HK1156252A

HK1156252A - Antifolate agent combinations in the treatment of cancer

Info

Publication number: HK1156252A
Application number: HK11110688.3A
Authority: HK
Inventors: Charles P. Theuer; Bonne Jean Adams
Original assignee: Tracon Pharmaceuticals, Inc.
Priority date: 2006-12-29
Filing date: 2007-12-21
Publication date: 2012-06-08

Description

Combined use of antifolates in cancer treatment

RELATED APPLICATIONS

Priority of the present application claims U.S. provisional patent application series No. 60/877,836 entitled "combined use of an antimetabolite in the treatment of cancer" filed on 29.12.2006 by Theuer et al, U.S. provisional patent application series No. 60/883,266 entitled "combined use of an antimetabolite in the treatment of cancer" filed on 3.1.2007 by Theuer et al, and U.S. provisional patent application series No. 60/883,959 entitled "combined use of an antimetabolite in the treatment of cancer" filed on 8.1.2007 by Theuer et al, to the maximum extent allowed by law, the foregoing being incorporated herein by reference in their entirety, including any accompanying drawings.

Field of the invention

The present invention relates generally to compounds having various utilities, including use in research, diagnosis and therapy. More particularly, compositions comprising methoxyamine and an antimetabolic anticancer agent, and methods of treating certain cancers by administering these compositions, are described and provided herein.

Background

Cancer is a worldwide problem. Therefore, there is great interest in finding new compositions and methods for treating cancer. There are three general categories of cancer treatment: chemotherapy, radiation therapy, and surgery. Treatment is often combined because combination therapy tends to increase the chances of cancer eradication compared to treatment strategies utilizing monotherapy. Typically, surgical removal of large tumor masses is followed by chemotherapy and/or radiation therapy.

Chemotherapeutic drugs can act in a variety of ways. For example, chemotherapeutic drugs may act by interfering with cell cycle progression or causing DNA strand breaks. If the cancer cells are unable to overcome the cell cycle block or cell damage caused by the therapeutic compound, the cancer cells will typically die by an apoptotic mechanism. The use of a single chemotherapeutic drug to treat cancer, with or without surgery or radiation therapy, has several disadvantages. Typically, cancer cells develop resistance to chemotherapeutic drugs. These resistance results in the need for higher drug doses and/or the re-spread of the cancer. Chemotherapeutic drugs may be toxic to the patient. Thus, there is an upper limit of implementable amounts that the patient can accept. However, if another drug can be developed to inhibit the pathway leading to drug resistance, then cancer cells can become susceptible to the effects of chemotherapeutic drugs.

The design of drugs to overcome cancer chemotherapy resistance should achieve the following goals: 1) finding a combination that reverses resistance, not just improves the activity of the chemotherapeutic drug against the tumor, and 2) finding another drug that does not increase the toxic effects of the first chemotherapeutic drug. Under these conditions, there is a need for extensive experimental testing of drugs known to have anti-cancer properties, together with other drugs that may have anti-cancer properties, or drugs that may otherwise increase the efficacy of the first drug. Unfortunately, these approaches to the combination of multiple anticancer drugs have so far proven to be highly unsuccessful.

Therefore, there is a lack of therapies that can reverse the resistance of cancer treatment to chemotherapy.

Summary of The Invention

The inventions described and claimed herein have various features and embodiments, including but not limited to those enumerated or described or referenced in the summary of the invention. The inventions described and claimed herein are not limited to or by the features or embodiments illustrated in this summary of the invention, which is for illustrative purposes only and is not limiting.

These and other aspects and embodiments of the invention described and claimed herein will be apparent from the entire application and claims, and should be considered as part of this written specification.

Disclosed herein are compositions and methods for treating certain cancers. Part of the present application is based on the heretofore unknown recognition that certain molecules targeting DNA abasic damage or AP (purine-free/pyrimidine-free) sites improve, increase or enhance the efficacy of antimetabolic anticancer drugs. In other embodiments, an inhibitor of the base excision pathway, such as methoxyamine, is used in combination with an antimetabolic anticancer agent. Antimetabolic anticancer drugs are chemotherapeutic drugs that are structurally similar to the substances (metabolites) required for normal biochemical reactions, yet differ sufficiently in structure to interfere with normal cellular functions, including cell division. Antifolates are a preferred class of antimetabolites. Antifolate anticancer drugs are chemotherapeutic drugs that are structurally similar to folic acid, however, their structural differences are sufficient to block folate activity and disrupt the folate-dependent mechanisms necessary for cell replication. These antifolate anticancer drugs include: pemetrexed, capecitabine, edatrexate, methotrexate, lometrexol, nolatrexed, ralitexed, PT523 and trimetrexate. The use of any antifolate anticancer drug in combination with a BER (base excision repair) inhibitor is contemplated. In one aspect, the method comprises providing i) a patient diagnosed with cancer, ii) a first formulation comprising an antifolate anticancer agent and iii) a second formulation comprising methoxyamine; administering the first formulation to the patient; and administering the second formulation to the patient, wherein methoxyamine is administered in an amount sufficient to enhance or increase the effect of the antifolate anticancer agent. The second formulation may be administered orally. In another aspect, the method comprises: providing i) a patient diagnosed with cancer, wherein the cancer is at least partially resistant to treatment with pemetrexed alone, ii) a first formulation comprising pemetrexed; and iii) a second formulation comprising methoxyamine; administering a first formulation to the patient; and administering said second formulation to said patient, wherein methoxyamine is administered in an amount sufficient to enhance said pemetrexed activity and overcome said drug resistance. In these methods, the methoxyamine and antifolate anticancer agent may be administered as a formulation. Furthermore, the methoxyamine and antifolate anticancer agents may be administered sequentially in any order. Furthermore, methoxyamine can be administered orally, and antifolate anticancer agents can be administered orally or intravenously. Moreover, the amount of methoxyamine may be an amount sufficient to sensitize cancer cells without causing undue sensitization of normal cells. Furthermore, a synergistic effect may be obtained by administering methoxyamine and an antifolate anticancer agent. Furthermore, the antifolate anticancer agent may be administered orally or intravenously, and the methoxyamine may be administered orally in an amount sufficient to enhance the activity of the antifolate anticancer agent not more than twice a day. Moreover, a patient may be selected for having a cancer that is at least partially resistant to treatment with an antifolate anticancer agent alone, and wherein the second formulation comprising methoxyamine is administered in an amount effective to enhance the antifolate anticancer agent activity and overcome the resistance. Moreover, the ratio of the methoxyamine to the antifolate anticancer agent may be from 1: 5 to 1: 500, more preferably from 1: 15 to 1: 40, even more preferably from about 1: 20 to about 1: 30. Moreover, the cancer may be selected from: carcinomas (carcinomas), melanomas, sarcomas, lymphomas, leukemias, astrocytomas, gliomas, malignant melanomas, chronic lymphocytic leukemia, lung cancer, colorectal cancer, ovarian cancer, pancreatic cancer, renal cancer, endometrial cancer, gastric cancer, liver cancer, head and neck cancer, and breast cancer. In a preferred embodiment, the antifolate anticancer agent is pemetrexed.

In another embodiment, an improved method is disclosed, in a method of treating cancer in a patient diagnosed with cancer comprising administering to the patient an antifolate anticancer agent, the improvement comprising administering to said patient methoxyamine in an amount sufficient to enhance toxicity of said antifolate anticancer agent. Also disclosed are anti-cancer formulations comprising a dosage form containing pemetrexed and a dosage form containing a synergistic amount of methoxyamine, and methods of using the formulations according to the disclosed methods of treatment. In another embodiment, improved use of methoxyamine for the use of an antifolate anticancer agent to treat cancer in a patient is disclosed, the improvement comprising the use of methoxyamine in an amount sufficient to enhance toxicity of said antifolate anticancer agent in said patient.

In one aspect, the invention is based on the previously unknown recognition that certain molecules that target AP sites, such as methoxyamine, are fully bioavailable when administered orally and can maintain the minimum effective concentration when administered orally once or twice daily. Anticancer drugs are usually administered as intravenous bolus injections (intravenous bolus) because they are rarely well absorbed in the gastrointestinal tract. Intravenous administration has a number of disadvantages. First, intravenous injection of chemotherapeutic agents is required in clinics or hospitals. Second, intravenous therapy is usually administered as a bolus, which results in very large but short drug exposure. Some anticancer drugs are most effective after sustained contact, which can be achieved by repeated oral doses. Particularly true for those drugs that inhibit the resistance mechanisms of chemotherapeutic drugs, the desired beneficial effect requires prolonged inhibition of the resistance pathway. Prolonged drug contact may be achieved by continuous intravenous administration. However, administration of anticancer drugs in a continuous infusion manner requires a complicated drug infusion device and intravenous catheterization. Oral administration avoids the requirement for continuous intravenous infusion and is the preferred route of administration for the patient. However, to our knowledge, BER inhibitors that reverse chemotherapeutic drug resistance and have near complete oral bioavailability as provided herein have not been developed to date.

Pemetrexed is a multi-targeted antifolate that acts in a manner mechanistically different from 5-FU and other early antimetabolites. Pemetrexed is unique in that it is a pyrrolopyrimidine antifolate analogue which is metabolized intracellularly to the higher polyglutamic acid form by folate polyglutamic acid synthase (FPGS). The pentaglutamic acid form is the major intracellular species, and pemetrexed polyglutamic acid has about 60 times the potency of the parent monoglutamic acid compound; pemetrexed polyglutamic acid also shows prolonged cell retention. Thus, the pharmacological effects of pemetrexed following bolus intravenous administration can last for many days.

Pemetrexed inhibits Thymidylate Synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide transformylase (GARFT), all folate-dependent enzymes involved in the de novo biosynthesis of thymidine and purine nucleotides. In contrast, 5-FU and other early antimetabolites predominantly inhibited TS only. Although the precise mechanism by which pemetrexed causes cell death is still unclear, it does not only involve TS inhibition. Thus, among the heterogeneous non-selective human colon Cancer Cell line panel (Cell line panel), the predictor of best sensitivity to 5FU is TS activity, and multiple sensitivity determinants, including FPGS activity and TS enzyme kinetics, are important for pemetrexed (van Triest B, Pinedo HM, van Hensbergen Y. Thymethylatynthase level as the main predictor parameter for sensitivity to 5-FU, butnot for Folate-based Thymidylate Synthase Inhibitors, in 13 Nonselect Colon Cancer Cell lines. Clin. Cancer. Res.1999; 5: 643-54). Further studies confirmed that the sensitivity of gastrointestinal cell lines to pemetrexed cannot be predicted by TS expression (Kim JH, Lee KW, Jung Y et al, cytoxic effects of pemetrexed enterstric cells. cancer Sci.2005; 96: 365-71).

The unique pharmacological activity of pemetrexed was clarified by in vitro studies of the activity of multiple cancer cell lines in comparison to 5-FU. In a series of 13 colon cancer cell lines, for example, pemetrexed is 18 to 627 times more potent than 5-FU (van Triest et al, 1999). The unique pharmacology and the fact that pemetrexed is believed to have a variety of mechanisms of action that are not yet fully understood makes it difficult to figure out how effective it would be when used in combination with other specific anti-cancer drugs to treat a particular cancer.

One aspect of the present invention is the discovery of unexpectedly improved efficacy of a combination of methoxyamine and an antifolate compound in the treatment of cancer. Accordingly, one embodiment described herein relates to a method comprising providing i) a patient diagnosed with cancer, ii) a first formulation comprising an antifolate anticancer agent and iii) a second formulation comprising methoxyamine; administering a first formulation to a patient; and administering to the patient a second formulation, wherein methoxyamine is administered in an amount sufficient to enhance or increase the effect (i.e., enhance activity) of the antifolate anticancer agent. Any antifolate anticancer drug may be used, provided that in certain embodiment methods 5-FU is specifically excluded. In typical embodiments, the anti-cancer agent may be selected from: pemetrexed, edatrexate, methotrexate, lometrexol, nolatrexed, raltrexed, PT523, trimetrexate, aminopterin, 5, 10-dideazatetrahydrofolic acid (ddatff), pirtrexin, raltitrexed, GW1843[ (S) -2- [5- [ (1, 2-dihydro-3-methyl-1-oxobenzo [ f ] quinazolin-9-yl) methyl ] amino-1-oxo-2-isoindolyl (isoindolynyl) ] -glutaric acid ], pharmaceutically acceptable salts thereof, and any combination thereof. In a more typical embodiment, the anti-cancer agent may be selected from: pemetrexed, edatrexate, methotrexate, lometrexol, nolatrexed, ralitexed, PT523, trimetrexate, aminopterin, pharmaceutically acceptable salts thereof, and any combination thereof. In a most typical embodiment, the anti-cancer agent may be pemetrexed and pharmaceutically acceptable salts thereof. For example, pemetrexed may be the disodium salt. In exemplary embodiments, pemetrexed may be the disodium salt heptahydrate.

In a typical non-limiting embodiment, the antifolate anticancer agent is pemetrexed. The methoxyamine and antifolate anticancer agents may be administered sequentially (in any order) or together as a formulation. For example, pemetrexed can be 200 to 1,000mg/m²The amount of body surface area per day, alternatively from 500 to 600mg/m²The body surface area is administered intravenously in daily amounts. In another embodiment, the ratio of methoxyamine to antifolate anticancer agent may be from 1: 5 to 1: 500.

