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Timing- and sequence-dependent synergism between etoposide and methotrexate or etoposide, methotrexate and 5-fluorouracil in advanced leukemia L1210
Cancer Letters, 29 (1985) 277-282 Elsevier Scientific Publishers Ireland Ltd.
277
TIMING AND SEQUENCE-DEPENDENT SYNERGISM BETWEEN ETOPOSIDE AND METHOTREXATE OR ETOPOSIDE, METHOTREXATE AND 5-FLUOROURACIL IN ADVANCED LEUKEMIA L1210*
H. OSSWALD”, R. HERRMANNb
and M. YOUSSEFa
aInstitute of Toxicology and Chemothempy, German Cancer Research Center, Im Neuenheimer Feld 280. D-6900 Heidelberg and bMedizinische Klinik, 1000 Berlin, Charlottenburg (F.R.G.) (Received 8 August 1985) (Accepted 3 October 1985)
SUMMARY
The optimal synergism in combination therapy of leukemia L1210 depends on the sequence and the timing of the agents used. Etoposide (20 mg/kg applied intravenously) 3 h before methotrexate (50 mg/kg administered subcutaneously) results in significantly improved therapeutic action. Simultaneous application of these drugs, or an exceeding of that interval, do not entail synergism. Similar to the results obtained with other transplantable murine tumors, the optimal interval between methotrexate and 5-fluorouracil treatments of leukemia L1210 amounts to 6 h. Sequential treatment with etoposide (20 mg/kg given intravenously) significantly improves the efficacy of combined methotrexate (50 mg/kg applied subcutaneously) and 5fluorouracil (80 mg/kg administered subcutaneously). The optimal synergism combining etoposide, methotrexate and fi-fluorouracil is achieved when the interval between etoposide and methotrexate amounts to 3 h followed 6 h later by 5-fluorouracil.
INTRODUCTION
Methotrexate (MTX) attracts continuing interest in respect to improving its chemotherapeutic action concerning membrane transport [ 1,2,9,17, 20,211, polyglutamination [7,9,10;11,18,19,21] or its affinity to dihydrofolate reductase [ 161. The augmentation of the MTX transport and polyglutamination by the semi-synthetic podophyllotoxin derivatives teniposide
*Dedicated to Professor Dr. Dietrich Schmiihl on the occasion of his 60th birthday. 0304-3635/85/$03.30 01985 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
278
(VM) or etoposide (VP) in vitro [ 211, motivated our in vivo experiments with advanced leukemia L1210. Therefore we tested the combination of VP with MTX in relation to the sequence and the interval aiming at a chemotherapeutic synergism. Moreover, it seemed of interest to question the possibility of chemotherapeutic synergism between VP and the sequential MTX 5flurorouracil (FU) combination [2]. The mechanism of action of that combination of drugs is still under discussion [3,4,8] . The MTX-FU combination attracts interest in the clinical treatment of mammary carcinoma resistant to conventional combinations [ 131; it also improves the response rate in metastatic colorectal cancer [14] when the interval between the sequential administration of MTX and FU exceeds 4 h. MATERIALS
AND METHODS
Female SPF B6D2F1mice (Charles River, Wiga, Sulzbach, F.R.G.) weighing 24-26 g, were housed in groups of 10 in Makrolon cages (type III). The animals received Altromin N pellets and water ad libitum. The mice were kept in a barrier room at 26°C and humidity of 60 f 5% with 8 changes of air/h. The parent line of L1210 leukemia was maintained in DBA/2 mice by transplanting 10s a&tic cells with transfers of 5 days. L1210 as&tic cells were inoculated intraperitoneally to the experimental groups at a dose of lo5 cells/BBDzF1 mouse. The onset of treatment was at the fourth day after implantation, and lasted for 2 weeks. Drug-treated groups and leukemic controls received saline solutions. The animals were observed daily and the dead mice were autopsied to determine the presence of ascites and hepatosplenomegaly or other pathologic changes of their organs. The treatment began at the 5th day after the tumor transplantation and lasted for 2 weeks. The antineoplastic agents were given at single doses of the individual weights of the mice (0.1 ml for 10 g body wt). The dilution of the ampouled etoposide solutions in saline needed particular precautions because of the limited stability of the drug at the concentrations used. The dilution of the etoposide ampoule solution began immediately before the intravenous injection. The stability of the prepared etoposide saline lasts for 12-14 min, a sufficient time to inject 10 mice intravenously. Our experience showed that diluted etoposide solutions lose their chemotherapeutic activity after 15-20 mm. After 20-30 min the salinediluted etoposide became turbid and separated a light suspension. Etoposide (Vepesid@ ) and methotrexate ampoules for clinical use were provided by Bristol-Myers (Neu-Isenburg, F.R.G.). 5-Fluorouracil substance was supplied by Mack (Illertissen, F.R.G.). The chemotherapeutic effect of the drugs or their combinations were determined by the increase of life span in percent (ILS) and by the mean survival time in days (MST). The differences of the chemotherapeutic action between the treated groups were calculated by the rank sum test at a significance level of P > 0.001.
