Elsevier

Biochemical Pharmacology

Volume 54, Issue 12, 15 December 1997, Pages 1341-1349
Biochemical Pharmacology

Research Papers
Renal Cellular Transport, Metabolism, and Cytotoxicity of S-(6-Purinyl)glutathione, a Prodrug of 6-Mercaptopurine, and Analogues

https://doi.org/10.1016/S0006-2952(97)00401-2Get rights and content

Abstract

The disposition of S-(6-purinyl)glutathione (6-PG) and its metabolites, including the antitumor agent 6-mercaptopurine (6-MP), was characterized in freshly isolated renal cortical cells from male F344 rats to assess the ability of the kidney to convert 6-PG to 6-MP. The intracellular transport and accumulation of 6-PG and 6-MP, the metabolism of 6-PG to 6-MP, and the potential cytotoxicity of 6-MP, 6-thioxanthine (6-ThXan), and 6-thioguanine (6-ThGua) were determined. 6-PG and 6-MP were accumulated by renal cortical cells by time- and concentration-dependent processes, reaching maximal levels of 14.2 and 1.52 nmol/106 cells, respectively, with 1 mM concentrations of each compound. Treatment with acivicin, an inhibitor of 6-PG metabolism by γ-glutamyltransferase, increased accumulation of 6-PG, and treatment with α-keto-γ-methiolbutyrate, a keto acid cosubstrate that stimulates activity of the cysteine conjugate β-lyase (β-lyase), which generates 6-MP, decreased accumulation of 6-PG. Incubation of renal cells with 10 mM 6-PG generated 6-MP at a rate of 2.4 nmol/min per 106 cells, demonstrating that the β-lyase pathway forms the desired product from the prodrug within the intact renal cell. Preincubation of cells with acivicin or aminooxyacetic acid, an inhibitor of the β-lyase, decreased the net formation of 6-MP, demonstrating further the function of the β-lyase. 6-MP, 6-ThXan, and 6-ThGua exhibited approximately equivalent cytotoxicity (45–55% release of lactate dehydrogenase with 1 mM at 2 hr) in isolated renal cells. Based on the known antitumor potency of these agents, this suggests that cytotoxicity and antitumor activity occur by distinct mechanisms. The high amount of accumulation of 6-PG and its subsequent metabolism to 6-MP, as compared with the relatively low amount of accumulation of 6-MP, in renal cells suggest that 6-PG can function as a prodrug and is a more effective delivery vehicle for 6-MP to renal cells than 6-MP itself. Administration of 6-PG may be an effective means of treating renal tumors or suppressing renal transplant rejection.

Section snippets

Materials

6-PG and DCVC were synthesized as previously described 11, 15. Purity was > 95%, as assessed by HPLC, FAB-MS, and 1H NMR. 6-MP, 6-ThXan, 6-ThGua, acivicin, AOAA, allopurinol, KMB, and collagenase (type I) were purchased from the Sigma Chemical Co. (St. Louis, MO). All other chemicals were of the highest purity available and were obtained from commercial sources. Solutions of acivicin, AOAA, allopurinol, KMB, or 6-PG were prepared in Krebs–Henseleit buffer or saline. Solutions of 6-MP, 6-ThXan,

Accumulation of 6-PG and 6-MP

Uptake and net intracellular accumulation of 0.1 to 1 mM 6-PG over a 60-min time course was measured in suspensions of renal cortical cells preincubated with either buffer (Fig. 2A), 0.25 mM acivicin to inhibit GGT (Fig. 2B), or 10 mM KMB to stimulate the β-lyase (Fig. 2C). Under control conditions with buffer, 6-PG accumulated to a maximum intracellular level of 14.2 nmol/106 cells at 15 min with 1 mM 6-PG and decreased thereafter to reach a level of 8.02 nmol/106 cells at 60 min. Net

Discussion

6-PG and 6-PC are metabolized both in vivo and in vitro to 6-MP in the kidneys of rats, and administration of 6-PG or 6-PC leads to the selective renal accumulation of 6-MP 9, 10, 11. These data suggested that 6-PG and 6-PC can function as prodrugs of the antitumor agent 6-MP. Additional studies showed that 6-CP, which also has antitumor activity, is metabolized in vivo to 6-PG and 6-MP, suggesting that metabolism of 6-CP through the GSH conjugation and β-lyase pathways may play a role in the

Acknowledgements

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK40725 (L. H. L.) and R01-DK44295 (A. A. E.). L. H. L. is a recipient of a Research Career Development Award (Grant K04-DK02090) from the National Institute of Diabetes and Digestive and Kidney Diseases. The authors also thank Mr. David A. Putt for his technical assistance.

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