Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
  • A61K38/482—Serine endopeptidases (3.4.21)
  • A61K38/484—Plasmin (3.4.21.7)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the present invention relates to the field of treating diseases associated with the abnormal growth of cells.
• the present invention relates to administration of low doses of therapeutic agents, such as endostatin, on an antiangiogenic schedule.
• angiogenesis means the generation of new blood vessels into a tissue or organ. Under normal physiological conditions, humans or animals undergo angiogenesis only in very specific restricted situations. For example, angiogenesis is normally observed in wound healing, fetal and embryonal development and formation of the corpus luteum, endometrium and placenta. However, angiogenesis also occurs under abnormal or undesired conditions such as during tumor development, growth and metastasis. This type of angiogenesis may also be referred to as uncontrolled angiogenesis. Both controlled and uncontrolled angiogenesis are thought to proceed in a similar manner. Endothelial cells and pericytes surrounded by a basement membrane form capillary blood vessels.
• Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukocytes.
• the endothelial cells which line the lumen of blood vessels, then protrude through the basement membrane.
• Angiogenic stimulants induce the endothelial cells to migrate through the eroded basement membrane.
• the migrating cells form a "sprout" off the parent blood vessel, where the endothelial cells undergo mitosis and proliferate.
• the endothelial sprouts merge with each other to form capillary loops, creating the new blood vessel.
• Persistent, unregulated angiogenesis occurs in a multiplicity of disease states, tumor metastasis and abnormal growth by endothelial cells.
• the diverse pathological disease states in which unregulated angiogenesis is present have been grouped together as angiogenic dependent or angiogenic associated diseases.
• the hypothesis that tumor growth is angiogenesis-dependent was first proposed in 1971 by M. Judah
• Tumor 'take' has occurred, every increase in tumor cell population must be preceded by an increase in new capillaries converging on the tumor.”
• Tumor 'take' is currently understood to indicate a pre- vascular phase of tumor growth in which a population of tumor cells occupying a few cubic millimeters volume and not exceeding a few million cells, can survive on existing host microvessels. Expansion of tumor volume beyond this phase requires the induction of new capillary blood vessels.
• Angiogenesis not only provides the increased nutrients and pathways for the removal of waste needed for the expansion of the tumor, but it also facilitates tumor metastasis by providing a route for tumor cells to leave the primary site and enter the bloodstream (Zetter, 1998).
• angiogenesis increases the entry of tumor cells into the bloodstream by providing an increased density of immature, highly permeable blood vessels that have thinner basement membranes and fewer intracellular junction complexes than normal mature vessels
• This extended treatment-free period also creates a high risk of selection for drug-resistance tumors due to the genetic instability and high mutation rate of neoplastic cells (Donehower et al., 1992).
• many antiangiogenic agents fail to completely regress or prevent the reoccurrence of a tumor.
• the angiogenesis inhibitor TNP-470 has been reported to slow the growth, but not to regress drug-sensitive Lewis lung carcinoma (Brem et al., 1993).
• compositions and methods for the treatment of diseases associated with the abnormal growth of cells that increase the efficiency of the chemotherapeutic and/or antiangiogenic agents used for treatment. More particularly, compositions and methods are needed for the treatment of diseases associated with the abnormal growth of cells that reduce the dose of chemotherapeutic or antiangiogenic agent required for treatment.
• compositions and methods for the treatment of cancer are needed that reduce the occurrence of drug resistant tumors.
• the present invention addresses the continuing need for improved methods of treatment of diseases associated with the abnormal growth of cells, and accordingly relates to compositions and methods for the treatment of diseases associated with the abnormal growth of cells.
• the present invention provides methods of administering low doses of therapeutic agents.
• the therapeutic agents as described herein are chemotherapeutic agents and antiangiogenic agents.
• the antiangiogenic agent is endostatin protein.
• the methods of the present invention include antiangiogenic scheduling of one or more chemotherapeutic agents, one or more antiangiogenic agents, and a combination of one or more chemotherapeutic agents and one or more antiangiogenic agents.
• the present invention additionally provides the chemotherapeutic and antiangiogenic agents used in the methods described herein.
• compositions and methods of the present invention also address the continuing need for methods of treatment that reduce the occurrence of drug resistant tumors.
• antiangiogenic scheduling refers to the administration of one or more therapeutic agents in such a manner as to reduce vascularization or re-vascularization of a tumor during a treatment schedule.
• the antiangiogenic schedule provides a sustained apoptosis of the vascular endothelial cells in the tumor bed.
• a preferred antiangiogenic schedule comprises the administration of a therapeutic agent every four to eight days, and a more preferred antiangiogenic schedule comprises the administration of a therapeutic agent every six days.
• a single chemotherapeutic agent such as cyclophosphamide is administered on an antiangiogenic schedule.