279
RESULTS
Our experiments with etoposide indicated a time-dependent lability of the diluted solutions of the VP ampoules and a decrease of the chemotherapeutic action. Our experience showed that the diluted ampouled agent should be used as soon as possible, but not after 12 min. The results are shown in Table 1. Due to the advanced stage of the leukemia L1210 a moderate tumor inhibition was observed by single doses of VP, MTX or FU, whereas the combined drugs gamed a synergism if the optimal sequence and interval were used. The simultaneous combination of VP and MTX possessed no additive chemotherapeutic effect (group 8). An interval of 3h (group 9) between VP and MTX induced a significant synergism, com-
TABLE 1 INFLUENCE OF THE SEQUENCE AND THE INTERVAL ON ETOPOSIDE METHOTREXATE OR ETOPOSIDE METHOTREXATE 5-FLUOROURACIL COMBINATIONS IN LEUKEMIA L1210 Group
Increase of life span in % (ILS)
Scheme of treatment (single doses per week) Interval (h)
Drug
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Control group MTX 50 mg/kg MTX 7 5 mg/kg FU 80 n&kg FU 120 mg/kg VP 20 mg/kg VP 30 mg/kg VP 20 mg/kg VP 20 mg/kg VP 20 mg/kg MTX 50 mg/kg MTX 50 mg/kg MTX 50 mg/kg MTX 50 mg/kg VP 20 mg/kg
16
VP
Drug
S.C. S.C. S.C. B.C. i.v. i.v. i.v. i.v. i.v. S.C. S.C. B.C. B.C. i.v.
20 mg/kg i.v.
3
MTX 50 mg/kg MTX 50 mgFg MTX 50 mg/kg VP 20 mg/kg VP 20 mg/kg FU 80 mg/kg FU 80 mg/kg MTX 50 mg/kg after 3 h FU 80 mg/kg MTX 50 mg/kg after 6 h FU 80 mg/kg
Mean survival time (MST, days ? S.D.)
s.c. s.c. S.C. i.v. i.v. S.C. S.C. S.C.
45 58 38 42 50 66 65 136 81 41 83 76 91 180
8.6 2 0.51 13.2 ? 1.31 13.6 + 1.07 11.9 + 1.59 12.2 + 1.55 12.9 + 1.91 14.3 ? 1.64 14.2 ? 2.35 20.3 f. 1.49 15.6 ? 2.46 12.1 + 1.66 15.7 + 2.41 15.1 ? 2.23 16.4 + 2.76 24.1 + 2.96
S.C. S.C.
212
26.8
f. 3.61
S.C.
10’ cells i.p. Onset of treatment: 5th day after transplantation. Duration of treatment: 2 weeks. Comparison of the differences of the chemotherapeutic resuts between the treated groups by the log rank sum test (significance level P > 0.001). Group 8 vs. group 9, P = 0.0002;group 13 vs. group 15, P= 0.00002;group 14 vs. group 16, P = 0.00002.
