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1016 Vol. 6, 1016–1023, March 2000 Clinical Cancer Research

Treatment Regimens Including the Multitargeted Antifolate LY231514 in Human Tumor Xenografts

Beverly A. Teicher,1 Victor Chen, Chuan Shih, covered through structure-activity relationship studies based on Krishna Menon, Patrick A. Forler, Val G. Phares, the novel antipurine antifolate lometrexol (1). MTA contains a and Tracy Amsrud pyrrole moiety in the place of the tetrahydropyridine ring of lometrexol, which results in a major shift in activity from the Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, inhibition of de novo purine biosynthesis to predominantly the Indiana 46285 inhibition of de novo thymidylate biosynthesis (2–4). MTA is an excellent substrate for mammalian folylpolyglutamate syn- ABSTRACT thase (5) and, in the polyglutamated form with three or more The scheduling of antifolate antitumor agents, including glutamyl residues, is a potent inhibitor of the enzymes TS, the new multitargeted autofolate LY231514 (MTA), with 5- dihydrofolate reductase, and GARFT (2). fluorouracil was explored in the human MX-1 breast carci- Mice have relatively high concentrations (about 1 ␮M)of noma and human H460 and Calu-6 non-small cell lung carci- circulating thymidine. Therefore, evaluation of antitumor com- noma xenografts to assess antitumor activity and toxicity (body pounds with tumors in mice may tend to underpredict both the weight loss). Administration of the antifolate (methotrexate, antitumor activity and toxicity of drugs that inhibit TS as com- MTA, or LY309887) 6 h prior to administration of 5-fluorou- pared with what may be expected in humans (6, 7). To com- racil resulted in additive growth delay of the MX-1 tumor pensate for this potential problem in studying antitumor activity when the antifolate was methotrexate or LY309887 and great- in mice, MTA was tested using a mutant tumor that was thy- er-than-additive tumor growth delay (TGD) when the antifo- midine kinase negative, murine lymphoma L5178/TK-/HX, and late was MTA. In the H460 tumor, the most effective regimens found to be very active (8). MTA was also found to be an were a 14-day course of MTA or LY309887 along with 5- effective antitumor agent against several human tumor xe- fluorouracil administered on the final 5 days. In addition, the nografts with normal thymidine kinase levels, including the simultaneous combination of MTA administered daily for 5 VRC5 colon carcinoma, the GC3 colon carcinoma, the BXPC3 days for 2 weeks with administration of gemcitabine resulted in pancreatic carcinoma, the LX-1 non-small cell lung carcinoma, greater-than-additive H460 TGD. MTA was additive with frac- and MX-1 breast carcinoma (1). In several studies, feeding a tionated radiation therapy in the H460 tumor when the drug low-folate diet and then repleting the animals modulated the was administered prior to each radiation fraction. MTA ad- folate levels in mice by administration of specific doses of folic ministered along with paclitaxel produced greater-than-addi- acid (9). Both the antitumor activity and toxicity of MTA could tive H460 TGD and additive responses along with vinorelbine be modulated in this manner and at certain folate levels, anti- and carboplatin. In the Calu-6 non-small cell lung carcinoma tumor activity toward specific tumors could be optimized. xenograft, MTA administered in combination with cisplatin or An important component in the development of a new anti- oxaliplatin was highly effective, whereas MTA administered in cancer drug is an understanding of its potential for inclusion in combination with cyclophosphamide, gemcitabine, or doxoru- combination treatment regimens. In recent studies, MTA was tested bicin produced additive responses. Administration of MTA in combination with cisplatin, methotrexate, 5-fluorouracil, pacli- along with paclitaxel or doxorubicin resulted in additive MX-1 taxel, docetaxel, doxorubicin, LY309887 [GARFT inhibitor (1, 2)], TGD. Thus, MTA appears to be especially effective in combi- and fractionated radiation therapy in vivo using the EMT-6 mam- nation therapies including 5-fluorouracil or an antitumor plat- mary carcinoma, the human HCT 116 colon carcinoma, and the inum complex. human H460 non-small cell lung carcinoma grown as xenografts in nude mice (10). Isobologram methodology was used to determine INTRODUCTION the additivity or synergy of the combination regimens. MTA administered with cisplatin, paclitaxel, docetaxel, or fractionated ra- MTA2 (N-(4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo- diation therapy produced additive to greater than additive tumor [2,3-d)-pyrimidin-5-yl)ethyl]-benzoyl]-L-glutamic acid) was dis- response by tumor cell survival assay and TGD. Although an additive tumor response was observed when MTA was administered with methotrexate, synergistic tumor responses were seen when MTA was administered with the GARFT inhibitor Received 6/8/99; revised 10/26/99; accepted 11/5/99. LY309887 or with the topoisomerase I inhibitor irinotecan. MTA The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked was administered in combination with full doses of each anticancer advertisement in accordance with 18 U.S.C. Section 1734 solely to agent studied, with no evidence of increased toxicity resulting from indicate this fact. the combination (10). 1 To whom requests for reprints should be addressed. Phone: (317) 276- In the current studies, MTA was administered alone, in 2739; Fax: (317) 277-6285; E-mail: [email protected]. 2 The abbreviations used are: MTA, LY231514; TS, thymidylate syn- combination with standard chemotherapeutic agents, with a thase; GARFT, glycinamide ribonucleotide formyltransferase; TGD, special focus on scheduling with 5-fluorouracil, gemcitabine, tumor growth delay. and platinum complexes, or with radiation therapy to tumor-

