Improved Regimen For Treating Cancer With 5-fluorouracil, 5,10-methylenetetrahydrofolate And Capecitabine

IMPROVED REGIMEN FOR TREATING CANCER WITH 5-FLUOROURACIL, 5,10-METHYLENETETRAHYDROFOLATE AND

CAPECITABINE

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Nos. 60/893,324, filed March 6, 2007, and 60/894,161, filed March 9, 2007, incorporated in their entirety herein.

BACKGROUND OF THE INVENTION

[0002] The common mode of action of intravenously administered 5-FU and oral fluoropyrimidines (e.g. capecitabine or XELOD A®) predicts similar efficacy of these two agents. Preclinical and clinical studies comparing 5-FU and XELODA treatment regimens confirm this. For example, Phase III clinical studies that compared 5-FU plus leucovorin treatment to XELODA treatment in metastatic colorectal cancer showed essentially equivalent time to tumor progression (4.6 and 4.7 months, respectively) and overall survival (12.9 and 12.8 months, respectively). As such, it has been suggested that XELODA is an acceptable replacement to 5-FU.

[0003] The choice of using 5-FU or XELODA often comes down to doctor or patient preference, with each drug having its own advantages and disadvantages. For example,

XELODA has the potential convenience factor of an oral drug. In contrast, the intravenous delivery of 5-FU requires patient visits to the clinic for dosing. However, the recommended dosing schedule (1250 mg/m2 twice a day comprising approximately 10 tablets per dose) and patient responsibility for self-administration of XELODA contributes to patient noncompliance. Under the controlled environment of the clinic, there is not as great an issue of patient noncompliance for 5-FU. Moreover, the safety profile of XELODA compared to 5-FU, including less XELOD A-induced diarrhea, nausea, and stomatitis have also been suggested as advantages of XELODA. However, XELODA causes hand-foot syndrome in a significant proportion of patients as soon as 11 days after initiation of treatment, requiring either dose reduction or interruption. In turn, dose reduction has been shown to potentially have a negative impact on time to tumor progression. In contrast, 5-FU does not cause a significant amount of hand foot syndrome.

[0004] Folinic acid (leucovorin) has been used as a modulator of 5-fluorouracil (5-FU) for cancer treatment, and leucovorin plus 5-FU has become the standard combination therapy for many cancer types, including colorectal cancer. However, leucovorin is inactive directly and must undergo several metabolic transformations to its active metabolite 5,10- methylenetetrahydrofolate (COF ACTOR®; or 5,10-CH2FH4) to be effective. In contrast, COFACTOR supplies 5,10-methylenetetrahydrofolate directly and has demonstrated enhancement of the anti-tumor effects of 5-FU in Phase I/II human clinical trials for colorectal and breast cancer. Furthermore, preclinical tumor xenograft models have previously shown that COFACTOR significantly enhances 5-FU anti-tumor activity when given as a daily treatment for 7 consecutive days. Furthermore, COFACTOR also reduced 5- FU induced weight loss compared to leucovorin in this treatment regimen.

[0005] Given the same mechanism of action, time to tumor progression, and survival following treatment with XELODA or 5-FU as individual drugs, it is not obvious to use 5-FU and XELODA together in a combination treatment. Thus, it is surprising to find in a human colorectal xenograft model that a combination regimen of sequential treatment of 5-FU plus COFACTOR on days 1 and 8 and XELODA on days 2-7 and 9-14 provides a synergistic effect in the treatment of colorectal cancer. The combination treatment of 5-FU plus COFACTOR and XELODA results in significantly better anti-tumor activity, prolonged animal survival, and less toxicity than XELODA treatment alone on days 1-14, or treatment with 5-FU plus leucovorin on days 1 and 8 and XELODA on days 2-7 and 9-14.

BRIEF SUMMARY OF THE INVENTION [0006] In one aspect, the present invention provides a method of potentiating the therapeutic effect of 5-fluorouracil (5-FU) and 5,10-methylenetetrahydrofolate upon tumor load in a patient hosting 5-FU sensitive tumors. The first step of the method involves cyclic administering to the patient a combination of 5-FU and 5,10 methyl enetetrahydro folate. The second step of the method involves administering capecitabine 1 day after the first step i where the administration of capecitabine is in an amount that potentiates the therapeutic effect of 5-fluorouracil by reducing the tumor load in the patient to a greater amount than treatment with 5-FU and 5,10-methylenetetrahydrofolate without capecitabine. [0007] In one embodiment of the method the present invention, the capecitabine is administered at a daily dosage of between 625 and 1250 mg/m2 twice a day. In another embodiment, the capecitabine is administered orally. In yet another embodiment, the administration of capecitabine occurs daily for between 1 to 6 days before the patient is again treated with 5-FU and 5, 10-methylenetetrahydrofolate.

[0008] In other embodiments of the present invention, the cyclic administering to the patient of a combination of 5-FU and 5,10 methyl enetetrahydrofolate occurs at least three times over a period of three weeks.

