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Increased uptake of chemotherapeutic agents into renal proximal tubular cells by coupling to amino acids
1717 Antitumor activity of six Sm-analogues which were in vitro more active than the parent drug (Sm) was studied in eight in vivo murine tumor models. In vivo potentiation of cisplatin antitumor activity was studied for Sm and three active analogues in s.c. implanted L1210 leukemia. Based on these studies Ethyldeshydroxy-sparsomycin (EdSm) was chosen for further preclinical investigation on the synergism between sparsomycins and cisplatin. Combined treatment of EdSm and cisplatin was studied in L1210 leukemia bearing mice. We investigated the optimal treatment conditions with regard to: doses, number of injections, treatment schedule, interval between administration of the two drugs, route of drug administration as well as site of tumor inoculation. Best results were obtained in the L1210 i.p. tumor model when drug treatment was ip on days 1, 5 and 9. Using nontoxic doses of EdSm (5 mg/kg) and cisplatinum (3 mg/kg) no differences in antitumor activity were observed after pretreatment, simultaueous treatment or posttreatment of cisplatin with EdSm. All schedules of combined drug treatment generated 4 to 6 cured mice in each group consisting of six mice. The enhancement of cisplatin antitumor activity by EdSm In vivo could be observed in L1210 leukemia, P388 leukemia and B16 melanoma, but also in a sparsomycin resistant L1210 subclone. P.th.O$6 ]
Increased uptake of chemotherapeutic agents into renal proximal tubular ceres by coupling to amino acids Koob, M., Seitz, A. and Dekant, W. Institut fur Toxikoiogie, Universitiit W'~zburg, Versbacher Str. 9, 8700 Wfirzburg, F.R.G.
Renal adenocarcinoma, one of the most common urologic cancers, can be treated by different methods with varying success. However, nephrectomy is the treatment usually chosen. A chemotherapeutic concept is not available yet, most likely due to a multidrug resistance of the tumor. Only occasional reports suggest a beneficial effect of chemotherapy. A new concept in chemotherapy is the use of prodrugs using specific target cell biochemistry to concentrate or liberate the chemotherapeutic agent in the target tissue and to avoid systemic adverse effects. The kidney possesses transport systems to concentrate amino acid derivatives, thus coupling chemotherapeutic agents to amino acids may result in an enrichement of the drug in the renal cell. Mercapturic acids and glutathione (GSH) de-ivatives are processed to cysteine S-conjugates by renal enzymes involved in mercapturic acid formation. Cysteine S-conjugates are substrates for renal fl-lyase, a pyridoxal phosphate dependent enzyme, thus this metabolic pathway may liberate the drug specifically in the kidney as target organ. To validate this concept, we compared the uptake of 6-mercaptopurine (MP) and the corresponding derivatives S-(pudnyl)glutathione and N-acetyl-S-purinyl-L-cysteine, as model ~,mpounds, into rat renal proximal tubuiar ceils. Uptake was studied with different concentratio~ over a period of 90 m~n; time dependent decrease of the S-conjugates in the incubation medium indicative of uptake into the cell was determined by HPLC analysis. Cellular uptake of MP amounted to 50 nmoles/107 cells within 90 rain. S-~urinyl)glutathione was taken up twice the rate of MP, whilst almost an eightfold increase in cellular uptake could be observed for N-acetyl-S-purinyl-L-cysteine. Renal accumulation of MP-derivatives w~re m,~kedly dimintLished by probenecid (1 mM), an inhibitor of the organic anion transports Furthermore, increasing medium concentrations could not enhance transport over 50 nmoles/2 x 10 e cells/30 min, indicating the involvement of an active transport system. The intracellular formation of MP was demonstrated by HPLC and UV-spectroscopy however, exact quantification was limited by the method used. Time dependent elimination of MP and derivatives from the incubation medi,im (2 x 106 cells/ml, cell viability determined by trypan blue exclusion was over 85~ during the course of the experiments).
MP (0.1 raM) MP-(3SH (0.1 raM) MP-N-Ac-CYS (0.1 mM)
0 rain
30
60
90
100~ 100~ 100~
97~ 95~ 55~
95% 85~ 30~
90~ 80% 20~
Increased uptake of chemotherapeutic agents into renal proximal tubular ceres by coupling to amino acids Koob, M., Seitz, A. and Dekant, W. Institut fur Toxikoiogie, Universitiit W'~zburg, Versbacher Str. 9, 8700 Wfirzburg, F.R.G.
Renal adenocarcinoma, one of the most common urologic cancers, can be treated by different methods with varying success. However, nephrectomy is the treatment usually chosen. A chemotherapeutic concept is not available yet, most likely due to a multidrug resistance of the tumor. Only occasional reports suggest a beneficial effect of chemotherapy. A new concept in chemotherapy is the use of prodrugs using specific target cell biochemistry to concentrate or liberate the chemotherapeutic agent in the target tissue and to avoid systemic adverse effects. The kidney possesses transport systems to concentrate amino acid derivatives, thus coupling chemotherapeutic agents to amino acids may result in an enrichement of the drug in the renal cell. Mercapturic acids and glutathione (GSH) de-ivatives are processed to cysteine S-conjugates by renal enzymes involved in mercapturic acid formation. Cysteine S-conjugates are substrates for renal fl-lyase, a pyridoxal phosphate dependent enzyme, thus this metabolic pathway may liberate the drug specifically in the kidney as target organ. To validate this concept, we compared the uptake of 6-mercaptopurine (MP) and the corresponding derivatives S-(pudnyl)glutathione and N-acetyl-S-purinyl-L-cysteine, as model ~,mpounds, into rat renal proximal tubuiar ceils. Uptake was studied with different concentratio~ over a period of 90 m~n; time dependent decrease of the S-conjugates in the incubation medium indicative of uptake into the cell was determined by HPLC analysis. Cellular uptake of MP amounted to 50 nmoles/107 cells within 90 rain. S-~urinyl)glutathione was taken up twice the rate of MP, whilst almost an eightfold increase in cellular uptake could be observed for N-acetyl-S-purinyl-L-cysteine. Renal accumulation of MP-derivatives w~re m,~kedly dimintLished by probenecid (1 mM), an inhibitor of the organic anion transports Furthermore, increasing medium concentrations could not enhance transport over 50 nmoles/2 x 10 e cells/30 min, indicating the involvement of an active transport system. The intracellular formation of MP was demonstrated by HPLC and UV-spectroscopy however, exact quantification was limited by the method used. Time dependent elimination of MP and derivatives from the incubation medi,im (2 x 106 cells/ml, cell viability determined by trypan blue exclusion was over 85~ during the course of the experiments).
MP (0.1 raM) MP-(3SH (0.1 raM) MP-N-Ac-CYS (0.1 mM)
0 rain
30
60
90
100~ 100~ 100~
97~ 95~ 55~
95% 85~ 30~
90~ 80% 20~