WO2006113536A2 - Use of pt523 for treating cancers - Google Patents

Abstract

The instant disclosure provides methods of treating or managing a cancer, or symptom thereof, using PT523 and pharmaceutical formulations thereof.

Description

USE OF PT523 FOR TREATING CANCERS

1. TECHNICAL FIELD

[0001] This application provides methods of treating or managing cancers, or symptoms thereof, including, for example, cervical cancer, non-small cell lung cancer, acute lymphocytic leukemia, and the like, using PT523 and pharmaceutical formulations thereof.

2. BACKGROUND

[0002] The dihydrofolate reductase (DHFR) inhibitor methotrexate (MTX), has been used as an anticancer drug since the 1950s. MTX is polyglutamated intracellularly which increases MTX retention (and its toxicity) in noncancerous cells and in many types of cancer cells. MTX-resistant cancers, either arising spontaneously or that acquired MTX-resistance in patients undergoing MTX therapy, typically have a reduced ability to form polyglutamated MTX. As resistance to MTX in cancer cells increases, MTX's toxicity to noncancerous cells prevents the administration of therapeutically effective dosages of MTX. MTX-resistance may be acquired in leukemias, such as acute lymphocytic leukemia, cervical cancers, lung cancers, including non-small cell lung cancer, colorectal cancers, breast cancers, and epidermoid cancers of the head and neck. [0003] It is important that a variety of therapeutic options are available to treat a cancer. For example, non-small cell lung cancer may recur or progress after standard first-line therapy of a platinum-based chemotherapeutic regimen (e.g., carbop latin or cisplatin), which requires a second or third line therapeutic option. Particularly desirable are methods that can be used without incurring resistance often found with classical DHFR inhibitors like MTX.

[0004] Scientists identified N^α-(4-amino-4-deoxypteroyl)-N^δ-hemiphthaloyl-L- ornithine (PT523) in the 1980s while assaying compounds in vitro for their potential at inhibiting growth of MTX-resistant tumor cells. See Rosowsky et al., 1988, J Med. Chem. 31:1332-1337; Rosowsky et al, 1989, Pteridines 1:91-98; U.S. Pat. No. 4,767,761. PT523 is a nonpolyglutamatable DHFR inhibitor that is efficiently carried into cells by the reduced folate carrier pathway. See Rosowsky, 1999, Curr. Med. Chem. 6:329-352, incorporated by reference herein in its entirety for all purposes.

[0005] U.S. Patent No. 6,989,386, issued January 24, 2006, discloses ammonium salts of PT523, inter alia, that possess a superior combination of aqueous solubility and improved chemical stability in pharmaceutical compositions as compared to the corresponding acidic form of PT523. [0006] Citation or identification of any reference in this or any other section of this application shall not be construed as an admission that such reference is available as prior art to the present invention.

3. SUMMARY OF THE INVENTION

[0007] The present disclosure provides methods for the treatment or management of a cancer or a symptom thereof, hi particular, the present methods utilize PT523 or liquid pharmaceutical formulations thereof. Administration of PT523 and formulations thereof can be useful therapy for delaying cancerous growth, reducing tumor sizes or cancerous cell numbers or prolonging survival of the subject afflicted with cancer. As suggested, for example, in the toxicology, pharmacokinetic and efficacy studies described herein, the

PT523 formulations and methods using these formulations, including the doses and dosing schedules provided herein, are likely to minimize toxic side-effects while providing beneficial therapeutic effects.

[0008] hi one aspect, the present invention provides methods that comprise administering PT523 to the subject thereby treating or managing a cancer or symptom thereof. The subject can be a mammal, preferably a human.

[0009] In some embodiments, the PT523 to be administered is a PT523 ammonium salt, hi certain preferred embodiments, a dissolved PT523 ammonium salt is administered to the subject.

[0010] In certain embodiments, PT523 is administered by a parenteral route. In some embodiments, PT523 is intravenously administered.

[0011] In certain embodiments, an amount of PT523 effective to treat or manage a cancer or symptom thereof is administered to a subject, wherein the effective amount of

PT523 is tolerable by the subject.

[0012] hi certain embodiments of the methods provided, about 0.2 mg/m² to about

900 mg/m², or between about 5 mg/m² to about 600 mg/m², or between about 30 mg/m² to about 400 mg/m², or between about 50 mg/m² to about 200 mg/m², or about 75.0 mg/m² to about 175 mg/m², or between about 9 mg/m² to about 200 mg/m², or between about

9 mg/m² to about 160 mg/m², or between about 9 mg/m² to about 100 mg/m², or between about 9 mg/m² to about 80 mg/m² of PT523 is administered.

[0013] In certain embodiments, the cancer to be treated or managed is an ovarian cancer, endometrial cancer, biliary cancer, esophageal cancer, mesothelioma, cervical cancer, vulvar cancer, gastric cancer, pancreatic cancer, breast cancer, prostate cancer, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, non-small cell lung cancer, melanoma, lymphoma, renal cancer, colon cancer, soft tissue sarcoma, head and neck squamous carcinoma, or symptom thereof.

[0014] In certain embodiments, a second therapeutic agent is administered to the subject. Optionally, a third therapeutical agent is administered to the subject. Typically, the second therapeutic agent or optional third therapeutic agent is a chemotherapeutic agent, such as, for example, cisplatin, carboplatin, paclitaxel, docetaxel, vinorebine, gemcitabine, irinotecan, topotcan, erlotinib, and the like, hi some embodiments the second therapeutic agent or third therapeutic agent is an epidermal growth factor receptor-tyrosine kinase

(EGFR-TK) inhibitor, for example, gefitinib or erlotinib. The second therapeutic agent or optional third therapeutic agent can be administered concurrently or sequentially with

PT523 administration.

[0015] In some embodiments, the second therapeutic agent or third therapeutic agent is an anti-angiogenesis inhibitor, a radioisotope or a radiolabled agent.

[0016] In certain embodiments, PT523 is administered to the subject in a liquid formulation comprising PT523 ammonium salt, mannitol, L-arginine and 5% dextrose in water.

[0017] In certain embodiments, PT523 is administered to the subject in a dosing schedule or "therapeutic cycle" generally comprising about two to about five weeks in which PT523 is administered on day one of the cycle followed by a number of days in which PT523 is not administered. For example, in a typical 28 day therapeutic cycle PT523 is administered on days 1, 8 and 15. Following the 28^th day of the cycle, a second cycle can be initiated, typically within 1 to 4 days. Dosing schedules for administration of PT523 are provided below.

[0018] In certain embodiments, the methods provided herein for the treatment or management of a cancer, further comprise administering folic acid or a folate binding protein binding agent to the subject before, during, and/or after the administration of PT523.

[0019] In some embodiments, the methods provide for the administration of cyanocobalamin (Vitamin B₁₂) to the subject prior to, during, or after PT523 is administered.

4. TERMINOLOGY

[0020] The term "about," as used herein, unless otherwise indicated, refers to a value that is no more than 10% above or below the value being modified by the term. For example, the term "about 5 mg/m²" means a range of from 4.5 mg/m² to 5.5 mg/m². As another example, "about 1 hour" means a range of from 54 minutes to 66 minutes. [0021] As used herein, the terms "manage," "managing", "management" and the like refer to the beneficial effects that a subject suffering from a cancer derives when the methods provided herein are practiced on that subject, but which do not result in a cure of the cancer. In certain embodiments, a subject is administered PT523 as described herein to "manage" a cancer so as to prevent or slow the progression or worsening of the cancer. For example, in some embodiments, a subject is administered PT523 as described herein to "manage" a cancer so as to prevent or slow tumor growth. In some embodiments, a subject is administered PT523 as described herein to "manage" a cancer so as to lengthen what would otherwise be the expected life span of the subject without being administered PT523 for the cancer.

[0022] The term "pharmaceutically acceptable," as used herein, refers to a composition, e.g., a carrier, diluent, excipient, vehicle or salt, approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly, in humans. [0023] The term "pharmaceutical formulation," as used herein, refers to composition comprising PT523, and a suitable carrier, diluent, excipient, or vehicle suitable for administration to a subject. The term includes, but is not limited to compositions for parenteral administration as described below.

[0024] As used herein, the terms "subject" and "patient" are used interchangeably, and can refer to a mammal (e.g., a mouse, rat, guinea pig, rabbit, cow, pig, horse, donkey, goat, sheep, camel, cat, dog), more preferably a primate (e.g., a monkey, ape, gorilla, chimpanzee), and most preferably a human.

[0025] As used herein, the terms "treat", "treatment", "treating" and the like refer to the eradication, reduction or amelioration of a cancer or symptom thereof that results from the administration of PT523 as described herein. In certain embodiments, a subject is administered PT523 as described herein to "treat" a cancer so as to result in tumor shrinkage or reduce cancer cell numbers. In certain embodiments, a subject is administered PT523 as described herein to "treat" a cancer so as to prevent or halt spread of a cancer. In certain embodiments, a subject is administered PT523 as described herein to "treat" a cancer so as to provide a beneficial clinical outcome, e.g., an increased subject survival time. 5. DETAILED DESCRIPTION OF FIGURES

[0026] Figure 1 provides body weight averages of athymic mice implanted with

A549 human lung cancer cells after treatment with PT523 alone or in combination with paclitaxel, cisplatin or TARCEV A^®.

[0027] Figure 2 provides percent changes in average body weight from Day 1 of treatment of athymic mice implanted with A549 human lung cancer cells after the treatment with PT523 alone or in combination with paclitaxel, cisplatin or TARCEV A^®.

[0028] Figure 3 presents tumor volumes in athymic mice implanted with A549 human lung cancer cells after the treatment with PT523 alone or in combination with paclitaxel, cisplatin or TARCEV A^®.

[0029] Figure 4 presents percent changes in tumor volumes in athymic mice implanted with A549 human lung cancer cells after the treatment with PT523 alone or in combination with paclitaxel, cisplatin or TARCEV A^®.

[0030] Figure 5 presents changes in tumor volumes in athymic mice implanted with

A549 human lung cancer cells after the treatment with PT523 alone.

