`Niyikiza
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 7,772,209 B2
`Aug. 10, 2010
`
`US007772209B2
`
`ANTIFOLATE COMBINATION THERAPIES
`
`W0
`
`WO 95/27723
`
`10/1995
`
`OTHER PUBLICATIONS
`
`Calvert H.: “Folate status and the safety pro?le of antifolates”, Semi
`nars in Oncology, 2002, 29/2 Suppl. 5, pp. 3-7, XP008005755.
`Calvert H.: “Future directions in the development of pemetrexed”,
`Seminars in Oncology, 2002, 29/2 Suppl. 5, pp. 54-61,
`XP008005744.
`Westerhof, et a1: “Carrier-and receptor-mediated transport of folate
`antagonists targeting folate-dependent enzymes: correlates of
`molecularstructure and biological activity”, Mol. Pharmacology,
`1995, 48(3), pp. 459-471, XP008005762.
`WorZalla, et a]: “Role of folic acid in modulating the toxicity and
`ef?cacy of the multitargeted antifolate, LY231514”, Anticancer
`Research (1998), 18(5A), pp. 3235-3239, XP008005757.
`Hanauske, et a1: “Pemetrexed disodium: A novel antifolate clinically
`active against multiple solid tumors”, Oncologist, Alphamed Press,
`US, vol. 4, No. 6, 2001, pp. 363-373, XP008005751.
`Bunn, et a1: “Vitamin B 12 and folate reduce toxicity of Alimta
`(pemetrexed disodium, LY 231514, MTA), a novel antifolate/
`antimetabolite”, Program/Proceedings-American Society of Clini
`cal Oncology, the Society, US, vol. 76A, No. 20, 2001, p. 300,
`XPO08005885.
`Dierkes, et al., Supplementation With Vitamin B 12 Decreases
`Homocystein and Methylmalonic Acid but Also Serum Folate in
`Patients With End-Stage Renal Disease. Metabolism. May 1999. vol.
`48, No. 5, pp. 631-635. See: abstract.
`Arsenyan et a1. (Abstract: Onkol. Nauchn., (1978) 12(10):49-54.
`John, et al. (Cancer 2000, 88: 1807-13).
`Poydock et al., “Growth-inhibiting effect of hydroxocobaltniin and
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`The Cecil Reference, Textbook of Medicine, 21st Edition (2000).
`Chapter 198. pp. 1060-1074.
`Poydock M. Effect of combined ascorbic acid and B- 12 on survival of
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`Clin Nutr 1991; 54: 1261S-5S.
`Poydock M, et a1. Mitogenic inhibition and effect on survival of mice
`bearing L1210 leukemia using a combination of dehydroascorbic
`acid and hydroxycobalamin. Am JClin Oncol 1985; 8: 2666-269.
`Poydock M, et a1. In?uence of Vitamins C and B12 on the Survival
`Rate of Mice Bearing Ascites Tumor. Expl Cell Biol 1982; 50:88-91.
`Toohey J. Dehydroascorbic acid as an anti-cancer agent. Cancer
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`Sallah S, et a1. Intrathecal methotrexate-induced megaloblastic ane
`mia in patients with acute leukemia. Archives of Pathology & Labo
`ratory Medicine 1999; 123(9): 774-777.
`NishiZaWaY, et al. Effects of methylcobalamin on the proliferation of
`androgen-sensitive or estrogen-sensitive malignant cells in culture
`and in vivo . International Journal for I/ltamin and Nutrition Research
`1997; 67(3):164-170.
`
`(Continued)
`Primary ExamineriKevin Weddington
`(74) Attorney, Agent, or FirmiEliZabeth A. McGraW
`
`(57)
`
`ABSTRACT
`
`A method of administering an antifolate to a mammal in need
`thereof, comprising administering an effective amount of said
`antifolate in combination With a methylmalonic acid loWer
`ing agent.
`
`(54)
`
`(75)
`
`(73)
`
`Inventor: Clet Niyikiza, Indianapolis, IN (US)
`
`Assignee: Eli Lilly and Company, Indianapolis,
`IN (US)
`
`(*)
`
`Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 162 days.
`
`(21)
`
`(22)
`
`(65)
`
`(62)
`
`(60)
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`(51)
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`(52)
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`(58)
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`(56)
`
`Appl. No.: 11/776,329
`
`Filed:
`
`Jul. 11, 2007
`
`Prior Publication Data
`
`US 2008/0032948 A1
`
`Feb. 7, 2008
`
`Related US. Application Data
`
`Division of application No. 11/288,807, ?led on Nov.
