[CANCER RESEARCH 42, 2081-2086, May 1982] 0008-5472/82/0042-0000$02.00 Optimal Scheduling of Methotrexate and 5-Fluorouracil in Human Breast Cancer1 Chris Benz, Tina Tillis, Ellen Tattelman, and Ed Cadman2 Departments of Medicine and Pharmacology, Yale School of Medicine, New Haven, Connecticut 06510 ABSTRACT tually die of disseminated disease (11, 25). The use of combi nation chemotherapy in disseminated breast cancer has im We have shown previously that methotrexate pretreatment proved objective response rates over single-agent therapy by of murine leukemia and human colon carcinoma cell cultures about 40%, yet there has been no improvement in overall results in augmented intracellular accumulation of 5-fluorour- survival and no clear superiority in palliative benefit over the acil metabolites. Both of these drugs are commonly used for use of sequential single-agent therapy (9, 18). These discour the treatment of women with breast cancer; thus, sequencing aging facts reflect the brevity of response durations following of methotrexate before 5-fluorouracil was evaluated in vitro systemic chemotherapy, 7 to 11 months, and low complete using a human mammary carcinoma cell line, 47-DN. Intracel response rates of about 15% (7). Perhaps more knowledgeable lular 5-fluorouracil accumulation was maximally increased 4- scheduling of multiple drugs given in combination will improve fold in cultures pretreated with 10 /ÕMmethotrexate for 24 hr. our therapeutic impact on breast cancer; basic laboratory This enhancement of 5-fluorouracil metabolism was associated studies may be able to provide the necessary rationale for with increased intracellular levels of 5-phosphoribosyl 1-pyro- devising such synergistic combinations. phosphate, resulting from the antipurine effect of methotrexate. MTX3 and FUra are 2 of the most commonly used drugs in Brief exposure to exogenous hypoxanthine at physiological the treatment of breast cancer. These 2 antimetabolites are concentrations reversed the biochemical synergism between usually administered simultaneously and often in combination methotrexate and 5-fluorouracil. Other antimetabolites associ with cyclophosphamide and prednisone, both in the adjuvant ated with elevations of 5-phosphoribosyl 1-pyrophosphate en setting and as treatment for disseminated disease (18). Our hanced intracellular accumulation of 5-fluorouracil up to 2.5- laboratory's biochemical studies in mouse leukemia and human fold. In cloning assays, 18 hr of methotrexate pretreatment colon carcinoma cell lines have shown that the antitumor effect followed by 5-fluorouracil resulted in optimal synergistic cyto- of combined MTX and FUra is synergistically enhanced when toxicity, which could be prevented if high concentrations of MTX precedes FUra administration in vitro (1, 5, 6). By inhibit leucovorin were given between methotrexate and 5-fluorouracil ing de novo purine synthesis and elevating intracellular pools administration. of PRPP, MTX enhances the intracellular accumulation and Since these results indicated that optimal breast tumor tox- metabolism of FUra, resulting in synergistic tumor cell kill. This icity in vitro was achieved by 18- to 24-hr sequencing of synergism does not occur when MTX and FUra are given methotrexate and 5-fluorouracil, a clinical toxicity study was simultaneously or when FUra treatment precedes MTX. Fur carried out to assess whether this drug schedule could be thermore, results in the human colon carcinoma cell line sug tolerated. Seven patients with advanced cancer were treated gest that the MTX pretreatment interval for optimal synergism with 21 courses of sequential therapy. No toxicity occurred with FUra is dependent on cellular growth rates (1). These with 38% of treatment courses; mild to moderate leukopenia studies have now been extended to a hormone-dependent and mucositis occurred with 29 and 38% of courses, respec human breast carcinoma cell line, 47-DN, and applied in the tively. Toxicity was related to retreatment interval and not design of a clinical trial assessing toxicity to optimally se- cumulative drug dose or elevated serum methotrexate levels. quenced MTX and FUra. These clinical results suggest that Phase II studies evaluating 24-hr-sequenced methotrexate and 5-fluorouracil in breast MATERIALS AND METHODS cancer are warranted. Cell Line, Drug, and Clonal Growth Assay. The hormone-dependent INTRODUCTION human mammary carcinoma, 47-DN, is a well-characterized (19) con tinuously growing monolayer cell line that under present culture con Studies of adjuvant chemotherapy in postmastectomy pa ditions doubles in 30 hr. Cells were grown in Roswell Park Memorial tients with positive axillary nodes suggest that combination Institute Tissue Culture Medium 1640 supplemented with 10% of fetal drug therapy can reduce the rate of recurrence by 20 to 40% calf serunrneonatal calf serum (1:1) (Grand Island Biological Co., and significantly increase survival for women with operable Grand Island, N. Y.). For indicated experiments, cells were grown in medium containing dialyzed fetal calf serum (Grand Island Biological breast cancer (10, 18, 25). Despite these optimistic projec Co.). Maximal 47-DN growth rate was obtained when 1 nw estradiol tions, present incidence rates show that one of every 13 women (Sigma Chemical Co., St. Louis, Mo.) and 0.2 IU insulin (Eli Lilly and will develop breast cancer, and more than one-half will even- Co., Indianapolis, Ind.) per ml were added to the culture medium. Stocks were passaged weekly, and single-cell suspensions were pre 1Supported by Grants CA-24187, CA-27130, and CA-08341 from the Na pared using a trypsin (0.05%):EDTA (0.02%) solution. Stock cultures tional Cancer Institute and Grant CH-145 from the American Cancer Society. 