Fig. 1 Growth delay of the human H460 non- small cell lung carcinoma grown as a xenograft in male nude mice after treatment with MTA (100 mg/kg) i.p., days (d) 7–13 or days 7–20; methotrexate (0.8 mg/kg) i.p., days 7–13 or days 7–20; GARFT inhibitor (LY309887; 30 mg/kg) i.p., days 7, 10, and 13 or days 7, 10, 13, 16, and 19; 5-fluorouracil (5-FU;30mg/ kg) i.p., days 7–11 or 16–20 alone or in combinations including an antifolate along with 5-fluorouracil administered on days 7–11 (early) or on days 16–20 (late). Rows, means from two independent experiments; bars, SE.

bearing mice to explore the potential interaction of MTA in When the animals were 7–8 weeks of age, they were exposed to combination anticancer treatment regimens. 4.5 Gy of total body radiation delivered using a GammaCell 40 irradiator (Nordion, Inc., Ottowa, Ontario, Canada). Twenty- MATERIALS AND METHODS four h later, MX-1, Calu-6, or H460 tumor cells (5 ϫ 106) Drugs prepared from a brie of several donor tumors were implanted s.c. in a 1:1 mixture of RPMI tissue culture medium and Ma- MTA and LY309887 (GARFT inhibitor) and gemcitabine trigel (Collaborative Biomedical Products, Inc., Bedford, MA) were obtained from Eli Lilly & Co. (2, 11, 12). Cisplatin, in a hind leg of the animals. MX-1 tumors grew to 500 mm3 in carboplatin, cyclophosphamide, methotrexate, 5-fluorouracil, 34.7 Ϯ 2.9 days, Calu-6 tumors grew to 500 mm3 in 19.0 Ϯ 3.4 paclitaxel, docetaxel, vinorelbine, and doxorubicin were pur- days, and H460 tumors grew to 500 mm3 in 14.0 Ϯ 0.8 days. chased from Sigma Chemical Co. (St. Louis, MO). Oxaliplatin was purchased from Nescott Fine Chemicals (Santiago, Chile). TGD Experiments Tumors H460 Experiments. Treatments were initiated on day 7 The Calu-6 human non-small cell lung adenocarcinoma post-tumor cell implantation, when the H460 tumors were ap- originated from a 61-year old female treated with radiation proximately 200 mm3 in volume. Animals were treated by with therapy in 1976 (13). The MX-1 breast carcinoma originated as MTA (100 mg/kg) i.p. injection on days 7–13 or days 7–20; a poorly differentiated mammary carcinoma in a 29-year old with methotrexate (0.8 mg/kg) by i.p. injection on days 7–13 or female. Calu-6 cells were purchased from American Type Cul- days 7–20; or with LY309887 (30 mg/kg) by i.p. injection on ture Collection (Manassas, VA). The MX-1 breast carcinoma days 7, 10, and 13 or days 7, 10, 13, 16, and 19 alone or along and H460 human non-small cell lung carcinoma were obtained with 5-fluorouracil (30 mg/kg) by i.p. injection on days 7–11 or from the National Cancer Institute-Fredrick Cancer Research 16–20. In another experiment, MTA (100 or 150 mg/kg) was Facility, Division of Cancer Treatment Tumor Repository. Each administered by i.p. injection on days 7–11 and 14–18, on days of the tumor cell lines is tumorigenic in nude mice. 16–22, or on days 16–30 alone or in combination with gemcit- Nude mice, male and female, were purchased from Charles abine (60 mg/kg) by i.p. injection on days 7, 10, 13, and 16. In River Laboratories (Wilmington, MA) at 5–6 weeks of age. a third experiment, MTA (100 mg/kg) was administered by i.p.