280
pared to the same sequence with an interval of 6 h (group 10) which did not meet any additive effect. A change in the used sequence (MTX prior to VP) resulted in a chemotherapeutic antagonism when the interval amounted to 3 h (group 11). The interval of 6 h between MTX and VP did not entirely fulfil the additive efficacy of these drugs (group 12). The sequential combination of MTX followed by FU 3 h later (group 13) does not act additively. However, an interval of 6 h after MTX leads to an additive chemotherapeutic effect (group 14). The sequential treatment with VP, MTX and FU applied at 3-h intervals achieved an overadditive tumor inhibition (group 15). Moreover, the sequential therapy with VP followed after 3 h with MTX and with FU after an additional 6 h resulted in a chemotherapeutic synergism in leukemia L1210. DISCUSSION
Vincristine stimulates net transport of MTX [ 5,6,12] in mammalian cells. One of the possible causes of the chemotherapeutic synergism between vincristine and MTX in L1210 leukemia-bearing mice is assumed to be the association of enhanced net MTX transport with augmented MTX inhibition of thymidylate biosynthesis [ 211. The rapidly formed MTX polyglutamates [8,10,11] in cells, which decrease the reversibility of the cytotoxic effect of MTX, should be the most important determinant of the antifolate action. The MTX polyglutamates are retained within the cells, particularly their longer chain forms [ 111. Marked differences have been observed in the accumulation of MTX polyglutamate derivatives between MTXaensitive tumor cells and the murine bone marrow granulocytic progenitor cells [ 71 or normal intestinal cells [ 5,9]. The longer retention of MTX polyglutamates within tumor cells [ 201 have been recognised as an important factor in MTX selectivity. Vincristine markedly blocks the energydependent MTX efflux by enhancing the exchangeable antifolate level within the cells and the formation of MTX polyglutamates in vitro [ 111. Unlike the vinca alkaloids, VP and VM do not influence microtubule assembly and function; one of their effects may be a premitotic block of the cell cycle [15]. However, probenecid can also block the MTX &flux and thereby augment MTX polyglutamate formation [ 11 J . In vitro VP and VM stimulated MTX transport and polyglutamate formation when these epipodophylltoxin derivatives were applied 5 min before adding MTX to cultures of Ehrlichcarcinoma cells [21]. When VM was removed [21] prior to the exposure to MTX, the stimulatory effect of VM on net MTX transport was eliminated indicating the reversibility of that effect. Our in vivo experiments show a marked synergism between VP and MTX in L1210 leukemia-bearing mice when the interval between the treatments amounts to 3 h.
281
There seems to be probably another mechanism of action in combining VP and MTX in vivo and in vitro but the in vitro experiments do not exceed 60 min. The results in vivo point at importance of the interval between BP and MTX. Also the sequential combinations of MTX and FU obtain a synergism when VP is applied 3 h before MTX. Further experiments are ongoing combining VP and FU. REFERENCES 1 Bender, R.A., Bleyer, W.A., Frisby, S.A. and Oliverio, V.T. (1975) Alteration of methotrexate uptake in human leukemia cells by other agents. Cancer Res., 35, 1305-1308. 2 Bertino, J.R., Sawicki, W.L., Lundquist. C.A. and Gupta, V.S. (1977) Scheduledependent antitumor effects of methotrexate and 5fluorouracil. Cancer Res., 37, 327-328. 3 Browman, G.P. (1984) Clinical application of the concept of methotrexate plus 5-FU sequence dependent “synergy”: How good is the evidence; Cancer Treat. Rep., 68, 466-469. 4 Cadman, E., Heimer, R. and Davis, L. (1979) Enhanced 5-fluorouracil nucleotide formation after methotrexate administration: explanation for drug synergism. Science (Wash. D.C.) 205, 1135-1137. 