United States Patent [191
`Grindey et al.
`
`1||||IIIIIIIIIllillllllllllllllllllllllllllllll1111lllllllllllllllllllllll
`
`USOO5217974A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,217,974
`Jun. 8, 1993
`
`[54] METHOD FOR TREATING
`GAR_TRANSFORMYLASE TUMORS IN
`gigggdlgs AND REDUCING MAMMALIAN
`
`4,997,838 3/1991 Akimoto et a1. .................. .. 514/258
`5,010,194 4/1991 Mueller et a1. .................... .. 544/258
`FOREIGN PATENT DOCUMENTS
`
`'
`[75] Inventors: Gerald B. Grindey, Indianapolis;
`Chuan Shih, Carmel, both of 1nd
`[73] Assignee: Eli Lilly and Company, Indianapolis,
`Ind'
`[21] Appl. No.: 940,568
`-
`:
`. 4, 1992
`[22] Flled
`Sep
`
`ntmuation 0'
`
`er.
`
`o.
`
`v,
`
`,
`
`I! ~
`
`,
`
`1
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`a '
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`[63] Co _ Related fUS'S' gpplglcjljzg imlmio 1992 ab n
`ionedzaéwgglll 15: ‘ggm‘guillhoighc’fs ieéér?iz'u
`'
`’ a an “e ’
`’
`"g"
`Pa" °f 5“ N°' 6mm’ Ma" 29’ 1991’ ab‘mdmd'
`[51] Int. Cl.5 ................... .- AUIN 43/40; AOIN 43/54;
`A61K 31/44; A61K 31/505
`[52] us. Cl. .................................. .. 514/260; 514/340;
`514/2272; 514/267; 514/269; 514/275;
`514/292; 514/293; 514/342; 514/443; 514/445;
`
`'
`
`[58] Field of Search ................... .. 514/260, 340, 227.2,
`514/267, 269, 275, 292, 293, 342, 443,445,468
`References Cited
`U.S. PATENT DOCUMENTS
`
`[56]
`
`514/468
`
`4,684,653 8/1987 Taylor et a1. ..................... .. 514/258
`4,833,145 5/1989 Taylor et a1. ..
`.1 514/258
`4,871,743 10/1989 Taylor et a1. ..
`.. 515/272
`4,882,334 11/1989 Shih et a1. .... .1
`.. 514/258
`4,902,796 2/1990 Taylor et a1. ..
`.... .. 544/279
`4,946,846 8/ 1990 Nomura et a1.
`.... _. 544/258
`4,996,206 2/1991 Taylor et a1. ..................... .. 514/258
`
`1093554 1/1981 Canada .
`409125 1/1991 European pat 0m _
`88/08844 11/1988 PCT 1m Appl. .
`OTHER PUBLICATIONS
`Young, et al., Proc. Amer. Assoc. Cancer Res, 31, 1053
`(1990).
`Muggia, et al., Proc. Amer. Soc. Clinical 011001051, 1,
`1285 (1990).
`Grindey, et al., Proceedings of the 82nd Annual Meet
`ingl o3f2the gtggericzm isSZOICéTES?) for Cancer Research,
`Internal Eli Lilly and Company Memo Entitled “Can
`cer Progress Conference Trip Report”.
`Derwent Abstract 453195 (abstracting DT2063027).
`Morgan, S. L., et a1., Arthritis and Rheumatism 33: 9-18
`(1990),
`Straw, et al_, Cancer Research, 44:3114-3119 (1984)_
`Temple, et al., Cancer Treatment Reports, 65:111'7-1119
`(1981),
`
`v0 _
`
`, p.
`
`,
`
`st.
`
`.
`
`,
`
`.
`
`N
`
`‘ L
`jrlma’y "Emmi? “hag M- NR1“?
`86"” 0’ "'"— ‘even ~ (‘mama my
`
`ABSTRACT
`[57]
`Administration of a folate binding protein binding agent
`in conjunction with use of an antitumor agent which is
`an inhibitor of glycinamide ribonucleotide transformy
`lase or other antifolate reduces the toxic effects of such
`agent and provides an enhanced therapeutic index.
`
`22 Claims, No Drawings
`
`Wockhardt Exhibit 1009 - 1
`
`

`
`1
`
`5,217,974
`
`METHOD FOR TREATING
`GAR-TRANSFORMYLASE TUMORS IN
`MAMMALS AND REDUCING MAMMALIAN
`‘
`TOXICITY
`
`This application is a continuation of application Ser.
