Enhanced Antitumor Activity for the Thymidylate Synthase Inhibitor 1843U89 Through Decreased Host Toxicity with Oral Folic Acid

Home , Fluorodeoxyuridylate

[CANCER RESEARCH 55. 61 17-6I25. December 15, 19951 Enhanced Antitumor Activity for the Thymidylate Synthase Inhibitor 1843U89 through Decreased Host Toxicity with Oral Folic Acid

Gary K. Smith,â€•2HerbertAmyx, Christine M. Boytos,' David S. Duch, Robert Ferone, and H. Robert Wilson

Divisions of Cell Biology 1G. K. S., D. S. D.], Biochemistry [C. M. B., R. F., H. R. W.], and Toxicology [H. A.], The Wellcome Research Labs, Burroughs Wellcome, Research Triangle Park. North Carolina 27709

ABSTRACT lates (9, 12, 19, 20), 1843U89 is resistant to leucovorin reversal (3). Leucovorin can influence antifolate activity by direct competition for The purpose of this investigation was to determine whether antitumor transport or by elevation of intracellular reduced folate levels (3, 21, selectivity of the third generation thymidylate synthase inhibitor 1843U89 22). In turn, elevated intracellular folates compete directly with com could be enhanced by a combination ofthe drug with folic acid. The effects offolic acid on toxicity of 1843U89 to the dog and mouse and on antitumor petitive and mixed inhibitors for binding to their respective target efficacy of 1843U89 in the mouse were studied. These data were compared enzymes, which decreases inhibition of the target enzyme (1, 9, to the effect of folic acid on the in vitro cell culture antitumor activity of 15â€”18).The cellular folates also compete with the antifolates for 1843U89. The sensitivity of eight cancer cell lines (three ovarian, one polyglutamation, which decreases the extent of antifolate polygluta colon, one ileocecal, one epidermoid, one osteosarcoma, and one breast mation and cellular retention (9, 15, 23). Since the monoglutamates of line) to 1843U89 was tested in vitro in the presence and absence of folic most antifolates are poorer inhibitors than the polyglutamates, de acid. Folic acid concentrations greater than 100 pM were required to creased polyglutamation directly decreases target enzyme inhibition decrease 1843U89 activity in seven of the cell lines. Only the activity in (1, 9, 12, 14, 15, 17â€”20).The effects all decrease the efficacy of the HCT-8, the ileocecal line, was reversed at folic acid concentrations below antifolates in the presence of leucovorin. Since the mono- and diglu 100 @tM.Oralfolic acid given 30 mm prior to an i.v. dose of 1843U89 tamate forms of 1843U89 are equipotent noncompetitive inhibitors of increased the maximally tolerated dose and the lethal dose of 1843U89, both in dogs and in thymidine-depleted mice. In mice, oral folic acid TS, the reduced folates do not compete with 1843U89 for binding to produced little or no effect upon the antitumor efficacy of 1843U89 in two TS, and the decreased polyglutamation of 1843U89 has less influence. of three tumor cell lines in vivo.HCT-8, the line that was sensitive to folate The main influence of leucovorin on 1843U89 efficacy then is reversal in vitro, was also sensitive in vivo. The results show that an oral through competition for transport on the reduced folate transporter (3). dose of folic acid 30 mm prior to i.v. 1843U89 can block mouse and dog These striking differences of 1843U89 from a variety of other intestinal toxicity without decreasing efficacy of 1843U89 in two of three antifolates led us to seek a means to use the differences to increase human tumor lines in the nude mouse. Thus, the data reported here antitumor selectivity. In the present studies, we have investigated the indicate that the antitumor selectivity of 1843U89 may be enhanced potential of combination chemotherapy with 1843U89 plus folic acid through a combination of 1843U89 with oral folic acid. to provide the additional selectivity. We report here the use of oral folic acid to block the dose-limiting gut toxicity of l843U89 but not INTRODUCTION its antitumor effects. A similar strategy was reported previously for the competitive inhibitor DDATHF (24, 25). Differences between the 1843U89 is a third generation TS3 inhibitor under clinical devel two strategies are discussed. The combination of folic acid with opment as an anticancer agent. The compound is cytocidal against a 1843U89 may provide a mechanism for enhanced clinical antiturnor variety of cancer cell types in culture (1â€”4)and is active in several selectivity. antitumor models in mice (3, 5â€”7).l843U89 displays several attrac tive features compared to other antifolates. In human cells, the major metabolite of 1843U89 is the diglutamate (8). Both 1843U89 itself MATERIALS AND METHODS and the diglutamate metabolite bind to TS with noncompetitive ki netics ( 1). The affinities of 1843U89 and the diglutamate for TS are Chemicals. l843U89 was synthesizedat the Weilcome ResearchLabora virtually identical (1). In comparison, the major cellular metabolites tories as described (2). It was solubilized with approximately 2 molar equiv for a variety of other antifolates, including MTX, DDATHF, 5-dea alents of NaOH in endotoxin-tested Dulbecco's PBS without calcium or zaacyclotetrahydrofolate, CB3717, and Tomudex, are the lii- to hexa magnesium (Sigma Chemical Co., St. Louis, MO), adjusted to pH 6.8â€”7.2,and was sterile filtered prior to use. Folic acid (Sigma Chemical Co.) for oral glutamates (9â€”15).Furthermore, these polyglutamates are competi administration was dissolved in water by adjusting the pH to 7.2 to 7.4 with tive or mixed inhibitors (with strong competitive components) of their NaOH. respective enzymes (1, 9, 15â€”18).Lastly, the affinities of these other Cell Culture. Humantumorcell lines were obtainedfrom the following antifolate polyglutamates are greater for their respective enzymes than sources: breast adenocarcinoma MCF7, ileocecal adenocarcinoma HCT-8, are those of the parent compounds (1, 9, 12, 15â€”18). ovarian carcinomas A2780, OVCAR-3 and SK-OV-3 and osteosarcoma 143 B These properties of l843U89 described above participate to pro TK (American Type Culture Collection, Rockville, MD); colon carcinoma duce another unusual feature of 1843U89. Compared to other antifo GC3TK (J. Houghton, St. Jude Children's Research Hospital, Memphis, TN); ileocecal adenocarcinoma HCT-8/TK (Y. Rustum, Roswell Park Cancer

