[CANCER RESEARCH 56. 2331-2335. May 15. 1996]
`
`Augmentation of the Therapeutic Activity of Lometrexol [(6-R)5,lO-Dideazatetra(cid:173)
`hydrofolate] by Oral Folic Acidl
`
`Teresa Alati, John F. Worzalla, Chuan Shih,z Jesse R. Bewley, Sidney Lewis, Richard G. Moran, and
`Gerald B. Grinder
`Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285 [T. A., J. F. W., C. S., J. R. B .. G. B. G.); Department of Radiology, Wishard Memorial Hospital.
`Indianapolis, Indiana 46202 {So L}; and Department of Pharmacology and Toxicology and the Massey Cancer Center, Medical College of Virginia. Richmond. Virginia 23298
`{R.G.M.}
`
`ABSTRACT
`
`Recent cUnical trials with lometrexol [(6R)-S,lO-dldeazatetrahydrofo(cid:173)
`late] have revealed a level of toxicity In humans that was not predicted on
`the basis of previous in YiIlO preclinical studies. Because standard labora(cid:173)
`tory animal diets contain high levels of folic acid relative to human folate
`intake, the toxicity and therapeutic activity of lometrexol was studied In
`mice under conditions of restricted dietary folate Intake. Remarkably, the
`lethality of this drug increased by three orders of magnitude In mildly
`folate-deficient mice, mimicking the unexpected toxicity seen In humans.
`Lometrexol had Umited therapeutic activity In folate-deficient mice bear(cid:173)
`Ing the C3H mammary adenocarcinoma, compared with the substantial
`therapeutic index for treatment of this tumor in animals on standard diet.
`When folic acid was administered p.o. to mice that were mildiy folate
`deficient, antitumor activity was again observed at nontoxic doses of
`lometrexol, and the range of lometrexol doses that allowed safe therapeu(cid:173)
`tic use of this drug Increased at higher dietary folate Intake. At a fixed
`dose of lometrexol, the antitumor effects in animals were dependent on the
`level of dietary folate and went through a distinct optimum. Excessively
`high folate Intake reversed the antitumor effects of lometrexol. Optimiza(cid:173)
`tion of the foUc acid content In the diet and of the lometrexol dosage are
`predicted to have substantial impact on the clinical activity of this ciass of
`drugs.
`
`INTRODUCTION
`
`Lometrexol4 is a very potent folate antimetabolite that has no
`measurable activity against either of the two previously exploited
`targets for chemotherapy in the folate metabolic pathway, dihydrofo(cid:173)
`late reductase and thymidylate synthase (1, 2). Rather, it is active as
`an antiproliferative agent by virtue of inhibition of the fIrst folate(cid:173)
`dependent enzyme of de novo purine synthesis, i.e., glycinamide
`ribonucleotide formyltransferase (3). Several lines of evidence indi(cid:173)
`cate that metabolism of lometrexol to polyglutamate forms by FPGS
`plays a major role in the action of this compound. Enzyme kinetic
`studies have demonstrated that the long chain polyglutamates have
`much lower kinetically determined Kjs (4). Cellular studies demon(cid:173)
`strate that the cytotoxic events initiated by lometrexol exposure occur
`or continue after drug exposure, apparently as a result of the accu(cid:173)
`mulation of polyglutamate metabolites (5). In vitro studies have
`shown that lometrexol is a very efficient substrate for human and
`mouse FPGS (3), and that polyglutamate metabolites accumulate
`rapidly and extensively in tumor cells exposed to drug (6). In addition.
`lometrexol binds very tightly to the folate receptor species studied
`thus far (7-9) and is an excellent substrate for the reduced folate
`transport system (9). Most of these characteristics distinguish lome-
`
`Received 10/24195; accepted 3/14196.
`The costs of publication of this article were defrayed in part by the payment of page
`charges. This article musl therefore be hereby marked advertisement in accordance with
`18 U.S.C. Section 1734 solely to indicate this fact.
`I Supported in part by NllI Grant CA 27605 (to R. G. M.).
`2 To whom requests for reprints should be addressed, at Lilly Research Laboratories,
`Lilly Corporate Center. 307 East McCarty Street. Indianapolis, IN 46285-0540.
`3 Deceased November 16, 1993.
