Optimal Scheduling of Methotrexate and 5-Fluorouracil in Human Breast Cancer1

Home , Fluorouracil

[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 /Ã•Mmethotrexate 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).

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Downloaded from cancerres.aacrjournals.org on September 24, 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 37Â°.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 /Â¿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. Bars, range of values from multiple experiments. 21 courses of therapy was: P.O. MTX (50 mg/sq m) every 6 hr 5 times; i.v. bolus FUra (600 mg/sq m) 1 hr after fifth dose of MTX; and P.O. LV 3.0 (10 mg/sq m) every 6 hr 6 times beginning 6 hr after the fifth MTX dose. Patients receiving more than one course were initially retreated 800 at 7 to 14 days. If toxicity developed, the retreatment interval and not drug dosage was altered. All patients had received prior treatment no u> less than 3 weeks before study entry, and 6 of the 7 had previously O 2.0 received 1-hr-sequenced MTX-FUra as part of an earlier study (13). 600 tt No patient was entered with preexisting neutropenia or creatinine clearance <65 ml/min. Toxicity and performance status were evalu o ated by the Eastern Cooperative Oncology Group criteria (24). Serum MTX levels were measured by the radioimmunoassay technique during Â§ all 21 courses of therapy. I.O 400 ^ Statistics. All experimentally derived charts and tables represent mean values of triplicate samples in single experiments, with S.D. <5% or as indicated. All experiments were repeated at least once for validity. Calculations were performed on a Hewlett-Packard 67 programmable 200 calculator. O IO 20 30 Time (hr) RESULTS Chart 2. Intracellular accumulation (O) and incorporation into RNA (â€¢)of [3H]FUra into human breast cancer cells, 47-DN, exposed to 100 JIM FUra at Clonal growth of 47-DN shows dose-dependent sensitivity to Time 0.

