RU2423114C2 - Combinations of antifolate agent in cancer treatment - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed is application of first composition, containing pemetrexede and second composition, containing methoxyamine, for treatment of patient with diagnosed cancer, selected from lung cancer, colorectal cancer, ovary cancer, pancreatic cancer, kidney cancer, endometrial cancer, stomach cancer, lover cancer and breast cancer.

EFFECT: methoxyamine enhanced, increased or potentiated chemotherapeutic effect of antifolate anti-cancer agent; also demonstrated is unexpected reversing resistance to pemetrexede.

16 cl, 8 dwg,1 tbl

Description

RELATED APPLICATIONS

This application claims priority to provisional application for US patent number 60/877836, filed December 29, 2006 Theuer et al. titled "ANTIMETABOLITE AGENT COMBINATIONS IN THE TREATMENT OF CANCER", US Provisional Patent Application No. 60/883266 filed January 3, 2007 Theuer et al. titled "ANTIMETABOLITE AGENT COMBINATIONS IN THE TREATMENT OF CANCER" and provisional patent application US number 60/883959, filed January 8, 2007 Theuer et al. entitled "ANTIMETABOLITE AGENT COMBINATIONS IN THE TREATMENT OF CANCER", all of which are incorporated by reference into this description of the invention in their entirety, including any graphic materials, fully permitted by law.

FIELD OF THE INVENTION

The present invention generally relates to compounds having various uses, including those in research, diagnosis, and therapy. More specifically, this specification describes and proposes compositions comprising methoxyamine and an antimetabolic anticancer agent, and methods for treating certain types of cancer by administering such compositions.

BACKGROUND OF THE INVENTION

Cancer is a global problem. For this reason, it is of vital interest to find new compositions and methods for treating cancer. Cancer treatment is divided into three main categories: chemotherapy, radiation therapy and surgery. Typically, therapies are combined, since a combination of therapies often increases the likelihood that the cancer will be eradicated compared to treatment strategies that use a single therapy. Usually, surgical removal of large tumor masses is followed by chemotherapy and / or radiation therapy.

Chemotherapeutic agents can act in different ways. For example, chemotherapeutic agents can act by interfering with the sequence of the cell cycle or by generating DNA strand breaks. If a cancer cell is not able to overcome the blockage of the cell cycle or damage to the cell caused by a therapeutic compound, then often the cell dies due to apoptotic mechanisms. The use of a single chemotherapeutic agent in the treatment of cancer in the presence or absence of surgery or radiation has several disadvantages. Typically, cancer cells develop resistance to a chemotherapeutic agent. Such resistance leads either to the need for higher dosages of the drug and / or to the resumption of the spread of cancer. Chemotherapeutic agents may be toxic to the patient. Therefore, there is a practice-based upper bound on the amount that a patient can receive. However, if a second agent can be developed to suppress the direction causing resistance, cancer cells may become sensitive to the actions of the chemotherapeutic agent.

The creation of a drug to overcome resistance to chemotherapeutic treatment of cancer should be approached with the goal of 1) finding a combination that reverses resistance, and not only enhancing the activity of a chemotherapeutic drug in relation to the effect on the tumor, and 2) finding a second drug that does not enhance toxic effects first chemotherapeutic agent. These conditions require a large amount of empirical testing of agents that are known to have anti-cancer properties, with agents that may either have anti-cancer properties or may supplement the first agent in another way. Unfortunately, such approaches are largely unsuccessful for combinations of many anti-cancer agents.

Therefore, there is a lack of therapies that reverse chemotherapy resistance to treat cancer.

SUMMARY OF THE INVENTION

The inventions described and claimed in this specification have many features and embodiments, including, but not limited to, those set forth or described, or referred to in this summary of the invention. The inventions described and claimed in this description of the invention are not limited by the features or embodiments specified in this summary of the invention, which are included only for illustration and not limitation.

These and other aspects and embodiments of the inventions described and claimed in this description of the invention will be apparent from and throughout the application and claims, all of which should be considered part of their written description.

Disclosed herein are compositions and methods useful in treating certain types of cancer. Part of this application is based on a previously unknown recognition that some molecules that target abasic damage or AR (apuric / apyrimidine) sites in DNA improve, increase or potentiate the effectiveness of antimetabolic anticancer agents. In another embodiment, a base removal pathway inhibitor such as methoxyamine is combined with an antimetabolic anticancer agent. An antimetabolic anticancer agent is a chemotherapeutic agent with a structure similar to the structure of a substance (metabolite) required for normal biochemical reactions, but differing enough to interfere with normal cell functions, including cell division. Antifolates are the preferred class of antimetabolic agents. An anti-folate anticancer agent is a chemotherapeutic agent with a structure similar to that of folic acid, but different enough to block folic acid activity and disrupt folate-dependent mechanisms necessary for cell replication. Such anti-folate anticancer agents include pemetrexed, capecitabine, edatrexate, methotrexate, lomerexol, nolatrexed, altitrexide, PT523 and trimerexate. The use of any anti-folate anticancer agent in combination with a BER inhibitor (excision repair by removing base) is contemplated. In one aspect, the method takes 1) a subject who is diagnosed with cancer, 2) a first composition containing an anti-folate anticancer agent, and 3) a second composition containing methoxyamine; administering said first composition to said subject; and administering said second composition to said subject, wherein methoxyamine is administered in an amount sufficient to improve or enhance the action of the anti-folate anticancer agent. The second composition can be administered orally. In another aspect, the method takes 1) a patient who has been diagnosed with cancer, said cancer being at least partially resistant to treatment with pemetrexed alone, 2) a first pemetrexed composition; and 3) a second composition containing methoxyamine; administering said first composition to said patient; and administering said second composition to said patient, wherein methoxyamine is administered in an amount sufficient to potentiate the activity of said pemetrexed and overcome said resistance. In these methods, methoxyamine and an anti-folate anticancer agent can be administered as a composition. In addition, methoxyamine and an anti-folate anticancer agent can be administered sequentially, in any order. Also, methoxyamine can be administered orally, and an anti-folate anticancer agent can be administered either orally or intravenously. Also, the amount of said methoxyamine may be an amount sufficient to sensitize cancer cells without over-sensitizing normal cells. Also, methoxyamine and an anti-folate anticancer agent can be administered to achieve a synergistic effect. Also, an anti-folate anticancer agent can be administered orally or intravenously, and said methoxyamine can be administered orally, not more than twice a day, in an amount sufficient to potentiate the activity of said anti-folate anticancer agent. A patient may also be selected having cancer that is at least partially resistant to treatment with a single anti-folate anti-cancer agent, and wherein said second methoxyamine-containing composition is administered in an amount effective to potentiate the activity of said anti-folate anti-cancer agent and overcome said resistance. In addition, the ratio of said methoxyamine to said antifolate anticancer agent may be from 1: 5 to 1: 500, more preferably from 1:15 to 1:40, and even more preferably from about 1:20 to about 1:30. In addition, the cancer can be selected from the group consisting of carcinomas, melanomas, sarcomas, lymphomas, leukemias, astrocytes, gliomas, malignant melanomas, chronic lymphocytic leukemia, lung cancers, colorectal cancers, ovarian cancers, pancreatic cancers, kidney cancers, endometrial cancers, stomach cancers, liver cancers, head and neck cancers, and breast cancers. In preferred embodiments, the antifolate anticancer agent is pemetrexed.

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

In one aspect, the present invention is based on a previously unknown recognition that certain molecules, such as methoxyamine, which target AP sites, are completely orally bioavailable and maintain minimum effective concentrations once or twice daily by oral administration. Anticancer agents are usually administered as an intravenous bolus, as they are rarely well absorbed from the gastrointestinal tract. Intravenous dosing has disadvantages. Firstly, intravenous injection during chemotherapy requires treatment in a doctor’s office or in a hospital. Secondly, intravenous therapy is usually prescribed as a bolus, which leads to a very high, but short-term exposure to the drug. Some anticancer agents may be most active after prolonged exposure, which can be achieved through repeated oral doses. This is especially true for agents that inhibit chemotherapeutic drug resistance mechanisms, where prolonged inhibition of resistance pathways may be necessary for the desired therapeutic effect. Prolonged exposure of the drug can be carried out using continuous intravenous administration. However, the administration of anticancer agents in the form of continuous infusions requires a sophisticated apparatus for drug infusion and intravenous catheterization. Oral administration eliminates the need for continuous intravenous infusion and is the route of administration preferred by patients. However, as far as the authors of the invention are aware, BEP inhibitors that reverse chemotherapy resistance and have almost complete oral bioavailability, as proposed in this description of the invention, have not been created to date.

Pemetrexed is a multi-purpose antifolate that acts in a manner whose mechanism is different from 5-FU (5-fluorouracil) and another earlier generation of antimetabolites. Pemetrexed is unique in that it is an anti-folate analog of pyrrolopyrimidine, which is intracellularly metabolized to higher polyglutamate forms using folyl-polyglutamate synthetase (FPGS). The pentaglutamate form is the predominant intracellular variety, and pemetrexed polyglutamates are about 60 times more active than the parent monoglutamate compound; pemetrexed polyglutamates are also continuously retained by the cell. Consequently, the pharmacological effects of pemetrexed persist for many days after intravenous administration of a bolus.

Pemetrexed inhibits thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyl transferase (GARFT), all folate-dependent enzymes involved in the de novo biosynthesis of thymidine and purine nucleotides. In contrast, 5-FU and other earlier generations of antimetabolites mainly inhibit only TS. The exact mechanism by which pemetrexed causes cell death has not yet been established, but involves more than just TS inhibition. Therefore, while TS activity was the best prognostic for 5FU sensitivity in a heterogeneous non-selective panel of a human colon cancer cell line, determinants of multiple sensitivity, including FPGS activity and TS enzymatic kinetics (van Triest B, Pinedo HM, were important for pemetrexed) , van Hensbergen Y. Thymidylate synthase level as the main predictor parameter for sensitivity to 5-FU, but not for Folate-based Thymidylate Synthase Inhibitors, in 13 Nonselected Colon Cancer Cell Lines. Clin. Cancer. Res. 1999; 5: 643- 54). An additional study confirmed that the sensitivity of gastrointestinal cell lines to pemetrexed cannot be predicted by TS expression (Kim JH, Lee KW, Jung Y et al. Cytotoxic effects of pemetrexed in gastric cancer cells. Cancer Sci. 2005; 96: 365 -71).

The unique pharmacological activity of pemetrexed manifested itself in an in vitro study of activity on many cancer cell lines compared to 5-FU. In a series of 13 colorectal cancer cell lines, for example, pemetrexed was 18 to 627 times more potent than 5-FU (van Triest et al, 1999). This unique pharmacology and the fact that pemetrexed is supposed to have a number of mechanisms of action that are not fully understood make it difficult to understand how effective it can be when combined with other specific anti-cancer agents for the treatment of specific types of cancer.

One aspect of the present invention is the discovery of an unexpected improvement in the treatment of cancer by the combined administration of methoxyamine with an antifolate compound. Thus, one embodiment described herein is directed to methods in which 1) a patient is diagnosed with a cancer, 2) a first composition containing an anti-folate anticancer agent, and 3) a second composition containing methoxyamine; administering the first composition to a patient; and administering a second composition to said patient, wherein methoxyamine may be administered in an amount sufficient to enhance or enhance the action (e.g., potentiation of activity) of the antifolate anticancer agent. Any anti-folate anticancer agent may be used provided that 5-FU is specifically excluded in specific embodiments of the method. In a typical embodiment, the anticancer agent may be selected from the group consisting of pemetrexed, edatrexate, methotrexate, lometrexol, nolatrexed, ralitrexed, PT523, trimethrexate, aminopterin, 5,10-didyazate tetrahydrofolate, (DDATTrexF, pRITTrextF, ) -2- [5 - [(1,2-dihydro-3-methyl-1-oxobenzo [f] quinazolin-9-yl) methyl] amino-1-oxo-2-isoindolin] -glutaric acid], their pharmaceutical salts and any combination. In a more typical embodiment, the anticancer agent may be selected from the group consisting of pemetrexed, edatrexate, methotrexate, lometrexol, nolatrexed, ralitrexed, PT523, trimerexate, aminopterin, their pharmaceutical salts, and any combination thereof. In a most typical embodiment, the anticancer agent may be pemetrexed and its pharmaceutically acceptable salts. For example, pemetrexed may be a disodium salt. In a typical embodiment, pemetrexed may be disodium heptahydrate.

In a typical, but not limiting embodiment, the anti-folate anticancer agent is pemetrexed. Methoxyamine and an anti-folate anticancer agent may be administered sequentially (in any order) or administered together as a composition. Pemetrexed can, for example, be administered intravenously at a dose of 200-1000 mg / m ² body surface area per day, or at a dose of 500-600 mg / m ² body surface area per day. In another embodiment, the ratio of methoxyamine to antifolate anticancer agent may be from 1: 5 to 1: 500.