`4 The abbreviations used are: lometrexol. (6R)-5,IO-dideaza-5,6.7.8-tetrahydrofolate;
`FPGS. folylpoIY-')I-glutamate synthetase.
`
`trexol from the prototypical folate antimetabolite used in cancer
`chemotherapy, methotrexate.
`Lometrexol was advanced to clinical trial as a result of its broad
`spectrum activity against a series of transplanted mouse tumors and of
`human tumor xenografts (10, 11), as well as a result of its novel
`mechanism of action and biochemical profIle (1-9). The early Phase
`I clinical trials (12-15) revealed several unexplained patterns of
`behavior of this drug in humans. The most surprising was the extreme
`potency of the compound; toxicity was observed at doses that were a
`small fraction of those predicted from the previous extensive animal
`studies. Toxicity to platelets, upon onset, was prolonged, and there
`was a distinctly cumulative nature to the hematopoietic toxicity (12,
`15). These toxicological characteristics have threatened the continued
`development of lometrexol; a schedule of lometrexol, used alone,
`suitable for a Phase II trial has not been found, despite evaluation of
`several schedules. Yet, in several of the reported Phase I trials of this
`drug, there have been anecdotal reports of substantial tumor responses
`(12-15).
`Several years ago, we had noted that the administration of folic acid
`to animals protected against the toxicity of this compound without
`compromising its antitumor activity. The previous preliminary reports
`of this work (16, 17) have prompted second generation Phase I trials
`that now appear to have demonstrated the protective effects of folic
`acid against lometrexol toxicity in humans (18, 19)5.6 and which are
`currently testing whether protocols involving the administration of
`folic acid will allow the toxicity of lometrexol to be clinically man(cid:173)
`ageable. In this report, we described the preclinical evidence that
`initiated these trials and describe the rather startling relationship
`between the level of intake of folic acid and the toxicity of lometrexol.
`
`MATERIALS AND METHODS
`
`Lometrexol was synthesized and purified as described previously (1, 3).
`Folic acid was purchased from Sigma Chemical Co. (St. Louis, MO). C3H
`female mice were purchased from Charles River Laboratories, Inc. (Wilming(cid:173)
`ton, MA) and weighed 20-23 g when used in these experiments. Mice were
`housed in temperature- and humidity-controlled rooms and were fed either a
`standard laboratory rodent chow or a folic acid-deficient diet containing 1 %
`succinylsulfathiazole; both diets were purchased from Ralston Purina Co. (St.
`Louis. MO). The average conlent of folates from natural sources in both diets
`was found to be 0.03 ppm, whereas the standard diet was analyzed to contain
`7.3 ppm of added folic acid. It was estimated that mice on a standard diet
`ingested 1 to 2 mglkglday of folaleS, while mice on a low folale diet ingested
`0.001 to 0.008 mglkglday. In some studies, folic acid was added to the drinking
`water and was solubilized with sodium bicarbonate; in other studies, solubi(cid:173)
`lized folic acid was adminiSlered once a day by oral gavage. Food and waler
`were provided ad libitum. Consumption of food (4 to 5 glday per mouse) or
`waler (4 to 5 mlIday per animal) was not appreciably different among groups
`of animals. The content of folales in serum and in washed RBC of mice fed
`each diet was delermined by a competitive binding assay procedure (Quant(cid:173)
`aphase folate radioassay kit; Sio-Rad Laboratories, Hercules, CAl using folic
`acid as a standard.
`
`5 A. Hilary Calvert, personal communication.
`6 J. Roberts. personal communication.
`
`2331
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1090-0001
`
`

`
`FOLIC ACID·ENHANCED DDATHF THERAPEUTICS
`
`100 &>~
`
`75
`
`«i
`>
`·E
`In = 50
`~
`
`;:I
`
`II)
`
`25
`
`0
`0.01
`
`0.1
`
`10
`
`100
`
`1000
`
`lometrexol, mglkg per day
`
`100
`
`75
`
`50
`
`25
`
`0
`
`Fig. I. The toxicity of lometrexol to C3H mice is increased by a folate·deficient diet.
`Mice were fed either a standard laboratory diet (II) or a folate-deficient diet for 2 weeks
`(6) or for 4 weeks (0) prior to the first dose of lometrexol and for the duration of the
`study. Groups of mice (10 animals/group) on each diet were given five daily doses of
`lometrexol i.p. at the indicated doses. The data present the percentage of animals alive 3
`weeks after the last dose of lometrexol, and each point represents the cumulative results
`from two such experiments.