[0009] In a further embodiment, the present invention provides the method where the tumor is colorectal cancer. In another embodiment, the tumor is a cancer of the breast. In another embodiment, the tumor is a cancer of the pancreas, hi another embodiment, the tumor is a cancer of the stomach.

[0010] In another embodiment, the present invention provides a method where the treatment further includes co-administration of a chemotherapeutic drug selected from the group consisting of cisplatin, oxaplatin, irinotecan, gemcitabine, bevacizumab and cetuximab.

[0011] In other embodiments, the present invention provides a method where the administration of 5-FU is between 250 and 1000 mg/m2 per treatment. In still other embodiments, the administration of 5,10 methylenetetrahydro folate is between 50 and 70 mg/m2 per treatment. [0012] In a second aspect, the present invention provides a method of reducing adverse side effects arising from treatment of 5-fluorouracil and 5,10-methylenetetrahydrofolate in a patient hosting 5-FU sensitive tumors. The first step of the method involves cyclic administering to the patient a combination of 5-FU and 5,10-methylenetetrahydrofolate. The second step of the method involves administering capecitabine 1 day after the first step i where the administration of capecitabine is in an amount that potentiates the therapeutic effect of 5-fluorouracil while reducing adverse side effects in the patient compared to side effects arising from treatment with 5-FU and 5,10-methylenetetrahydrofolate without capecitabine.

[0013] In a further embodiment, the present invention provides the method of reducing having the preferred embodiments described above. [0014] In a third aspect, the present invention provides a method of reducing adverse side effects of a treatment regimen consisting of daily doses of capecitabine in a patient with cancer. The first step of the method involves cyclic administering to the patient a combination of 5-FU and 5,10-methylenetetrahydrofolate. The second step of the method involves administering capecitabine 1 day after the first step i where the cyclic administration of 5-FU and 5,10-methylenetetrahydrofolate followed by the administration of capecitabine reduces the adverse effects of a treatment regimen consisting of daily doses of capecitabine alone.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1. Figure 1 is a graphical representation of treatment regimens 1-4: 1) Water, PO daily, 14 days (days 1-14); 2) XELODA, PO daily, 14 days (days 1-14); 3) Leucovorin plus 5-FU, IP, weekly (days 1 and 8) and XELODA, PO daily, (days 2-7 and 9- 14); and 4) COFACTOR plus 5-FU, IP, weekly (days 1 and 8) and XELODA, PO daily, (days 2-7 and 9-14).

[0016] Figure 2. Figure 2 is a graphical representation showing tumor growth kinetics for all four treatment regimens. The COFU→XELODA treatment (regimen no. 4) shows slower growth kinetics as compared to the other three treatment regimens.

[0017] Figure 3. Figure 3 is a graphical representation of the percent of mice surviving over time for each of the four different regimen. The graph shows that COFU→XELODA treatment (regimen no. 4) prolonged mouse survival compared to the other regimens. The median survival of COFU→XELODA treated mice (60 days) was significantly longer (p < 0.05) than FOFU→XELODA (regimen no. 3, 39 days) and XELODA alone (38 days). In addition, the XELODA and FOFU→XELODA treatment did not significantly change survival compared to control (water) treated mice (44.5 days).

[0018] Figure 4. Figure 4 is a graphical representation of weight loss in mice, showing a small changes in body weight for COFU→XELODA treatment as compared to treatment with water alone. Treatment with FOFU→XELODA and XELODA alone, showed significantly more weight loss as compared to treatment with water. [0019] Figure 5. Figure 5 shows a graphical representation of the NCI common toxicity criteria weight loss grading. Treatment with COFU→XELODA showed less grade 1 toxicity at day eight and no grade 2 toxicity at day 15. In contrast, both XELODA alone and FOFU→XELODA showed greater grade 1 toxicity at day 8 and up to grade 2 weight loss toxicity at day 15.

DETAILED DESCRIPTION OF THE INVENTION I. General

[0020] The treatment of colorectal cancer with 5, 10-methylenetetrahydro folate (5,10-CH2FH4) in combination with either 5-fluorouracil (5-FU) or capecitabine (XELOD A™), is known to improve the treatment of cancer while lowering toxicity as compared to treatment with the individual drugs. However, the efficacy of each treatment is no better than the other, as predicted by the same mechanism of action of each drug. The present invention provides the surprising discovery that treatment of cancer with a combination of 5-FU and 5,10-CH2FH4 approximately once a week, followed by treatment with capecitabine on the days in between the 5-FU and 5,10-CH2FH4 combination treatment, results in lower toxicity, fewer side effects and improved patient survival. [0021] The present invention provides a novel regimen for potentiating the therapeutic effect of 5-FU and 5,10-CH2FH4 upon tumor load in a patient hosting 5-FU sensitive tumors. The method comprises a first step of cyclic administering to the patient a combination of 5- FU and 5,10-CH2FH4. The second step involves administering capecitabine 1 day after the first step where the administration of capecitabine is in an amount that potentiates the therapeutic effect of 5-FU by reducing the tumor load in the patient to a greater amount than treatment with 5-FU and 5,10-CH2FH4 without capecitabine.