[0031] Figure 6 presents changes in tumor volumes in athymic mice implanted with

A549 human lung cancer cells after the treatment with PT523 alone or in combination with paclitaxel.

[0032] Figure 7 presents changes in tumor volumes in athymic mice implanted with

A549 human lung cancer cells after the treatment with PT523 alone or in combination with cisplatin.

[0033] Figure 8 presents changes in tumor volumes in athymic mice implanted with

A549 human lung cancer cells after the treatment with PT523 alone or in combination with

TARCEVA^®.

6. DETAILED DESCRIPTION OF THE INVENTION

[0034] In one aspect, the present invention provides methods for the treatment or management of a cancer or symptom thereof in a subject in need of such treatment or management. These methods are based, for example, on the observation of the particular toxicology, pharmacokinetic and in vivo efficacy studies of PT523 described below. Safe and effective doses for the administration of pharmaceutical formulations described herein have been determined, and the methods described herein provide a novel approach to the treatment or management of a cancer or symptom thereof in a subject. Preparation of PT523 and formulations suitable for administration to a subject, preferably a human, are described in Section 6.1. [0035] In certain embodiments, the methods provided for the treatment or management of a cancer or symptom thereof in a subject in need of such treatment or management comprise administering an effect amount of a dissolved PT523 ammonium salt thereby treating or managing the cancer or symptom thereof. [0036] hi general, the cancer to be treated or managed can be a metastatic or inoperable malignancy, neoplasm, carcinoma, leukemia, sarcoma, tumor, solid tumor, lymphoma, or any cancer for which there is no known curative or standard or standard palliative therapy, or for which standard therapy has failed.

[0037] In some embodiments, PT523 is administered to the subject as a first line therapy for the treatment or management of a cancer or symptom thereof. In some embodiments, PT523 is administered as a second or third line therapy. [0038] hi certain embodiments, PT523 is administered to the subject in need of treatment or management of cancer, or symptom thereof, resistant to treatment or management with methotrexate, aminopterin, or pemetrexed.

[0039] In certain embodiments, the cancer or symptom thereof to be treated or managed is an ovarian cancer, endometrial cancer, biliary cancer, esophageal cancer, mesothelioma, cervical cancer, vulvar cancer, gastric cancer, pancreatic cancer, breast cancer, prostate cancer, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, non-small cell lung cancer, melanoma, lymphoma, renal cancer, colon cancer, soft tissue sarcoma, head and neck squamous carcinoma, or symptom thereof. [0040] hi preferred embodiments, the cancer to be treated or managed is a non-small cell lung cancer or a symptom thereof. Generally, the non-small lung cancer can be histologically or cytologically confirmed prior to administration of PT523. hi some embodiments the non-small lung cancer is a recurrent disease or one for which first line therapy has failed.

[0041] hi preferred embodiments, the cancer to be treated or managed is a leukemia such as acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, or a symptom thereof. Generally, the leukemia can be histologically or cytologically confirmed prior to administration of PT523. hi some embodiments the leukemia is a recurrent or refractory disease or one for which first line therapy has failed. 6.1. Preparation of PT523 and Pharmaceutical Formulations Thereof

[0042] PT523 suitable for administration to a subject may be obtained by any synthetic technique known to those of skill in the art or can be obtained commercially (e.g., Nerviano Medical Sciences S.r.L, Nerviano, Italy). The structure and exemplary syntheses ofPT523 are described in, for example, Rosowsky et al., 1988, J Med. Chem. 31 :1332- 1337, Rosowsky et al, 1989, Pteridines 1:91-98, and U.S. Pat. Nos. 4,767,761 and 6,989,386, which are incorporated herein by reference in their entireties for all purposes. [0043] In preferred embodiments, a PT523 ammonium salt, or pharmaceutical formulations thereof, are administered to the subject in the methods provided herein. Preparation of PT523 ammonium salts and compositions comprising PT523 ammonium salts are described, for example, in U.S. Pat. No. 6,989,386, issued January 24, 2006, and WO 2004/094427 Al, which are incorporated herein by reference in their entireties for all purposes. .

[0044] In some embodiments where PT523 is administered to a subject for the treatment or management of a cancer or symptom thereof, PT523 is a reconstituted or dissolved form of a PT523 ammonium salt. For example, PT523 can be a reconstituted or dissolved form of a monoammonium salt, diammonium salt, other incremental ammonium salt, mixtures of monoammonium salt, diammonium salt, and/or other incremental ammonium salt, and so forth, without limitation. In some embodiments, the molecular formula of PT523 is C₂₇H₂₇N₉O₆-NH₃.

[0045] In some embodiments, PT523 in an ionic form, preferably in a pharmaceutical ionic form, is administered to a subject for the treatment or management of a cancer or symptom thereof. Counter ions to ionic PT523, or pharmaceutical formulations thereof, can be, for example, inorganic cations, such as Na⁺, K⁺, Ca²⁺, Cs⁺, Sr⁺, Zn²⁺, Ba²⁺, Mg²⁺, Al²⁺, or Li⁺; aliphatic amines, for example, (n-Bu)₄ N⁺, tetraethylammonium, tetramethylammonium, meglumine, D-glucosamine, D-galactosamine, D-glucamine, piperazine, tromethamine, choline, diethylamine, 4-phenylcyclohexylamine, benzathine, 2-aminoethanol, benethamine, adamantanamine, guanidine, 1-methyl-guanidine, 1-ethyl- guanidine, tryptamine, 5-methoxytryptamine, olamine, diolamine; or amino acids such as L-arginine, L-lysine, cysteine, L-ornithine, L-histidine, serine. Other exemplary counter ions include but not limited to mono, bis and trialkyl amines like phenothiazine-propanamine, dimethylaminoethanol, imidazoleethanamine, methyl ammonium, dimethyl ammonium, trimethyl ammonium, ethyl ammonium, diethyl ammonium, triethyl ammonium, cyclohexyl ammmonium, dicyclohexyl ammonium, tricyclohexyl ammonium, benzyl ammonium, dibenzyl ammonium, tribenzyl ammonium, N-methyl-N-b enzylammonium, alpha-methylb enzylammonium, alpha-methyl- N-benzylbenzylammonium, alpha-metliyl-alpha-naplithylmetliylammonium, benzhydrylammonium, N-methylpiperazinium, N-methyl tryptammonium, N,N,-dimethyltryptammonium, pyrollidinium, piperidinium, N-methylpiperidinium, N-benzylpiperidinium, 4-amino- 1 -benzylpiperidinium, 1 -acetylpiperazinium, morpholinium, N-methyl, and N-ethyl morpholinium, 4-aminocyclohexanol, 3-acetamidopyrollidine, N-acetylethylenediamine, 1 -adamantanemethylamine, l-(l-adamantyl)ethylamine, N-(l-adamantyl)ethylenediamine, beta-alanine amide, allylamine, acetamidine, aminoacetamidine, 2-(2-aminoethoxy)ethanol, 2-amino-2-ethyl- 1,3 -propanediol, R- and S- 2-aminobutane, 3-amino-l-propanol, R- and S-l-amino-2-propanol, R- and S-2-amino-l-butanol, 4-amino- 1-butanol, cyclobutylamine, 2-aminocyclohexanol, 4-aminocyclohexylamine, cis- and trans-2-aminocyclohexanol, cis- and trans-2-aminomethyl- 1 -cyclohexanol, cis-2-amino- 1 -cyclohexanecarboxamide, 1 -amino-2-ethylhexane, 6-amino-2-methyl-2-heptanol, 2-amino-2-methyl- 1 ,3 -propanediol, 2-amino-2-methyl-l-propanol, 2-amino-4-phenylbutane, (1R,2R) and (lS,2S)-2-amino- 1-phenylpropanediol, R- and S-3-amino-3-phenyl-l-propanol, R- and S-2-phenylglycinol, 2-phenylethylamine, R- and S- phenylalanilol, R- and S- valinol, 2-amino- 1,3 -propanediol, 3-amino-l,2-propanediol, 2-amino- 1-benzyloxybutane, 1-aminoindane, and (1R,2S- and 1 S,2R)-cis- 1 -amino-2-indanol.

[0046] In certain embodiments, the form of PT523 administered to the subject consists of no more than 4% of the D-enantiomeric form. In some embodiments, the form of PT523 administered to the subject consists of no more than 3%, 2%, 1% or 0.5% of the D-enantiomeric form.

[0047] In certain embodiments, PT523 or pharmaceutical formulation thereof is administered orally, parenterally or topically to the subject.

[0048] Liquid pharmaceutical formulations of PT523, its ammonium salts or other salts provided herein, comprise PT523 in a suitable pH, osmolality, tonicity, purity and sterility to allow safe administration to a subject. Liquid pharmaceutical formulations of PT523 can be prepared, for example, by dissolving, dispersing, reconstituting, solubilizing or otherwise mixing PT523 and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension. [0049] Solutions or suspensions used for parenteral, oral, intradermal, subcutaneous, or topical administration of PT523 or pharmaceutical formulations thereof can include any known pharmaceutically acceptable components as exemplified by, but not limited to, the following: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, arginines, citrates and phosphates; and agents for the adjustment of tonicity such as sodium chloride, sucrose or dextrose.

[0050] Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances. [0051] If administered intravenously, suitable carriers include physiological saline, phosphate buffered saline (PBS) or dextrose and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.