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Â¡onfor over 30 hr in these cells as described in other human of FUra accumulation occurs after 24-hr pretreatment with 10 monolayer cultures (1). This intracellular accumulation was JIMMTX (Table 2). Adding hypoxanthine back to cells optimally also accompanied by a proportionate increase in FUra incor pretreated with MTX totally reversed this augmentation in FUra poration into cellular RNA. After 6 hr of exposure to 100 /IM accumulation (Table 3). FUra, the total amount of FUra metabolites concentrated intra- Cloning studies were performed to demonstrate synergistic cellularly is greater than 20-fold that of FUra in the medium, growth inhibition in 47-DN cells treated sequentially by MTX suggesting that these biochemical results are not aberrant and FUra. In studies describing our monolayer cloning tech consequences of dying cells. For convenience in subsequent nique, we showed that HCT-8 and 47-DN cells required greater experiments, the 6-hr time point was used as a measure of the than 6 and 12 hr of MTX pretreatment, respectively, prior to rate of intracellular FUra accumulation. FUra exposure to produce greater than additive cytotoxicity The effect of MTX on the rate of intracellular FUra accumu (2). These results were reproduced in 47-DN cells cultures lation is represented in Chart 3A. A 24-hr exposure to 10 fiM using several concentrations of MTX and FUra, chosen to MTX resulted in the greatest enhancement of intracellular FUra mimic possible clinical use of these agents. Table 4 illustrates accumulation. Longer and shorter MTX pretreatment periods this syngergistic cytotoxicity, as well as the influence of LV on were less effective. This enhancement of FUra accumulation sequenced MTX-FUra synergism in 47-DN cells. MTX was was also dependent on MTX concentration with maximum present in the cell cultures for 24 hr, and FUra was added enhancement observed after 10 JUMMTX, a dose which com during each of four 6-hr intervals during the MTX exposure. pletely inhibits clonal growth after 24 hr (Chart 1). The en Greater than additive cytotoxicity was observed only when hancement of intracellular FUra accumulation following MTX FUra was added after 18 hr of exposure to MTX. When LV was exposure was observed at concentrations of FUra ranging from added during exposure to MTX, MTX-FUra synergism was 1 tO 100/iM. observed only when the LV followed administration of FUra. LV Biochemical studies in L1210 and HCT-8 cells have shown at 100 U.Mtotally reversed the inhibition of thymidylate synthesis that enhanced FUra accumulation is associated with increased by 0.1 Â¡IMMTX (as measured by incorporation of [3H]deoxyu- amounts of intracellular PRPP, which are the result of de novo ridine into DNA) but did not prevent the biochemical or growth- purine synthesis inhibition by MTX (1, 5, 6). In these cell lines, inhibitory effects of higher concentrations of MTX. >90% of the accumulated FUra is present as ribonucleotides. MTX-pretreated 47-DN cells also had increased PRPP levels, Table 1 Enhancement of intracellular accumulation of FUra (100fnm)in which were maximal by 24 hr and paralleled the increased pretreated 4 7-DN rates of intracellular FUra accumulation (Chart 36). In both % of control* control and MTX-pretreated 47-DN, 70 to 75% of accumulated Pretreatment (24 hr) FUra was present as soluble 5-fluorouridine triphosphate and Methylmercaptopurine riboside (0.1 UM) 178 AzaserinedOfiM) 195 >98% as total FUra-containing ribonucleotides; MTX-pre 6-Diazc-5-oxc-L-norleucine (10 MM) 246 treated cells increased incorporation of FUra into RNA by 2- to L-Alanosine (10 KM) 136 3-fold. Other drugs, known to inhibit de novo purine synthesis Control = 2.26 Â±0.61 (S.D.) nmol/10e cells/6 hr. and increase intracellular pools of PRPP in L1210 and HCT-8 cells (1, 4), also enhanced FUra accumulation in 47-DN cells Table 2 from 1.3- to 2.5-fold (Table 1). Enhancement of intracellular accumulation of FUra (100 pu) in pretreated 47-DN Hypoxanthine is capable of consuming intracellular pools of Cultures were grown in Roswell Park Memorial Institute Tissue Culture Medium PRPP and reducing FUra accumulation in MTX-pretreated 1640 supplemented with 10% dialyzed fetal calf serum, insulin (0.2 ID/ml), and L1210 cells (5). It is normally present in both human and bovine estradici (1 UM) serum (16) and can be removed by dialysis. When 47-DN cells of control8Interval1 at following pretreatment are grown in hypoxanthine-free medium, optimal enhancement MTX concentration (MM)0.01 2 hr 24hr93 A. [3HJFUro Accumulation B Intracellular PRPP 0.10 100 149 1.00 150 209 10.0% 178 249 a Control = 2.34 nmol/10' cells/6 hr. 4000

Table 3 Effect of hypoxanthine on intracellular accumulation of FUra in pretreated 4 7-DN Cultures were grown in Roswell Park Memorial Institute Tissue Culture Medium 1640 supplemented with 10% dialyzed fetal calf serum, insulin (0.2 III/ml), and estradici (1 nw) 1110 (JIM)Hypoxanthine,Pretreatment m (% of control0)55 2 12 24 0 3 18 24 3 ru 24hr0 Time (hr) Time (hr) mo 10.0 1010 63 Chart 3. The effect of MTX exposure on intracellular FUra accumulation and PRPP levels of human breast cancer cells, 47-DN. Monolayer cell cultures were 1.0 214 425 exposed to 10 /IM MTX for the indicated time periods before adding 100 Â¡Ã•M16- 0.10MTX, 10 3H]FUra (20 Ci/mmol) and determining the intracellular FUra accumulation. The 10Accumulation 350 ' Control = 1.43 nmol/10" cells/6 hr. intracellular PRPP levels were also measured at the indicated times.