`
`The C3H mammary adenocarcinoma was implanted s.c. in the lateral flank
`of each mouse I day prior to the first dose of drug treatment. Lometrexol was
`solubilized in either 2.5% GAF Emulphor EL620 (Warren-Graham, Cock(cid:173)
`eysville, MD) or PBS and was administered i.p. daily for 5 days, beginning I
`day after tumor implantation. Control mice received an equivalent volume of
`vehicle alone. In some experiments, mice were placed on a folate-deficient
`mouse diet for 2-4 weeks prior to tumor implantation. The growth of this
`tumor was not significantly different in animals fed the standard or folate(cid:173)
`deficient diets. Tumor dimensions were estimated by external calipers inter(cid:173)
`faced with a computer, allowing tumor weights and inhibition of tumor growth
`to be calculated according to the formula (20): tumor weight (mg) = width2
`(mm) X length (mm) X 0.5, and stored electronically (21).
`Tumor weights were measured 10 days after the first dose of lometrexol. In
`each experiment, there were at least 10 animals/treatment group, and each
`result was confirmed in at least two independent experiments performed
`several weeks apart. Animals were observed for at least 21 days after the last
`dose of lometrexol before scoring drug-induced lethality.
`
`RESULTS
`
`Enhanced Lethality of Lometrexol to Mice witb Dietary Re(cid:173)
`striction of Folic Acid. The toxicity of lometrexol seen in early
`clinical trials was observed at surprisingly low doses (6-15 mg/m2
`;
`Refs. 12-15), considering the toxicity previously seen in mice (LDso
`of greater than 175 mglkg, when administered daily five times) and
`dogs (maximum tolerated dose of 5 mglkg given once weekly for six
`weeks)? Given that standard laboratory mouse diets contain high
`levels of folic acid, it was suspected that this unexpected poor pre(cid:173)
`diction of the clinical toxicity pattern from previous animal studies
`was due to a difference in the folate status of laboratory chow-fed
`mice and that of patients with advanced cancer. Hence, the toxicity of
`lometrexol to C3H mice was studied after dietary deprivation of folic
`acid. In this study, mice fed a diet of laboratory chow ad libitum were
`found to have RBC folate levels of 836 ± 133 ng/ml and serum folate
`contents of 43.4 ± 14.1 ng/ml. After 2 weeks on a diet formulated
`without added folic acid, folate levels fell to 418 ± 61 ng/ml in RBC
`and to 10.2 ± 1.8 ng/ml in serum, levels comparable to those con(cid:173)
`sidered normal in humans (22, 23). Serum and RBC folate contents
`remained quite constant with longer periods on this folate-deficient
`diet up to at least lO weeks; after 4 weeks, serum folates and RBC
`folates remained at levels (9.4 ± 1.4 and 396 ± 11 ng/ml, respec(cid:173)
`tively) similar to those measured at 2 weeks.
`
`7 Unpublished toxicology studies on file (number D 00788; Eli Lilly and Company,
`Indianapolis, IN).
`
`As in previous studies (10), lometrexol was toxic on a daily (five
`times) schedule only at rather high doses (LDso, 300 mglkg per day)
`for mice fed standard laboratory chow (Fig. 1). However, with fre(cid:173)
`quent administration of lometrexol to animals that had been on a diet
`deficient of folic acid for 2 weeks, the toxicity of this drug was
`observed at lOOO-fold lower doses (LDso' 0.3 mglkg per day; Fig. I).
`Maintaining the mice on this folate-deficient diet for 2 additional
`weeks did not result in any further sensitization of the mice to
`lometrexol. This degree of increased toxicity is even more remarkable
`in view of the fact that 2 weeks of folate-deficient diet only decreased
`the content of folates in RBC, an indicator of tissue stores of folates,
`to about one-half of the control levels (see above).
`The therapeutic activity of lometrexol against transplanted mouse
`tumors and human xenograft systems has been reported previously to
`be substantial in studies performed in animals fed standard mouse
`diets (10, II). The therapeutic activity of this drug against the C3H
`mammary adenocarcinoma allowed complete suppression of the
`growth of tumor when measured II days after tumor inoculation (Fig.