• a single antiangiogenic agent such as endostatin protein, angiostatin protein or TNP-470 is administered on an antiangiogenic schedule.
• a combination of chemotherapeutic and antiangiogenic agents are administered on an antiangiogenic schedule.
• compositions and methods for the treatment of diseases associated with the abnormal growth of cells that are improved over the prior art.
• compositions and methods for antiangiogenic scheduling It is another object of the present invention to provide compositions and methods for antiangiogenic scheduling. It is another object of the present invention to provide compositions and methods for the treatment of diseases associated with the abnormal growth of cells that provide a more sustained apoptosis of the vascular endothelial cells in the tumor bed.
• Figure l (a-b).
• Figure 1 is a graph showing antiangiogenic versus conventional scheduling of cyclophosphamide for drug-resistant Lewis lung carcinoma. Open triangles - control saline, open circles - conventional schedule (150 mg/kg every other day for 3 doses (white arrows, total 450 mg/kg) every 21 days), closed circles - antiangiogenic schedule (170 mg/kg every 6 days, CTX, thin black arrows), closed squares - antiangiogenic schedule of cyclophosphamide and TNP-470 (170 mg/kg cyclophosphamide and 12.5 mg/kg TNP-470 administered on the same day of the 6 day cycle for 7 cycles, CTX + TNP, thick black arrows).
• Figure 2 is a table showing the in vitro anti- endothelial effects of activated cyclophosphamide on bovine capillary endothelial cell migration, survival and proliferation, apoptosis and cell cycle distribution.
• 4- hydroperoxycyclophosphamide which spontaneously converts to 4-HC in aqueous solution, was added at the indicated concentrations (Cone). Values shown are the mean ⁇ the standard error of the mean.
• Relative cell number refers to remaining, adherent endothelial cells (>600 fi) from an initial plating of 12,500 cells.
• Apoptosis was determined as a percentage of 10,000 intact (gated) cells by fluorescent flow cytometry. Cell cycle analysis was determined similarly using the ModFit LT software.
• Figure 3(a-b) is a graph showing endothelial cell apoptosis precedes tumor cell apoptosis in cyclophosphamide- resistant Lewis lung carcinoma.
• Apoptotic rates on the antiangiogenic schedule of cyclophosphamide for endothelial cells are denoted as open circles, dashed line and apoptotic rates on the antiangiogenic schedule of cyclophosphamide for tumor cells are denoted as closed circles, solid line.
• Day 0 reflects analysis of 2 control tumors harvested at 100-200 mm 3 .
• Day 0 reflects analysis of two control tumors harvested at 100-200 mm 3 .
• Figure 3(b) is a graph showing tumor cell apoptotic rates in drug-resistant Lewis lung carcinoma treated with the conventional schedule (open circles dotted line, white arrows) and antiangiogenic schedule (closed circles, solid line, black arrows).
• Day 0 reflects analysis of 2 control tumors harvested at 100-200 mm 3 .
• Figure 4 is a graph showing growth of drug-resistant Lewis lung carcinoma in p53 +/+ (dashed line) versus p53
• the present invention provides compositions and methods for the treatment of diseases associated with the abnormal growth of cells, and more specifically cancer.
• the compositions and methods of the present invention decrease the dose administration of antiangiogenic and chemotherapeutic agents.
• the present invention also addresses the continuing need for methods of treatment that reduce the occurrence of drug resistant tumors.
• the present invention provides methods of antiangiogenic scheduling of a therapeutic agent.
• antiangiogenic agents are administered to an individual in need of treatment at a low dose.
• antiangiogenic agent refers to a composition that is capable of reducing the formation or growth of blood vessels.
• antiangiogenic agents include, but are not limited to, endostatin protein, angiostatin protein, TNP-470, angiozyme, anti-VEGF, apra (CT2584), bay 12-9566, benefin, BMS275291, bryostatin- 1 (SC339555), CAI, carboxyamido-imidazole, CM 101, combretastatin, dexrazoxane
• ICRF187 DMXAA, EMD 121974, flavopiridol, GTE, IM862, interferon-alpha, interlukin-12, inhibitors of matrix metalloproteinases such as marimastat, metaret, metastat, MMI- 270, neovastat (AE-941), octreotide (somatostatin), paclitaxel (taxol), penicllamine, photopoint, PI-88, prinomastat (AG-3340), purlytin, PTK787, squalamine, suradista (FCE26644), SU101, SU5416, SU6668, tamoxifen (nolvadex), tetrathiomolybdate, thalidomide, vitaxin and xeloda (capecitabine), cycloogenase, platelet factor 4 (PF-4), an N-terminally truncated proteolytically cleaved PF
• a low dose of endostatin protein is administered to an individual.
• endostatin protein refers to a C-terminal region fragment of a collagen molecule that has antiangiogenic activity in vivo.
