United States Patent [19J
`Grindey et al.
`
`[54]
`
`[75]
`
`[73]
`
`METHOD FOR TREATING
`GAR-TRANSFORMYLASE TUMORS IN
`MAMMALS AND REDUCING MAMMALIAN
`TOXICITY
`Inventors: Gerald B. Grindey, Indianapolis;
`Chuan Shih, Carmel, both of Ind.
`Assignee: Eli Lilly and Company, Indianapolis,
`Ind.
`
`[21]
`
`[22]
`
`Appl. No.: 940,568
`Sep. 4, 1992
`
`Filed:
`
`[63]
`
`[51]
`
`[52]
`
`[58]
`
`[56]
`
`Related U.S. Application Data
`
`Continuation of Ser. No. 911,429, Jul. 10, 1992, aban(cid:173)
`doned, which is a continuation of Ser. No. 750,841,
`Aug. 26, 1991, abandoned, which is a continuation-in(cid:173)
`pan of Ser. No. 677,031, Mar. 29, 1991, abandoned.
`
`Int. CI.s ..................... AOlN 43/40; AOIN 43/54;
`A61K 31/44; A61K 31/505
`U.S. Cl ..................................... 514/260; 514/340;
`514/227.2; 514/267; 514/269; 514/275;
`514/292; 514/293; 514/342; 514/443; 514/445;
`514/468
`Field of Search ..................... 514/260, 340, 227 .2,
`514/267, 269, 275, 292, 293, 342, 443,445,468
`
`References Cited
`U.S. PATENT DOCUMENTS
`4,684,653 8/1987 Taylor et al. ....................... 514/258
`4,833,145 5/1989 Taylor et al. ....................... 514/258
`4,871,743 10/1989 Taylor et al. ....................... 515/272
`4,882,334 11/1989 Shih et al. ........................... 514/258
`4,902,796 2/1990 Taylor et al. ....................... 544/279
`4,946,846 8/1990 Nomura et al. ..................... 544/258
`4,996,206 2/1991 Taylor et al. ....................... 514/258
`
`11111111111111111111111111111111 lllll lllll 111111111111111111111111111111111
`US005217974A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,217,974
`Jun.8, 1993
`
`4,997,838 3/1991 Akimoto et al. .................... 514/258
`5,010,194 4/1991 Mueller et al. ...................... 544/258
`
`FOREIGN PATENT DOCUMENTS
`1093554 1/1981 Canada .
`409125 1/1991 European Pat. Off. .
`88/08844 11/1988 PCT Int'! Appl. .
`
`OTHER PUBLICATIONS
`Young, et al., Proc. Amer. Assoc. Cancer Res., 31, 1053
`(1990).
`Muggia, et al., Proc. Amer. Soc. Clinical Oncology, 1,
`1285 (1990).
`Grindey, et al., Proceedings of the 82nd Annual Meet(cid:173)
`ing of the American Association for Cancer Research,
`vol. 32, p. 384, Abst. 1921 (1991).
`Internal Eli Lilly and Company Memo Entitled "Can(cid:173)
`cer Progress Conference Trip Report".
`Derwent Abstract 45319S (abstracting DT2063027).
`Morgan, S. L., et al., Arthritis and Rheumatism 33: 9-18
`(1990).
`Straw, et al., Cancer Research, 44:3114-3119 (1984).
`Temple, et al., Cancer Treatment Reports, 65: 1117-1119
`(1981).
`
`Primary Examiner-Nathan M. Nutter
`Attorney, Agent, or Firm-Steven A. Fontana; Leroy
`Whitaker
`
`[57]
`ABSTRACT
`Administration of a fol ate binding protein binding agent
`in conjunction with use of an antitumor agent which is
`an inhibitor of glycinamide ribonucleotide transformy(cid:173)
`lase or other antifolate reduces the toxic effects of such
`agent and provides an enhanced therapeutic index.
