein R is a reactive group which reacts with the reactive site of a post proline cleaving enzyme.
2. The method of claim 1, wherein the compound of Formula I is an agent of Formula II:
Figure imgf000090_0002
wherein m is an integer between 0 and 100, inclusive; A and A1 are L- or D- naturally or non- naturally occurring amino acid residues or a peptide or peptidomimetic such that when A is an amino acid residue and m > 1 each A in Am may be a different amino acid residue different from every other A in Am and when A is a peptide or peptidomimetic m is 1 ; the C bonded to B is in the L- or R-configuration; and each X1 and X2 is, independently, a hydroxyl group or a group capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH.
3. The method of claim 1, wherein the compound of Formula I is a compound of Formula III:
Figure imgf000090_0003
wherein m is an integer between 0 and 100, inclusive; A and Ai are L- or D- naturally or non- naturally occurring amino acid residues or a peptide or a peptidomimetic such that when m >1 A in each repeating bracketed unit is independently selected; the C bonded to B is in the L- or R-configuration; and each X1 and X2 is, independently, a hydroxyl group or a group capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH.
4. The method of claim 1 , wherein the compound of Formula I is Ile-boroPro.
5. The method of claim 1, wherein the compound of Formula I is Val-boroPro.
6. The method of claim 1 , wherein the compound of Formula I is provided as a mixture of linear and cyclic forms.
7. The method of claim 1 , wherein the cancer is a carcinoma.
8. The method of claim 1, wherein the cancer is non-small cell lung cancer (NSCLC).
9. The method of claim 1 , wherein the cancer is a tumor.
10. The method of claim 1 , wherein the cancer is a metastasis.
11. The method of claim 1 , wherein the cancer is a refractory cancer.
12. The method of claim 11, wherein the refractory cancer is refractory to a platinum- containing therapy.
13. The method of claim 1 , wherein the subj ect has not undergone an anti-cancer therapy selected from the group consisting of surgery, radiation and chemotherapy.
14. The method of claim 1, wherein the subject has undergone an anti-cancer therapy selected from the group consisting of surgery, radiation and chemotherapy.
15. The method of claim 1 , further comprising treating the subj ect with a therapy selected from the group consisting of surgery and radiation.
16. The method of claim 15, wherein the compound of Formula I and pemetrexed are administered before, during, and/or after treating the subject with a therapy selected from the group consisting of surgery and radiation.
17. The method of claim 15, wherein the compound of Formula I and pemetrexed are administered before and after treating the subject with the therapy selected from the group consisting of surgery and radiation.
18. The method of claim 15, wherein the compound of Formula I and pemetrexed are administered before and during treating the subject with the therapy selected frorii the group consisting of surgery and radiation.
19. The method of claim 15, wherein the compound of Formula I and pemetrexed are administered during and after treating the subject with the therapy selected from the group consisting of surgery and radiation.
20. The method of claim 1 , wherein the compound of Formula I is administered via a route of administration different from that of pemetrexed.
21. The method of claim 1 , wherein the compound pf Formula I is administered orally.
22. The method of claim 1 or 21, wherein pemetrexed is administered parenterally.
23. The method of claim 1 , wherein the compound of Formula I is administered to the subject after pemetrexed administration.
24. The method of claim 1 , wherein the compound of Formula I is administered to the subject about 24 hours after the administration of pemetrexed.
25. The method of claim 1 , wherein pemetrexed is administered on day 1 of a 22 day cycle.
26. The method of claim 25, wherein the compound of Formula I is administered on days 2-15 of the 22 day cycle.
27. The method of claim 1, wherein pemetrexed is administered on days 1-3 of a 24 day cycle.
28. The method of claim 27, wherein the compound of Formula I is administered on days 4-17 of the 24 day cycle.
29. The method of claim 26 or 28, wherein days 18-24 of the 24 day cycle are a rest period.
30. The method of claim 26 or 28, wherein the 22 or 24 day cycle is repeated.
31. The method of clam 30, wherein the 22 or 24 day cycle is repeated once or twice.
32. The method of claim 1, wherein the compound of Formula I is administered at a dose of about 50 - 1000 micrograms per day or 0.001 - 0.01 mg/kg/day.