• collagen molecules include, but are not limited to, collagen XVIII, collagen XV and collagen IV.
• endostatin proteins may also be found in U.S. Patent No. 5,854,205 which is hereby incorporated by reference.
• the present invention provides methods of administering low doses of endostatin protein for the treatment of angiogenesis-related diseases and conditions.
• endostatin protein is administered at a dose of less than approximately 0.3 mg/kg/day, more preferably, between approximately 0.01 to 0.1 mg/kg/day and most preferably, between approximately 0.03 to 0.08 mg/kg/day.
• endostatin protein is administered to an individual wherein tumor development has progressed to a late stage at a dose of between approximately 1 to 20 mg/kg/day, more preferably between approximately 2 to 10 mg/kg/day, and most preferably between approximately 4 to 6 mg/kg/day.
• angiostatin protein refers to a kringle region fragment of a plasminogen molecule that has antiangiogenic activity in vivo.
• angiostatin proteins may be found in U.S. Patent No. 5,837,682 and U.S. Patent No. 5,854,221 which are hereby incorporated by reference.
• Plasminogen contains five kringle region fragments, denoted kringles 1-5, as well as inter-kringle regions. It is to be understood that the term “angiostatin protein” refers to any single kringle region, any combination of kringle regions, or any kringle regions in addition to any inter-kringle regions that retain antiangiogenic activity in vivo.
• angiostatin protein is approximately kringle regions 1-3, kringle regions 1-3.5, kringle regions 1-4 or kringle regions 1-4.5 of human plasminogen. In another preferred embodiment, angiostatin protein comprises kringle regions 1-4.5 of human plasminogen.
• endostatin protein and angiostatin protein also include shortened proteins wherein one or more amino acid is removed from either or both ends of an endostatin protein or an angiostatin protein, respectively, or from an internal region of either protein, yet the proteins retain angiogenesis inhibiting activity in vivo.
• endostatin protein and angiostatin protein also include lengthened proteins or peptides wherein one or more amino acids is added to either or both ends of an endostatin protein or an angiostatin protein, respectively, or to an internal location, yet the proteins retain angiogenesis inhibiting activity in vivo. Labeling endostatin protein and angiostatin protein with other radioisotopes or chemicals such as ricin may also be useful in providing a molecular tool for destroying the target cells containing endostatin protein or angiostatin protein receptors.
• angiostatin protein and "endostatin protein” are angiostatin protein and endostatin protein derivatives.
• An angiostatin protein derivative includes a protein having the amino acid sequence of a kringle region fragment of a plasminogen that has antiangiogenic activity.
• An angiostatin protein also includes a peptide having a sequence corresponding to an antiangiogenic angiostatin fragment of a kringle region fragment of a plasminogen.
• an "antiangiogenic angiostatin fragment” is defined to be a peptide whose amino acid sequence corresponds to a subsequence of a kringle region fragment of a plasminogen, referred to as an "antiangiogenic angiostatin subsequence".
• An endostatin protein derivative includes a protein having the amino acid sequence of a C-terminal region fragment of a collagen molecule that has antiangiogenic activity.
• An endostatin protein also includes a peptide having a sequence corresponding to an antiangiogenic endostatin fragment of a C- terminal region fragment of a collagen molecule.
• an “antiangiogenic endostatin fragment” is defined to be a peptide whose amino acid sequence corresponds to a subsequence of a C- terminal region fragment of a collagen molecule, referred to as an "antiangiogenic endostatin subsequence".
• a “subsequence” is a sequence of contiguous amino acids found within a larger sequence.
• a subsequence is generally composed of approximately at least 70%, more preferably 80%, and most preferably 90% of the larger sequence.
• Angiostatin and endostatin protein derivatives also include a protein or peptide having a modified sequence in which one or more amino acids in the original sequence or subsequence have been substituted with a naturally occurring amino acid residue or amino acid residue analog (also referred to as modified amino acid). Such substitutions may modify the bioactivity of angiostatin protein and angiostatin protein, such as by increasing or decreasing the angiogenesis inhibiting activity, and produce biological or pharmacological agonists or antagonists.
• Suitable angiostatin and endostatin derivatives have modified sequences which are substantially homologous to the amino acid sequence of an angiostatin and endostatin protein, respectively, or to an antiangiogenic subsequence of an angiostatin and endostatin protein, respectively.
• amino acid residue is a moiety found within a protein or peptide and is represented by -NH-CHR-CO-, wherein R is the side chain of a naturally occurring amino acid.
• amino acid residue and “amino acid” are used interchangeably.
• An “amino acid residue analog” includes D or L configurations having the following formula: -NH-CHR-CO-, wherein R is an aliphatic group, a substituted aliphatic aromatic 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.
