US 200600794.80A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0079480 A1 Niyikiza (43) Pub. Date: Apr. 13, 2006

(54) NOVELANTIFOLATE COMBINATION (60) Provisional application No. 60/215.310, filed on Jun. THERAPES 30, 2000. Provisional application No. 60/235,859, filed on Sep. 27, 2000. Provisional application No. (76) Inventor: Clet Niyikiza, Indianapolis, IN (US) 60/284.448, filed on Apr. 18, 2001. Correspondence Address: Publication Classification EL LILLY & COMPANY PATENT DIVISION (51) Int. Cl. P.O. BOX 6288 A6II 3/74 (2006.01) INDIANAPOLIS, IN 46206-6288 (US) A6II 3/525 (2006.01) (52) U.S. Cl...... 514/52: 514/251 (21) Appl. No.: 11/288,807 (22) Filed: Nov. 29, 2005 (57) ABSTRACT Related U.S. Application Data A method of administering an antifolate to a mammal in (62) Division of application No. 10/297,821, filed on Dec. need thereof, comprising administering an effective amount 5, 2002, filed as 371 of international application No. of said antifolate in combination with a methylmalonic acid PCT/US01/14860, filed on Jun. 15, 2001. lowering agent. US 2006/00794.80 A1 Apr. 13, 2006

NOVELANTFOLATE COMBINATION others, such as Lometrexol and raltitrexed. (Jackman A. L. THERAPES Calvert A H Folate-Based Thymidylate Synthase Inhibitors as Anticancer Drugs. Ann Oncol 1995;6(9):871-881; Lao 0001 Potentially, life-threatening toxicity remains a havini S, Wedge S. R. Lind MJ, et al. A phase I clinical major limitation to the optimal administration of antifolates. study of the antipurine antifolate Lometrexol (DDATHF) (see, generally, Antifolate Drugs in Cancer Therapy, edited given with oral folic acid. Invest New Drugs 1996:14:325 by Jackman, Ann L. Humana Press, Totowa, N.J., 1999.) In 335; and Maughan TS, James R D, Kerr D, et al., on behalf Some cases, a Supportive intervention is routinely used to of the British MRC Colorectal Cancer Working Party. Pre permit safe, maximal dosing. For example, Steroids. Such as liminary results of a multicenter randomized trial comparing dexamethone, can be used to prevent the formation of skin 3 chemotherapy regimens (deCramont, Lokich, and raltitr rashes caused by the antifolate. (Antifolate, pg. 197.) exed) in metastatic colorectal cancer. Proc ASCO 0002 Antifolates represent one of the most thoroughly 1999:18:Abst 1007.) studied classes of antineoplastic agents, with aminopterin 0004 Initially, folic acid was used as a treatment for initially demonstrating clinical activity approximately 50 toxicities associated with GARFTI see, e.g. U.S. Pat. No. years ago. Methotrexate was developed shortly thereafter, 5,217.974. Folic acid has been shown to lower homocys and today is a standard component of effective chemothera teine levels (see e.g. Homocysteine Lowering Trialists peutic regime for malignancies such as lymphoma, breast Collaboration Lowering blood homocysteine with folic acid cancer, and bead and neck cancer. (Bonnadonna G, Zambetti based supplements: meta-analysis of randomized trials. BMJ M. Valagussa P. Sequential or alternating doxorubicin and 1998:316:894-898 and Naurath H J, Joosten E. Riezler R, CMF regimens in breast cancer with more than three posi Stabler S. P. Allen R H, Lindenbaum J. Effects of vitamin tive nodes: Ten year results. JAMA 1995:273(7):542-547: B12, folate and vitamin B6 supplements in elderly people Bonnadonna G. Valagussa P. Moliterni A, Zambetti M. with normal serum vitamin concentrations. Lancet Brambilla C. Adjuvant cyclophosphamide, methotrexate, 1995:346:85-89), and homocysteine levels have been shown and fluorouracil in node-positive breast cancer: The results to be a predictor of cytotoxic events related to the use of of 20 years of follow-up. N Engl J Med 1995:332(14):901 GARFT inhibitors, see e.g. U.S. Pat. No. 5,217.974. How 906; and Hong W K. Schaefer S. Issell B, et al. A prospective ever, even with this treatment, cytotoxic activity of GARFT randomized trial of methotrexate versus cisplatin in the inhibitors and antifolates as a class remains a serious con treatment of recurrent squamous cell carcinoma of the head cern in the development of antifolates as pharmaceutical and neck. Cancer 1983:52:206-210.) Antifolates inhibit one drugs. The ability to lower cytotoxic activity would repre or several key folate-requiring enzymes of the thymidine and purine biosynthetic pathways, in particular, thymidylate sent an important advance in the use of these agents. synthase (TS), dihydrofolate reductase (DHFR), and glyci 0005 Surprisingly and unexpectedly, we have now dis namide ribonucleotide formyltransferase (GARFT), by covered that certain toxic effects such as mortality and competing with reduced folates for binding sites of these nonhematologic events, such as skin rashes and fatigue, enzymes. (Shih C, Habeck L. L. Mendelsohn L. G. Chen VJ, caused by antifolates, as a class, can