[0022] One embodiment of the present invention relates to the use Of S5IO-CH2FH4 as a modulator of 5-FU in cancer chemotherapy. 5,10-CH2FH4 increases response rates to 5-FU as a result of increasing the inhibition of TS by the 5-FU metabolite, FdUMP, in tumors. Thus, 5,10-CH2FH4 can be used to inhibit the growth of tumors when used in combination with 5-FU, or with other drugs which are metabolized to FdUMP including floxuridine (FUDR), ftorafur (tegafur), and Doxifluridine™ (5'-deoxyfluorouridine) and capecitabine.

[0023] The mechanism of action of 5, 10-CH2FH4 is promotion of TS inhibition by FdUMP in fluoropyrimidine-treated tumors, which can occur by increasing the rate of formation and stability of TS-FdUMP-5,10-CH2FH4 and TS-FdUMP FH4 ternary complexes. [0024] The reduced toxicity of 5 -FU (or an analog or prodrug thereof) when combined with 5,10-CH2FH4 can permit drug regimens in which 5,10-CH2FH4 and 5-FU (or an analog or prodrug thereof) are used in combination with one or more additional anti-cancer drugs that would be prohibitively toxic in the absence OfCH2FH4. II. Definitions

[0025] "5-FU sensitive tumors" refers to tumors that can be treated by administration of 5-FU. The administration of 5-FU to the tumor results in one or more of the following: growth inhibition of the tumor; decrease in size of the tumor; abatement; remission; diminishing of symptoms or making the symptom, injury, pathology or condition more tolerable to the patient; or decreasing the frequency or duration of the symptom or condition.

[0026] "Cyclic administering" refers to a treatment protocol involving repeated administration of a primary drug treatment to a patient at set intervals. The interval can be days or weeks. Preferably, the interval is several days, or about a week. During the interval between treatments, a secondary drug treatment can be administered to the patient. Typically, cyclic administering refers to the administration of at least two primary drug treatments.

[0027] "Efficacy" of an anticancer treatment or chemotherapy regimen is determined by its anti-tumor or anti-cancer cell effects and ability to improve clinical results of treatment, such as, for example, remission, time to progression, response rate, and survivorship. Accepted methods of assessing the efficacy of an anticancer treatment or chemotherapy regimen are well-established in the field of cancer treatment. For example, anti-cancer effects can be assessed by detecting cancer cells or markers, for example in serum or plasma. Examples of tumor proteins or antigens that can be detected include CEA for colon cancer and CA 19-9 for pancreatic cancer. For solid tumors, anti-tumor effects can be measured by monitoring tumor size and the change in tumor size over time.

[0028] "Patient" refers to animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. Li certain embodiments, the patient is a human.

[0029] "Potentiating the therapeutic effect" refers to increasing or multiplying the therapeutic effect of a drug by administering the drug in combination with, or simultaneous to, another drug. The method of the present invention is a method of potentiating the therapeutic effect of 5-FU and 5, 10-methylenetetrahydro folate administered in combination.

[0030] "Toxicity" refers to harmful effects of an entity on the cells, tissues, organs, or systems of the body. Toxic effects result from biochemical reactions of the entity with the cells or tissues of the subject being treated, and can be general or specific, involving a particular system or organ. Toxicity can include, as non-limiting examples, increased lacrimation; mucositis; esophagopharyngitis; neurological toxicity, such as parasthesias, insomnia, and dizziness; gastrointestinal toxicity, such as nausea, vomiting, and diarrhea; weight loss toxicity; cardiac toxicity; dermatological toxicity, including alopecia, sweating, and rashes; and hematological toxicity, such as, but not limited to, neutropenia, thrombocytopenia, lymphopenia, and leucopenia. Clinical definitions of toxicity parameters can be found in the National Cancer Institute's Common Toxicity Criteria (version 3) or in the World Health Organization Toxicity Criteria.

[0031] "Tumor load" refers to the amount or size of a tumor in a patient. III. Method of Potentiating the Therapeutic Effect of 5-FU, 5,10-CH2FH4 and Capecitabine

[0032] The present invention provides a novel regimen for potentiating the therapeutic effect of 5-fluorouracil (5-FU) and 5,10-methylenetetrahydrofolate (5,10-CH2FH4) upon tumor load in a patient hosting 5-FU sensitive tumors. The combination of 5-FU, 5,10-CH2FH4 and capecitabine results in a synergistic effect for the treatment of 5-FU sensitive tumors. The synergistic effect is afforded by the cyclic administering to a patient a combination of 5-FU and 5,10-CH2FH4. The cyclic administering involves the administration of 5-FU and 5,10-CH2FH4 on one day, followed by a subsequent administration of 5-FU and 5,10-CH2FH4 several days later. Typically, the interval is about one week. On the days when the 5-FU and 5,10-CH2FH4 are not being administered, an oral dose of capecitabine is administered.