5 Chello, P.L., Sirotnak, F.M. and Dorick, D.M. (1979) Different effects of vincristine on methotrexate uptake by L1210 cells and mouse intestinal epithelia in vitro and in vivo. Cancer Res., 39,.2106-2112. 6 Chello, P.L., Sirotnak, F.M., Dorick, D.M. and Moccio, D.M. (1979) Scheduledependent synergism of methotrexate and vincristine against murine L1210 leukemia. Cancer Treat. Rep., 63,1889-1894. 7 Fabre, J., Fabre, G. and Goldman, J.D. (1984) Polyglutamation, an important element in methotrexate cytotoxicity and selectivity in tumor versus murine granulocitic progenitor. Cancer Res., 44,3190-3195. 8 Fernandes, D.J. and Bertino, J.R. (1980) 5-Fluorouracil methotrexate synergy: enhancement of 5fluorodeoxyuridylate binding to thymidylate synthetase by dihydropteroylpolyglutamates. Pmt. No. 72. Acad. Sci. U.S.A., 77, 5663-5667. 9 Fry, D.W., Anderson, L.A., Borst, M. and Goldman, J.D. (1983) Analysis of the role of membrane transport and polyglutamation of methotrexate in gut and the Ehrlich tumor in vivo as factors in drug sensitivity and selectivity. Cancer Res., 43, 10871092. 10 Fry, D.W., Yalowich, J.C. and Goldman, J.D. (1982) Rapid formation of polyglutamyl derivatives of methotrexate and their association with dihydrofolate reductase as assessed by high pressure liquid chromatography in the Ehrlich ascites tumor cell in vitro. J. Biol. Chem., 257,1890-1896. 11 Fry, D.W., Yalowich, J.C. and Goldman, I.D. (1982) Augmentation of the intracellular levels of polyghrtamyl derivatives of methotrexate by vincristine and probenecid in Ehrlich ascites tumor cells. Cancer Res., 42, 2532-2536. 12 Fyfe, M.J. and Goldman, I.D. (1973) Characteristics of the vincristine-induced augmentation of methotrexate uptake in Ehrlich ascites tumor cells. J. Biol. Chem., 248,5067-5073. 13 Herrmann. R., Manegold, C., Schroeder, M., Tlgges, F.-J., Bar&h, H., Jungi, F. and Fritze, D. (1984) Sequential methotrexate and 5-FU in breast cancer resistant to the conventional application of the drugs. Cancer Treat. Rep., 68, 1279-1281. 14 Herrmann, R., Spehn, J., Beyer, J.H., von Franque, U., Schmieder, A., Holzmann, K. and Abel, U. (1984) Sequential methotrexate and 5-fluorouracil: improved response rate in metastatic colorectal cancer. J. Clin. Oncol., 2, 591-594.
282 15 Issel, B.F. and Crooke, S.T. (1979) Etoposide (VP-16-213). Cancer Treat. Rev., 6, 107-124. 16 Matherly, L.H., Anderson, L.A. and Goldman, I.D. (1984) Role of the cellular oxidation-reduction state in methotrexate binding to dihydrofolate reductase and dissociation induced by reduced folates. Cancer Res., 44,2325-2330. 17 Moccio, D.M., Sirotnak, F.M., Samuels, L.L., Ahmed, T., Yagoda, A., DeGraw, J.I. and Piper, J.R. (1984) Similar specificity of membrane transport for folate analogues and their metabolites by murine and human tumor cells: a clinically directed study. Cancer Res., 44,352-357. 18 Rosenblatt, D.S., Whitehead, V.M., Vera, N., Pottier, A., DuPont, M. and Vuchich, M.J. (1978) Prolonged inhibition of DNA synthesis associated with the accumulation of methotrexate polyglutamates by cultured human cells Mol. Pharmacol., 14,11431147. 19 Schilsky, R.L., Bailey, B.D. and Chabner, B.A. (1980) Methotrexate polyglutamate synthesis by cultured human breast cancer cells. Proc. Natl. Acad. Sci. U.S.A., 77, 2919-2922. 20 Sirotnak, F.M. (1980) Correlates of folate analog transport, pharmacokinetics, and selective antitumor action. Pbarmacol. Ther., 8,71-103. 21 Yalowich, J.C., Fry, D.W. and Goldman, I.D. (1982) Teniposide (VM-26) and etoposide (VP-16-213)-induced augmentation of methotrexate transport and polyglutamylation in Ehrlich ascites cells in vitro. Cancer Res., 42, 3648-3653.