On the other hand, methoxyamine may be administered orally in an amount sufficient to sensitize the cancer without causing undue sensitization of normal tissues. In a non-limiting preferred embodiment, methoxyamine is administered orally to provide a greatly enhanced bioavailability relative to other orally administered anticancer agents. In other non-limiting preferred embodiments, methoxyamine is administered orally such that it is capable of maintaining a minimum effective concentration when administered once or twice daily. One way to measure oral bioavailability is to compare the levels obtained with those obtained by intravenous administration of methoxyamine. Thus, in another aspect of the invention, methoxyamine is administered orally to achieve a bioavailability of at least 50% relative to intravenous administration, at least 60% relative to intravenous administration, at least 70% relative to intravenous administration, at least 75% relative to intravenous administration, at least 80% relative to intravenous administration, at least 85% relative to intravenous administration, at least 90% relative to intravenous administration, at least 95% relative to intravenous administration, or approximately equivalent to intravenous administration. It is important to recognize that in addition to the unexpectedly high bioavailability obtained with oral administration compared to intravenous administration of methoxyamine, a more desirable pK profile compared to intravenous administration of methoxyamine was also obtained. In another aspect of the invention, oral administration of methoxyamine after once or twice daily administration maintains the lowest effective concentration due to a half-life in plasma of > 4 hours. This advantage allows for desirable oral dosing regimens of methoxyamine, including once or twice daily dosing. While intravenous administration of pemetrexed in combination with oral administration of methoxyamine is a preferred, non-limiting embodiment, other routes of administration for each anticancer agent are also contemplated.

In another aspect of the invention, methods of treating certain cancers that are resistant to treatment with anticancer drugs are provided. Accordingly, there is also provided a method comprising:

providing i) a patient diagnosed with a cancer, wherein the cancer is resistant to treatment with pemetrexed alone, ii) a first formulation comprising an antifolate anticancer agent; and iii) a second formulation comprising methoxyamine;

administering a first formulation to a patient; and

administering to the patient a second formulation wherein the methoxyamine may be administered in an amount sufficient to potentiate or increase the effect (i.e. enhance toxicity) of the antifolate anticancer agent. In one embodiment, the antifolate anticancer agent may be pemetrexed. The methoxyamine and antifolate anticancer agents may be administered sequentially (in any order) or together as a formulation. Pemetrexed can be 200 to 1,000mg/m²The amount of body surface area per day, alternatively from 500 to 600mg/m²The body surface area is administered intravenously in daily amounts. Pemetrexed and nailThe ratio of oxyamines may be from 1: 5 to 1: 500. In another embodiment, the amount of methoxyamine may be administered orally in an amount sufficient to sensitize the cancer without causing undue sensitization of normal tissues. In another embodiment, the amount of methoxyamine may be administered orally once daily or twice daily in an amount sufficient to sensitize the cancer without causing undue sensitization of normal tissues. Although oral administration of methoxyamine is an unexpectedly preferred route of administration, other types of administration are possible.

Another embodiment relates to a method of treating cancer by providing a first and a second formulation, wherein the first formulation comprises an antifolate and the second formulation comprises orally administered methoxyamine. The first formulation comprising the antifolate may be administered by conventional routes of administration, including intravenous administration. A non-limiting preferred antifolate is pemetrexed. Accordingly, in one embodiment, the antifolate is pemetrexed and the second formulation comprising methoxyamine is administered orally in an amount to have a synergistic effect compared to pemetrexed alone.

In another aspect, the method can be used to treat cancer resistant to pemetrexed alone. According to these embodiments, pemetrexed is administered in an amount that reverses resistance to (and is therefore synergistic with) antifolates used alone. Thus, in one embodiment, the methoxyamine is administered orally in an amount effective to enhance or increase pemetrexed toxicity and overcome cancer resistance to pemetrexed treatment. For example, the efficacy of pemetrexed to treat cancer may be reduced during the treatment cycle due to increased drug resistance. The administration of methoxyamine can prevent the development of resistance, providing a greater additive effect than the treatment of cancer with methoxyamine or pemetrexed alone.

Also provides a method comprising

Providing i) a patient diagnosed with cancer, wherein the cancer may be selected from: cancer, melanoma, sarcoma, lymphoma, leukemia, astrocytoma, glioma, malignant melanoma, chronic lymphocytic leukemia, lung cancer, colorectal cancer, ovarian cancer, pancreatic cancer, renal cancer, endometrial cancer, gastric cancer, liver cancer, head and neck cancer, and breast cancer, wherein the cancer is resistant to pemetrexed alone treatment, ii) a first formulation comprising an antifolate anticancer agent and iii) a second formulation comprising methoxyamine;

administering a first formulation to the patient; and

administering to said patient a second formulation wherein methoxyamine may be administered in an amount sufficient to enhance or increase the effect of the antifolate anticancer agent. The methoxyamine and antifolate anticancer agent may be administered continuously or together as a formulation. For example, methoxyamine may be administered first, and antifolate anticancer drugs may be administered last, or antifolate anticancer drugs may be administered first, and methoxyamine may be administered last.

In a typical embodiment, the antifolate anticancer agent may be pemetrexed. Pemetrexed can be 200 to 1,000mg/m²The amount of body surface area per day, or at 500 to 600mg/m²The body surface area is administered intravenously in daily amounts. The ratio of pemetrexed to methoxyamine may be from 1: 5 to 1: 500. In another embodiment, the amount of methoxyamine can be administered orally in an amount sufficient to cause the cancer cells to be susceptible (i.e., sensitive) to anticancer drug treatment without causing undue damage to normal cells. In another embodiment, the amount of methoxyamine may be administered orally once or twice daily in an amount sufficient to sensitize the cancer without causing undue sensitization of normal tissues.

Another embodiment may be a formulation comprising methoxyamine and an antifolate anticancer agent, wherein the methoxyamine may be administered in an amount sufficient to enhance toxicity of the antifolate anticancer agent. Preferably, the antifolate anticancer agent is pemetrexed.

In another embodiment, the ratio of methoxyamine to antifolate anticancer agent in any of the methods described above may be from 1: 5 to 1: 500.

In another embodiment, another anticancer agent may be administered before or after treatment with methoxyamine and an antifolate anticancer agent in any of the methods described above.

In another embodiment, a method of treating cancer in a patient diagnosed with cancer is described comprising administering to the patient an antimetabolic anticancer drug, the method having the following improvements: administering methoxyamine to the patient in an amount sufficient to enhance toxicity of the antimetabolic anticancer agent. The antimetabolic anticancer agent may be an antifolate anticancer agent. The antifolate anticancer agent may be pemetrexed, and the ratio of the methoxyamine to the antifolate anticancer agent may be 1: 5 to 1: 500. The cancer may be resistant to treatment with pemetrexed alone.

Brief description of the drawings

FIGS. 1A-B show the effect of Pemetrexed and MX on DNA strand breaks as determined by basic (FIG. 1A) and neutral (FIG. 1B) comet assay.

FIGS. 1C-D show a comparison of the comet tail length after treatment of cells with pemetrexed alone or MX alone, and pemetrexed plus MX, as determined by basic (FIG. 1C) and neutral (FIG. 1D) comet assays.

FIG. 2 shows a graph of mean plasma MX concentrations in male Sprague-Dawley rats at various time points after intravenous and oral bolus MX administration at an amount of 20mg/kg body weight.

FIG. 3 shows a graph of mean plasma MX concentrations in female Sprague-Dawley rats at various time points after intravenous and oral bolus MX administration at an amount of 20mg/kg body weight.

Fig. 4A shows a graph of the relative amount of AP sites detected in H460 cells after 24 hours of treatment with pemetrexed and MX.

Fig. 4B shows a graph of the relative amount of AP sites detected in H460 cells at 24 hours, 48 hours, and 72 hours.

FIG. 5A shows a schematic for the preparation of DNA substrates with regular AP sites or MX-AP sites.

FIG. 5B shows the resistance of MX-linked AP sites to the cleavage by AP-endonuclease (APE).

FIG. 6 shows the effect of pemetrexed in combination with MX on DNA double strand breaks and apoptosis.

FIG. 7 shows the effect of pemetrexed in combination with MX on BER protein levels in H460 cells.

FIG. 8 shows the effect of pemetrexed and MX on moderate volumes of NCI-H460, A549, HCT116 and MDA-MB-468 tumors grown in nude mice.

Detailed Description

Certain embodiments of the present invention generally relate to novel compositions comprising methoxyamine and an antifolate anticancer agent, and the use of these compositions to treat certain cancers.

Definition of

Unless otherwise indicated, the following terms have the following meanings when used herein and in the appended claims. Terms not defined below or elsewhere in the specification have art-recognized meanings.

As used in the specification and claims, the terms "agent" and "drug" as used herein refer to a compound, mixture of compounds, biological macromolecule, or extract from biological material such as bacteria, plants, fungi, or cells or tissues of an animal, particularly a mammal, suspected of having therapeutic properties. The agent or drug may be purified, substantially purified, or partially purified.

As used herein for the purpose of the specification and claims, the term "antimetabolite" refers to chemotherapeutic agents that are structurally similar to substances required for normal biochemical reactions (metabolites such as nucleosides), but differ in structure insufficiently to interfere with the normal function of cells, including cell division.

As used in the specification and claims, the term "antifolate" as used herein refers to a chemotherapeutic drug that is structurally similar to folic acid, yet differs in structure insufficiently to block the activity of folic acid and disrupt the folate-dependent mechanisms required for cell replication. As used herein, antifolates are a class of antimetabolites.

As used in the specification and claims, the term "antineoplastic agent" as used herein refers to chemotherapeutic drugs used to inhibit or prevent the maturation and proliferation of tumors (tumors) that can become malignant by targeting DNA.

As used in the specification and claims, the term "stain" as used herein refers to any method known to those skilled in the art for better revealing, distinguishing, or identifying a particular component and/or characteristic of a cell.

As used in the specification and claims, the terms "operably linked", "operable order" and "operably linked" as used herein refer to the joining of nucleic acid sequences in such a way as to produce a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule. The term also refers to amino acid sequences that are linked in such a way as to produce a functional protein.

As used in the specification and claims, the term "antigen" as used herein refers to a protein, glycoprotein, lipoprotein, lipid, or other substance that is reactive with an antibody that is specific for a portion of the molecule.

For the purposes of the specification and claims, the term "morphology" as used herein is suitably intended to refer to the visual appearance of a cell or organism when viewed using the eye, optical microscope, confocal microscope or electron microscope.

For the purposes of the specification and claims, the terms "subject", "individual" and "patient" as used herein refer to a human or other animal, such as a farm or laboratory animal (e.g., guinea pig or mouse), which is capable of suffering from a naturally occurring or artificially induced cell cycle (influenced) determined disease, including but not limited to cancer.

The term "reversal of resistance" refers to the use of a second agent in combination with an initial chemotherapeutic agent to reduce tumor volume to a statistically significant level (e.g., p < 0.05) as compared to the tumor volume of an untreated tumor in situations where the initial chemotherapeutic agent alone does not reduce tumor volume to a statistically significant level as compared to the untreated tumor volume. This is typically applied to tumor volume measurements when untreated tumors are grown in logarithmic rhythm.

The term "enhancing" as used herein means enhancing (enhance) or increasing (increase) the beneficial activity or efficacy of an anticancer agent over that expected from the anticancer agent alone or the enhancer alone.

The term "sensitize" as used herein refers to altering cancer cells or tumor cells in a manner that allows an anticancer drug or radiation therapy to more effectively treat the associated neoplastic disease. In some embodiments, normal cells are not affected to the extent that chemotherapy or radiation therapy does not cause undue damage to normal cells.

The term "synergistic effect" as used herein means that the combined effect of two or more anticancer drugs or chemotherapeutic drugs may be greater than the sum of the individual effects of the individual anticancer drugs or chemotherapeutic drugs. For example, the combined effect of a BER inhibitor such as methoxyamine and an anticancer drug such as pemetrexed is stronger than the sum of the individual effects of methoxyamine and pemetrexed alone.

The term "therapeutically effective amount" means that amount of a test compound that will produce a desired response, e.g., a biological or medical response of a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.

As used in the specification and in the claims, the term "wild-type" (wt) cell or cell line as used herein refers to a cell or cell line that retains the characteristics normally associated with the cell or cell line type for the physiological process or morphological characteristics being examined. The cells or cell lines are allowed to have non-wild type characteristics in physiological processes or morphological characteristics that are not to be detected, as long as they do not significantly affect the process or characteristic being detected.

The term "pharmaceutically acceptable salt" refers to a salt of a compound that does not cause significant irritation to the organism to which it is administered and does not diminish the biological activity and properties of the compound. In some embodiments, the salt is an acid addition salt of the compound. Pharmaceutically acceptable salts can be obtained by reacting the compounds with inorganic acids such as hydrohalic acids (e.g., hydrochloric or hydrobromic acid), sulfuric acid, nitric acid, phosphoric acid, and the like. Pharmaceutically acceptable salts may also be obtained by reacting the compounds with organic acids such as aliphatic or aromatic carboxylic or sulphonic acids, for example acetic, succinic, lactic, malic, tartaric, citric, ascorbic, nicotinic, methanesulphonic, ethanesulphonic, p-toluenesulphonic, salicylic or naphthalenesulphonic acids. Pharmaceutically acceptable salts may also be obtained by reacting a compound with a base to form, for example, an ammonium salt, an alkali metal salt such as a sodium or potassium salt, an alkaline earth metal salt such as a calcium or magnesium salt, an organic base such as dicyclohexylamine, N-methyl-D-glucamine, tris (hydroxymethyl) methylamine, C₁-C₇Salts of alkylamines, cyclohexylamine, triethanolamine, ethylenediamine, and salts with amino acids such as arginine, lysine, and the like.