Fig. 2 Growth delay of the human H460 non- small cell lung carcinoma grown as a xenograft in male nude mice after treatment with MTA (100 or 150 mg/kg) i.p., days (d) 7–11 and 14–18, days 16–22, or days 16–30; gemcitabine (GEM; 60 mg/kg) i.p., days 7, 10, 13, and 16 alone or in simultaneous or sequential combinations. Rows, means from two independent experiments; bars, SE.

injection on days 7–11 and 14–18 alone or along with oxali- MTA (150 mg/kg) administered by i.p. injection on days 7–11, platin (12.5 mg/kg) by i.p. injection on day 7; oxaliplatin with methotrexate (0.8 mg/kg) administered by i.p. injection on (5 mg/kg) by i.p. injection on days 7 and 14; cisplatin (10 days 7–11, with 5-fluorouracil (30 mg/kg) administered by i.p. mg/kg) by i.p. injection on day 7; docetaxel (22 mg/kg) by i.v. injection on days 7–11, or with combinations of these agents injection on days 8, 12, and 16; paclitaxel (24 mg/kg) by i.v. administered simultaneously or sequentially, with 6 h between injection on days 8, 10, 12, and 15; vinorelbine (10 mg/kg) by drug injections. In another experiment, MTA (100, 150, or 200 i.p. injection on day 8; or carboplatin (50 mg/kg) by i.p. injec- mg/kg) was administered by i.p. injection on days 7–11 alone or tion on day 8. In the fourth experiment, MTA (100 mg/kg) was along with paclitaxel (24 mg/kg) administered by i.v. injection administered by i.p. injection on days 7–11 and 14–18 or days on days 7, 9, 11, and 13 or along with doxorubicin (1.75 mg/kg) 8, 11, 14, and 17 alone or along with fractionated radiation administered by i.p. injection on days 7–11. therapy (2, 3, or 4 Gy; GammaCell 40, Nordion Inc., Ottawa, The progress of each tumor was measured twice per week Ontario, Canada) delivered on days 7–11 and 14–18. until it reached a volume of 4000 mm3. Tumor volumes were Calu-6 Experiments. Treatments were initiated on day calculated as the volume of a hemi-ellipsoid based on tumor diam- 7 post-tumor cell implantation, when the Calu-6 tumors were eter measurements made using calipers in two dimensions. TGD approximately 200 mm3 in volume. Animals were treated was calculated as the time taken by each individual tumor to reach with MTA (100 mg/kg) administered by i.p. injection on 500 mm3 compared with the time in the untreated controls. Each days 7–11 and days 14–18 alone or along with cisplatin (10 treatment group included 5 animals, and each experiment was done mg/kg) by i.p. injection on day 7; cyclophosphamide (125 twice; therefore the number of animals per condition was 10. TGD mg/kg) by i.p. injection on days 7, 9, and 11; gemcitabine times (days) are the means Ϯ SE for the treatment group compared (60 mg/kg) by i.p. injection on days 7, 10, 13, and 16; with those for the control group (14, 15). Toxicity of the treatment doxorubicin (1.75 mg/kg) by i.p. injection on days 7–11; regimens was assessed using change in body weight over the oxaliplatin (12.5 mg/kg) by i.p. injection on day 7; or oxali- course of the experiments. Body weights were measured twice per platin (5 mg/kg) by i.p. injection on days 7 and 14. week at the same time as tumor diameter measurements. MX-1 Experiments. Treatments were initiated on day 7 All in vivo studies were performed in accordance with NIH post-tumor cell implantation, when the MX-1 tumors were ap- and American Accreditation Association of Laboratory Animal proximately 50 mm3 in volume. Animals were treated with Care guidelines.