277
TIMING AND SEQUENCE-DEPENDENT SYNERGISM BETWEEN ETOPOSIDE AND METHOTREXATE OR ETOPOSIDE, METHOTREXATE AND 5-FLUOROURACIL IN ADVANCED LEUKEMIA L1210*
H. OSSWALD”, R. HERRMANNb
and M. YOUSSEFa
aInstitute of Toxicology and Chemothempy, German Cancer Research Center, Im Neuenheimer Feld 280. D-6900 Heidelberg and bMedizinische Klinik, 1000 Berlin, Charlottenburg (F.R.G.) (Received 8 August 1985) (Accepted 3 October 1985)
SUMMARY
The optimal synergism in combination therapy of leukemia L1210 depends on the sequence and the timing of the agents used. Etoposide (20 mg/kg applied intravenously) 3 h before methotrexate (50 mg/kg administered subcutaneously) results in significantly improved therapeutic action. Simultaneous application of these drugs, or an exceeding of that interval, do not entail synergism. Similar to the results obtained with other transplantable murine tumors, the optimal interval between methotrexate and 5-fluorouracil treatments of leukemia L1210 amounts to 6 h. Sequential treatment with etoposide (20 mg/kg given intravenously) significantly improves the efficacy of combined methotrexate (50 mg/kg applied subcutaneously) and 5fluorouracil (80 mg/kg administered subcutaneously). The optimal synergism combining etoposide, methotrexate and fi-fluorouracil is achieved when the interval between etoposide and methotrexate amounts to 3 h followed 6 h later by 5-fluorouracil.
INTRODUCTION
Methotrexate (MTX) attracts continuing interest in respect to improving its chemotherapeutic action concerning membrane transport [ 1,2,9,17, 20,211, polyglutamination [7,9,10;11,18,19,21] or its affinity to dihydrofolate reductase [ 161. The augmentation of the MTX transport and polyglutamination by the semi-synthetic podophyllotoxin derivatives teniposide
*Dedicated to Professor Dr. Dietrich Schmiihl on the occasion of his 60th birthday. 0304-3635/85/$03.30 01985 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
278
(VM) or etoposide (VP) in vitro [ 211, motivated our in vivo experiments with advanced leukemia L1210. Therefore we tested the combination of VP with MTX in relation to the sequence and the interval aiming at a chemotherapeutic synergism. Moreover, it seemed of interest to question the possibility of chemotherapeutic synergism between VP and the sequential MTX 5flurorouracil (FU) combination [2]. The mechanism of action of that combination of drugs is still under discussion [3,4,8] . The MTX-FU combination attracts interest in the clinical treatment of mammary carcinoma resistant to conventional combinations [ 131; it also improves the response rate in metastatic colorectal cancer [14] when the interval between the sequential administration of MTX and FU exceeds 4 h. MATERIALS
AND METHODS
Female SPF B6D2F1mice (Charles River, Wiga, Sulzbach, F.R.G.) weighing 24-26 g, were housed in groups of 10 in Makrolon cages (type III). The animals received Altromin N pellets and water ad libitum. The mice were kept in a barrier room at 26°C and humidity of 60 f 5% with 8 changes of air/h. The parent line of L1210 leukemia was maintained in DBA/2 mice by transplanting 10s a&tic cells with transfers of 5 days. L1210 as&tic cells were inoculated intraperitoneally to the experimental groups at a dose of lo5 cells/BBDzF1 mouse. The onset of treatment was at the fourth day after implantation, and lasted for 2 weeks. Drug-treated groups and leukemic controls received saline solutions. The animals were observed daily and the dead mice were autopsied to determine the presence of ascites and hepatosplenomegaly or other pathologic changes of their organs. The treatment began at the 5th day after the tumor transplantation and lasted for 2 weeks. The antineoplastic agents were given at single doses of the individual weights of the mice (0.1 ml for 10 g body wt). The dilution of the ampouled etoposide solutions in saline needed particular precautions because of the limited stability of the drug at the concentrations used. The dilution of the etoposide ampoule solution began immediately before the intravenous injection. The stability of the prepared etoposide saline lasts for 12-14 min, a sufficient time to inject 10 mice intravenously. Our experience showed that diluted etoposide solutions lose their chemotherapeutic activity after 15-20 mm. After 20-30 min the salinediluted etoposide became turbid and separated a light suspension. Etoposide (Vepesid@ ) and methotrexate ampoules for clinical use were provided by Bristol-Myers (Neu-Isenburg, F.R.G.). 5-Fluorouracil substance was supplied by Mack (Illertissen, F.R.G.). The chemotherapeutic effect of the drugs or their combinations were determined by the increase of life span in percent (ILS) and by the mean survival time in days (MST). The differences of the chemotherapeutic action between the treated groups were calculated by the rank sum test at a significance level of P > 0.001.