be significantly reduced Schultz RM. Multiple folate enzyme inhibition: Mechanism by the presence of a methylmalonic acid lowering agent, of a novel pyrrolopyrmidine-based antifolate LY231514 without adversely affecting therapeutic efficacy. The present (MTA). Advan Enzyme Regul, 1998; 38:135-152 and Shih invention thus provides a method for improving the thera C, Chen VJ, Gossett L S. et al. LY231514, a pyrrolo2,3- peutic utility of antifolate drugs by administering to the host dpyrimidine-based antifolate that inhibits multiple folate undergoing treatment with a methylmalonic acid lowering requiring enzymes. Cancer Res 1997:57:1116-1123.) Sev agent. We have discovered that increased levels of methyl eral antifolate drugs are currently in development. Examples malonic acid is a predictor of toxic events in patients that of antifolates that have thymidylate synthase inhibiting receive an antifolate drug and that treatment for the (“TSI) characteristics include 5-fluorouracil and Tomu increased methylmalonic acid, Such as treatment with Vita dex(R). An example of an antifolate that has dihydrofolate min B12, reduces mortality and nonhematologic events, reductase inhibiting (“DHFRI) characteristic is Methotrex Such as skin rashes and fatigue events previously associated ate(R). An example of an antifolate that has glycinamide with the antifolate drugs. ribonucleotide formyltransferase inhibiting (“DHFRI) 0006 Additionally, we have discovered that the combi characteristics is Lometrexol. Many of these antifolate drugs nation of a methylmalonic acid lowering agent and folic acid inhibit more than one biosynthetic pathway. For example synergistically reduces the toxic events associated with the Lometrexol is also an inhibitor of dihydrofolate reductase administration of antifolate drugs. Although, the treatment and pemetrexed disodium (AlimtaR), Eli Lilly and Company, and prevention of cardiovascular disease with folic acid in Indianapolis, Ind.) has demonstrated thymidylate synthase, combination with vitamin B12 is known, the use of the dihydrofolate reductase, and glycinamide ribonucleotide combination for the treatment of toxicity associated with the formyltransferase inhibition. administration of antifolate drugs was unknown heretofore. 0003) A limitation to the development of these drugs is 0007. The present invention relates to a method of admin that the cytotoxic activity and subsequent effectiveness of istering an antifolate to a mammal in need thereof, com antifolates may be associated with substantial toxicity for prising administering an effective amount of said antifolate Some patients. Additionally antifolates as a class are asso in combination with a methylmalonic acid lowering agent. ciated with sporadic severe mylosuppression with gas 0008 Furthermore, the present invention relates to a trointestinal toxicity which, though infrequent, carries a high method of reducing the toxicity associated with the admin risk of mortality. The inability to control these toxicities led istration of an antifolate to a mammal comprising adminis to the abandonment of clinical development of some anti tering to said mamma an effective amount of said antifolate folates and has complicated the clinical development of in combination with a methylmalonic acid lowering agent. US 2006/00794.80 A1 Apr. 13, 2006

0009 Furthermore, the present invention relates to a 0020. As used herein, the term “effective amount” refers method of inhibiting tumor growth in mammals comprising to an amount of a compound or drug, which is capable of administering to said mammals an effective amount of an performing the intended result. For example, an effective antifolate in combination with a methylmalonic acid lower amount of an antifolate drug that is administered in an effort ing agent. to reduce tumor growth is that amount which is required to 0010 Furthermore, the present invention relates to a reduce tumor growth method of administering an antifolate to a mammal in need 0021. As used herein, the term “toxicity” refers to a toxic thereof, comprising administering an effective amount of event associated with the administration on an antifolate. said antifolate in combination with a methylmalonic acid Such events include, but are not limited to, neutropenia, lowering agent and a FBP binding agent A preferred FBP thrombopenia, toxic death, fatigue, anorexia, nausea, skin binding agent is folic acid. rash, infection, diarrhea, mucositis, and anemia. For further explanation of the types of toxicity experienced by patients 0011 Furthermore, the present invention relates to a receiving antifolates, see, generally, Antifolate Drugs in method of reducing the toxicity associated with the admin Cancer Therapy. Preferably, toxicity refers to toxic death, istration of an antifolate to a mammal comprising adminis fatigue, neutropenia, thrombopenia, and tering to said mammal an effective amount of said antifolate in combination with a methylmalonic acid lowering agent 0022. As used herein, the term “nonhematologic event and a FBP binding agent. A preferred FBP binding agent is refers to the occurrence of skin rash or fatigue due to the folic acid. administration of an antifolate. 