`
`22 Claims, No Drawings
`
`Teva – Fresenius
`Exhibit 1009-00001
`
`

`
`1
`
`5,217,974
`
`2
`available salt or ester thereof. The invention more par(cid:173)
`ticularly provides a method for reducing the mamma(cid:173)
`lian toxicity of a GAR-transformylase inhibitor or other
`antifolate which binds to a FBP which comprises ad(cid:173)
`ministering a toxicity-reducing amount of a FBP bind(cid:173)
`ing agent or a physiologically-available salt or ester
`thereof to. the mammal receiving treatment. In particu(cid:173)
`lar, there is provided a method for reducing the toxicity
`10 of a GAR-transformylase inhibitor or other antifolate
`which binds to a FBP in a mammal which comprises
`pretreating the mammal with an amount of a compound
`selected from folic acid, (6R)-5-methyl-5,6,7,8-tetrahy-
`15 drofolic acid, and (6R)-5-formyl-5,6,7,8-tetrahydrofolic
`acid, or a physiologically-available salt or ester thereof,
`sufficient to have substantially blocked the FBP before
`administration of the antifolate. In the most preferred
`embodiment of the invention, Lometrexol is adminis(cid:173)
`tered to a subject suffering from a solid tumor or other
`type of cancer and in need of treatment after pretreat-
`mei:it with folic acid, thereby reducing toxic effects of
`Lometrexol while maintaining good antitumor activity.
`
`METHOD FOR TREATING
`GAR· TRANSFORMYLASE TUMORS IN
`MAMMALS AND REDUCING MAMMALIAN
`TOXICITY
`
`This application is a continuation of application Ser.
`No. 07/911,429 filed Jul. 10, 1992, now abandoned,
`which is a continuation application Ser. No. 07/750,841,
`filed Aug. 26, 1991, now abandoned, which is a con(cid:173)
`tinuation-in-part of application Ser. No. 07/677,031
`filed Mar. 29, 1991 and now abandoned.
`
`5
`
`BACKGROUND OF THE INVENTION
`Lometrexol is the generic name given to 5, 10-
`dideazatetrahydrofolic acid, also
`referred
`to as
`DDA THF. Lometrexol is a member of a new class of
`antitumor agents which have been found to specifically 20
`inhibit glycinamide ribonucleotide (GAR) transformy·
`lase, an enzyme required in the initial stages of purine
`biosynthesis, see J. Med. Chem., 28, 914 (1985). Several
`of these GAR·transformylase inhibitors are described, 25
`along with their antitumor utilities, 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. GAR-transformylase inhibitors are also
`known to be useful in treating conditions such as gout,
`psoriasis, mycosis fungoides, autoimmune disorders, 30
`rheumatoid arthritis and other inflammatory disorders,
`and during organ transplantation and other related im(cid:173)
`munosuppressant related conditions.
`Lometrexol has been studied clinically and shown to 35
`be a potent antitumor agent, especially against solid
`tumors such as colorectal, lung, breast, head and neck
`and pancreatic; Young et al., Proc. Amer. Assoc. Cancer
`Research, 31, 1053 (1990). Like most other antitumor
`agents, Lometrexol exhibits some undesirable side ef- 40
`fects, in addition to its efficacy against tumors; Muggia
`et al., Proc. Amer. Soc. Clinical Oncology. 9, 1285 (1990).
`Typical side effects observed to date include anorexia,
`weight loss, mucositis, leukopenia, anemia, hypoac- 45
`tivity and dehydration.
`We have now discovered that the toxic effects of
`lometrexol and related GAR-transformylase inhibitors
`and other antifolate agents which bind to folate binding
`protein (FBP) (see, e.g., Kane, et al., Laboratory Investi· 50
`gation, 60, 737 (1989)) can be significantly reduced by
`the presence of a FBP binding agent, without adversely
`affecting therapeutic efficacy. The present invention
`thus provides a method for improving the therapeutic 55
`utility of GAR-transformylase inhibitors and other an(cid:173)
`tifolates by co-administering a FBP binding agent to the
`host under going treatment.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`The invention provides a method for reducing the
`toxicity of GAR-transformylase inhibitor or other an(cid:173)
`tifolates that bind to a FBP that is found in biological
`systems by the prior administration of a FBP binding
`agent or a physiologically-available salt or ester thereof.
`GAR-transformylase inhibitors and related antifolates
`are those compounds which effectively inhibit the bio(cid:173)
`logical actions of the enzyme known as glycinamide
`ribonucleotide transformylase. This enzyme is well
`known to be required in the initial stages of purine bio(cid:173)
`synthesis in mammals, which is implicated in DNA
`synthesis. Interruption of this biosynthetic pathway
`causes a disturbance in DNA synthesis and conse-
`quently causes cell death. Any compound which is
`shown to inhibit the GAR-transformylase or other fo(cid:173)
`late-requiring enzyme is subject to treatment in accor(cid:173)
`dance with this invention.