`No. 07/911,429 ?led Jul. 10, 1992, now abandoned,
`which is a continuation application Ser. No. 07/ 750,841,
`?led Aug. 26, 1991, now abandoned, which is a con
`tinuation-in-part of application Ser. No. 07/677,031
`?led Mar, 29, 1991 and now abandoned.
`
`BACKGROUND OF THE INVENTION
`
`2
`available salt or ester thereof. The invention more par
`ticularly provides a method for reducing the mamma
`lian toxicity of a GAR-transformylase inhibitor or other
`5 antifolate which binds to a FBP which comprises ad
`ministering a toxicity-reducing amount of a FBP bind—
`ing agent or a physiologically-available salt or ester
`thereof tothe mammal receiving treatment. In particu- I
`lar, there is provided a method for reducing the toxicity
`of a GAR-transforrnylase 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
`drofolic acid, and (6R)~5-formyl-5,6,7,8-tetrahydrofolic
`acid, or a physio]ogically-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
`tered to a subject suffering from a solid tumor or other
`type of cancer and in need of treatment after pretreat
`ment with folic acid, thereby reducing toxic effects of
`Lometrexol while maintaining good antitumor activity.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`The invention provides a method for reducing the
`toxicity of GAR-transformylase inhibitor or other an
`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
`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
`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
`late-requiring enzyme is subject to treatment in accor
`dance with this invention.
`Typical GAR-transformylase inhibitors include the
`pyrido[2,3-d]pyrimidine derivatives described by Tay
`lor et al. in US. 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 US. Pat. No. 4,997,838. Antifo
`late compounds which can be employed in this inven
`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
`GAR-transformylase inhibitors and antifolates are also
`included within the scope of this invention, and such
`compounds can be determined by routine evaluation of
`either their ability to interact with and inhibit the sub
`ject enzyme or to bind to the FBP.
`In a preferred embodiment of the invention, folic acid
`is administered to a subject subsequently receiving an
`agent defined by the formula
`
`Lometrexol is the generic name given to 5,10
`dideazatetrahydrofolic acid, also referred to as
`DDATHF. Lometrexol is a member of a new class of
`antitumor agents which have been found to speci?cally
`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,
`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,
`rheumatoid arthritis and other in?ammatory disorders,
`and during organ transplantation and other related im
`munosuppressant related conditions.
`Lometrexol has been studied clinically and shown to
`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
`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
`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
`gation, 60, 737 (1989)) can be signi?cantly 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
`utility of GAR-transformylase inhibitors and other an
`tifolates by co-adrninistering a FBP binding agent to the
`host under going treatment.
`
`35
`
`40
`
`45
`
`50
`
`SUMMARY OF THE INVENTION
`
`In one aspect of this invention, we provide a method
`of inhibiting the growth of GAR-transformylase
`dependent tumors in mammals comprising administer
`ing to said mammals an effective amount of a GAR
`transformylase inhibitor or other antifolate which binds
`to a FBP in combination with a toxicity-reducing
`amount of a FBP binding agent, or a physiologically
`
`65
`
`Wockhardt Exhibit 1009 - 2
`
`

`
`3
`
`5,217,974
`
`4
`
`R‘
`
`R2
`I
`(CHM-B
`/
`N \ Q c
`/“\ ’
`/|
`“2N
`N
`
`Z
`
`‘
`
`coNncncn;cn2c00n
`l
`coon
`
`x
`
`wherein
`R1 is hydroxy or amino;
`R2 is hydrogen, methyl, ethyl, or propynyl;
`B is —CH-- or —N—;
`n is l, 2 or 3;
`’
`Z is nitrogen or carbon;
`A is pyrido, tetrahydropyrido, pyrrolo, dihydropyr
`rolo, cyclopentyl or cyclohexyl;
`X is hydrogen or halo; and pharmaceutically accept
`able salts thereof.
`In a particularly preferred embodiment of the inven
`tion, lometrexol is utilized as the GAR-transformylase
`inhibitor.