`29, 2005, noW abandoned, Which is a division of appli
`cation No. 10/297,821, ?led as application No. PCT/
`US01/14860 on Jun. 15, 2001 , noW Pat. No. 7,053,065.
`
`Provisional application No. 60/215,310, ?led on Jun.
`30, 2000, provisional application No. 60/235,859,
`?led on Sep. 27, 2000, provisional application No.
`60/284,448, ?led on Apr. 18, 2001.
`
`Int. Cl.
`(2006.01)
`A61K 31/70
`(2006.01)
`A61K 31/685
`(2006.01)
`A61K 31/50
`(2006.01)
`A61K 31/525
`(2006.01)
`A61K 31/519
`US. Cl. ......................... .. 514/52; 514/77; 514/249;
`514/251; 514/265.1
`Field of Classi?cation Search ................. .. 514/ 52,
`514/77, 249, 251, 265.1
`See application ?le for complete search history.
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
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`5/2006 NiyikiZa et al.
`2003/0216350 A1 11/2003 Allen et a1.
`2003/0225030 A1 12/2003 Allen et a1.
`2004/0005311 A1
`1/2004 Pitrnan
`
`FOREIGN PATENT DOCUMENTS
`
`EP
`
`0 546 870
`
`6/1993
`
`22 Claims, No Drawings
`
`Wockhardt Exhibit 1001 - 1
`
`
`
`US 7,772,209 B2
`Page 2
`
`OTHER PUBLICATIONS
`
`Tsao C, et al. In?uence of cobalamin on the survival of mice bearing
`ascites tumor. Pathobiology 1993; 61(2): 104-8.
`Kamei T, et al. Experimental study of the therapeutic effects of folate,
`vitamin A, and vitamin B12 on squamous metaplasia of the bronchial
`epithelium. Cancer 1993; 71(8): 2477-83.
`ShimiZu N, et al. Experimental study of antitumor effect of methyl
`B12. Oncology 1987; 44(3): 169-73.
`Herbert, V. The role of vitamin B12 and folate in carcinogenesis.
`Advances in Experimental Medicine and Biology 1986; 206 (Essent.
`Nutr. Carcinog.), 293-311.
`Kroes A, et al. Effects of 5-?uorouracil treatment of rat leukemia With
`concomitant inactivation of cobalamin. Anticancer Research 1986;
`6(4): 737-42.
`Kroes A, et al. Enhanced therapeutic effect of methotrexate in experi
`mental rat leukemia after inactivation of cobalamin (vitamin B12) by
`nitrous oxide. Cancer Chemotherapy and Pharmacology 1986;
`17(2): 114-20.
`Barak A. Vitamin B 12 as a possible adjunct in prevention of
`methotrexate hepatotoxicity. Biochemical Archives 1985; 1(3): 139
`42.
`Herbert V. The inhibition and promotion of cancers by folic acid,
`vitamin B12, and their antagonists. ACS Symposium Series (1985);
`277(Xenobiot. Metab.: Nutr. Eff.), 31-6.
`Alimta, NDA 021462, Approved Label of Jul. 2, 2009.
`“Clinical Chemistry: principle, procedures, correlations,” 3rd edi
`tion, 1996, published by Lippincott: pp. 618-627.
`Fluorouracil, Physicians Desk References, (c) 1998, pp. 2463-2464.
`Hammond, L., et al., “A phase I and pharmacokinetic (PK) study of
`the multitarget antifol (MTA) LY231514 With folic acid, ” American
`Society of Clinical Oncology (ASCO) Meeting Abstract No. 866
`(1998).
`Kisliuk, RL., 1984. “The Biochemistry of Folates.” In Sirotnak (Ed),
`Folate Antagonists as Therapeutic Agents. pp. 2-68. Harcourt Brace
`Jovanovich, Publishers.
`
`Kisliuk, RL., 1999. “Folate Biochemistry in RElation to Antifolate
`Selectivity.” In Jackson (Ed.), Antifolate Drugs in Cancer Therapy.
`pp. 13-36. Humana Press, New Jersey.
`Leucovorin, Physicians Desk Reference, (c) 1999. pp. 1389-1391.