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Seven patients were treated with a schedule of MTX, FUra, DISCUSSION and LV (see "Materials and Methods") designed according to the sequencing schedule that produced optimal tumor toxicity Chart 4 schematically illustrates the mechanism of enhanced in vitro. In addition, drug doses were chosen which could be FUra accumulation following MTX pretreatment. MTX inhibits administered conveniently and would achieve cytotoxic serum dihydrofolate reducÃase preventing regeneration of the re concentrations. The toxicity experienced from 24-hr-se- duced folate (tetrahydrofolate) pool necessary for one-carbon quenced MTX and FUra is shown in Table 5. There were no transfer in thymidylate synthesis and in de novo purine synthe- episodes of thrombocytopenia or deterioration in renal func tion. Four of 7 patients tolerated more than 2 courses at an de novo Pyrimidine novo Purine average treatment interval of 23 days. Two of 21 courses were Synthesis! Synthesis^^Â©iij^**^"^ associated with severe toxicity (Eastern Cooperative Oncology PRPPOÃŽofotÂ« Group Grades III to IV), and this occurred with the second PÂ»01Â«Â®Furo/^^^J^Â»PUMP!FUDP treatment course in both patients. Mild to moderate toxicity (Eastern Cooperative Oncology Group Grades I to II) occurred in 11 courses, usually after the first or second treatment, and

8 treatment courses were associated with no toxicity whatso FdUMPOMP Â» ""^xlf?!, .I ever. Mean serum MTX levels 1 hr prior to and 1 hr after the ^^^~~"~- . ^ /FUMP -> 41K)!s fifth dose of MTX were 1.07 Â±0.74 S.D. and 2.10 Â±0.92 *1 | dUMP Â¿r 1 Â¡IM,respectively. Occurrence of toxicity was unrelated to either 1Ã¶ /JUDP RNA /X ^x^^b^iiIMPAMP cumulative drug doses or greater than average serum MTX Jf1*3/ ^dUDP (i\ levels. x'uRNAJHP tj^v

dTMP1dTTPde Table 4 Effect of MTX, FUra, and LV on donai growth of 4 7-DN GMP1 % of control growth 1AOP GOP/ \P--Â»dCTP ' 1 TreatmentMTX, FUra90 JIM Â«-DNA /l\ATP ' .Â» C hrLV. 0.1 fiM, 24 Â±9a1 dATP dGTP GTP 6hrFUra,100/iM, 00 Â±1090 6hrMTX -.FUra61st2nd3rd4thMTX Â±990 Â±324 RNA DNA RNA Â±960 Â±510 Chart 4. Proposed interaction of MTX and FUra. Broken arrows, multiple Â±673 Â±120 enzymatic steps. Enzymes (circled numbers): 1, amidophosphoribosyltransfer- Â±742 ase; 2, phosphoribosyl glycineamide formyltransferase; 3, phosphoribosyl ami- Â±479 -Â»LV +FUracBefore noimidazole carboxamide formyltransferase; 4, thymidylate synthetase; 5, dihydrofolate reducÃase;6,orotate phosphoribosyttransferase; and 7,phosphoribosyl FUraWith Â±869 FUraAfter pyrophosphate synthetase. MTX inhibits Enzyme 5, and dTMP synthesis contin Â±649 ues until the tetrahydrofolate pools no longer support the methyl transfer to FUra1 Â±5lOjiMFUra24 dUMP. Because of this reduction in tetrahydrofolate pools, purine synthesis is * Mean Â±S.D. also inhibited. 5-Fluorodeoxyuridine monophosphate directly inhibits Enzyme 4 b FUra added during each of 4 6-hr intervals during a 24-hr 0.1 UM MTX in the presence of tetrahydrofolate. FUMP, 5-fluorouridine monophosphate; exposure. FUDP, 5-fluorouridine diphosphate; FdUMP, 5-fluorodeoxyuridine monophos- c LV (100 /IM, 6 hr) added before, with, or after FUra. The FUra was given phate; FUTP, 5-fluorouridine triphosphate; OMP, orotidine monophosphate; FH4. only during the fourth interval of MTX exposure in the experiment with LV. tetrahydrofolate; FHÂ¡,dihydrofolate.