279
RESULTS
Our experiments with etoposide indicated a time-dependent lability of the diluted solutions of the VP ampoules and a decrease of the chemotherapeutic action. Our experience showed that the diluted ampouled agent should be used as soon as possible, but not after 12 min. The results are shown in Table 1. Due to the advanced stage of the leukemia L1210 a moderate tumor inhibition was observed by single doses of VP, MTX or FU, whereas the combined drugs gamed a synergism if the optimal sequence and interval were used. The simultaneous combination of VP and MTX possessed no additive chemotherapeutic effect (group 8). An interval of 3h (group 9) between VP and MTX induced a significant synergism, com-
TABLE 1 INFLUENCE OF THE SEQUENCE AND THE INTERVAL ON ETOPOSIDE METHOTREXATE OR ETOPOSIDE METHOTREXATE 5-FLUOROURACIL COMBINATIONS IN LEUKEMIA L1210 Group
Increase of life span in % (ILS)
Scheme of treatment (single doses per week) Interval (h)
Drug
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Control group MTX 50 mg/kg MTX 7 5 mg/kg FU 80 n&kg FU 120 mg/kg VP 20 mg/kg VP 30 mg/kg VP 20 mg/kg VP 20 mg/kg VP 20 mg/kg MTX 50 mg/kg MTX 50 mg/kg MTX 50 mg/kg MTX 50 mg/kg VP 20 mg/kg
16
VP
Drug
S.C. S.C. S.C. B.C. i.v. i.v. i.v. i.v. i.v. S.C. S.C. B.C. B.C. i.v.
20 mg/kg i.v.
3
MTX 50 mg/kg MTX 50 mgFg MTX 50 mg/kg VP 20 mg/kg VP 20 mg/kg FU 80 mg/kg FU 80 mg/kg MTX 50 mg/kg after 3 h FU 80 mg/kg MTX 50 mg/kg after 6 h FU 80 mg/kg
Mean survival time (MST, days ? S.D.)
s.c. s.c. S.C. i.v. i.v. S.C. S.C. S.C.
45 58 38 42 50 66 65 136 81 41 83 76 91 180
8.6 2 0.51 13.2 ? 1.31 13.6 + 1.07 11.9 + 1.59 12.2 + 1.55 12.9 + 1.91 14.3 ? 1.64 14.2 ? 2.35 20.3 f. 1.49 15.6 ? 2.46 12.1 + 1.66 15.7 + 2.41 15.1 ? 2.23 16.4 + 2.76 24.1 + 2.96
S.C. S.C.
212
26.8
f. 3.61
S.C.
10’ cells i.p. Onset of treatment: 5th day after transplantation. Duration of treatment: 2 weeks. Comparison of the differences of the chemotherapeutic resuts between the treated groups by the log rank sum test (significance level P > 0.001). Group 8 vs. group 9, P = 0.0002;group 13 vs. group 15, P= 0.00002;group 14 vs. group 16, P = 0.00002.