Table 1 Growth delay of the human H460 non-small cell lung carcinoma grown as a xenograft in male nude mice after treatment with MTA in combination with other cytotoxic anticancer therapies Tumor growth delay (days) Maximum body weight loss (%) Alone ϩMTA Alone ϩMTA MTA (100 mg/kg) d7–11; 14–18a 3.2 Ϯ 0.3 8.0 Cisplatin (10 mg/kg) d7 4.0 Ϯ 0.3 12.8 Ϯ 1.1 12.0 17.0 Carboplatin (50 mg/kg) d7 2.9 Ϯ 0.3 6.6 Ϯ 0.5 20.0 30.0 Oxaliplatin (12.5 mg/kg) d7 5.2 Ϯ 0.4 6.5 Ϯ 0.6 8.0 15.0 Oxaliplatin (5 mg/kg) d7; 14 7.7 Ϯ 0.7 7.9 Ϯ 1.0 13.0 19.5 Paclitaxel (24 mg/kg) d8, 10, 12, 15 10.8 Ϯ 1.1 14.0 Ϯ 1.3 13.0 19.0 Docetaxel (22 mg/kg) d8, 12, 16 7.9 Ϯ 0.9 14.6 Ϯ 1.6 27.0 36.0 Vinorelbine (10 mg/kg) d7 5.9 Ϯ 0.5 7.6 Ϯ 0.8 8.0 21.5 2Gyϫ 10 10.7 Ϯ 0.6 12.3 Ϯ 0.8 16.0 17.0 3Gyϫ 10 13.9 Ϯ 1.0 17.8 Ϯ 1.4 16.0 17.0 4Gyϫ 10 23.8 Ϯ 1.9 26.7 Ϯ 2.2 16.0 17.0 a d, day(s).

Fig. 3 Growth delay of the human Calu-6 non-small cell lung carcinoma xenograft grown in female nude mice after treatment with MTA (100 mg/kg) i.p., days (d) 7–11 and 14–18; cisplatin (10 mg/kg) i.p., day 7; cyclophosphamide (CTX; 125 mg/kg) i.p., days 7, 9, and 11; gemcitabine (60 mg/kg) i.p., days 7, 10, 13, and 16; doxorubicin (1.75 mg/kg) i.p., days 7–11; oxaliplatin (12.5 mg/kg) i.p., day 7, or (5 mg/kg) i.p., days 7 and 14, alone or in combinations including MTA. Rows, means of two independent experiments; bars, SE.