0023. As used herein, the term “in combination with 0012 Furthermore, the present invention relates to a refers to the administration of the methylmalonic acid low method of inhibiting tumor growth in mammals comprising ering agent, the antifolate drug, and optionally the folic acid; administering to said mammals an effective amount of an in any order such that sufficient levels of methylmalonic acid antifolate in combination with a methylmalonic acid lower lowering agent and optionally folic acid are present to ing agent and a FBP binding agent. A preferred FBP binding reduce the toxicity of an antifolate in a mammal. The agent is folic acid administration of the compounds maybe simultaneous as a 0013 Furthermore, the present invention relates to the single composition or as two separate compositions or can use of a methylmalonic acid lowering agent, alone or in be administered sequentially as separate compositions such combination with a FBP binding agent, in the preparation of that an effective amount of the agent first administered is in a medicament useful in lowering the mammalian toxicity of the patient’s body when the second and/or third agent is an antifolate. A preferred FBP binding agent is folic acid. administered. The antifolate drug may be administered to the mammal first, followed by treatment with the methylma 0014 Furthermore, the present invention relates to the lonic acid lowering agent. Alternatively, the mammal may use of a methylmalonic acid lowering agent in the prepara be administered the antifolate drug simultaneously with the tion of a medicament useful in lowering the mammalia methylmalonic acid lowering agent. Preferably, the mammal toxicity associated with an antifolate, and the medicament is is pretreated with the methylmalonic acid lowering agent administered in combination with an antifolate. and then treated with the antifolate. If folic acid is to be 0.015 Furthermore, the present invention relates to the administered in addition to the methylmalonic acid lowering use of a methylmalonic acid lowering agent in the prepara agent, the folic acid may be administered at any time prior, tion of a medicament useful in lowering the mammalian post, or simultaneously to the administration of either the toxicity associated with an antifolate, and the medicament is methylmalonic acid lowering agent or the antifolate. Pref administered in combination with an antifolate and a FBP erably, the mammal is pretreated with the methylmalonic binding agent. acid, and then treated with folic acid, followed by treatment with the antifolate compound. 0016 Furthermore, the present invention relates to the 0024. The terms “antifolate” and “antifolate drug” refer use of a methylmalonic acid lowering agent in the manu to a chemical compound which inhibits at least one key facture of a medicament for use in a method of inhibiting folate-requiring enzyme of the thymidine or purine biosyn tumor growth in mammals, which method comprises admin thetic pathways, preferably thymidylate synthase (“TS), istering said methylmalonic acid lowering agent in combi dihydrofolate reductase (DHFR), or glycinamide ribo nation with an antifolate. nucleotide formyltransferase (“GARFT), by competing 0017 Furthermore, the present invention relates to a with reduced folates for binding sites of these enzymes. product containing a methylmalonic acid lowering agent, an Preferred examples of antifolates include 5-fluorouracil, as antifolate and optionally a FBP binding agent as a combined manufactured by Glaxo; Tomudex(R), as manufactured by preparation for the simultaneous, separate or sequential use Zeneca; Methotrexate R, as manufactured by Lederle; Lom in inhibiting tumour growth etrexolor, as manufactured by Tularik; pyrido 2,3-dipyrimi dine derivatives described by Taylor et al in U.S. Pat. Nos. 0018. The current invention concerns the discovery that 4,684,653, 4,833,145, 4,902,796, 4,871,743, and 4,882,334; administration of a methylmalonic acid lowering agent in derivatives described by Akimoto in U.S. Pat. No. 4,997, combination with an antifolate drug reduces the toxicity of 838; thymidylate synthase inhibitors as found in EPO appli the said antifolate drug. cation 239,362; and most preferred, Pemetrexed Sodium 0019. The term “inhibit” as it relates to antifolate drugs (ALIMTA), as manufactured by Eli Lilly & Co. refers to prohibiting, alleviating, ameliorating, halting, 0.025 The terms “methylmalonic acid” and “MMA” refer restraining, slowing or reversing the progression of, or to a structural isomer of Succinic acid present in minute reducing tumor growth amounts in healthy human urine. US 2006/00794.80 A1 Apr. 13, 2006