`Methotrexate, Physicians Desk Reference, (c) 1999. pp. 1397-1413.
`Morgan, et al., “Folic acid supplementation prevent de?cient blood
`folate levels and hyperhomocysteinemia during long-term, low dose
`methotrexate therapy for rheumatoid arthritis: implications for car
`diovascular disease prevent,” J. Rheumatol. 25:441-446. (1998).
`NiyikiZa, C., et al., “LY231514 (MTA): relationship of vitamin
`metabolite pro?le to toxicity,” American Society of Clinical Oncol
`ogy (ASCO) Meeting Abstract No. 2139 (1998).
`Raltitrexed, The Complete Drug Reference, Martindale, 32nd Ed.,
`Pharmaceutical Press, London, pp. 560, 1990.
`Shih, C., et al., “LY231514, a Pyrrolo[2,3-d]pyrimidine-based
`Antifolate that Inhibits Multiple Folate-requiring Enzymes,” Cancer
`Research. 57: 1 1 16-1123. 1997.
`Shih, C., et al., “Preclinical Pharmacology Studies and the Clinical
`Development of a Novel Multitargeted Antifolate, MTA
`(LY231514),” In Jackson (Ed.), Antifolate Drugs in Cancer Therapy.
`pp. 13-36 Humana Press, New Jersey, 1998.
`Volkov, I., “The master key effect of vitamin B12 in treatment of
`malignancyiA potential therapy.”, Medical Hypotheses. 70:324
`328. 2008.
`Maysishecheva, N.V., et al.:“AntitumorActivity of Methotrexate
`When Used in Combination With Cobalamine Derivatives”,
`Eksperimentalnaya Onkologija (1982), vol. 4, No. 5:29-33.
`McDonald, A.C., et al.: “Clinical Phase I Study of LY231514, a
`Multitargeted Antifolate, Administered by Daily x 5 q 21 Schedule”,
`Annals of Oncology (1996), vol. 7:85, Abstract No. 291.
`Sofyina, Z.P., et al.: “Possibility of Potentiating the Antineoplastic
`Action of Folic Acid Antagonist by Methylcobalamine Analogs”,
`Vestnik Akademii Medicinskich Nauk SSSR (1979), vol. 1: 72-78.
`
`* cited by examiner
`
`Wockhardt Exhibit 1001 - 2
`
`
`
`1
`ANTIFOLATE COMBINATION THERAPIES
`
`US 7,772,209 B2
`
`2
`inability to control these toxicities led to the abandonment of
`clinical development of some antifolates and has complicated
`the clinical development of others, such as Lometrexol and
`raltitrexed. (Jackman A L, Calvert A H Folate-Based
`Thymidylate Synthase Inhibitors as Anticancer Drugs. Ann
`Oncol 1995; 6(9):871-881; Laohavinij S, Wedge S R, Lind M
`J, et al. A phase I clinical study of the antipurine antifolate
`Lometrexol (DDATHF) given With oral folic acid. Invest NeW
`Drugs 1996; 14:325-335; and Maughan T S, James R D, Kerr
`D, et al., on behalf of the British MRC Colorectal Cancer
`Working Party. Preliminary results of a multicenter random
`ized trial comparing 3 chemotherapy regimens (deGramont,
`Lokich, and raltitrexed) in metastatic colorectal cancer. Proc
`ASCO 1999; 18:Abst 1007.) Initially, folic acid Was used as
`a treatment for toxicities associated With GARFTI see, eg
`US. Pat. No. 5,217,974. Folic acid has been shoWn to loWer
`homocysteine levels (see eg Homocysteine LoWering Trial
`ist’s Collaboration. LoWering blood homocysteine With folic
`acid based supplements: meta-analysis of randomized trials.
`BMJ 1998; 316:894-898 and Naurath H J, Joosten E, Riezler
`R, Stabler S P, Allen R H, Lindenbaum J. Effects of vitamin B
`12, folate and vitamin B6 supplements in elderly people With
`normal serum vitamin concentrations. Lancet 1995; 346:85
`89), and homocysteine levels have been shoWn to be a pre
`dictor of cytotoxic events related to the use of GARFT inhibi
`tors, see eg US. Pat. No. 5,217,974. HoWever, evenWith this
`treatment, cytotoxic activity of GARFT inhibitors and anti
`folates as a class remains a serious concern in the develop
`ment of antifolates as pharmaceutical drugs. The ability to
`loWer cytotoxic activity Would represent an important
`advance in the use of these agents.