A. Patients in need

[0033] Patients that benefit from the methods of the present invention are those patients suffering from a 5-FU sensitive cancer, such as, for example, colorectal carcinoma, pancreatic, breast, head and neck, esophageal cancer, or stomach cancer. In some preferred embodiments of the present invention, the patient has a tumor type that in current practice is not commonly treated with 5-FU, such as, but not limited to ovarian cancer or cervical cancer.

[0034] In some embodiments of the present invention, treating a cancer patient with 5,10- CH2FH4, 5-FU (or an analog or prodrug thereof), and capecitabine can reduce the rate of tumor growth in a cancer patient when compared with treating the patient with capecitabine in the absence of 5,10-CH2FH4 and 5-FU (or an analog or prodrug thereof), or when compared with treating a patient with 5-FU (or an analog or prodrug thereof) and capecitabine in the absence of 5,10-CH2FH4.

[0035] In other embodiments of this aspect of the present invention, treating cancer patients with 5,10-CH2FH4, 5-FU (or an analog or prodrug thereof), and capecitabine can increase the survivorship of cancer patients when compared with treating cancer patients with capecitabine in the absence Of S5IO-CH2FH4 and 5-FU (or an analog or prodrug thereof) or when compared with treating cancer patients with 5-FU (or an analog or prodrug thereof) and capecitabine in the absence of 5,10-CH2FH4. [0036] The treatment methods of the present invention provide improved treatment of cancer, lower toxicity, lower side effects and improved patient survival. In one respect, the synergistic effect afforded by the use of 5,10-CH2FH4 and 5-FU in combination with capecitabine, allows better efficacy in the treatment of cancer along with lower dosing levels of the drugs. Accordingly, patients sensitive to the side effects and toxicity of the individual drugs will benefit from the combination treatment as the cancer can be effectively treated at lower doses, thus reducing the side effects to the patient. In another respect, the lower side effects and toxicity afforded by the use of 5,10-CH2FH4 and 5-FU in combination with capecitabine can allow dose escalation of one or more of the drugs in the treatment regimen to allow improved efficacy. In total, the use Of S5IO-CH2FH4 and 5-FU in combination with capecitabine allows a wider dosing range of drugs while maintaining or enhancing overall efficacy of treatment.

B. Formulations

[0037] Formulations of 5-FU are commercially available from American Pharmaceutical Partners, Inc. The formulations are available in a variety of sizes (10 mL to 100 mL). Typically concentrations for the formulations are 50 mg/mL. Formulations of 5-FU comprise 5-FU in water, with the pH adjusted to 9.2 using sodium hydroxide. [0038] Formulations of capecitabine are available from Roche. The formulations are available as 150 mg and 500 mg film-coated tablets. The core of the tablets comprise the following excipients: anhydrous lactose, croscarmellose sodium, hypromellose, microcrystalline cellulose and magnesium stearate. The tablet coating comprises the following excipients: titanium dioxide, yellow and red iron oxide, and talc.

[0039] Formulations of 5, 10-CH2FH4 comprise 5, 10-CH2FH4 in combination with citric acid and ascorbic acid, at an essentially neutral pH. As used herein, the term "essentially neutral pH" means a pH of 7.0 ± 0.2. The relative amounts of citric acid and ascorbic may vary, without substantially affecting the stability of the composition, from about 0.75:1 to about 2.25:1 weight ratio of citric acid to ascorbic acid. In accordance with a preferred embodiment, citric acid and ascorbic acid are present at a weight ratio of about 1.5:1.

[0040] The formulation of 5, 10-CH2FH4 comprises the steps of (a) preparing a solution of citric acid and ascorbic acid at an essentially neutral pH; and (b) dissolving 5,10-CH2FH4 in the solution. Preferably, the solution of citric acid and ascorbic acid is chilled to 10° C and kept chilled at this temperature until all of 5,10-CH2FH4 has gone into solution. In step (a), the essentially neutral pH of the solution of citric acid and ascorbic acid is obtained by adjusting and/or buffering the pH of the solution in any manner known in the art, such as with NaOH or HCl. The relative amounts of citric acid and ascorbic may vary, without substantially affecting the stability of the composition, from about 0.75:1 to about 2.25:1 weight ratio of citric acid to ascorbic acid. Once all of the 5,10-CH2FH4 has gone into solution, the formulated 5,10-CH2FH4 may then be filled into vials and lyophilized.

C. Administration

[0041] In practicing the methods of the present invention, the pharmaceutical compositions can be used alone, or in combination with other therapeutic or diagnostic agents. The pharmaceutical compositions can be administered to the patient in a variety of ways, including topically, parenterally, intravenously, colonically, rectally or intraperitoneally. Preferably, the pharmaceutical compositions are administered parenterally, intravenously, or orally. The additional anticancer drugs used in the combination protocols of the present invention can be administered separately or one or more of the anticancer drugs used in the combination protocols can be administered together. Where one or more anticancer drug is administered separately, the timing and schedule of administration of each drug can vary. [0042] For example, bolus injection of each drug can be given once weekly for a number of weeks. Preferably, 5,10-CH2FH4 is administered prior to 5-FU or 5-FU analog or prodrug. For example, the patient can receive the 5,10-CH2FH4 dose from about 10 minutes to about four hours prior to receiving the 5- FU dose. The protocol for the combination therapy is not limiting, and can include any feasible administration protocols with respect to frequency, duration, and dosage.