[0052] Parenteral preparations of PT523 can be enclosed in ampules, disposable syringes or single or multiple dose vials made of glass, plastic or other suitable material. [0053] Suitable PT523 pharmaceutical formulations can be dry formulations that include freeze dried or lyophilized powders for long term storage followed by reconstitution in a suitable diluent prior to administration. Typically, PT523 can be supplied in containers containing about 25 mg or about 50 mg of PT523 in a lyophilized powder form. In preferable embodiments, a dry PT523 pharmaceutical formulation comprises PT523 in the form of an ammonium salt. PT523 can be reconstituted or dissolved in sterile water or other pharmaceutically acceptable solvent prior to administration to the subject. [0054] Other dry pharmaceutical formulations of PT523 include those wherein a composition comprising PT523 is compressed into tablet or pill form suitable for oral administration or compounded into a sustained release formulation. When the dry formulation is intended for oral administration but PT523 is to be delivered to epithelium in the intestines, it is preferred that the formulation be encapsulated with an enteric coating to protect the formulation and prevent premature release of PT523 therein. A variety of enteric coatings are known in the art, and any suitable coating, or combinations of coatings, can be employed. [0055] As those skilled in the art will appreciate, the pharmaceutical formulations of

PT523 can be placed into any suitable dosage form. Pills and tablets represent some of such dosage forms. The pharmaceutical formulations can also be encapsulated into any suitable capsule or other coating material, for example, by compression, dipping, pan coating, spray drying, etc. Suitable capsules include those made from gelatin and starch. In turn, such capsules can be coated with one or more additional materials, for example, and enteric coating, if desired.

[0056] In certain embodiments of the methods provided herein, PT523 or a pharmaceutical formulation thereof is administered as a pill, e.g., a capsule, tablet, caplet or the like, that is suitable for oral administration. Numerous capsule manufacturing, filling, and sealing systems are well-known in the art. Preferred capsule dosage forms can be prepared from gelatin and starch. Gelatin has been the traditional material, and the dosage forms are generally produced by well known dip molding techniques. After manufacture, gelatin capsules are filled with the desired composition and then sealed. A more recently developed alternative to gelatin dosage forms are capsules produced from starch. Starch capsules (typically made from potato starch) afford several advantages compared to gelatin capsules, including pH-independent dissolution, better suitability for enteric coating, water in the dosage form is tightly bound to the starch (and is thus less likely to migrate into the composition encapsulated in the dosage form), and the absence of animal-derived ingredients (which may be antigenic or contaminated with pathogens). Starch capsules are odorless and rigid, and exhibit similar dissolution properties as compared to gelatin capsules.

[0057] Containers of dry or liquid pharmaceutical formulations can be labeled to identify to identify the formulation contained therein and other info useful to health care providers and subjects in the treatment of cancers or symptoms thereof including, but not limited to, instructions for use, dose, dosing interval, duration, indication, contraindications, warnings, precautions, handling and storage instructions and the like. 6.2. Doses and Dosing Schedules

[0058] The amount of PT523 to be administered to a subject in the methods provided can be determined based on subject's body surface area (BSA). For example, in calculating BSA, actual heights and weights of the subject can be used, with no adjustment to "ideal" body weight. For instance, BSA (m²) can be calculated as follows:

[0059] In a non-limiting embodiment of the methods provided wherein PT523 is administered to a subject, the dose administered can be between about 0.2 mg/m² to about

900 mg/m², or between about 5 mg/m² to about 600 mg/m², or between about 30 mg/m² to about 400 mg/m², or between about 50 mg/m² to about 200 mg/m², or about 75.0 mg/m² to about 175 mg/m², or between about 9 mg/m² to about 200 mg/m², or between about

9 mg/m² to about 160 mg/m², or between about 9 mg/m² to about 100 mg/m², or between about 9 mg/m² to about 80 mg/m².

[0060] In certain embodiments of the methods provided, PT523 is administered in an amount effective to treat or manage the cancer of symptom thereof wherein the amount of PT523 administered is tolerable by the subject. By tolerable, it is meant that the side effects of PT523 are reversible (e.g., nonlethal) and relatively short-lasting (i.e., the subject should be free of side effects within minutes or by 6-15 days). In some embodiments, doses of PT523 are tolerable if at some time after PT523 administration, the subject's absolute neutrophil count (ANC) is equal to or exceeds 1000/mm³, platelet count is equal to or exceeds 50,000/mm³ and/or serum creatinine concentration is equal to or less than 1.5 mg/dL.

[0061] A dose of PT523 or pharmacological formulations thereof can be administered at once to a subject, or may be divided into a number of smaller doses to be administered at intervals of time.

[0062] In certain embodiments, PT523 is administered once per day for at least one day. In some embodiments, PT523 is administered to the subject once per day for two, three, four, five or more consecutive days.

[0063] In certain embodiments, PT523 is administered to the subject once a week.

For example, PT523 can administered to the subject about once per week for at least about two consecutive weeks, about three consecutive weeks, about four consecutive weeks, about five consecutive weeks or more consecutive weeks.

[0064] In certain embodiments, PT523 is administered in a dosing schedule or

"therapeutic cycle." In a therapeutic cycle PT523 is administered on day 1 of the cycle and the cycle concludes with one or more days of no PT523 administration. During one or more days during the cycle, PT523 can be administered. For example, in a 28-day cycle, PT523 can be administered to the subject on days 1, 8 and 15 followed by 13 days of no PT523 administration, to constitute one cycle. As another example, in a 22-day cycle, PT523 can be administered about once per week for two weeks (for instance on day 1 and on day 8 or day 15) followed by at least a week of no administration, to constitute one cycle.

[0065] In certain embodiments, PT523 or a pharmaceutical formulation thereof is administered to the subject in one, two, three, four or five infusions over a therapeutic cycle of 21 days. PT523 or a pharmaceutical formulation thereof can, for example, be administered on days 1 and 8 of a 21 day cycle. As another example, PT523 or a pharmaceutical formulation can be administered from dayl to day 5 followed by sixteen days of no administration in a 21 day cycle.

[0066] In some embodiments, PT523 or a pharmaceutical formulation thereof is administered to the subject for five days, followed by a period of no administration of

PT523 or a pharmaceutical formulation thereof.

[0067] m certain embodiments, PT523 or a pharmaceutical formulation thereof is administered to the subject in two to four infusions over a therapeutic cycle of about two to about six weeks. In some embodiments the duration of a therapeutic cycle is about three weeks, about four weeks, about five weeks or about seven weeks.

[0068] It will be understood that where a dose of PT523 to be administered in a cycle is specified, then the dose of PT523 to be administered represents the total dose administered to the subject in the cycle. For example, 30 mg/m² PT523 administered on days 1, 8 and 15 in a 28-day cycle means that 10 mg/m² PT523 is administered on days 1, 8 and 15.

[0069] Methods of treatment or management of a cancer or a symptom thereof in a subject can comprise any number of therapeutic cycles. In certain embodiments, PT523 is administered in two or more therapeutic cycles. Therapeutic cycles can begin one after the other or may be separated by one and up to four days.

[0070] In some embodiments, the following criteria should be observed in the subject before a second or later dose of PT523 is administered to the subject: absolute neutrophil count (ANC) ≥lOOO/mm³; platelets >50,000/mm³; and serum creatinine <1.5 mg/dL. A second or later dose of PT523 can be administered to a subject in which their values for ANC, platelet count and serum creatinine concentration exceed these numbers.

[0071] In general, subjects who demonstrate objective tumor response continue on a therapeutic cycle. Response to PT523 administration and tumor regression can be evaluated, for example, using the international criteria proposed by the Response Evaluation criteria in Solid Tumors (RECIST). See Therasse et al, 2000, J. Natl Cancer Inst. 92:205-

16. Changes in only the largest diameter (uni-dimensional measurement) of the tumor lesions are used in the RECIST criteria. Measurements are made with conventional techniques (CT, MRI, X-ray, spiral CT scan), or if superficial lesions are to be observed, e.g., skin lesions or nodules apparent beneath skin, or palpable lymph nodes, these may be measured directly, for example, using a ruler.

[0072] In the methods provided, PT523 or pharmaceutical formulations thereof can be parenterally, intradermally, or topically administered. In certain embodiments, PT523 or a pharmaceutical formulation thereof is administered parenterally.

[0073] The teπn parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intraarterial injection, or infusion techniques.

[0074] In preferred embodiments, PT523 or a pharmaceutical formulation thereof is administered intravenously to the subject. For example, continuous intravenous infusions can be administered with the aid of an i.v. drip, infusion pump or device. In certain embodiments, PT523 can be administered in a combination of continuous intravenous infusions and intravenous push infusion ("bolus doses"). In some embodiments, PT523 is delivered through a free running peripheral intravenous or central venous catheter, without the use of an inline filter, using an infusion pump or delivered via either gravity drip or i.v. push. Typically, PT523 can be administered in an about five minute infusion. PT523 can administered into a single arm of the subject. However, any suitable vessel can be used for infusion, including peripheral vessels such as the vessels in the antecubital fossa of the arm or a central line into the chest. In preferred embodiments, the composition is infused into the cephalic or median cubital vessel at the antecubital fossa in the arm of the subject.

6.3. Supportive Care

[0075] In certain embodiments of the methods provided wherein PT523 is administered to a subject, folic acid, a folate binding protein binding agent, and/or cyanocobalamin (vitamin B₁₂) is administered prior to, during, and/or after PT523 administration to the subject. Without intending to be bound by any particular theory or mechanism, it is believed that administration of folic acid or folate binding protein binding agent and cyanocobalamin reduce toxicities without affecting the efficacy of PT523 administered to a subject.

[0076] Folate binding protein binding agents are known to those skilled in the art.

See, e.g., U.S. Patent Nos. 5,217,974 and 6,579,877, incorporated herein by reference in their entireties.

[0077] In certain embodiments, folic acid, a folate binding protein binding agent or cyanocobalamin are administered to the subject one, two, three, four, five, six, seven or eight days before PT523 is administered to the subject.

[0078] In some embodiments, folic acid, a folate binding protein binding agent or cyanocobalamin are administered to the subject on the same day on which PT523 is administered to the subject.

[0079] In some embodiments, folic acid, a folate binding protein binding agent or cyanocobalamin are administered to the subject between one day to one week, or between one week to three weeks after PT523 is administered to the subject.

[0080] Folic acid, a folate binding protein binding agent or cyanocobalamin can be administered to the subject in weekly intervals or in daily intervals.

[0081] Typically, about 350 μg to about 1000 μg folic acid (or an equivalent amount of a folate binding protein binding agent) can be administered daily to the subject before, during and/or after PT523 administration. Folic acid is generally taken orally.