Damage to DNA is minimized by enzymes that recognize errors, eliminate errors, and replace damaged DNA with corrected nucleotides. DNA damage occurs when a single strand break is introduced, a base is removed from a previously unpaired partner, a base is covalently modified, a base is converted to another base that does not properly pair with a partner base, or a covalent bond is introduced between complementary strand bases. The excision repair system removes mismatched or damaged bases from the DNA strand and then synthesizes new DNA to replace them. Base Excision Repair (BER) begins during DNA replication and allows correction of damaged/mismatched bases before replication is complete.

The Base Excision Repair (BER) action is initiated by a DNA glycosylase that removes the N-glycoside (base-sugar) bond, releases the damaged base and creates an abasic site (e.g., a purine-free or pyrimidine-free (AP) site). An apurinic or Apyrimidinic (AP) site results from the loss of a purine or pyrimidine residue, respectively, from DNA (deoxyribonucleic acid). Uracil residues can be formed by spontaneous deamination of cytosine and can lead to a C → T transition if not repaired. There is also a glycosylase that recognizes and excises hypoxanthine (the deamination product of adenine). Other glycosylases eliminate alkylated bases (e.g., 3-methyladenine, 3-methylguanine and 7-methylguanine), open ring purines, oxidation damaged bases, and UV light dimers in some organisms. Uracil DNA Glycosylase (UDG) is an example of a DNA glycosylase. The BER protein level of UDG was affected by combined treatment with pemetrexed and MX (fig. 7).

The AP site is further treated with a5 '-3' endonuclease (AP endonuclease (APE)) which cleaves the phosphodiester bonds bonded on both sides of the damaged purine or pyrimidine base. By cleaving the phosphodiester bond at the AP site, the AP endonuclease causes strand breakage.

PARP helps to handle DNA strand breaks caused during BER. PARP is a DNA nick monitoring protein (nick surveyability protein) that binds weakly to BER intermediates when single nucleotide BER proceeds normally to completion. In contrast, when the single nucleotide BER is stopped by the block in the cleavage step, PARP and AP endonucleases (APE), DNA polymerase β and FEN-1 are strongly bound to the BER intermediate.

In mammalian cells, 5 '-deoxyribose phosphate sugar (5' -deoxyribosougar phosphate) is removed by the intrinsic AP lyase (dRP) activity of DNA polymerase β (pol β). The DNA polymerase also fills the gap with new nucleotides.

Finally, DNA ligase covalently attaches the 3' end of the new species to the original species. Thus, the wild-type sequence was restored.

Topoisomerase I and II are also involved in DNA repair because they recognize spontaneous AP sites, forming stable cleavable complexes. Topoisomerase II inhibitors promote DNA cleavage and other chromosomal aberration reactions, including sister chromatid exchange.

Some embodiments described herein may relate to a method comprising:

providing i) a patient diagnosed with cancer, ii) a first formulation comprising an antifolate anticancer agent and iii) a second formulation comprising methoxyamine;

administering a first formulation to a patient; and administering said second formulation to said patient, wherein methoxyamine may be administered in an amount sufficient to enhance or increase the effect of the antifolate anticancer agent. Any antifolate anticancer drug may be used, provided that in certain embodiments of the method, 5-FU is specifically excluded.

In typical embodiments, the anti-cancer agent may be selected from: pemetrexed, capecitabine, edatrexate, methotrexate, lometrexol, nolatrexed, raltrexed, PT523, trimetrexate, aminopterin, 5, 10-dideazatetrahydrofolate (ddatff), pirtrexin, raltitrexed, GW1843[ (S) -2- [5- [ (1, 2-dihydro-3-methyl-1-oxobenzo [ f ] quinazolin-9-yl) methyl ] amino-1-oxo-2-isoindolyl ] -glutaric acid ], pharmaceutically acceptable salts thereof, and any combination thereof. In a more typical embodiment, the anti-cancer agent may be selected from: pemetrexed, capecitabine, edatrexate, methotrexate, lometrexol, nolatrexed, ralitexed, PT523, trimetrexate, aminopterin, pharmaceutically acceptable salts thereof, and any combination thereof. In a most typical embodiment, the anti-cancer agent may be pemetrexed and pharmaceutically acceptable salts thereof. For example, pemetrexed may be in the form of the disodium salt. In exemplary embodiments, pemetrexed may be the disodium salt heptahydrate.

In certain embodiments, the present invention contemplates the use of an anti-cancer agent and a BER inhibitor that cause AP site formation.

In typical embodiments, the anti-cancer agent may be selected from: pemetrexed, capecitabine, edatrexate, methotrexate, lometrexol, nolatrexed, raltrexed, PT523, trimetrexate, aminopterin, 5, 10-dideazatetrahydrofolate (ddatff), pirtrexin, raltitrexed, GW1843[ (S) -2- [5- [ (1, 2-dihydro-3-methyl-1 oxybenzo [ f ] quinazolin-9-yl) methyl ] amino-1-oxo-2-isoindolyl ] -glutaric acid ], pharmaceutically acceptable salts, and any combination thereof. In a more typical embodiment, the anti-cancer agent may be selected from: pemetrexed, capecitabine, edatrexate, methotrexate, lometrexol, nolatrexed, ralitexed, PT523, trimetrexate, aminopterin, pharmaceutically acceptable salts thereof, and any combination thereof. In a most typical embodiment, the anti-cancer agent may be pemetrexed and pharmaceutically acceptable salts thereof. For example, pemetrexed may be the disodium salt. In exemplary embodiments, pemetrexed may be the disodium salt heptahydrate.

In typical embodiments, the BER inhibitor may be selected from: methoxyamine, etoposide (VP-16, VP-16-123), meso (meso) -4, 4 '- (2, 3-butanediyl) -di- (2, 6-piperazinedione) (ICRF-193, dioxopiperazine), Doxorubicin (DOX), amsacrine (4', 9-acridinylmethanesulfonyl-m-methoxyaniline (anisidide); mMSA), paroxetine, nalidixic acid, oxolinic acid, neomycin, coumaromycin A1, fostrexasin, teniposide, mitoxantrone, daunorubicin, N- [ 2-dimethylamino) ethyl]Acridine-4-carboxamide (DACA), meparoline (merbarone), mepacrine, ellipticine, epipodophyllotoxin, ethidium bromide, epirubicin, pirarubicin, 3 '-deamino-3' -morpholino-13-deoxy-10-hydroxycarminomycin; 2 ', 3' -dipentafluorophenoxyacetyl-4 ', 6' -ethylene-beta-D-glucoside (F11782; fluorinated lipophilic epidophylloid) of the 2N-methylglucamine salt of 4 '-phosphate-4' -dimethyl epipodophyllotoxin, doxorubicin, actinomycin D, anthracyclines (e.g., 9-aminoanthracyclines), pyrazoline acridine (PZA), camptothecin, topotecan, pharmaceutically acceptable salts and solvates thereof and any combination thereof. In more typical embodiments, the BER inhibitor may be selected from: methoxyamine (MX), N-ethylmaleimide, O⁶-benzylguanine, pharmaceutically acceptable salts thereof and any combination thereof. In a most typical embodiment, the BER inhibitor may be Methoxyamine (MX) or a salt thereof.

In one embodiment, the BER inhibitor may be a compound having formula IA compound of structure (I):formula I

Wherein X is O or NH,

y is O, S, or NH,

z is absent or represents O, S, or NH,

r represents hydrogen or a hydrocarbon moiety, and

a pharmaceutically acceptable salt thereof.

In some embodiments, the BER inhibitor may be used to treat a patient or subject having a neoplastic disease. For example, the neoplastic disease may be a cancer selected from: cancer, melanoma, sarcoma, lymphoma, leukemia, astrocytoma, glioma, malignant melanoma, chronic lymphocytic leukemia, lung cancer, prostate cancer, colorectal cancer, ovarian cancer, pancreatic cancer, renal cancer, endometrial cancer, gastric cancer, liver cancer, head and neck cancer.

In some embodiments, the BER inhibitor may be used to treat a patient or individual with a neoplastic disease who is being treated with an anti-cancer drug.

In typical embodiments, the BER inhibitor may be selected from: methoxyamine, etoposide (VP-16, VP-16-123), meso (meso) -4, 4 '- (2, 3-butanediyl) -di- (2, 6-piperazinedione) (ICRF-193, dioxopiperazine), Doxorubicin (DOX), amsacrine (4', 9-acridinylmethanesulfonyl-m-methoxyaniline; mMSA), paroxetine, nalidixic acid, oxolinic acid, neomycin, coumaromycin A1, fostretin, teniposide, mitoxantrone, daunorubicin, N- [ 2-dimethylamino) ethyl]Acridine-4-carboxamide (DACA), mebutalone, mepacrine, ellipticine, epipodophyllotoxin, ethidium bromide, epirubicin, pirarubicin, 3 '-deamino-3' -morpholino-13-deoxy-10-hydroxycarminomycin; 2 ', 3' -bis-pentafluorophenoxyethyl of 4 '-phosphate-4' -dimethyl epipodophyllotoxin 2N-methylglucamine saltAcyl-4 ', 6' -ethylene- β -D glucoside (F11782; fluorinated lipophilic epidophylloid), doxorubicin, actinomycin D, an anthracycline (e.g., 9-aminoanthracycline), pyrazoline acridine (PZA), camptothecin, topotecan, pharmaceutically acceptable salts thereof, and any combination thereof. In more typical embodiments, the BER inhibitor may be selected from: methoxyamine (MX), N-ethylmaleimide, O⁶-benzylguanine, pharmaceutically acceptable salts thereof and any combination thereof. In a most typical embodiment, the BER inhibitor may be Methoxyamine (MX) or a salt thereof.

In typical embodiments, the anti-cancer agent may be selected from: pemetrexed, capecitabine, edatrexate, methotrexate, lometrexol, nolatrexed, raltrexed, PT523, trimetrexate, aminopterin, 5, 10-dideazatetrahydrofolate (ddatff), pirtrexin, raltitrexed, GW1843[ (S) -2- [5- [ (1, 2-dihydro-3-methyl-1-oxobenzo [ f ] quinazolin-9-yl) methyl ] amino-1-oxo-2-isoindolyl ] -glutaric acid ], pharmaceutically acceptable salts thereof, and any combination thereof. In a more typical embodiment, the anti-cancer agent may be selected from: pemetrexed, capecitabine, edatrexate, methotrexate, lometrexol, nolatrexed, ralitexed, PT523, trimetrexate, aminopterin, pharmaceutically acceptable salts thereof, and any combination thereof. In a most typical embodiment, the anti-cancer agent may be pemetrexed and pharmaceutically acceptable salts and solvates thereof. For example, pemetrexed may be the disodium salt. In exemplary embodiments, pemetrexed may be the disodium salt heptahydrate.

In some embodiments, the BER inhibitor and the anti-cancer agent may be administered to the individual in combination. For example, the BER inhibitor and the anticancer agent may be administered to the individual together in the form of a parenteral formulation. Alternatively, the BER inhibitor and the anticancer agent may be administered to the individual together in an oral formulation, such as a solid dosage formulation.

In some embodiments, the BER inhibitor and the anti-cancer agent may be administered to the individual sequentially, wherein the anti-cancer agent is administered first to the individual, followed by administration of the BER inhibitor. For example, the anticancer agent may be administered to the individual in the form of a parenteral formulation, such as an intravenous formulation, or an oral formulation, such as a solid dosage form formulation, followed by administration of the BER inhibitor in the form of a parenteral formulation, such as an intravenous formulation, or an oral formulation, such as a solid dosage form formulation.

Alternatively, in some embodiments, the BER inhibitor and the anti-cancer agent may be administered to the individual sequentially, wherein the BER inhibitor is administered first to the individual, followed by the anti-cancer agent. For example, the BER inhibitor may be administered to the individual in the form of a parenteral formulation, such as an intravenous formulation, or an oral formulation, such as a solid dosage form formulation, followed by administration of the anti-cancer agent in the form of a parenteral formulation, such as an intravenous formulation, or an oral formulation, such as a solid dosage form formulation.

In some embodiments, the anti-cancer drug and BER inhibitor may produce a greater anti-cancer effect than the anti-cancer effect of each drug alone. For example, the combined anti-cancer effect of the anti-cancer agent and the BER inhibitor may be greater than the sum of the anti-cancer effects of the anti-cancer agent and the BER inhibitor used alone.

Compounds useful as BER inhibitors, e.g. Methoxyamine (MX), N-ethylmaleimide, O⁶-benzylguanine having the structure of formula I:formula I

Wherein X is O or NH,

y is O, S, or NH,

z is absent or represents O, S, or NH, and

r represents hydrogen or a hydrocarbon moiety,

and pharmaceutically acceptable salts thereof.