Received 7/19/95; accepted 10/16/95. Institute, Buffalo, NY); and human epidermoid carcinoma KB3â€”l(Dr. The costs of publication of this article were defrayed in part by the payment of page Michael Gottesman, National Cancer Institute, Bethesda, MD). Cells were charges. This article must therefore be hereby marked advertisement in accordance with propagated as monolayer cultures as described (3). The basal medium for all 18 U.S.C. Section 1734 solely to indicate this fact. cell lines was folate-free RPMI 1640 (GIBCO-BRL) supplemented with 10 nM I Present address: Department of Cancer Biology, GlaxoWellcome, S Moore Drive, Research Triangle Park, NC 27709. calcium leucovorin and 10% dialyzed FCS [except A2780 and OVCAR-3, 2 To whom requests for reprints should be addressed, at Department of Cancer which contained 10% heat-inactivated (30 mm at 56Â°C) FCS that had been Biology, GlaxoWellcome, 5 Moore Drive, Research Triangle Park, NC 27709. Phone: depleted of thymidine by an incubation for 15 mm at 37Â°C with 1 unit/ml (919)315-4179;Fax:(919)315-3321. Escherichia coli thymidine phosphorylase]. MCF7, A2780, and OVCAR-3 3 The abbreviations used are: TS, thymidylate synthase; MTX, methotrexate; DDATHF, 5,10-dideazatetrahydrofolate, lomotrexol; diglutamate of I843U89, 1843U89 media contained 10 ,@g/ml insulin (Sigma 1-1882); the plates for these three with one additional glutamate; PEG-TPase, polyethylene glycol-thymidine phosphoryl cell lines were coated with 10 pg/mI PepTite-2000 (Telios Pharmaceutical ase; RTCB, residual tumor cell burden; MTD, maximum tolerated dose. Research Products) for 1 h to aid cell attachment. 6117

In Vitro Protection Studies. Cell growth inhibition and reversal experi Length and width of implanted fragments were measured at the time of surgery ments were done as described (3). The ability of folic acid or leucovorin to and again at autopsy. i843U89 was administered to mice by the i.p. route in block the cytotoxicity of 1843U89 in several human tumor cell lines was also volumes of either 10 or 20 mI/kg. At each dose interval, mice were weighed, assessed by a clonogenic assay. Log phase cells were allowed to attach and doses were adjusted according to individual body weight to the nearest overnight to 60-mm plates before a 24-h exposure to 1843U89 Â±folic acid or gram. In all cases, five mice were used per group. Antitumor activity was leucovorin (0.01â€”100 ,tM). After the 24-h drug exposure, drug was removed, assessed in three ways. Differences between various treatments were deter the plates were washed with 3 ml of sterile Dulbecco's PBS without calcium mined by Kruskal-Wallis analysis (SAS, Cary, NC) of relative tumor volumes or magnesium, and 3 ml of fresh media containing the appropriate levels of of individual mice by group; values of P < 0.05 were used to indicate statistical folic acid or leucovorin (but not 1843U89) were added. The plates were significance. The percentage of growth inhibition was calculated from group incubated 10â€”14days at 37Â°Cin 95% air/5% CO2. the media was removed, median doublings of treated and control tumors, treatment initiation to sacri and the plates were stained with a solution of 10 mg/mi crystal violet in 10% fice. Histological evaluation of tumors in early studies indicated that 1843U89 formaldehyde, 5% acetic acid, and 60% methanol (5-mm stain, water rinse, might effect lysis of tumor cells with varying degrees of replacement with scar and air dry). Colonies of >0.1 mm were counted on an image analyzer (Artek tissue. Accordingly, the RTCB of each lesion was also assessed and scored as Systems Corp.; Model 982B). All determinations were run in triplicate. The follows by an individual unfamiliar with the particular study: histological inoculum density was adjusted to obtain 200â€”300 colonies on the control score = 0, no effect of therapy evident from histological evaluation of lesion plates, and the 1843U89 concentration was chosen from a previous titration to (RTCB > 60%); histological score = 1,equivocal to minimal effect (RTCB 40 yield â€”80% inhibition of colony formation. to 60%); histological score = 2, moderate to strong effect (RTCB 5% to 40%); Animals. Male CD-i and female BALB/c athymic mice mice were pur and histological score = 3, strong effect (RTCB < 5%). chased from Charles River Laboratories (Wilmington, DE) and used for all studies. Mice were housed in polycarbonate, filter-capped micro-isolator cages RESULTS and given sterilized food and water ad libitum. Female purebred beagles at least 10 months of age were purchased from Marshall Farms, USA, Inc. (North In vitro Effects of Folic Acid or Leucovorin on 1843U89. Based Rose, NY) and used for all studies. Beagles were housed individually in cages upon three-way mutual competition for transport among 1843U89, and given Prolab Canine 1600 Certified Diet (Agway, Inc.) and municipal tap leucovorin, and MTX but not folic acid, we have reported that water ad libitum. All animals were maintained in a temperature (22Â°CÂ±1Â°C)-, humidity (50 Â±[0%)-, and photoperiod (12-h iight/i2-h dark)-controiled room 1843U89 appears to enter MOLT-4 human T-cell leukemia on the with 10 to 15 air changes/h. reduced folate carrier (3). We have now also observed similar mutual Dog Protection Studies. 1843U89 was administerediv. by slow bolus transport competition among 1843U89, MTX, and leucovorin but not infusion via the cephalic vein once daily for five consecutive days. From a 20 folic acid in MCF7 breast adenocarcinoma and SW480 and WiDr mg/mi solution of 1843U89, 0.3, 0.6 and 0.9 ml/kg were dosed to obtain 6, 12, colon carcinoma cell lines, which suggests broader tumor use of this and 18 mg/kg, respectively. Oral foiic acid was given 30 to 40 mm prior to transporter for 1843U89 (data not shown). Since folic acid does not 1843U89. To facilitate rapid ingestion of the entire dose, folic acid was given compete for this 1843U89 tumor cell transport and the products of by gavage by #12 gelatin capsules as a 50 mg/mI solution. Animals were cellular metabolism of folic acid, the reduced folates, do not compete monitored during the dosing period and for 30 days after the dose. for 1843U89 binding to TS (1), folic acid should not effectively Mouse Protection Studies. The ability of folic acid to block weight loss reverse 1843U89 cytotoxicity in tumor cells. Table 1 shows the ability and death was determined in mice given 1843U89. The high plasma thymidine levels in mice, which prevent the toxicity of thymidylate synthase inhibitors, of folic acid and leucovorin to reverse the inhibition of clonogenic was reduced by treating the mice with a conjugate of PEG-TPase. PEG-TPase growth by l843U89 in eight separate human tumor lines. HCT-8 was (Weilcome Research Laboratories, Research Triangle Park, NC) was admin the most sensitive line to folic acid reversal, requiring 28 p.Mfor 50% istered at 2500 units/kg, i.p., on days 1 and 4, 1.5 h prior to 1843U89, which reversal of l843U89 activity; all others required over 100 p@Mfor50% was dosed at 200 and 400 mg/kg, i.p., bid X 7 days. Folic acid was dosed p.o. reversal. In contrast to folic acid, leucovorin, which does compete for at 300 mg/kg 30 mm prior to each 1843U89dose. In all cases, five mice were 1843U89 transport in the @Mrange,reversed 1843U89 cytotoxicity used per group. The mice were maintained as described above and monitored more efficiently (Table 1). To further investigate this resistance of for weight change and death. 1843U89 to folic acid or leucovorin reversal, reversal of 1843U89 and Subrenal Capsule Tumor Growth Assay. Priorto their use in vivo, all another TS inhibitor, Tomudex, by the two agents were compared in cell lines were verified to be free of Mycoplasma bacteria and adventitial murine viral pathogens by mouse antibody production test (Charles River three cell lines (Table 2). Since Tomudex has been reported to be Biotechnical Laboratories). Cell preparation, implantation, and evaluation pro efficiently reversed by leucovorin (3), we expected Tomudex activity cedures of cell lines and tumors in in vivo studies have been described to be more sensitive to both folic acid and leucovorin. As can be seen elsewhere (3, 26). Briefly, cells were implanted as fragments under the renal in Table 2, both 100 and 10 @LMleucovorinvery efficiently reversed capsule of [8â€”22g male CD-i athymic mice by published procedures (26). the activity of Tomudex but only weakly reversed the activity of

