US007772209B2
`
`(12) United States Patent
`Niyikiza
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,772,209 B2
`Aug. 10, 2010
`
`(54) ANTIFOLATE COMBINATION THERAPIES
`
`WO
`
`WO 95/27723
`
`10/1995
`
`(75)
`
`Inventor: Clet Niyikiza, Indianapolis, IN (US)
`
`(73) 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) Appl. No.: 111776,329
`
`(22) Filed:
`
`Jul. 11, 2007
`
`(65)
`
`Prior Publication Data
`
`US 2008/0032948 A1
`
`Feb. 7, 2008
`
`Related U.S. Application Data
`
`(62) Division of application No. 11/288,807, filed on Nov.
`29, 2005, now abandoned, which is a division of appli-
`cation No. 10/297,821, filed as application No. PCT/
`US01/14860 on Jun. 15, 2001, now Pat. No. 7,053,065.
`
`(60) Provisional application No. 60/215,310, filed on Jun.
`30, 2000, provisional application No. 60/235,859,
`filed on Sep. 27, 2000, provisional application No.
`60/284,448, filed onApr. 18, 2001.
`
`(51)
`
`(52)
`
`(58)
`
`(56)
`
`Int. CI.
`A 61K 31/70
`(2006.01)
`A61K 31/685
`(2006.01)
`A61K 31/50
`(2006.01)
`A61K 31/525
`(2006.01)
`A61K 31/519
`(2006.01)
`U.S. CI ............................ 514/52; 514/77; 514/249;
`514/251; 514/265.1
`Field of Classification Search ................... 514/52,
`514/77, 249, 251,265.1
`See application file for complete search history.
`
`References Cited
`
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`
`(Continued)
`
`Primary Examine~Kevin Weddington
`(74) Attorney, Agent, or Firm~lizabeth 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.
`
`EP
`
`0 546 870
`
`6/1993
`
`22 Claims, No Drawings
`
`Sandoz Inc.
`Exhibit 1001-0001
`
`JOINT 1001-0001
`
`

`
`US 7,772,209 B2
`Page 2
`
`OTHER PUBLICATIONS
`
`Tsao C, et al. Influence of cobalamin on the survival of mice bearing
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`Hammond, L., et al., "A phase I and pharmacokinetic (PK) study of
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`Selectivity." In Jackson (Ed.), Antifolate Drags in Cancer Therapy.
`pp. 13-36. Humana Press, New Jersey.
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`folate levels and hyperhomocysteinemia during long-term, low dose
`methotrexate therapy for rheumatoid arthritis: implications for car-
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`
`* cited by examiner
`
`Sandoz Inc.
`Exhibit 1001-0002
`
`JOINT 1001-0002
`
`

`
`US 7,772,209 B2
`
`1
`ANTIFOLATE COMBINATION THERAPIES
`
`This application is a divisional of application Ser. No.
`11/288,807, filed 29 Nov., 2005 now abandoned, which is a
`divisional of application Ser. No. 10/297,821 filed 12 May,
`2002, now U.S. Pat. No. 7,053,065, which claims priority
`under 35 USC 371, for PCT/US01/14860, filed 15 Jun., 2001,
`which claims the priority of U.S. provisional applications No.
`60/215,310, filed 30 Jun., 2000, No. 60/235,859, filed 27
`Sep., 2000, and No. 60/284,448, filed 18 Apr., 2001.
`Potentially, life-threatening toxicity remains a maj or limi-
`tation to the optimal administration of antifolates. (see, gen-
`erally, Antifolate Drugs in Cancer Therapy, edited by Jack-
`man, Ann L., Humana Press, Totowa, N.J., 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. (Antifolate, 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 R
`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, Moliterni A, Zambetti M, Brambilla C. Adjuvant
`cyclophosphamide, methotrexate, and fluorouracil in node-
`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 biosynthetic pathways,
`in particular, thymidylate synthase (TS), dihydrofolate reduc-
`tase (DHFR), and glycinamide ribonucleotide formyltrans-
`ferase (GARFT), by competing with reduced folates for bind-
`ing sites of these enzymes. (Shih C, Habeck L L, Mendelsohn
`L G, Chen V 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 dxugs are currently in
`development. Examples of antifolates that have thymidylate
`synthase inhibiting ("TSI") characteristics include 5-fluorou-
`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 dxugs 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 ofantifolates
`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
`
`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
`5 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
`10 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
`15 a treatment for toxicities associated with GARFTI see, e.g.
`U.S. Pat. No. 5,217,974. Folic acid has been shown to lower
`homocysteine levels (see e.g. Homocysteine Lowering Trial-
`ist’s Collaboration. Lowering blood homocysteine with folic
`acid based supplements: meta-analysis of randomized trials.
`2o 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-
`25 dictor ofcytotoxic events related to the use ofGARFT inhibi-
`tors, see e.g.U.S. Pat. No. 5,217,974. However, even with 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
`3o 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-
`35 folates, as a class, can be significantly reduced by the pres-
`ence of a methylmalonic acid lowering agent, without
`adversely affecting therapeutic efficacy. The present inven-
`tion thus provides a method for improving the therapeutic
`utility of antifolate drugs by administering to the host under-
`4o 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 dxug and that treatment for the increased methyl-
`malonic acid, such as treatment with vitamin B12, reduces
`45 mortality and nonhematologic events, such as skin rashes and
`fatigue events previously associated with the antifolate dxugs.
`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-
`5o 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 dxugs 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
`6o 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
`65 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.
`
`55
`
`Sandoz Inc.
`Exhibit 1001-0003
`
`JOINT 1001-0003
`
`