`2). The activity of lometrexol against the C3H tumor allowed com(cid:173)
`plete suppression of the growth of this tumor at a range of doses that
`were without toxicity. However, when the antitumor activity of lome(cid:173)
`trexol was studied in mice with serum folate levels brought to the
`range of those in humans by 2 weeks of dietary restriction of folic acid
`intake, only limited therapeutic effects were observed, and the doses
`which partially suppressed tumor growth could not be increased
`without toxic effects (Fig. 2). In a series of experiments, even mod(cid:173)
`erate inhibition of tumor growth was not observed in mice fed folate(cid:173)
`deficient diets at any lometrexol dose without lethality to at least 2 of
`lO animals (Fig. 2). The therapeutic index of lometrexol to animals
`fed a folate-deficient diet, measured by the shift between the dose(cid:173)
`response curves for antitumor effects and toxicity, was less than a
`factor of two, whereas that for mice fed a diet with "standard," i.e.,
`high, levels of folic acid was more than a factor of 100.
`Reinstatement of tbe Tberapeutic Activity of Lometrexol witb
`Orally-Administered Folic Acid. Increasing amounts of folic acid
`were administered to folate-deficient mice to reconstitute the thera(cid:173)
`peutic activity observed in standard laboratory animal diets and to
`study the relationship between intake of folic acid and the therapeutic
`ratio of lometrexol. In initial studies, folic acid was added to the
`drinking water of mice after they had been on a folate-deficient diet
`for 2-3 weeks. Mice bearing the C3H mammary tumor that were
`treated with folic acid in the drinking water at the lowest dose studied,
`
`/0
`
`Ol)
`
`~
`-5
`~
`8
`...
`~
`= 0
`;e
`;S
`
`"-
`0
`
`'.:::1
`
`100
`
`75
`
`50
`
`25
`
`0
`
`,::/¢>
`.... 0
`0.1
`
`----. 100
`
`~
`S-
`~
`~
`oQ
`
`75
`
`50
`
`25
`
`0
`
`10
`
`100
`
`lometrexol, mg/kg per day
`
`Fig. 2. Inhibition of the growth of the C3H mammary adenocarcinoma by lometrexol
`in mice fed a standard or a folate·deficient diet. Mice were fed either a standard diet (e,
`~ or a folate-deficient diet (0. 0) for 2 weeks prior to inoculation with tumor and for
`the duration of the study. Treatment with five daily doses of lometrexol was initiated on
`the day after tumor inoculation. Tumor dimensions were measured 10 days after the first
`dose of lometrexol. Each treatment group in an experiment contained 10 mice. and the
`data shown represents the sum of data from two such experiments. 0 and e. tumor
`inhibition; 0 and. lethality.
`2332
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1090-0002
`
`

`
`FOLIC ACID·ENHANCED DDATHF THERAPEUTICS
`
`~.~""""'-6
`
`100
`
`100
`
`~ 75
`
`25
`
`~ .s
`~ ... 0
`~ 50
`.....
`0
`o:l
`0
`'::1
`
`I
`
`75
`
`i
`50 ~
`
`lfI
`
`25
`
`0
`
`~
`
`0
`0.1
`
`.... 0·· ...
`100
`
`10
`
`lometrexol, mg/kg per day
`
`sensitive to the lethality of this drug than observed in animals fed
`standard laboratory diet. and the therapeutic index of lometrexol
`against mouse tumors was substantially reduced by such a dietary
`restriction. Clinical experience with lometrexol to date has indicated
`similar effects of administered folates on the toxicity of this drug as is
`documented herein in mice. Thus. it has been shown that patients who
`respond to lometrexol with progressive myelosuppression can be
`rescued from undue toxicity by the administration of the reduced
`folate. leucovorin (12. 15). In a clinical trial in which lometrexol was
`given without folic acid on a once-every-3-week schedule. cumulative
`hematopoietic toxicity was seen that would preclude repetitive dosing.