TablecultureConcentration 1 Reversal of 1843U89 cytotoxicity by folic acid or leucovorin in cells in

50%reversal required for @siCell of 1843U89 cytotoxicity % reversal of 1843U89 cytotoxicity with 1 or 10 agentÂ°Folic reversal

LeucovorinFolic acid

p@SK-OV-3Ovarian>100lineType(p@M) acid Leucovorin 1 @M 10 @.sM 1 @.LM10 6395A2780Ovarian>100 0.6 0 0 3163OVCAR-3Ovarian>100 5 0 13 4151HCT-8Ileocecal28 10 5 10

9498KB3-lEpidermoid>100 0.07 25 44

0l43B 3.7 0 4GC3/TKColon>100TKOsteosarcoma>100 5.0 0 1253MCF7Breast>100 8 0 7 2125a 32 0 3 Reversalof80%inhibition by the drug; thus, 50% reversal leads to 40% inhibition remaining. Partial (80%) inhibition was chosen to be able to detect small reversal effects. 6118

Table 2 Effect offolic acid or leucovorin on cell growth inhibition byU89Reversal Tomudex and 1843 agent100

@Mleucovorin10 acidIC50@sMleucovorin25 @Mfolic

FoldDrugIC50 FoldIC50 FoldIC50 increaseTomudexSW4800.4>55,000 (nM)(nM) increase(riM) increase(nM)

20GC3TK2.1>1,000 >1lo,000a10,000 25,00tr7 26HCT-81.432 >5001,000 50052.8 231843U89SW480130

1.2GC3TK0.341 30Â°1 1â€•1.2 3HCT-80.822.5 1.6 343.4 101 3a From Duch etal. (3).

1843U89 in either GC3TK or SW480 cells. Furthermore, 25 ,LM The ability of folic acid to protect against the lethal toxicity of folic acid increased the IC50 of Tomudex approximately 20-fold in all l843U89 was tested in beagle dogs given a lethal dose of drug with three of SW480, GC3TK, and HCT-8 cells, while these conditions or without prior treatment with oral folic acid. On a 5-day dosing only increased the 1843U89 IC50 1- to 3-fold. Thus, the efficacy of schedule, 6 mg/kg/thy 1843U89 i.v. was a 100% lethal dose in dogs 1843U89 in cell culture is less sensitive than Tomudex to reversal by (Table 3). By contrast, when 50 mg/kg oral folic acid was given 30 either leucovorin or folic acid, and folic acid is the less effective mm prior to the i.v. doses of 1843U89 (Table 4), all of the animals reversing agent. survived both this dose and twice this dose (6 and 12 mg/kg/day Effect of Oral Folic Acid on Toxicity of 1843U89 in the Beagle l843U89, respectively). However, even with the oral 50 mg/kg folic Dog. As shown in Table 3, the MTh for i.v. 1843U89 on a daily acid doses, five daily doses of 18 mg/kg/day l843U89 i.v. were fatal 5-day schedule in the beagle dog was between 2 and 6 mg/kg/day; at to all dogs treated. Importantly, dose-limiting toxicity at 18 mg/kg 2 mg/kg, toxicity was mild, but 6 mgfkg was lethal. Gastrointestinal 1843U89 with 50 mg/kg folic acid was intestinal and comparable to toxicity, including severe diarrhea and maturation arrest enteritis, was that seen with 6 mg/kg 1843U89 alone. Thus, oral administration of dose limiting. Hematological toxicity was observed but was consis folic acid protects against the lethal toxicity of i.v. 1843U89, thereby tently mild and never dose limiting (data not shown). Thus, a mech raising the MTh for the drug more than 2-fold and potentially greater anism to decrease the gastrointestinal toxicity should result in an than 6-fold [MTh for 1843U89 alone is between 2 and 6 mg/kg increased MTD for the drug. (Table 3); MiD for l843U89 plus folic acid is between 12 and 18 mg/kg]. In other experiments (data not shown), it was found that five daily Table 3 Toxicity offive daily iv. doses of 1843U89 in beagledogf'1843U89 doses of up to 100, 200, or 500 mg/kg/day of folic acid were well dose tolerated and effective in protection against a 10 mg/kg/day dose of (mg/ks/day)2 l843U89. The minimal dose of folic acid to provide protection to all 6Mâ€• dogs was between 10 and 50 mg/kg/day since a dose of 10 mg/kg/day FÃ˜C FM of folic acid provided protection to three of four dogs receiving the 10 mg/kg/day 1843U89. However, the use of multiple daily doses of folic 2cClinical Ã˜C 2c acid did permit the reliable use of lower individual doses; thus, 16.7 signs: hypothermia, labored breathing, No No Yes Yes retching. righting reflex slow or absent, pale or 10 mg/kg folic acid three times daily (30 mm before and 3 and 6 gingiva, prostration, salivation, and h after the 1843U89 dose) each protected four of four dogs receiving lacrimationDiarrhea, 10 mg/kg/day 1843U89. emesis, and/or decreased activity, Yes Yes Yes Yes Leucovorin at 10 to 200 mg/kg/day could replace folic acid and and/ordehydrationBody protect all dogs receiving 10 mg/kg/day 1843U89. As will be shown YesFoodweight decrease Yes Yes Yes below, leucovorin more effectively reversed 1843U89 antitumor ef ficacy and was, therefore, not pursued further. YesGrossconsumption decrease Yes Yes Yes Effect of Oral Folic Acid on Weight Loss and Death of Mice pathology: dark red discoloration of the No No Yes Yes Administered 1843U89. In contrast to human cells, l843U89 is not lining and contents of intestinal tract, and/or efficiently transported into mouse cells (3). In addition, high circulat severe, reddened mucosal lining or brown, watery contents in stomach; and/or ing thymidine levels in mice decrease the efficacy and toxicity of all longitudinal dark red streaking of the colon, TS inhibitors in mice (3, 4, 27, 28). As such, 1843U89 has very little and/or severe thymic hemorrhage; and/or toxicity in mice (data not shown; see also Refs. 3 and 4). Nonetheless, hemorrhageHistopathology:minimal to severe injection site if mice are first depleted of thymidine by prior administration of a maturation arrest enteritis of No No Yes Yes polyethylene glycol conjugate of thymidine phosphorylase (PEG intestinal tract; severe thymus atrophy, congestion; and/or bile stasis in liver; TPase), high doses of 1843U89 can produce weight loss and death minimal to severe involution of the white (Fig. 1). This effect is presumably due to depletion, by PEG-TPase, of pulp of spleen; mild to moderate involution circulating thymidine available for salvage since circulating thymi nodesMaturationof cervical and mesenteric lymph dine levels drop from 0.7â€”1.5,.LMto<0.03 p.M.4The toxicity produced arrest of myeloid elements in bone No No Yes No by this regimen appears to be gut toxicity, as shown by bloody marrowa FourdogsweretreatedateachdoselevelofI843U89,twomalesandtwofemales.diarrhea and histopathology (data not shown). To test whether folic b M, male; F, female. C Deaths. 4 R. Ferone, unpublished data. 6119