• Suitable substitutions for amino acid residues in the sequence of the angiostatin and endostatin proteins described herein include conservative substitutions that result in angiogenic angiostatin and endostatin protein derivatives.
• a conservative substitution is a substitution in which the substituting amino acid (naturally occurring or modified) is structurally related to the amino acid being substituted.
• "Structurally related" amino acids are approximately the same size and have the same or similar functional groups in the side chains.
• Group I includes leucine, isoleucine, valine, methionine and modified amino acids having the following side chains: ethyl, rc-propyl n-butyl.
• Group I includes leucine, isoleucine, valine and methionine.
• Group II includes glycine, alanine, valine and a modified amino acid having an ethyl side chain.
• glycine alanine, valine and a modified amino acid having an ethyl side chain.
• Group II includes glycine and alanine.
• Group III includes phenylalanine, phenylglycine, tyrosine, tryptophan, cyclohexylmethyl, and modified amino residues having substituted benzyl or phenyl side chains.
• Preferred substituents include one or more of the following: halogen, methyl, ethyl, nitro, -NH 2 , methoxy, ethoxy and -CN.
• Group III includes phenylalanine, tyrosine and tryptophan.
• Group IV includes glutamic acid, aspartic acid, a substituted or unsubstituted aliphatic, aromatic or benzylic ester of glutamic or aspartic acid (e.g., methyl, ethyl, w-propyl iso- propyl, cyclohexyl, benzyl or substituted benzyl), glutamine, asparagine, -CO-NH-alkylated glutamine or asparagine (e.g., methyl, ethyl, n-propyl and w ⁇ -propyl) and modified amino acids having the side chain -(CH 2 ) 3 -COOH, an ester thereof (substituted or unsubstituted aliphatic, aromatic or benzylic ester), an amide thereof and a substituted or unsubstituted N-alkylated amide thereof.
• glutamic acid e.g., methyl, ethyl, w-propyl
• Group IV includes glutamic acid, aspartic acid, methyl aspartate, ethyl aspartate, benzyl aspartate and methyl glutamate, ethyl glutamate and benzyl glutamate, glutamine and asparagine.
• Group V includes histidine, lysine, ornithine, arginine, N-nitroarginine, ⁇ -cycloarginine, ⁇ -hydroxyarginine, N-amidinocitruline and 2-amino-4-guanidinobutanoic acid, homologs of lysine, homologs of arginine and homologs of ornithine.
• Group V includes histidine, lysine, arginine and ornithine.
• a homolog of an amino acid includes from 1 to about 3 additional or subtracted methylene units in the side chain.
• Group VI includes serine, threonine, cysteine and modified amino acids having C1-C5 straight or branched alkyl side chains substituted with -OH or -SH, for example, - CH 2 CH 2 OH, -CH 2 CH 2 CH 2 OH or -CH 2 CH 2 OHCH 3 .
• Group VI includes serine, cysteine or threonine.
• suitable substitutions for amino acid residues in the amino acid sequences described herein include "severe substitutions" that result in angiostatin and endostatin protein derivatives that are antiangiogenic.
• the side chain of the substituting amino acid can be significantly larger (or smaller) than the side chain of the amino acid being substituted and/or can have functional groups with significantly different electronic properties than the amino acid being substituted.
• severe substitutions of this type include the substitution of phenylalanine or cyclohexylmethyl glycine for alanine, isoleucine for glycine, a D amino acid for the corresponding L amino acid or -NH-CH[(-CH 2 ) 5 -COOH]-CO- for aspartic acid.
• a functional group may be added to the side chain, deleted from the side chain or exchanged with another functional group.
• Examples of severe substitutions of this type include adding an amine or hydroxyl, carboxylic acid to the aliphatic side chain of valine, leucine or isoleucine, exchanging the carboxylic acid in the side chain of aspartic acid or glutamic acid with an amine or deleting the amine group in the side chain of lysine or ornithine.
• the side chain of the substituting amino acid can have significantly different steric and electronic properties that the functional group of the amino acid being substituted. Examples of such modifications include tryptophan for glycine, lysine for aspartic acid and -(CH 2 ) COOH for the side chain of serine. These examples are not meant to be limiting.
• Substantial homology exists between two amino acid sequences when a sufficient number of amino acid residues at corresponding positions of each amino acid sequence are either identical or structurally related such that a protein or peptide having the first amino acid sequence and a protein or peptide having the second amino acid sequence exhibit similar biological activities.
• sequence homology there is substantial sequence homology among the amino acid sequences when at least 30%, more preferably at least 40%, and most preferably at least 50%, of the amino acids in the first amino acid sequence are identical to or structurally related to the second amino acid sequence. Homology is often measured using sequence analysis software, e.g., BLASTIN or BLASTP.
• endostatin protein and
• angiostatin protein also refer to fusion proteins containing endostatin protein and/or angiostatin protein.