In single nucleotide BER, deoxyribose phosphate (dRP) at the abasic site is removed by the lyase activity of DNA polymerase β. Compounds such as methoxyamine react with the aldehyde at the abasic site, rendering it resistant to the β -elimination step of the dRP lyase mechanism, thus blocking the single nucleotide BER.

In some embodiments, suitable compounds can prevent the substrate of the AP endonuclease from being easily cleaved. Anticancer drugs can act by binding to the AP site and preventing APE-mediated phosphodiester bond cleavage. Other compounds that bind to the AP site and prevent APE-mediated phosphodiester bond cleavage include O-benzylhydroxylamine; ethyl aminooxyacetate; aminooxyacetic acid; ethyl aminooxyacetate; h₂N-OCHMeCO₂H; a carboxymethoxyamine; aminooxyacetic acid; HN ═ C (NH)₂)SCH₂CH₂ONH₂；H₂N-O(CH₂)₃SC(NH₂)＝NH；MeOC(O)CH(NH₂)CH₂O-NH₂；H₂NOCH₂CH(NH₂)CO₂H; paracaseolin acid; h₂N-O(CH₂)₄O-NH₂(ii) a O- (p-nitrobenzyl) hydroxylamine; 2-amino-4- (aminooxymethyl) thiazole; 4- (aminooxymethyl) thiazole; o, O' - (O-phenylenedimethylene) dihydroxylamine; 2, 4-dinitrophenoxyamine; o, O' - (m-phenylenedimethylene) dihydroxylamine; o, O' - (p-phenylenedimethylene) dihydroxylamine; h₂C＝CHCH₂O-NH₂；H₂N-O(CH₂)₄O-NH₂；H₃C(CH₂)₁₅O-NH₂Dimethyl diethyl 2, 2' - (1, 2-ethanediyl) bis (3-aminooxy) butenedioate; a compound having any of the following structures:

and pharmaceutically acceptable salts of any of these compounds.

Compounds useful as BER inhibitors include PARP inhibitors such as 4-amino-1, 8-naphthalimide (ANI), PD128763, 3-AB, 6-AN, and 8-hydroxy-2-methyl-quinazolin-4-, [³H]Ketone (NU-1025).

Compounds useful as BER inhibitors include DNA polymerase inhibitors (e.g., DNA polymerase β, γ, or ε) such as veronicin, aphidicolin, 2 ', 3' -dideoxycytidine triphosphate (ddCTP), 2 ', 3' -dideoxythymidine triphosphate (ddTTP), 2 ', 3' -dideoxyadenosine triphosphate (ddATP), 2 ', 3' -dideoxyguanosine triphosphate (ddGTP), 1- β -D-arabinofuranosyl cytosine (Ara-C), caffeine, cytarabine (arabinocytidine), and bleomycin.

Compounds useful as BER inhibitors include DNA ligase inhibitors (e.g., DNA ligase I, II or III) such as ursolic acid and oleanolic acid, elaeostearic acid, norbrnstic acid, swertirchiside, frangulan, auricularia pepper base chloride and bleomycin. XRCC1 is a protein partner for DNA ligase III, and XRCC1 inhibitors such as 3-AB are also used as BER inhibitors.

Topoisomerase II inhibitors induce DNA cleavage and other chromosomal aberration responses, including sister chromatid exchange. Compounds useful as BER inhibitors also include: topoisomerase II inhibitors, for example etoposide (VP-16, VP-16-123), meso-4, 4 '- (2, 3-butanediyl) -di- (2, 6-piperazinedione) (ICRF-193, dioxopiperazine), Doxorubicin (DOX), amsacrine (4', 9-acridinylcarbamoylm-methoxyaniline; mMSA), paroxetine, nalidixic acid, oxolinic acid, neomycin, coumaromycin A1, fostretin, teniposide, mitoxantrone, daunorubicin, N- [ 2-dimethylamino) ethyl ] acridine-4-carboxamide (DACA), mebutan, mepacrine, ellipticine, epidophyllotoxin, ethidium bromide, epirubicin, pirarubicin, 3 '-deamino-3' -morpholino-13-deoxy-10-hydroxycarminol (DACA) A mycin; 2 ', 3' -dipentafluorophenoxyacetyl-4 ', 6' -ethylene-beta-D-glucoside (F11782; fluorinated lipophilic epipodophylloid), doxorubicin, actinomycin D, an anthracycline (e.g., 9-aminoanthracycline), and pyrazoline acridine (PZA) of 4 '-phosphate-4' -dimethyl epipodophyllotoxin 2N-methylglucamine salt. Topoisomerase I inhibitors, such as camptothecin and topotecan, can also be used as BER inhibitors.

In some embodiments, other enzyme inhibitors, whether known in the art or hereafter proven, and inhibitors of other elements of the BER pathway, such as DNA alkyltransferases, may be used in the compositions and methods of the present embodiments without departing from the scope and spirit of the present embodiments.

In certain embodiments, the invention concerns the use of anti-cancer agents that cause the formation of AP sites, such as pemetrexed and BER inhibitors (except topoisomerase inhibitors) such as methoxyamine.

In typical embodiments, the anti-cancer agent may be selected from: pemetrexed, capecitabine, edatrexate, methotrexate, lometrexol, nolatrexed, ralitexed, PT523 and trimetrexate. In a more typical embodiment, the anti-cancer agent may be pemetrexed and pharmaceutically acceptable salts thereof. For example, pemetrexed may be the disodium salt. In exemplary embodiments, pemetrexed may be the disodium salt heptahydrate.

In some embodiments, the anti-cancer agent can be at about 25mg/m²To about 5,000mg/m²Dosing of body surface area. For example, the dose may be about 25mg/m²To about 200mg/m²A body surface area; the dosage may be about 150mg/m²To about 500mg/m²A body surface area; the dosage may be about 400mg/m²To about 1000mg/m²A body surface area; the dosage may be about 900mg/m²To about 5,000mg/m²A body surface area; the dosage may be about 200mg/m²To about 1,000mg/m²A body surface area; alternatively, the dosage may be about 500mg/m²To about 600mg/m²Body surface area. Antifolates are a non-limiting preferred class of anticancer agents. In some embodiments, the anti-cancer agent may be selected from: pemetrexed, capecitabine, edatrexate, methotrexate, lometrexol, nolatrexed, ralitexed, PT523 and trimetrexate. In a more typical embodiment, the anti-cancer agent may be pemetrexed and pharmaceutically acceptable salts thereofA salt. For example, pemetrexed may be the disodium salt. In exemplary embodiments, pemetrexed may be the disodium salt heptahydrate.

In some embodiments, the ratio of BER inhibitor to anticancer agent may be from about 1: 2 to about 1: 10000. For example, the ratio of BER inhibitor to anticancer agent may be from about 1: 2 to about 1: 100; the ratio of BER inhibitor to anticancer agent may be from about 1: 50 to about 1: 500; the ratio of BER inhibitor to anticancer agent may be from about 1: 450 to about 1: 10000; the ratio of BER inhibitor to anticancer agent may be from about 1: 5 to about 1: 500; the ratio of BER inhibitor to anticancer agent may be from about 1: 10 to about 1: 50; the ratio of BER inhibitor to anticancer agent may be from about 1: 15 to about 1: 40; alternatively, the ratio of BER inhibitor to anticancer agent may be from about 1: 20 to about 1: 30. In typical embodiments, the BER inhibitor may be selected from: methoxyamine (MX), N-ethylmaleimide, O⁶-benzylguanine, pharmaceutically acceptable salts thereof and any combination thereof. In a more typical embodiment, the BER inhibitor may be Methoxyamine (MX).

In some embodiments, the BER inhibitor is administered in an amount sufficient to enhance or increase the effect of the anti-cancer agent.

In typical embodiments, the anti-cancer agent may be selected from: pemetrexed, capecitabine, edatrexate, methotrexate, lometrexol, nolatrexed, raltrexed, PT523, trimetrexate, aminopterin, 5, 10-dideazatetrahydrofolate (ddatff), pirtrexin, raltitrexed, GW1843[ (S) -2- [5- [ (1, 2-dihydro-3-methyl-1-oxobenzo [ f ] quinazolin-9-yl) methyl ] amino-1-oxo-2-isoindolyl ] -glutaric acid ], pharmaceutically acceptable salts thereof, and any combination thereof. In a more typical embodiment, the anti-cancer agent may be selected from: pemetrexed, capecitabine, edatrexate, methotrexate, lometrexol, nolatrexed, ralitexed, PT523, trimetrexate, aminopterin, pharmaceutically acceptable salts thereof, and any combination thereof. In a most typical embodiment, the anti-cancer agent may be pemetrexed and pharmaceutically acceptable salts thereof. For example, pemetrexed may be the disodium salt. In exemplary embodiments, pemetrexed may be the disodium salt heptahydrate.

In typical embodiments, the BER inhibitor may be selected from: methoxyamine, etoposide (VP-16, VP-16-123), meso-4, 4 '- (2, 3-butanediyl) -bis- (2, 6-piperazinedione) (ICRF-193, dioxopiperazine), Doxorubicin (DOX), amsacrine (4', 9-acridinylcarbamoyl-m-methoxyaniline; mMSA), pazestin, nalidixic acid, oxolinic acid, neomycin, coumaromycin A1, fostretin, teniposide, mitoxantrone, daunorubicin, N- [ 2-dimethylamino) ethyl]Acridine-4-carboxamide (DACA), mebutalone, mepacrine, ellipticine, epipodophyllotoxin, ethidium bromide, epirubicin, pirarubicin, 3 '-deamino-3' -morpholino-13-deoxy-10-hydroxycarminomycin; 2 ', 3' -dipentafluorophenoxyacetyl-4 ', 6' -ethylene-beta-D-glucoside (F11782; fluorinated lipophilic epidophylloid) of the 2N-methylglucamine salt of 4 '-phosphate-4' -dimethyl epipodophyllotoxin, doxorubicin, actinomycin D, anthracyclines (e.g., 9-aminoanthracyclines), pyrazoline acridine (PZA), camptothecin, topotecan, pharmaceutically acceptable salts thereof and any combination thereof. In more typical embodiments, the BER inhibitor may be selected from: methoxyamine (MX), N-ethylmaleimide, O⁶-benzylguanine, pharmaceutically acceptable salts thereof and any combination thereof. In a most typical embodiment, the BER inhibitor may be Methoxyamine (MX) or a salt thereof. For example, the BER inhibitor may be methoxylamine hydrochloride (MX).

In some embodiments, the ratio of BER inhibitor to anticancer agent may be from about 1: 2 to about 1: 10000. For example, the ratio of BER inhibitor to anticancer agent may be from about 1: 2 to about 1: 100; the ratio of BER inhibitor to anticancer agent may be from about 1: 50 to about 1: 500; the ratio of BER inhibitor to anticancer agent may be from about 1: 450 to about 1: 10000; the ratio of BER inhibitor to anticancer agent may be from about 1: 5 to about 1: 500; the ratio of BER inhibitor to anticancer agent may be from about 1: 10 to about 1: 50; the ratio of BER inhibitor to anticancer agent may be from about 1: 15 to about 1: 40; alternatively, the ratio of BER inhibitor to anticancer agent may be from about 1: 20 to about 1: 30. In the typicalIn embodiments of (a), the BER inhibitor may be selected from: methoxyamine (MX), N-ethylmaleimide, O⁶-benzylguanine, pharmaceutically acceptable salts thereof and any combination thereof. In a more typical embodiment, the BER inhibitor may be Methoxyamine (MX). In a most typical embodiment, the BER inhibitor may be Methoxyamine (MX) and the anti-cancer agent may be pemetrexed. For example, the pemetrexed may be the disodium salt of pemetrexed. In exemplary embodiments, pemetrexed may be the disodium salt heptahydrate.

Some embodiments provide methods of treating cancer, comprising

Providing a first formulation comprising an anti-cancer agent and a second formulation comprising a BER inhibitor, which may be administered separately or as a combined formulation;

selecting a subject diagnosed with cancer, wherein said cancer is resistant to treatment with an anti-cancer drug, alone or in combination with other anti-cancer drugs;

administering the first formulation and the second formulation;

wherein the amount of the first agent and the amount of the second agent may be such amounts that when administered to the subject, the anticancer effect may be greater than the anticancer effect of the first agent alone.

In some embodiments, the first formulation may comprise an anti-cancer agent selected from the group consisting of: pemetrexed, capecitabine, edatrexate, methotrexate, lometrexol, nolatrexed, ralitexed, PT523, trimetrexate, aminopterin, pharmaceutically acceptable salts thereof, and any combination thereof. In a typical embodiment, the anti-cancer agent may be pemetrexed. In some embodiments, the second formulation may comprise a BER inhibitor selected from the group consisting of: methoxyamine (MX), N-ethylmaleimide, O⁶-benzylguanine, pharmaceutically acceptable salts thereof and any combination thereof. In typical embodiments, the BER inhibitor may be methoxyamine.