Table 4 Effect of 50 mg/kg oralfolic acid pretreatment on the toxicity of 5-daydaily i.v.1843U89dogsÂ°1in beagle

843U89 dose (mg/kg/day) (mg/kg/day)50b650b12Folic Acid dose

50b@b1850bM@FM 50b

FMF0d0d0d

@d2d2dNoNoNo

Severe clinical signs: altered stool with blood, frequent emesis, decreased activity, cool to the NoYesYes touch, blood and/or stains around anogenital area, salivation, labored breathing, body tremors, and nystagmus

Mild clinical signs: occasional altered stool without blood, occasional emesis, and salivation Yes Yes Yes Yes No No

Body weight decrease Yes Yes Yes Yes Yes Yes

Food consumption decrease Yes Yes Yes Yes Yes Yes

Histopathology: mild. minimal. or very minimal maturation arrest enteritis of the cecum, colon, Yes Yes Yes Yes No No and/or ileum

Gross pathological changes noted in the 6- and 12-mg/kg/day group: red streaking of the colon, approximately 5 mm raised reddened areas at the junction of the colon with the ileum.

Gross pathological changes noted in the 18-mg/kg/day group only: dark red discoloration and congestion of the mucosa of the duodenum, jejunum, ileum, cecum, and colon, minimal white streaking of the renal cortex.

Histopathological changes noted in the 18-mg/kg/day group only: moderate to severe maturation arrest enteritis of the duodenum, jejunum, ileum, cecum, and colon. Maturation arrest was noted in the bone marrow smears from two of the three dogs humanely sacrificed. Kidney congestion, minimal to mild dilated tubules, casts, very minimal focal tubular epithelial necrosis, and very minimal tubular epithelial regeneration.

Note Effects in all parameters were reversed or reversing at the end of the postdose recovery period in all surviving animals. aFourdogsweretreatedateachdoselevelof1843U89,twomalesandtwofemales. h Folic acid dose (mg/kg/day).

( M, male; F, female. (I Deaths.

acid can protect in this model of l843U89 toxicity, drug was admin 23 istered to TPase-pretreated mice via i.p. injection twice daily for 7 days with or without oral 300 mg/kg folic acid 30 mm prior to l843U89. The data in Fig. 1 show that folic acid prevented the 20% 22 weight loss caused by 200 mg/kg 1843U89. A higher dose of 1843U89, 400 mg/kg, was lethal to all mice by day 10 in the absence of folic acid, but in the presence of the protectant, 80% of the animals 21 survived (Fig. 2). Thus, folic acid can protect against the lethal toxicity of l843U89 in mice as well as dogs. Effect of Folic Acid on the Antitumor Efficacy of 1843U89. To 20 determine the effect of folic acid or leucovorin on the in vivo antitu .@ C) mor efficacy of 1843U89, three human tumor lines were grown under a the renal capsule of mice and treated with 1843U89 with or without 19 a C) prior oral doses of folic acid or leucovorin. These tumor lines a (GC3TK colon carcinoma, HCT-8f1'K ileocecal adenocarcinoma, a and 143B TK osteosarcoma) all lack thymidine kinase, preventing 4 18 thymidine salvage. GC3TK is the most sensitive of the three cell lines to 1843U89. Table 5 shows that 3 to 10 mg/kg of I843U89 twice daily for 5 days 17 greatly inhibited tumor growth and led to cell kill and regression of GC3TK (all measured on day 10, experiments 1 through 4). In this table, the term histological score (measured as described in â€œMaterials 16 and Methodsâ€•)is introduced and used as a measure of the extent of cell kill caused by drug; values for histological score of 2 to 3 indicate extensive tumor cell kill. As can be seen in Table 5, most treatment schedules produced histological scores of 2 to 3, indicating a substan 0 2 4 6 8 10 12 14 16 tial antitumor effect. The effect of folic acid upon therapy of GC3TK Day by 1843U89 is also shown in Table 5. Addition of 50 to 500 mg/kg Fig. 1. Effect of oral folic acid on the weight loss of mice given 200 mg/kg 1843U89. folic acid p.o. 30 mm prior to l843U89 did not change either growth BALB/C female mice were dosed, i.p., on days I and 4 with 2500 units of PEG-TPase/kg. inhibition or tumor cell kill (Table 5, experiments 1 through 4). Since On days 1 through 7, the mice were dosed, i.p., with 200 mg l843U89/kg, twice daily, 5.5 h apart alone (â€¢)or along with oral predoses of folic acid (LI). For animals receiving the in vitro leucovonn reversed 1843U89 activity in GC3TK only oral folic acid protection, 300 mg folic acid/kg were dosed 0.75 to 0.5 h prior to all poorly, although more efficiently than folic acid, leucovorin was also l843U89 doses. 6120

tested on the activity of 1843U89 in this tumor. The addition of oral 200 mg/kg leucovorin had little effect (Table 5, experiments 2 and 3). 100 Since multiple doses of folic acid were also protective in the dog, we investigated the effect of multiple daily doses of folic acid on 90 therapy in mice. Four separate conditions were tested: (a) 1843U89 alone once daily for 5 days; (b) folic acid 30 rain prior to the 1843U89 80 dose; (c) folic acid 30 mm prior to and 3 h after the 1843U89 dose; and (d) folic acid 30 mm prior to and 3 and 6 h after the 1843U89 dose. The results are displayed in Table 5, experiment 5. In this 70 experiment, 50 mg/kg of 1843U89 alone once daily for 5 days inhibited tumor growth and led to cell kill and regression of GC3TK. 60 Administration of the single or multiple daiiy oral doses of 366 mg/kg a folic acid did not change this antitumor efficacy, although multiple doses may have produced some decrease in cell kill, as measured by @m the histological score. For this experiment, the effect of the folic acid administrations upon tumor outgrowth to day 21 was also measured. This is shown in Fig. 3. No significant outgrowth was observed in the 1843U89 treated tumors, while control tumors increased their volume 30 14-fold. Folic acid once or twice daily had no effect on the antitumor activity of 1843U89. Thus, folic acid produced no measurable reversal 20 of 1843U89 efficacy in GC3TK. This effect of folic acid on 1843U89 therapy of GC3TK@ was 10 compared with the effect on therapy with two other anticancer agents under clinical development, DDATHF and Tomudex (12, 19). Since both of these agents are reversed by folates in cell culture more 0 efficiently than is 1843U89, we wished to determine whether a similar 0 2 4 6 8 10 12 14 16 reversal would be observed in vivo. The results of this experiment are Day shown in Fig. 4. Both Tomudex and DDATHF inhibited the growth of Fig. 2. Effect of oral folic acid on the lethality of 400 mg/kg 1843U89 in mice. GC3TK, but upon addition of folic acid, a modest reversal of the BALB/C female mice were dosed, i.p., on days 1 and 4 with 2500 units of PEG-TPase/kg. efficacy of each drug was observed. As before, no such reversal was On days 1 through 7, the mice were dosed, i.p., with 400 mg 1843U89/kg, twice daily, 5.5 h apart alone (@) or along with oral predoses of folic acid (0). For animals receiving the seen for 1843U89. The effect of folic acid upon the action of these oral folic acid protection, 300 mg folic acid/kg were dosed 0.75 to 0.5 h prior to all three agents was further substantiated with measurement of tumor cell 1843U89 doses. kill, as determined by the histological score. Folic acid had no effect on cell kill by 1843U89 but decreased cell kill by both DDATHF and