33. The method of claim 32, wherein the dose is delivered in two administrations per day.
34. The method of claim 1 , wherein pemetrexed is administered at a dose of about 500 mg/m2 per day.
35. The method of claim 1 , wherein pemetrexed is administered at a sub-therapeutic dose.
36. The method of claim 1 , wherein pemetrexed is administered at a dose of less than 500 mg/m per day.
37. The method of claim 9, wherein the size or volume of the tumor is reduced by at least 50% from pretreatment size or volume.
38. The method of claim 1, further comprising administering cisplatin, carboplatin, gemcitabine, paclitaxel or docetaxel to the subject.
39. The method of claim 1 , further comprising administering an antigen to the subject.
40. The method of claim 39, wherein the antigen is targeted to a tissue or a cell.
41. The method of claim 39, wherein the antigen is a cancer antigen.
42. The method of claim 41, wherein the cancer antigen is PGP9.5, H/Ley/Leb, B7-DC, or B7-H1.
43. The method of claim 1, further comprising administering an antibody or an antigen- binding antibody fragment to the subject.
44. The method of claim 43, wherein the antibody or antibody fragment is an anti-a5β 1 integrin antibody, an anti-EGFR antibody, or alpha IR-3.
45. The method of claim 44, wherein the anti-a5βl integrin antibody is Volocimixab.
46. The method of claim 44, wherein the anti-EGFR antibody is Nimotuzumab (TheraCIM).
47. The method of claim 1, further comprising administering an adjuvant to the subject.
48. The method of claim 1, wherein the effective amount of the compound of Formula I is less than 75% of the effective amount of the agent when administered as a single agent.
49. The method of claim 1 or 46, wherein the effective amount of pemetrexed is less than 75% of the effective amount of the agent when administered as a single agent.
50. A composition comprising a compound of Formula I and pemetrexed in an effective amount to reduce tumor size or volume by at least 50% of pretreatment size or volume.
51. The composition of claim 50, further comprising a pharmaceutically acceptable carrier.
52. A method for treating a subject having cancer comprising administering to a subject in need thereof erlotinib and a compound of Formula I: PR wherein P is a targeting group which binds to the reactive site of a post proline- cleaving enzyme, and wherein R is a reactive group which reacts with the reactive site of a post proline cleaving enzyme, in an effective amount to reduce the cancer load by at least 60% compared to pretreatment cancer load.
53. A method for treating a subject having cancer comprising administering to a subject in need thereof an effective amount of erlotinib and a compound of Formula I:
PR wherein P is a targeting group which binds to the reactive site of a post proline- cleaving enzyme, and wherein R is a reactive group which reacts with the reactive site of a post proline cleaving enzyme, and wherein the effective amount of erlotinib and/or the compound of Formula I is less than 75% of the effective amount of erlotinib and/or the compound of Formula I when administered as a single agent.
54. A method for treating a subject having cancer comprising administering to a subject in need thereof an effective amount of erlotinib and a compound of Formula I : PR wherein P is a targeting group which binds to the reactive site of a post proline- cleaving enzyme, and wherein R is a reactive group which reacts with the reactive site of a post proline cleaving enzyme, and wherein the erlotinib is administered once daily and the compound of Formula I is administered twice daily.
55. The method of claim 53, wherein the compound of Formula I is an agent of Formula II:
Figure imgf000096_0001
wherein m is an integer between 0 and 100, inclusive; A and A1 are L- or D- naturally or non- naturally occurring amino acid residues or a peptide or peptidomimetic such that when A is an amino acid and m > 1 each A in Am may be a different amino acid residue different from every other A in Am and when A is a peptide or peptidomimetic m is 1 ; the C bonded to B is in the L- or R-configuration; and each X1 and X2 is, independently, a hydroxyl group or a group capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH.
56. The method of claim 53, wherein the compound of Formula I is a compound of Formula III:
Figure imgf000096_0002
wherein m is an integer between 0 and 10, inclusive; A and A1 are L- or D- naturally occurring or non-naturally occurring amino acid residues or a peptide or a peptidomimetic such that when m>l A in each repeating bracketed unit is independently selected; the C bonded to B is in the R configuration; and each X1 and X2 is, independently, a hydroxyl group or a group capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH.