In some embodiments, the anti-cancer agent can be about 25mg/m²To about 5,000mg/m²The amount of body surface area is administered. For example, the dose may be about 25mg/m²To about 200mg/m²A body surface area; the dosage may be about 150mg/m²To about 500mg/m²A body surface area; the dosage may be about 400mg/m²To about 1000mg/m²A body surface area; the dosage may be about 900mg/m²To about 5,000mg/m²A body surface area; the dosage may be about 200mg/m²To about 1,000mg/m²A body surface area; alternatively, the dosage may be about 500mg/m²To about 600mg/m²Body surface area. In some embodiments, the anti-cancer agent may be selected from: pemetrexed, capecitabine, edatrexate, methotrexate, lometrexol, nolatrexed, ralitexed, PT523 and trimetrexate. In a more typical embodiment, the anti-cancer agent may be pemetrexed and pharmaceutically acceptable salts thereof. For example, pemetrexed may be the disodium salt. In exemplary embodiments, pemetrexed may be the disodium salt heptahydrate.

Some embodiments provide methods of treating cancer, comprising

selecting a subject diagnosed with cancer;

administering the first formulation and the second formulation:

wherein the amount of the first agent and the amount of the second agent may be such amounts that, when administered to the subject, the anticancer effect is greater than the additive anticancer effect of the first agent comprising the anticancer agent and the second agent comprising the BER inhibitor.

In some embodiments, the anti-cancer agent can be at about 25mg/m²To about 5,000mg/m²Dosing of body surface area. For example, the dose may be about 25mg/m²To about 200mg/m²A body surface area; the dosage may be about 150mg/m²To about 500mg/m²A body surface area; the dosage may be about 400mg/m²To about 1000mg/m²A body surface area; the dosage may be about 900mg/m²To about 5,000mg/m²A body surface area; the dosage may be about 200mg/m²To about 1,000mg/m²A body surface area; alternatively, the dosage may be about 500mg/m²To about 600mg/m²Body surface area. In some embodiments, the anti-cancer agent may be selected from: pemetrexed, capecitabine, edatrexate, methotrexate, lometrexol, nolatrexed, ralitexed, PT523 and trimetrexate. In a more typical embodimentIn the formula, the anticancer drug may be pemetrexed and its pharmaceutically acceptable salt. For example, pemetrexed may be the disodium salt. In exemplary embodiments, pemetrexed may be the disodium salt heptahydrate.

Another aspect of embodiments of the present invention is the unexpected effect that certain BER inhibitors act in a synergistic manner in combination with certain antifolates to unexpectedly reverse the resistance of certain antifolates. Thus, in a non-limiting preferred embodiment, the antimetabolic anticancer agent is an antifolate anticancer agent. These antifolates disrupt folate-dependent metabolic processes essential for cell replication. Antifolates differ from other chemotherapeutic drugs in that they disrupt cellular processes involved in folate metabolism. This includes inhibition of folate dependent enzymes, including but not limited to Thymidylate Synthase (TS). Disruption of the folate-dependent process results in inappropriate DNA replication and apoptosis in rapidly dividing cells, including cancer cells. In one embodiment, the ratio of MX to antimetabolic anticancer agent is about 1: 5 to 1: 500. In certain embodiments, the ratio of MX to antimetabolic anticancer agent is from about 1: 10 to about 1: 100, from about 1: 25 to about 1: 75, from about 1: 15 to about 1: 40, or from about 1: 20 to about 1: 30. In addition, can be connectedThe combination of MX and an antimetabolic anticancer agent is preceded or followed by another anticancer agent.Pharmaceutical composition

It will be understood that the compositions provided herein may be in any form that allows the compositions to be administered to a patient. For example, the composition may be in the form of a solid, liquid or gas (e.g., an aerosol). Other suitable routes of administration include, but are not limited to, oral administration, topical administration, parenteral administration (e.g., sublingual or buccal administration), sublingual administration, rectal administration, vaginal administration, and intranasal administration. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal (intraspecific), intracavernosal, intrathecal, intrameatal, intraurethral injection or infusion techniques. The pharmaceutical composition is prepared in a manner that allows the active ingredient it contains to be bioavailable after administration to a patient. The compositions for administration to a patient are in the form of one or more dosage units, wherein, for example, a tablet may be a single dosage unit and a container of one or more compounds of the invention in aerosol form may contain a plurality of dosage units.

In another aspect, the present disclosure relates to a pharmaceutical composition comprising a physiologically acceptable surfactant, carrier, diluent, excipient, smoothing agent, suspending agent, film-forming material, and coating aid, or a combination thereof; and the compounds disclosed herein. Acceptable carriers or diluents for therapeutic use are well known in the Pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing co., Easton, PA (1990), the entire contents of which are incorporated herein by reference. Preservatives, stabilizers, dyes, sweeteners, flavoring agents, and the like may be provided in the pharmaceutical composition. For example, sodium benzoate, ascorbic acid and parabens may be added as preservatives. In addition, antioxidants and suspending agents may be used. In various embodiments, alcohols, esters, sulfated fatty alcohols, and the like may be used as surfactants; sucrose, glucose, lactose, starch, crystalline cellulose, mannitol, light anhydrous silicate, magnesium aluminate, magnesium methyl silicate aluminate, synthetic aluminum silicate, calcium carbonate, sodium bicarbonate, calcium hydrogen phosphate, carboxymethylcellulose calcium, etc. may be used as the excipient; magnesium stearate, talc, hardened oil, etc. can be used as a smoothing agent; coconut oil, olive oil, sesame oil, peanut oil, soybean can be used as suspending agent or lubricant; cellulose acetate phthalate as a carbohydrate such as a derivative of cellulose or sugar, or a methyl acetate-methacrylate copolymer as a derivative of polyethylene may be used as a suspending agent; and plasticizers such as phthalates and the like may be used as suspending agents.

The term "pharmaceutical composition" refers to a mixture of a compound disclosed herein with other chemical ingredients, such as a diluent or carrier. The pharmaceutical composition facilitates administration of the compound to an organism. A variety of compound administration techniques exist in the art including, but not limited to, oral administration, injection administration, aerosol administration, parenteral administration, and topical administration. Pharmaceutical compositions can also be obtained by reacting the compounds with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.

The term "carrier" is defined as a compound that facilitates entry of the compound into a cell or tissue. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier because it facilitates the uptake of various organic compounds by cells or tissues of an organism.

The term "diluent" is defined as a compound diluted in water that is capable of dissolving the compound of interest and stabilizing the biologically active form of the compound. Salts dissolved in buffer solutions are used in the art as diluents. One commonly used buffer solution is phosphate buffered saline because it mimics the saline environment in human blood. Since buffer salts can control the pH of a solution at low concentrations, buffer diluents rarely affect the biological activity of a compound.

The term "physiologically acceptable" is defined as a carrier or diluent that does not diminish the biological activity and properties of the compound.

The pharmaceutical compositions described herein may be administered to a human patient as such, or in a composition in which they are mixed with other active ingredients, or suitable carriers or excipients, as a combination therapy. Formulations and techniques for administration of the compounds of the present application may be found in "Remington's Pharmaceutical Sciences," Mack Publishing co., Easton, PA, 18th edition, 1990.

Suitable routes of administration may include, for example, oral, rectal, transmucosal, topical or enteral administration; parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, and intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections. The compounds may also be administered in sustained or controlled release dosage forms, including depot injections, osmotic pumps, pills, transdermal (including electrotransport) patches, and the like, for sustained and/or timed, pulsatile administration at a predetermined rate.

The pharmaceutical compositions of the present invention may be prepared in a manner known per se, for example by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting methods.

The pharmaceutical compositions used in the present invention may thus be prepared in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which enable the active compounds to be readily processed into preparations which can be used pharmaceutically. The appropriate formulation depends on the chosen route of administration. Any of the well known techniques, carriers and excipients that are appropriate and known in the art may be used; such as those described above in Remington's Pharmaceutical Sciences.

Injections may be prepared in conventional forms such as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or emulsions. Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride and the like. In addition, if desired, the injectable pharmaceutical composition may contain minor amounts of nontoxic auxiliary substances such as wetting agents, pH buffering agents and the like. Physiologically compatible buffers include, but are not limited to Hanks's solution, Ringer's solution, or physiological saline buffer. If desired, absorption enhancing agents (e.g., liposomes) can be used.

For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation.

Pharmaceutical preparations for parenteral administration, for example by bolus injection (bolus injection) or continuous infusion, comprise aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or other organic oils such as soybean oil, grapefruit oil or almond oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, for example sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Formulations for injection may be presented in unit dosage form with an additional preservative, for example, in ampoules or in multi-dose containers. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents (formulating agents) such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

For oral administration, the compounds can be readily formulated by combining the active compound with pharmaceutically acceptable carriers well known in the art. These carriers enable the compounds of the present invention to be formulated as tablets, pills, lozenges, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient in need of treatment. Pharmaceutical preparations for oral use can be obtained by the following method: combining the active compound with a solid excipient, optionally grinding the resulting mixture, and treating the mixture of granules, if desired, with subsequent addition of suitable auxiliaries, to obtain tablets or dragee cores. In particular, suitable excipients are fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations are, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. The dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions (lacquer solutions), and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablet or lozenge coating in order to identify or characterize different compositions of active compound doses. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablet or lozenge coating in order to identify or characterize different compositions of active compound doses.

Pharmaceutical preparations which can be used orally include push-fit (push-fit) capsules made of gelatin, and soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, for example fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition to this, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use in the present invention are conveniently delivered in the form of an aerosol packaged in a pressurized pack or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. For pressurized aerosols, the dosage unit may be determined by providing a valve to deliver a determined amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

Further disclosed herein are various pharmaceutical compositions well known in the pharmaceutical art for uses including intraocular, intranasal, and intra-aural delivery. Penetrants suitable for such uses are generally known in the art. Pharmaceutical compositions for intraocular delivery include aqueous ophthalmic solutions of the active compound in water-soluble form, such as eye drops, or in the form of gellan gum (Shedden et al, clin. ther., 23 (3): 440-50(2001)) or hydrogels (Mayer et al, Ophthalmologica, 210 (2): 101-3 (1996)); ophthalmic ointments; ophthalmic suspensions, e.g., microparticles, drug-containing small polymeric particles suspended in a liquid carrier medium (Joshi, a., j. ocul. pharmacol., 10 (1): 29-45(1994)), lipid-soluble formulations (Alm et al, prog.clin.biol.res., 312: 447-58(1989)), and microspheres (Mordenti, toxicol.sci., 52 (1): 101-6 (1999)); and an ocular insert. All references mentioned above are incorporated herein by reference in their entirety. These appropriate pharmaceutical formulations are most often and most preferably made in sterile, isotonic and buffered form for stability and comfort. Pharmaceutical compositions for intranasal delivery may also include drops and sprays that are often formulated to mimic nasal secretions in various ways to ensure that normal ciliary action is maintained. As disclosed in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing co., Easton, PA (1990), the entire contents of which are incorporated herein by reference, and well known to those skilled in the art, suitable formulations are most often and most preferably isotonic, lightly buffered to maintain a pH of 5.5 to 6.5, most often and most preferably containing an antimicrobial preservative and a suitable Pharmaceutical stabilizer. Pharmaceutical formulations for intra-aural delivery include suspensions and ointments for intra-aural topical application. Typical solvents for otic formulations include glycerin and water.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described above, the compounds of the present invention may also be formulated as depot preparations. Such long acting formulations may be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (e.g. as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g. as a sparingly soluble salt.

For hydrophobic compounds, a suitable pharmaceutical carrier may be a co-solvent system comprising benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase. A commonly used co-solvent system is the VPD co-solvent system, which is 3% w/v benzyl alcohol, 8% w/v of the non-polar surfactant Polysorbate 80^TMAnd 65% w/v polyethylene glycol 300, and added to the solution of the specified volume composition with absolute ethanol. Naturally, the compositional proportions of the co-solvent system can be varied significantly without compromising solubility and toxicity characteristics. Moreover, the co-solvent composition may vary: for example, other low toxicity non-polar surfactants may be substituted for POLYSORBATE 80^TMThe use is carried out; the content of polyethylene glycol can vary in size; other biocompatible polymers may be substituted for polyethylene glycol, such as polyvinylpyrrolidone; other sugars or polysaccharides may be substituted for glucose.

Alternatively, other delivery systems for hydrophobic drug compounds may be used. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethyl sulfoxide can also be used, although usually at the expense of greater toxicity. In addition, these compounds can be delivered using a sustained release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained release materials have been identified and are well known to those skilled in the art. Sustained release capsules can release compounds over a period of weeks to over 100 days, depending on their chemical nature. Depending on the chemical nature and biological stability of the therapeutic agent, additional strategies may be used to stabilize the protein.

Drugs intended for intracellular administration may be administered using techniques well known to those of ordinary skill in the art. For example, these drugs may be encapsulated in liposomes. All molecules present in the aqueous solution are incorporated inside the water at the time of liposome formation. Because the liposome is fused to the cell membrane, the liposome components are isolated from the external microenvironment and efficiently delivered into the cytoplasm. The liposomes may be coated with a tissue specific antibody. The liposomes will be selectively targeted to and absorbed by the desired organ. Alternatively, small hydrophobic organic molecules may be administered directly intracellularly.