`
`15
`
`20
`
`As noted above, the drug products which can be
`employed in the present invention include other antifo
`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
`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
`ter, Clin. Chem., 19 (10), 1101-1105 (1973). Typical
`antifolates evaluated in the referenced procedure have
`the following folate binding constants presented in
`Table I below:
`‘
`
`Binding Constant
`(ng/ml)
`
`TABLE I
`
`3
`
`Z
`
`x
`
`coNncncnzcnzcoon
`coon
`
`on
`
`N
`
`/
`)\
`
`nzN
`
`N
`
`(cn2),,
`
`|
`
`N
`n
`
`n = 3
`
`x n
`
`n = z
`
`x _ Z-F
`
`it = 2
`
`x s-F
`
`on
`
`N \
`
`/"\ ’
`HzN
`N
`
`N
`n
`
`OH
`N /
`x
`HZN
`N
`
`coNncncnzcnzcoon
`
`coon
`
`coNncncnzcnzcoon
`
`coon
`
`conncncnzcnzcoon
`coon
`
`Tnzczcn
`cnz-N
`
`CONHCIIHCHZCHZCOOH
`coon
`
`12.5
`
`no
`
`30.7
`
`250.8
`
`24.5
`
`26.0
`
`‘2'0
`
`Wockhardt Exhibit 1009 - 3
`
`

`
`5,217,974
`
`TABLE I-continued
`
`‘im
`CH2—N
`
`S
`
`CONHCHCHZCHZCOOH
`COOl-l
`
`Binding Constant
`(ng/ml)
`
`245
`
`20
`
`25
`
`As used in this invention, the term “FBP binding
`agent” refers to folic acid, (6R)-5-methyl-5,6,7,8-tet
`rahydrofolic acid, or (6R)-5-formyl-5,6,7,8-tetrahy
`drofolic acid. 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 b-position-they
`are 10-20 fold more efficient in binding the folate bind
`ing 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 compounds are usually pre
`pared as a mixture with their natural form (65) of diaste
`reomers by non-stereoselective reduction from the cor
`responding dehydro precursors followed by separation
`through chromatographic or enzymatic techniques. See
`e.g., PCT Patent Application Publication WO 880844
`(also Derwent Abstract 88-368464/51) and Canadian
`Patent 1093554.
`30
`Folic acid is a vitamin which is required by mammals
`for proper regeneration of the blood-forming elements
`and their functioning, and as a coenzyme is involved in
`intermediary metabolic processes in which one-carbon
`units are transferred. These reactions are important in
`interconversions of various amino acids and in purine
`and pyrimidine synthesis. Folic acid is commonly sup
`plied to diets of humans via consumption of food
`sources such as liver, kidney, dry beans, asparagus,
`mushrooms, broccoli, lettuce, milk and spinach, as well
`40
`as by vitamin supplements. The minimum amount of
`folic acid commonly required by normal adults is about
`0.05 mg/day. According to this invention, folic acid, or
`a physiologically-available salt or ester thereof, is ad
`ministered to a human subject at a dose of about 0.5
`mg/day to about 30 mg/day to diminish the toxic ef
`fects of a GAR-transformylase inhibitor or other antifo
`late also being administered to such subject. In a pre
`ferred embodiment, folic acid will be administered at
`about 1 to about 5 mg/day together with the normal
`dosing of GAR-transformylase inhibitor such as lomet
`rexol.
`Based upon'the relative binding constants for the
`respective compounds, it will be expected that approxi
`mately 1 mg/day to 90 mg/day (preferably approxi
`mately 2-15 mg/day) of (6R)-S-methyl-S,6,7,8-tetrahy
`drofolic acid or about 5-300 mg/day (preferably about
`10-50 mg/day) of (6R)-5-formyl-5,6,7,8-tetrahydrofolic
`acid, or their respective physiologically-available salt or
`ester thereof, will be employed with the GAR-transfor
`mylase inhibitor.
`“Physiologically-available salt" refers to potassium,
`sodium, lithium, magnesium, or preferably a calcium
`salt of the FBP binding agent. “Physiologically-availa
`ble .
`.
`. ester” refers to esters which are easily hydro
`lyzed upon administration 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 a physiological]y-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 admin
`istered orally, preferably as a sustained release formula
`tion, 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 administered to the subject
`mammal prior to treatment with the GAR-transformy
`lase inhibitor or other antifolate. Pretreatment with the
`suitable amount of FBP binding agent from about 1 to
`about 24 hours is usually suf?cient to substantially bind
`to and block the folate binding protein prior to adminis
`tration of the GAR-transformylase inhibitor or other
`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,
`multiple dosing of the FBP binding agent can be em
`ployed 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 pretreatment.