• angiostatin protein and endostatin protein are recombinantly fused together into a single protein molecule.
• Endostatin protein and angiostatin protein may also be recombinantly fused to a Fc portion of an antibody.
• a therapeutic agent is administered on an antiangiogenic schedule.
• antiangiogenic schedule refers to the administration of one or more therapeutic agents in such a manner as to reduce vascularization or re-vascularization of a tumor during a treatment schedule.
• the term "antiangiogenic scheduling” includes, but is not limited to, modification of the timing of the administration of a therapeutic agent in order to reduce vascularization or re- vascularization of a tumor during a treatment schedule, modification of the amount of the administration of a therapeutic agent in order to reduce vascularization or re-vascularization of a tumor during a treatment schedule, and modification of the formulation of a therapeutic agent in order to reduce vascularization or re-vascularization of a tumor during a treatment schedule.
• the antiangiogenic schedule provides a relatively sustained apoptosis of the vascular endothelial cells in the tumor bed.
• the antiangiogenic schedule comprises the administration of a therapeutic agent every four to eight days, and more preferably every six days.
• the antiangiogenic schedule comprises the administration of cyclophosphamide every six days.
• doxil is administered on an antiangiogenic schedule of every six days.
• the antiangiogenic schedule comprises the administration of a therapeutic agent by continuous infusion.
• the antiangiogenic schedule comprises the administration of 5-fluorouracil or 6-mercaptopurine by continuous infusion.
• the antiangiogenic schedule comprises the administration of a therapeutic agent that is encapsulated into liposomes or conjugated to vascular integrin-binding peptides.
• the antiangiogenic schedule comprises the administration of a liposomal encapsulation of doxorubicin (doxil) or doxorubicin conjugated to vascular integrin-binding peptides.
• doxorubicin doxil
• doxorubicin conjugated to vascular integrin-binding peptides doxorubicin conjugated to vascular integrin-binding peptides.
• therapeutic agent refers to a pharmaceutical composition, a nutraceutical composition or radiation. Examples of pharmaceutical compositions include, but are not limited to, chemotherapeutic agents and antiangiogenic agents.
• composition refers to a natural, or non-synthetic, composition that provides health benefits to an individual to whom the composition is administered.
• the methods include antiangiogenic scheduling of one or more chemotherapeutic agents, one or more antiangiogenic agents, and a combination of one or more chemotherapeutic agents and one or more antiangiogenic agents.
• the present invention additionally provides the chemotherapeutic and antiangiogenic agents used in the methods described herein.
• chemotherapeutic agents are administered on an antiangiogenic schedule to an individual in need of such a treatment.
• chemotherapeutic agent refers to a chemical composition used for the treatment or control of a disease in an individual.
• chemotherapeutic agents include, but are not limited to, 6-mercaptopurine, dacarbazine, 1-asparaginase, procarbazine, raloxifen, tamoxifen, antimetabolites such as cytarabine, 5-fluorocil, gemcitabine, cladribine, fludarabine, pentostatin, and hyroxyurea, plant alkaloids such as docetaxel, paclitaxel, vinblastine, vincristine, and vinorelbine, topoisomerase inhibitors such as daunorubicin, doxorubicin, idarubicin, etoposide, teniposide, dactinomycin and mitoxantrone, alkylating agents such as busulfan, chlorambucil, cyclophosphamide, ifosfamide, mecholorethamine, melphalan, thiotepa, carmustine, lomustine, carb
• cyclophosphamide is administered on an antiangiogenic schedule to an individual in need of such a treatment.
• cyclophosphamide is administered to an individual in need of such a treatment on an antiangiogenic schedule, wherein the antiangiogenic schedule comprises administration every four to eight days, and more preferably every six days.
• the six day antiangiogenic schedule of cyclophosphamide ( 1 ) increased endothelial cell apoptosis in the tumor bed; (2) demonstrated long-term suppression of the growth of cyclophosphamide-resistant Lewis lung carcinoma, a significant improvement over the conventional schedule; (3) eradicated drug- sensitive Lewis lung carcinoma by avoiding acquired drug- resistance, an outcome not possible with a conventional schedule; and (4) eradicated the majority of drug-resistant Lewis lung carcinomas when combined with another angiogenesis inhibitor, TNP-470.
• a combination of chemotherapeutic and antiangiogenic agents are administered on an antiangiogenic schedule.
• cyclophosphamide and TNP-470 are administered on an antiangiogenic schedule to an individual in need of such a treatment.
• the cyclophosphamide and TNP-470 are administered on the same day of a six-day treatment cycle.
• the compositions and methods described above are useful for the treatment of diseases and conditions related to the abnormal growth or proliferation of cell.
• the compositions and methods of the present invention are useful for the treatment of metastatic and angiogenesis-dependent cancers and other angiogenesis-related diseases.