0026. The term “methylmalonic acid lowering agent' relevant circumstances, including the condition to be treated, refers to a substrate, which lowers the concentration of the chosen route of administration, the actual agent admin methylmalonic acid in a mammal. A preferred example of istered, the age, weight, and response of the individual such a substrate is vitamin B12. For methods of determining patient, and the severity of the patient’s symptoms, and methylmalonic acid and Substrates therefore, see, e.g., Mat therefore the above dosage ranges are not intended to limit char D B, Feussner J. R. Millington D S, et al. Isotope the scope of the invention in any way. In some instances dilution assay for urinary methylmalonic acid in the diag dosage levels below the lower limit of the aforesaid range nosis of vitamin B12 deficiency. A prospective clinical may be more than adequate, while in other cases still larger evaluation Ann Intern Med 1987: 106: 707-710; Norman E doses may be employed without causing any harmful side J. Morrison J. A. Screening elderly populations for cobal effect. amin (vitamin B12) deficiency using the urinary methylma 0030) The term “FBPbinding agent” as used herein refers lonic acid assay by gas chromatography mass spectrometry. to a folic binding protein binding agent which includes folic Am J Med 1993; 94: 589-594; Norman E. J. Gas Chroma acid, (6R)-5-methyl-5,6,7,8-tetrahydrofolic acid, and (6R)- tography mass spectrometry screening of urinary methyl 5-formyl-5,6,7,8-tetrahydrofolic acid, or a physiologically malonic acid: early detection of vitamin B12 (cobalamin) available salt or ester thereof. This latter compound is the deficiency to prevent permanent neurologic disability. (6R)-isomer of leucovorin as disclosed in J. Am. Chem. GC/MS News 1984; 12:120-129; Martin D C, Francis J, Soc., 74, 4215 (1952). Both of the tetrahydrofolic acid Protetch J. Huff F.J. Time dependency of cognitive recovery compounds are in the unnatural configuration at the 6-po with cobalamin replacement: report of a pilot study. JAGS sition. They are 10-20 fold more efficient in binding the 1992; 40: 168-172; Norman E. J. Cronin C. Cobalamin folate binding protein compared with their respective (6S)- deficiency. Neurol 1996: 47: 310-311; Rasmussen K, isomer, see Ratnam, et. al., Folate and Antifolate Transport Moelby I, Jensen M. K. Studies on methylmalonic acid in in Mammalian Cells Symposium, Mar. 21-22, 1991, humans; Savage DG, Lindenbaum J. Stabler SP. Allen R H. Bethesda, Md. These compounds are usually prepared as a Sensitivity of methylmalonic acid and total homocysteine mixture with their natural form (6S) of diastereomers by determination for diagnosing cobalamin and folate defi non-stereo selective reduction from the corresponding dehy ciency. Am J Med 1994: 96: 239-246. dro precursors followed by separation through chromato 0027. The term “vitamin B12” refers to vitamin B12 and graphic or enzymatic techniques. See e.g. PCT Patent Appli its pharmaceutical derivatives, such as hydroxocobalamin, cation Publication WO 880844 (also Derwent Abstract cyano-10-chlorocobalamin, aquocobalamin perchlorate, 88-368464/51) and Canadian Patent 1093554. See, e.g. aquo-10-chlorocobalamin perchlorate, azidocobalamin, Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, chlorocobalamin, and cobalamin. Preferably the term refers Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, to vitamin B12, cobalamin, and chlorocobalamin. and Choline (2000), 8 Folate, pp. 196-305. 0028. The dosage generally will be provided in the form 0031 “Physiologically-available salt” refers to potas of a vitamin Supplement, namely as a tablet administered sium, Sodium, lithium, magnesium, or preferably a calcium orally, such as a Sustained release formulation, as an aqueous salt of the FBP binding agent. “Physiologically-available . . Solution added to drinking water, or as an aqueous parenteral ... ester” refers to esters which are easily hydrolyzed upon formulation. Preferably the methylmalonic acid lowering administration to a mammal to provide the corresponding agent is administered as an intramuscular injection formu FBP binding agent free acid, such as C-C alkyl esters, lation. Such formulations are known in the art and are mixed anhydrides, and the like. commercially available. 