Cyanocobalamin can be orally administered daily to the subject in, for example, about

100 μg doses. In certain embodiments, folic acid, folate binding protein binding agent and/or cyanocobalamin are administered by parenteral, subcutaneous, or intramuscular delivery.

[0082] Generally, subjects to whom PT523 is administered in the methods provided can be given blood and platelet transfusions, narcotics or standard anti-emetic therapy as appropriate.

6.4. Combination Therapy

[0083] In certain embodiments of the methods provided wherein PT523 is administered to a subject, a second therapeutic agent, optionally a third therapeutic agent, is administered to the subject.

[0084] The use of the term "combination therapy" or "combination cancer therapy" does not limit the order in which agents or treatments are administered to a subject in the methods provided. For example, the agents of the combination therapy can be administered concurrently, sequentially in any order or cyclically to a subject. In some embodiments, two components of a combination therapy are administered concurrently to a subject. [0085] Components of combination therapy can be administered to a subject in the same pharmaceutical composition. Alternatively, components of combination therapies can be administered to a subject in separate pharmaceutical compositions, and these separate compositions may be administered by the same or by different routes of administration, including, for example, oral, parenteral, or topical.

[0086] In preferable embodiments, the second therapeutic agent, and/or optional third therapeutic agent are administered to subject according to their respective standard or art-recognized doses and dosing schedules.

[0087] In some embodiments, a second therapeutic agent, and/or optional third therapeutic agent, is selected for its additive effects with PT523 on the treatment or management of a cancer or symptom thereof.

[0088] In some embodiments, a second therapeutic agent, and/or optional third therapeutic agent, is selected for its synergistic effects with PT523 on the treatment or management of a cancer or symptom thereof.

[0089] Examples of agents that can be used a second therapeutic agent or third therapeutic agent in the methods provided herein, include chemotherapeutic agents and toxins. For illustration, a second therapeutic agent or therapeutic agent can be abrin, anthramycin (AMC), asparaginase, auristatin E, 5-azacytidine, azathioprine, bleomycin, busulfan, buthionine sulfoximine, camptothecin, carboplatin, carmustine (BSNU), CC- 1065, chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin, daunorubicin, decarbazine, docetaxel, doxorubicin, an estrogen, 5-fluorodeoxyuridine, fluosol, 5-fluorouracil, gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU), mechlorethamine, melphalan, 6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone, nitroimidazole, plicamycin, procarbizine, pseudomonas exotoxin, ricin A, streptozotocin, tenoposide, 6-thioguanine, thioTEPA, topotecan, triapazamine, vinblastine, vincristine, vinorelbine, VP- 16 and VM-26.

[0090] hi certain embodiments, a second therapeutic agent or optional third therapeutic agent is selected from the agents consisting of cisplatin, carboplatin, cetuximab, cyclosphamide, docetaxel, doxorubicin, erlotinib (TARCEV A^®), etopside, gefitinib, gemcitabine hydrochloride, ifosfamide, irinotecan, lomustine, paclitaxel, topotecan, and vinorelbine tartrate.

[0091] In some embodiments, wherein PT523 is administered in a combination therapy to a subject to treat or manage a cancer or symptom thereof, wherein the cancer or symptom thereof to be treated or managed is a non-small cell lung cancer or symptom thereof, the second therapeutic agent or optional third therapeutic agent is selected from the agents consisting of cisplatin, carboplatin, cyclosphosphamide (CYTOXAN^®), docetaxel, doxorubicin, etopside, erlotinib (TARCEV A^®), gemcitabine hydrochloride, ifosfamide, irinotecan, lomustine (CEENU^®), paclitaxel, topotecan (HYCAMTIN™), and vinorelbine tartrate.

[0092] In certain embodiments wherein the cancer or symptom thereof to be treated or managed in a combination therapy is a non-small cell lung cancer or symptom thereof, the second therapeutic agent or optional third therapeutic agent is an epidermal growth factor receptor-tyrosine kinase (EGFR-TK) inhibitor. In some embodiments, the EGFR-TK inhibitor is gefitinib, erlotinib, or cetuximab.

[0093] In certain embodiments where a second therapeutic agent, and/or optional third therapeutic agent are administered to the subject, the second therapeutic agent or and/or optional third therapeutic agent is a radioisotope or radiolabled agent.

[0094] In some embodiments, the second therapeutic agent or and/or optional third therapeutic agent is an anti-angiogenic agent, such as, for example, bevacizumab

(AVASTIN™),

7. EXAMPLES

[0095] In the following sections, results are presented of studies on the in vivo efficacy of PT523 alone and in combination with known anticancer agents in art-recognized animal models of cancer, PT523 stability in solution, binding of PT523 to plasma components, the pharmacokinetics of PT523 as characterized in mice, rats, dogs and human, the toxicokinetics of PT523 in dogs utilizing GLP (Good Laboratory Practices) and rats, and the treatment of non-small cell lung cancer and leukemia with PT523 in humans.

7.1. Jn vivo Efficacy of PT523 alone and in combination with Known Anticancer Agents in Animal Models of Cancer

7.1.1 In vivo efficacy of PT523 in AS283 human lymphoma xenograft

[0096] The activity of PT523 was determined in an in vivo efficacy model by the

National Cancer Institute as part of its Rapid Access to Intervention Development ("RAID") program. The effects of PT523 on the growth of AS283 human lymphoma xenografts in severe combined immunodeficient (SCID) mice are summarized in Table 1. [0097] AS283 lymphoma cells were implanted subcutaneously in SCED/NCr mice on Day 0, and treatment was begun on Day 9. PT523 was injected daily in 0.05 M Tris buffer (0.1 mL/10 g body weight) for five days. The vehicle itself did not inhibit tumor growth, but caused some weight loss when given i.v. versus i.p.

Table 1: In Vivo Activit of PT523 A ainst AS283 Human Lymphoma Xenografts in SCIP Mice

[0098] When administered daily for five days as a 54 mg/kg intraperitoneal injection starting on Day 9, PT523 produced a 97% reduction in size of the AS283 xenograft on Day 26 as compared with controls, with one of six mice found to be tumor-free on Day 40. When the dose was increased to 80 mg/kg (qd x 5), three out of six mice died of drug toxicity and there were no Day 40 survivors. Essentially complete suppression of growth of the AS283 xenograft was observed when the 54 mg/kg (qd x 5) dose of PT523 was administered intravenously, and there were no toxic deaths. Another group of six mice received 80 mg/kg on this schedule and in this group there was one toxic death and one 40 day survivor.

7.1.2 In vivo efficacy of PT523 in

PC-3 human prostate carcinoma xenografts

[0099] The effects of PT523 on the growth of PC-3 human prostate carcinoma in athymic nude mice were studied in a second in vivo efficacy model. The results of this study are shown in Table 2.

[00100] PC-3 prostate carcinoma cells were implanted subcutaneously in athymic nude mice on Day 0, and treatment was begun on Day 13. PT523 was injected daily in 0.05 M Tris buffer (0.1 mL/10 g body weight). The PC-3 tumor caused approximately 20% weight loss in mice not treated with drug or vehicle. The vehicle itself did not inhibit tumor growth.

[00101] Intraperitoneal PT523 injections of 54 and 80 mg/kg (daily for five days) produced growth inhibition of the tumor xenograft of 60% (Day 29) and 63% (Day 22), respectively. When the treatment was given by the intravenous route, the 54 mg/kg dose resulted in a 40% reduction in tumor size. Doses of 80 mg/kg resulted in toxic deaths in three of six animals. In general, there was a greater weight loss in mice with the PC-3 xenografts in comparison to those with AS283 xenografts. However, untreated PC-3 mice also experienced considerable weight loss, and suggesting that the weight loss in the drug- treated group was probably due to the tumor rather than the drug.

7.1.3 In vivo efficacy of PT523 alone and in combination in A549 human non-small cell lung cancer xenografts

[00102] This examples show the effects of PT523 alone and in combination with three known anticancer agents on the growth of A549 human non-small cell lung cancer xenografts in athymic nude mice.

[00103] Study Design: A549 human non-small cell lung cancer cells (10⁶ viable cells in 100 μl) were implanted into 210 athymic nude mice via subcutaneous injection. After cell implantation, the animals were monitored daily for tumor growth. When the tumors became palpable, the volume of each tumor was measured with a caliper. Tumor volume was calculated using the formula: V= L x W x H x^π/₆, where L, W, and H represented length, width, and height of the tumor respectively. [00104] Day 1 was designated as the first day of PT523 treatment and was started when tumor volumes reached a mean value of approximately 60mm³. On Day 1, 160 animals with optimal tumor volumes (~60mm³) were randomized into 16 groups and the rest excluded from the study. The descriptions of the 16 groups are shown in Table 3. [00105] On Day 1, treatment with all drugs began according to the study design in

Table 3 below. PT523 was prepared daily in sterile deionized water. Paclitaxel was dissolved in a mixture of DMSO/Cremophore E1/H₂O (10:18 :72). Cisplatin was dissolved in DMSO first and diluted to final volume with sterile H₂O and kept DMSO concentration at 2 %. TARCEV A^® tablets were purchased from a commercial vendor. For dosing, the tablets were grinded to a powder form and dissolved in 0.5% hydroxypropylcellulose in sterile saline. PT523, paclitaxel, and cisplatin were administered via i.v. injection once weekly and TARCEV A^® was given orally daily via gavaging. The total treatment period was 4 weeks. Dosing regimen is described in Table 3 below.

[00106] Tumor volumes were measured every 3-4 days and body weights once a week for 30 days. Animals were observed daily for adverse clinical signs related to the treatment received. Unscheduled sacrifices were performed when tumor volumes reached > 1,500 mm3, or loss of > 25% of the original body weight, or tumors developed ulceration, or animals became moribund.

Table 3 Study Design

[00107] Statistical analysis: ANOVA and Dunnett's multiple comparison tests along with T-tests were conducted using GraphPad's InStat biostatistics program (Version 3.0). The control group was compared to all other treatment groups. A probability value (p) of less than 0.05 was considered to be statistically significant.