`Surprisingly and unexpectedly, We have noW discovered
`that certain toxic effects such as mortality and nonhemato
`logic events, such as skin rashes and fatigue, caused by anti
`folates, as a class, can be signi?cantly reduced by the pres
`ence of a methylmalonic acid loWering agent, Without
`adversely affecting therapeutic ef?cacy. The present inven
`tion thus provides a method for improving the therapeutic
`utility of antifolate drugs by administering to the ho st under
`going treatment With a methylmalonic acid loWering agent.
`We have discovered that increased levels of methylmalonic
`acid is a predictor of toxic events in patients that receive an
`antifolate drug and that treatment for the increased methyl
`malonic acid, such as treatment With vitamin B12, reduces
`mortality and nonhematologic events, such as skin rashes and
`fatigue events previously associated With the antifolate drugs.
`Additionally, We have discovered that the combination of a
`methylmalonic acid loWering agent and folic acid synergisti
`cally reduces the toxic events associated With the administra
`tion of antifolate drugs. Although, the treatment and preven
`tion of cardiovascular disease With folic acid in combination
`With vitamin B 12 is knoWn, the use of the combination for the
`treatment of toxicity associated With the administration of
`antifolate drugs Was unknoWn heretofore.
`The present invention relates to a method of administering
`an antifolate to a mammal in need thereof, comprising admin
`istering an effective amount of said antifolate in combination
`With a methylmalonic acid loWering agent.
`Furthermore, the present invention relates to a method of
`reducing the toxicity associated With the administration of an
`antifolate to a mammal comprising administering to said
`mammal an effective amount of said antifolate in combina
`tion With a methylmalonic acid loWering agent.
`Furthermore, the present invention relates to a method of
`inhibiting tumor groWth in mammals comprising administer
`ing to said mammals an effective amount of an antifolate in
`combination With a methylmalonic acid loWering agent.
`
`20
`
`25
`
`35
`
`This application is a divisional of application Ser. No.
`11/288,807, ?led 29 Nov., 2005 noW abandoned, Which is a
`divisional of application Ser. No. 10/297,821 ?led 12 May,
`2002, now US. Pat. No. 7,053,065, Which claims priority
`under 35 USC 371, for PCT/US01/14860, ?led 15 Jun., 2001,
`Which claims the priority of US. provisional applications No.
`60/215,310, ?led 30 Jun., 2000, No. 60/235,859, ?led 27
`Sep., 2000, and No. 60/284,448, ?led 18 Apr., 2001.
`Potentially, life-threatening toxicity remains a major limi
`tation to the optimal administration of antifolates. (see, gen
`erally, Antifolale Drugs in Cancer Therapy, edited by Jack
`man, Ann L., Humana Press, Totowa, NJ, 1999.) In some
`cases, a supportive intervention is routinely used to permit
`safe, maximal dosing. For example, steroids, such as dexam
`ethone, can be used to prevent the formation of skin rashes
`caused by the antifolate. (Anlifolale, pg 197.)
`Antifolates represent one of the most thoroughly studied
`classes of antineoplastic agents, With aminopterin initially
`demonstrating clinical activity approximately 50 years ago.
`Methotrexate Was developed shortly thereafter, and today is a
`standard component of effective chemotherapeutic regimens
`for malignancies such as lymphoma, breast cancer, and head
`and neck cancer. (Bonnadonna G, Zambetti M, Valagussa P.
`Sequential or alternating doxorubicin and CMF regimens in
`breast cancer With more than three positive nodes: Ten year
`results. JAMA 1995;273(7):542-547; Bonnadonna G, Vala
`gussa P, Molitemi A, Zambetti M, Brambilla C. Adjuvant
`cyclophosphamide, methotrexate, and ?uorouracil in node
`30
`positive breast cancer: The results of 20 years of folloW-up. N
`Engl J Med 1995; 332(14):901-906; and Hong W K, Schaefer
`S, Issell B, et al. A prospective randomized trial of methotr
`exate versus cisplatin in the treatment of recurrent squamous
`cell carcinoma of the head and neck. Cancer 1983; 52:206
`210.) Antifolates inhibit one or several key folate-requiring
`enzymes of the thymidine and purine bio synthetic pathWays,
`in particular, thymidylate synthase (TS), dihydrofolate reduc
`tase (DHFR), and glycinamide ribonucleotide formyltrans
`ferase (GARFT), by competing With reduced folates forbind
`ing sites of these enzymes. (Shih C, Habeck L L, Mendelsohn
`L G, ChenV J, Schultz R M. Multiple folate enzyme inhibi
`tion: Mechanism of a novel pyrrolopyrimidine-based anti
`folate LY231514 (MTA). Advan Enzyme Regul, 1998;
`38:135-152 and Shih C, Chen V J, Gossett L S, et al.