[0043] Following treatment of the 5,10-CH2FH4 and 5-FU combination, capecitabine can be administered orally on a daily basis until the next 5,10-CH2FH4 and 5-FU combination treatment, approximately about one week. The treatment time for capecitabine can be as low as 1 day, and as high as 6 days. Preferably, capecitabine can be administered for about 6 days. After the capecitabine treatment, another treatment of 5,10-CH2FH4 and 5-FU can be administered. This can be again followed by treatment with capecitabine for about as low as 1 day and as high as about 6 days. Accordingly, capecitabine can be administered only on days when the 5,10-CH2FH4 and 5-FU combination are not administered. One of skill in the art will appreciate that other treatment regimens are useful in the present invention.

[0044] Some examples of anticancer drug protocols that use capecitabine are described in Blum JL, et al. "Multi center phase II study of capecitabine in paclitaxel-refractory metastatic breast cancer." J Clin Oncol 1999; 17: 485-93; in Hoff et al. "Comparison of oral capecitabine versus intravenous fluorouracil plus leucovorin as first-line treatment in 605 patients with metastatic colorectal cancer: results of a randomized phase III study." J Clin Oncol 2001;19(8):2282-92; and in Van Cutsem E, et al. "Oral capecitabine compared with intravenous fluorouracil plus leucovorin in patients with metastatic colorectal cancer: results of a large phase III study." J Clin Oncol 2001; 19 (21):4097-106; all of which areiierein incorporated by reference, in particular for disclosure of chemotherapy regimens using capecitabine. The present invention includes administering 5,10-CH2FH4 in protocols that include capecitabine to reduce toxicity of capecitabine treatment.

[0045] A number of chemotherapy protocols that combine 5-FU with one or more anticancer drugs are known in the field of cancer therapy. "Randomized trial of intensive cyclophosphamide, epirubicin, and fluorouracil chemotherapy compared with cyclophosphamide, methotrexate, and fluorouracil in premenopausal women with node- positive breast cancer." J Clin Oncol 1998; 16(8): 2651-8; herein incorporated by reference, particularly for disclosure of anticancer protocols that use 5-FU.) Anticancer protocols that include 5-FU in combination with one or more additional drugs (other than a folate cofactor) also include therapies for breast cancer that include cyclophosphamide, doxorubicin, and fluorouracil (see, for example, Bennett JM, Muss HB, Doroshaw JH, et al. "A randomized multicenter trial comparing mitoxantrone, cyclophosphamide, and fluorouracil with doxorubicin, cyclophosphamide, and fluorouracil in the therapy of metastatic breast cancer." J Clin Oncol 1988;6(10):1611-20; herein incorporated by reference, in particular for disclosure of anticancer protocols that include 5-FU.) The present invention includes the addition of 5,10-CH2FH4 to chemotherapy regimens such as these to reduce the toxicity of the chemotherapy regimens. [0046] The regimen of the present invention can be used to treat 5-FU sensitive cancers in combination with a chemotherapeutic drug selected from platins, topoisomerase inhibitors, angiogenesis inhibitors, and epidermal growth factor receptor inhibitors (e.g., cisplatin, oxaplatins, irinotecan, bevacizumab and cetuximab).

[0047] Yet other types of protocols to which 5,10-CH2FH4 can be added to reduce the toxicity of treatment are anticancer protocols that combine 5-FU with mitomycin C, such as that disclosed in Keane TJ, Cummings BJ, O'Sullivan B, Payne D, Rawlinson E, MacKenzie R, Danjoux C, Hodson I. "A randomized trial of radiation therapy compared to split course radiation therapy combined with mitomycin C and 5 -fluorouracil as initial treatment for advanced laryngeal and hypopharyngeal squamous carcinoma." IJ Radiation Oncology Biol Phys, 1993:25 (4):613-8; incorporated by reference, in particular for disclosure relating to anticancer protocols that include 5-FU, and others that combine the use of carboplatin with 5- FU as disclosed in Calais G, Alfonsi M, Bardet E, et al. "Randomized trial of radiation therapy versus concomitant chemotherapy and radiation therapy for advanced-stage oropharynx carcinoma." J Natl Cancer Inst 1999; 91 :2081-6, herein incorporated by reference, in particular for disclosure relating to anticancer protocols that include 5-FU. In these treatments, anticancer treatment protocols include radiation therapy in addition to chemotherapy.