280
pared to the same sequence with an interval of 6 h (group 10) which did not meet any additive effect. A change in the used sequence (MTX prior to VP) resulted in a chemotherapeutic antagonism when the interval amounted to 3 h (group 11). The interval of 6 h between MTX and VP did not entirely fulfil the additive efficacy of these drugs (group 12). The sequential combination of MTX followed by FU 3 h later (group 13) does not act additively. However, an interval of 6 h after MTX leads to an additive chemotherapeutic effect (group 14). The sequential treatment with VP, MTX and FU applied at 3-h intervals achieved an overadditive tumor inhibition (group 15). Moreover, the sequential therapy with VP followed after 3 h with MTX and with FU after an additional 6 h resulted in a chemotherapeutic synergism in leukemia L1210. DISCUSSION
Vincristine stimulates net transport of MTX [ 5,6,12] in mammalian cells. One of the possible causes of the chemotherapeutic synergism between vincristine and MTX in L1210 leukemia-bearing mice is assumed to be the association of enhanced net MTX transport with augmented MTX inhibition of thymidylate biosynthesis [ 211. The rapidly formed MTX polyglutamates [8,10,11] in cells, which decrease the reversibility of the cytotoxic effect of MTX, should be the most important determinant of the antifolate action. The MTX polyglutamates are retained within the cells, particularly their longer chain forms [ 111. Marked differences have been observed in the accumulation of MTX polyglutamate derivatives between MTXaensitive tumor cells and the murine bone marrow granulocytic progenitor cells [ 71 or normal intestinal cells [ 5,9]. The longer retention of MTX polyglutamates within tumor cells [ 201 have been recognised as an important factor in MTX selectivity. Vincristine markedly blocks the energydependent MTX efflux by enhancing the exchangeable antifolate level within the cells and the formation of MTX polyglutamates in vitro [ 111. Unlike the vinca alkaloids, VP and VM do not influence microtubule assembly and function; one of their effects may be a premitotic block of the cell cycle [15]. However, probenecid can also block the MTX &flux and thereby augment MTX polyglutamate formation [ 11 J . In vitro VP and VM stimulated MTX transport and polyglutamate formation when these epipodophylltoxin derivatives were applied 5 min before adding MTX to cultures of Ehrlichcarcinoma cells [21]. When VM was removed [21] prior to the exposure to MTX, the stimulatory effect of VM on net MTX transport was eliminated indicating the reversibility of that effect. Our in vivo experiments show a marked synergism between VP and MTX in L1210 leukemia-bearing mice when the interval between the treatments amounts to 3 h.
281
There seems to be probably another mechanism of action in combining VP and MTX in vivo and in vitro but the in vitro experiments do not exceed 60 min. The results in vivo point at importance of the interval between BP and MTX. Also the sequential combinations of MTX and FU obtain a synergism when VP is applied 3 h before MTX. Further experiments are ongoing combining VP and FU. REFERENCES 1 Bender, R.A., Bleyer, W.A., Frisby, S.A. and Oliverio, V.T. (1975) Alteration of methotrexate uptake in human leukemia cells by other agents. Cancer Res., 35, 1305-1308. 2 Bertino, J.R., Sawicki, W.L., Lundquist. C.A. and Gupta, V.S. (1977) Scheduledependent antitumor effects of methotrexate and 5fluorouracil. Cancer Res., 37, 327-328. 3 Browman, G.P. (1984) Clinical application of the concept of methotrexate plus 5-FU sequence dependent “synergy”: How good is the evidence; Cancer Treat. Rep., 68, 466-469. 4 Cadman, E., Heimer, R. and Davis, L. (1979) Enhanced 5-fluorouracil nucleotide formation after methotrexate administration: explanation for drug synergism. Science (Wash. D.C.) 205, 1135-1137. 