• metastatic disease is metastatic cancer.
• Angiogenesis-related diseases include, but are not limited to, angiogenesis-dependent cancer, including, for example, solid tumors, blood born tumors such as leukemias, and tumor metastases; benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osier-Webber Syndrome; myocardial angiogenesis; plaque neo vascularization; telangiectasia; hemophiliac joints; angiofibroma; and wound granulation.
• angiogenesis-dependent cancer including, for example, solid tumors, blood born tumors such as leukemias, and tumor
• the HA binding proteins of the present invention are useful in the treatment of disease of excessive or abnormal stimulation of endothelial cells. These diseases include, but are not limited to, intestinal adhesions, atherosclerosis, scleroderma, and hypertrophic scars, i.e., keloids. They are also useful in the treatment of diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa) and ulcers (Helobacter pylori).
• the antiangiogenic agents described above can be provided as isolated and substantially purified proteins and protein fragments. Both the antiangiogenic and chemotherapeutic agents described herein can be provided in pharmaceutically acceptable formulations using formulation methods known to those of ordinary skill in the art.
• the antiangiogenic and chemotherapeutic agents described above may be a solid, liquid or aerosol. Examples of solid therapeutic compositions include pills, creams, and implantable dosage units. The pills may be administered orally and the therapeutic creams may be administered topically.
• the implantable dosage units may be administered locally, for example at a tumor site, or may be implanted for systemic release of the therapeutic angiogenesis- modulating composition, for example subcutaneously.
• liquid compositions include formulations adapted for injection subcutaneously, intravenously, intraarterially, and formulations for topical and intraocular administration.
• aerosol formulations include inhaler formulations for administration to the lungs.
• the formulations may be administered by any route, including but not limited to, the topical, transdermal, intraperitoneal, intracranial, intracerebroventricular, intracerebral, intravaginal, intrauterine, oral, rectal or parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular) route.
• the antiangiogenic and chemotherapeutic agents may be incorporated into biodegradable polymers allowing for sustained release of the compound, the polymers being implanted in the vicinity of where drug delivery is desired, for example, at the site of a tumor or implanted so that the antiangiogenic or chemotherapeutic agent is slowly released systemically.
• biodegradable polymers and their use are described, for example, in detail in Brem et al., "Interstitial chemotherapy with drug polymer implants for the treatment of recurrent gliomas" J. Neurosurg. 74:441-446 ( 1991 ).
• Osmotic minipumps may also be used to provide controlled delivery of high concentrations of antiangiogenic and chemotherapeutic agents through cannulae to the site of interest, such as directly into a metastatic growth or into the vascular supply to that tumor.
• the dosage of the antiangiogenic and chemotherapeutic agents of the present invention will depend on the disease state or condition being treated and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound.
• the present invention provides compositions and methods for administering a low dose of chemotherapuetic and/or antiangiogenic agent.
• a “low dose” refers to a dosage between approximately 0.001 mg/kilogram per day to 0.3 mg/kilogram per day, more preferably 0.01 to 0.1 mg/kilogram per day, and most preferably 0.03 to 0.08 mg/kilogram per day.
• an antiangiogenic agent is administered to a human or animal on an antiangiogenic schedule, wherein the antiangiogenic schedule comprises administration every four to eight days, and more preferably every six days.
• the present invention has application for both human and veterinary use.
• the methods of the present invention contemplate single as well as multiple administrations, given either simultaneously or over an extended period of time.
• the therapeutic agent formulations may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). Suitable pharmaceutical carriers and excipients are known to those skilled in the art, however, an example of a suitable pharmaceutical excipient is water. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
• Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
• the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules or vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use.
• Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
• Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients, particularly mentioned above, the formulations of the present invention may include other agents conventional in the art having regard to the type of formulation in question. Optionally, cytotoxic agents may be incorporated or otherwise combined with the therapeutic agents described herein, or biologically functional protein fragments thereof, to provide dual therapy to the patient.
• Example 1 Eradication of drug-sensitive Lewis lung carcinoma by the antiangiogenic schedule of cyclophosphamide and prevention of acquired drug resistance
• treatment of a drug- sensitive Lewis lung carcinoma comprising administration of cyclophosphamide on an antiangiogenic schedule eradicated tumors in 100% of mice.
• drug- resistant Lewis lung carcinoma was explanted into tissue culture as described for the cyclophosphamide-resistant breast cancer cell line, EMT-6/CTX (Teicher et al., 1990).
• the drug-resistant Lewis lung carcinoma and the original, drug-sensitive Lewis Lung carcinoma were screened for mouse hepatitis virus and other pathogens, and stored in aliquots in liquid nitrogen.
• cells were thawed and passaged once in C57B 16/J mice (Jackson Labs, ME).