0032. The FBP binding agent to be utilized according to this invention can be in its free acid form, or can be in the 0029. The skilled artisan will appreciate that the methyl form of a physiologically-acceptable Salt or ester which is malonic lowering agents are effective over a wide dosage converted to the parent acid in a biological system. The range. For example, when cobalamin is used as the meth dosage generally will be provided in the form of a vitamin ylmalonic lowering agent, the dosage of cobalamin may fall Supplement, namely as a tablet administered orally, prefer within the range of about 0.2 ug to about 3000 g of ably as a Sustained release formulation, as an aqueous cobalamin from once daily for a month to once every nine Solution added to drinking water, an aqueous parenteral weeks for a year. Preferably, cobalamin will be dosed as an intramuscular injection of about 500 ug to about 1500 ug formulation, e.g., an intravenous formulation, or the like. administered from about every 24 hours to about every 1680 0033. The FBP binding agent is usually administered to hours. Preferably, it is an intramuscular injection of about the subject mammal prior to treatment with the antifolate. 1000 ug administered initial from about 1 to about 3 weeks Pretreatment with the suitable amount of FBP binding agent prior to administration of the antifolate and repeated from from about 1 to about 24 hours is usually sufficient to about every 24 hours to about every 1680 hours, regardless substantially bind to and block the folate binding protein of when treatment with the antifolate is started and contin prior to administration of the antifolate. Although one single ued until the administration of the antifolate is discontinued. dose of the FBP binding agent, preferably an oral adminis Most preferred is an intramuscular injection of about 1000 tration of folic acid, should be sufficient to load the folate ug administered initially from about 1 to about 3 weeks prior binding protein, multiple dosing of the FBP binding agent to the first administration of the antifolate and repeated can be employed for periods up to weeks before treatment every 6 to 12 weeks, preferably about every 9 weeks, and with the active agent to ensure that the folate binding protein continued until the discontinuation of the antifolate admin is sufficiently bound in order to maximize the benefit derived istrations. However, it will be understood that the amount of from Such pretreatment. the methylmalonic acid lowering agent actually adminis 0034. In the especially preferred embodiment of this tered will be determined by a physician, in the light of the invention, about 0.1 mg to about 30 mg, most preferably US 2006/00794.80 A1 Apr. 13, 2006

about 0.3 mg to about 5 mg. of folic acid is administered increases in tumor growth delay to 22 days and 23 days at orally to a mammal about 1 to 3 weeks post administration the ALIMTA doses of 100 mg/kg and 150 mg/kg, respec of the methylmalonic acid lowering agent and about 1 to tively. The tumor growth delays with ALIMTA and vitamin about 24 hours prior to the parenteral administration of the B12 (165 mg/kg) treatment were 22 days and 24 days at amount of an antifolate. However, it will be understood that ALIMTA doses of 100 mg/kg and 150 mg/kg, respectively. the amount of the methylmalonic acid lowering agent actu ally administered will be determined by a physician, in the 0041) Body weight was used as a general measure of light of the relevant circumstances, including the condition toxicity for each of the treatment regimes. The body weight to be treated, the chosen route of administration, the actual loss pattern reflected the treatment regimens with weight agent administered, the age, weight, and response of the decrease during the treatment times of days 7 through 11 and individual patient, and the severity of the patient’s Symp 14 through 18 with some weight recovery during the inter toms, and therefore the above dosage ranges are not intended vening two days. The weight loss due to ALITMA was dose to limit the scope of the invention in any way. In some dependent but overall minor (3%). Folic acid alone at either instances dosage levels below the lower limit of the afore 6 mg/kg or 60 mg/kg did not cause weight loss, in fact folic said range may be more than adequate, while in other cases acid treated animals maintained weight and gained weight still larger doses may be employed without causing any over the course of the experiment better than the control harmful side effect. animals. The animals treated with ALIMTA (100 mg/kg) and folic acid (60 mg/k-g) gained weight (about 20%) over the 0035) In general, the term “pharmaceutical” when used as course of the experiment. an adjective means Substantially non-toxic to living organ isms. 0042 Administration of vitamin B12 did not prevent weight gain in the annals over the time course of the Methods experiment. The animals treated with ALIMTA (100 mg/kg) 0036) To assess the effect of a methylmalonic acid low along with vitamin B12 gained weight while those