Table 5 Clinical toxicity sfudy of 24-hr sequence^ MTX and FUra with LV rescue gradea/courseancePatientToxicity

PatientPrimary sta- cancerBreastBreastBreastBreastMycosiscourses1221124Total 0, 0,00, 0, 0,0II, 0, 0,00, 0, 0,00, I0,IV, I, 0, 0, 00,00,0,0II,I, 0. 0, 0, 00,01,0,00.00,0,00Mucositis0,I, 0, I, 0, 00,0II.II, 1,II, 0, III0,0,0I, 1.0II. fungoidesMycosis III, 00,0,00Diarrhea0, 0II. fungoidesHead 0,00Nausea-Emesis0, II,00 and necktus" courses462323121LeukopeniaII. No. of courses Grade8Leukopenia0

13 I 4 1 3 2 II 2 2 0 6 III 1 0 0 0 IV 1Nausea-Emesis18 0Diarrhea18 0Mucositis13 0 Eastern Cooperative Oncology Group criteria (24).

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1982 American Association for Cancer Research. Optimal Scheduling of MTX and FUra in Human Breast Cancer sis. The consequence of the reduction in de novo purine with 27 and 38% of courses, respectively (Table 5). synthesis is increased accumulation of the cosubstrate, PRPP, Since LV can rapidly reverse the biochemical effects of MTX, required for the initial biochemically committed step in the decreasing both intracellular pools of PRPP and rate of FUra purine pathway. The increased availability of PRPP in MTX- accumulation (5), it is important to establish the optimal clinical treated cells can be used by orotate phosphoribosyltransferase timing of LV administration. Our experiments (Table 4) support to transform FUra to 5-fluorouridine monophosphate, which the theoretical concept that LV should be given after FUra can in turn be converted to other intracellular nucleotides. administration. Circulating levels of "salvage" purines in human plasma can There are at least 2 toxic intracellular metabolites of FUra; fluorodeoxyuridylate, which inhibits thymidylate synthetase potentially prevent the antitumor effects of de novo purine and therefore DNA synthesis, and 5-fluorouridine triphosphate, inhibitors and abrogate the sequence-dependent synergism of which is incorporated into cellular RNA. Intracellular accumu MTX-FUra (5, 15, 16). The kinetic characteristics of the en lation and phosphorylation of FUra was enhanced up to 4-fold zymes necessary to salvage hypoxanthine, inosine, or adeno- in MTX-pretreated 47-DN cell cultures and was associated with sine vary considerably from tumor to tumor (3, 8, 21, 31 ); it is synergistic cytotoxicity. The 18- to 24-hr MTX pretreatment of interest that the normal physiological range for plasma interval necessary to maximally enhance FUra accumulation hypoxanthine is between 0.1 and 10 Â¡IM(16), the same con and toxicity is longer than that observed in both HCT-8 (6 to centration range that reverses FUra accumulation in MTX-pre 12 hr) and L1210 (3 hr) cells which have shorter doubling treated 47-DN (Table 3). Thus, agents known to decrease times. The critical role of this pretreatment interval has not excretion or increase circulating levels of hypoxanthine such been reported previously (12, 22, 27) and would be expected as allopurinol, probenecid, sulfinpyrazone, and salicylates (14) to be important in the design of clinical protocols. might also reduce the likelihood of antitumor activity by MTX- When regeneration of the tetrahydrofolate pool is blocked by FUra therapy. Stem cell assays have been used to try to MTX, continued synthesis of thymidylate and DNA depletes the determine the plasma hypoxanthine concentrations that might tetrahydrofolate pool and subsequently reduces de novo purine selectively protect bone marrow precursors from MTX toxicity synthesis. The fact that MTX substantially alters folate pools (16). Studies comparing the selectivity of sequential MTX-FUra and reduces purine synthesis only in cells which are synthesiz toxicity by stem cell assay of normal human marrow and freshly ing DNA has important therapeutic implications. Tumors with resected breast cancer specimens would be of considerable longer doubling times might have proportionately fewer cells interest and further help in the rational design of clinical treat synthesizing DNA during any given interval of MTX