[0048] The present invention provides a treatment regimen of first administering on the same day, 5,10-CH2FH4 and 5-FU. Following administration of 5-FU and 5,10-CH2FH4, capecitabine can be administered on days when 5,10-CH2FH4 and 5-FU are not administered. Capecitabine can be administered for at least about 1 day, or up to about 6 days. Preferably, capecitabine can be administered for about 6 days. After the capecitabine treatment, another treatment of 5,10-CH2FH4 and 5-FU can be administered. This can be again followed by treatment with capecitabine for about as low as 1 day and as high as about 6 days. Typically, capecitabine can be administered on a daily basis for about 14 days consecutive days, followed by a week without treatment of capecitabine. [0049] In clinical studies, number of lesions, tumor size, and tumor growth rate can be monitored by radiography, tomography, and, where possible, direct measurement of tumor mass. Anti-tumor effects can also be measured using molecular biology and biochemistry techniques, such as ELISA, PCR, western blotting, or immunocytochemistry.

[0050] The pharmaceutically effective amount of a composition required as a dose will depend on the route of administration, the type of cancer being treated, and the physical characteristics of the patient. The dose can be tailored to achieve a desired effect, but will depend on such factors as body surface area, weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize.

[0051] Those skilled in the art of cancer treatment and chemotherapy would be able to determine optimal dosages and regimens for 5,10-CH2FH4, 5-FU and capecitabine using well- established protocols for evaluating toxicity and efficacy for individual patients. Some preferred treatments of cancer patients with 5-FU and 5,10-CH2FH4 are regimens using from 10 milligrams to 1 gram Of S5IO-CH2FH4 per m2, preferably from 20 milligrams to 500 milligrams of 5,10-CH2FH4 per m2, and more preferably from about 30 milligrams to about 250 milligrams of 5,10-CH2FH4 per m2. For example, a preferred dose of 5,10-CH2FH4 can be from about 50 to about 70 milligrams per m2.

[0052] Administration of 5-FU can be performed at a variety of dosages less than about 5 gram per m2, e.g., from about 25 milligrams to about 5 grams per m2, e.g., from about 50 milligrams to 2.5 grams per m , e.g., from about 100 milligrams to about 1 gram per m . For example, a preferred dose of 5-FU can be from about 250 to about 1000 milligrams per m2.

[0053] The daily dosage of capecitabine, for example, can be less than about 7500 mg per m2, e.g., from about 500 mg to about 7500 mg per m2, e.g., from about 1000 mg to about 5000 mgs per m2, e.g., from about 1500 mg to about 3000 mg per m2. The dose can be divided into one to six (preferably two) administrations per day. [0054] The foregoing are general guidelines only that can be expanded or altered based on, for example, cancer type and grade, patient age, health status, and sex, the particular drugs used in combination, the route and frequency of administration, and experimental and clinical findings using a multidrug combination. Capecitabine can be administered as an oral formulation or by any other feasible means.

IV. Examples

Example 1. Formulation of 5,10-CH2FH4

[0055] This example provides a representative method of formulating a lyophilized composition of 5,10-CH2FH4 at an essentially neutral pH comprising citric acid and ascorbic acid.

[0056] Procedure:

1. Sparge WFI with filtered Nitrogen Gas, NF for 30 min.

2. Record tare wt of 100 mL plastic bottle.

3. Weigh out citric acid, ascorbic acid and about 90 g N2 sparged water. Mix to dissolve. 4. Adjust pH to 7.0 ± 0.1 with IN NaOH or HCl.

5. Chill the solution to 100C

6. Add 5,10-CH2FH4, mix to dissolve.

7. Record pH (7.0 ± 0.2).

8. Add more water to HO g final weight (or 100 mL). Record wt. 9. Pass through a 0.2-micron filter while keeping the solution chilled as possible.

10. Fill into vials (2mL or 100 mg 5,10-CH2FH4 calcium salt per vial) while keeping the solution chilled as possible.

11. Freeze dry.

12. Seal vials under slight vacuum with nitrogen in the headspace. 13. Crimp the vials. Example 2. Treatment of colon carcinoma with 5-FU/5,l 0-CH2FH4 + Capecitabine

[0057] This Example provides a new regimen for the treatment of cancer using existing anti-cancer drugs and protocols. The combination of existing drugs and protocols in a novel regimen has surprisingly resulted in improved treatment of colorectal cancer, as well as a reduction in drug toxicity and adverse side effects. The novel regimen of the instant invention also improved patient survival.

[0058] Human colon carcinoma HT-29 cells were injected into mice and treated according to the following regimens:

1) Water, PO daily, 14 days (days 1-14); 2) XELODA, PO daily, 14 days (days 1-14);

3) Leucovorin plus 5-FU, IP, weekly (days 1 and 8) and XELODA, PO daily, (days 2- 7 and 9-14); and

4) COFACTOR plus 5-FU, IP, weekly (days 1 and 8) and XELODA, PO daily, (days 2-7 and 9-14). For further clarity, the individual treatment regimens are presented graphically (Figure 1).

[0059] Mice were female 6-8 week old nude mice (nu/nu) were obtained from Harlan, Inc. Four mice per cage were maintained in isolated, hepa-fϊlter-ventilated cages.