57. The method of claim 52, 53, or 54, wherein the compound of Formula I is Ile-boroPro.
58. The method of claim 52, 53, or 54, wherein the compound of Formula I is VaI- boroPro.
59. The method of claim 52, 53, or 54, wherein the compound of Formula I is provided as a mixture of linear and cyclic forms.
60. The method of claim 52, 53, or 54, wherein the cancer is a carcinoma.
61. The method of claim 52, 53, or 54, wherein the cancer is non-small cell lung cancer (NSCLC).
62. The method of claim 52, 53, or 54, wherein the cancer is a tumor.
63. The method of claim 52, 53, or 54, wherein the cancer is a metastasis.
64. The method of claim 52, 53, or 54, wherein the cancer is a refractory cancer.
65. The method of claim 64, wherein the refractory cancer is refractory to cisplatin, carboplatin and/or gemcitabine.
66. The method of claim 52, 53, or 54, wherein the subject has undergone an anti-cancer therapy selected from the group consisting of surgery, radiation and chemotherapy.
67. The method of claim 52, 53, or 54, wherein the compound of Formula I is administered orally.
68. The method of claim 52, 53, 54 or 67, wherein erlotinib is administered orally.
69. The method of claim 52 or 54, wherein the compound of Formula I is administered at a dose of about 50 - 1000 micrograms per day or 0.001 - 0.01 mg/kg/day.
70. The method of claim 52 or 54, wherein erlotinib is administered at a dose of about 150 mg per day.
71. The method of claim 52, 53 or 54, further comprising administering pemetrexed, cisplatin, carboplatin, gemcitabine, or docetaxel to the subject.
72. The method of claim 52, 53 or 54, further comprising administering an antigen to the subject.
73. The method of claim 72, wherein the antigen is targeted to a tissue or a cell.
74. The method of claim 72, wherein the antigen is a cancer antigen.
75. The method of claim 73, wherein the cancer antigen is PGP9.5, H/Ley/Leb, B7-DC, or B7-H1.
76. The method of claim 52, 53 or 54, further comprising administering an antibody or an antigen-binding antibody fragment to the subject.
77. The method of claim 76, wherein the antibody or antibody fragment is an anti-a5β 1 integrin antibody, an anti-EGFR antibody, or alpha IR-3.
78. The method of claim 77, wherein the anti-a5β 1 integrin antibody is Volocimixab.
79. The method of claim 77, wherein the anti-EGFR antibody is Nimotuzumab (TheraCIM).
80. The method of claim 52, 53 or 54, further comprising administering an adjuvant to the subject.
81. A composition comprising a compound of Formula I and erlotinib in an effective amount to reduce tumor size or volume by at least 60% of pretreatment size or volume.
82. The composition of claim 81 , further comprising a pharmaceutically acceptable carrier.
83. A method for treating a subject having cancer comprising administering to a subject in need thereof an effective amount of docetaxel and a compound of Formula I:
PR wherein P is a targeting group which binds to the reactive site of a post proline- cleaving enzyme, and wherein R is a reactive group which reacts with the reactive site of a post proline cleaving enzyme, wherein docetaxel is administered on day 1 of a 22-day cycle, and wherein the compound of Formula I is administered on days 2-15 of the 22 day cycle.
84. The method of claim 83, wherein docetaxel is administered at a dose of about 75 mg/m2.
85. The method of claim 83, wherein the compound of Formula I is administered orally at a dose of about 200-300 micrograms.
86. The method of claim 83-85, wherein the 22 -day cycle is repeated.
87. The method of claims 83-86, wherein the 22 day cycle is repeated two times, three times, four times, five times, or six times.
88. The method of claim 83, wherein the subject has been previously administered a regimen comprising a platinum-containing compound, and optionally gemcitabine or a taxane.
89. The method of claim 88, wherein the subject has been previously administered a regimen comprising a platinum-containing compound and optionally gemcitabine or a taxane at least 1 month, at least 2 months, at least 3 months, at least 4 months, or at least 5 months prior to treatment with docetaxel and the compound of Formula I.