Table 5 Effect offolic acid or leucovorin on the antitumor activity of 1843U89 against GC3TKTcolon tumor? doseGrowthHistol.Dosemg/kgScheduleNameâ€•mg/kgScheduleinhib.doseProtectant

Dosel843U89 Experiment no. (%)â€˜@score'@10bid 5NoneN/AN/A>1002.010bidx h>1002.03.2bidX5NoneN/AN/A891.83.2bidX5FA500â€”0.5h781.8x 5FA500â€”0.5

210.0bid 5NoneN/AN/A>1002.610.0bidX h>1002.210.0bid x 5FA50â€”0.5 h>1001.83.2bid x 5LV200â€”0.5 5NoneN/AN/A>1002.03.2bidX5FA50â€”0.5h>1002.33.2bidX5LV200â€”0.5h>1001.8340.0bidx

5NoneN/AN/A>1003.040.0bidx h>1003.012.7bid x 5LV300â€”0.5 5NoneN/AN/A>1002.812.7bidx h>1002.24.0bid x 5LV300â€”0.5 5NoneN/AN/A>1002.44.0bidx h>1002.3410.0bid x 5LV300-0.5

5NoneN/AN/A>1002.410.0bidx h>1002.4550.0qd x 5FA366â€”0.5

h>1002.650.0qd X 5PBSN/Aâ€”0.5 h>1003.050.0qd X 5FA366â€”0.5 +3h>1001.950.0qd X 5FA366â€”0.5h, X 5FA366â€”0.5h,+3,6h>1002.0

a Therapy was from days 3 to 7 post implant, and tumors were measured on day 10. b FA, folic acid; LV, leucovorin; N/A, not applicable.

C Inhib., inhibition. Tumors with greater than 100% growth inhibition regressed upon therapy. d Histol., histology. Score = 0, no effect of therapy evident from histological evaluation of lesion; score = 1, equivocal to minimal effect; score = 2, moderate to strong effect; score = 3, strong effect. 6121

in an outgrowth assay. In this experiment, 50 mg/kg 1843U89 alone completely inhibited growth measured on day 10, and little outgrowth occurred over the 21 days. With the addition of a twice daily oral folic acid dose of 366 mg/kg, a substantial delay in outgrowth was still apparent on day 10; however, significant reversal was apparent by day 21.

DISCUSSION

It is well accepted that a variety of classical folate-based antitumor a E agents in use or currently under development are reversed by folates 0 such as folic acid or the reduced folates (5-formyltetrahydrofolate, 0 leucovorin, is the reduced folate commonly used; Refs. 9, 12, 19, and E 20). These folates are often used to â€œrescueâ€•apatient from severe I- toxicity after therapy with a large dose of MTX (9). However, since there is little selectivity between cancer and normal cells with the drugs, both tumor cells and normal cells are rescued from the action of the antifolate. The effect of both folic acid and leucovorin in the rescue of cancer cells from these classical antifolates is clearly shown with the glyci namide ribonucleotide transformylase inhibitors DDATHF and 5-dea zaacyclotetrahydrofolate and the TS inhibitor Tomudex (Table 2; Refs. 12, 19, 20, 29, and 30). These compounds and/or their active forms, the polyglutamated compounds, are all competitive or mixed inhibitors (with respect to the folate substrate) of their respective Day target enzymes (12, 19, 20). Therefore, folic acid and leucovorin, which are efficiently metabolized to the reduced folate substrates by Fig. 3. Effect of folic acid on the efficacy of 1843U89 in GC3TK tumors. Tumors were grown under the renal capsule. Animals were untreated (U), dosed i.p. with 50 mg/kg normal and tumor cells, reverse the toxicity of these antitumor agents 1843U89 once daily (0), dosed i.p. with 50 mg/kg 1843U89 once daily 30 thin after an in both normal and cancer cells. oral dose of 366 mg/kg folic acid (s), or dosed i.p. with 50 mg/kg 1843U89 once daily with oral doses of 366 mg/kg folic acid given 30 mm prior and 3 h after the 1843U89 dose (@)on days3 through7 postimplant. 8

Tomudex. The histological score for DDATHF decreased from 1 to 0, and that for Tomudex decreased from 3.0 to 2.0. In contrast, the 7 histological score for 1843U89 was 2.6 with or without folic acid. The effect of folic acid upon l843U89 therapy of l43B TK osteosarcoma is shown in Table 6. Thirty to 100 mg/kg l843U89 6 twice daily for 5 days inhibited the growth of the 143B TK ; the addition of 366 mg/kg of oral folic acid 30 mm prior to the i.p. doses 5 of l843U89 had no effect on this inhibition. To explore further the a effect of folic acid upon 1843U89 therapy of l43B TK, an out E 0 growth experiment was performed. In this experiment, 300 mg/kg >4 l843U89 once daily (days 3 through 7) were tested with and without 0 E single daily oral doses of 366 mg/kg folic acid (Fig. 5). Measured on 3 day 10, the efficacy of treatment with l843U89 alone was not signif â€” icantly different from treatment with folic acid. When these partially inhibited tumors were examined on day 21, both those that received l843U89 alone and those that received l843U89 plus folic acid 2 showed outgrowth, and the tumors that were treated with l843U89 plus folic acid were significantly larger (Fig. 5). The effect of leucovorin on the efficacy of 1843U89 in 143B TK was also determined. It can be seen in Table 6 that 200 mg/kg leucovorin partially reversed the efficacy of 100 mg/kg 1843U89 0 P (twice daily for 5 days) against this tumor. Since leucovonn did not none 50 mg/kg I 0 mg/kg 10 mg/kg reverse the inhibition of GC3TK by i843U89, leucovorin reversal DDATHF Tomudex 1843U89 appearsto be tumordependent. Therapy Fifty mg/kg of 1843U89 once daily partially inhibited the growth of Fig. 4. Effect of oral folic on the efficacy of DDATHF, Tomudex, or l843U89 in the ileocecal adenocarcinoma HCT-8/TK (Table 7). Administration GC3TK tumors. Tumors were grown under the renal capsule. Animals were dosed i.p. of single or multiple oral doses of 366 mg/kg folic acid produced little with DDATHF, Tomudex, or 1843U89 either alone (U) or 30 mm following a 366-mg/kg reversal of this growth inhibition as measured on day 10 (Table 7). oral dose of folic acid ([I) on days 3 through 7 postimplant. DDATHF was dosed once daily; Tomudex and l843U89 were dosed twice daily. Tumors were measured on day 10. However, the data in Fig. 6 show that folic acid very effectively For comparison of drug effects with and without folic acid, P 0.083 for DDAThF, reversed the antitumor activity of 1843U89 when measured on day 21 P = 0.0090 for Tomudex, and P = 0.462 for l843U89. 6122

tumorsâ€•ITable 6 Effec: offolic acid orleucovorin on the antitumor activity of 1843U89 against 143B TK osteosarcoma

doseDoseDose 843U89 doseProtectant

(%)C100bidmg/kgScheduleNameâ€•mg/kgScheduleGrowth inhib. mm>100100bid x 5NoneN/Aâ€”30 mm>100100bid X 5FA366â€”30 mm523 X 5LV200â€”30 mm6031.6bid x SNoneN/Aâ€”30 mm5731.6bid X 5FA366â€”30 I.6bid x 5LV200â€”30 mm59

â€˜ITherapy was from days 3 to 7 post implant, and tumors were measured on day 10. b FA, folic acid; LV, leucovorin; N/A, not applicable.