treated ering agent, alone or in combination with folic acid on the with ALUMTA (150 mg/kg) along with vitamin B12 main antitumor efficacy of an antifolate in a human tumor tained weight over the course of the experiment. Xenograft model, female nude mice bearing human MX-1 0043. In conclusion, administration of Super-physiologic breast carcinoma were treated with ALIMTA alone or along but non-toxic doses of the vitamins, folic acid and Vitamin with super-physiologic doses of folic acid or vitamin B12 B12, did not alter the antitumor activity of ALIMTA in the (cobalamin). human MX-1 breast carcinoma xenograft tumor in nude 0037. The animals were maintained on sterilized standard mice and did not increase the toxicity of ALIMTA as lab chow ad libitum and sterilized water ad libitum. The determined by body weight measurements of the animals. human MX-1 tumor cells (5x10) obtained from donor 0044) The effect of vitamin B12, alone or in combination tumors were implanted Subcutaneously in a thigh of female with folic acid, on antifolates can be demonstrated in stan nude mice 8- to 10-weeks old. Beginning on day 7 post dard tests commonly utilized to determine the antitumor tumor cell implantation, the animals were treated with activity and toxic effects of the antifolates themselves. In ALIMTA (100 mg/kg or 150 mg/kg) once daily on days 7 one such test, mice are inoculated with the C3H strain of through 11 and 14 through 18 by intraperitoneal injection mammary adenocarcinoma by inserting a 2 mm by 2 mm alone or along with folic acid (6 mg/kg or 60 mg/kg) and/or section of tumor into the axillary region of the mice by vitamin B12 (165 mg/kg) by intraperitoneal injection on the trocar. The timing of administering the methylmalonic acid same schedule. lowering agent, alone or in combination with the folic acid, 0038 Tumor response was monitored by tumor volume and the antifolate may be varied. Ten animals are used at measurements twice weekly over the course of the experi each dosage level. Antitumor activity is assessed on day ten ment. Toxicity was monitored by body weight measure (when day one is first dosage of antifolate) by measuring the ments made at the same time as the tumor Volume measure length and width of the tumor growth using Vernier calipers, ments. Tumor growth delay was the difference in days for and the activity is expressed as a percent inhibition of tumor the treated and the controls tumors to reach 1000 mm. growth. 0.039 The human MX-1 breast carcinoma xenograft was 0045 When the antifolate is administered to infected responsive to treatment with ALIMTA with doses of 100 mice which are maintained on a diet totally free of vitamin mg/kg and 150 mg/kg producing tumor growth delays of 17 B12 and optionally folic acid for two weeks prior to and days and 21 days, respectively. Folic acid was administered during treatment, it exhibits moderated antitumor activity at to the animals alone at two doses 6 mg/kg and 60 mg/kg on very low doses, but also causes severe toxicity at a very low the same schedule as ALIMTA and produced tumor growth dose (measured as death of mice). delays of 7 days and 12 days, respectively. Vitamin B12 administered alone at a dose of 165 mg/kg resulted in a 0046. A test group of mice are maintained on a vitamin tumor growth delay of 12 days. B12 and optionally folic acid free diet for two weeks before treatment. Vitamin B12 and optionally folic acid is then 0040 Combinations of ALIMTA at each of the two doses administered during the treatment by intramuscular injection were administered along with each of the vitamins as of 0.0003% vitamin B12 (weight/volume) and optionally simultaneous combination regimens. Administration of folic providing the animals drinking water containing 0.0003% acid (6 mg/kg) along with ALIMTA did not alter the tumor folic acid (weight/volume). This concentration translates to growth delay produced from that obtained with ALIMTA about 1.75 mg of vitamin B12 and optionally folic acid per alone. The addition of folic acid at the higher dose (60 square meter of body Surface per day. As the foregoing mg/kg) along with each dose of ALIMTA resulted in small results indicate, addition of the indicated level of vitamin US 2006/00794.80 A1 Apr. 13, 2006