In yet another aspect, methoxyamine can be orally administered in an amount sufficient to sensitize cancer without causing excessive sensitization of normal tissue. In a non-limiting preferred embodiment, methoxyamine is administered orally so that it has substantially enhanced bioavailability compared to other anticancer agents orally administered. In other non-limiting preferred embodiments, methoxyamine is administered orally so that it maintains a minimum effective concentration when dosed once or twice a day. One way to measure oral bioavailability is to compare achieved levels compared to intravenously administered methoxyamine. Thus, in yet another aspect of the present invention, methoxyamine is orally administered with a bioavailability of at least 50% compared with intravenous administration, at least 60% compared with intravenous administration, at least 70% compared to intravenous administration of at least at least 75% compared with intravenous administration, at least 80% compared with intravenous administration, at least 85% compared with intravenous administration, at least 90% compared with intravenous administration, at least 95% compared with intravenous administration, or approximately equivalent bioavailability with intravenous administration. It is important to recognize that in addition to the unexpectedly high degree of bioavailability achieved by oral administration, a more desirable pK profile is obtained compared to the intravenous administration of methoxyamine compared to the intravenous administration of methoxyamine. In yet another aspect of the present invention, an orally administered methoxyamine maintains a minimum effective concentration after administration once or twice a day due to a plasma half-life> 4 hours. This advantage provides the possibility of the desired oral dosing regimen for methoxyamine, including administration once or twice a day. While the intravenous administration of pemetrexed combined with the oral administration of methoxyamine are preferred non-limiting embodiments, other routes of administration are contemplated for each anticancer agent.

In yet another aspect of the invention, methods of treating certain types of cancer that are resistant to treatment with a single anti-cancer agent are provided. In accordance with this, methods are also proposed in which:

take 1) a patient who is diagnosed with cancer, where the cancer may be resistant to treatment with pemetrexed alone, 2) the first composition containing an anti-folate anticancer agent; and 3) a second composition containing methoxyamine;

administering the first composition to a patient; and

the second composition is administered to the patient, and methoxyamine can be administered in an amount sufficient to improve or enhance the effect (i.e., potentiation of toxicity) of the antifolate anticancer agent. In one embodiment, the anti-folate anticancer agent may be pemetrexed. Methoxyamine and an anti-folate anticancer agent can be administered sequentially (in any order) or administered together as a composition. Pemetrexed can be administered intravenously at a dose of 200-1000 mg / m ² body surface area per day, or at a dose of 500-600 mg / m ² body surface area per day. The ratio of pemetrexed to methoxyamine can range from 1: 5 to 1: 500. In yet another embodiment, the amount of methoxyamine can be administered orally in an amount sufficient to sensitize the cancer without causing excessive sensitization of normal tissue. In yet another embodiment, this amount of methoxyamine can be administered orally or once a day or twice a day in an amount sufficient to sensitize the cancer without causing excessive sensitization of normal tissue. While oral administration of methoxyamine is unexpectedly a preferred route of administration, other types of administration are possible.

Another embodiment is directed to a method of treating cancer, wherein the first and second composition is taken, wherein said first composition comprises antifolate and the second composition contains orally administered methoxyamine. The first antifolate-containing composition may be administered by conventional routes of administration, including intravenous. A non-limiting preferred antifolate is pemetrexed. Accordingly, in one embodiment, the antifolate is pemetrexed, and the second composition containing methoxyamine is orally administered in an amount that is synergistically effective compared to pemetrexed treatment.

In yet another aspect, the method can be used to treat cancers that are resistant to pemetrexed alone. According to these embodiments, pemetrexed is administered in an amount that reverses resistance (and therefore is synergistic) to a single antifolate. Thus, in one embodiment, methoxyamine is orally administered in an amount effective to improve or increase the toxicity of pemetrexed and overcome the cancer resistance to treatment with pemetrexed. For example, the effectiveness of pemetrexed for the treatment of cancer may be reduced due to the development of resistance during the treatment cycle. Administration of methoxyamine can circumvent developed resistance by providing a more than additive effect on cancer treatment with either methoxyamine or pemetrexed alone.

Also proposed methods in which:

1) a patient is diagnosed with a cancer, where the cancer can be selected from the group consisting of carcinomas, melanomas, sarcomas, lymphomas, leukemias, astrocytes, gliomas, malignant melanomas, chronic lymphocytic leukemia, lung cancers, colorectal cancers, ovarian cancer, pancreatic cancer, kidney cancer, endometrial cancer, gastrointestinal cancer, liver cancer, head cancer s and neck and breast cancers, and where the cancer may be resistant to treatment with pemetrexed alone, 2) a first composition containing an anti-folate anticancer agent and 3) a second composition containing methoxyamine;

administering a first composition to said patient; and

administering a second composition to said patient, wherein methoxyamine can be administered in an amount sufficient to improve or increase the effect of the anti-folate anticancer agent. Methoxyamine and an anti-folate anticancer agent can be administered sequentially or administered together as a composition. For example, methoxyamine may be administered first and then anti-folate anticancer agent may be administered last, or anti-folate anti-cancer agent may be administered first and methoxyamine may be administered last.

In a typical embodiment, the anti-folate anticancer agent may be pemetrexed. Pemetrexed can be administered intravenously at a dose of 200-1000 mg / m ² body surface area per day, or at a dose of 500-600 mg / m ² body surface area per day. The ratio of pemetrexed to methoxyamine can range from 1: 5 to 1: 500. In yet another embodiment, this amount of methoxyamine can be orally administered in an amount sufficient to ensure that the cancer cells are susceptible to treatment with an anticancer agent (i.e., sensitize) without causing undue damage to normal cells. In yet another embodiment, this amount of methoxyamine can be administered orally either once or twice a day in an amount sufficient to sensitize the cancer without causing excessive sensitization of normal tissue.

Another embodiment may be a composition comprising methoxyamine and an anti-folate anti-cancer agent, where methoxyamine can be administered in an amount sufficient to potentiate the toxicity of the anti-folate anti-cancer agent. Preferably, the anti-folate anticancer agent is pemetrexed.

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

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

In yet another embodiment, a method is described for treating cancer in a patient diagnosed with cancer, comprising administering an antimetabolite anticancer agent to a patient, having the following improvement: administering methoxyamine to a patient in an amount sufficient to potentiate the toxicity of said antimetabolite anticancer agent. The anti-metabolic anti-cancer agent may be an anti-folate anti-cancer agent. The antifolate anticancer agent may be pemetrexed, and the ratio of said methoxyamine to the antifolate anticancer agent may be from 1: 5 to 1: 500. Cancer may be resistant to treatment with pemetrexed alone.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

On figa-B shows the effect of pemetrexed and MX on the breakdown of DNA strands using alkaline (Figa) and neutral (Fig.1B) comet analysis.

FIG. 1B-D shows a comparison of the length of the comet tails of cells treated with one pemetrexed or one MX and pemetrexed plus MX in cells subjected to alkaline (FIG. 1B) and neutral (FIG. 1G) comet analysis.

Figure 2 presents a graph showing the average concentration of MX in the plasma of male Sprague-Dawley rats at different times after dosing one bolus of MX by intravenous and oral administration at a concentration of 20 mg / kg body weight.

Figure 3 presents a graph showing the value of the concentration of MX in plasma obtained from female Sprague-Dawley rats at different points in time after dosing one bolus MX by intravenous and oral administration at a concentration of 20 mg / kg body weight.

FIG. 4A is a graph showing the relative number of AP sites detected in H460 cells 24 hours after treatment with pemetrexed and MX.

FIG. 4B is a graph showing the relative number of AP sites detected in H460 cells after 24 hours, 48 hours, and 72 hours.

Figures 5A and 5B show that MX-linked AP sites cannot be repaired by AP endonuclease.

On figa shows a diagram of the production of DNA substrates with regular AP sites or MX-AP sites. The block diagram shows the receipt of positive specific substrates of oligonucleotides containing the AP site or MX linked AP site

FIG. 5B shows that MX-linked AP sites are resistant to cleavage by AP endonuclease (APE). APE has the reactivity to cleave the AP site, but not the AP-MX site, which indicates that the AP-MX site is not repaired by APE

Figure 6 shows the effect of a combination of pemetrexed and MX on DNA double strand breaks and apoptosis. The combination of pemetrexed and TRC2 enhances double-stranded DNA breaks and apoptosis, which is independent of the B12 pathway. Cells were treated with 6 mM MX and harvested at 24 h.

Figure 7 shows the effect of the combination of pemetrexed and MX on protein levels with BEP in H460 cells. Protein levels of BER in H460 cells are presented before and after treatment with pemetrexed or a combination. Cells were treated with MX at a concentration of 6 mM and harvested at 24 h.

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

DETAILED DESCRIPTION OF THE INVENTION

Some embodiments of the invention mainly relate to new compositions containing methoxyamine and an anti-folate anticancer agent, and to the treatment of certain types of cancer using these compositions.

Definitions

Unless otherwise indicated, the following terms have the following meanings when used in this description of the invention and in the attached claims. Those terms that are not defined below or elsewhere in the description have their own meaning recognized in the prior art.

The terms "agent" and "drug" when used in this description for the purposes of the description and claims mean chemical compounds, mixtures of chemical compounds, biological macromolecules, or extracts obtained from biological materials (such as bacteria, plants, fungi or animals, in particular a mammal), cells or tissues that are believed to possess therapeutic properties. The agent or drug may be purified, substantially purified or partially purified.

The term "antimetabolite" is used in this description for the purposes of the description and claims to denote a chemotherapeutic agent with a structure similar to that of a substance (metabolite, for example nucleoside) required for normal biochemical reactions, but differing enough to interfere with normal cell functions including cell division.

The term "antifolate" is used in this description of the invention for the purposes of the detailed description and claims, to refer to a chemotherapeutic agent with a structure similar to that of folic acid, but different enough to block folic acid activity and disrupt folate-dependent mechanisms necessary for cellular replication. When used in this description of the invention, antifolates are one class of antimetabolites.

The term "antineoplastic" is used in this description of the invention for the purposes of the detailed description and claims, to refer to a chemotherapeutic agent intended to inhibit or prevent the maturation and proliferation of neoplasms (tumors) that can become malignant through DNA targeting.

The term "staining" is used in this description of the invention for the purpose of a detailed description and claims, to refer to any number of methods known in this field, which are used to better visualize, distinguish or identify specific component (s) and / or characteristics (i) cells or cells.

The term “in functional combination”, “in functional order” and “functionally linked” is used in this description for the purposes of the detailed description and claims to refer to the connection of nucleic acid sequences in such a way that a nucleic acid molecule capable of controlling transcription of this gene and / or synthesis of the desired protein molecule. The term also refers to joining amino acid sequences in such a way that a functional protein is obtained.

The term "antigen" is used in this description of the invention for the purposes of the description and claims to refer to a protein, glycoprotein, lipoprotein, lipid or other substance that is reactive with respect to an antibody specific for a portion of a molecule.

The term "morphology" is used in this description for the purposes of the description and claims to denote the visual characteristics of a cell or organism when viewed with the naked eye, by optical microscopy, confocal microscopy or electron microscopy, as appropriate.

The term “subject”, “individual” and “patient” is used in this description for the purposes of the description and claims to refer to a person or other animal, such as farm animals or laboratory animals (eg, guinea pig or mouse), capable of having a disease, determined by the cell cycle (influenced by the cell cycle), either natural or induced, including cancer, but without limiting it.

The term "reverses resistance" means that the use of the second agent in combination with the main chemotherapeutic agent can cause a significant decrease in tumor volume at a level of statistical significance (for example, p <0.05) when compared with the volume of untreated tumor in the case when the main chemotherapeutic agent does not have the ability to cause a statistically significant decrease in tumor volume compared to the volume of untreated tumor. The term is mainly applied to measurements of the volume of tumors performed during the period of time when the untreated tumor grows logarithmically rhythmically.

The term "potentiating" as used herein means an improvement or increase in the useful activity or effectiveness of an anticancer agent compared to what would be expected from a single anticancer agent or one potentiating agent.

The term “sensitization”, as used herein, means a change in cancer cells or tumor cells in a manner that enables more effective treatment of the associated neoplastic disease with an anticancer agent or radiation therapy. In some embodiments, normal cells are not exposed to the extent that unnecessarily traumatizes normal cells through chemotherapy or radiation therapy.

The term "synergistic effect" when used in this description of the invention means that the combined effect of two or more anticancer agents or chemotherapeutic drugs may be more significant than the sum of the individual effects of anticancer agents or chemotherapeutic drugs themselves. For example, the combined effect of a BEP inhibitor such as methoxyamine and an anticancer agent such as pemetrexed may be greater than the sum of the individual effects of methoxyamine and pemetrexed per se.

The term “therapeutically effective amount” means the amount of a compound of interest that will elicit the desired response, for example, the biological or medical response of a tissue, system, animal or person that is sought, for example, by a researcher, veterinarian, doctor or other clinician.

The term "wild type" (wt) of a cell or cell line is used in this description for the purposes of the description and claims to refer to a cell or cell line that retains the properties normally associated with this type of cell or cell line, in relation to the physiological process or morphological characteristics that check. It is allowed that the cell or cell line has properties of the off-type in relation to the physiological process or morphological properties that have not been tested, as long as they do not significantly affect the process or property being tested.