`Typical GAR-transformylase inhibitors include the
`pyrido[2,3-d]pyrimidine derivatives described by Tay(cid:173)
`lor et al.
`in U.S. Pat. Nos. 4,684,653, 4,833,145,
`4,902,796, 4,871,743 and 4,882,334. Another series of
`GAR-transformylase inhibitors has recently been de-
`scribed by Akimoto in U.S. Pat. No. 4,997,838. Antifo(cid:173)
`late compounds which can be employed in this inven(cid:173)
`tion include thymidylate synthase inhibitors as found in
`EPO Patent Application 239,362. All of the foregoing
`references are incorporated herein by reference for
`their teaching of the structure and synthesis of typical
`GAR-transformylase inhibitors and antifolates. Other
`SUMMARY OF THE INVENTION
`60 GAR-transformylase inhibitors and antifolates are also
`In one aspect of this invention, we provide a method
`included within the scope of this invention, and such
`compounds can be determined by routine evaluation of
`of inhibiting
`the growth of GAR-transformylase-
`either their ability to interact with and inhibit the sub-
`dependent tumors in mammals comprising administer-
`ing to said mammals an effective amount of a GAR- 65 ject enzyme or to bind to the FBP.
`transformylase inhibitor or other antifolate which binds
`In a preferred embodiment of the invention, folic acid
`to a FBP in combination with a toxicity-reducing
`is administered to a subject subsequently receiving an
`amount of a FBP binding agent, or a physiologically-
`agent defined by the formula
`
`Teva – Fresenius
`Exhibit 1009-00002
`
`

`
`3
`
`5,217,974
`
`4
`
`wherein
`R 1 is hydroxy or amino;
`R2 is hydrogen, methyl, ethyl, or propynyl;
`B is -CH- or -N-;
`n is 1, 2 or 3;
`Z is nitrogen or carbon;
`A is pyrido, tetrahydropyrido, pyrrolo, dihydropyr(cid:173)
`rolo, cyclopentyl or cyclohexyl;
`X is hydrogen or halo; and pharmaceutically accept(cid:173)
`able salts thereof.
`In a particularly preferred embodiment of the inven- 20
`tion, lometrexol is utilized as the GAR-transformylase
`inhibitor.
`
`As noted above, the drug products which can be
`employed in the present invention include other antifo(cid:173)
`lates which are capable of binding to folate binding
`protein. Folic acid itself has a binding constant (ng/ml)
`of 1.8, and lometrexol has a binding constant of 9.7 to
`15 bovine FBP. Any GAR-transformylase inhibitor or
`other antifolate that binds at less than about 500 ng/ml
`can be utilized in the method of this invention. The
`folate binding constant for drug products can be readily
`determined by the general procedure of Dunn and Fos(cid:173)
`ter, C/in. Chem., 19 (10), 1101-1105 (1973). Typical
`antifolates evaluated in the referenced procedure have
`the following folate binding constants presented in
`Table I below:
`
`TABLE I
`
`Binding Constant
`(ng/ml)
`
`n=3 X=H
`n = 2
`X = 2-F
`n = 2
`X = 3-F
`
`N
`
`A
`
`H2N
`
`N
`
`OH
`
`N
`)l
`H2N
`N
`
`N
`H
`
`N
`H
`
`s
`
`CONHCHCH2CH2COOH
`I
`COOH
`
`12.5
`14.0
`30.7
`
`250.8
`
`24.5
`
`26.0
`
`12.0
`
`Teva – Fresenius
`Exhibit 1009-00003
`
`

`
`5
`
`5,217,974
`
`6
`
`TABLE I-continued
`
`'DOH
`
`N
`~
`HJC
`N
`
`CHJ~
`CH2-~--Z s )--coNH?HCH2CH2COOH
`
`COOH
`
`Binding Constant
`(ng/ml)
`
`245
`
`The FBP binding agent to be utilized according to
`As used in this invention, the term "FBP binding
`this invention can be in its free acid form, or can be in ·
`agent" refers to folic acid, (6R)-5-methyl-5,6,7,8-tet-
`the form of a physiologically-acceptable salt or ester
`rahydrofolic acid, or (6R)-5-formyl-5,6,7,8-tetrahy-
`drofolic acid. This latter compound is the (6R)-isomer 15 which is converted to the parent acid in a biological
`of Ieucovorin as disclosed in J. Am. Chem. Soc., 74, 4215
`system. The dosage generally will be provided in the
`(1952). Both of the tetrahydrofolic acid compounds are
`form ofa vitamin supplement, namely as a tablet admin-
`in the unnatural configuration at the 6-position-they
`istered orally, preferably as a sustained release formula-
`are 10-20 fold more efficient in binding the folate bind-
`tion, as an aqueous solution added to drinking water, an
`ing protein compared with their respective (6S)- 20 aqueous parenteral formulation, e.g., an intravenous
`isomer-see Ratnam, et. al., Folate and Antifolate
`formulation, or the like.