Data Analysis Statistical comparisons for the TGD assays were carried For determination of additivity, isobolograms were gener- out with the Dunnett multiple comparisons test after a signifi- ated for the special case in which the dosage of one agent is held cant effect was found by ANOVA (21, 22). constant. This method allowed determination of additive effect for different levels of the variable agent (16–20). The radiation RESULTS dose-modifying factor was calculated as the ratio of the radia- The doses and schedules for the standard chemotherapeutic tion dose required to produce 20 days of TGD in the treated and agents in these studies are standard, widely used regimens for control groups. each agent. The doses and schedules used for MTA in these

Fig. 4 Growth delay of the human MX-1 breast carcinoma xenograft grown in female nude mice after treatment with 5-fluorouracil (5-FU;30mg/ kg) i.p., days (d) 7–11; MTA (150 mg/kg) i.p., days 7–11; or methotrexate (MTX; 0.8 mg/kg) i.p., days 7–11. The antifolate (MTA or MTX) was administered simultaneously with each dose of 5-fluorouracil, or the antifolate was administered 6 h prior to each dose of 5-fluorouracil. Columns, means of two independent experiments; bars, SE.

studies were determined to be optimal for MTA in earlier more toxic regimen as determined by weight loss, especially studies (1, 6–8). The human H460 non-small cell lung carci- with the higher dose of MTA. noma xenograft is a relatively quickly growing tumor undergo- Antitumor platinum complexes and antitubulin agents are ing log-linear growth and reaching a volume of about 4000 mm3 widely used in the treatment of non-small cell lung carcinoma. in 36 days post-tumor cell (5 ϫ 106) implantation s.c. in a hind Combination regimens with MTA and cisplatin, carboplatin, leg of male nude mice. Each of the antifolates (MTA, metho- and oxaliplatin were studied. The antitumor platinum complexes trexate, and 309887) produced a duration-dependent TGD in the cisplatin and carboplatin were administered once on the first day H460 lung carcinoma (Fig. 1). 5-Fluorouracil was also an active of the treatment regimen, and the antitumor platinum complex, antitumor agent against the H460 tumor-producing TGDs that oxaliplatin, was administered once or once per week for 2 were dependent upon the tumor burden at the initiation of weeks. Although, among the antitumor platinum complexes treatment. For combination regimens, 5-fluorouracil was admin- tested, oxaliplatin produced the greatest TGD in the H460 istered on days 7–11 along with each antifolate on days 7–13, or xenograft, the combination regimen of MTA and cisplatin pro- 5-fluorouracil was administered on days 16–20 along with each duced the greatest TGD (P Ͻ 0.01; Table 1). Paclitaxel was a antifolate in the longer regimen on days 7–20. The most effec- more effective single agent than docetaxel or vinorelbine against tive regimens were the combination of the longer course of the H460 tumor. The combination regimen of MTA with simul- MTA treatment along with 5-florouracil administration on days taneous administration of paclitaxel or docetaxel produced a 16–20 and the combination of the longer course of 309887 greater TGD than the combination of MTA with vinorelbine. treatment along with 5-fluorouracil administration on days 16– Each of the combination regimens was more toxic than the 20. The least effective combination regimens were those that single-agent treatments. Cisplatin and oxaliplatin were less toxic included methotrexate. Administration of 5-fluorouracil early in in combination with MTA than was carboplatin, and paclitaxel these combination regimens resulted in greater weight loss than was less toxic in combination with MTA than was docetaxel. administering 5-fluorouracil later. Radiation therapy is also widely used for the treatment of The antitumor activity of MTA against the H460 tumor is non-small cell lung carcinoma. Radiation therapy was delivered dependent upon dose, duration, and tumor burden. In a daily for locally to the H460 xenograft tumor-bearing limb in fractions of 5 days regimen given for 2 weeks, the higher dose of 150 mg/kg 2, 3, or 4 Gy administered daily for 5 days for 2 weeks. per dose was more effective than the lower dose of 100 mg/kg Radiation therapy produced increasing TGD with increasing per dose (Fig. 2). Gemcitabine, administered every 3rd day for dose of radiation (Table 1). The effect of adding treatment with four doses, was also an active antitumor agent against the H460 MTA to fractionated radiation therapy was investigated. MTA tumor. The simultaneous combination of MTA administered on administration enhanced the TGD produced by radiation therapy the weekly schedule with administration of gemcitabine resulted and did not add to the toxicity of the radiation therapy. in greater-than-additive TGD of the H460 tumor. However, The Calu-6 non-small cell lung carcinoma was grown as a beginning administration of MTA at the completion of the s.c. xenograft in female nude mice. MTA was an active antitu- gemcitabine regimen resulted in a less effective therapeutic mor agent against the Calu-6 tumor. Combinations of MTA with regimen. Simultaneous administration of MTA and gemcita- the antitumor platinum complexes cisplatin and oxaliplatin were bine, although a more effective anticancer regimen, was also a tested, with the antitumor platinum complex being administered