Additional therapeutic or diagnostic agents may be added to the pharmaceutical composition. Alternatively or additionally, the pharmaceutical composition may be combined with other compositions comprising other therapeutic or diagnostic agents.Method of administration

The compound or pharmaceutical composition may be administered to the patient by any suitable means. Non-limiting examples of methods of administration include (a) administration by oral route, including administration in capsules, tablets, granules, sprays, syrups, or other such forms; (b) by non-oral route, such as rectal, vaginal, intraurethral, intraocular, intranasal or intraaural administration, which includes administration in the form of aqueous suspensions, oily preparations and the like, or in the form of drops, sprays, suppositories, ointments and the like; (c) administration by injection, subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, intradermal injection, intraorbital injection, intravesicular injection, intraspinal injection, intrasternal injection, and the like, including infusion pump delivery; (d) local administration, e.g., by injection directly into the kidney or heart region, e.g., by depot implant administration; and (e) topical administration; when the skilled person considers it suitable to contact the compounds of the invention with living tissue, and other methods of administration.

Pharmaceutical compositions suitable for administration include compositions comprising an effective amount of the active ingredient to achieve the intended purpose. The therapeutically effective amount of a compound disclosed herein as required by dose will depend upon the route of administration, the type of animal being treated, including humans, and the physical characteristics of the particular animal being treated. The dosage can be specifically tailored to achieve the desired effect, but will depend on such factors as body weight, diet, concurrent medication and other factors that will be recognized by those skilled in the medical arts. More particularly, a therapeutically effective amount refers to an amount of a compound effective to prevent, alleviate or ameliorate symptoms of a disease or prolong the survival of a subject. Determining a therapeutically effective amount is within the ability of those skilled in the art, particularly in light of the teachings specifically disclosed herein.

Useful in vivo dosages and particular modes of administration will vary according to the age, weight and species of mammal being treated, the particular compound used and the particular use for which the compound is used, as will be apparent to those skilled in the art. One skilled in the art can use routine pharmacological methods to determine an effective dosage level, i.e., a dosage level required to achieve a desired result. Generally, human clinical use of the product begins with lower dosage levels, which are escalated until the desired effect is achieved. Alternatively, using established pharmacological methods, acceptable in vitro studies can be used to establish useful dosages and routes of administration for compositions identified by the methods of the invention.

In non-human animal studies, the use of effective products begins with higher dosage levels, which are gradually reduced until the desired effect is no longer obtained or the undesirable side effects disappear. The dosage may vary within wide limits depending on the desired effect and the therapeutic indication. Generally, the dose may be from about 10 micrograms/kg to 100mg/kg body weight, preferably from about 100 micrograms/kg to 10mg/kg body weight. Alternatively, the dose may be based and calculated on the patient surface area, as will be appreciated by those skilled in the art.

The exact formulation, route of administration and dosage of the pharmaceutical compositions of the present invention may be selected by the individual physician in accordance with the condition of the patient. (see, e.g., Fingl et al, 1975, "the pharmaceutical Basis of Therapeutics", the entire contents of which are incorporated herein by reference, especially Chapter 1, page 1). Generally, the dosage of the composition administered to the patient may range from about 0.5 to 1000mg/kg of patient body weight. The dosage may be administered in a single or a series of two or more administrations over one or more days, depending on the patient's needs. When a human dose of the compound has been determined for at least some conditions, the present invention will use the same dose, or a dose that is about 0.1% to 500%, more preferably about 25% to 250% of the determined human dose. ED that can be derived from animal toxicity studies and efficacy studies when there is no established human dose, as is the case for newly discovered pharmaceutical compounds₅₀Or ID₅₀Values, or other suitable values derived from in vitro or in vivo studies, infer suitable human dosages.

It should be noted that the attending physician knows how and when to terminate, interrupt or adjust administration due to toxicity or organ dysfunction. Conversely, if the clinical response is not sufficient (except for toxicity), the attending physician will also know to adjust the dosing to higher levels. The size of the dose administered during the treatment of the condition of interest will vary with the severity of the condition to be treated and the route of administration. For example, the severity of a condition can be assessed in part by standard prognostic evaluation methods. Moreover, the dosage and approximate frequency of administration will vary according to the age, weight and response of the individual patient. In veterinary medicine similar protocols to those discussed above may be used.

Although the exact dosage will be determined on a "drug-by-drug" basis, in most cases some generalizations of the dosage may be made. For adult human patients, for example, the daily dosage regimen may be 0.1mg/m of each active ingredient²To 2000mg/m²The daily oral dosage of body surface area is usually 1mg/m²To 500mg/m²Body surface area per day, e.g. 5mg/m²To 200mg/m²Body surface area was daily. In other embodiments, the intravenous, subcutaneous, or intramuscular dose of each active ingredient is 0.01mg/m²To 100mg/m²The body surface area is usually 0.1mg/m per day²To 60mg/m²Body surface area per day, for example, 1mg/m can be used²To 40mg/m²Body surface area daily dose. When administered as a pharmaceutically acceptable salt, the dosage can be calculated as the free base. In some embodiments, the composition is administered from 1 to 4 times daily. Alternatively, the compositions of the invention may be administered by continuous intravenous infusion, the dosage of each active ingredient preferably being up to 1000mg/m²Body surface area was daily. As will be appreciated by those skilled in the art, in certain instances, it may be desirable to administer the compounds disclosed herein in amounts that exceed or are well outside the preferred dosage ranges described above in order to effectively and aggressively treat a particularly malignant disease or infection. In some embodiments, the compound will be administered over a period of continuous treatment, for example, a week or more, or months or years.

The amount and interval of administration can be adjusted individually to provide plasma levels of the active moiety that are sufficient to maintain a therapeutic effect or Minimum Effective Concentration (MEC). The MEC for each compound will vary, but can be estimated from in vitro data. The dose required to obtain a MEC will depend on the individual characteristics and route of administration. In any case, HPLC assays or bioassays may be used to determine plasma concentrations.

Dosing intervals may also be determined using MEC values. The composition should be administered using a regimen that maintains plasma levels above MEC for 10-90% of the time, typically 30-90%, most typically 50-90%.

For topical administration or selective absorption, the effective local concentration of the drug may not be related to the plasma concentration.

The amount of the composition administered depends on the subject being treated, the weight of the subject, the severity of the affliction, the mode of administration and the judgment of the prescribing physician.

The potency and toxicity of the compounds disclosed herein can be assessed using known methods. For example, the toxicology of a particular compound or subgroup of compounds sharing certain chemical moieties can be determined by measuring in vitro toxicity to a cell line, e.g., a mammalian cell line, preferably a human cell line. The results of such studies can often predict toxicity to animals, such as mammals, or more particularly, humans. Alternatively, toxicity of a particular compound in an animal model can be determined using known methods, e.g., mouse, rat, rabbit, or monkey. The potency of a particular compound can be determined using several recognized methods, such as in vitro methods, animal models, or human clinical trials. There are recognized in vitro models for almost every condition, including but not limited to cancer, cardiovascular disease, and various immune dysfunction. Similarly, acceptable animal models can be used to determine the efficacy of chemicals used to treat these conditions. When selecting a model to determine efficacy, one of skill in the art can be guided by the state of the art to select an appropriate model, dosage, and route of administration, and dosage regimen. Of course, human clinical trials can also be used to determine the efficacy of compounds for humans.

If desired, the compositions of the present invention may be presented in a pack or dispenser device which may contain one or more unit doses containing the active ingredient. The package may for example comprise a metal or plastic foil, such as a blister pack (blister pack). The packaging or dispensing device may also be accompanied by instructions for administration. The packaging or dispensing device may also be accompanied by a notice associated with the container in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, wherein the notice is responsive to approval by the agency of the form of human or veterinary pharmaceuticals. Such a notice may be, for example, a label approved by the U.S. food and drug administration for a prescription drug, or an approved product insert (production insert). Compositions comprising the compounds of the present invention may also be prepared in a compatible pharmaceutical carrier and placed in an appropriate container for indication of the indication being treated.

Throughout the specification, any detailed description of a particular compound should be understood to include that compound and any (other) pharmaceutically acceptable salts thereof.

In the first step of BER, a battery of glycosylases recognizes an abnormal base such as N³mA and N⁷mG (O' Connor et al, "Isolation and construction of expression of a cDNA of a 3-methyaladine DNA glycosylase" EMBO J.9: 3337. times.3342, 1990; Samson et al, "Cloning and catalysis of a 3-methyaladine DNA glycosylase cDNA from human cells maps to chromosome 16" Proc. Natl.Acad. Sci. USA 88: 9127. times.9131, 1991), T: G (Neddermann et al, "functional expression of soluble human transcription in 11(IL-11) epitope and expression of viral IL-11. times.130. 7. times.7. purine of guanine and adenine, such as guanine 7. times.7. adenine and 7. times.7. for example: a (Vollberg et al "Isolation and characterization of the human uracil DNA glycosylation gene" Proc. Natl.Acad.Sci.USA 86: 8693 8697, 1989; Olsen et al "Molecular Cloning of human uracil-DNA glycosylation enzyme," a high bound DNA replication enzyme "EMBO J.8: 3121. 3125, 1989; Radiella et al" Cloning and characterization of hG 1; a human homolog of the G1 gene of Saccharomyces cerevisiae "Proc. Natl.Acad.Sci.Sci.USA 94.8010-1997, 1997; Rosentissue et al" Cloning and characterization of nucleic acid hybridization of Natl.7435. Natl.7434. USA). After the N-glycosidic bond is enzymatically or naturally hydrolyzed and the abnormal base is released, AP (purine-free/pyrimidine-free) endonuclease hydrolyzes the phosphodiester backbone 5' resulting in damage and dRpase (a DNA deoxyribose phosphodiesterase whose activity is associated with polymerase β) cleaves the residual dRp creating a nucleotide gap. The gap is filled by DNA polymerase beta and the nick is sealed by DNA ligase. This approach is called short patch BER. BER InterchangeablePathways include DNA synthesis to fill gaps of 2 to 13 nucleotides. This long patch repair requires Proliferating Cell Nuclear Antigen (PCNA) and PCNA-dependent DNA polymerase (Wilson "Mammalian base extension repair and DNA polymerase beta" MutationRes 407: 203-215, 1998).

Poly- (ADP-ribose) -poly (PARP) acts as a nick sensor for DNA strand breaks either by itself or by interacting with XRCC1, participating in BER. PARP binds to damaged DNA, resulting in automated ribosylation. The modified protein is then released and allows other proteins to enter and repair DNA strand breaks (Wilson "major base expression replication and DNA polymerase beta" Mutation Res.407: 203-215, 1998; Molinete et al, "Over production of the poly (ADP-rib) polymerase DNA-binding domain blocks amplification-induced DNA replication synthesis in major molecules ls" EMBO J.12: 2109-2117, 1993; Caldeott et al, "XRCCI polypeptide interactions with DNA polymerase beta and position poly (ADP-rib) molecules, and DNA ligand III a non molecular ' k-sensor ' in vitro ' acquisition 4394: 1996). Thus, PARP is involved in BER in both short and long patch repair after nick formation. BER appears to be the most effective in the alternative (long patch repair) pathway. Figure 6 shows that the combination of pemetrexed and MX increases the formation of fragmented PARP. The increase in DNA double strand breaks and apoptosis is independent of the Bcl-2 pathway.

Generally, the nomenclature used hereinafter and the laboratory procedures in cell culture, tissue culture, tumor biology and molecular genetics described below are those well known and commonly employed in the art. Standard techniques are used for cell culture methods, assay design and compound formulation and nomenclature. General chemical reactions and purification steps were performed according to the manufacturer's instructions. These techniques and procedures are generally accomplished in accordance with conventional procedures and various general references in the art (see generally Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., and Current Protocols in Molecular Biology (1996) John Wiley and sons, Inc., N.Y., incorporated herein by reference), which are incorporated herein by reference. All information contained therein is incorporated herein by reference.

Pemetrexed (2- [4- [2- (4-amino-2-oxo-3, 5, 7-triazabicyclo [ 4.3.0)]Non-3, 8, 10-trien-9-yl) ethyl]Benzoyl radical]Aminoglutaric acid) (ALIMTA)^TM；Eli Lilly&Co.) is an antimetabolite chemotherapeutic drug having the structure:

pemetrexed is an antifolate antineoplastic agent that acts by disrupting the folate-dependent mechanism required for cell replication. Pemetrexed inhibits Thymidylate Synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide transformylase (GARFT), enzymes involved in the de novo biosynthesis of thymidine and purine nucleotides. Pemetrexed is approved by the FDA for the treatment of non-small cell lung cancer and for the treatment of malignant pleural mesothelioma in combination with cisplatin (a platinum-based chemotherapeutic drug). The recommended dose for treating mesothelioma (in combination with cisplatin) or non-small cell lung cancer is about 500mg/m²Body surface area was administered daily as an intravenous infusion over 10 minutes on the first day of each 21-day cycle. For therapeutic combinations with methoxyamine, a typical dosage range for pemetrexed is usually 200mg/m²To 1,000mg/m²Body surface area per day, or 500mg/m²To 600mg/m²Body surface area per day; a typical dosage range for methoxyamine is 1 to 200mg/m²Body surface area per day, alternatively 6 to 120mg/m²Body surface area was daily. In one embodiment, pemetrexed is administered in the same manner as described above; however, intravenous infusion may be performed over 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, or more, and may be performed on day 1, plus one or more additional days of a given cycle, which may be 1 week, 2 weeks, 3 weeks, 1 month, or more.

Examples

Materials and methods

Chemicals and reagents. Methoxyamine (MX) was purchased from Sigma (st. MX was dissolved in sterile water (pH 7.0).