`LY231514, a pyrrolo[2,3-d]pyrimidine-based antifolate that
`inhibits multiple folate-requiring enzymes. Cancer Res 1997;
`57:1116-1123.) Several antifolate drugs are currently in
`development. Examples of antifolates that have thymidylate
`synthase inhibiting (“TSI”) characteristics include 5-?uorou
`racil and Tomudex®. An example of an antifolate that has
`dihydrofolate reductase inhibiting (“DHFRI”) characteristic
`is Methotrexate®. An example of an antifolate that has gly
`cinamide ribonucleotide
`formyltransferase
`inhibiting
`(“GARFTI”) characteristics is Lometrexol. Many of these
`antifolate drugs inhibit more than one biosynthetic pathWay.
`For example Lometrexol is also an inhibitor of dihydrofolate
`reductase and pemetrexed disodium (Alimta®, Eli Lilly and
`Company, Indianapolis, Ind.) has demonstrated thymidylate
`synthase, dihydrofolate reductase, and glycinamide ribo
`nucleotide formyltransferase inhibition.
`A limitation to the development of these drugs is that the
`cytotoxic activity and subsequent effectiveness of antifolates
`may be associated With substantial toxicity for some patients.
`Additionally antifolates as a class are associated With spo
`radic severe mylosuppression With gastrointestinal toxicity
`Which, though infrequent, carries a high risk of mortality. The
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`Wockhardt Exhibit 1001 - 3
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`US 7,772,209 B2
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`3
`Furthermore, the present invention relates to a method of
`administering an antifolate to a mammal in need thereof,
`comprising administering an effective amount of said anti
`folate in combination With a methylmalonic acid lowering
`agent and a FBP binding agent. A preferred FBP binding
`agent is folic acid.
`Furthermore, the present invention relates to a method of
`reducing the toxicity associated With the administration of an
`antifolate to a mammal comprising administering to said
`mammal an effective amount of said antifolate in combina
`tion With a methylmalonic acid loWering agent and a FBP
`binding agent. A preferred FBP binding agent is folic acid.
`Furthermore, the present invention relates to a method of
`inhibiting tumor groWth in mammals comprising admini ster
`ing to said mammals an effective amount of an antifolate in
`combination With a methylmalonic acid loWering agent and a
`FBP binding agent. A preferred FBP binding agent is folic
`acid.
`Furthermore, the present invention relates to the use of a
`methylmalonic acid loWering agent, alone or in combination
`With a FBP binding agent, in the preparation of a medicament
`useful in loWering the mammalian toxicity of an antifolate. A
`preferred FBP binding agent is folic acid.
`Furthermore, the present invention relates to the use of a
`methylmalonic acid loWering agent in the preparation of a
`medicament useful in loWering the mammalian toxicity asso
`ciated With an antifolate, and the medicament is administered
`in combination With an antifolate.
`Furthermore, the present invention relates to the use of a
`methylmalonic acid loWering agent in the preparation of a
`medicament useful in loWering the mammalian toxicity asso
`ciated With an antifolate, and the medicament is administered
`in combination With an antifolate and a FBP binding agent.
`Furthermore, the present invention relates to the use of a
`methylmalonic acid loWering agent in the manufacture of a
`medicament for use in a method of inhibiting tumor groWth in
`mammals, Which method comprises administering saidmeth
`ylmalonic acid loWering agent in combination With an anti
`folate.
`Furthermore, the present invention relates to a product
`containing a methylmalonic acid loWering agent, an anti
`folate and optionally a FBP binding agent as a combined
`preparation for the simultaneous, separate or sequential use in
`inhibiting tumour groWth.
`The current invention concerns the discovery that admin
`istration of a methylmalonic acid loWering agent in combi
`nation With an antifolate drug reduces the toxicity of the said
`antifolate drug.