( Inhib., inhibition. Tumors with greater than 100% growth inhibition regressed upon therapy.

By contrast, l843U89 and its diglutamate are strong noncompeti Competition with folic acid for tumor cell transport does not occur tive inhibitors of the target enzyme TS (1). As such, the reduced folate in this concentration range, although since folic acid does not bind substrates generated in cells from folic acid or leucovorin do not effectively to the reduced folate transporter in the tumor (3). Thus, compete for 1843U89 binding to TS. Thus, folic acid and, to a lesser therapy with 1843U89 was less sensitive to folic acid reversal. Indeed, extent, leucovorin do not efficiently reverse cytotoxicity of 1843U89 data in Tables 5â€”7indicated little effect of folic acid upon 1843U89 (Ref. 3; Tables 1 and 2). We reasoned then that the folic acid or efficacy in GC3TK, l43B TK, or HCT-81TK when measured at leucovorin might not reverse the in vivo antitumor activity of day 10. However, results of the 21-day tumor outgrowth studies (Figs. l843U89. 3, 4, and 6) revealed smaller effects than could be observed in the The dose-limiting toxicity of l843U89 in the dog was maturation simple growth inhibition and regression assay. The outgrowth assay arrest ententis (Table 3). We sought to block this toxicity by prevent clearly revealed that HCT-8/TK efficacy was compromised by the ing the entry of 1843U89 into these cells. 1843U89 enters tumor cells folic acid protection, which was not readily seen in the growth on a saturable transporter, which it shares with leucovorin but not folic inhibition assay. However, even with this more sensitive assay, no acid (3). This requirement for saturable transport of l843U89 into effect of folic acid was seen on l843U89 therapy of GC3TK, and a tumor cells suggested that, similar to other antifolates, entry of significant but modest effect of folic acid was seen on suboptimal 1843U89 into gut cells would also be (at least partially) by a saturable 1843U89 therapy of 143B TK. This insensitivity to folic acid rever process (31â€”38).Both folic acid and leucovorin have been reported to sal in vivo is to be compared with the effect of folic acid on therapy bind to or be transported into immature and mature gut cells compet of GC3TK with either DDATHF or Tomudex. The less sensitive itively with MTX. In brush border membrane vesicles, the affinity for all three compounds appears to be similar (3 1, 33â€”38).In the imma ture cells of the crypt, the affinities for MTX and leucovorin are 300 higher than for folic acid (32). Based upon structure activity comparisons for these transporters, it was assumed that l843U89 would also be transported into the gut cells on the same carriers. Since the transporters are saturable, trans 250 ported compounds compete with each other for binding and transport on the carriers and inhibit the uptake of one another (31â€”38).Thus, intestinal folic acid or leucovonn were expected to block the entry of l843U89 into gut cells and in turn block the gastrointestinal toxicity 200 of the drug. To provide high local folic acid or leucovorin concentra a tions in the gut, we chose oral dosing of protectant given 30 mm E before the iv. dose of l843U89. This regimen was intended to 0 > 150 saturate the gut transporter(s) with folic acid or leucovorin prior to any 0 exposure to 1843U89, thereby preventing 1843U89 binding and trans E I- port. We show in the current work that the host toxicity, either dog or mouse, can indeed be greatly ameliorated by the protocol, but whether 100 the protection is truly working by the mechanism described is uncer tam. Clearly, additional studies are needed to confirm the detailed mechanism of action of this protection, including studies on 1843U89 transport into normal, immature human crypt cells. 50 Leucovorin can block 1843U89 intestinal toxicity, but as shown in Tables I and 6 (see also Ref. 3), leucovorin is also more effective than folic acid in reversing the antitumor efficacy of l843U89 both in vivo and in vitro. At least part of the antitumor reversal due to leucovorin Lr. is likely due to competition for l843U89 transport on the reduced 3 10 21 folate transporter in these cells. This conclusion is based upon the Day observation that 5 to 10 @tMleucovorin were required for partial Fig. 5. Effect of folic acid on the efficacy of 1843U89 in I43BTK tumors. Tumors reversal of 1843U89 in most (although not all) cell lines in vitro (Ref. were grown under the renal capsule. Animals were untreated (U). dosed i.p. with l843U89 3 and Table I). At these concentrations, leucovorin is near or above its alone at 300 mg/kg once daily (0), or dosed i.p. with 1843U89 at 300 mg/kg once daily 30 mm following a 366-mg/kg oral dose of folic acid (1l@)ondays 3 through 7 postimplant. Km for tumor cell transport and will, therefore, efficiently compete For comparison of the effect of l843U89 with and without folic acid, P = 0.917 at day with l843U89 (3). 10 and P = 0.043 at day 21. 6123

FOLIC ACID INTESTINAL PROTECFION FOR 1843U89

Table 7 Effect offolic acid on the antitumor activity of 1843U89 against HCT-8ffK work reported here, no effort was made to make animals folate ileocecal tu,norsâ€• deficient. All diets included folate, and we have reported that mice on Protectant doseGrowth this diet have normal folate levels (40). In addition, folic acid doses (%)CNameâ€•SchedulePBS Dose inhib. used here are far above those used in the studies with DDATHF (5 â€”O.5h82FA N/A mg/patient equals approximately 2.5 mg/m2 compared to the 1000 â€”0.5h72FA 366 mg/m2 used here). Thus, it appears likely that the mechanism of folic +3h52FA 366 â€”O.5h, acid protection is different in each case. â€”O.Sh,+3,6h64a 366 The data reported here indicate that in dogs or mice, in the presence l843U89 was dosed once daily on days 3 to 7 at 50 mg/kg. b FA, folic acid; N/A, not applicable. of folic acid, 1843U89 can be safely dosed at otherwise lethal levels C Inhib., inhibition. Tumors with greater than 100% growth inhibition regressed upon of the drug, allowing either additional dose escalation or better toler therapy. ated lower doses. Furthermore, a single oral dose of 366 mg/kg folic acid has little or no effect on the antitumor efficacy of 1843U89 in the 140 human tumor xenografts of colon carcinoma GC3TK and osteosar coma 143B TK in vivo (doses of 50 mg/kg in the dog and 366 mg/kg in the mouse are approximately equal mg/m2 doses, 1000 and 1100 120i[ mg/m2,respectively).In the ileocecaladenocarcinoma,HCT-8ffK, there was partial reversal by multiple oral doses of the protectant. These folic acid-sensitive tumors may be less common since, in cell 10011 culture, HCT-8 was the only one of the eight lines tested that was sensitive to folic acid reversal. In conclusion, the results of these experiments show that an oral dose of folic acid 30 mm prior to l843U89 can block both mouse and @ 80@ @ I dog intestinal toxicity. This regimen does not, however, reverse effi 0 cacy of l843U89 against two of three human tumor xenografts in the 0 nude mouse. Thus, the data reported here from experiments with cells E ,E 60 in culture and with mice and dogs indicate that the antitumor selec tivity of 1843U89 may be enhanced (host toxicity reduced) through combination therapy with oral folic acid. The true test of the utility of

@ 40 I@ :i@@ the combination in humans is under way.