`
`US 7,772,209 B2
`
`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 administer-
`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 said meth-
`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 &rug.
`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 eflbrt 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, Antifolate Drugs in Cancer
`Therapy. Preferably, toxicity refers to toxic death, fatigue,
`neutropenia, thrombopenia, and mucositis.
`
`4
`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
`5 administration of the methylmalonic acid lowering agent, the
`antifolate drug, and optionally the folic acid; in any order
`such that sufficient 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
`10 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 first administered is in the patient’s body when
`the second and/or third agent is administered. The antifolate
`15 drug may be administered to the mammal first, followed by
`treatment with the methylmalonic acid lowering agent. Alter-
`natively, the mammal may be administered the antifolate dxug
`simultaneously with the methylmalonic acid lowering agent.
`Preferably, the mammal is pretreated with the methylmalonic
`20 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
`25 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-
`30 requiting 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
`35 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 U.S. Pat. Nos. 4,684,
`653, 4,833,145, 4,902,796, 4,871,743, and 4,882,334; deriva-
`4o tives described by Akimoto in U.S. 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
`45 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
`5o 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
`deficiency. A prospective clinical evaluation. Ann Intern Med
`55 1987; 106: 707-710; Norman E J, Morrison JA. Screening
`elderly populations for cobalamin (vitamin B12) deficiency
`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
`6o screening of urinary methylmalonic acid: early detection of
`vitamin B12 (cobalamin) deficiency to prevent permanent
`neurologic disability. GC/MS News 1984; 12:120-129; Mar-
`tin D C, Francis J, Protetch J, HuffF J. Time dependency of
`cognitive recovery with cobalamin replacement: report of a
`65 pilot study. JAGS 1992; 40: 168-172; Norman E J, Cronin C.
`Cobalamin deficiency. Neurol 1996; 47:310-311 ; Rasmus-
`sen K, Moelby I, Jensen M K. Studies on methylmalonic acid
`
`Sandoz Inc.
`Exhibit 1001-0004
`
`JOINT 1001-0004
`
`

`
`US 7,772,209 B2
`
`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 defi-
`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 B 12, 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 smiled 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 ~tg to about 3000 ~tg 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 ~tg to about 1500 ~tg administered from
`about every 24 hours to about every 1680 hours. Preferably, it
`is an intramuscular injection of about 1000 ~tg 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 ~tg administered initially
`from about 1 to about 3 weeks prior to the first 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 configuration at the 6-position. They are
`10-20 fold more efficient in binding the folate binding protein
`compared with their respective (6S)-isomer, see Ratuam, 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 e.g. PCT Patent Application Publication WO
`
`6
`880844 (also Derweut Abstract 88-368464/51) and Canadian
`Patent 1093554. See, e.g. Dietary Reference Intakes for Thia-
`min, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12,
`Pantothenic Acid, Biotin, and Choline (2000), 8 Folate, pp.
`5 196-305.
`"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 administra-
`10 tion to a mammal to provide the corresponding FBP binding
`agent free acid, such as C1 -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
`15 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
`2o 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
`25 about 1 to about 24 hours is usually sufficient 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 sufficient to load the folate binding protein,
`30 multiple dosing 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 sufficiently bound in
`order to maximize the benefit derived from such pretreat-
`ment.
`35 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
`4o 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
`45 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
`5o 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.
`
`55
`
`Methods
`To assess the effect of a methylmalonic acid lowering
`agent, alone or in combination with folic acid on the antitu-
`mor efficacy of an antifolate in a human tumor xenograft
`6o model, female nude mice bearing human MX-1 breast carci-
`noma were treated with ALIMTA alone or along with super-
`physiologic doses of folic acid or vitamin B 12 (cobalamin).
`The animals were maintained on sterilized standard lab
`chow ad libitum and sterilized water ad libitum. The human
`65 MX- 1 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-
`
`Sandoz Inc.
`Exhibit 1001-0005
`
`JOINT 1001-0005
`
`

`
`US 7,772,209 B2
`
`7
`tion, the animals were treated