[00108] Results: Throughout the entire study most animals remained healthy and active. No overt toxic effect was observed in any treatment group. Several animals were euthanized early due to ulceration of their tumors.

[00109] There was a slight decrease in body weight during the beginning of the study in most groups from days 9-16. However by the end of the study they regained their lost weights as shown in Figures 1 and 2.

[00110] The mean tumor growth rates of all groups and percent change of tumor volumes from Day 1 are presented in Figures 3 and 4. The individual data for each animal in all groups are presented in Table 4. The results in Figures 3, 4 and 5 show that treatment of animals with PT523 alone at 15 mg/kg (low dose), 25 mg/kg (medium dose), and 35 mg/kg (high dose) inhibited A549 tumor growth. The change of tumor volumes from Day 1 of the groups treated with 25 and 35 mg/kg of PT523 was significantly lower than that of the control (P < 0.05).

[00111] The inhibitory effect of PT523 in combination with the three known anticancer agents was significantly different when compared with control. PT523 at 15, 25, and 35 mg/kg in combination with paxlitaxel at 7.5 mg/kg significantly inhibited tumor growth with P values < 0.05, 0.01, and 0.05 respectively. See Figure 6. PT523 at 25 mg/kg in combination with 5 mg/kg of cisplatin did not significantly inhibit the tumor growth when compared with control. However, PT523 at 15 mg/kg or 35 mg/kg in combination with 5 mg/kg of cisplatin significantly inhibited the tumor growth with the P values < 0.01 and 0.05 respectively. See Figure 7. PT523 at 15 and 25 mg/kg in combination with 50 mg/kg of TARCEV A^® inhibited tumor growth with the P values < 0.05 and 0.01 respectively. PT523 at the high dose level (35 mg/kg) in combination with 50 mg/kg of TARCEV A^®, while inhibiting tumor growth compared to control, did not show significant inhibition of tumor growth likely due to the large variation of tumor volumes in this group. See Figure 8. [00112] Throughout the entire study, no obvious toxic effect was seen in the treatment groups as indicated by the body weight measurements.

[00113] These results demonstrate that compound PT523 inhibits the growth of A549 human non-small cell lung carcinoma xenografts in athymic nude mice. The inhibition on change of tumor growth from Day 1 of the study was statistically significant. Further, PT523 in combination with paclitaxel, cisplatin, and TARCEV A^® showed significant inhibitory effects on tumor growth. The greatest inhibition was obtained in groups treated with PT523 in combination with TARCEVA^®.

Ul

7.2. PT523 Stability in Solution and Plasma

[00114] A liquid cliromatography/mass spectroscopy/mass spectroscopy

("LS/MS/MS") technique for detecting and quantifying PT523 was developed and validated for mouse, rat, dog, and human plasma. The technique provided the required sensitivity, selectivity, and accuracy for the analysis of PT523 in these biological fluids. The mean percentage accuracy of the method over the concentration range of PT523 of 1 to 500 ng/mL ranged between 94% and 104%. The percent coefficient of variation for concentrations above 2.5 ng/mL was less than 10%. The percent coefficient of variation for the lowest concentration (1 ng/mL) ranged from 1.4 to 17%. [00115] The stability of PT523 was assessed in 1.4% NaHCO₃ (an isotonic concentration), NaOH/saline (prepared by dilution of 0. IN NaOH into 0.9% saline), and Tris base (3.4% Tromethamine; also isotonic). PT523 was found to be stable at concentrations ranging from 1 to 10 mg/mL in 1.4% NaHCO₃ for at least 72 hours at both 4⁰C and at room temperature (longer time points were not assessed). PT523 was found to be stable in NaOH/saline at a concentration of 10 mg/mL of PT-523 for at least 168 hours at -20⁰C, 4⁰C, or 37⁰C maintained under dark conditions (all temperatures tested), reduced lights (room temperature), or ordinary fluorescent lighting (room temperature). PT523 was found to be stable in 3.4% tromethamine at a concentration of 10 mg/mL of PT523 for at least 168 hours at -2O⁰C, 4⁰C, or 37°C maintained under dark conditions (all temperatures tested), reduced lights (room temperature), or ordinary fluorescent lighting (room temperature). Separate studies using sunlight showed that placing the formulation in front of a sunlit window inside the laboratory resulted in rapid degradation of the formulation within two hours. Hence, PT523 was observed to degrade rapidly when exposed to sunlight.

[00116] The in vitro stability of PT523 was evaluated in mouse, rat, dog and human plasma at concentrations of 50 ng/mL (0.87 μM) and 100 μg/mL (174.2 μM). Serial analysis of the solutions by LS/MS/MS revealed no evidence of degradation of PT523 in plasma of any species following incubation at 37 °C for 4 hours, room temperature for approximately 24 hours, 4 °C for approximately 24 hours, or when frozen at -20 ⁰C for 22 days.

7.3. PT523-Plasma Protein Binding

[00117] Plasma protein binding was determined for mouse, rat, dog, and human plasma. Each species of plasma was mixed with PT523 at a final concentration of 0.1, 1, or iuu μg/nij./. Aiier preparation, each mixture was incubated for approximately 15 minutes at 37⁰C. Aliquots of each plasma mixture were centrifuged, and the filtrate was analyzed by LC/MS/MS for PT523 concentration. Results are shown in Table 5.

^a PBS: phosphate buffered saline (pH 7.4) ^b Concentration of PT-523 measured in the plasma or PBS mixtures prior to filtration. ^c fb (%): Percent bound fraction ^d NA: not applicable. All samples were prepared in triplicate except replicates for PBS.

[00118] The extent of binding of PT523 appeared to be different in plasma from the various species. For mouse plasma, plasma protein binding from 49% to 58% of added PT523 was observed, and appeared to be independent of PT-523 concentration. In rat plasma, protein binding ranged from 65% to 77%. For dog plasma, protein binding of PT523 displayed a slight concentration dependency, and was approximately 84% at a PT523 concentration of 0.1 μg/mL, and 70-73% at higher concentrations of 1 and 100 μg/mL. The concentration dependency was confirmed in two separate experiments. The results shown in Table 5 provide the results of one of the experiments. The extent of protein binding was highest in human plasma, and ranged from 91% to 93% at the three concentrations of PT-523 tested.

7.4. Pharmacokinetic Studies

[00119] Initial pharmacokinetic studies were conducted in CD2F1 mice to confirm the suitability and ruggedness of the LC/MS/MS method for analyzing PT523 in plasma. The results of the initial studies indicated that the LC/MS/MS method was suitable for the analysis of plasma samples obtained after in vivo administration of PT523. ^'1^'0^'0^'12^'Ol tlϊe pharmacokinetics of PT523 were characterized in CD₂Fl mice, male

Fischer-344 rats and beagle dogs following single intravenous administration (mice, rats and dogs) or repeated intravenous administration (dogs). Tables 6 and 7 summarize the pharmacokinetic data with PT523 in various species.

Table 6: Pharmacokinetic Parameters of PT523 in Mice and Rats After I.V. Administration

^a Value is the extrapolated concentration at 0 minutes. ^b NA= not applicable

[00121] The pharmacokinetics of PT523 were characterized in mice by giving CD₂Fl mice a single i.v. (tail vein) dose of 5 or 15 mg/kg of PT523 at an injection volume of 0.1 mL/lOg body weight. At various times after post-dosing, three mice per dose group were sacrificed and blood was collected from the brachial artery of mice anesthetized with isoflurane. In addition, for each dose group, urine and feces were collected from 5 mice maintained in a single metabolism cage at 0-6 hours and 6-24 hours post-dosing. PT523 was formulated by dissolving PT523 in 0.1N NaOH, and diluting the solution to the appropriate volume with 0.9% saline. The solution was filtered through a 0.45 μm syringe filter. The concentration and homogeneity of the dosing formulations was also assessed. The formulations were determined to be within 95% of target. Plasma, urine and feces samples were subsequently analyzed for concentrations of PT523 and its 7-hydroxy metabolite concentrations.

[00122] The pharmacokinetic parameters calculated from compartmental analysis of the plasma concentration time-data in mice showed that after an i.v. dose of 5 mg/kg, the data were best fit to a two-compartment kinetic model. Results are summarized in Table 6. The calculated area under the curve (AUC) value was 256 μg-min/mL. The apparent half- lives of PT523 in plasma were 4.1 and 20.1 minutes. The estimated clearance and volume of distribution at steady state (Vd_ss) were 19.5 mL/min/kg and 375 mL/min, respectively. [00123] For mice given an i.v. dose of 15 mg/kg, the plasma PT523 concentration- time data were best fit to a three-compartment model. The calculated AUC value was 791 μg-min/mL, which was approximately three-fold higher than the value determined following administration of the three-fold lower dose of 5 mg/kg. Computed half-lives for PT523 were 6.6, 22.0, and 478 minutes. The clearance and Vd_ss were similar to those determined after administration of the lower i.v. dose, and were 19.0 mL/min/kg and 556 mL/kg, respectively.

[00124] Urine and fecal elimination of PT523 over a 24 period was observed in mice administered an i.v. dose of 5 or 15 mg/kg. For mice administered 5 mg/kg i.v., the total excretion of unmetabolized PT523 accounted for 45% of the dose; of this amount, 19.5% was eliminated in urine and 27% in feces within 24 hours after dosing (Table 6). During this same interval, the amount eliminated in urine and feces as 7-hydroxy-PT523 accounted for only 0.3% of the dose. For mice administered 15 mg/kg i.v., 18% of the dose was eliminated in urine as unmetabolized PT523 within 24 hours of dosing, and 34% was eliminated in feces; thus the total 24-hour recovery of PT-523 represented 52% of the dose (Table 6). The amount eliminated in urine and feces as 7-hydroxy-PT523 during this same interval accounted for 0.5% of the dose. The urinary clearance of PT523 was estimated to be 4 mL/min/kg after administration of an i.v. dose of 5 or 15 mg/kg. The fecal clearance of PT523 was slightly higher, and was 6 or 7 mL/min/kg after a dose of 5 or 15 mg/kg, respectively. After administration of either dose level, the urinary and fecal clearances of 7-hydroxy-PT523 were higher than those for PT523, and ranged between 10 and 22 mL/min/kg.