Western Blotting (Western Blotting) was used to detect PARP cleavage. Cell extracts were lysed by SDS-PAGE (12% polyacrylamide) for 1 hour at 150V in a Bio-Rad minigel apparatus. Proteins were transferred to PVDV membranes using a Bio-Rad micro transfer blotting cell (mini Trans-Blot cell) for 1 hour under 100V conditions. Blotted membranes were blocked with 5% milk powder in 15TBS buffer and probed with anti-PARP antibody C2-10(Trevigen, Gaithersburg, Md.) for 2 hours. After three 5 min washes with TBS-Tween20 (0.05%), the blots were incubated with a second antibody anti-mouse HRP-anti-IgG for 1 h (Amersham Life Science, Arlington Height Ill.). Antibody binding was visualized with ECL according to the manufacturer's instructions (Amersham Life Science, Arlington Heights, il.).

Tumors in nude mice. Tumor cells (5X 10)⁶) Injected into the flank of 6-8 week old female athymic HSD nude mice. Animals received 5 daily intraperitoneal injections of: saline, pemetrexed alone (150mg/kg), MX alone (4mg/kg), or a combination of pemetrexed with MX (150mg/kg) and MX (4mg/kg) in a 26.7: 1.0 ratio of pemetrexed to MX. Tumors were measured with calipers using the National Cancer Institute formula: l (mm) xI²(mm)/2, wherein L is the maximum diameter of the tumor and I is the minimum diameter of the tumor. When the tumor volume reaches about 100-³Tumor-bearing mice were randomly assigned for either the control group or the treatment group (6-9 mice/group).

Example 1

FIG. 8 shows that in the human NCI-H460 NSCLC cell line model, the A549 NSCLC cell line model, the HCT116 colon cancer cell line model, and the MDA-MB-468 breast cancer cell line model, all of the groups receiving Methoxyamine (MX) + pemetrexed exhibited stronger tumor growth delays than the group receiving pemetrexed alone. MX reversed the resistance of the NCI-H460 NSCLC cell line and the HCT116 colon cancer cell line to the effect of pemetrexed chemotherapy.

Example 2

To determine the effect of pemetrexed + Methoxyamine (MX) on the number of AP sites generated relative to pemetrexed alone, the AP sites formed by pemetrexed and blocked by MX were determined using aldehyde-reactive probe (ARP) reagent. ARP and MX have similar reactivity towards AP sites, and they react especially with aldehyde groups, which are a ring-opened form of AP sites. Such assays are described in Liu et al, (Molecular Cancer Therapeutics 2: 1061-1066, 2003) and operate essentially as described in Nakamura et al, (Cancer Res.58: 222-225, 1998). H460 cells were treated with pemetrexed (0, 100, 200 or 400 μm) for 24 hours. Then, DNA (15. mu.g) was extracted from the cells, and cultured with 1mM ARP at 37 ℃ for 10 minutes. The DNA was precipitated and washed with ethanol, then resuspended in TE buffer (10mM Tris-HCl, pH 7.2, 1mM EDTA) and denatured at 100 ℃ for 5 minutes. The DNA was then rapidly frozen on ice and mixed with an equal amount of ammonium acetate (2M). The single stranded DNA was then immobilized on a nitrocellulose membrane using a vacuum filtration device. The membrane was incubated with streptavidin-conjugated horseradish peroxidase for 30 minutes at room temperature and rinsed with wash buffer (20mM Tris-HCl, 1mM EDTA, 0.26M NaCl, 1% Tween-20). The ARP-AP site was visualized with ECL reagent (Amersham, Piscataway, N.J.). The results are shown in 4A. 100. Pemetrexed at doses of 200 and 400 μ M induced the formation of AP sites, with the degree of AP site induction being proportional to the pemetrexed dose (fig. 4A). The addition of MX to pemetrexed significantly reduced the number of detectable AP sites compared to pemetrexed alone. The combined effect of 200 μ M pemetrexed and 6 mmx was observed at 24 hours, 48 hours and 72 hours. The results are shown in fig. 4B. Over time, both the pemetrexed alone group and the pemetrexed in combination with MX reduced the number of detectable AP sites.

In summary, pemetrexed induces the formation of AP sites, and the combination of pemetrexed and 100 μ M MX reduces detectable AP sites to control levels, which is not the reason for the absence of AP sites, but rather the AP sites are occupied by MX, rendering them unavailable for APR use. The occupancy of the AP site is time-dependent, indicating that sustained MX levels are necessary for maximal effect.

Example 3

DNA strand break assays were used to determine the ability of pemetrexed and Methoxyamine (MX) to increase tumor cell death mediated by apoptosis and DNA strand break. Comet assay assays are described in Liu et al (supra) based on the ability of denatured, fragmented DNA fragments to migrate out of cells under the influence of an electric field. Undamaged DNA moves slower when current is passed through it and remains within the nuclear region. Intracellular DNA damage was assessed based on an assessment of the shape of the DNA "comet" tail and migration distance (Helma et al, Mutat. Res.466: 9-15, 2000). Cells were harvested after 4 hours of exposure to 200 μ M pemetrexed, 6mM MX, or 200 μ M pemetrexed +6mM MX, respectively, and washed with PBS. A suspension of H460 cells (1X 10) was mixed at 42 ℃ in a ratio of 1: 10(v/v)⁵/ml cold PBS) was mixed with 1% low gel temperature agarose and 75 μ l was quickly transferred with a pipette onto a comet slide (CometSlide) (Trevigen, inc. After the low gel temperature agarose had been placed, the slides were submerged in a pre-cooled lysis buffer (10mM Tris-HCl, pH 10.5-11.5, 2.5M NaCl, 100mM EDTA, containing 1% Triton X-100 added prior to use) for 1 hour at 4 ℃. After lysis, the slides were washed with distilled water, vertically arranged in an electrophoresis tank, and immersed in an alkaline buffer (50mM NaOH, pH 12-12.5, 1mM EDTA) for 30 minutes. The slides were then electrophoresed in alkaline (pH > 13, 300mM NaOH, 1mM EDTA) and neutral solution (1XTBE) at 18V (0.6V/cm), 250mA for 25 minutes. Alkaline electrophoresis detects single-and double-stranded DNA breaks from AP sites and other alkaline labile DNA adducts, while neutral electrophoresis mainly detects double-stranded DNA breaks. The slides were removed, washed with neutralization buffer (0.5M Tris-HCl, pH 7.5) for 10 minutes, then PBS, after which they were air dried overnight at room temperature. Silver staining kit (Tre) was used according to the manufacturer's instructionsvigen) stained the DNA. Comets were observed using an Olympus microscope. Images were captured with a digital camera and analyzed using NIH image software.

Comet images are shown in FIGS. 1A (alkaline assay) and 1B (neutral assay). After treatment with pemetrexed and MX, different comets were observed with a tail length of about 4 times the length treated with MX alone and about 2 times the length treated with pemetrexed alone (fig. 1C-D).

Results from xenografting validation studies, AP site assays and comet assays show that MX acts as a structural modulator of the AP site, enhancing the therapeutic effect of the antimetabolite pemetrexed by reversing resistance to chemotherapy, thereby producing a synergistic effect.

Example 4

The effect of AP or MX-AP sites on topoisomerase II-mediated DNA cleavage was analyzed. The position-specific apurinic site is introduced by the following steps: replacement of a single nucleoside with deoxyuridine at the topoisomerase II cleavage site followed by removal of the uracil base with uracil-DNA glycosylase yielded an AP site that was further cultured with MX to create a MX-AP site (fig. 5A).

First, it was determined whether APE had a different effect between the regular AP site and the MX-AP site, which are located at positions specific for topoisomerase II cleavage. The results show that APE cleaves the regular AP site but not the MX-bound AP site (FIG. 5B), however, both the AP and MX-AP sites can be cleaved by topoisomerase II, indicating that the MX-AP site stimulates topoisomerase II-mediated DNA cleavage.

Example 5

Oral and intravenous bioavailability of MX was studied by administration to Sprague Dawley rats (Non-GLP) by rapid perfusion (singlebolus). A study described below was performed to assess the bioavailability of Methoxyamine (MX) at safe dose levels by comparing pharmacokinetic parameters of MX following rapid perfusion oral and intravenous administration.

Test animalSprague Dawley rats 7-10 weeks old weighing 250-350g in 30 males and 30 females were used during the study.

Formulations and concentrations for administrationA single dose solution was prepared on the day of dosing, the test article was adjusted to 98% purity, and 816.77mg of MX was dissolved in 5% glucose in a 200-mL volumetric flask in order to obtain a concentration of 4.00mg/mL "active" MX. The prepared solution was divided into two amber bottles on average for oral administration or intravenous administration. Aliquots were taken for analysis at the time of preparation and after administration, transported on dry ice, and stored at temperatures < -70 ℃.

DosingAll animals were weighed on the day of dosing. The dose for each animal was based on this body weight. A constant dose volume of 5mL/kg was used. Intravenous doses were injected into the tail vein by rapid infusion using a 3-mL syringe connected to a 26Gx1 "needle. The intravenous dose was administered at a rate of about 2 mL/min. Oral doses were administered in a rapid infusion manner using an 18Gx2 "delivery needle connected to a 3-mL syringe.

Blood sample collectionBlood samples were taken at 5, 15, 30 minutes and 1,2, 4, 6, 8, 12 and 24 hours post-dose, with blood samples taken at two time points per animal. Blood samples were collected via the jugular vein at earlier time points and via the ventral vein at sacrifice at later time points. Blood transfer from draw syringe (drawing syring) to containing K₃-EDTA as anticoagulant in a 2mL blood collection tube and inverted to mix them. In use of CO₂Abdominal venous blood collection was performed immediately after euthanasia.

Plasma sample preparation and storage conditionsBlood tubes were placed on wet ice (wet ice) prior to centrifugation to prepare plasma. The whole blood sample was centrifuged at 3000rpm for 10 minutes at 4 ℃. The plasma was aspirated into tubes, initially placed on dry ice, and subsequently stored at a temperature of < -70 ℃.

Mass Spectrum (MS)Mass spectrometric detection was performed by electrospray ionization with positive turbo spray (positive turbo spray) according to the specifications set forth below. MS instrument: applied Biosystems 3000HPLC instrument: agilent 1100 Series Binary Pump autosampler: LEAP Technologies CTC-PAL electrospray ionization (ESI) conditions: temperature: 500 ℃ nebulizer gas: nitrogen CAD gas: and (3) nitrogen DP: 40 curtains of gas (CUR): 10 collision gas: 6 ion spray voltage (IS): 5000 Exit Potential (EP): 10 NEB: 12, monitor: an analyte: 207.0/149.3 and 207.0/178.4 atomic weight IS (nitrobenzyl coumarin): 354.2/296.0 and 354.2/163.1 atomic weight HPLC conditions (performed on Agilent 1100): mobile phase A: 0.1% aqueous formic acid mobile phase B: 0.1% formic acid in acetonitrile solution column: thermo Aquasil C18, 50x3mm guard column: thermo Aquasil C18, 10x4mm flow rate: 1.0mL/min injection volume: gradient of 50 μ L:time (% by min) B0.0 0％2.0 10％2.2 90％4.5 90％4.6 0％5.6 0％

Plasma sample LC-MS/MS analysisIndividual plasma samples were thawed at ambient temperature and 250 μ L aliquots were taken for analysis if dilution was not required. Samples taken from intravenously dosed animals at 5 minutes to 1 hour and samples taken from orally dosed animals at 15 minutes to 8 hours required dilution 5 to 40 fold to fall within the linear range of the method (1 to 1,000 ng/mL). For samples that need to be diluted, appropriate amounts were taken and mixed with blank rat plasma containing the same anticoagulant to achieve a total volume of 250 μ L. The quantification was corrected for dilution factor. Plasma for LC-MS/MS analysis was prepared as follows: ● mixing the blood plasmaVortex for 30 seconds and centrifuge at 14,000rpm for 10 minutes to allow for the precipitation of interfering particles. ● A100. mu.L aliquot was taken from the supernatant and placed in a 1.5mL microcentrifuge tube. ● to the 100. mu.L plasma aliquot was added 310. mu.L of water: formic acid (2: 1), 30. mu.L of an aqueous solution of warfarin (IS) (10. mu.g/mL), and 100. mu.L of a 2: 1 aqueous: formic acid solution of diethylaminobenzaldehyde (10mg/mL) and mixed well. ● the mixture was then incubated in a water bath at 80 ℃ for 2 hours. After incubation, the supernatant was transferred to HPLC bottles for LC-MS/MS quantification.

Methoxyamine (MX) pharmacokinetic and bioavailability analysisPharmacokinetic analysis Methoxyamine (MX) Pharmacokinetic (PK) profiles and oral bioavailability were determined in male and female Sprague Dawley rats following intravenous and oral administration of MX by bolus infusion at a dose of 20mg/kg body weight. There were 30 rats (15 males and 15 females) per route of administration for pharmacokinetic analysis. To obtain MX pharmacokinetic profiles, plasma samples were taken at the time of pre-dose nominal sampling (nominal sampling), and at 5, 15, 30 minutes and 1,2, 4, 6, 8, 12 and 24 hours after dosing. To maintain normal health, each rat was bled a maximum of two times at predetermined time points to generate plasma. Representative MX concentrations were obtained by averaging values from three rats of the same sex and route of administration at each time point. For the average dose, the average actual sampling time was correspondingly from three rats of the same sex and administration route at each nominal time point. Mean plasma MX concentration, mean dose, and mean actual sampling time were used for pharmacokinetic analysis for each route of administration.