The term "pharmaceutically acceptable salt" refers to a salt of a compound that does not cause significant irritation in the body into which it is administered and does not invalidate the biological activity and properties of the compound. In some embodiments, the salt is an acid addition salt of a compound. Pharmaceutical salts can be prepared by reacting the compound with inorganic acids such as hydrohalic acid (e.g. hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid, phosphoric acid and the like. Pharmaceutical salts can also be prepared by reacting the compound with an organic acid, such as aliphatic or aromatic carboxylic or sulfonic acids, for example, acetic, succinic, lactic, maleic, tartaric, citric, ascorbic, nicotinic, methanesulfonic, ethanesulfonic, para-toluenesulfonic, salicylic acid or acid. Pharmaceutical salts can also be prepared by reacting the compound with a base to form a salt, such as 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 salt, such as dicyclohexylamine, N -methyl-D-glucamine, tris (hydroxymethyl) methylamine, C ₁ -C ₇ alkylamine, cyclohexylamine, triethanolamine, ethylenediamine and salts with amino acids such as arginine, lysine and the like.

DNA damage is minimized by enzymes that recognize errors, remove them and replace the damaged DNA with the correct nucleotides. DNA damage occurs when a single-stranded break is introduced, the base is removed, leaving its former partner unpaired, the base is covalently modified, the base is converted to another that is not properly paired with a paired base, or a covalent bond is introduced between the bases on opposite strands. Excision repair systems remove an incorrectly paired or damaged base from a DNA strand and then synthesize new DNA to replace it. Excision repair by removal of bases (BER) is initiated during DNA replication and allows repairing damaged bases / mismatched bases before replication is complete.

Excision repair by removing base (BER) is initiated by DNA glycosylase, which eliminates N-glycosidic (base-sugar) bonds, releasing the damaged base and forming an abasic site (e.g., apurine or apyrimidine (AP) site). An apuric or apyrimidine (AP) site results from the loss of a purine or pyrimidine residue, respectively, from DNA (deoxyribonucleic acid). Uracil residues can form from spontaneous deamination of cytosine and can lead to C → T movement if not repaired. There is also glycosylase that recognizes and removes hypoxanthine, a product of adenine deamination. Other glycosylases remove alkylated bases (such as 3-methyladenine, 3-methylguanine and 7-methylguanine), open-ring purines, oxidatively damaged bases and, in some organisms, UV (ultraviolet) photodimers. Uracil DNA Glycosylase (UDG) is an example of DNA glycosylase. Protein levels of BER UDG are affected by treatment with a combination of pemetrexed and MX (FIG. 7).

It is then processed using 5'-3 'endonuclease (AP-endonuclease (ARE)), which cuts the phosphodiester bond on both sides of the damaged purine or pyrimidine base. AP endonucleases introduce chain breaks by cleavage of phosphodiester bonds at AP sites.

PARP helps process DNA strand breaks induced during BER. PARP is a protein that controls single-stranded DNA breaks that weakly binds to BER intermediates when a single nucleotide BER proceeds normally until completion. In contrast, when a single nucleotide BER is stopped by blocking in the removal step, PARP binds strongly to BER intermediates as well as to AP endonuclease (APE), pol β DNA, and FEN-1.

In mammalian cells, 5'-deoxyribose sugar phosphate is removed using endogenous AR-lyase (dRP) activity of (transcriptional) DNA polymerase β (pol β). The DNA polymerase enzyme also fills the gaps with new nucleotides.

As a result, DNA ligase covalently binds the 3'-end of the new substance with the old substance. Thus, the wild-type sequence is restored.

Topoisomerases I and II are also involved in DNA repair, as they recognize spontaneous AP sites and form stable cleavable complexes. Topoisomerase II inhibitors stimulate DNA cleavage and other chromosomal aberrations, including sister chromatid exchanges.

As described herein, certain embodiments may be directed to methods in which:

take 1) a patient diagnosed with cancer, 2) a first composition containing an anti-folate anticancer agent and 3) a second composition containing methoxyamine;

administering the first composition to a patient; and administering a second composition to said patient, wherein methoxyamine can be administered in an amount sufficient to improve or enhance the effect of the anti-folate anticancer agent. Any anti-folate anticancer agent may be used provided that 5-FU is specifically excluded in some embodiments of the method.

In a typical embodiment, the anticancer agent may be selected from the group consisting of pemetrexed, capecitabine, edatrexate, methotrexate, lometrexol, nolatrexed, ralitrexed, PT523, trimerexate, aminopterin, 5,10-diderexazetetrahydrofolic acid (WAT, pyriteta43, pyrite рит RAT S) -2- [5 - [(1,2-dihydro-3-methyl-1-oxobenzo [f] quinazolin-9-yl) methyl] amino-1-oxo-2-isoindolin] -glutaric acid], their pharmaceutical salts and any combination. In a more typical embodiment, the anticancer agent may be selected from the group consisting of pemetrexed, capecitabine, edatrexate, methotrexate, lometrexol, nolatrexed, ralitrexed, PT523, trimerexate, aminopterin, their pharmaceutical salts, and any combinations thereof. In a most typical embodiment, the anticancer agent may be pemetrexed and its pharmaceutically acceptable salts. For example, pemetrexed may be a disodium salt. In a typical embodiment, pemetrexed may be disodium heptahydrate.

In some embodiments of the present invention, the use of an anticancer agent that induces the formation of AP sites and a BER inhibitor is contemplated.

In a typical embodiment, the anticancer agent may be selected from the group consisting of pemetrexed, capecitabine, edatrexate, methotrexate, lometrexol, nolatrexed, ralitrexed, PT523, trimerexate, aminopterin, 5,10-diderexazetetrahydrofolic acid (WAT, pyriteta43, pyrite рит RAT S) -2- [5 - [(1,2-dihydro-3-methyl-1-oxobenzo [f] quinazolin-9-yl) methyl] amino-1-oxo-2-isoindolin] -glutaric acid], their pharmaceutical salts and any combination. In a more typical embodiment, the anticancer agent may be selected from the group consisting of pemetrexed, capecitabine, edatrexate, methotrexate, lometrexol, nolatrexed, ralitrexed, PT523, trimerexate, aminopterin, their pharmaceutical salts, and any combinations thereof. In a most typical embodiment, the anticancer agent may be pemetrexed and its pharmaceutically acceptable salts. For example, pemetrexed may be a disodium salt. In a typical embodiment, pemetrexed may be disodium heptahydrate.

In a typical embodiment, the BER inhibitor may be selected from the group consisting of methoxyamine, etoposide (VP-16, VP-16-123), meso-4,4 '- (2,3-butanediyl) bis- (2,6- piperazinedione) (ICRF-193, bis-dioxopiperazine), doxorubicin (DOX), amsarkin (4 ′, 9-acridinylaminomethanesulfone-m-anisidide; mAMSA), pazelliptin, nalidixic acid, oxolinic acid, novobiocinum, tenamer, kume mitoxantrone, daunorubicin, N- [2-dimethylamino) ethyl] acridine-4-carboxamide (DACA), merbarone, quinacrine, ellipticins, epipodophyllotoxins, ethidium bromide, epirubicin, pyrara itsina, 3'-deamino-3'-morpholino-13-deoxo-10-hydroxy-carminomycin; 2 ′ ′, 3 ′ ′ - bis-pentafluorophenoxyacetyl-4 ′, 6′-ethylidene-beta-D-glycoside 4′-phosphate-4′-dimethylepipodophyllotoxin 2N-methyl-glucamine salt (F11782; fluorinated lipophilic epipodophylloid), adriamycin actinomycin D, anthracyclines (such as 9-amino-anthracycline), pyrazoloacridine (PZA), camptothecin, topotecan, their pharmaceutical salts and solvates, and any combinations. In a more typical embodiment, the BER inhibitor may be selected from the group consisting of methoxyamine (MX), N-ethyl maleimide, O ⁶ -benzylguanine, pharmaceutically acceptable salts thereof, and any combination thereof. In a most typical embodiment, the BER inhibitor may be methoxyamine (MX) or its salts.

In one embodiment, the BER inhibitor may be compounds having structures of formula I:

where X represents O or NH,

Y represents O, S or NH,

Z is absent or represents O, S or NH,

R represents hydrogen or a hydrocarbon group.

and their pharmaceutically acceptable salts.

In some embodiments, a BER inhibitor can be used to treat a patient or subject having a neoplastic disease. For example, a neoplastic disease may be cancer selected from the group consisting of carcinomas, melanomas, sarcomas, lymphomas, leukemias, astrocytes, gliomas, malignant melanomas, chronic lymphocytic leukemia, lung cancers, prostate cancers, colorectal cancers, ovarian cancers, cancers pancreatic diseases, cancer of the kidneys, cancer of the endometrium, cancer of the gastrointestinal tract, cancer of the liver, cancer of the head and neck.

In some embodiments, a BER inhibitor can be used to treat a patient or individual having a neoplastic disease that is being treated with an anticancer agent.

In a typical embodiment, the BER inhibitor may be selected from the group consisting of methoxyamine, etoposide (VP-16, VP-16-123), meso-4,4 ′ - (2,3-butanediyl) bis- (2,6- piperazinedione) (ICRF-193, bis-dioxopiperazine), doxorubicin (DOX), amsarkin (4 ′, 9-acridinylaminomethanesulfone-meta-aniside; mAMSA), pazelliptin, nalidixic acid, oxolinic acid, novobiocinin, cumene, cumene, cumene, cumene mitoxantrone, daunorubicin, N- [2-dimethylamino) ethyl] acridine-4-carboxamide (DACA), merbarone, quinacrine, ellipticins, epipodophyllotoxins, ethidium bromide, epirubicin, p iraubicin, 3′-deamino-3′-morpholino-13-deoxo-10-hydroxy-carminomycin; 2 ′ ′, 3 ′ ′ - bis-pentafluorophenoxyacetyl-4 ′, 6′-ethylidene-beta-D-glycoside 4′-phosphate-4′-dimethylepipodophyllotoxin 2N-methyl-glucamine salt (F11782; fluorinated lipophilic epipodophylloid), adriamycin actinomycin D, anthracyclines (such as 9-amino-anthracycline), pyrazoloacridine (PZA), camptothecin, topotecan, their pharmaceutical salts, and any combinations thereof. In a more typical embodiment, the BER inhibitor may be selected from the group consisting of methoxyamine (MX), N-ethyl maleimide, O ⁶ -benzylguanine, their pharmaceutically acceptable salts, and any combinations thereof. In a most typical embodiment, the BER inhibitor may be methoxyamine (MX) or its salts.

In a typical embodiment, the anticancer agent may be selected from the group consisting of pemetrexed, capecitabine, edatrexate, methotrexate, lomerexol, nolatrexed, ralitrexed, PT523, trimerexate, aminopterin, 5,10-didezase-tetrahydrofolic acid F, pyrite 18, DDAT [(S) -2- [5 - [(1,2-dihydro-3-methyl-1-oxobenzo [f] quinazolin-9-yl) methyl] amino-1-oxo-2-isoindolin] -glutaric acid] , their pharmaceutical salts and any combinations. In a more typical embodiment, the anticancer agent may be selected from the group consisting of pemetrexed, capecitabine, edatrexate, methotrexate, lometrexol, nolatrexed, ralitrexed, PT523, trimerexate, aminopterin, their pharmaceutical salts, and any combinations thereof. In a most typical embodiment, the anticancer agent may be pemetrexed and their pharmaceutically acceptable salts and solvates. For example, pemetrexed may be a disodium salt. In a typical embodiment, pemetrexed may be disodium heptahydrate.

In some embodiments, the BER inhibitor and anticancer agent can be administered to an individual in combination. For example, a BER inhibitor and an anticancer agent can be administered to an individual together in a parenteral composition. Alternatively, the BER inhibitor and anticancer agent may be administered to the individual together in an oral composition, such as a solid dosage composition.

In some embodiments, the BER inhibitor and anticancer agent can be administered to an individual sequentially, wherein the individual first receives an anticancer agent and then receives a BER inhibitor. For example, an individual can be given an anticancer agent in a parenteral composition, such as an intravenous composition, or in an oral composition such as a solid dosage composition, and then a BER inhibitor in a parenteral composition such as an intravenous composition, or in an oral composition such as a solid metered composition.

Alternatively, in some embodiments, the BER inhibitor and anticancer agent can be administered to the individual sequentially, with the BER inhibitor being first given to the individual and then the anticancer agent being given. For example, an individual may be given a BER inhibitor in a parenteral composition, such as an intravenous composition, or in an oral composition, such as a solid dosage composition, and then given an anticancer agent in a parenteral composition, such as an intravenous composition, or in an oral composition, such as a solid metered composition.

In some embodiments, the anticancer agent and the BER inhibitor can cause an anticancer effect greater than the individual anticancer effects of the individual agents. For example, the combined anticancer effect of an anticancer agent and a BER inhibitor may be more significant than the combined anticancer effect of an anticancer agent and a BER inhibitor when used individually.

Thus, compounds useful as BER inhibitors, such as methoxyamine (MX), N-ethyl maleimide, O ⁶ -benzylguanine and compounds having structures of formula I:

where Y represents O, S, or NH,

Z represents the absence or presence of O, S, or NH, and

R represents hydrogen or a hydrocarbon group,

and their pharmaceutically acceptable salts.

In a single nucleotide BER, deoxyribose phosphate (dRP) at the abasic site is removed through pol β DNA lysis activity. Compounds, such as methoxyamine, interact with the aldehyde of the abasic site, making them immune to the β-elimination step of the dRP-lyase mechanism, thus blocking the single nucleotide BER.