[00125] For pharmacokinetic studies in rats, male Fisher-344 rats were given a single i.v. (tail vein) dose of 5 or 15 mg/kg of PT523 at an injection volume of 2 mL/kg. At various times after post-dosing, blood was collected from a jugular cannula from six rats per dose group, with the samples being taken from the animals in each group at alternating time points, hi addition, urine and feces were collected from a separate group of three rats per dose group maintained in a metabolism cage at either 0-6 hours, 6-12 hours, and 12-24 hours post-dosing (15 mg/kg) or 0-6 and 6-24 hours (5 mg/kg). [00126] The data were best fit to a three-compartment model. Calculated AUC values were 438,789 and 1,160,677 ng-min/mL for the 5 and 15 mg/kg doses, respectively (Table 6). Estimated half-lives for 5 mg/kg were 3.2, 15.6, and 45.0 minutes; for 15 mg/kg, the half-lives were 4.6, 22.8, and 183 minutes. Values for clearance after 5 or 15 mg/kg were 11.4 and 12.9 mL/min/kg, respectively. The Vd_ss was estimated to be 240 mL/kg for the 5 mg/kg dose and 299 mL/min for the 15 mg/kg dose.

[00127] Excretion data for PT523 after the i.v. doses of 5 or 15 mg/kg were collected. For the three rats administered 5 mg/kg, the mean total excretion of unchanged PT523 accounted for 87% of the dose (range: 75% to 92%); of this amount, a mean value of 31% was eliminated in the urine and 56% in the feces within 24 hours post-dosing (Table 6). During the same interval, the amount of 7-hydroxy-PT523 eliminated in urine and feces accounted for only 0.04% of the dose.

[00128] For the three rats administered 15 mg/kg, a mean value of 22% of the dose

(range: 20% to 26%) was eliminated in urine as unchanged PT523 within 24 hours post-dosing, and 32% of the dose (range: 27% to 38%) was eliminated in feces; thus, the mean total 24-hour recovery of PT523 represented 54% of the administered dose (range: 52% to 58%). The urinary and fecal elimination of 7-hydroxy-PT523 during this same interval accounted for only 0.05% of the dose.

[00129] The urinary clearance of PT523 was estimated to be 3 mL/min/kg in rats.

The fecal clearance of PT523 was 6 and 4 mL/min/kg after 5 and 15 mg/kg, respectively. The urinary and fecal clearance of 7-hydroxy-PT523 could not be calculated for 5 mg/kg, but for 15 mg/kg, the urinary and fecal clearance of 7-hydroxy-PT523 was very high, estimated at 143 and 58 mL/min/kg, respectively.

[00130] Pharmacokinetics and dose range-finding studies were performed in groups of one male and one female Beagle dogs. The groups were given a single intravenous dose of 0, 2, 5, 7.5, or 15 mg/kg (0, 40, 100, 150 and 300 mg/m², respectively) of PT523 in a 1 mL/kg dose volume. PT523 was formulated in Tris base/saline. The study was conducted in two phases since the initial doses of 7.5 and 15 mg/kg produced moribundity or mortality.

[00131] Blood samples were collected for pharmacokinetics from a peripheral vein

(noninjection site) of each dog at time points 2 min, 5 min, 10 min, 20 min, 30 min, 60 min, 90 min, 120 min, 4 h, 8 h, 12 h and 24 h. Each blood sample was mixed with EDTA and placed on ice. The samples were centrifuged, and the plasma was separated and stored frozen at approximately -2O⁰C until analyzed for PT523 concentration using LC/MS. The data were analyzed using WIN NONLDSf^® Software (Pharsight Corp., Mountain View CA), using a three-compartment model with first order elimination. Pharmacokinetic parameters calculated from the observed plasma concentration-time data for PT523 are summarized in Table 7.

NC = not calculated; M = male; F = female.

^§ These doses resulted in moribundity and/or mortality

[00132] As suggested in Table 7, AUC increased proportionally to dose. The estimated half-lives for the three phases of elimination of PT523 were similar among dogs in each dose group, and were 1.8-5.9 minutes, 70.6-90.3 minutes, and 194-514 minutes for the a phase, β phase, and 7 phase, respectively. The clearance and volume of distribution at steady state (Vd_ss) of PT-523 were also independent of the dose level and ranged from 2.5- 4.1 mL/min/kg and 317-553 mL/kg, respectively. There was no evidence of saturation of elimination or metabolism of PT523. The long terminal phase of elimination (up to 514 minutes) was considered to be possibly related to high plasma protein binding. The estimated Vd_ss for dogs, 317 to 533 mL/kg, was smaller than total body water; hence, the apparent limited distribution of PT523 in dogs might have also been related to the extent of plasma protein binding in that only free, unbound drag would have been available to distribute into tissues.

[00133] hi another study, groups of beagle dogs were dosed once daily by bolus i.v. administration at approximately the same time each day for five days. Groups of animals were sacrificed at intervals of 8 and 33 days. [00134] Blood samples were collected for pharmacokinetics from a peripheral vein

(noninjection site) of each dog at the time points prior to dosing, 2 min, 15 min, 30 min, and 6 Iu- and Day 1 and on 2 min post-dosing on Day 5. Each blood sample was mixed with EDTA and placed on ice. The samples were centrifuged, and the plasma was separated and stored frozen at approximately -2O⁰C until analyzed for PT523 concentration using HPLC/MS/MS. Data were analyzed using WIN NONLIN^® Software using both compartmental and noncompartmental methods. The analyses subsequently determined that the data were poorly fitted to a compartmental model; hence, only the noncompartmental analyses were calculated.

[00135] At 0.002 mg/kg/day, the only measurable timepoints were 2 and 15 minutes.

After 30 minutes, plasma concentrations of PT523 approached or were below the limit of quantitation. Pharmacokinetic parameters calculated from noncompartmental analysis of the plasma concentration-time data for PT523 at the 0.02 mg/kg/day dose are shown in Table 7.

[00136] Plasma concentrations of PT523 on Study Day 1 at 0.02 mg/kg/day ranged from 108 to 211 ng/niL at 2 minutes post-dosing. Thereafter, plasma drug concentrations declined biphasically, and were between 3.93 and 6.02 ng/mL at 3 hours post-dosing. On Day 5, plasma concentrations of PT523 at 2 minutes post-dosing were approximately 2-fold higher than on Day 1, and ranged from 237 to 475 ng/mL. No 7-hydroxy PT-523 was detectable at any timepoint.

7.4.1 Pharmacokinetic Studies in humans

[00137] For pharmacokinetic studies in humans, twenty four adult patients with refractory solid tumors and with normal renal and hepatic function were given a 5 -minute i.v. infusion every seven days for three weeks. Plasma samples were obtained at -5, 4, 10, 15, 20, 30, 45 min; 1, 2, 3, 4, 6, 8, 24, and 48 h after starting the first weekly infusion. Urine was collected and pooled from 0-8, 8-24, and 24-48 h. An LC/MS assay was used to measure PT523 in plasma and urine. Interday accuracy and precision were both <15% at the lowest concentrations measured in plasma (0.50 ng/mL) and urine (50 ng/mL). Pharmacokinetic parameters were estimated by standard noncompartmental methods. [00138] Data was obtained from groups of at least three patients receiving doses of 5,

6.7, 9, 12, and 16 mg/m². The PT523 concentration in plasma decreased in a polyexponential manner and the terminal log-linear phase was achieved 4-6 h after dosing, hi the 7 patients receiving doses of 16 mg/m², the mean peak drug concentration in plasma (C_max) was 5,650 ± 300 ng/mL and the median plasma concentration 48 h after dosing was 1.7 ng/mL^range,^" i7(f-^'23.4 ng/mL). The apparent biological half-life (t_1/2jZ), total body clearance (CL) and apparent volume of distribution at steady-state (V_ss) were all independent of the dose. Mean ± SD values of PK parameters for the entire cohort of 24 patients were: CL, 1.16 ± 0.31 L/h/m²; t_1/2iZ, 5.3 ± 1.2 h; V_ss, 7.7 ± 1.5 L/m². The mean amount of the dose excreted as unchanged drug in urine over 48 h was 40 ± 15%. [00139] These results show that PT523 exhibits linear PK with moderate interpatient variability when administered as a 5 min i.v. infusion at doses of 5 - 16 mg/m². Renal clearance is a major route of elimination.

7.5. Toxicology of PT523

[00140] The toxicological assessment of PT523 was performed in Fisher 344 rats and beagle dogs. Administration of PT523 as a single i.v. injection and a repeated dosing schedule involving an i.v. injection given on 5 consecutive days was evaluated in both species. Animals were examined for clinical signs of toxicity, clinical pathology parameters (i.e., complete blood counts and serum chemistries) and gross and microscopic histopathological changes. Objectives included determination of the target organ toxicity and its reversibility following administration of PT523.

[00141] In one study, PT523 prepared in Tris base/saline was administered to male rats as a single i.v. (tail vein) bolus administration using a slow push. Doses of PT523 ranged from 5 mg/kg to 380 mg/kg.

[00142] Each rat was observed for clinical signs of toxicity at least once daily from

Day 1 until the day of sacrifice. On dosing days, the rats were observed at approximately 1 hour post-dosing. The rats were also observed twice daily for signs of mortality/moribundity.

[00143] All rats were subjected to a complete gross necropsy. A full panel of tissues were collected and fixed in 10% neutral buffered formalin for histological examination. [00144] Single intravenous injection of PT523 at doses < 120 mg/kg (< 720 mg/m²) were well tolerated by rats. Drug-related toxicity observed after administering PT523 at doses ranging from 140 to 380 mg/kg (840-2,280 mg/m²) included body weight loss, moderate hematological changes, gross lesions in the large intestines and enlarged mandibular lymph nodes. A 3-10% reduction in group mean body weights was observed between Days 1 and 4 for rats given a single dose of 180 mg/kg or above. Additional weight losses (3-16% decrease in group mean body weights) was observed between Day 4 and 8 at doses of 240 mg/kg and above. [00145] Mortality occurred in 1/3 rats seven days after the administration of a

240 mg/kg dose and in 1/3 rats eight days after receiving a dose of 380 mg/kg. Clinical evidence of toxicity observed at dose levels > 240 mg/kg included hunched posture, emaciation and diarrhea.