`Transport in Mammalian Cells Symposium, Mar. 21-22,
`The FBP binding agent is administered to the subject
`1991, Bethesda, Md. These compounds are usually pre-
`mammal prior to treatment with the GAR-transformy-
`pared as a mixture with their natural form (6S) of diaste-
`lase inhibitor or other antifolate. Pretreatment with the
`reomers by non-stereoselective reduction from the cor- 25 suitable amount of FBP binding agent from about I to
`responding dehydro precursors followed by separation
`about 24 hours is usually sufficient to substantially bind
`through chromatographic or enzymatic techniques. See
`to and block the folate binding protein prior to adminis-
`e.g., PCT Patent Application Publication WO 880844
`tration of the GAR-transformylase inhibitor or other
`(also Derwent Abstract 88-368464/51) and Canadian
`antifolate. Although one single dose of the FBP binding
`Patent 1093554.
`30 agent, preferably an oral administration of folic acid,
`Folic acid is a vitamin which is required by mammals
`should be sufficient to load the folate binding protein,
`for proper regeneration of the blood-forming elements
`multiple dosing of the FBP binding agent can be em-
`and their functioning, and as a coenzyme is involved in
`ployed for periods up to weeks before treatment with
`intermediary metabolic processes in which one-carbon
`the active agent to ensure that the folate binding protein
`units are transferred. These reactions are important in 35 is sufficiently bound in order to maximize the benefit
`interconversions of various amino acids and in purine
`derived from such pretreatment.
`and pyrimidine synthesis. Folic acid is commonly sup-
`In the especially preferred embodiment of this inven-
`plied to diets of humans via consumption of food
`tion, about I mg to about 5 mg of folic acid is adminis-
`sources such as liver, kidney, dry beans, asparagus,
`tered orally to a mammal about 1 to about 24 hours
`mushrooms, broccoli, lettuce, milk and spinach, as well 40 prior to the parenteral administration of the amount of
`as by vitamin supplements. The minimum amount of
`lomotrexol which is normally required to attain the
`folic acid commonly required by normal adults is about
`desired therapeutic benefit. Although greater or addi-
`0.05 mg/day. According to this invention, folic acid, or
`tional doses of folic acid or another FBP binding agent
`a physiologically-available salt or ester thereof, is ad-
`are also operable, the above parameters will usually
`ministered to a human subject at a dose of about 0.5 45 bind the folate binding protein in an amount sufficient to
`mg/day to about 30 mg/day to diminish the toxic ef-
`reduce the toxicity effects normally seen upon lomo-
`fects of a GAR-transformylase inhibitor or other antifo-
`trexol administration above.
`late also being administered to such subject. In a pre-
`It should be noted that the FBP binding agent is not
`ferred embodiment, folic acid will be administered at
`an antitumor agent and that the pretreatment of a mam-
`about 1 to about 5 mg/day together with the normal 50 mal with a FBP binding agent is not a synergistic or
`dosing of GAR-transformylase inhibitor such as lomet-
`potentiating effect. Rather, by having substantially
`bound the folate binding protein with a FBP binding
`rexol.
`Based upon the relative binding constants for the
`agent prior to administration of the GAR-transformy-
`lase inhibitor or other antifolate, the toxic effects of
`respective compounds, it will be expected that approxi-
`mately 1 mg/day to 90 mg/day (preferably approxi- 55 such subsequent treatment are greatly reduced without
`mately 2-15 mg/day) of (6R)-5-methyl-5,6,7,8-tetrahy-
`affecting the therapeutic efficacy.