In some embodiments, suitable compounds may prevent the AP endonuclease substrate from being prone to cleavage. Anticancer agents can act by binding to AP sites and preventing APE-mediated cleavage of phosphodiester bonds. Other compounds that can bind to AP sites and prevent APE-mediated cleavage of phosphodiester bonds include O-benzylhydroxylamine; ethylaminooxyacetate; aminooxyacetic acid; ethyl amino hydroxyacetate; H ₂ N-OCHMeCO ₂ H; carboxymethoxyamine; aminooxyacetic acid; HN = C (NH ₂ ) SCH ₂ CH ₂ ONH ₂ ; H ₂ NO (CH ₂ ) ₃ SC (NH ₂ ) = NH; MeOC (O) CH (NH ₂ ) CH ₂ O-NH ₂ ; H ₂ NOCH ₂ CH (NH ₂ ) CO ₂ H; canalin; H ₂ NO (CH ₂ ) ₄ O-NH ₂ ; O- (para-nitrobenzyl) hydroxylamine; 2-amino-4- (aminooxymethyl) thiazole; 4- (aminooxymethyl) thiazole; O, O '- (ortho-phenylenedimethylene) dihydroxylamine; 2,4-dinitrophenoxyamine; O, O '- (meta-phenylenedimethylene) dihydroxylamine; O, O '- (para-phenylenedimethylene) dihydroxylamine; H ₂ C = CHCH ₂ O-NH ₂ ; H ₂ NO (CH ₂ ) ₄ O-NH ₂ ; H ₃ C (CH ₂ ) ₁₅ O-NH ₂ , 2,2 '- (1,2-ethanediyl) bis (3-aminooxy) fumaric acid dimethyldiethyl ether; compounds 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- [ ³ N] he (NU-1025).

Compounds useful as BER inhibitors include DNA polymerase inhibitors (e.g., S β, γ or ε DNA polymerases), such as prunazin, aphidicolin, 2 ′, 3′-dideoxycytidine triphosphate (ddCTP), 2 ′, 3 ′ dideoxythymidine triphosphate (ddTTP), 2 ′, 3′-dideoxyoxyadenosine triphosphate (ddATP), 2 ′, 3′-dideoxyguanosine triphosphate (ddGTP), 1-beta-D-arabinofuranosylcytosine (Ara-C) thiocinine, caffeine, caffeine.

Compounds useful as BER inhibitors include DNA ligase inhibitors (e.g., DNA ligases I, II, or III) such as ursolic and oleanolic acids, aleuritic acid, protolichesteric acid, swertifrancheside, fulvopumerin, fagaronin bleomycin. XRCC1 is a protein partner of DNA ligase III, and XRCC1 inhibitors such as 3-AB are also useful as BER inhibitors.

Topoisomerase II inhibitors induce DNA cleavage and other chromosomal aberrations, including sister chromatid exchanges. Compounds useful as BER inhibitors also include topoisomerase II inhibitors such as etoposide (VP-16, VP-16-123), meso-4,4 ′ - (2,3-butanediyl) bis- (2.6 -piperazinedione) (ICRF-193, bis-dioxopiperazine), doxorubicin (DOX), amsarkin (4 ′, 9-acridinylaminomethanesulfone-meta-anisidide; mAMSA), pazelliptin, nalidixic acid, oxolinic acid, fumiocycin, noviobicin, tenibicin aminocycin, , mitoxantrone, daunorubicin, N- [2-dimethylamino) ethyl] acridine-4-carboxamide (DACA), merbarone, quinacrine, ellipticins, epipodophyllotoxins, ethidium bromide, epirubicin, pyrarubicin 3'-deamino-3'-morpholino-13-deoxo-10-hydroxy-carminomycin; 2 ′ ′, 3 ′ ′ - bis-pentafluorophenoxyacetyl-4 ′, 6′-ethylidene-beta-D-glycoside 4′-phosphate-4′-dimethylepipodophyllotoxin 2N-methyl-glucamine salt (F11782; fluorinated lipophilic epipodophylloid), adriamycin, actinomycin D, anthracyclines (such as 9-aminoanthracycline) and pyrazoloacridine (PZA). Topoisomerase I inhibitors such as camptothecin and topotecan can also be used as BER inhibitors.

In some embodiments, other enzyme inhibitors, either known in the art or identified below, as well as inhibitors of other elements of the BER pathway, such as DNA alkyl transferase, can be used in the compositions and methods without departing from the scope and essence of the present embodiments.

In some embodiments, the present invention contemplates the use of an anticancer agent, such as pemetrexed, which induces the formation of AP sites, and a BER inhibitor (other than a topoisomerase inhibitor), such as methoxyamine.

In a typical embodiment, the anticancer agent may be selected from the group consisting of pemetrexed, capecitabine, edatrexate, methotrexate, lometrexol, nolatrexed, ralitrexed, PT523 and trimerexate. In a more typical embodiment, the anticancer agent may be pemetrexed and its pharmaceutically acceptable salts. For example, pemetrexed may be a disodium salt. In a typical embodiment, pemetrexed may be disodium heptahydrate.

In some embodiments, the anticancer agent can be administered at a dose of from about 25 mg / m ² to about 5000 mg / m ² body surface area. For example, the dose may be from about 25 mg / m ² to about 200 mg / m ² body surface area; the dose may be from about 150 mg / m ² to about 500 mg / m ² body surface area; the dose may be from about 400 mg / m ² to about 1000 mg / m ² body surface area; the dose may be from about 900 mg / m ² to about 5000 mg / m ² body surface area; the dose may be from about 200 mg / m ² to about 1000 mg / m ² body surface area; or the dose may be from about 500 mg / m ² to about 600 mg / m ² body surface area. Antifolates are a non-limiting preferred class of anticancer agents. In some embodiments, the anticancer agent can be selected from the group consisting of pemetrexed, capecitabine, edatrexate, methotrexate, lometrexol, nolatrexed, ralitrexed, PT523 and trimerexate. In a more typical embodiment, the anticancer agent may be pemetrexed and its pharmaceutically acceptable salts. For example, pemetrexed may be a disodium salt. In a typical embodiment, pemetrexed may be disodium heptahydrate.

In some embodiments, the ratio of the BER inhibitor to the 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; or the ratio of the BER inhibitor to the anticancer agent may be from about 1:20 to about 1:30. In a typical embodiment, the BER inhibitor may be selected from the group consisting of methoxyamine (MX), N-ethyl maleimide, O ⁶ -benzylguanine, their pharmaceutically acceptable salts, and any combinations 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 improve or enhance the effect of the anticancer agent.

In a typical embodiment, the anticancer agent may be selected from the group consisting of pemetrexed, capecitabine, edatrexate, methotrexate, lomerexol, nolatrexed, ralitrexed, PT523, trimerexate, aminopterin, 5,10-didezase-tetrahydrofolic acid F, pyrite 18, DDAT [(S) -2- [5 - [(1,2-dihydro-3-methyl-1-oxobenzo [f] quinazolin-9-yl) methyl] amino-1-oxo-2-isoindolin] -glutaric acid] , their pharmaceutical salts and any combinations. In a more typical embodiment, the anticancer agent can be selected from the group consisting of pemetrexed, capecitabine, edatrexate, methotrexate, lomerexol, nolatrexed, ralitrexed, PT523, trimerexate, aminopterin, their pharmaceutical salts and any combination thereof. In a most typical embodiment, the anticancer agent may be pemetrexed and its pharmaceutically acceptable salts. For example, pemetrexed may be a disodium salt. In a typical embodiment, pemetrexed may be disodium heptahydrate.

In a typical embodiment, the BER inhibitor may be selected from the group consisting of methoxyamine, etoposide (VP-16, VP-16-123), meso-4,4 ′ - (2,3-butanediyl) bis- (2,6- piperazinedione) (ICRF-193, bis-dioxopiperazine), doxorubicin (DOX), amsacrine (4 ′, 9-acridinylaminomethanesulfone-m-aniside; mAMSA), pazelliptin, nalidixic acid, oxolinic acid, novobiocinin, tenamer, kume mitoxantrone, daunorubicin, N- [2-dimethylamino) ethyl] acridine-4-carboxamide (DACA), merbarone, quinacrine, ellipticins, epipodophyllotoxins, ethidium bromide, epirubicin, pyra rubicin, 3′-desamino-3′-morpholino-13-deoxo-10-hydroxy-carminomycin; 2 ′ ′, 3 ′ ′ - bis-pentafluorophenoxyacetyl-4 ′, 6′-ethylidene-beta-D-glycoside 4′-phosphate-4′-dimethylepipodophyllotoxin 2N-methyl-glucamine salt (F11782; fluorinated lipophilic epipodophylloid), adriamycin actinomycin D, anthracyclines (such as 9-aminoanthracycline), pyrazoloacridine (PZA), camptothecin, topotecan, their pharmaceutical salts, and any combinations thereof. In a more typical embodiment, the BER inhibitor may be selected from the group consisting of methoxyamine (MX), N-ethyl maleimide, O ⁶ -benzylguanine, pharmaceutically acceptable salts thereof, and any combination thereof. In a most typical embodiment, the BER inhibitor may be methoxyamine (MX) or its salts. For example, the BER inhibitor may be methoxyamine hydrochloride (MX).

In some embodiments, the ratio of the BER inhibitor to the anticancer agent may be from about 1: 2 to about 1: 10000. For example, the ratio of the BER inhibitor to the anticancer agent may be from about 1: 2 to about 1: 100: the ratio of the BER inhibitor to the anticancer agent can 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; or the ratio of the BER inhibitor to the anticancer agent may be from about 1:20 to about 1:30. In a typical embodiment, the BER inhibitor may be selected from the group consisting of methoxyamine (MX), N-ethyl maleimide, O ⁶ -benzylguanine, their pharmaceutically acceptable salts, and any combinations 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 anticancer agent may be pemetrexed. For example, pemetrexed may be a pemetrexed disodium salt. In a typical embodiment, pemetrexed may be disodium heptahydrate.

In some embodiments, a cancer treatment method is provided, wherein

take a first composition containing an anticancer agent and a second composition containing a BER inhibitor, which can be administered individually or as a combination composition;

selecting a subject who is diagnosed with cancer, wherein said cancer is resistant to treatment with an anticancer agent alone or in combination with other anticancer agents;

the specified first composition and the specified second composition;

where the amount of said first composition and the amount of said second composition may be such that upon administration to said subject, the anticancer effect may be more significant than the anticancer effect of only one first composition.

In some embodiments, the first composition may comprise an anticancer agent selected from the group consisting of pemetrexed, capecitabine, edatrexate, methotrexate, lomerexol, nolatrexed, ralitrexed, PT523, trimerexate, aminopterin, their pharmaceutical salts and any combination thereof. In a typical embodiment, the anticancer agent may be pemetrexed. In some embodiments, the second composition may comprise a BER inhibitor selected from the group consisting of methoxyamine (MX), N-ethyl maleimide, O ⁶ -benzylguanine, pharmaceutically acceptable salts thereof, and any combinations thereof. In a typical embodiment, the BER inhibitor may be methoxyamine.

In some embodiments, the anticancer agent can be administered at a dose of from about 25 mg / m ² to about 5000 mg / m ² body surface area. For example, the dose may be from about 25 mg / m ² to about 200 mg / m ² body surface area; the dose may be from about 150 mg / m ² to about 500 mg / m ² body surface area; the dose may be from about 400 mg / m ² to about 1000 mg / m ² body surface area; the dose may be from about 900 mg / m ² to about 5000 mg / m ² body surface area; the dose may be from about 200 mg / m ² to about 1000 mg / m ² body surface area; or the dose may be from about 500 mg / m ² to about 600 mg / m ² body surface area. In some embodiments, the anticancer agent may be selected from the group consisting of pemetrexed, capecitabine, edatrexate, methotrexate, lometrexol, nolatrexed, ralitrexed, PT523 and trimerexate. In a more typical embodiment, the anticancer agent may be pemetrexed and its pharmaceutically acceptable salts. For example, pemetrexed may be a disodium salt. In a typical embodiment, pemetrexed may be disodium heptahydrate.

In some embodiments, the ratio of the BER inhibitor to the 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; or the ratio of the BER inhibitor to the anticancer agent may be from about 1:20 to about 1:30. In a typical embodiment, the BER inhibitor may be selected from the group consisting of methoxyamine (MX), N-ethyl maleimide, O ⁶ -benzylguanine, their pharmaceutically acceptable salts, and any combinations thereof. In a more typical embodiment, the BER inhibitor may be methoxyamine (MX).

In some embodiments, a cancer treatment method is provided wherein a first composition containing an anticancer agent and a second composition containing a BER inhibitor are taken, which can be administered individually or as a combination composition:

a subject is diagnosed with a cancer;

the specified first composition and the specified second composition;

where the amount of said first composition and the amount of said second composition can be such that when administered to said subject, the anticancer effect can be significantly greater than the total anticancer effect of the first composition containing the anticancer agent and the second composition containing the BER inhibitor.

In some embodiments, the first composition may comprise an anticancer agent selected from the group consisting of pemetrexed, capecitabine, edatrexate, methotrexate, lomerexol, nolatrexed, ralitrexed, PT523, trimerexate, aminopterin, their pharmaceutical salts and any combination thereof. In a typical embodiment, the anticancer agent may be pemetrexed. In some embodiments, the second composition may comprise a BER inhibitor selected from the group consisting of methoxyamine (MX), N-ethyl maleimide, O ⁶ -benzylguanine, pharmaceutically acceptable salts thereof, and any combinations thereof. In a typical embodiment, the BER inhibitor may be methoxyamine.