B12 to the diet of a subject receiving an antifolate results in 0054 3. A dose of folic acid between 350 ug and 1000 excellent antitumor activity at low doses, with little or no ug is acceptable if neither option #1 or option # 2 is toxic effects. available. 0047 The foregoing tests establish that for tumor bearing 0055 For purposes of this study, patients should take oral rice maintained on a vitamin B12 and optionally folic acid folic acid daily beginning approximately 1 to 3 weeks before free diet prior to and during treatment with an antifolate, the treatment with ALIMTA plus cisplatin or cisplatin alone and toxicity of the antifolate is very large, with 1 mg/kg/day continuing daily until discontinuation from Study therapy. being lethal to the majority of the mice, and lower antitumor activity is observed at non-toxic drug doses. Very low doses 0056 2. Vitamin B12 of vitamin B12 partially reverses drug toxicity and improved 0057 Vitamin B12 will be obtained and administered as antitumor activity. Larger doses of vitamin B12 reduce a 1000 ug intramuscular injection A vitamin B12 injection antifolate toxicity even more significantly. Pretreatment of must be administered approximately 1 to 3 weeks before the mouse with vitamin B12 and then administering folic treatment with ALIMTA and should be repeated approxi acid prior to administering the antifolate demonstrates a mately every 9 weeks until the patient discontinues from striking reduction in toxicity, almost eliminating the anti study therapy. folate toxicity completely. Thus, the use of vitamin B12 in 0058. Folic acid supplementation, 350-600 ug or equiva combination with an antifolate reduces drug toxicity without lent should be taken orally daily beginning approximately 1 adversely affecting antitumor activity, and the use of vitamin to 3 weeks prior to the first dose of MTA plus cisplatin and B12 in conjunction with folic acid synergistically reduces continue daily until the patient discontinues from Study drug toxicity. therapy. A vitamin B12 injection, 1000 ug, must be given 0.048. In a typical clinical evaluation involving cancer intramuscularly approximately 1 to 3 weeks prior to the first patients, all of whom have histologically or cytologically dose of ALIMTA and should be repeated approximately confirmed diagnosis of cancer, an antifolate is administered every 9 weeks until the patient discontinues from study in combination with vitamin B12. Vitamin B12 is adminis therapy. tered as a 1000 ug intramuscular injection 1-3 weeks prior 0059 Compare presupplementation homocysteine and to treatment with the antifolate, and 1000 ug intramuscular injection of vitamin B12 is made approximately every 9 methylmalonic acid levels to a) the level immediately prior weeks until the patient discontinues from therapy. The to the initial dose of study drug, and b) to the level antifolate is administered in four doses over a two week immediately prior to the second dose of study drug (i.e., period by rapid intravenous injection, followed by two after a full cycle of Supplementation), and compare the weeks of non-therapy. Dosing is made on days 1, 4, 8 and prevalence of specific toxicities experienced in up to the first 11 of any two week period. Patients will have an initial seven cycles of therapy in patients who have been Supple course of therapy at a dose of 5 mg/m/dose, and depending mented from baseline to the prevalence seen in the earlier upon the toxic effects observed in the initial course, their patients (n=246) who were not supplemented (Farber et al.) Subsequent courses may be at the same dose, or may be 0060 Toxicity may be compared in specific patients in escalated to 6 mg/m, or may be attenuated to 4 mg/m. non-Supplemented cycles versus Supplemented cycles 0049. In preparation for the foregoing clinical study, pilot (cross-over patients). studies in humans have established that vitamin B12 given 0061 The data to be compared are: to patients receiving Alimta has effected reduced side effects due to the Alimta. One to two weeks prior to administration 0062 1) Patient numbers and baseline demographic of ALIMTA urine is collected and blood is drawn from a data for those supplemented from baseline. human Subject; and vitamin metabolite levels, methylma 0063. 2) Homocysteine and methylmalonic acid levels, lonic acid and homocysteine, are determined. Homocysteine levels at baseline, prior to first dose, prior to second levels are determined in blood by a fluorescent polarization dose, and prior to each therapy cycle depending of the immunoassay kit manufactured by Abbot Laboratories. type of cancer under study. Methylmalonic acid levels are determined by urine levels using a 24 hour urine collection kit available from Biolab 0064 3) Grade 3 and 4 hematologic toxicity in these Medical Unit (a United Kingdom company). Additionally fully Supplemented patients. urine and blood may be collected one week prior to admin 0065 4) Grade 3 and 4 nonhematologic toxicity in istration of ALIMTA (after at least 5 days of folic acid these fully supplemented patients. Supplementation and at least 1 week vitamin B12 Supple mentation), and up to 4 days prior to every cycle. 0066. The grading of toxicities in chemotherapuetic clini cal trials is well known to a person of skill in the art. Method of Administration and Dosing Procedures: Examples of fatigue and skin rash grading are provided 0050) 1. Folic Acid: below. 0051. Folic acid will be supplied as one of the following Fatigue Grading - options, with preference in order from option #1 to option H3: 0067 Neuromotor 0.052 1. 350-600 ug folic acid. Grade 0 none or no change 0053 2. A multivitamin containing folic acid in the Grade 1 subjective weakness; no objective findings range of 350 ug to 600 g is acceptable if option #1 is Grade 2 mild objective weakness without significant impair not available. ment of function US 2006/00794.80 A1 Apr. 13, 2006