`drofolic acid or about 5-300 mg/day (preferably about
`The effect of folic acid on GAR-transformylase in-
`10-50 mg/day) of (6R)-5-formyl-5,6,7,8-tetrahydrofolic
`hibitors has been demonstrated in standard tests com-
`acid, or their respective physiologically-available salt or
`monly utilized to determine the antitumor activity and
`ester thereof, will be employed with the GAR-transfor- 60 toxic effects of the GAR-transformylase inhibitors
`mylase inhibitor.
`themselves. In one such test, mice are inoculated with
`"Physiologically-available salt" refers to potassium,
`the C3H strain of mammary adenocarcinoma by insert-
`sodium, lithium, magnesium, or preferably a calcium
`ing a 2 mm by 2 mm section of tumor into the axillary
`salt of the FBP binding agent. "Physiologically-availa-
`region of the mice by trocar. In all experiments, lomet-
`ble ... ester" refers to esters which are easily hydro- 65 rexol was administered intraperitoneally once a day for
`lyzed upon administration to a mammal to provide the
`five consecutive days, starting on the day following
`corresponding FBP binding agent free acid, such as
`tumor implantation. Ten animals were used at each
`C 1-C4 alkyl esters, mixed anhydrides, and the like.
`dosage level. Antitumor activity was assessed on day
`
`Teva – Fresenius
`Exhibit 1009-00004
`
`

`
`7
`ten by measuring the length and width of the tumor
`growth using vernier calipers, and the activity was
`expressed as a percent inhibition of tumor growth.
`When lometrexol was administered to infected mice
`which are maintained on a diet totally free of folic acid 5
`for two weeks prior to and during treatment, it exhib-
`ited moderate antitumor activity at very low doses, but
`also caused severe toxicity at a very low dose (measured
`as death of mice). These data are presented in Table II
`below.
`
`5,217,974
`
`8
`human dose of about 30 mg/day), good antitumor activ(cid:173)
`ity of lometrexol is observed at higher dose levels.
`These results are shown in Table IV below:
`TABLE IV
`
`Antitumor Activity and Toxicity of Lometrexol
`in c3H Mice after Two Weeks on Folate-Free Diet
`Plus Addition of 0.003% Folate to Drinking Water
`Lometrexol Dose Antitumor Activity
`Toxicity
`(% Inhibition)
`(mg/kg)
`(Mice Dead/Total Mice)
`IO ---6"-.2-5""-----9-l<J<-o------0-/-10---'--
`12.5
`89%
`25
`97%
`50
`96%
`
`TABLE II
`Antitumor Activity and Toxicity of Lometrexol
`in C3H Mice after Two Weeks on Folate-Free Diet
`Toxicity
`(Mice Dead/Total Mice) 15
`
`0110
`0/10
`0/10
`
`A test group of mice were maintained on a folic acid
`free diet for two weeks before treatment. Felic acid was
`then administered during the treatment by providing
`the animals drinking water containing 0.0003% folic
`acid (weight/volume). This concentration translates to
`about 1.75 mg of folic acid per square meter of body
`surface per day; since the animals consume about 4 ml
`of water each day.
`
`0.0003 grams
`lOOml.
`
`X -1..!!!L_ = 0.000012 grams
`day
`day
`
`0.012 milligrams
`day
`
`The average size of a mouse is 0.00687 m2
`
`0.012 grams X
`day
`
`1
`0.00687 ml
`
`= 1.75 mg/m2/day
`
`For a human subject of about I. 73 m2 size, this translates
`to an adult human dosage of about 3.0 mg/day. The
`effect of the foregoing folate dosage on the activity and
`toxicity of lometrexol is shown in Table III below:
`TABLE III
`Antitumor Activity and Toxicity of Lometrexol
`in C3H Mice after Two Weeks on Folate-Free Diet
`Plus Addition of 0.0003% Folate to Drinking Water
`Antitumor Activity
`Lometrexol Dose
`Toxicity
`(% Inhibition)
`(Mice Dead/Total Mice)
`(mg/kg)
`0/10
`13%
`0.125
`0/10
`26%
`0.25
`0/10
`48%
`0.5
`0/10
`97%
`1.0
`0/10
`98%
`2.0
`4/10
`99%
`4.0
`
`SO
`
`As the foregoing results indicate, addition of the indi(cid:173)
`cated level of folic acid to the diet of a subject receiving
`lometrexol results in excellent antitumor activity at low 60
`doses, with little or no toxic effects.