In some embodiments, the anticancer agent can be administered at a dose of from about 25 mg / m ² to about 5000 mg / m ² body surface area. For example, the dose may be from about 25 mg / m ² to about 200 mg / m ² body surface area; the dose may be from about 150 mg / m ² to about 500 mg / m ² body surface area; the dose may be from about 400 mg / m ² to about 1000 mg / m ² body surface area; the dose may be from about 900 mg / m ² to about 5000 mg / m ² body surface area; the dose may be from about 200 mg / m ² to about 1000 mg / m ² body surface area; or the dose may be from about 500 mg / m ² to about 600 mg / m ² body surface area. In some embodiments, the anticancer agent may be selected from the group consisting of pemetrexed, capecitabine, edatrexate, methotrexate, lometrexol, nolatrexed, ralitrexed, PT523 and trimerexate. In a more typical embodiment, the anticancer agent may be pemetrexed and its pharmaceutically acceptable salts. For example, pemetrexed may be a disodium salt. In a typical embodiment, pemetrexed may be disodium heptahydrate.

In some embodiments, the ratio of the BER inhibitor to the 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 the BER inhibitor to the anticancer agent may be from about 1: 5 to about 1: 500: the ratio of the BER inhibitor to the anticancer agent can 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; or the ratio of the BER inhibitor to the anticancer agent may be from about 1:20 to about 1:30. In a typical embodiment, the BER inhibitor may be selected from the group consisting of methoxyamine (MX), N-ethyl maleimide, O ⁶ -benzylguanine, their pharmaceutically acceptable salts, and any combinations thereof. In a more typical embodiment, the BER inhibitor may be methoxyamine (MX).

Another aspect of the present embodiment is an unexpected demonstration that the evidence that some BER inhibitors act synergistically in combination with some antifolates with unexpected reversal of resistance to certain antifolates. Thus, in non-limiting preferred embodiments, the anti-metabolic anti-cancer agent is an anti-folate anti-cancer agent. These antifolates interrupt folate-dependent metabolic processes essential for cell replication. Antifolates differ from other chemotherapeutic agents in that they act by interrupting the cellular processes involved in folate metabolism. This action includes, but is not limited to, inhibition of folate-dependent enzymes, including thymidylate synthase (TS). Interruption of folate-dependent processes leads to improper DNA replication and apoptosis of rapidly dividing cells, including cancer cells. In one embodiment, the ratio of MX to antimetabolic anticancer agent is from about 1: 5 to 1: 500. In some 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, a second anticancer agent may be administered before or after the combination of MX and the antimetabolite anticancer agent.

Pharmaceutical Compositions

It should be understood that the compositions proposed in this description of the invention may be in any form that allows the composition to be administered to a patient. For example, the composition may be in the form of a solid, liquid, or gas (e.g., aerosol). Other suitable routes of administration include, but are not limited to, oral, topical, parenteral (e.g., sublingual or buccal), sublingual, rectal, vaginal and intranasal. The term parenteral, as used herein, includes subcutaneous injections, intravenous, intramuscular, intrasternal, intracavernous, intrathecal, intracanal, intraurethral injection or infusion methods. The pharmaceutical composition is prepared in the form of a preparation so as to ensure bioavailability of the active ingredients contained therein when the composition is administered to a patient. Compositions to be administered to a patient take the form of one or more dosage units, where, for example, a tablet may be a single dosage unit, and a container for one or more of the compounds of the invention in aerosol form may contain multiple dosage units.

In yet another aspect, the present disclosure relates to a pharmaceutical composition comprising physiologically acceptable surfactants, carriers, diluents, excipients, smoothing agents, suspension agents, film-forming agents and coating excipients, or a combination thereof; and a compound disclosed herein. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical field and are described, for example, in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, PA (1990), which is incorporated herein by reference in its entirety. its fullness. Preservatives, stabilizers, colorants, sweeteners, flavors, flavoring agents and the like can be presented in the pharmaceutical composition. For example, sodium benzoate, ascorbic acid, and para-hydroxybenzoic acid esters can be added as preservatives. In addition, antioxidants and suspending agents can be used. In various embodiments, alcohols, esters, sulfated aliphatic alcohols and the like can be used as surface-active agents; as excipients, sugar, glucose, lactose, starch, crystalline cellulose, mannitol, light anhydrous silicate, magnesium aluminate, magnesium aluminum metasilicate, synthetic aluminum silicate, calcium carbonate, sodium hydrogen carbonate, calcium hydrogen phosphate, calcium carboxymethyl cellulose and the like can be used; magnesium stearate, talc, hydrogenated fat and the like can be used as smoothing agents; coconut oil, olive oil, sesame oil, peanut oil and soy can be used as suspension agents or lubricants; cellulose acetate phthalate in the form of a carbohydrate derivative such as cellulose or sugar, or a methyl acetate-methacrylate copolymer in the form of a polyvinyl derivative can be used as suspension agents; and plasticizers, such as phthalate esters and the like, can be used as suspension agents.

The term “pharmaceutical composition” refers to a mixture of a compound disclosed herein with other chemical components, such as diluents or carriers. The pharmaceutical composition facilitates the administration of the compound to the body. There are numerous methods for administering a compound in the art, including, but not limited to, oral, injectable, aerosol, parenteral, and topical administration. Pharmaceutical compositions can also be prepared 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, para-toluenesulfonic acid, salicylic acid and the like.

The term “carrier” defines a chemical compound that facilitates the incorporation of the compound into cells or tissues. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier since it facilitates the absorption of many organic compounds by cells or body tissues.

The term “diluent” defines chemical compounds diluted with water, which dissolves the compounds of interest, and also stabilizes the biologically active form of the compound. Salts dissolved in buffered solutions are used as diluents in the art. One commonly used buffered solution is phosphate buffered saline, as it mimics the saline conditions of human blood. Since buffer salts can control the pH of a solution at low concentrations, a buffered diluent rarely alters the biological activity of a compound.

The term "physiologically acceptable" defines a carrier or diluent that does not invalidate the biological activity and properties of the compound.

The pharmaceutical compositions described herein can be administered to a human patient alone, or in pharmaceutical compositions where they are mixed with other active ingredients, as in combination therapy, or with suitable carriers or excipients (excipients). Methods of preparation and administration of the compounds of this application can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, 18th Edition, 1990.

Suitable routes of administration may, for example, include oral, rectal, transmucosal, topical or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal or intraocular injections. The compounds can also be administered in sustained or controlled release dosage forms, including depot injections, osmotic pumps, tablets, transdermal (including electrotransfer) patches, and the like, for sustained and / or time-varying, pulsed administration at a predetermined rate.

The pharmaceutical compositions of the present invention can be prepared by a method that is known per se, for example, by conventional mixing, dissolving, granulating, dragee-making, grinding into powder, emulsification, encapsulation, capture or tabletting processes.

Thus, pharmaceutical compositions for use in accordance with the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers containing excipients and excipients that facilitate the processing of the active compounds into pharmaceutical preparations. A suitable composition depends on the chosen route of administration. Any well-known methods, carriers and excipients can be used as suitable and as understood in the art, for example, Remington's Pharmaceutical Sciences above.

Suitable for injection compositions can be obtained in conventional forms either as liquid solutions or suspensions, solid forms suitable for solution or suspension in a liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, decitin, albumin, monosodium glutamate, cysteine hydrochloride and the like. In addition, if desired, injectable pharmaceutical compositions may contain minor amounts of non-toxic adjuvants, such as wetting agents, pH buffering agents and the like. Physiologically compatible buffers include, but are not limited to, Hank's solution, Ringer's solution, or physiological saline. If required, absorption enhancing agents (e.g. liposomes) can be used.

For transmucosal administration in the composition, penetrating liquids suitable for the barrier through which it must be penetrated can be used.

Pharmaceutical compositions for parenteral administration, for example by bolus injection or continuous infusion, contain aqueous solutions of the active compounds in a water-soluble form. In addition, suspensions of the active compounds can be prepared in the form of suitable oily injectable suspensions. Suitable lipophilic solvents or media include fatty acids, such as sesame oil, or other organic oils, such as soybean, grapefruit or almond oils, or synthetic fatty acid esters, such as ethyl oleate, or triglycerides, or liposomes. Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to enable the preparation of high concentration solutions. Compositions for injection can be presented in unit dosage form, for example, in ampoules or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous media, and may contain drug-forming agents, such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form before use for dilution with a suitable medium, for example sterile pyrogen-free water.

For oral administration, the compounds can be easily formulated by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers provide the ability to prepare the compounds of the invention in the form of a preparation, such as tablets, pills, dragees, capsules, liquids, gels, syrups, pastes, suspensions, and the like, for oral administration to a patient to be treated. Pharmaceutical preparations for oral administration can be obtained by combining the active compounds with a solid excipient, possibly grinding the resulting mixture and processing the mixture of granules after adding suitable excipients, if necessary, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents, such as crosslinked polyvinylpyrrolidone, agar or alginic acid or a salt thereof, such as sodium alginate, may be added. Dragee cores are offered with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may possibly contain gum arabic, talc, polyvinylpyrrolidone, carbopol, polyethylene glycol, and / or titanium dioxide, varnish solutions and suitable organic solvents or solvent mixtures. Dyes or pigments can be added to the coatings of tablets or dragees to identify or to distinguish between different combinations of doses of the active compound. To this end, concentrated sugar solutions may be used, which may possibly contain gum arabic, talc, polyvinylpyrrolidone, carbopol, polyethylene glycol, and / or titanium dioxide, varnish solutions, and suitable organic solvents or solvent mixtures. Dyes or pigments can be added to the coatings of tablets or dragees to identify or to distinguish between different combinations of doses of the active compound.

Pharmaceutical preparations that can be administered orally include tightly filled capsules made from gelatin, as well as soft, sealed capsules made from gelatin and a plasticizer such as glycerol or sorbitol. Tightly filled capsules may contain the active ingredients in admixture with an excipient such as lactose, binders such as starch) and / or lubricants such as talc or magnesium stearate, and possibly stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin or liquid polyethylene glycols. In addition, you can add stabilizers. All compositions for oral administration should be in amounts suitable for such administration.

Compositions for buccal administration may take the form of tablets or lozenges formulated in the usual manner.

For administration by inhalation, the compounds for use in the present invention are conveniently delivered in the form of a supplied aerosol spray from a pressurized package or nebulizer using a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined using a valve to release a metered amount. Capsules and ampoules of, for example, gelatin for use in an inhaler or insufflator may be prepared containing a powder mixture of a compound and a suitable powder base, such as lactose or starch.

In addition, various pharmaceutical compositions well known in the pharmaceutical field for applications that include intraocular, intranasal and intraoral delivery are disclosed herein. Suitable penetrating agents for these applications are generally known in the art. Pharmaceutical compositions for intraocular delivery include aqueous ophthalmic solutions of the active compounds in a water-soluble form, such as eye drops, or in gellan gum (Shedden et al., Clin. Ther. 23 (3): 440-50 (2001)) or hydrogels ( Mayer et al., Ophtalmologica, 210 (2): 101-3 (1996)); eye ointments; ophthalmic suspensions, such as microparticles, drug-containing small polymer particles that are suspended in a carrier liquid (Joshi, A., J. Ocul. Pharmacol., 10 (1): 29-45 (1994)), lipid-soluble compositions ( Alm et al., Prog. Clin. Biol. Res., 312: 447-58 (1989), and microspheres (Mordenti, Toxicol. Sci., 52 (1): 101-6 (1999)); and ocular tabs. All of the above references are incorporated into this description by reference in their entirety. Such suitable pharmaceutical compositions are most often and preferably prepared sterile, isotonic and buffered for stability and comfort. Pharmaceutical compositions for intranasal delivery may also include drops and sprays, often formulated to mimic, in many respects, nasal secrets to ensure that the ciliary effect remains normal. As disclosed in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA (1990), which is incorporated herein by reference in its entirety and is well known to those skilled in the art, suitable compositions are most often and preferably are isotonic, slightly buffered to maintain a pH of 5.5 to 6.5, and most often and preferably contain antimicrobial preservatives and suitable drug stabilizers. Pharmaceutical compositions for intra-auricular delivery include suspensions and ointments for topical application to the ear. Conventional solvents for such ear compositions contain glycerin and water.

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

In addition to the compositions described previously, the compounds can also be prepared as a depot preparation. Such long acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or with ion exchange resins, or as sparingly soluble derivatives, for example, such as sparingly soluble salts.

For hydrophobic compounds, a suitable pharmaceutical carrier may be a co-solvent system containing 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 a solution of 3% w / v. benzyl alcohol, 8% wt./about. the non-polar surfactant Polysorbate 80 ™ and 65% w / v. polyethylene glycol 300, brought to the desired volume with absolute ethanol. Of course, the proportions in the co-solvent system can vary significantly without invalidating their solubility and toxicity characteristics. In addition, the characteristics of the components of the cosolvents may vary: for example, other low toxic non-polar surfactants may be used instead of POLYSORBATE 80 ™; the fractional size of polyethylene glycol may vary; other biocompatible polymers, for example polyvinylpyrrolidone, can replace polyethylene glycol; and other sugars or polysaccharides can replace dextrose.