`even at the lowest dose studied. 15 mg/m2 (12). However. an ongoing
`clinical trials indicates that doses of at least 130 mg/m2 can be
`repetitively administered every 3 weeks if patients are concurrently
`given oral folic acid at 5 mg daily for 7 days before and after each
`lometrexol dose. Previous clinical trials have found low tolerance of
`lometrexol on a once a week for a 3-week schedule. with even the
`dose found acceptable for three doses on this schedule (6 mg/m2
`)
`poorly tolerated upon additional doses (15). A second ongoing trial6
`indicates that patients given oral folic acid daily at 5 mg/m2 can
`tolerate eight weekly doses of lometrexol of at least 5 mg/m2 with no
`obvious cumulative toxicity; this is the first time that frequent admin(cid:173)
`istration of lometrexol has been found possible in humans. Hence,
`repetitive administration of lometrexol in humans on either a weekly
`or on an every-3-week schedule appears to be feasible with (but not
`without) the coadministration of folic acid. and higher doses of
`lometrexol appear practical with folic acid administration. Although it
`remains to be determined whether the events responsible for these
`major shifts in the toxicity of lometrexol caused by folic acid are the
`
`100·
`
`80·
`
`~ 60·
`'" -5
`.!:!
`~
`
`40·
`
`20·
`
`2000·
`
`1500·
`
`.nnn
`lJVV
`
`"nn
`
`f\
`
`if
`.i OIl
`'0
`~
`5
`S a
`
`folic acid. mg/kg
`lometrexol. mg/kg
`
`0
`0
`
`t
`t
`t
`
`T
`o 0.6
`60 600 2000
`6
`12.5 12.5 12.5 12.5 12.5 12.5
`
`Fig. 3. Enhancement of the therapeutic effects of lometrexol against the C3H mam(cid:173)
`mary adenocarcinoma in folate-deficient mice by oral folic acid. C3H female mice were
`fed a folate·deficient diet for 2 weeks prior to the inoculation of tumor and for the duration
`of the experiment. Folic acid was added to the drinking water I day prior to the first
`treatment with lometrexol and was continued for the duration of the experiment at 0.0003
`(0,11), 0.003 (~), or 1% (0) (w/v). Lometrexol was administered daily for 5 days i.p. at
`the indicated doses, and the size of each tumor was estimated 10 days after the first
`treatment with lometrexol. Each group consisted of 10 mice. 0, D. ~: tumor inhibition;
`., lethality. All animals receiving 0.003% or I % folic acid in drinking water tolerated
`lometrexol at any of the doses noted.
`
`0.0003% (w/v). demonstrated higher tolerance of lometrexol given
`i.p. for 5 consecutive days (Fig. 3) compared to the toxicity of
`lometrexol in folate-deficient animals (Fig. 2). For instance. no le(cid:173)
`thality was observed at 1 or 2 mglkg of 10metrexoVday for animals
`with 0.0003% folic acid in the drinking water (Fig. 3). whereas doses
`as low as 0.4 mg/kg per day were lethal to some animals in the
`absence of folic acid in the drinking water (Fig. 2). This amount of
`folic acid intake. which was equivalent to a folic acid dose of 0.6
`mglkg/day. allowed complete suppression of tumor growth at lome(cid:173)
`trexol doses of 1 and 2 mglkg/day. but higher doses (4 mglkg/day) had
`substantial toxicity. At 0.003% folic acid in the drinking water. which
`resulted in an intake equivalent to 6 mglkg of folic acid/day. a broad
`range of lometrexol doses allowed complete suppression of this tumor
`without toxicity (Fig. 3); this intake of folic acid in animals fed
`folate-deficient diets appeared to mimic the therapeutic response seen
`in animals on standard laboratory diets. However. at high levels of
`folic acid intake (1 % in the drinking water. equivalent to 2000
`mglkg/day intake of folic acid). the antitumor activity of the drug was
`completely blocked at all doses of lometrexol studied (Fig. 3). The
`dependence of toxicity and antitumor activity on folic acid intake was
`studied more closely at a fixed dose of lometrexol (12.5 mglkg/day
`daily five times). Again. this dose of lometrexol was uniformly lethal
`to animals fed a folate-deficient diet for 2 weeks. but 6 mglkg of folic
`acid given daily by oral gavage completely protected the animals and
`allowed substantial antitumor activity (Fig. 4). Ten-fold increments in
`the dose of folic acid progressively reversed the antitumor activity of
`this fixed dose of lometrexol until. at very high doses of folic acid. the
`antitumor effects of this dose of lometrexol were negated. Hence. folic
`acid supplementation of animals allowed a substantial antitumor ac(cid:173)
`tivity without toxicity. and the window of folic acid intake compatible
`with low toxicity and high antitumor activity appeared sufficiently
`wide to allow therapeutic use of the combination of lometrexol with
`oral folic acid.