[0060] The human colon carcinoma HT-29 was obtained from American Type Culture Collection (ATCC). Cells were maintained in complete media (DMEM containing 10% fetal bovine serum, 2 mM 1-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin) in a 370C, 5% CO2 humidified incubator. Cells were passaged every 2-3 days prior to use in vivo.

[0061] 5-Fluorouracil (5-FU) and leucovorin (folinic acid; 5-formyltetrahydrofolate pentahydrate) were obtained from Sigma- Aldrich. COFACTOR (fotrexorin; 5,10- methylenetetrahydofolate) was obtained from Merck Eprova AG. XELOD A™ (Capecitabine) was obtained from Roche.

[0062] HT-29 cells were prepared for subcutaneous implantation as follows. Tissue culture flasks of semi-confluent HT-29 cells were washed once with phosphate buffered saline (PBS) followed by cell detachment with trypsin. Detached cells were then washed once in complete media followed by one wash with PBS. Finally, cells were diluted to 107 cells/mL in PBS. Mice were inoculated subcutaneously with 106 HT-29 cells (100 μL). Two weeks following tumor implantation, when mean tumor volumes reached approximately 90-100 mm , mice were randomized to 20 mice per group and drug treatments were initiated. Tumor volume was calculated using the following formula: tumor volume = (Length x Width2)/2. Mice were euthanized by CO2 followed by cervical dislocation either when a tumor reached >2cm in diameter or upon tumor ulceration.

[0063] The drugs were dosed as follows. 5-FU, Leucovorin, and COFACTOR were each injected intraperitoneally (IP) at 0.6 mg/mouse/drug (approximately 30 mg/kg body weight; 90 mg/m2). XELODA™ was delivered by oral gavage (PO) at 7.5 mg/mouse (approximately 375 mg/kg body weight; 1125 mg/m2). Water (200 μL/mouse) was delivered by oral gavage. Mice were randomized to four separate treatment groups (Table 1):

Table 1. Treatment Groups

Group # Treatment Mice/group

1 Water 20

2 XELODA 20

3 Leucovorin plus 5-FU plus XELODA (FOFU - > XELODA) 20

4 COFACTOR plus 5-FU plus XELODA (COFU - → XELODA) 20

Total 80

[0064] Tumor growth inhibition. Sequentially measured tumor volumes were plotted versus time (Figure 2). COFU-→XELODA resulted in greater inhibition of tumor growth compared to the other drug regimens. On day 26, the mean tumor volume of COFU→XELODA treated mice (288.5 mm3 ± 32.6, mean ± SEM) was significantly less (p < 0.05) than both XELODA alone (461.0 mm3 ± 50.4) and FOFU→XELODA (447.8 mm3 ± 46.5) treatment groups. In contrast, XELODA and FOFU→XELODA treatment did not significantly inhibit tumor growth any more than control (water) treated mice (495.1 mm3 ± 61.8). [0065] In addition to tumor inhibition differences at day 26, tumor growth inhibition by COFU→XELODA was also detected at early time points following treatment initiation. As soon as day 8 of treatment, the mean tumor volume of COFU-→XELODA treated mice (139.6 mm3 ± 10.8) was significantly less than both XELODA alone (196.7 mm3 ± 13.7) and FOFU→XELODA (202.1 mm3 ± 16.8) treatment groups. [0066] Animal Survival. Percent mice surviving over time was plotted (Figure 3).

Consistent with the tumor inhibition results, COFU-→XELODA treatment prolonged mouse survival compared to the other drug regimens. The median survival of COFU→XELODA treated mice (60 days) was significantly longer (p < 0.05) than FOFU→XELODA (39 days) and XELODA alone (38 days). In contrast, XELODA and FOFU→XELODA treatment did not significantly change survival compared to control (water) treated mice (44.5 days). [0067] The cause of death for the majority of mice was euthanasia due to tumor ulceration or tumor diameters >2cm. However, a small percentage of mice in the XELODA only treatment group (15%) and FOFU→XELODA (5%) died following the onset of clinical signs of illness or potentially drug-related toxicity (e.g. weight loss and lethargy), hi contrast, there were no deaths secondary to clinical signs of illness or drug-related toxicity in either the COFU→XELODA or control (water) treatment groups.

[0068] Drug-related toxicity. Mouse body weights were followed over the course of treatment as a biologic readout of drug toxicity (Figure 4). The percent change in body weight was determined on days 8 and 15 of the study (% change body weight = [(day 8 or 15 body weight)/(day 1 body weight)-l]*100). Compared to the mean percent change body weight of control (water) treated mice on day 8 (0.94% increase ± 0.68) or day 15 (-0.06% decrease ± 0.71), COFU→XELODA treatment did not cause significant changes (p > 0.05) in body weight on either day 8 (-1.30% decrease ± 0.59) or day 15 (-1.76% decrease ± 0.83). In contrast, FOFU→XELODA treatment caused significantly more (p < 0.05) weight loss on day 8 (-2.16% decrease ± 0.87) and day 15 (-4.92% decrease ± 1.21) compared to control treated mice. Furthermore, the maximum severity of weight loss following

COFU→XELODA treatment (-5.95% and -9.66% on days 8 and 15, respectively) was less than the maximum severity of weight loss following FOFU→XELODA treatment (-9.13% and -19.58% on days 8 and 15, respectively).