Fig. 5 Growth delay of the human MX-1 breast carcinoma xenograft grown in female nude mice after treatment with MTA (100, 150, or 200 mg/kg) i.p., days (d) 7–11; paclitaxel (PAC; 24 mg/kg) i.v., days 7, 9, 11, and 13; doxorubicin (DOX; 1.75 mg/kg) i.p., days 7–11, alone or in combinations including MTA. Rows, means of two independent experiments; bars, sSE.

simultaneously at the beginning of the MTA treatment course tered by i.p. injection on days 7–11 produced a TGD of 7.5 Ϯ (Fig. 3). Treatment regimens including MTA with either cispla- 0.5 days in the MX-1 tumor (Fig. 4). The simultaneous combi- tin or oxaliplatin were highly effective, producing greater-than- nation of methotrexate and 5-fluorouracil resulted in antagonism additive TGD of the Calu-6 tumor (P Ͻ 0.01 for both cisplatin between the two agents, whereas the simultaneous combination and oxaliplatin combinations). The antitumor alkylating agent of MTA and 5-fluorouracil produced additive TGD in the MX-1 cyclophosphamide was a highly effective antitumor agent tumor. When the administration of methotrexate preceded 5-fluo- against the Calu-6 tumor. The simultaneous combination of rouracil treatment, an additive TGD for the combination was MTA treatment and cyclophosphamide was additive against the observed. On the other hand, when the administration of MTA Calu-6 tumor. Gemcitabine was also an active single agent preceded 5-fluorouracil treatment, a greater-than-additive TGD against the Calu-6 tumor. The simultaneous combination of resulted (P Ͻ 0.01). The combination treatment regimen of MTA and gemcitabine was additive in TGD. Doxorubicin was MTA followed by 5-flurorouracil was most effective in maxi- an active single antitumor agent against the Calu-6 tumor. mizing tumor response and produced less weight loss than the Combination regimens, including MTA with doxorubicin, were other combination regimens; thus, this sequential regimen had a highly effective treatments against the Calu-6 tumor. Mice bearing the Calu-6 tumor were less prone to weight loss by the better therapeutic index than the other combination treatments. combination regimens than mice bearing the H460 tumor; how- Administration of MTA over a dosage range resulted in ever, in each case, the combination regimens were more toxic increasing TGD with increasing dose of MTA in the MX-1 than the single-agent therapies. tumor (Fig. 5). Paclitaxel is a very effective single agent against The MX-1 human breast carcinoma was grown as a s.c. the MX-1 tumor. The simultaneous combination of MTA and xenograft in female nude mice. MTA (150 mg/kg) administered paclitaxel resulted in additive TGD of the two antitumor agents by i.p. injection on days 7–11 produced a TGD of 3.0 Ϯ 0.3 over the dosage range of MTA. Doxorubicin was an active days in the MX-1 tumor. Methotrexate (0.8 mg/kg) administered antitumor agent against the MX-1 tumor. The simultaneous by i.p. injection on days 7–11 produced a TGD of 2.8 Ϯ 0.3 combination of MTA and doxorubicin resulted in additive TGD days in the MX-1 tumor. 5-Fluorouracil (30 mg/kg) adminis- of the two agents, with increasing TGD with increasing dose of