Alternatively, other delivery systems may be used for hydrophobic pharmaceutical compounds. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. You can also use some organic solvents, such as dimethyl sulfoxide, although usually due to greater toxicity. In addition, compounds can be delivered using a sustained release system, such as semipermeable matrices of solid hydrophobic polymers containing a therapeutic agent. Various sustained release substances have been created and are well known to those skilled in the art. Slow-release capsules may, depending on their chemical nature, release compounds over a period of several weeks up to over 100 days. Depending on the chemical nature and biological stability of the therapeutic reagent, additional protein stabilization strategies can be used.

Agents intended for intracellular administration can be administered using techniques well known to those skilled in the art. For example, such agents may be encapsulated in liposomes. All molecules present in an aqueous solution at the time of liposome formation are included in the aqueous interior. The liposomal content is protected from the external microenvironment, and since liposomes merge with cell membranes, it is effectively delivered to the cell cytoplasm. The liposome may be coated with a tissue-specific antibody. Liposomes will be targeted to the desired organ and selectively captured by it. Alternatively, small hydrophobic organic molecules can be directly introduced intracellularly.

Additional therapeutic or diagnostic agents may be included in the pharmaceutical compositions. Alternatively or additionally, the pharmaceutical compositions may be combined with other compositions that contain other therapeutic or diagnostic agents.

Administration Methods

The compounds or pharmaceutical compositions may be administered to a patient by any suitable means. Non-limiting examples of methods of administration include, but are not limited to: (a) administration by oral route, the administration comprising administration in a capsule, tablet, granule, spray, syrup, or other similar forms; (b) administration by non-oral routes such as rectal, vaginal, intraurethral, intraocular, intranasal or intra-auricular, the administration comprising administration in the form of an aqueous suspension, oil preparation or the like, or in the form of drops, spray, suppository, cream, ointment or the like; (c) administration by injection subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, intraocularly, intraarticular, intraspinal, intrasternal or the like, including delivery by an infusion pump; (d) administration locally, such as by injection directly into the region of the kidneys or heart, for example, via depot implantation; and (e) topical administration; it seems appropriate for those skilled in the art to bring the compound of the invention into contact with living tissue.

Pharmaceutical compositions suitable for administration include compositions wherein the active ingredients are contained in an amount effective to achieve their intended use. A therapeutically effective amount of the compounds disclosed herein as a dose will depend on the route of administration, the type of animal, including the person to be treated, and the physical characteristics of the particular animal in question. The dose can be calculated to achieve the desired effect, but will depend on factors such as weight, diet, concomitant treatment and other factors that experts in the medical field will recognize. More specifically, a therapeutically effective amount means an amount of a compound effective to prevent, alleviate or alleviate the symptoms of the disease or prolong the life of the subject to be treated. The definition of a therapeutically effective amount is indeed within the scope of the skill of those skilled in the art, especially in view of the detailed description provided herein.

It will be apparent to those skilled in the art that the useful in vivo dosage administered and the particular route of administration will vary depending on the age, weight and type of mammal to be treated, the particular compounds used and the particular benefit for which these compounds are used. Determination of effective dosage levels, which are dosage levels necessary to achieve the desired result, can be performed by a person skilled in the art using conventional pharmacological methods. Typically, clinical applications of products to humans begin at lower dosage levels, with an increase in dosage levels until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish useful doses and routes of administration of the compositions identified by the present methods using established pharmacological methods.

In studies not in humans, the use of potential products begins at higher dosage levels, with a decrease in dosage until the desired effect is no longer achieved or until the elimination of harmful side effects. Dosage can vary widely depending on the desired effects and therapeutic indications. Typically, dosages may be from about 10 micrograms / kg to 100 mg / kg body weight, preferably from about 100 micrograms / kg to 10 mg / kg body weight. Alternatively, dosages may be based and calculated based on the surface area of the patient, as understood by those skilled in the art.

The exact preparation, route of administration and dosage of the pharmaceutical compositions of the present invention can be selected by the attending physician taking into account the patient's condition (see, for example, Fingl et al. 1975, in The Pharmacological Basis of Therapeutics, which is incorporated herein by reference in its entirety, with a specific reference to chap. 1, p. 1). Typically, the dose range of a composition administered to a patient may be from about 0.5 to 1000 mg / kg of patient body weight. The dosage may be a single dose or a series of two or more, given in a course of one or more days, as necessary for the patient. In cases where dosages of the compounds for humans have been established for at least some conditions, the same dosages, or dosages that are about 0.1% -500%, more preferably about 25% -250% of the established dosage, are used in the present invention. for a person. If a dosage for humans has not been established, as will be the case with newly discovered pharmaceutical compounds, a suitable dosage for humans may be inferred from the values of ED ₅₀ or ID ₅₀ , or other suitable values obtained from in vitro or in vivo studies, as suitable in toxicity studies and animal efficacy studies.

It should be noted that the attending physician must know how and when to stop, interrupt or adjust the administration due to toxicity or organ dysfunctions. On the other hand, the attending physician should also know how to adjust treatment to higher levels if the clinical response was not adequate (excluding toxicity). The amount of the administered dose in the management of the disorder of interest will vary depending on the severity of the condition to be treated and on the route of administration. The severity of the condition can, for example, be partially assessed using standard prognostic analysis methods. In addition, the dose and, possibly, the frequency of doses will also vary depending on the age, body weight and immune response of the individual patient. A program comparable to that discussed above can be used in veterinary medicine.

Although the exact dosage will be determined depending on the particular drug, in most cases some generalizations regarding dosage may be made. The daily dosage regimen for an adult human patient may be, for example, an oral dose of 0.1 mg / m ² to 2000 mg / m ² body surface area per day of each active ingredient, usually 1 mg / m ² to 500 mg / m ² body surface area per day, for example from 5 mg / m ² to 200 mg / m ² body surface area per day. In other embodiments, an intravenous, subcutaneous or intramuscular dose of each active ingredient from 0.01 mg / m ² to 100 mg / m ² body surface area per day, usually from 0.1 mg / m ² to 60 mg / m ² area, can be used. body surface per day, for example from 1 mg / m ² to 40 mg / m ² body surface area per day. In cases where a pharmaceutically acceptable salt is administered, the dosage can be based on the free base. In some embodiments, the composition is administered 1-4 times per day. Alternatively, the compositions of the invention can be administered by continuous intravenous infusion, preferably with a dose of each active ingredient up to 1000 mg / m ² body surface area per day. Those skilled in the art will understand that in specific situations it may be necessary to administer the compounds disclosed herein in amounts that exceed or even significantly exceed the above preferred dosage range in order to effectively and actively treat particularly aggressive diseases or infections. In some embodiments, the compounds will be administered during the continuous treatment period, for example for a week or more, or for months or years.

The dosage and interval can be adjusted individually to obtain concentrations of the active group in plasma sufficient to maintain modulating effects, or a minimum effective concentration (MEC). MES will vary for each compound, but can be determined from in vitro data. The dosages necessary to achieve the MES will depend on the individual characteristics and route of administration. However, HPLC or bioassay can be used to determine plasma concentrations.

Dosing intervals can also be determined using the MEC value. Compositions should be administered using a regimen that maintains plasma levels above MEC for 10-90% of the time, usually 30-90% and most typically 50-90%.

In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to the plasma concentration.

The amount of composition administered may depend on the subject being treated, the subject's weight, severity of the disease, route of administration and judgment of the prescribing physician.

Compounds disclosed herein can be evaluated for efficacy and toxicity using known methods. For example, the toxicology of a particular compound or subgroup of compounds having some common chemical fragments can be ascertained by determining in vitro toxicity to a cell line, such as a mammalian and preferably human cell line. The results of such studies are often predictive of toxicity to animals, such as mammals, or more specifically to humans. Alternatively, the toxicity of specific compounds in an animal model, such as mice, rats, rabbits or monkeys, can be determined using known methods. The effectiveness of a particular compound can be established using various recognized methods, such as in vitro methods, animal models, or human clinical trials. There are generally recognized in vitro models for almost every class of condition, including but not limited to cancer, cardiovascular disease, and various immune dysfunctions. Similarly, acceptable animal models can be used to establish the effectiveness of chemicals in the treatment of such conditions. When choosing a model for determining effectiveness, a person skilled in the art can be guided by the prior art to select the appropriate model, dose and route of administration and mode. Of course, human clinical trials can also be used to determine the effectiveness of a compound in humans.

Compositions may be presented, if desired, in a package or spray device, which may contain one or more dosage forms containing the active ingredient. The package may, for example, contain metal or plastic foil, for example, a blister pack. The packaging or spray device may be accompanied by instructions for administration. The packaging or dispenser may also be accompanied by a notice associated with the container, in the form prescribed by the government agency that regulates the manufacture, use or sale of pharmaceuticals, the notification reflecting the approval by that institution of this form of medicine for human administration or veterinary use. Such a notice, for example, may be a label approved by the U.S. Food and Drug Administration for prescription drugs, or an approved package insert. You can also get compositions containing the compound according to the invention, prepared in the form of a preparation in a compatible pharmaceutical carrier, placed in a suitable container, and labeled for the treatment of this condition.

Throughout the description of the invention, any reference to a particular compound should be understood as encompassing that compound and any pharmaceutically acceptable salts thereof (other).

In the first stage of BER, a number of glucosidases recognize abnormal bases, such as N ³ mA and N ⁷ mG (O'Connor et al. "Isolation and structure of a cDNA expressing a mammalian 3-methyladenine-DNA glycosylase" EMBO J. 9: 3337- 3342, 1990; Samson et al. "Cloning and characterization of a 3-methyladenine DNA glycosylase with DNA from human cells whose gene maps to chromosome 16" Proc. Natl. Acad. Sci. USA 88: 9127-9131, 1991), erroneous base pairing T: G (Neddermann et al. "Functional expression of soluble human interleukin-11 (IL-11) receptor alpha and stoichiometry of in vitro IL-11 receptor complexes with gp130" J. Biol. Chem. 271: 12767-12774 , 1996), and deaminated bases such as hypoxanthine / oxidized 8-oxo-7,8-dihydroguanine or uracil: A (Vollberg et al. "Isolation and characterization of the human u racil DNA glycosylase gene "Proc. Natl. Acad. Sci. USA 86: 8693-8697, 1989; Olsen et al." Molecular cloning of human uracil-DNAglycosylase, a highly conserved DNA repair enzyme "EMBO J. 8: 3121-3125 . 1989; Radicella et al. "Cloning and characterization of hOGG1, a human homolog of the OGG1 gene of Saccharomyces cerevisiae" Proc. Natl. Acad. Sci. USA 94.8010-8015, 1997; Rosenquist et al. "Cloning and characterization of a mammalian 8-oxoguanine DNA glycosylase" Proc. Natl. Acad. Sci. USA 94: 7429-7434, 1997). After enzymatic or spontaneous hydrolysis of the N-glycosidic bond and the release of the wrong base, the AR (apuric / apyrimidine) endonuclease hydrolyzes the phosphodiester backbone 5 ′ to damage, and dRpase (DNA deoxyribophosphodiesterase and its activity is linked to polymerase β), removes the residual d nucleotide. DNA polymerase β fills the gap, and DNA ligase cross-links a single-stranded gap. Such a path is called BER short sequences. An alternative way for BER involves DNA synthesis to fill the gap of 2-13 nucleotides. This long-sequence repair requires proliferating cell nuclear antigen (PCNA) and PCNA-dependent DNA polymerase (Wilson "Mammalian base excision repair and DNA polymerase beta" Mutation Res. 407: 203-215, 1998).

Poly- (ADP-ribose) -polymerase (PARP) acts as a single-stranded DNA strand break detector on its own or through interaction with XRCC1 and is involved in BER. PARP binds damaged DNA, which leads to autobosylation. The modified protein is then released and allows other proteins to reach and repair DNA strand breaks (Wilson "Mammalian base excision repair and DNA polymerase beta" Mutation Res. 407: 203-215, 1998; Molinete et al. "Over production of the poly (ADP- ribose) polymerase DNA-binding domain blocks alkylation-induced DNA repair synthesis in mammalian cells "EMBO J. 12: 2109-2117, 1993; Caldecott et al." XRCCI polypeptide interacts with DNA polymerase β and possibly poly (ADP-ribose) polymerase , and DNA ligase III is a novel molecular 'nick-sensor' in vitro (Nucleic Acids Res. 24: 4387-4394, 1996). Thus, PARP is involved in BER after the formation of a single chain rupture in both short and long repair sequences. It turns out to be the most active in the alternative (long-sequence repair) BER pathway. Figure 6 shows that the combination of pemetrexed and MX enhances the formation of cut PARP. The increase in DNA double strand breaks and apoptosis are independent of the Bcl-2 pathway.

In general, the terminology used below and laboratory techniques for cell culture, tissue culture, tumor biology, and molecular genetics described below are well known and commonly used in the art. Conventional techniques are used for cell culture methods, experiment design, and compound preparation in the form of a preparation and terminology. In general, chemical interactions and purification steps are carried out according to the manufacturer's instructions. Techniques and methods are generally performed according to generally accepted methods in the art and various general references (see, mainly, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor , NY, and Current Protocols in Molecular Biology (1996) John Wiley and Sons, Inc., NY, which are incorporated herein by reference), which are presented throughout this document. All information contained therein is incorporated into this description by reference.