Grade 3 objective weakness with impart of function groups. Seventeen of these patients received ALIMTA, but did not receive vitamin B12 or folic acid, as described supra. Grade 4 paralysis The remaining patients received treatment with vitamin Rash Grading— B12, folic acid, and ALIMTA, as described supra. Of patients who received the combination treatment, 8 out of 45 0068 Skin responded to the chemotherapy. Of patients who did not Grade 0 none or no change receive the combination treatment, but rather, received only treatment with ALIMTA, only 1 out of 17 patients Grade 1 scattered macular or papular eruption or erythema responded. that is asymptomatic Grade 2 scattered macular or papular eruption or erythema with pruritus or other associated eruption symptoms 1-2. (canceled) 3. A method of inhibiting tumor growth in humans com Grade 3 generalized symptomatic macular, papular, or prising administering to said human an effective amount of vesicular eruption pemetrexed disodium in combination with a methylmalonic acid lowering agent selected from the group consisting of Grade 4 exfoliative dermatitis or ulcerating dermatitis vitamin B. hydroxocobalamin, cyano-10-chlorocobalamin 0069. The vitamins (both folic acid and B12) to be used aquocobalamin perchlorate, aquo-10-chlorocobalamin per in the following studies may be obtained from Zenith Gold chlorate, azidocobalamin, chlorocobalamin, cobalamin, and Line, Centrum, Folvite, or in Canada Apo-Fohic. Cyanoco cyanocobalamin wherein the methylmalonic acid lowering balamin is used as the methylmalonic acid lowering agent in agent is administered prior to the first administration of these studies. pemetrexed and the methylmalonic acid lowering agent 0070 Current and past clinical trials show a 4% drug administration is repeated from about every 6 weeks to related death total, 50% grade 3/4 neutropenia, 7% grade 4 about every 12 weeks. thrombocytopenia, and 10% grade 3/4 diarrheas and 4-33. (canceled) mucositis in patients administered ALIMTA and folic acid as 34. The method of claim 3 wherein the human is further described in U.S. Pat. No. 5,217.974. Vitamin B12 supple treated with folic acid. mentation with ALIMTA has a moderate effect on drug 35. The method of claim 38 wherein about 500 ug to about related toxicity, lowering drug related deaths to 3% and 1000 g of the methylmalonic acid lowering agent is admin severe toxicities by about 25%. The combination of vitamin istered. B12 and folic acid with ALIMTA has lowered the drug 36. The method of claim 35 wherein about 1000 ug of the related deaths to <1% in over 480 so treated. The combi methylmalonic acid lowering agent is administered. nation of vitamin B12 and folic acid has lowered the drug 37. The method of any one of claims 3,34, or 35 wherein related grade 3/4 toxic events, see Table 1. the methylmalonic acid lowering agent is administered by an intramuscular injection, orally, or as a parenteral. TABLE 1. 38. The method of claim 37 wherein the methylmalonic acid lowering agent is administered by an intramuscular Percent of occurrences Percent of occurrences post injection. prior to B12/folic acid B12/folic acid treatment treatment (N = 246) (N = 78) 39. The method of claim 38 wherein the methylmalonic acid lowering agent is administered orally. Hematologic 37% 6.4% Toxicity/ 40. A method of inhibiting tumor growth in humans Non-Hematologic comprising administering an effective amount of pemetr Toxicity exed disodium in combination with about 500 ug to about Neutropenia 32% 2.6% 1500 ug of a methylmalonic acid lowering agent selected Mucositis 59 1.3% Diarrhea 6% 2.6% from the group consisting of vitamin B12, hydroxocobal Neutropenia and 3% O% amin, cyano-10-chlorocobalamin, aquocobalamin perchlor Mucositis ate, aquo-10-chlorocobalamin perchlorate, azidocobalamin, Neutropenia and 3% O% Diarrhea chlorocobalamin, cobalamin, and cyanocobalamin wherein Neutropenia and 296 O% the treatment with the methylmalonic acid lowering agent is Infection administered by an intramuscular injection or orally and is repeated from about every 24 hours to about every 1680 hours, until treatment with pemetrexed disodium has ended. 0071 Additionally, sixty-two chemonaive patients requiring chemotherapeutic treatment were divided into two k k k k k