5 Chello, P.L., Sirotnak, F.M. and Dorick, D.M. (1979) Different effects of vincristine on methotrexate uptake by L1210 cells and mouse intestinal epithelia in vitro and in vivo. Cancer Res., 39,.2106-2112. 6 Chello, P.L., Sirotnak, F.M., Dorick, D.M. and Moccio, D.M. (1979) Scheduledependent synergism of methotrexate and vincristine against murine L1210 leukemia. Cancer Treat. Rep., 63,1889-1894. 7 Fabre, J., Fabre, G. and Goldman, J.D. (1984) Polyglutamation, an important element in methotrexate cytotoxicity and selectivity in tumor versus murine granulocitic progenitor. Cancer Res., 44,3190-3195. 8 Fernandes, D.J. and Bertino, J.R. (1980) 5-Fluorouracil methotrexate synergy: enhancement of 5fluorodeoxyuridylate binding to thymidylate synthetase by dihydropteroylpolyglutamates. Pmt. No. 72. Acad. Sci. U.S.A., 77, 5663-5667. 9 Fry, D.W., Anderson, L.A., Borst, M. and Goldman, J.D. (1983) Analysis of the role of membrane transport and polyglutamation of methotrexate in gut and the Ehrlich tumor in vivo as factors in drug sensitivity and selectivity. Cancer Res., 43, 10871092. 10 Fry, D.W., Yalowich, J.C. and Goldman, J.D. (1982) Rapid formation of polyglutamyl derivatives of methotrexate and their association with dihydrofolate reductase as assessed by high pressure liquid chromatography in the Ehrlich ascites tumor cell in vitro. J. Biol. Chem., 257,1890-1896. 11 Fry, D.W., Yalowich, J.C. and Goldman, I.D. (1982) Augmentation of the intracellular levels of polyghrtamyl derivatives of methotrexate by vincristine and probenecid in Ehrlich ascites tumor cells. Cancer Res., 42, 2532-2536. 12 Fyfe, M.J. and Goldman, I.D. (1973) Characteristics of the vincristine-induced augmentation of methotrexate uptake in Ehrlich ascites tumor cells. J. Biol. Chem., 248,5067-5073. 13 Herrmann. R., Manegold, C., Schroeder, M., Tlgges, F.-J., Bar&h, H., Jungi, F. and Fritze, D. (1984) Sequential methotrexate and 5-FU in breast cancer resistant to the conventional application of the drugs. Cancer Treat. Rep., 68, 1279-1281. 14 Herrmann, R., Spehn, J., Beyer, J.H., von Franque, U., Schmieder, A., Holzmann, K. and Abel, U. (1984) Sequential methotrexate and 5-fluorouracil: improved response rate in metastatic colorectal cancer. J. Clin. Oncol., 2, 591-594.
282 15 Issel, B.F. and Crooke, S.T. (1979) Etoposide (VP-16-213). Cancer Treat. Rev., 6, 107-124. 16 Matherly, L.H., Anderson, L.A. and Goldman, I.D. (1984) Role of the cellular oxidation-reduction state in methotrexate binding to dihydrofolate reductase and dissociation induced by reduced folates. Cancer Res., 44,2325-2330. 17 Moccio, D.M., Sirotnak, F.M., Samuels, L.L., Ahmed, T., Yagoda, A., DeGraw, J.I. and Piper, J.R. (1984) Similar specificity of membrane transport for folate analogues and their metabolites by murine and human tumor cells: a clinically directed study. Cancer Res., 44,352-357. 18 Rosenblatt, D.S., Whitehead, V.M., Vera, N., Pottier, A., DuPont, M. and Vuchich, M.J. (1978) Prolonged inhibition of DNA synthesis associated with the accumulation of methotrexate polyglutamates by cultured human cells Mol. Pharmacol., 14,11431147. 19 Schilsky, R.L., Bailey, B.D. and Chabner, B.A. (1980) Methotrexate polyglutamate synthesis by cultured human breast cancer cells. Proc. Natl. Acad. Sci. U.S.A., 77, 2919-2922. 20 Sirotnak, F.M. (1980) Correlates of folate analog transport, pharmacokinetics, and selective antitumor action. Pbarmacol. Ther., 8,71-103. 21 Yalowich, J.C., Fry, D.W. and Goldman, I.D. (1982) Teniposide (VM-26) and etoposide (VP-16-213)-induced augmentation of methotrexate transport and polyglutamylation in Ehrlich ascites cells in vitro. Cancer Res., 42, 3648-3653.