• mice harboring drug-resistant Lewis lung carcinomas received 170 mg/kg of cyclophosphamide subcutaneously every six days for two cycles and then the tumors were allowed to grow for transfer.
• Tumor inoculations, drug injections (including ondansetron and dexamethasone), and tumor measurements were carried out as described in Boehm et al., 1997 using 28-30 gram adult male C57B 16/J mice (Jackson Labs, ME) free of viral pathogens.
• Mice in these experiments were fed a "Western Type" diet, 42% calories from fat (TD 88137, Harlan Teklad, Madison, WI) to ameliorate weight loss. These food pellets were placed on the floor of the cage.
• Cyclophosphamide was then administered daily or every 3, 4, 5, 6, 7, or 8 days to mice bearing the drug-resistant Lewis lung carcinoma.
• the dosing schedules were more widely spaced and more sustained than similar non-conventional schedules reported previously for Lewis lung carcinoma (Humphreys et al., 1970;
• Cyclophosphamide administered at 170 mg/kg every six days proved more effective in controlling tumor growth than other cyclophosphamide schedules tested (including schedules with a higher dose-intensity, e.g. 135 mg/kg every 4 days, data not shown).
• Figure 1 (a) may be read as follows.
• mice treated on the conventional schedule lost 21% of body weight, which was regained prior to the next treatment cycle.
• the antiangiogenic schedule there was no net tumor growth for 36 days and weight loss was less than 5%. Following the first seven cycles (36 days) of therapy on the antiangiogenic schedule, tumor growth occurred at a slow rate.
• Cyclophosphamide induces apoptosis of endothelial cells in vitro
• the ability of cyclophosphamide to inhibit endothelial cell proliferation and induce apoptosis in vitro was determined as follows. For proliferation studies, 12,500 bovine adrenal capillary endothelial cells in DMEM and 10% bovine calf serum were plated onto gelatinized 24-well plates in quadruplicate. For apoptosis and cell cycle determinations, 2 x 10 6 cells were similarly split into TISO flasks. Sixteen hours later, the media was aspirated and replaced with DMEM and 5% bovine calf serum with or without 5 ng/ml bFGF (Scios, Mountain View, CA) as indicated.
• the cells were trypsinized and enumerated for proliferation as described in O'Reilly et al., 1994 or washed with phosphate buffered saline and incubated with annexin-fluorescein as per the Apo Alert Annexin V apoptosis detection kit (Clontech, Palo Alto,
• capillary endothelial cells were exposed for 16 hours to 4-HC in vitro at concentrations similar to those obtained in vivo (Kachel et al., 1994).
• the 4-HC drug induced a concentration-dependent cell cycle arrest and apoptosis of capillary endothelial cells (Figure 2).
• Lower concentrations 0.1 ⁇ g/ml 4- HO
• mice corneas were implanted with bFGF pellets which stimulated corneal neovascularization over 5-6 days (O'Reilly et al., 1994).
• cyclophosphamide controls drug-resistant Lewis lung carcinoma through endothelial cell inhibition is that cyclophosphamide induces in vivo apoptosis of endothelial cells prior to in vivo apoptosis of tumor cells.
• cell turnover was analyzed in drug- resistant tumors as follows.
• mice harboring drug-resistant Lewis lung carcinoma were injected with 0.5 ml of 10 mM BrdU (Boehringer- Mannheim, Indianapolis, IN) in phosphate buffered saline intraperitoneally one hour prior to being euthanized with Metofane (Mallinckrodt Veterinary, Mundelein, IL) followed by cervical subluxation.
• Metofane Methyl Veterinary, Mundelein, IL
• mice on the conventional schedule tumors on days 5, 7, 12, 14, 17, and 21 were analyzed for proliferation and apoptosis as previously described (Brem et al., 1993).
• mice on the antiangiogenic schedule tumors were analyzed on days 6.5, 7, 8, 10, and 12.
• Tumors were resected and immediately fixed in cold buffered formalin, incubated overnight at 4°C, then changed into cold phosphate buffered saline, and paraffin embedded within 24 hours of excision. Tumor sections of 5 microns were deparaffinized.
• Antigen retrieval included lOmM EDTA pH 6.0 at 70°C for 5 minutes and cooling to room temperature for 20 minutes, followed by digestion with proteinase K (Boehringer Mannheim, Indianapolis, IN) 10 ⁇ g/ml in 0.1 M Tris pH 7.4 at 37°C for 20 minutes.
• Double immunofluorescence staining with von Willebrand factor antibody and TUNEL assay was employed to discriminate endothelial cell apoptosis from tumor cell apoptosis.
• TUNEL assay was performed according to the fluorescein ApopTag kit (Oncor, Gaithersburg, MD). Slides were incubated with rabbit anti-human von Willebrand factor polyclonal antibody (Dako, Carpinteria, CA) 1 :500 overnight at 4°C. Biotinylated anti-rabbit secondary antibody was added, followed by Texas Red-avidin and anti-digoxigenin-fluorescein. Sections were co- stained with Hoechst 33258 (Sigma, St. Louis, MO).