Pemetrexed (2- [4- [2- (4-amino-2-oxo-3,5,7-triazabicyclo [4.3.0] nona-3,8,10-trien-9-yl) ethyl] benzoyl] aminopentadicarboxylic acid) (ALIMTA ™; Eli Lilly & Co.) is an antimetabolic chemotherapeutic agent that has the following structure:

Pemetrexed is an antifolate antineoplastic agent that detects its effect by disrupting the folate-dependent mechanisms necessary for cell replication. Pemetrexed inhibits thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyl transferase (GARFT), enzymes involved in de novo biosynthesis of thymidine and purine nucleotides. Pemetrexed is approved by the FDA for the treatment of non-small cell lung cancer and is approved in combination with cisplatin (a platinum-based chemotherapy drug) for the treatment of malignant pleural mesothelioma. The recommended dose for the treatment of mesothelioma (in combination with cisplatin) or non-small cell lung cancer is approximately 500 mg / m ² body surface area per day, administered as an intravenous infusion over 10 minutes on 1 day of every 21-day cycle. For use in combination treatment with methoxyamine, typical dosage ranges of pemetrexed are generally from 200 mg / m ² to 1000 mg / m ² body surface area per day, or from 500 mg / m ² to 600 mg / m ² body surface area in day; and typical dosage ranges of methoxyamine are from 1 to 200 mg / m ² body surface area per day, or 6-120 mg / m ² body surface area per day. In one embodiment, pemetrexed is administered in the same manner as described above; however, intravenous infusion can be performed for 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes or more, and can be performed in 1 day, plus one or more additional days of this cycle, which can be 1 week, 2 weeks , three weeks, one month or longer.

EXAMPLES

Substances and methods

Chemicals and reagents. Methoxyamine (MX) was purchased from Sigma (ST. Louis, Mo.). MX was dissolved in sterilized water (pH 7.0).

Western blotting to determine PARP cleavage. Cell extracts were separated by SDS-PAGE (polyacrylamide gel electrophoresis with sodium dodecyl sulfate) (12% polyacrylamide) in a Bio-Rad mini-gel instrument at 150 V for 1 h. Proteins were transferred onto PVDV membranes using a Bio- mini-blotter Rad for 1 h at 100 V. Blotted membranes were blocked with 5% milk powder in 15 TBS buffer and then tested for 2 h with anti-PARP antibody C2-10 (Trevigen, Gaithersburg, Md.). After three 5-minute washes with TBS-Tween 20 (0.05%), the blots were incubated with secondary antibodies, anti-IgG against murine HRPs, for 1 h (Amersham Life Science, Arlington Height III.). Antibody binding was observed using ECL (Electro-Generated Chemiluminescence) according to the manufacturer's instructions (Amersham Life Science, Arlington Heights, III.).

Tumors of "naked" mice. Tumor cells (5 × 10 ⁶ ) were injected into the barrel of a female nude mouse HSD 6-8 weeks old. Animals received five daily intraperitoneal injections of either saline, one pemetrexed (150 mg / kg), one MX (4 mg / kg), or a combination of pemetrexed (150 mg / kg) and MX (4 mg / kg) with a ratio of pemetrexed to MX 26.7: 1.0. Tumors were measured using calipers using the formula of the National Cancer Institute: V = L (mm) × I ² (mm) / 2, where L is the largest diameter and I is the smallest diameter of the tumor. When the volume of nodules reached approximately 100-150 mm ³ , tumor-bearing mice were randomly assigned to control or treatment groups (6-9 mice / group).

EXAMPLE 1

Fig. 8 shows that in all groups receiving methoxyamine (MX) + pemetrexed, there was a more significant inhibition of tumor growth compared to groups receiving only pemetrexed in the human cell line model NCl-H460 NSCLC, cell line model A549 NSCLC, HCT116 colorectal cancer cell line models and MDA-MB-468 breast cancer cell line models. MX reversed the resistance of the NCl-H460 NSCLC cell line and the HCT116 colorectal cancer cell line to the effects of pemetrexed chemotherapy.

EXAMPLE 2

To measure the effect of pemetrexed + methoxyamine (MX) compared with one pemetrexed on the number of generated AP sites, a reagent-aldehyde reactive assay (ARP) was used to measure AP sites formed using pemetrexed and blocked MX. ARP and MX have a similar ability to react with AP sites and interact specifically with the aldehyde group, an open-ring form of AP sites. The analysis is described in Liu et al. (Molecular Cancer Therapeutics 2: 1061-1066, 2003) and was carried out essentially as described by 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 μg) was extracted from the cells and incubated with 1 mM ARP at 37 ° C for 10 min. DNA was precipitated and washed with ethanol, then resuspended in TE buffer (10 mM Tris-HCl, pH 7.2, 1 mM EDTA) and denatured at 100 ° C for 5 min. Then the DNA was rapidly cooled on ice and mixed with an equivalent amount of ammonium acetate (2 M). Then single-stranded DNA was immobilized on a nitrocellulose membrane using a vacuum filtering device. The membrane was incubated with streptavidin-conjugated horseradish peroxidase at room temperature for 30 minutes and washed with wash buffer (20 mM Tris-HCl, 1 mM EDTA, 0.26 M NaCl, 1% Tween-20). ARP-AP sites were visualized using ECL reagents (Amersham, Piscataway, NJ). The results are shown in FIG. 4A. Pemetrexed in doses of 100, 200 and 400 μM induced the formation of AP sites with a degree of induction of AP sites proportional to the dose of pemetrexed (Fig. 4A). Adding MX to pemetrexed significantly reduced the number of detectable AP sites compared to pemetrexed alone. The effects of a combination of 200 μM pemetrexed and 6 mM MX were evaluated at 24 hours, 48 hours and 72 hours. The results are shown in Fig. 4B. Over time, the number of detectable AP sites decreased with both pemetrexed and pemetrexed in combination with MX.

Thus, pemetrexed induces the formation of AP sites, although the combination of pemetrexed and 100 μM MX lowered the detected AP sites to control levels, which is not due to the lack of AP sites, but due to the occupation of MX sites, which makes them inaccessible ARP Retention of AP sites is time-dependent, indicating that constant MX levels are required for maximum effect.

EXAMPLE 3

DNA strand break analysis was performed to determine the ability of pemetrexed and methoxyamine (MX) to enhance tumor cell death mediated by apoptosis and DNA strand break. Comet analysis is described in Liu et al. (supra.) and is based on the ability of fragments of denatured, cut DNA to move out of the cell under the influence of an electric field. Intact DNA moves more slowly and remains within the nucleus when an electric current is passed. DNA damage was assessed in the cell based on the shape of the cometary DNA tail and distance of movement (Helma et al., Mutat. Res. 466: 9-15, 2000). Cells were harvested and washed with PBS (phosphate buffered saline) after exposure to 200 μM pemetrexed, 6 mm MX, or 200 μM pemetrexed + 6 mm MX, each for 4 hours. A suspension of H460 cells (1 × 10 ⁵ / ml cold PBS) was mixed with 1% low-melting agarose at 42 ° C at a ratio of 1:10 (v / v), and 75 μl was immediately measured with a pipette on a CometSlide (Trevigen, Inc. , Gaithersburg, MD). When the fusible agarose solidified, the slides were immersed in a pre-cooled lysis buffer (10 mM Tris-HCl, pH 10.5-11.5, 2.5 M NaCl, 100 mM EDTA containing 1% Triton X-100, added just before use) at 4 ° C for 1 h. After lysis, the slides were washed with distilled water, placed longitudinally in an electrophoresis tank and immersed in alkaline buffer (50 mM NaOH, pH 12-12.5, 1 mM EDTA) for 30 min. Then the slides were subjected to electrophoresis in both alkaline (pH> 13, 300 mM NaOH, 1 mM EDTA) and in a neutral solution (1 X TBE) for 25 min at 18 V (0.6 V / cm), 250 mA . Alkaline electrophoresis determines both single- and double-stranded DNA breaks arising from AP sites, as well as other unstable DNA adducts in an alkaline medium, while neutral electrophoresis determines mainly double-stranded DNA breaks. The slides were removed and washed with neutralizing buffer (0.5 M Tris-HCl, pH 7.5) for 10 min, then PBS, then allowed to air dry overnight at room temperature. DNA was stained using a silver staining kit (Trevigen) following the manufacturer's instructions. The comets were visualized using an Olympus microscope. Images were captured using a digital camera and analyzed using the NIH image processing program.

Comet images are shown in FIGS. 1A (alkaline analysis) and 1B (neutral analysis). After treatment with pemetrexed and MX, clearly distinguishable “comets” were observed, and the tail length was approximately 4 times greater than in the case of MX alone and approximately twice as long as in the case of pemetrexed (Fig. 1B-1G).

The results of a study of the effectiveness of xenograft, analysis of AP sites and comet analysis show that MX acts as a structural modulator of AP sites, enhancing the therapeutic effect of the antimetabolic agent pemetrexed by reversing chemotherapy resistance, thereby creating a synergistic effect.

EXAMPLE 4

Analysis of the effect of the AP site or MX-AP site on DNA cleavage mediated by topoisomerase II. A position-specific apurine site was turned on by replacing a single nucleoside with deoxyuridine at the topoisomerase II cleavage site and then removing the uracil base with uracil DNA glycosylase to form AP sites, which were then incubated with MX to obtain MX AP sites (Fig. 5A )

First, it was determined whether APE has a different effect for regular AP sites and MX-AP sites located in a position-specific site with respect to digestion with topoisomerase II. The results show that APE is able to cleave regular AP sites faster than MX-linked AP sites (Fig. 5B), however, both AP and MX-AP sites are cleaved by topoisomerase II, suggesting that MX-AP- sites are able to stimulate topoisomerase II-mediated DNA cleavage.

EXAMPLE 5

Study of MX oral and intravenous bioavailability by administering a single bolus to Sprague Dawley rats (non-GLP). The studies described below were performed to evaluate the bioavailability of methoxyamine (MX) at a safe dose level by comparing the pharmacokinetic parameters after unipolar oral and intravenous administration of MX.

The studied animals. The study used thirty males and thirty female Sprague Dawley rats, 250-350 g with an age of 7-10 weeks.

Receiving and concentration of a dose. A single dose solution was obtained on the day of the dose, adjusted in accordance with 98% purity of the study drug, to achieve a concentration of 4.00 mg / ml of “active” MX, 816.77 mg of MX was dissolved in 5% dextrose in 200 ml volumetric flask. The resulting solution was equally divided into two yellow vessels, intended for either oral or intravenous administration. Aliquots were selected for analysis upon receipt and after dosing, transferred to dry ice and stored at a temperature of less than -70 ° C.

Dose Administration All animals were weighed on the day of the dose. Individual doses for animals were based on his body weight. A constant dose volume of 5 ml / kg was used. Intravenous doses were administered via a single bolus injection into the tail vein using a 3 ml syringe attached to a 26G × 1 needle. An intravenous dose was administered at a rate of approximately 2 ml / min. Oral doses were administered as a single bolus using an 18G × 2 feeding needle. "attached to a 3 ml syringe.

Blood sampling. Blood samples were taken at 5, 15, 30 min and 1, 2, 4, 6, 8, 12 and 24 hours after administration, with sampling at two points in time for each animal. Blood samples were taken from the jugular vein for an earlier point in time and from the abdominal vein when killing for a later point in time. Blood was transferred from a blood sampling syringe into a 2 ml blood collection tube containing K ₃ -EDTA as an anticoagulant and turned upside down for mixing. Blood sampling from the abdominal vein was performed immediately after euthanasia using CO ₂ .

Obtaining a plasma sample and storage conditions. A blood tube was placed on wet ice, then centrifuged to obtain plasma. Whole blood samples were centrifuged at 3,000 rpm at 4 ° C for 10 minutes. Plasma was collected in test tubes in test tubes and first placed on dry ice and then stored at a temperature below -70 ° C.

Mass spectrometry (MS). Mass spectrometric determination was performed using electrospray ionization by positive turbo atomization, according to the specifications listed below.

Instrument for MS: Applied Biosystems 3000

HPLC (High Performance Liquid Chromatography) Instrument: Agilent 1100 Series Binary Pump

Auto Sampler: LEAP Technologies CTC-PAL

Electrospray Ionization Conditions (ESI):

Temperature: 500 ° C

Spray Gas: Nitrogen

gas for CAD (collisional dissociation): nitrogen

DP: 40

Shielding gas (CUR): 10

Collision gas: 6

Ion Spray Voltage (IS): 5000

Output Voltage (EP): 10

NEB: 12

Monitor:

Analyte substance: 207.0 / 149.3 and 207.0 / 178.4 amu

IS (acenocoumarol): 354.2 / 296.0 and 354.2 / 163.1 amu

HPLC Terms (on Agilent 1100):

Mobile phase A: 0.1% formic acid in water

Mobile phase B: 0.1% formic acid in acetonitrile

Column: Thermo Aquasil C18, 50 × 3 mm

Protective column: Thermo Aquasil C18, 10 × 4 mm

Gas flow: 1.0 ml / min

The volume of the injected sample: 50 μl

Gradient:

Time (min) %AT 0,0 0% 2.0 10% 2.2 90% 4,5 90% 4.6 0% 5,6 0%

Plasma sample for LC-MS / MS analysis. Individual plasma samples were thawed at ambient temperature, and an aliquot of 250 μl was taken for analysis if dilution was not required. Samples taken in a period of time from 5 minutes to 1 hour from animals with intravenous administration, or from 15 minutes to 8 hours from animals with an oral dose, required 5-40-fold dilution to get into the linear range of the method (1-1000 ng / ml). For samples requiring dilution, a suitable volume was taken and mixed with untreated rat plasma containing the same anticoagulant to give a final volume of 250 μl. Quantitative results were adjusted using a dilution factor. Plasma for analysis of LC-MS / MS was prepared as follows:

- Aliquots of the plasma were vortexed for 30 s and centrifuged at 14,000 rpm for 10 min to precipitate interfering particles.