REFERENCES

1. Dcv, I. K., Dallas, W. S., Ferone, R., Hanlon, M., McKee, D. D., and Yates, B. B. Mode of binding of folate analogs to thymidylate synthase: evidence for two asym metric but interactive substrate binding sites. J. Biol. Chem., 269: 1873â€”1882,1994. 2. Pendergast, W., Dickerson, S. H., Dcv, I. K., Ferone, R., Duch, D. S., and Smith, G. K. Benzo[/]quinazoline inhibitors of thymidylate synthase: methyleneamino 3 10 21 linked arylglutamate derivatives. J. Med. Chem., 37: 838â€”844, 1994. 3. Duch, D. S., Banks, S. D., Dcv, I. K., Dickerson, S. H., Ferone, R., Heath, L. S., Day Humphreys, J., Knick, V., Pendergast, W., Singer, S., Smith, G. K., Waters, K., and Wilson, H. R. Biochemical and cellular pharmacology of l843U89, a novel benzo Fig. 6. Effect of folic acid on the efficacy of l843U89 in HCT8/TK tumors. Tumors were grown under the renal capsule. Animals were untreated (U), dosed i.p. with l843U89 quinazoline inhibitor of thymidylate synthase. Cancer Res., 53: 810â€”818, 1993. 4. Banks, S. D., Waters, K. A., Barrett. L. L., Dickerson, S., Pendergast, W., and Smith, alone at 50 mg/kg once daily (0). or dosed i.p. with l843U89 at 50 mg/kg once daily 30 mm following a 366-mg/kg oral dose of folic acid (lIlt)on days 3 through 7 postimplant. G. K. Destruction of WiDr multicellular tumor spheroids with the novel thymidylate synthase inhibitor l843U89 at physiological thymidine concentrations. Cancer Chemother. Pharmacol., 33: 455â€”459, 1994. 5. Wilson, H. R., Heath, L. S., Knick, V. C., Koszalka, G. W., and Ferone, R. In vivo 10-day growth inhibition assay demonstrated modest reversal of ther antitumor activity of 1843U89, a new antifolate thymidylate synthase inhibitor. Proc. apy with these latter two compounds by folic acid. Thus, 1843U89 Am. Assoc. Cancer Res., 33: 407, 1992. 6. Wilson, H. R., Ferone, R., Boytos, C. M., Heath, L S., Jones, W., Knick, V. C., and therapy appears unique in its low sensitivity to folic acid, as predicted Waters, K. Enhanced l843U89 in vivoactivityby coadministrationof methoxypolyeth from previous reports on folic acid reversals of antifolates (29, 30) as yleneglycol-thymidine phosphorylase. Proc. Am. Assoc. Cancer Re&, 36: 376, 1995. well as the data presented in Table 2. 7. Boytos, C. M., Ferone, R., Jones, W. A., Rudolph, S. K., Franklin, S. L., and Houle, C. D. In vivo antitumor activity of 1843U89 against experimental liver metastases in While this work was ongoing, a host protection scheme based upon the nude mouse. Proc. Am. Assoc. Cancer Res., 36: 376, 1995. folic acid was proposed for the competitive glycinamide ribonucle 8. Ferone, R., Hanlon, M. H., Waters, K. A., and Dcv, I. K. Influence of intracellular otide transformylase inhibitor DDAThF (24) and is being tested in the polyglutamation on the cytotoxicity of the thymidylate synthase inhibitor 1843U89. Proc. Am. Assoc. Cancer Res., 34: 274, 1993. clinic (25). Importantly, these authors have argued and provided 9. Allegra, C. J. Antifolates. In: B. A. Chabner and J. M. Collins (eds.), Cancer evidence to suggest that this system functions only under conditions Chemotherapy: Principles and Practice, pp. 110â€”153.Philadelphia: J. B. Lippincott Co., 1990. of folate deficiency. The mechanism for this protection is thought to 10. Pizzomo, G., Sokoloski, J. A., Cashmore, A. R., Moroson, B. A., Cross, A. D., and be folate repletion of the folate-binding protein in normal cells. This Beardsley, G. P. Intracellular metabolism of 5, l0-dideaza-5,6,7,8-tetrahydrofolic acid folate repletion is thought to then prevent DDATHF from entering a in human leukemic cell lines. Mol. Pharmacol., 39: 85-89, 1991. I 1. Hanlon, M. H., Ferone, R., Mullm, R. J., and Keith, B. R. In vivo and in vitro variety of normal cells by prevention of the binding of DDATHF to metabolism of 5-deazaacyclotetrahydrofolate and acyclic tetrahydrofolate analogue. the folate-binding protein (24). Prevention of toxicity by this strategy Cancer Res., 50: 3207â€”3211, 1990. 12. Jackman, A. L., Taylor, G. A., Gibson, W., Kimbell, R., Brown, M., Calvert, A. H., requires very small amounts of folic acid (5 mg/patient in the Judson, I. R., and Hughes, L. R. ICI D1694, a quinazoline antifolate thymidylate clinic), presumably due to the high affinity of folic acid for the synthase inhibitor that is a potent inhibitor of L1210 cell growth in vitro and in vivo: binding protein (39). Indeed folic acid levels in drinking water above a new agent for clinical study. Cancer Res., 51: 5579â€”5586, 1991. 13. Manteuffel-Cymborowska, M., Kaminska, B., and Grzelakowska-Sztaberg, B. Dose 0.003% reversed the antitumor efficacy in this system in mice (24), dependent retention and polyglutamation ofN'Â°-propargyl-5,8-dideazafolic acid (CB presumably due to the competitive effects discussed above. In our 3717) in the tumour-bearing mouse. Anticancer Res., 8: 791â€”796,1988. 6124