`The term “inhibit” as it relates to antifolate drugs refers to
`prohibiting, alleviating, ameliorating, halting, restraining,
`sloWing or reversing the progression of, or reducing tumor
`groWth.
`As used herein, the term “effective amount” refers to an
`amount of a compound or drug, Which is capable of perform
`ing the intended result. For example, an effective amount of
`an antifolate drug that is administered in an effort to reduce
`tumor groWth is that amount Which is required to reduce
`tumor groWth.
`As used herein, the term “toxicity” refers to a toxic event
`associated With the administration on an antifolate. Such
`events include, but are not limited to, neutropenia, throm
`bopenia, toxic death, fatigue, anorexia, nausea, skin rash,
`infection, diarrhea, mucositis, and anemia. For further expla
`nation of the types of toxicity experienced by patients receiv
`ing antifolates, see, generally, Anlifolale Drugs in Cancer
`Therapy. Preferably, toxicity refers to toxic death, fatigue,
`neutropenia, thrombopenia, and mucositis.
`
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`As used herein, the term “nonhematologic event” refers to
`the occurrence of skin rash or fatigue due to the administra
`tion of an antifolate.
`As used herein, the term “in combination With” refers to the
`administration of the methylmalonic acid loWering agent, the
`antifolate drug, and optionally the folic acid; in any order
`such that suf?cient levels of methylmalonic acid loWering
`agent and optionally folic acid are present to reduce the tox
`icity of an antifolate in a mammal. The administration of the
`compounds maybe simultaneous as a single composition or
`as tWo separate compositions or can be administered sequen
`tially as separate compositions such that an effective amount
`of the agent ?rst administered is in the patient’s body When
`the second and/ or third agent is administered. The antifolate
`drug may be administered to the mammal ?rst, folloWed by
`treatment With the methylmalonic acid loWering agent. Alter
`natively, the mammal may be administered the antifolate drug
`simultaneously With the methylmalonic acid loWering agent.
`Preferably, the mammal is pretreated With the methylmalonic
`acid loWering agent and then treated With the antifolate. If
`folic acid is to be administered in addition to the methylma
`lonic acid loWering agent, the folic acid may be administered
`at any time prior, post, or simultaneously to the administra
`tion of either the methylmalonic acid loWering agent or the
`antifolate. Preferably, the mammal is pretreated With the
`methylmalonic acid, and then treated With folic acid, fol
`loWed by treatment With the antifolate compound.
`The terms “antifolate” and “antifolate drug” refer to a
`chemical compound Which inhibits at least one key folate
`requiring enZyme of the thymidine or purine biosynthetic
`pathWays, preferably thymidylate synthase (“TS”), dihydro
`folate reductase (“DHFR”), or glycinamide ribonucleotide
`formyltransferase (“GARFT”), by competing With reduced
`folates for binding sites of these enZymes. Preferred examples
`of antifolates include Tomudex®, as manufactured by Zen
`eca; Methotrexate®, as manufactured by Lederle; Lometr
`exol®, as manufactured by Tularik; pyrido[2,3-d]pyrimidine
`derivatives described by Taylor et al in US. Pat. Nos. 4,684,
`653, 4,833,145, 4,902,796, 4,871,743, and 4,882,334; deriva
`tives described by Akimoto in US. Pat. No. 4,997,838;
`thymidylate synthase inhibitors as found in EPO application
`239,362; and most preferred, Pemetrexed Disodium (AL
`IMTA), as manufactured by Eli Lilly & Co.
`The terms “methylmalonic acid” and “MMA” refer to a
`structural isomer of succinic acid present in minute amounts
`in healthy human urine.
`The term “methylmalonic acid loWering agent” refers to a
`substrate, Which loWers the concentration of methylmalonic
`acid in a mammal. A preferred example of such a substrate is
`vitamin B12. For methods of determining methylmalonic
`acid and substrates therefore, see, e.g., Matchar D B, Feuss
`ner J R, Millington D S, et al. Isotope dilution assay for
`urinary methylmalonic acid in the diagnosis of vitamin B12
`de?ciency. A prospective clinical evaluation. Ann lntem Med
`1987; 106: 707-710; Norman E J, Morrison J A. Screening
`elderly populations for cobalamin (vitamin B12) de?ciency
`using the urinary methylmalonic acid assay by gas chroma
`tography mass spectrometry. Am J Med 1993; 94: 589-594;
`Norman E J. Gas Chromatography mass spectrometry
`screening of urinary methylmalonic acid: early detection of
`vitamin B12 (cobalamin) de?ciency to prevent permanent
`neurologic disability. GC/MS NeWs 1984; 12: 120-129; Mar
`tin D C, Francis J, Protetch J, Huff F J. Time dependency of
`cognitive recovery With cobalamin replacement: report of a
`pilot study. JAGS 1992; 40: 168-172; Norman E J, Cronin C.