- An aliquot of 100 μl was taken from the supernatant and placed in a 1.5 ml microcentrifuge tube.

- To a 100 μl aliquot of plasma was added 310 μl of a mixture of H ₂ O: formic acid (2: 1), 30 μl of acenocoumarol (IS) in H ₂ O (10 μg / ml) and 100 μl of diethylaminobenzaldehyde in a solution of 2: 1 H ₂ O : formic acid (10 mg / ml) and mixed well.

- Then the mixture was incubated in a water bath at 80 ° C for 2 hours. After incubation, the supernatant was transferred to an HPLC vial for quantification by LC-MS / MS.

Analysis of the pharmacokinetics and bioavailability of methoxyamine (MX). Pharmacokinetic analysis

Pharmacokinetic (PK) profiles and oral bioavailability of methoxyamine (MX) were determined on male and female Sprague Dawley rats after a single bolus dose of MX by intravenous and oral administration at a concentration of 20 mg / kg body weight. Thirty rats (15 males and 15 females) were used in the pharmacokinetic analysis for each dosing method. In order to determine the pharmacokinetic characteristics of MX, plasma samples were obtained at predetermined sampling times before dosing 5, 15, 30 minutes, and 1, 2, 4, 6, 8, 12, and 24 hours after dosing. To maintain a normal state of health, blood was withdrawn from each rat a maximum of two times at a given point in time to obtain plasma. A typical MX concentration was obtained by averaging the values at each time point for three rats of the same sex and the dosing method. For the average dose, the average actual sampling time was obtained, respectively, from three rats of the same sex and the dosing method for each nominal time. The average plasma concentration of MX, the average dose, and the average actual sampling time were used for the pharmacokinetic analysis of each dosing method.

Curves of average plasma MX concentration versus time of averaged sample were plotted for each gender and dosing method using Microsoft Excel 2000-SR1 ™ (Figure 2-3). Plasma MX concentrations, recorded as below the quantifiable limits (BQL) or undetectable, if any, are considered 0.00 ng / ml for pharmacokinetic modeling purposes.

Pharmacokinetic parameter analysis was performed using non-compartmental modeling using WinNonlin 5.1 (Pharsight Corporation, Mountain View, CA). Pharmacokinetic parameters included the maximum plasma concentration (C _max ), the maximum plasma concentration time (T _max ), the half-life (t _1/2 ), the area under the plasma concentration curve as a function of time from time zero to the last measured plasma concentration ( AUC _last ), and the area under the plasma concentration curve versus time versus time zero extrapolated to infinity (AUC _0-∞ ). For comparison, AUC _0-∞ resulted in a nominal total dose of MX 5 mg (AUC _0-∞ 5). For pharmacokinetic parameters, the abbreviations indicated above were used.

Bioavailability analysis. Absolute oral bioavailability was determined using the ratio of oral administration of methoxyamine (MX) to intravenous AUC _0-∞ (reduced to a total dose of 5 mg MX) using Microsoft Excel 2000-SR1 ™ using the following formula (MX = TRC102):

Actual MX dose levels were 20.1 mg / kg, 20.1 mg / kg for males and rats in the intravenous dosing group, and 19.9 mg / kg, 20.0 mg / kg for males and female rats in the oral groups, respectively. dosing.

Pharmacokinetics and bioavailability. Table 1 shows the pharmacokinetic parameters of methoxyamine (MX) for male and female Sprague Dawley rats after dosing a single MX bolus by intravenous and oral administration at a concentration of 20 mg / kg body weight.

Table 1 Pharmacokinetic parameters of MX in the plasma of male and female Sprague Dawley rats after dosing a single bolus of MX by intravenous (BB) and oral administration at a concentration of 20 mg / kg body weight Male Female BB oral BB oral The average total dose of MX (mg) 5.63 5.56 4.73 4.61 C _max (ng / ml) 15510 2205 10965 2959 T _max (h) 0.08 1,0 0.08 0.50 t _1/2 (h) 5.2 4.2 4.6 5.7 AUS _last (ng / ml · h) 12518 13596 12971 11643 AUC _0-∞ (ng / ml · h) 12706 13811 13142 12029 AUC _0-∞ (ng / ml · h) ^a 11284 12420 13892 13047 Bioavailability (%) 110 94 F 1,1 0.94 ^a AUC _0-∞ 5 (ng / ml · h) is obtained by normalizing AUC _0-∞ (ng / ml · h) to a total MX dose of 5 mg.

For male rats, plasma concentrations of MX were measured over a full 24-hour controlled period for both intravenous and oral administration. Visual inspection of the curve of the dependence of the intravenous average concentration of MX in the plasma of males on the average time indicates a phase of rapid distribution, which was completed within approximately 2 hours after dosing. C _max for the intravenous route was 15.510 ng / ml and was achieved immediately upon completion of the bolus administration. Systemic exposure to MX, as indicated by AUC _last. and AUC _0-∞ , was 12.518 ng / ml · h and 12.706 ng / ml · h, respectively. The intravenous AUC _0-∞ , reduced to a total nominal dose of 5 mg (AUC _0-∞ 5), was 11.284 ng / ml · h.

For male rats in the oral dosing method, MX absorption was rapid, with T _{max of} 1.0 hour. The C _max value of 2.205 ng / ml was significantly less than with the intravenous dosing route. Systemic exposure to MX, as indicated by oral AUC _last. and AUC _0-∞ , respectively 13.596 ng / ml · h and 13.811 ng / ml · h. The oral AUC _0-∞ , reduced to a nominal total dose of 5 mg (AUC _0-∞ 5), was 12.420 ng / ml · h. The elimination half-lives were short and similar for the two dosing routes (BB: 5.2 h, oral: 4.2 h). The calculated absolute oral bioavailability in male Sprague Dawley rats was 110 percent.

For female rats, there were quantitatively determined plasma concentrations of MX over a full 24-hour sampling period for both the intravenous and oral dosing methods. A visual examination of the curve of the dependence of the intravenous average concentration of MX in the plasma of females on the average time indicates a phase of rapid distribution, which ends in the same way as in male rats, within about 2 hours after dosing. C _max for the intravenous method, C _max was 10.965 ng / ml and started immediately after the completion of the bolus administration. Systemic exposure to MX, as indicated by AUC _last. and AUC _0-∞ , respectively, were 12.971 ng / ml · h and 13.142 ng / ml · h. The intravenous AUC _0-∞ , reduced to a nominal total dose of 5 mg (AUC _0-∞ 5), was 13.892 ng / ml · h.

For female rats with the oral dosing route, MX absorption was fast, with T _max 0.5 hours. The C _max value of 2959 ng / ml was significantly lower than with the intravenous method, but similar to the value for male rats with the oral dosing method. Systemic exposure to MX, as indicated by AUC _last. and AUC _0-∞ , respectively, were 11.643 ng / ml · h and 12.029 ng / ml · h, respectively. The oral AUC _0-∞ , reduced to a nominal total dose of 5 mg (AUC _0-∞ 5), was 13.047 ng / ml · h. The half-life was short and similar for the two dosing methods (BB: 4.6 hours, oral: 5.7 hours) and comparable to that in male rats. The calculated absolute bioavailability in female Sprague Dawley rats was 94 percent.

In rats that were dosed with 20 mg / kg body weight (BW), either intravenously or orally, there were no clinical signs of toxicity.

In both male and female rats, Sprague Dawley MX, administered at a concentration of 20 mg / kg body weight via a single oral bolus, was rapidly (T _max 0.5-1.0 h) and completely absorbed with a systemic absolute bioavailability of approximately 100 percent. Although oral C _max MX is significantly less than BB C _max . systemic exposure to MX, as indicated by AUC _last. and AUC _0-∞ , similarly with two dosing methods. In addition, serum levels exceeding the target C _max are associated with activity in murine models of human cancer (50 ng / ml) after oral dosing at time points, which allows the use of an oral dosing regimen once or twice a day.

These data are important because they indicate that methoxyamine is completely bioavailable when administered orally and has a half-life of 4-6 hours, which allows the achievement of minimally effective concentrations when dosed once or twice a day. Both of these observations are unexpected. Most anticancer agents are not orally bioavailable in amounts sufficient to allow oral dosing. It should be noted that attempts to administer other specific anti-cancer drugs orally resulted in significantly lower bioavailability than achieved in this case. See, for example, bleomycin, carboplatin, cisplatin, oxaliplatin, paxitaxel, ralitrexed (antifolate), topotecan, vinblastine, vincristine, vinorelbine, all of which had a bioavailability of less than 50% (Chu E and DeVita VT. Physicians Cancer Chemotherapy Drug manual 2002. Boston: Jones and Bartlett Publishers, 2002). Secondly, the demonstrated half-life is longer than expected for a small molecule with a molecular weight of less than 100 D, which readily interacts with aldehydes that may be present in plasma, and longer than the expected half-life from plasma, allowing the maintenance of drug levels ( above the minimum effective concentration) with an oral dosing regimen once or twice a day. Full bioavailability and a half-life of 4-6 hours may provide the possibility of oral dosing using the administration scheme once or twice a day, which can be convenient for cancer patients.

Specialists in the art will understand from the foregoing that various modifications of the above methods and compositions can be made without deviating from the essence and scope of the invention. Accordingly, the invention can be embodied in other specific forms without deviating from the essence or its essential features. The present embodiments and examples, therefore, should be considered as illustrative and non-limiting in all respects, and all changes that fall within the meaning and range of equivalence of the claims are therefore intended to be encompassed by it.

Thus, it should be understood that there is no intention to use such terms and expressions to exclude any equivalents of the features shown and described, or their elements, but it should be recognized that various modifications are possible within the scope of the invention as claimed. It should also be understood that each of the narrower variants and subgeneric groups included in the general description of the invention also forms part of the invention. This includes a basic description of the invention with a condition or negative restriction that excludes any object from the class of objects, regardless of whether the excluded material is specifically mentioned here.

All patents, publications, scientific articles, websites and other documents and materials mentioned or noted in this description of the invention are a sign of competence of specialists in the field of technology to which the invention belongs, and each such reference document and material is included in this description inventions by reference to the same extent as if they were incorporated by reference in their entirety individually or as set forth herein in their entirety.

Claims (16)

1. The use of 1) a first composition containing pemetrexed, and 2) a second composition containing methoxyamine, for treating a patient diagnosed with cancer selected from lung cancer, colorectal cancer, ovarian cancer, pancreatic cancer, kidney cancer, endometrial cancer, stomach cancer, liver cancer and breast cancer. 2. The use of claim 1, wherein said cancer is non-small cell lung cancer, colorectal cancer, or breast cancer. 3. The use according to claim 1, where the specified pemetrexed is prepared in the form of a preparation for intravenous or oral administration. 4. The use according to claim 1, where the specified methoxyamine is prepared in the form of a preparation for intravenous or oral administration. 5. The use according to claim 1, where the specified methoxyamine is prepared in the form of a preparation for oral administration. 6. The use according to claim 1, wherein said methoxyamine and said pemetrexed are administered sequentially. 7. The use according to claim 1, where the specified methoxyamine is administered before pemetrexed. 8. The use according to claim 1, where the specified pemetrexed is administered before methoxyamine. 9. The use of claim 1, wherein said pemetrexed in said first composition is pemetrexed disodium heptahydrate. 10. The use according to claim 1, wherein said methoxyamine is prepared in the form of a preparation in an amount sufficient to sensitize said cancer to said pemetrexed. 11. The use according to claim 1, wherein said methoxyamine and said pemetrexed achieve a synergistic effect when administered to a patient. 12. The use according to claim 1, where the specified methoxyamine is administered once or twice a day. 13. The use according to claim 1, where the specified patient is selected as having cancer, partially or completely resistant to treatment with one pemetrexed, and where the specified second drug containing methoxyamine, is prepared in the form of a drug in an amount effective to potentiate the activity of pemetrexed and overcome the indicated resistance when administered to the patient. 14. The use according to claim 1, where the ratio of methoxyamine to pemetrexed is a ratio selected from: from about 1: 2 to about 1: 10000; from about 1: 2 to about 1: 100; from about 1:50 to about 1: 500; from about 1: 450 to about 1: 10000; from about 1:10 to about 1:50; from about 1: 5 to about 1: 500; from about 1:15 to about 1:40; and from about 1:20 to about 1:30. 15. The use according to claim 1, where the ratio of methoxyamine to pemetrexed is from about 1: 2 to about 1: 100. 16. The use according to any one of the preceding claims 1 to 15, wherein said pemetrexed is formulated in a dosage range selected from: from about 25 mg / m ² to about 200 mg / m ² body surface area; from about 150 mg / m ² to about 500 mg / m ² body surface area; from about 400 mg / m ² to about 1000 mg / m ² body surface area; from about 900 mg / m ² to about 5000 mg / m ² body surface area; from about 200 mg / m ² to about 1000 mg / m ² body surface area; and from about 500 mg / m ² to about 600 mg / m ² body surface area.

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