• Drug-resistant tumor growth inhibition by cyclophosphamide is linked to endothelial cell p53
• endothelial cells are the main targets of cyclophosphamide in drug-resistant Lewis lung carcinoma.
• p53-null mice Donehower et al., 1992. While the endothelial cells in these mice should be as susceptible to cyclophosphamide-mediated DNA damage (as p53 +/+ endothelium), they should be unable to fully repair this DNA damage or undergo apoptosis without the arrest of cell cycle mediated by p53.
• tumor growth in p53 +/+ mice would depend on p53-normal endothelial cells, while tumor growth in p53 -/- mice would depend on p53-null endothelial cells.
• Figure 4 compares the growth of drug-resistant Lewis lung carcinoma in p53 +/+ mice and p53 -/- mice treated on an antiangiogenic schedule wherein cyclophosphamide was administered every six days at 170 mg/kg. While tumor growth in p53 +/+ mice was completely suppressed for at least six 6-day cycles of cyclophosphamide, tumor growth in p53 -/- mice was not affected by the first dose of cyclophosphamide. Tumor cell proliferation, and tumor cell and endothelial cell apoptosis of these tumors in p53 -/- mice were all equivalent to untreated control tumors until day six.
• cyclophosphamide is not directly cytotoxic to tumor cells per se, as evidenced by the initial rapid tumor growth in p53 -/- mice, and therefore, cyclophosphamide must be inhibiting tumor growth mainly through its cytotoxic effect on endothelial cells in the tumor bed.
• cyclophosphamide would inhibit endothelial cell migration (see Figure 2), cause endothelial cell G, arrest (see Figure 2) and induce endothelial cell apoptosis (see Figures 2 and 3a), thus resulting in a balance of tumor cell proliferation and apoptosis (see Figures la and 3b) during the initial 36 days.
• TNP-470 (a gift from TAP Holdings Inc., Deerfield, IL) was obtained as a lyophilized powder of 100 mg drug and 726 mg G2-beta-cyclodextrin. 10.3 mg of lyophilized powder was reconstituted with 0.95 ml sterile 5% glucose in water just prior to administration. The dose of TNP-470 was lowered to l/7th of the dose used in the prior art, because of the occurrence of severe weight loss when TNP-470 was combined with the antiangiogenic schedule of cyclophosphamide.
• TNP-470 This lower dose of TNP-470, 12.5 mg/kg every 6 days, was administered on the same day, or day 1, 2, or 4 following administration of cyclophosphamide (170 mg/kg). All drugs were administered subcutaneously, and TNP-470 was injected subcutaneously at 0.01 ml/g body weight on the opposite flank 30 minutes after cyclophosphamide. The combination of cyclophosphamide and TNP-470 on the same day of the six-day cycle proved most efficacious (data not shown).
• mice Following 7 cycles of cyclophosphamide on the antiangiogenic schedule with TNP-470, approximately 70% of treated mice received daily subcutaneous administered of dextrose/saline (10% glucose in 75 mM NaCI) for 3-5 days, because during this brief time period the mice ate and drank poorly.
• dextrose/saline 10% glucose in 75 mM NaCI
• Table 1 depicts the long-term survival of 5/6 mice treated with the antiangiogenic schedule of cyclophosphamide and TNP-470.
• the arrow and note on the graph reflect the recurrence of 1/6 drug-resistant tumor 18 days off therapy.
• the results of five separate experiments with the antiangiogenic schedule of cyclophosphamide and TNP-470 are detailed in Table 1.
• Days refer to days following the discontinuance of therapy on day 36 and the # symbol refers to experiments wherein cages, food and water were not sterilized by autoclaving.
• the mouse corneal angiogenesis assay was used to screen for antiangiogenic schedules of other chemotherapeutic agents.
• 5-fluorouracil Roche Laboratories, Nutley, NJ
• 6- mercaptopurine ribose phosphate Sigma, St. Louis, MO
• Doxorubicin gave 31 ⁇ 4% and Doxil 42 ⁇ 3%inhibition of angiogenesis (data not shown).
• Endostatin protein is a potent endogenous inhibitor of angiogenesis and tumor growth.
• Dose response analysis of the efficacy of recombinant human endostatin protein in the early stage of B 16BL6 experimental metastasis model revealed significant inhibition of tumor progression at doses as low as 0.05 mg/kg/day. This low dose administration was 2000 fold less than the dose of an administration of 100 mg/kg/day reported in the prior art to treat primary human xenografts and subcutaneous murine tumors.
• tumor progression was inhibited with the administration of endostatin protein at 5 mg/kg/day.

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