2 Recipient of a Cancer Research Award from the American Cancer Society. To whom requests for reprints should be addressed. 3The abbreviations used are: MTX. methotrexate; FUra, 5-fluorouracil; PRPP, Received September 29, 1981; accepted January 29, 1982. 5-phosphoribosyl 1-pyrophosphate; LV, leucovorin (5-formyltetrahydrofolate). MAY 1982 2081 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1982 American Association for Cancer Research. C. Benz et al. and cloning studies were grown in 75-sq cm sterile plastic culture 6 hr of FUra and 24 hr of MTX (Chart 1) over the indicated flasks (Costar, Data Packaging, Cambridge, Mass.) with 25 ml of medium in 5% CO2 incubators at 37°.Cell counts were performed on concentration ranges. When dialyzed serum is used in the culture medium (free of hypoxanthine and thymidine), FUra and a Model ZBI Coulter Counter (Coulter Electronics, Inc., Hialeah, Fla.); MTX toxicity are enhanced by approximately 20 and 50%, clonal growth and drug sensitivity assays were performed using a monolayer technique and an automated colony counter (Biotran II; New respectively. Since 100 /¿MFUrais both therapeutically achiev Brunswick Scientific Co., Inc., Edison, N. J.) as described in detail able (20) and necessary to completely inhibit DNA synthesis in 47-DN (measured by incorporation of [3H]deoxyuridine into elsewhere (1, 2). All drugs were purchased from Sigma with the exception of [6-3H]FUra (20 Ci/mmol) which was obtained from Mo- DNA), this concentration was used in all biochemical assays. ravek Biochemicals (City of Industry, Calif.), and 6-diazo-5-oxo-i_-nor- Although 100 /IM FUra resulted in virtually complete growth leucine and u-alanosine, which were obtained from the Division of inhibition of 47-DN by 6 hr, Chart 2 shows that FUra at this Cancer Treatment, National Cancer Institute (Bethesda, Md.). concentration accumulated intracellularly in a near-linear fash- Biochemical Assays. The intracellular accumulation of FUra was measured by our previously described microfuge method (1). Briefly, cell cultures were exposed to 100 JIM [6-3H]FUra and at the indicated times harvested by rapid trypsinization (0.05% solution) and counted. A 0.1-ml suspension of these cells was placed in a 0.5-ml plastic microfuge tube and immediately centrifuged at 10,000 rpm for 15 sec to sediment the cells through a silicone-oil interphase into 0.04 ml of 5% perchloric acid. The drug-containing medium remained above this silicone-oil interphase. Triplicate samples in microfuge tubes were then frozen quickly in an ethylene glycol:dry ice bath and cut at the inter- phases of perchloric acid, oil, and medium. The radioactivity contained in the perchloric tip represented the total amount of intracellular FUra and metabolities accumulated, recorded as nmol FUra per 106 cells. The oil fraction contained no radioactivity; the third fraction was the medium. The amount of radiolabeled FUra incorporated into cellular RNA was quantitated by standard alkaline hydrolysis of perchlorate- insoluble cell precipitates; and soluble mono-, di-, and triphosphate ribonucleotides of FUra were quantitated by high-pressure liquid chro- matography (Partisil SAX column with a linear phosphate buffer gra dient), as described previously (1 ). Intracellular pools of PRPP in control and MTX-treated 47-DN cells were quantitated by a standard enzymatic assay using the enzyme 3 "o - adenine phosphoribosyltransferase which was isolated from suspen sion cultures of L1210 (1 ). Lysed cell extracts of 47-DN cells containing measurable amounts of PRPP were incubated with [3H]adenine (Sigma) and the isolated L1210 enzyme. The amount of [3H]AMP formed, which was dependent upon the available PRPP (which provided the phos- phorylated ribose for conversion of adenine to AMP), was quantitated by performing simultaneous control assays with known quantities of PRPP, and values were reported as ng PRPP per 106 cells. Chart 1. Clonal growth of treated human mammary carcinoma, 47-DN. Dupli cate cultures were seeded with 5 x 104 cells as described (2), and the mean Clinical Study. A limited study to determine toxicity of 24-hr-se- colony counts were recorded. The effects of FUra and MTX on the number of quenced administration of MTX and FUra was carried out in 7 patients monolayer colonies were measured and expressed as a percentage of control with advanced cancer (Table 5). Schedule and dose of drugs for the growth.