14. Sikora, E., Jackman, A. L., Newell, D. R., and Calvert, A. H. Formation and retention 26. Bogden, A. E., Kelton, D. E., Cobb, W. R., and Esber, H. J. A rapid screening method and biological activity of N'Â°-propargyl-5,8-dideazafolic acid (CB3717) polygluta for testing chemotherapeutic agents against human tumor xenografts. In: D. E. mates in L1210 cells in vitro. Biochem. Pharmacol., 37: 4047â€”4054, 1988. Houchens and A. Ovejera (eds.), Proceedings: Symposium on the Use of Athymic 15. Ward, W. H. J., Kimbell, R., and Jackman, A. L. Kinetic characteristics ofICI Di694: (Nude) Mice in Cancer Research, pp. 231â€”250.New York: Gustav Fisher, 1987. a quinazoline antifolate which inhibits thymidylate synthase. Biochem. Pharmacol., 27. Nottebrock, H., and Then, R. Thymidine concentrations in serum and urine of 43: 2029â€”2031, 1992. different animal species and man. Biochem. Pharmacol., 26: 2175â€”2179, 1977. 16. Keyomarsi, K., and Moran, R. G. Quinazoline folate analogs inhibit the catalytic 28. Jackman, A. L., Taylor, G. A., Calvert, A. H., and Harrap, K. R. Modulation of activity of thymidylate synthase but allow binding of 5-fluorodeoxyuridylate. J. Biol. antimetabolite effects. Biochem. Pharmacol., 33: 3269â€”3275, 1984. Chem.,265:19163â€”19169,1990. 29. Erba, E., Sen. S., Vikhanskaya, F. L, and D'Incalci, M. Mechanism of cytotoxicity 17. Habeck, L. L., Leitner, T. A., Shackelford, K. A., Gossett, L. S., Schultz, R. M., of 5,10-dideazatetrahydrofolic acid in human ovarian carcinoma cells in vitro and Andis, S. L., Shih, C., Grindey, G. B., and Mendelsohn, L. G. A novel class of modulation of the drug activity by folic or folinic acid. Br. J. Cancer, 69: 205â€”211, monoglutamated antifolates exhibits tight-binding inhibition of human glycinamide 1994. ribonucleotide formyltransferase and potent activity against solid tumors. Cancer 30. Gaumont, Y., Kisliuk, R., Parsons, J. C., and Greco, W. R. Quantitation of folic acid Res.,54:1021â€”1026,1994. enhancement of antifolate synergism. Cancer Res 52: 2228â€”2235, 1992. 18. Smith, G. K., Banks, S. D., Bigham, E. C., Cohn, N. K., Duch, D. S., Edelstein, M. P., 31. Said, H. M., Ghishan, F. K., and Redha, R. Folate transport by human intestinal brush Ferone, R., Hanlon, M. H., Heath, L. S., Humphreys, J., Kelley, J. L, Knick, V., border membrane vesicles. Am. J. Physiol., 252: 229â€”236, 1987. McLean, E. W., Mullin, R. J., Singer, S., and Wilson, H. R. In vitro and in vivo 32. SirOtnak, F. M., Moccio, D. M., and Yang, C. H. Similar characteristics of folate activity of the GAR transformylase inhibitor 5-deazaacyclotetrahydrofolate. In: H-C., analogue transport in vitro in contrast to dihydrofolate reductase levels in epitheial Curtius, S. Ghisla, and N. Blau (eds.), Chemistry and Biology of Pteridines: Pteridines cells at different stages of maturation of mouse small intestine. Cancer Res., 44: and Folic Acid Derivatives, pp. 1015-1022. Berlin: Waiter de Gruyter, 1990. 5204â€”5211, 1984. 19. Beardsley, G. P., Moroson, B. A., Taylor, E. C., and Moran, R. G. A new folate 33. Chello, P. L, Sirotnak, F. M., Dorick, D. M., and Donsbach, R. C. Therapeutic antimetabolite, 5,lO-dideaza-5,6,7,8-tetrahydrofolate, is a potent inhibitor of de novo relevance of differences in the structural specificity of the transport systems for folate purine synthesis. J. Biol. Chem., 264: 328â€”333, 1989. 20. Kelley,J. L, McLean,E. W., Cohn, N. IC. Edelstein,M. P., Ouch, D. S., Smith,G. K., analogs in L12l0 tumor cells and in isolated murine intestinal epitheial cells. Cancer Hanlon, M. H., and Ferone, R. Synthesis and biological activity ofan acycic analogue of Ret., 37: 4297â€”303,1977. 5,6,7,8-tetrahydrofolic acid, n-[4-[[3-(2, 4-diamino-l,6-dihydro-6.oxo-5-pyrimidinyl)pro 34. Zimmerman, J. Folic acid transport in organ-cultured mucosa of human intestines. pyllamino]benzoyl]-L-glutamic acid. J. Med. (1cm., 33: 561â€”567,1990. Gastroenterology, 99: 964-972, 1990. 21. Keyomarsi,K., and Moran, R. G. Folinic acid augmentationof the effects of fluoropy 35. Rosenberg, I. H., Zimmerman, J., and Selhub, J. Folate transport. Chemioterapia, 4: rimidines on murine and human leukemic cells. Cancer Ret., 46: 5229â€”5235,1986. 354â€”358, 1985. 22. Keyomarsi, K., and Moran, R. G. Mechanism of the cytotoxic synergism of fluoro 36. Schron,C. M.,pH modulationofthe kineticsof rabbitjejunal,brush-borderfolate pyrimidines and folinic acid in mouse leukemic cells. J. Biol. Chem., 263: 14402â€” transport. J. Membr. Biol., 118: 259â€”267, 1990. 14409, 1988. 37. Zimmerman, J. Methotrexate transport in the human intestine: evidence for hetero 23. Kennedy, D. G., Van den Berg, H. W., Clarke, R., and Murphy, R. F. The effect of geneity. Biochem. Pharmacol., 43: 2377â€”2383, 1992. leucovorin on the synthesis of methotrexate poly-gamma-glutamates in the MCF7 38. Zimmerman, J. Drug interactions in intestinal transport offolic acid and methotrexate: human breast cancer cell line. Biochem. Pharmacol., 34: 2897â€”2903, 1985. further evidence for the heterogeneity of folate transport in the human small intestine. 24. Gnndey, G. B., Alati, T., and Shih, C. Reversal of the toxicity but not antitumor Biochem. Pharmacol., 44: 1839â€”1842, 1992. activity of lomotrexol by folic acid. Proc. Am. Assoc. Cancer Res., 32: 324, 1991. 39. Henderson, G. B. Folate-binding proteins. Annu. Rev. Nutr., 10: 319â€”335, 1990. 25. Wedge, S. R., Laohavinij, S., Taylor, G. A., Newell, D. R., Charlton, C. J., Proctor, 40. Jansen, M., Dykstra, M., Lee, J. I., Stables, J., Topley, P., Knick, V. C., Muffin, R. J., M., Chapman, F., Simmons, D., Oakley, A., Gumbrell, L, and Calvert, A. H. Duch, D. S., and Smith, 0. K. Effect of purine synthesis inhibition in WiDr spheroids Modulation of lomotrexol toxicity by oral folic acid administration: a Phase I study. in vitro or in WiDr or colon 38 tumors in vivo: complete growth inhibition but not Proc. Am. Assoc. Cancer Ret., 34: 274, 1993. regression. Biochem. Pharmacol., 47: 1067â€”1078,1994.

6125

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1995 American Association for Cancer Research. Enhanced Antitumor Activity for the Thymidylate Synthase Inhibitor 1843U89 through Decreased Host Toxicity with Oral Folic Acid

Gary K. Smith, Herbert Amyx, Christine M. Boytos, et al.

Cancer Res 1995;55:6117-6125.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/55/24/6117

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/55/24/6117. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.