MTA. There was increasing weight loss in the paclitaxel and MTA was given along with gemcitabine than if gemcitabine was doxorubicin regimens as the dose of MTA was increased. administered prior to MTA (Fig. 2). Gemcitabine has been shown to be a potent radiation sensitizer both in vitro and in vivo (30–36), whereas in the DISCUSSION human H460 non-small cell lung carcinoma xenograft, MTA The folate pathway continues to be a target for anticancer was additive with radiation (Table 1). MTA has been tested drug development because it is vital to cell survival and appears previously in combination with radiation therapy against the to be one of the few aspects of cellular metabolism in which human HCT116 colon carcinoma xenograft and found to have there is little or no redundancy. A major challenge in the clinical an additive effect with radiation in that tumor (10). MTA also application of antifolates comes in establishing a therapeutic had a primarily additive effective in combination with fraction- index for these agents in tumor cells versus sensitive normal ated radiation therapy against the human H460 non-small cell tissues such as the bone marrow. One possibility for increasing lung carcinoma. The limitation of the application of this finding therapeutic potential is to develop treatment regimens in which to clinical trial may be the scheduling of the MTA administra- the antifolate is used along with another anticancer agent, re- tion along with the radiation therapy, because MTA was admin- sulting in a greater effect on the tumor cells than on the normal istered prior to each radiation fraction. tissues. The schedule dependence of in vivo treatment combi- Combination of MTA with each of the antitumor plati- nations of methotrexate and 5-fluorouracil were recognized in num complexes (cisplatin, carboplatin, and oxaliplatin) re- the 1970s using the murine C3H mammary adenocarcinoma, sulted in additive to greater-than-additive tumor response in where administering the methotrexate 6 h prior to 5-fluorouracil both the H460 and the Calu-6 non-small cell lung carcinoma resulted in the greatest tumor response (23). These early studies xenografts. In these regimens, the antitumor platinum com- were confirmed and extended to murine colon 38 and several plex was administration as a single dose along with the first human colon carcinoma xenografts, where it was determined dose of MTA (Table 1 and Fig. 3). It also appears that with that administration of the antifolate 4–8 h prior to the 5- careful scheduling, MTA can be highly effective when ad- fluorouracil maximized tumor cell killing without increasing ministered along with antitubulin agents, including paclitaxel bone marrow toxicity from that of the antifolate alone (24–26). and docetaxel. Although many of the combination regimens In the human MX-1 breast carcinoma xenograft, the same pat- produced greater weight loss in the animals than did the tern pertained whether the antifolate was methotrexate or MTA; single agents, especially in combinations with 5-fluorouracil, however, the increased tumor response obtained when MTA increased antitumor activity appeared more important than the weight loss. preceded 5-fluorouracil was much greater than that obtained From these preclinical in vivo results, it may be concluded with methotrexate. The pharmacological interaction between that MTA can be combined with other anticancer therapies to 5-fluorouracil and an antifolate has been the subject of many therapeutic advantage. Given the prolonged terminal half-life in studies (27). Two biochemical mechanisms have been put for- patients, the scheduling of MTA with other agents could possi- ward to account for the observed additivity. The first relates to bly be adjusted to sequential days without loss of the beneficial increased 5-fluorouracil metabolism to the 5-fluoro-UMP therapeutic interaction between the agents. through condensation with phosphoribosyl PPi, a metabolite that accumulates following the inhibition of de novo purine biosynthesis. The second relates to enhanced TS inhibition through REFERENCES formation of a ternary complex consisting of TS, 5-fluoro- 1. Shih, C., and Thornton, D. E. Preclinical pharmacology studies and dUMP and an antifolate. 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Beverly A. Teicher, Victor Chen, Chuan Shih, et al.

Clin Cancer Res 2000;6:1016-1023.

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