`Cobalamin de?ciency. Neurol 1996; 47: 310-311; Rasmus
`sen K, Moelby l, Jensen M K. Studies on methylmalonic acid
`
`Wockhardt Exhibit 1001 - 4
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`US 7,772,209 B2
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`20
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`25
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`30
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`35
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`5
`in humans; Savage D G, Lindenbaum J, Stabler S P, Allen R
`H. Sensitivity ofmethylmalonic acid and total homocysteine
`determination for diagnosing cobalamin and folate de?
`ciency.Am J Med 1994; 96: 239-246.
`The term “vitamin B12” refers to vitamin B12 and its
`pharmaceutical derivatives, such as hydroxocobalamin,
`cyano-10-chlorocobalamin, aquocobalamin perchlorate,
`aquo-10-chlorocobalamin perchlorate,
`aZidocobalamin,
`chlorocobalamin, and cobalamin. Preferably the term refers
`to vitamin B12, cobalamin, and chlorocobalamin.
`The dosage generally Will be provided in the form of a
`vitamin supplement, namely as a tablet administered orally,
`such as a sustained release formulation, as an aqueous solu
`tion added to drinking Water, or as an aqueous parenteral
`formulation. Preferably the methylmalonic acid loWering
`agent is administered as an intramuscular injection formula
`tion. Such formulations are knoWn in the art and are commer
`cially available.
`The skilled artisan Will appreciate that the methylmalonic
`loWering agents are effective over a Wide dosage range. For
`example, When cobalamin is used as the methylmalonic loW
`ering agent, the dosage of cobalamin may fall Within the
`range of about 0.2 pg to about 3000 pg of cobalamin from
`once daily for a month to once every nine Weeks for a year.
`Preferably, cobalamin Will be dosed as an intramuscular
`injection of about 500 pg to about 1500 pg administered from
`about every 24 hours to about every 1680 hours. Preferably, it
`is an intramuscular injection of about 1000 pg administered
`initially from about 1 to about 3 Weeks prior to administration
`of the antifolate and repeated from about every 24 hours to
`about every 1680 hours, regardless of When treatment With
`the antifolate is started and continued until the administration
`of the antifolate is discontinued. Most preferred is an intra
`muscular injection of about 1000 pg administered initially
`from about 1 to about 3 Weeks prior to the ?rst administration
`of the antifolate and repeated every 6 to 12 Weeks, preferably
`about every 9 Weeks, and continued until the discontinuation
`of the antifolate administrations. HoWever, it Will be under
`stood that the amount of the methylmalonic acid loWering
`agent actually administered Will be determined by a physi
`cian, in the light of the relevant circumstances, including the
`condition to be treated, the chosen route of administration, the
`actual agent administered, the age, Weight, and response of
`the individual patient, and the severity of the patient’ s symp
`toms, and therefore the above dosage ranges are not intended
`to limit the scope of the invention in any Way. In some
`instances dosage levels beloW the loWer limit of the aforesaid
`range may be more than adequate, While in other cases still
`larger doses may be employed Without causing any harmful
`side effect.
`The term “FBP binding agent” as used herein refers to a
`folic binding protein binding agent Which includes folic acid,
`(6R)-5-methyl-5,6,7,8-tetrahydrofolic acid, and (6R)-5
`formyl-5,6,7,8-tetrahydrofolic acid, or a physiologically
`available salt or ester thereof. This latter compound is the
`(6R)-isomer of leucovorin as disclosed in J. Am. Chem. Soc.,
`74, 4215 (1952). Both of the tetrahydrofolic acid compounds
`are in the unnatural con?guration at the 6-position. They are
`10-20 fold more ef?cient in binding the folate binding protein
`compared With their respective (6S)-isomer, see Ratnam, et.