exposure, ment for breast cancer utilizing these 2 agents. resulting in less opportunity for synergism with FUra. Variations greater than 10-fold have been observed among growth rates ACKNOWLEDGMENTS of primary human breast tumors and their metastatic lesions (29). This could influence clinical response rates to sequential We would like to thank Dr. Rabindranth Nayak at Columbia University for MTX-FUra therapy. providing the 47-DN cells, Barbara Stanley and Joan Gesmonde for their tissue culture assistance, and the following people for their aid in their work and Clinical trials of MTX and FUra sequenced 1 hr apart in preparation of this manuscript: Robert Heimer, Lee Newcomer, Arlene Cashmore. patients with advanced breast, colorectal, and head and neck and Hillary Raeffer. cancers have shown encouraging responses with little if any host toxicity (13,17, 26, 30). The long doubling times of breast REFERENCES tumors (29) and our experimental data with 47-DN cells sug 1. Benz. C., and Cadman. E. Modulation of 5-fluorouracil metabolism and gest that pretreatment intervals >18 hr are necessary to pro cytotoxicity by antimetabolite pretreatment in human colorectal adenocar- cinoma, HCT-8. Cancer Res., 41: 994-999, 1981. duce optimal cytotoxic synergism between MTX and FUra. 2. Benz, C., Schoenberg, M., Choti. M., and Cadman, E. Schedule-dependent Studies trying to demonstrate the clinical utility of prolonging cytotoxicity of methotrexate and 5-fluorouracil in human colon and breast MTX pretreatment intervals might show that increased host tumor cell lines. J. Clin. Invest., 66. 1162-1165, 1980. 3. Berlino, J., and Allaudeen, H. Biochemical abnormalities in some human toxicity negates the therapeutic advantage of enhanced tumor neoplasms; the leukemias. In: T. Symington and R. Carter (eds.), Scientific cell kill. One small uncontrolled study which gave patients Foundations of Oncology, pp. 77-85. Chicago: Year Book Medical Publish ers, Inc., 1976. comparable doses of MTX and FUra sequenced 4 hr apart 4. Cadman, E., Benz, C., Heimer, R., and O'Shaughnessy, J. The effect of de reported unacceptable myelosuppression and attributed this novo purine synthesis inhibitors on 5-fluorouracil metabolism and cytotox toxicity to the longer MTX pretreatment interval (28). Another icity. Biochem. Pharmacol., 30: 2469-2472. 1981. study, comparing MTX-FUra schedules in tumor-bearing mice, 5. Cadman, E., Heimer, R., and Benz, C. The influence of methotrexate pre treatment on 5-fluorouracil metabolism in L1210 cells. J. Biol. Chem., 256: found that 24-hr sequencing was maximally tumoricidal but 1695-1704, 1981. also increased early toxic deaths more than 6-fold over the 6. Cadman, E., Heimer, R., and Davis, L. Enhanced 5-fluorouracil nucleotide formation after methotrexate administration: explanation for drug synergism. other 2 drug schedules (23). Science (Wash. D. C.), 205: 1135-1137, 1979. In light of these studies, we conducted a Phase I study to 7. Carbone, P. P. Chemotherapy in the treatment strategy of breast cancer. determine whether 24-hr-sequenced MTX-FUra could be tol Cancer (Phila.), 36 (Suppl.): 633-637, 1975. 8. Chang, C., Brockman, R., and Bennett, L. Adenosine kinase from Li 210 erated. Although the dose of drugs administered was identical cells. J. Biol. Chem., 255: 2366-2371, 1980. to that used in previous studies with 1-hr drug sequencing (13, 9. Chlebowski, R. T., Irwin, L. E., Pugh, R. P., Sadoff, L.. Hestorff, R.. Wiener, 26), MTX was administered p.o. in divided doses over 24 hr for J. M., and Bateman, J. R. Survival of patients with metastatic breast cancer treated with either combination or sequential chemotherapy. Cancer Res., patient convenience to sustain serum MTX levels above 1 /IM. 39:4503-4506, 1979. The administration of LV was scheduled 6 hr after i.v. bolus 10. Cooper, R. G., Holland, J. F., and Glidewell. O. 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Chris Benz, Tina Tillis, Ellen Tattelman, et al.

Cancer Res 1982;42:2081-2086.

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