`In the especially preferred embodiment of this inven
`tion, about 1 mg to about 5 mg of folic acid is adminis
`tered orally to a mammal about 1 to about 24 hours
`prior to the parenteral administration of the amount of
`lomotrexol which is normally required to attain the
`desired therapeutic bene?t. Although greater or addi
`tional doses of folic acid or another FBP binding agent
`are also operable, the above parameters will usually
`bind the folate binding protein in an amount sufficient to
`reduce the toxicity effects normally seen upon lomo
`trexol administration above.
`It should be noted that the FBP binding agent is not
`an antitumor agent and that the pretreatment of a mam
`mal with a FBP binding agent is not a synergistic or
`potentiating effect. Rather, by having substantially
`bound the folate binding protein with a FBP binding
`agent prior to administration of the GAR-transformy
`lase inhibitor or other antifolate, the toxic effects of
`such subsequent treatment are greatly reduced without
`affecting the therapeutic efficacy.
`The effect of folic acid on GAR-transformylase in
`hibitors has been demonstrated in standard tests com
`monly utilized to determine the antitumor activity and
`toxic effects of the GAR-transformylase inhibitors
`themselves. In one such test, mice are inoculated with
`the C3H strain of mammary adenocarcinoma by insert
`ing a 2 mm by 2 mm section of tumor into the axillary
`region of the mice by trocar. In all experiments, lomet
`rexol was administered intraperitoneally once a day for
`?ve consecutive days, starting on the day following
`tumor implantation. Ten animals were used at each
`dosage level. Antitumor activity was assessed on day
`
`35
`
`45
`
`50
`
`60
`
`65
`
`Wockhardt Exhibit 1009 - 4
`
`

`
`5,217,974
`.
`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
`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.
`
`8
`human dose of about 30 mg/day), good antitumor activ
`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 C3l-I Mice after Two Weeks on Folate-Free Diet
`Plus Addition of 0.003% Folate to Drinking Water
`Lometrexol Dose Antitumor Activity
`Toxicity
`(mg/kg)
`(% Inhibition)
`(Mice Dead/Total Mice)
`6.25
`91%
`0/10
`12.5
`89%
`0/l0
`25
`97%
`0/10
`50
`96%
`0/10
`
`10
`
`TABLE In
`Antitumor Activity and Toxicity of Lometrexol
`in C3H Mice after Two Weeks on Folate-Free Diet
`Lometrexol Dose Antitumor Activity
`Toxicity
`(mg/kg)
`(% Inhibition)
`(Mice Dead/Total Mice)
`0.0625
`0%
`0/ 10
`0.125
`0%
`0/10
`0.25
`21%
`0/10
`0.5
`88%
`0/ 10
`1.0
`100%
`8/10
`
`The foregoing data establish that for tumor bearing
`mice maintained on a folic acid free diet prior to and
`during treatment with lometrexol, the toxicity of lomet
`rexol is very large, with 1 mg/kg/day being lethal to
`the majority of the mice, and lower antitumor activity is
`observed at non-toxic drug doses. Very low doses of
`folic acid (about 1 to 2 mg/day for an adult human)
`partially reversed drug toxicity and improved antitu
`mor activity. Larger doses of folic acid (up to about 30
`mg/day for an adult human) dramatically reduced
`lometrexol toxicity and markedly improved antitumor
`activity. Thus, the use of folic acid in combination with
`a GAR-transformylase inhibitor markedly reduces drug
`toxicity without adversely affecting antitumor activity.
`In a typical clinical evaluation involving cancer pa
`tients, all of whom have histologically or cytologically
`con?rmed diagnosis of cancer, lometrexol is adminis
`tered in combination with folic acid. Lometrexol is
`administered in four doses over a two week period by
`rapid intravenous injection, followed by two weeks of
`non-therapy. Dosing is made on days 1, 4, 8 and 11 of
`any two week period. Patients will have an initial
`course of therapy at a dose of 5 mg/mz/dose, and de
`' pending upon the toxic effects observed in the initial
`course, their subsequent courses may be at the same
`40
`dose, or may be escalated to 6 mg/mz, or may be attenu
`ated to 4 mg/ml.
`These patients will also receive orally 1 mg/day of
`folic acid, beginning the day before they are started on
`the ?rst course of lometrexol, and continuing through
`out their exposure to the drug. Such dosage of folic acid
`will be given once daily, generally in the morning
`hours.