`al., Folate and Antifolate Transport in Mammalian Cells
`Symposium, Mar. 21-22, 1991, Bethesda, Md. These com
`pounds are usually prepared as a mixture With their natural
`form (6S) of diastereomers by non-stereoselective reduction
`from the corresponding dehydro precursors folloWed by
`separation through chromatographic or enzymatic tech
`niques. See eg PCT Patent Application Publication W0
`
`40
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`50
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`55
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`60
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`65
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`6
`880844 (also DerWent Abstract 88-368464/51) and Canadian
`Patent 1093554. See, eg Dietary Reference Intakes for Thia
`min, Ribo?avin, Niacin, Vitamin B6, Folate, Vitamin B12,
`Pantothenic Acid, Biotin, and Choline (2000), 8 Folate, pp.
`1 96-305.
`“Physiologically-available salt” refers to potassium,
`sodium, lithium, magnesium, or preferably a calcium salt of
`the FBP binding agent. “Physiologically-available .
`.
`. ester”
`refers to esters Which are easily hydrolyZed upon admini stra
`tion to a mammal to provide the corresponding FBP binding
`agent free acid, such as C 1 -C4 alkyl esters, mixed anhydrides,
`and the like.
`The FBP binding agent to be utiliZed according to this
`invention can be in its free acid form, or can be in the form of
`a physiologically-acceptable salt or ester Which is converted
`to the parent acid in a biological system. The dosage generally
`Will be provided in the form of a vitamin supplement, namely
`as a tablet administered orally, preferably as a sustained
`release formulation, as an aqueous solution added to drinking
`Water, an aqueous parenteral formulation, e.g., an intravenous
`formulation, or the like.
`The FBP binding agent is usually administered to the sub
`ject mammal prior to treatment With the antifolate. Pretreat
`ment With the suitable amount of FBP binding agent from
`about 1 to about 24 hours is usually su?icient to substantially
`bind to and block the folate binding protein prior to admin
`istration of the antifolate. Although one single dose of the
`FBP binding agent, preferably an oral administration of folic
`acid, should be su?icient to load the folate binding protein,
`multiple do sing of the FBP binding agent can be employed for
`periods up to Weeks before treatment With the active agent to
`ensure that the folate binding protein is suf?ciently bound in
`order to maximiZe the bene?t derived from such pretreat
`ment.
`In the especially preferred embodiment of this invention,
`about 0.1 mg to about 30 mg, most preferably about 0.3 mg to
`about 5 mg, of folic acid is administered orally to a mammal
`about 1 to 3 Weeks post administration of the methylmalonic
`acid loWering agent and about 1 to about 24 hours prior to the
`parenteral administration of the amount of an antifolate.
`HoWever, it Will be understood that the amount of the meth
`ylmalonic acid loWering agent actually administered Will be
`determined by a physician, in the light of the relevant circum
`stances, including the condition to be treated, the chosen
`route of administration, the actual agent administered, the
`age, Weight, and response of the individual patient, and the
`severity of the patient’s symptoms, and therefore the above
`dosage ranges are not intended to limit the scope of the
`invention in any Way. In some instances dosage levels beloW
`the loWer limit of the aforesaid range may be more than
`adequate, While in other cases still larger doses may be
`employed Without causing any harmful side effect.
`In general, the term “pharmaceutical” When used as an
`adjective means substantially non-toxic to living organisms.
`
`Methods
`To assess the effect of a methylmalonic acid loWering
`agent, alone or in combination With folic acid on the antitu
`mor e?icacy of an antifolate in a human tumor xenograft
`model, female nude mice bearing human MX-l breast carci
`noma Were treated With ALIMTA alone or along With super
`physiologic doses of folic acid or vitamin B12 (cobalamin).
`The animals Were maintained on steriliZed standard lab
`choW ad libitum and steriliZed Water ad libitum. The human
`MX-l tumor cells (5x106) obtained from donor tumors Were
`implanted subcutaneously in a thigh of female nude mice 8- to
`10-Weeks old. Beginning on day 7 post tumor cell implanta
`
`Wockhardt Exhibit 1001 - 5
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`US 7,772,209 B2
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`7
`tion, the animals Were treated With ALIMTA (100 mg/kg or
`150 mg/kg) once daily on days 7 through 11 and 14 through
`18 by intraperitoneal injection alone or along With folic acid
`(6 mg/kg or 60 mg/kg) and/or vitamin B12 (165 mg/kg) by
`intraperitoneal injection on the same schedu