Using Microsoft Excel 2000-SR1^TMMean plasma MX concentrations versus mean sampling time curves were created for each sex and route of administration (fig. 2-3). For purposes of pharmacokinetic modeling, plasma MX concentrations reported below the quantifiable range (BQL) or undetectable, if any, were considered to be 0.00 ng/mL.

Use of none by WinNonlin 5.1(Pharsight Corporation, Mountain View, Calif.)The compartmental model was subjected to pharmacokinetic parameter analysis. Pharmacokinetic parameters include maximum plasma concentration (C)_max) Time of maximum plasma concentration (T)_max) Clearance half-life (t)_1/2) Area under the plasma concentration versus time curve from time zero to last measurable plasma concentration (AUC)_last) And the area under the plasma concentration versus time curve extending from time zero to infinity (AUC)_0-∞). For comparison, AUC_0-∞Normalized to the 5mg nominal total MX dose (AUC)_0-∞5). Pharmacokinetic parameters are abbreviated in the manner set forth previously.

Bioavailability assayUsing Microsoft Excel 2000-SR1^TMOral and intravenous Methoxyamine (MX) AUC using the following equation_0-∞Absolute oral bioavailability (MX ═ TRC102) was determined by the ratio of (MX normalized to total dose 5 mg):the actual MX-dose levels in the intravenous group were 20.1mg/kg and 20.1mg/kg for male and female rats, respectively, and 19.9mg/kg and 20.0mg/kg for male and female rats, respectively, in the oral group.

Pharmacokinetics and bioavailabilityThe Methoxyamine (MX) pharmacokinetic parameters of male and female Sprague Dawley rats after intravenous and oral fast perfusion administration at a dose of 20mg/kg body weight are listed in Table 1. Table 1 plasma MX pharmacokinetic parameters of male and female Sprague Dawley rats after intravenous and oral bolus MX administration at a dose of 20mg/kg body weight.^aAUC_0-∞5(ng/mL × hr) is determined by comparing the AUC_0-∞(ng/mL hr) normalized to a total dose of 5mg MX.

For both intravenous and oral administration to male rats, there was a quantifiable concentration of MX in plasma over the entire 24-hour sampling period. Visual inspection of male veinThe mean plasma MX concentration versus mean time curve shows a rapid distribution phase, which is completed approximately 2 hours after dosing. Intravenous route C_max15,510ng/mL and was generated immediately upon completion of bolus administration. Such as AUC_lastAnd AUC_0-∞Systemic MX exposure was shown to be 12,518ng/mL hr and 12,706ng/mL hr, respectively. Intravenous AUC adjusted to a nominal total dose of 5mg_0-∞(AUC_0-∞5) 11,284ng/mL × hr.

For male rats on the oral route of administration, MX is rapidly absorbed and T_maxIt was 1.0 hour. 2,205ng/mL of C_maxIs considered to be lower than the intravenous route. Such as AUC orally_lastAnd AUC_0-∞Systemic MX exposure was shown to be 13,596ng/mL hr and 13,811ng/mL hr, respectively. Adjusted to an oral AUC of 5mg for a nominal total dose_0-∞(AUC_0-∞5) 12,420ng/mL × hr. The elimination half-life is short, similarly between the two routes of administration (intravenous: 5.2hr, oral: 4.2 hr). The calculated absolute bioavailability was 110% for male Sprague Dawley rats dosed orally.

For both intravenous and oral dosing female rats, there was a quantifiable MX plasma concentration over the entire 24 hour sampling period. Visual inspection of the female venous mean plasma MX concentration versus mean time curve, similar to male rats, showed a rapid phase of distribution, which occurred approximately 2 hours after dosing. Intravenous route C_max10,965ng/mL, appeared immediately upon completion of bolus administration. Such as AUC_lastAnd AUC_0-∞Systemic MX exposure was shown to be 12,971ng/mL hr and 13,142ng/mL hr, respectively. Intravenous AUC adjusted to a nominal total dose of 5mg_0-∞(AUC_0-∞5) 13,892 ng/mL × hr.

For female rats on the oral route of administration, MX absorption is rapid and T_maxIt was 0.5 hour. 2959ng/mL of C_maxSignificantly lower than the intravenous route, which is also similar to male rats on the oral route of administration. Such as AUC orally_lastAnd AUC_0-∞Systemic MX exposure was shown to be 11,643ng/mL hr and 12,029ng/mL hr, respectively.Adjusted to an oral AUC of 5mg for a nominal total dose_0-∞(AUC_0-∞5) 13,047ng/mL × hr. The elimination half-life was short, similarly between the two routes of administration (intravenous: 4.6hr, oral: 5.7hr), and comparable to male rats. The absolute bioavailability calculated for female Sprague Dawley rats was 94%.

Rats dosed either intravenously or orally at 20mg/kg Body Weight (BW) showed no signs of clinical toxicity.

MX administered orally by bolus infusion at a dose of 20mg/kg BW was rapidly initiated (T) in male and female Sprague Dawley rats_max0.5-1.0hr) and complete absorption, absolute systemic bioavailability of about 100%. Despite oral MX C_maxIs significantly lower than vein C_maxBut as AUC_lastAnd AUC_0-∞Systemic MX exposure is shown to be similar between the two routes of administration. Furthermore, serum levels exceeded target C associated with activity in a human cancer mouse model at various time points following oral administration_maxA value (50ng/mL) that would allow for a once-a-day or twice-a-day dosing regimen.

These data are significant in that they indicate that methoxyamine is fully orally bioavailable and has a half-life of 4-6 hours, which allows once-a-day or twice-a-day dosing to achieve the lowest effective concentration. These findings are unexpected. Most anticancer drugs are not bioavailable in sufficient quantities to allow oral administration. It should be noted that attempts to administer other particular anticancer drugs orally have resulted in much lower bioavailability than herein. See, e.g., bleomycin, carboplatin, cisplatin, oxaliplatin, paclitaxel, raltitrexed (an antifolate), topotecan, vinblastine, vincristine, vinorelbine, all of which have a bioavailability of less than 50% (Chu E and devta vt. physics' cancer chemistry Drug manual 2002. Boston: Jones and Bartlett Publishers, 2002), and second, demonstrate a half-life that is longer than the expected half-life of small molecules with a molecular weight < 100 daltons, which readily react with aldehydes that may be present in plasma, and a longer than expected plasma half-life allows for sustained Drug levels (above the minimum effective concentration) on a once-a-day or twice-a-day oral dosing regimen. Complete bioavailability and a half-life of 4 to 6 hours may allow for convenient once-a-day or twice-a-day dosing methods for cancer patients to be administered orally.

It will be apparent to those skilled in the art from this disclosure that various modifications of the above-described methods and compositions can be made without departing from the spirit and scope of the invention. Accordingly, the present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The present embodiments and examples are, therefore, to be considered in all respects as illustrative and not restrictive, and all changes coming within the meaning and equivalency range of the appended claims are therefore intended to be embraced therein.

It is, therefore, to be understood that no equivalents of the features shown and described or portions thereof are intended to be excluded in the use of such terms and expressions, but it is recognized that various modifications are possible within the scope of the invention claimed. It will also be understood that various narrower species and generic groups falling within the generic disclosure also form part of the invention. This includes the generic recitation of the invention with a proviso or negative limitation removing any subject matter from the generic disclosure, regardless of whether or not the excised material is specifically recited herein.

All patents, publications, scientific articles, websites and other documents and materials cited or mentioned herein are indicative of the level of skill of those skilled in the art to which this invention pertains and, therefore, each cited document and material is hereby incorporated by reference to the same extent as if it had been individually incorporated by reference or set forth herein in its entirety.

Claims

1. A method, comprising:

providing i) a subject diagnosed with cancer, ii) a first formulation comprising an antifolate anticancer agent and iii) a second formulation comprising methoxyamine;

administering the first formulation to the subject; and

administering to the subject the second formulation, wherein methoxyamine is administered in an amount sufficient to enhance or increase the effect of the antifolate anticancer agent.

2. The method of claim 1, wherein the second formulation is administered orally.

3. A method, comprising:

providing i) a patient diagnosed with cancer, wherein the cancer is at least partially resistant to treatment with pemetrexed alone, ii) a first formulation comprising pemetrexed; and iii) a second formulation comprising methoxyamine;

administering the first formulation to the patient; and

administering to the patient the second formulation, wherein methoxyamine is administered in an amount sufficient to enhance the activity of the pemetrexed and overcome the drug resistance.

4. The method of any one of claims 1-3, wherein said methoxyamine and said antifolate anticancer agent are administered as a formulation.

5. The method of any one of claims 1-3, wherein said methoxyamine and said antifolate anticancer agent are administered sequentially, in any order.

6. The method of any one of claims 1-3, wherein said methoxyamine is administered orally and said antifolate anticancer agent is administered orally or intravenously.

7. The method of any one of claims 1-3, wherein the amount of methoxyamine is an amount sufficient to sensitize cancer cells without causing undue sensitization of normal cells.

8. The method of any one of claims 1-3, wherein said methoxyamine and said antifolate anticancer agent are administered to obtain a synergistic effect.

9. The method of any one of claims 1-3, wherein said antifolate anticancer agent is administered orally or intravenously, and said methoxyamine is administered orally in an amount sufficient to enhance the activity of said antifolate anticancer agent no more than twice daily.

10. The method of any one of claims 1-3, wherein a patient is selected having a cancer that is at least partially resistant to treatment with an antifolate anticancer drug alone, and wherein said second formulation comprising methoxyamine is administered in an amount effective to enhance the activity of said antifolate anticancer drug and overcome said resistance.

11. The method of any one of claims 1-3, wherein the ratio of methoxyamine to the antifolate anticancer agent is from 1: 5 to 1: 500.

12. The method of claim 11, wherein the ratio of said methoxyamine to said antifolate anticancer agent is from 1: 15 to 1: 40.

13. The method of claim 11, wherein the ratio of said methoxyamine to said antifolate anticancer agent is from about 1: 20 to 1: 30.

14. The method of any one of claims 1-13, wherein the cancer is selected from the group consisting of: carcinomas, melanomas, sarcomas, lymphomas, leukemias, astrocytomas, gliomas, malignant melanomas, chronic lymphocytic leukemia, lung cancer, colorectal cancer, ovarian cancer, pancreatic cancer, renal cancer, endometrial cancer, gastric cancer, liver cancer, head and neck cancer, and breast cancer.

15. The method of any one of claims 1-14, wherein the antifolate anticancer agent is pemetrexed.

16. In a method of treating cancer in a patient diagnosed with cancer, comprising administering to the patient an antifolate anticancer agent,

the improvement of administering methoxyamine to a patient in an amount sufficient to enhance the toxicity of said antifolate anticancer agent.

17. The improved method of claim 16, wherein said methoxyamine and said antifolate anticancer agent are administered as a formulation.

18. The improved method of claim 16, wherein said methoxyamine and said antifolate anticancer agent are administered sequentially, in any order.

19. The improved method of claim 16, wherein said methoxyamine is administered orally and said antifolate anticancer agent is administered orally or intravenously.

20. The improved method of claim 16, wherein said amount of methoxyamine is an amount sufficient to sensitize cancer cells without causing undue sensitization of normal cells.

21. The improved method of claim 16, wherein said methoxyamine and said antifolate anticancer agent are administered to achieve a synergistic effect.

22. The improved method of claim 16, wherein said antifolate anticancer agent is administered orally or intravenously, and methoxyamine is administered orally once or twice daily in an amount sufficient to enhance the activity of said antifolate anticancer agent.

23. The improved method of claim 16, wherein a patient having a cancer that is at least partially resistant to treatment with an antifolate anticancer drug alone is selected, and wherein said second agent comprising methoxyamine is administered in an amount effective to enhance the activity of said antifolate anticancer drug and overcome said resistance.

24. The improved method of claim 23, wherein the ratio of said methoxyamine to said antifolate anticancer agent is from 1: 5 to 1: 500.

25. The improved method of claim 24, wherein the ratio of said methoxyamine to said antifolate anticancer agent is from 1: 15 to 1: 40.

26. The improved method of claim 25, wherein the ratio of said methoxyamine to said antifolate anticancer agent is from about 1: 20 to 1: 30.

27. The improved method of any one of claims 16-26, wherein the cancer is selected from the group consisting of: carcinomas, melanomas, sarcomas, lymphomas, leukemias, astrocytomas, gliomas, malignant melanomas, chronic lymphocytic leukemia, lung cancer, colorectal cancer, ovarian cancer, pancreatic cancer, renal cancer, endometrial cancer, gastric cancer, liver cancer, head and neck cancer, and breast cancer.

28. The improved method of any one of claims 16-27, wherein said antifolate anticancer agent is pemetrexed.

29. An anti-cancer formulation comprising a dosage form comprising pemetrexed and a dosage form comprising a synergistic amount of methoxyamine.

30. A method of using the formulation of claim 29, comprising administering the formulation according to the method of any one of claims 1-28.

31. Use of methoxyamine for the treatment of cancer in a patient with an antifolate anticancer agent, comprising the use of methoxyamine in an amount sufficient to potentiate the toxicity of said antifolate anticancer agent in said patient.