`In preparation for the foregoing clinical study, pilot
`studies in humans have established that folic acid given
`to patients receiving lometrexol has effected reduced
`side effects due to the lometrexol. Speci?cally, in one
`subject who had a nasalpharyngeal carcinoma, who was
`supplimented with folic acid at 0.5 to 1.0 mg/day,
`lometrexol was well tolerated for up to 12 months of
`therapy. Moreover, this patient has no clinical evidence
`of disease after the 12 months of therapy. These data are
`consistent with the animal studies reported above.
`We claim:
`.
`1. A method of inhibiting the growth of GAR-trans
`formylase-dependent tumors in mammals comprising
`administering to said mammals an effective amount of a
`GAR-transformylase inhibitor which binds to a folate
`binding protein in combination with a toxicity-reducing
`amount of a folate binding protein binding agent se
`lected from folic acid, (6R)-5-methyl-5,6,7,8-tetrahy
`drofolic acid, and (6R)-5-formyl-5,6,7,8-tetrahydrofolic
`acid, or a physiologically-available salt or ester thereof.
`
`A test group of mice were maintained on a folic acid
`free diet for two weeks before treatment. Folic 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.
`
`25
`
`0.0003 grams
`I00 ml.
`
`4 ml.
`day
`
`0.000012 grams _
`day
`
`0.012 milligrams
`day
`
`The average size of a mouse is 0.00687 m2
`
`0.012 rams X _l_T : L75 mE/mZ/day
`day
`0.00687 m
`
`30
`
`3 LII
`
`For a human subject of about 1.73 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 C3I-I Mice after Two Weeks on Folate-Free Diet
`Plus Addition of 0.0003% Folate to Drinking Water
`Lometrexol Dose Antitumor Activity
`Toxicity
`(mg/kg)
`(% Inhibition)
`(Mice Dead/Total Mice)
`0.125
`13%
`O/ 10
`0.25
`26%
`0/ 10
`0.5
`48%
`0/ 10
`1.0
`97%
`0/ 10
`2.0
`98%
`0/ 10
`4.0
`99%
`4/10
`
`45
`
`As the foregoing results indicate, addition of the indi
`cated level of folic acid to the diet of a subject receiving
`lometrexol results in excellent antitumor activity at low
`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
`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
`/volume) of folic acid (which translates to an adult
`
`60
`
`65
`
`Wockhardt Exhibit 1009 - 5
`
`

`
`5,217,974
`
`5
`
`15
`
`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
`protein binding agent is (6R)-5-methyl-5,6,7,8-tetrahy
`drofolic acid or a physiologically~avai1able 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~
`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
`prises pretreating the mammal with an amount of a
`folate binding protein binding agent selected from folic
`acid, (6R)-5-methyl-5,6,7,B-tetrahydrofolic acid, and
`(6R)-5-formyl-5,6,7,8-tetrahydr0folic acid, or a physio
`logically available salt or ester thereof, suf?cient to
`have substantially blocked the folate binding protein
`before administration of the antifolate.
`17. The method of claim 16 wherein the GAR-trans
`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
`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
`ing protein binding agent is folic acid, or a physiologi
`cally-available salt or ester thereof.
`22. The method of claim 17 wherein the folate bind
`ing protein binding agent is folic acid, or a physiologi
`cally-available salt or ester thereof.
`
`9
`2. The method of claim 1 wherein the GAR-transfor
`mylase inhibitor is a pyrido[2,3-d]pyrimidine.
`3. The method of claim 2 wherein the GAR-transfor
`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 folic acid.
`6. The method of claim 5 wherein the folic acid is
`administered at,a dose of about 0.5 mg/day to about 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
`mg/day.
`8. A method for reducing mammalian toxicity of a
`GAR-transforrnylase- inhibitor which binds to a folate
`binding protein consisting of administering a toxicity
`reducing amount of a folate binding protein binding
`agent selected from folic acid, (6R)-5-methyl-5,6,7,8
`tetrahydrofolic acid, and (6R)e5-formyl-5,6,7,8-tetrahy
`drofolic acid, or a physiologically-available salt or ester
`thereof, to the mammal receiving treatment with the
`GAR-transforrnylase inhibitor‘
`9. The method of claim 8 wherein the GAR-transfor
`mylase inhibitor is a pyrido[2,3-d]pyrimidine.
`10. The-method of claim 9 wherein the GAR-trans
`forrnylase 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
`administered at a dose of about 0.5 mg/day to about 30
`mg/day.
`
`25
`
`30
`
`35
`
`45
`
`55
`
`65
`
`Wockhardt Exhibit 1009 - 6