[00146] Hematology assessments were made on Days 4, 5, 8 and/or 16. Drug related changes in hematologic parameters observed on days 4, 5 or 8 included decreased mean neutrophil count at doses >20 mg/kg; decreased reticulocyte count at doses >30 mg/kg; decreased RBC, hematocrit and hemoglobin at doses >140 mg/kg; Decreased platelet counts on Days 4 or 8 at 180 to 380 mg/kg, with minimum values ranging from 7% (380 mg/kg) to 70% (200 mg/kg) of controls; decreased WBC at 180, 240, 280, 300, 340, and 380 mg/kg, with minimum group mean WBC values observed at Day 8, respectively, of 59%, 29%, 29%, 35%, 15%, and 31% of the group mean values in controls. The maximum tolerated dose (MTD) of PT-523 was considered to be 200 mg/kg. A summary of some toxicology results in rats is shown in Table 8.

[00147] PT523 ammonium salt dissolved in Tris base/saline was administered in a single i.v. dose (see Table 8 for doses) using a dosing volume of 1 mL/kg to groups of one male and one female beagle dogs. Drug-induced toxicity was characterized by gastrointestinal symptoms, body weight loss, decreased leukocyte, neutrophil and lymphocyte counts, increased fibrinogen concentration, and histopathologic changes (lymphoid depletion in the lymph nodes, thymus, tonsil and spleen; bone marrow depletion; mucosal necrosis in the stomach and intestines). Results of this study are summarized in Table 8.

[00148] Treatment-related effects on hematological parameters included the following changes. Decreases in WBC counts at 5 mg/kg (male, Day 4), 7.5 mg/kg (female, Days 4 and 8), and 15 mg/kg. The WBC counts were 19% to 54% of the corresponding baseline values. Decreases in neutrophil counts at 2 mg/kg (male, Day 8), 7.5 mg/kg (female, Day 8), and 15 mg/kg (male Day 4, female, Days 4 and 6). The decreased neutrophil values ranged from 18% to 52% of the corresponding baseline values. Increases in RBC counts (values 198% to 143% of baseline values) at 2 mg/kg (Day 4), 5 mg/kg (female only, Days 4 and 8), 7.5 mg/kg (male, Days 2, 4, and 6), and 15 mg/kg (male, Day 4; female, Days 4 and 6). The changes were considered consistent with hemoconcentration due to dehydration or splenic concentration, and were usually accompanied by increases in BUN. [00149] Decreases in sodium observed on Days 4 or 6 at 5 mg/kg (female), 7.5, and

15 mg/kg with minimum values ranging from 81% to 93% of baseline values. Decreases in chloride were also observed on Day 4 or 6 in the 2 mg/kg (female), 5 mg/kg (female), 7.5, and 15 mg/kg groups with minimum values ranging form 65% to 93% of baseline. [00150] Decreases in total protein at 2 mg/kg (female), 5 mg/kg, 7.5 mg/kg (female only), and 15 mg/kg with minimum values ranging from 77% to 89% of baseline. The decreases in total protein were considered related to intestinal loss associated with intestinal necrosis.

[00151] A no-observable effect-level (NOEL) was not determined for PT523 in dogs in this study. Toxicity was observed at all dose levels, including the lowest dose (2 mg/kg). A dose of 7.5 mg/kg was associated with moribundity necessitating humane sacrifice, and a dose of 15 mg/kg resulted in mortality. The maximum tolerated dose of PT523 administered as a single IV bolus dose to dogs was considered to be 5 mg/kg (Table 8). [00152] Table 8 summarizes the results of the toxicology studies with PT523 in rats and dogs.

Table 8: Summary of Results of Toxicology Studies with PT523

[00153] Another study was undertaken to assess the target organ toxicity of PT523 and to assess the reversibility of toxic effects in rats given five consecutive daily i.v. doses of PT523 ammonium salt.

[00154] Male and female Fischer-344 rats were randomly assigned to four treatment groups of 10 males and 10 females each, and were dosed once daily as indicated in Table 8 by bolus i.v. administration at approximately the same time each day. Dosing formulations were made in Tris base/saline and administered in a dosing volume of 2 mL/kg. [00155] Animals were observed at least once daily for adverse clinical signs of toxicity on Day 1 through day of death or scheduled sacrifice (Day 8 or Day 33). On Days 1-5, these observations were made approximately one hour post-dosing. The rats were observed for mortality/moribundity once daily during quarantine and twice daily during the study periods.

[00156] Body weights were recorded on Days -3, 1-5 (prior to dosing), 8, 15, 22, and

33. hi addition, body weights were recorded prior to the moribund sacrifice of animals. For clinical pathology, blood samples were collected from surviving rats on Days 8, 15, 22, and 33. A blood sample was also obtained from rats sacrificed moribund on Days 10 and 11. The samples were analyzed for selected hematology and clinical chemistry parameters. All rats were subjected to a complete necropsy, and a full panel of tissues was collected and processed for histopathological examination.

[00157] No adverse clinical signs were noted at 3 mg/kg/day. Drug-related clinical signs of toxicity were observed in the higher dose groups and included piloerection, hunched posture, diarrhea, ataxia, cold to touch, and emaciation at 6 and 10 mg/kg/day. The observations at 10 mg/kg/day were generally more severe than at 6 mg/kg/day. In addition, isolated observations of dyspnea, tachypnea, cold to touch, emaciation, hypoactivity, jaundice, prostration and other incidental findings were observed at 10 mg/kg/day. The observations were most pronounced during the first two weeks of study, since there were no apparent drug-related signs of toxicity observed in the surviving male and female rats at 10 mg/kg/day between Day 16 and the day of scheduled sacrifice (Day 33).

[00158] Administration of PT523 by single daily i.v. dosing for 5 consecutive days to rats resulted in mortality at doses of 6 and 10 mg/kg/day. In surviving rats, the toxicity of PT523 appeared to be reversible. The maximum tolerated dose on this schedule (single daily x 5 days) was considered to be 3 mg/kg/day; the total dose administered to these rats over 5 days was 15 mg/kg (Table 8).

[00159] A study was undertaken to determine the toxicity of five daily i.v. doses of

PT523 administered to dogs in a dosing volume of 1 mL/kg. Results indicated that dogs given single daily i.v. doses of PT523 ranging from 0.01 to 1.0 mg/kg/day showed that mortality and/or moribundity occurred at doses of 0.05 mg/kg/day and above. Treatment- related toxicity was observed at 0.05 mg/kg/day and above characterized by gastrointestinal toxicity (emesis/bloody emesis and diarrhea/bloody diarrhea), body weight loss, clinical pathological changes (including decreased leukocytes, neutrophils, and lymphocytes, increases in RBC, HGB, AHCT< BUN, creatinine, globulin, ALT, ALP, total bilirubin, and fibrinogen, decreased glucose, total protein, and albumin), and histopathological changes in the lymph tissue, small and large intestines and stomach. The no observable effect level of PT-523 on a qd x 5 dose schedule was 0.01 mg/kg/day. The maximum tolerated dose of PT-523 was not determined in this study, but appears to be between 0.01 and 0.05 mg/kg/day. Gastrointestinal toxicity was dose limiting.

[00160] Toxicology analyses were also performed on the dogs that received five daily doses of 0.02 or 0.002 mg/kg/day of PT523 as described in Paragraphs [00133] to [00136] above. No adverse clinical signs were observed in any dogs in the 0 or 0.002 mg/kg/day dose groups. There were no treatment-related changes in body temperature. Clinical chemistry results did not indicate any treatment-related changes.

[00161] In this study, no mortality was observed in a five day repeated dose toxicity study of PT523 given by single daily intravenous injection to beagle dogs. Toxicity observed at 0.002 mg/kg/day (five days) was limited to histopathological changes in the lymph nodes, thymus, and tonsils. Toxicity at the higher dose of 0.02 mg/kg/day was manifested by signs of gastrointestinal toxicity and histopathological changes (lesions in the lymph nodes, thymus, tonsils, and small intestine) at 0.02 mg/kg/day. The toxicity of PT523 appeared to be reversible. The results of this study indicated that the no-observable adverse effect level was less than 0.002 mg/kg/day. The maximum tolerated dose appeared to be 0.02 mg/kg/day (Table 8).

7.6. Treatment/Management of Non-Small Lung Cancer with PT523

7.6.1 Treatment of non-small lung cancer using a 21-day therapeutic cycle

[00162] This example provides results of a PT523 dose escalation study where

PT523 was administered as a 5-minute infusion on days 1 and 8 on a 21 -day therapeutic cycle to patients with relapsed or refractory non-small lung cancer. [00163] Patient population: Fifteen patients with histologically or cytologically confirmed NSCLC stage III / IV or recurrent / refractory disease, that failed two or more lines of therapy other than PT523 were enrolled. Patients were within grades 0-2 of the Eastern Cooperative Oncology Group (ECOG) Performance Status grades (for information regarding grades on ECOG Performance Status, see the ECOG web site at http://www.ecog.org/general/perf_stat.html) and had signed informed consent documents. Patient baseline and disease characteristics are summarized in Table 9. Table 9: Baseline Demographic and Disease Characteristics (n=15)

[00164] Design: The starting dose was 13.5 .

m_™»g/ Λm_™» 2 per cycle), and patients received folic acid and vitamin B₁₂ supplementation. Dose escalation was as follows: 13.5 mg/m² (27 mg/m² per cycle); 27 mg/m² (54 mg/m² per cycle); 54 mg/m² (108 mg/m² per cycle); 90 mg/m² (180 mg/m² per cycle); 135 mg/m² (270 mg/m² per cycle), using a standard 3 + 3 design. Intrapatient dose escalation was made if no toxicity greater than grade 2 was observed, no evidence of disease progression as defined by the RECIST criteria was observed, and other eligibility criteria. Enrollment into the next highest dose level was completed with all subjects completing a full cycle of therapy without any dose limiting toxicity defined as ANC < 500/μ.L for greater than seven days, or ANC < 1000/μL and fever > 38.5 ⁰C; platelet nadir < 25,000 K/μL, or bleeding requiring transfusion, and grade 3 non-hematologic toxicity > 14 days.