`
`DISCUSSION
`
`Fig. 4. Reversal of the lethality (top panel) and antitumor activity (bottom panel) of
`lometrexol by oral folic acid. Mice were maintained on a folate-deficient diet for 2 weeks
`We have demonstrated that oral folic acid dramatically decreases
`prior to the inoculation of the C3H mammary adenocarcinoma and for the duration of the
`the toxicity of lometrexol to mice brought to serum and erythrocyte
`experiment. Treatment with lometrexol began I day later and continued for five daily i.p.
`folate levels typical of the normal human population. Animals fed a
`injections at 12.5 mglkglday. Folic acid was administered by oral gavage beginning the
`day of tumor inoculation and continuing until tumor size was estimated. 6 days after the
`low folate diet for a short period became more than l000-fold more
`last dose of lometrexol.
`2333
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1090-0003
`
`

`
`FOLIC ACID-ENHANCED DDATHF THERAPEUTICS
`
`Table I Folate content of diet and maximum tolerated dose (MTD) of lometrexol
`Daily folate ingestion (mg/m2) MID lometrexol (mg/m2)
`600
`3000
`0.6
`
`Schedule
`
`DailyX5
`Days 1,4, 7, to
`Daily X 5
`
`0.00~.025
`
`Ref.
`
`Fig. I
`UnpUblished data
`Fig. I
`
`Species
`
`Diet
`
`Mouse
`
`Standard mouse chow
`
`Dog
`
`Man
`
`Folate-deficient for 2 weeks
`
`Standard lab chow
`
`Recommended daily allowance
`Early Phase I trials
`
`Ongoing trials
`
`1-2
`
`0.25
`Normal diet
`
`Normal diet + 5
`Normal diet + 3-5
`
`100
`
`Once a week X 6
`
`?
`None found «3)
`
`None found «15)
`>5
`>130
`
`Once a week X 3, followed by a 2-week rest,
`then redose
`Every 3 weeks
`Weekly X 8
`Every 3 weeks
`
`24
`
`15
`
`12
`21
`20
`
`animals (28).8 These observations lead to the hypothesis that the
`substantial and unexpected toxicity of lometrexol in humans not given
`concurrent folic acid and in folate-deficient mice is due to the seques(cid:173)
`tration of drug in hepatic tissue, with the subsequent slow release of
`drug to the circulation at toxicologically relevant concentrations.
`Thus, in animals not protected by folic acid preconditioning, a bolus
`administration of drug appears to result in a peak level of lometrexol
`in the blood, followed by the equivalent of a long-term infusion of
`parent drug.
`The mechanism of this accumulation of lometrexol in liver involves
`metabolism to polyglutamate forms by the enzyme FPGS. Liver is
`rich in this critical enzyme (29), and hepatic lometrexol in folate(cid:173)
`deficient mice has been found to be almost exclusively higher poly(cid:173)
`glutamates.8 A recent report (30) has indicated that the metabolism of
`lometrexol to polyglutamates is controlled by the folate content of
`tissues, presumably by a direct feedback effect on FPGS. Hence,
`preadministration of folic acid to animals probably blocks the hepatic
`accumulation of lometrexol by causing an expansion of the folate
`content of hepatic tissue, an effect reported to occur at the dietary
`intake shown to block lometrexol toxicity (Fig. 4; Ref. 31). This
`competition between lometrexol and cellular folates for polyglutama(cid:173)
`tion may be a general phenomenon; the accumulation of methotrexate
`as polyglutamates has been reported to result in lower folate pools in
`liver and erythrocytes of patients treated chronically with this drug
`(32,33).