[0069] Data transformed to NCI Common Toxicity Criteria for weight loss are also plotted for days 8 and 15 (Figure 5). This data is consistent with the differences detected in percent body weight changes. On day 8, COFU→XELODA treatment resulted in a smaller percentage (6%) of grade 1 toxicity compared to either XELODA alone (15%) or FOFU→XELODA (20%). Furthermore, on day 15, grade 1 weight loss was the maximum toxicity grade seen in COFU→XELODA treated mice. In contrast, both XELODA alone and FOFU→XELODA caused up to grade 2 weight loss toxicity.

[0070] Statistical analysis of data was performed using GraphPad Prism scientific software. Bonferonni's t test was used to compare mean tumor volumes between multiple groups. Dunnett's multiple comparison test was used to compare mean body weights between treatment groups. The Logrank test was used to determine statistical differences between group survival curves.

[0071] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications can be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.

WHAT IS CLAIMED IS:

L A method of potentiating the therapeutic effect of 5-fluorouracil (5-FU) and 5, 10-methylenetetrahydro folate upon tumor load in a patient hosting 5-FU sensitive tumors, the method comprising: i) cyclic administering to the patient a combination of 5-FU and 5,10 methylenetetrahydrofolate; and, ii) administering capecitabine 1 day after step i where the administration of capecitabine is in an amount that potentiates the therapeutic effect of 5-fluorouracil by reducing the tumor load in the patient to a greater amount than treatment with 5-FU and 5,10-methylenetetrahydrofolate without capecitabine.

2. The method of claim 1 where the capecitabine is administered at a dosage of between 625 and 1250 mg/m2 twice a day.

3. The method of claim 1 where the capecitabine is administered orally.

4. The method of claim 1 where the administration of capecitabine occurs daily for between 1 to 6 days before the patient is again treated with 5-FU and 5,10-methylenetetrahydrofolate.

5. The method of claim 1 where the cyclic administering to the patient of a combination of 5-FU and 5,10 methylenetetrahydrofolate occurs at least three times over a period of three weeks.

6. The method of claim 1 where the tumor is colorectal cancer.

7. The method of claim 1 where the tumor is a cancer of the breast, pancreas or stomach.

8. The method of claim 1 where the treatment further includes co- administration of a chemotherapeutic drug selected from the group consisting of cisplatin, oxaplatin, irinotecan, gemcitabine, bevacizumab and cetuximab.

9. The method of claim 1 where the administration of 5-FU is between 250 and 1000 mg/m2 per treatment.

10. The method of claim 1 where the administration of 5,10 methylenetetrahydrofolate is between 50 and 70 mg/m2 per treatment.

11. A method of reducing adverse side effects arising from treatment of 5- fluorouracil and 5, 10-methylenetetrahydro folate in a patient hosting 5-FU sensitive tumors the method comprising: i) cyclic administering to the patient a combination of 5-FU and 5,10-methylenetetrahydrofolate; and, ii) administering capecitabine 1 day after step i where the administration of capecitabine is in an amount that potentiates the therapeutic effect of 5 -fluorouracil while reducing adverse side effects in the patient compared to side effects arising from treatment with 5-FU and 5,10-methylenetetrahydrofolate without capecitabine.

12. The method of claim 11 where the tumor is colorectal cancer.

13. The method of claim 11 where the tumor is a cancer of the breast, pancreas or stomach.

14. The method of claim 11 where the capecitabine is administered at a daily dosage of between 625 and 1250 mg/m2 twice a day.

15. The method of claim 11 where the administration of capecitabine occurs daily for between 1 to 6 days before the patient is again treated with 5-FU and 5,10-methylenetetrahydrofolate.

16. The method of claim 11 where the cyclic administering to the patient a combination of 5-FU and 5,10-methylenetetrahydrofolate occurs at least three times over a period of three weeks.

17. The method of claim 11 where the treatment further includes co- administration of a chemotherapeutic drug selected from the group consisting of cisplatin, oxaplatin, irinotecan, gemcitabine, bevacizumab and cetuximab.

18. The method of claim 11 where the administration of 5-FU is between 250 and 1000 mg/m2 per treatment.

19. The method of claim 1 where the administration of 5,10-methylenetetrahydrofolate is between 50 and 70 mg/m2 per treatment.

20. A method of reducing adverse side effects of a treatment regimen consisting of daily doses of capecitabine in a patient with cancer, the method comprising: i) cyclic administering to the patient a combination of 5-FU and 5,10-methylenetetrahydrofolate; and, ii) administering capecitabine 1 day after step i where the cyclic administration of 5-FU and 5,10-methylenetetrahydrofolate followed by the administration of capecitabine reduces the adverse effects of a treatment regimen consisting of daily doses of capecitabine alone.

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