[00165] Results: Patients completed a median of 3 cycles (ranging from 1 cycle to 8 cycles). Four patients (27% of patients) completed four or more cycles. Thirteen patients (87% of patients) completed two or more cycles. Antitumor activity is provided in Table 10.

Table 10: Antitumor Activity

[Oθi66] Clinical benefit was observed in ten patients (67%), two patients with partial responses ("PR") and 8 patients with stabilized disease progression ("SD") out of the fifteen patients enrolled (see Table 11 for additional detail on these patients). The duration of PR and SD has not been reached.

Table 11: Characteristics of Patients with Disease Control

[00167] The most commonly reported adverse event by the patients was mucositis and myelosuppression was observed. See Table 12 for additional details concerning adverse events. Treatment was discontinued in one patient who reported drug-related toxicity. Table 12: Adverse Events (n=15)

[00168] Three new patients (one female and two males) with confirmed non-small cell lung cancer at stage III/IV or recurrent/refractory disease and within ECOG PS 0-2, who failed two-lines of therapy with or without an EGFR TK inhibito were enrolled in the study described above (to make eighteen patients in total).

[00169] PT523 was administered as a 5-minute infusion on days 1 and 8 on a 21-day therapeutic cycle using the same dose escalation design (27-270 mg/m²/cycle) as described above. All patients received folic acid and vitamin B₁₂. Safety assessments were performed by standard laboratory and clinical DLT definitions. Responses were assessed based on RECIST criteria. Plasma PT523 concentrations were analyzed by a LC/MS/MS method. Concentration-time profiles of PT523 were analyzed by noncompartmental methods using WINNONLIN^® software.

[00170] The eighteen patients completed a median of 3.4 cycles (ranging from 1 cycle to 7 cycles). The tentative maximum tolerated dose is 90 mg/m²/dose. Mucositis was reported to be the predominate dose limiting toxcity. [00171] Clinical benefit was observed in eleven patients (61%): two patients with partial response and nine patients with stabilized disease progression out of eighteen patients enrolled.

[00172] PT523 exhibited linear pharmacokinetics profile across the 5 dose levels evaluated. Pharmacokinetics data for sixteen patients showed that C_max and AUCo-∞ exhibited linear PK and Overall mean values (± SD) for CL was 1.31 ± 0.31 L/hr/m²; for

Ti_/2_z was 6.5 ± 1.4 h, for V_ss was 8.9 ± 2.5 L/m².

[00173] These results demonstrate that PT523 has acceptable tolerability in human patients being well tolerated over multiple therapeutic cycles. Clinical benefit in patients with relapsed or refractory non-small lung cancer was observed. Thus, this example demonstrates a therapeutic benefit for PT523 administration in patients with cancer.

7.6.2 Treatment of non-small lung cancer in a patient on a 28-day therapeutic cycle

[00174] The following exemplifies a method for the treatment/management of a non-small lung cancer in a patient on a 28-Day therapeutic cycle (PT523 administered on Days 1 and 8) using a 40 mg/m² PT523 dosage.

[00175] Preparation of PT523: PT523 monoammonium salt is prepared in a sterile lyophilized powder for injection in vials containing 25 mg of PT523, 50 mg of mannitol, United States Pharmacopoeia (USP), and 28 mg of L-arginine, USP. The powder is packaged in 20 cc amber molded vials, with 20 mm lyo stoppers and red, flip-off aluminum seals. Vials of the sterile lyophilized powder for injection can be kept refrigerated (2-8°C). [00176] Dosage Determination: The patient's body surface area (BSA) is determined using actual heights and weights with no adjustment to "ideal" weight. The appropriate volume of reconstituted PT523 to be administered is calculated using the patient's BSA and the reconstituted concentration of PT523 to result in 20 mg/m² of reconstituted PT523 per single infusion {e.g., on Day 1 or Day 8).

[00177] PT523 Administration: On Day 1 and Day 8, each vial of 25 mg PT523 for

Injection is reconstituted with 5 mL of Water for Injection, USP to yield a clear, yellow solution with a drug concentration of 5 mg/mL and a pH of 9.1 to 9.6. The reconstituted vials are stored refrigerated for up to 7 days, or at room temperature for up to 24 hours, if necessary. The appropriate amount of reconstituted PT523 for Injection is diluted in 50 mL of 5% Dextrose Injection, USP for intravenous administration, which is used within 8 hours. [00178] On Days 1 and 8, each dose of PT523 is administered by i.v. infusion over

5 minutes. The drug is delivered through a free running peripheral intravenous catheter or central venous catheter, without the use of an inline filter, using an infusion pump. All materials in the fluid path of the medication bag and infusion set are constructed from medical grade PVC.

[00179] If the Day 8 schedule dose must be delayed, the delay should be no more than one day.

[00180] The patient is administered folic acid (1 mg PO qd) and cyanocobalamin

(100 μg PO qd) supplementation starting at least 1 day prior to the first infusion of PT523 and continuing through the duration of the cycle.

[00181] ^• A second cycle or third cycle is initiated if the following parameters are observed in the patient: ANC > 1000/mm³; platelets > 50,000/mm³; serum creatinine < 1.5 mg/dL.

[00182] The patient is kept on treatment as long as clinical benefit is observed.

7.7. Treatment/Management of Leukemia or Mvelodysplastic Syndrome with PT523

7.7.1 Treatment of leukemia using a 21-day therapeutic cycle

[00183] This example provides results of a PT523 dose escalation study where

PT523 was administered as a 5-minute infusion on days 1-5 on a 21-day therapeutic cycle to patients with relapsed or refractory leukemia or myolodysplastic syndrome. [00184] Patient population: Fourteen patients including seven patients with acute myelogenous leukemia, five patients with acute lymphocytic leukemia, one patient with chromic lymphocytic leukemia in the blastic phase, and one patient with hair cell leukemia were enrolled. These patients had been treated with several therapies and no standard therapies had resulted in a durable remission. Patients were within grades 0-2 of the Eastern Cooperative Oncology Group (ECOG) Performance Status grades (for information regarding grades on ECOG Performance Status, see the ECOG web site at http://www.ecog.org/general/perf_stat.html) and had signed informed consent documents. Median age of these patients were 48 years ranging from 11 years old to 76 years old. [00185] Design: The starting dose was 0.2mg/m² (1 mg/m² per cycle), and patients received folic acid and vitamin B₁₂ supplementation. Dose escalation was as follows: 0.2 mg/m² (1 mg/m² per cycle); 0.4 mg/m² (2 mg/m² per cycle); 0.6 mg/m² (3 mg/m² per cycle); 0.8 mg/m² (4 mg/m² per cycle) using a standard design. Intrapatient dose escalation was made if no toxicity greater than grade 2 was observed, no evidence of disease progression as defined by the RECIST criteria was observed, and other eligibility criteria. Enrollment into the next highest dose level was completed with all subjects completing a full cycle of therapy without any dose limiting toxicity defined as ANC < 500/μL for greater than seven days, or ANC < 1000/μL and fever > 38.5 ⁰C; platelet nadir < 25,000 K/μL, or bleeding requiring transfusion, and grade 3 non-hematologic toxicity > 14 days.

[00186] Results: Patients completed a median of one cycle (ranging from 1 cycle to

3 cycles). Clinical benefit was observed in one patient. No patients have developed by dose limiting toxicities or experienced a drug related serious adverse event.

[00187] These results demonstrate that PT523 at the tested doses was well tolerated in this heavily pretreated leukemia patient population. Clinical benefit in patients with relapsed or refractory leukemia was observed. Thus, this example demonstrates a therapeutic benefit for PT523 administration in patients with cancer.

[00188] All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.

[00189] While the invention has been described in terms of various preferred embodiments, the skilled artisan will appreciate that various modifications, substitutions, omissions, and changes may be made without departing from the spirit thereof. Accordingly, it is intended that the scope of the present invention be limited solely by the scope of the following claims, including equivalents thereof.

Claims

What is Claimed is:

1. A method for the treatment or management of a cancer or symptom thereof in a subject in need thereof comprising administering about 9.0 mg/m² to about 200 mg/m² of PT523 to the subject thereby treating or managing the cancer or symptom thereof.

2. The method of claim 1, wherein the cancer is an ovarian cancer, endometrial cancer, biliary cancer, esophageal cancer, mesothelioma, cervical cancer, vulvar cancer, gastric cancer, pancreatic cancer, breast cancer, prostate cancer, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), non-small cell lung cancer, melanoma, lymphoma, renal cancer, colon cancer, soft tissue sarcoma, head and neck squamous carcinoma, or symptom thereof.

3. The method of claim 1, further comprising co-administering a second therapeutic agent to the subject.

4. The method of claim 3, wherein the cancer is non-small cell lung cancer and the second therapeutic agent to be administered is an epidermal growth factor receptor- tyrosine kinase (EGFR-TK) inhibitor.

5. The method of claim 1, wherein the subject is human.

6. The method of claim 1, wherein PT523 is administered as a liquid pharmaceutical formulation comprising a dissolved PT523 ammonium salt and a pharmaceutically acceptable carrier, diluent, excipient or vehicle.

7. The method of claim 6, wherein PT523 is administered by an intravenous route.

8. The method of claim 1 , wherein the about 9.0 mg/m² to about 200 mg/m² of PT523 is administered in two to four infusions over a therapeutic cycle of about two to about five weeks.

9. The method of claim 8, wherein PT523 is administered in two or more therapeutic cycles.

10. The method of claim 1 , further comprising administering folic acid or a folate binding protein binding agent and cyanocobalamin to the subject.