`Larger doses of folic acid appear to have an even
`more dramatic effect on the antitumor activity and
`toxicity of the GAR-transformylase inhibitor. For ex(cid:173)
`ample, when mice were maintained on a folate acid-free
`diet for two weeks before treatment with lometrexol,
`and then given water containing 0.003% (weight(cid:173)
`/volume) of folic acid (which translates to an adult
`
`Teva – Fresenius
`Exhibit 1009-00005
`
`

`
`5,217,974
`
`9
`2. The method of claim 1 wherein the GAR-transfor(cid:173)
`mylase inhibitor is a pyrido[2,3-d]pyrimidine.
`3. The method of claim 2 wherein the GAR-transfor(cid:173)
`mylase inhibitor is lometrexol.
`4. The method of claim 1 wherein the folate binding
`protein binding agent is folic acid.
`5. The method of claim 3 wherein the folate binding
`protein binding agent is folfo acid.
`6. The method of claim 5 wherein the folic acid is
`administered at,a dose of about 0.5 mg/day to ab~ut 30
`mg/day.
`7. The method of claim 5 wherein the folic acid is
`administered at a dose of about 1 mg/day to about 5 15
`mg/day.
`8. A method for reducing mammalian toxicity of a
`GAR-transformylase inhibitor which binds to a folate
`binding protein consisting of administering a toxicity 2o
`reducing amount of a folate binding protein binding
`agent selected from folic acid, (6R)-5-methyl-5,6,7,8-
`tetrahydrofolic acid, and (6R)-5-formyl-5,6,7,8-tetrahy(cid:173)
`drofolic acid, or a physiologically-available salt or ester
`. .
`. h h 25
`h
`I
`thereof, to t e mamma rece1vmg treatment wit
`t e
`GAR-transformylase inhibitor.
`9. The method of claim 8 wherein the GAR-transfor(cid:173)
`mylase inhibitor is a pyrido[2,3-d]pyrimidine.
`10. The method of claim 9 wherein the GAR-trans- 30
`formylase inhibitor is lometrexol.
`11. The method of claim 8 wherein the folate binding
`protein binding agent is folic acid.
`12. The method of claim 11 wherein folic acid is 35
`administered at a dose of about 0.5 mg/day to about 30
`mg/day.
`
`10
`13. The method of claim 12 wherein folic acid is
`administered at a dose of about 1 mg/day to about 5
`mg/day.
`14. The method of claim 8 wherein the folate binding
`5 protein binding agent is (6R)-5-methyl-5,6, 7,8-tetrahy(cid:173)
`drofolic acid or a physiologically-available salt or ester
`thereof.
`15. The method of claim 8 wherein the folate binding
`protein binding agent is (6R)-5-formyl-5,6,7,8-tetrahy(cid:173)
`IO drofolic acid or a physiologically-available salt or ester
`thereof.
`16. A method for reducing the toxicity of a GAR-
`transformylase inhibitor or other antifolate which binds
`to a folate binding protein in a mammal which com(cid:173)
`prises pretreating the mammal with an amount of a
`folate binding protein binding agent selected from folic
`acid, (6R)-5-methyl-5,6,7,8-tetrahydrofolic acid, and
`(6R)-5-formyl-5,6,7,8-tetrahydrofolic acid, or a physio(cid:173)
`logically available salt or ester thereof, sufficient to
`have substantially blocked the folate binding protein
`before administration of the antifolate.
`17. The method of claim 16 wherein the GAR-trans(cid:173)
`formylase inhibitor is lomotrexol.
`18. The method of claim 16 wherein the folate bind-
`ing protein binding agent is folic acid.
`19. The method of claim 18 wherein the folic acid is
`administered about 1 to about 24 hours prior to adminis(cid:173)
`tration of the antifolate.
`20. The method of claim 19 wherein a dose of about
`0.5 mg to about 30 mg of folic acid is administered.
`21. The method of claim 10 wherein the folate bind(cid:173)
`ing protein binding agent is folic acid, or a physiologi(cid:173)
`cally-available salt or ester thereof.
`22. The method of claim 17 wherein the folate bind(cid:173)
`ing protein binding agent is folic acid, or a physiologi(cid:173)
`cally-available salt or ester thereof.
`* * * * *
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Teva – Fresenius
`Exhibit 1009-00006