`The administration of folic acid to folate-deficient animals clearly
`reverses both the lethal toxicity (Fig. 1) and the inhibitory effects of
`lometrexol on tumor (Figs. 3 and 4), but toxicity and therapeutics are
`affected at different doses of folic acid. There is a competitive rela(cid:173)
`tionship between the lethality of lometrexol and the dose of folic acid
`given, i.e .• the more folic acid given to mice, the higher the dose of
`lometrexol which is toxic (Fig. 3). Likewise, the more folic acid
`administered, the higher the dose of lometrexol required for therapeu(cid:173)
`tic effects. Hence, it becomes of practical significance how to properly
`find the combination of drug and folic acid dosages for optimal
`therapeutic effect without toxic risk. It appears that the minimal folic
`acid conditioning needed to avert toxicity will allow the optimal
`therapeutic effects (Fig. 4).
`
`same in mice and humans, the folate-deficient mouse and the folate(cid:173)
`repleted mouse are clearly important model systems for the study of
`this class of drugs and appear to predict clinical behavior of lome(cid:173)
`trexol.
`Table 1 compares the intake of folates in the diet of mouse, dog,
`and humans and relates these levels to the maximal tolerated dose of
`lometrexol. The daily intake of laboratory mice and dogs is essentially
`the same when fed commercial laboratory chows, and the tolerance of
`both species for lometrexol appears more similar when expressed in a
`dosage per unit surface area than per kg of body weight. Nevertheless,
`the differences in schedules that have been studied in mice, dogs, and
`humans makes a direct comparison tenuous, given that the toxicity of
`lometrexol to mice is clearly schedule dependent, with the maximum
`tolerated dose in mice for an intermittent schedule five times higher
`than that for a daily schedule (Table 1). However, extrapolating
`between the dosage schedules used in dogs and mice, it appears that
`dogs are substantially more sensitive to lometrexol than mice, in spite
`of the similar daily intake of folate in the two species. The minimal
`daily intake recommended for humans is 400 ILg which, for a 6O-kg
`human, would amount to about 0.25 mg/m2
`, substantially less than
`that ingested daily in mice or dogs fed standard laboratory diets. The
`fact that patients with active malignancies most often have plasma and
`RBC folate levels that are below normal and are often less than
`one-half the lower threshold of normal (24) suggests that folate intake
`in a population of patients on Phase 1 trials is likely to be less than that
`at recommended daily allowance, i.e., less than 0.25 mg/m2
`• The two
`ongoing trials are being performed at folate intake levels at least
`equivalent to those in experimental animals fed commercial labora(cid:173)
`tory diets (Table 1). However, it should be noted that all of the studies
`performed to date in mice and dogs have supplemented the diet with
`folic acid, similar to the conditions in the current clinical trials, but
`that folic acid is a "drug store artifact" not normally found in the
`human diet. The folate compounds ingested in normal human diets are
`typically fully reduced, methylated or formylated, polyglutamyl fo(cid:173)
`lates that are metabolized during adsorption and passage through the
`liver to, principally, 5-methyltetrahydrofolate (25). Folic acid follows
`a similar metabolic pathway during absorption and passage through
`the liver, but these processes are easily saturable, with free folic acid
`appearing in the circulation at higher oral intake (25, 26).
`What events are responsible for the alteration in lometrexol toxicity
`induced by folic acid remains a critical and current question. Studies ACKNOWLEDGMENTS
`at Lilly Research Laboratories have shown previously that a gamma
`phase of elimination of lometrexol can be detected using a competi(cid:173)
`tive particle concentration fluorescence immunoassay (27) in folate(cid:173)
`deficient but not folate-replete mice. Using this assay, the levels
`detected for several days in mouse plasma following single bolus
`lometrexol were sufficient to inhibit the growth of most mammalian
`cells in culture. In addition, the accumulation of lometrexol in the
`8 L. L. Habeck, S. H. Clay, R. C. Pohland, J. F. Worza1la, C. Shih, K. G. Bemis, and
`L. G. Mendelsohn. Whole body disposition and polyglutamate distribution of the GAR
`livers of folate-deficient mice has been reported, and this accumula-
`transformylase inhibitors L Y30887 and lometrexol in mice: effect of low folate diet,
`tion was diminished by the administration of folic acid to these
`submitted for publication.
`2334
`
`We thank Drs. A. Hilary Calvert, Sarah Freemantle, Franco Muggia, and
`John Roberts for their helpful critiques of this manuscript. We also thank
`Pamela Rutherford, Tracy Self, and Karla Theobald for their excellent tech-
`nical assistance.
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1090-0004
`
`

`
`FOUC ACID-ENHANCED DDATHF THERAPEUTICS
`
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