90. The method of claim 83, wherein the cancer is non-small cell lung cancer (NSCLC).
91. A method for treating a subject having cancer comprising administering to a subject in need thereof an effective amount of docetaxel and a compound of Formula I :
PR wherein P is a targeting group which binds to the reactive site of a post proline- cleaving enzyme, and wherein R is a reactive group which reacts with the reactive site of a post proline cleaving enzyme, and wherein the subject has been previously treated with a regimen comprising a platinum-containing compound and optionally an antimetabolite or a taxane.
92. A kit comprising a first container comprising a compound of Formula 'I and a second container comprising pemetrexed.
93. The kit of claim 92, wherein the compound of Formula I is formulated for oral administration.
94. The kit of claim 93, wherein the compound of Formula I is in a unit dosage of about 200 micrograms.
95. The kit of claim 93 , wherein the compound of Formula I is in a unit dosage of about 300 micrograms.
96. The kit of claim 92-95, wherein the compound of Formula I is formulated as a tablet, pill, capsule or caplet.
97. The kit of claim 92-96 further comprising a daily dispenser.
98. The kit of claim 92, wherein pemetrexed is formulated for parenteral administration.
99. The kit of claim 98, wherein pemetrexed is formulated for intravenous or intramuscular administration.
100. The kit of claim 98, wherein pemetrexed is in a unit dosage of about 850 mg.
101. The kit of claim 97, wherein the day of the month is printed on the daily dispenser.
102. The kit of claim 92 or 101 , wherein the first container is a blister package having day indicia and time indicia and wherein there are two time indicia for every day indicium.
103. The kit of claim 92, wherein the kit includes a one-day supply of pemetrexed, a 14-day supply of a compound of Formula I, and a 7-day supply of pills containing no medication.
104. The kit of claim 92, wherein the kit includes a 3 -day supply of pemetrexed, a 14-day supply of a compound of Formula I5 and a 7-day supply of pills containing no medication.
105. The kit of claim 92-104 further comprising instructions of use.
106. The kit of claim 105, wherein the instructions of use comprise instructing a health care provider to administer pemetrexed on day 1 of a 22 day cycle, the compound of Formula I on days 2-15 of the 22 day cycle, and the pills containing no medication on days 16-22 of the 22 day cycle.
107. The kit of claim 105, wherein the instructions of use comprise instructing a health care provider to administer pemetrexed on days 1-3 of a 24 day cycle, the compound of Formula I on days 4-17 of the 24 day cycle, and the pills containing no medication on days 18-24 of the 24 day cycle.
108. The kit of claim 106, wherein the instructions of use instruct the health care provider to administer pemetrexed intravenously over a period of about 10 minutes.
109. A kit comprising a first container comprising a compound of Formula I and a second container comprising erlotinib.
110. The kit of claim 109, wherein the compound of Formula I is formulated for oral administration.
111. The kit of claim 110, wherein the compound of Formula I is in a unit dosage of about 200 micrograms.
112. The kit of claim 110, wherein the compound of Formula I is in a unit dosage of about 300 micrograms.
113. The kit of claim 109 - 112, wherein the compound of Formula I is formulated as a tablet, pill, capsule or caplet.
114. The kit of claim 109 - 113 farther comprising a daily dispenser.
115. The kit of claim 109, wherein erlotinib is formulated for oral administration.
116. The kit of claim 115, wherein erlotinib is in a unit dosage of about 2000 mg.
117. The kit of claim 115, wherein erlotinib is in a unit dosage of about 4000 mg.
118. The kit of claim 114, wherein the day of the month is printed on the daily dispenser.
119. The kit of claim 109 or 114, wherein the first container is a blister package having day indicia and time indicia and optionally two time indicia for every day indicium. W 2
-101-
120. The kit of claim 109 or 114, wherein the second container is a blister package having day indicia and optionally time indicia.
121. The kit of claim 109, wherein the kit includes a 24-day supply of erlotinib, a 24-day supply of a compound of Formula I, and a 7-day supply of pills containing no medication.
122. The kit of claim 109-121 further comprising instructions of use.
123. The kit of claim 122, wherein the instructions of use comprise instructing a subject or a health care provider to administer erlotinib on days 1 -24 of a 31 day cycle, the compound of
Formula I on days 1-24 of the 31 day cycle, and the pills containing no medication on days 25-31 of the 31 day cycle.
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