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Intracellular Biotransformation of Platinum Compounds with the 1,2-Diaminocyclohexane Carrier Ligand in the L1210 Cell Line¹

Department of Biochemistry and Nutrition, School of Medicine [S. K. M., G. G., S. G. C.], and Division of Medicinal Chemistry, School of Pharmacy [S. D. W.], University of North Carolina, Chapel Hill, North Carolina 27599

We have previously reported the development of a two-column high performance liquid chromatography system for separation of platinum(II) complexes with the 1,2-diaminocyclohexane (DACH) carrier ligand (Mauldin et al., Anal. Biochem., 157: 129, 1986). Here we report the application of this technique to the study of the intracellular biotransformations of (DL)-trans-1,2-diaminocyclohexanedichloroplatinum(II) [PtCl₂(trans-DACH)] and (DL)-trans-1,2-diaminocyclohexanemalonatoplatinum(II) [Pt(mal)(trans-DACH)] in the L1210 cell line. The two-column high performance liquid chromatography system allowed separation and identification of both parent drugs and intracellular biotransformation products containing glutathione, methionine, cysteine, arginine, lysine, aspartate or glutamate, and serine or threonine. With the exception of the platinum-glutathione complex, the relative abundance of each biotransformation product was independent of drug concentration. The relative abundance of the platinum-glutathione biotransformation product increased with increasing platinum concentration, suggesting that platinum drugs cause an increase in intracellular glutathione levels in a dose-dependent manner. This hypothesis was verified by direct measurement of intracellular glutathione levels. In continuous uptake experiments, the intracellular levels of the parent compounds peaked between 2 and 5 h and declined to negligible levels by 24 h. In pulse-chase experiments, the chemical t for PtCl₂(trans-DACH) and Pt(mal)(trans-DACH) inside the cell at 37°C was determined to be 12–15 and 21–28 min, respectively. This is far shorter than previously determined rates for the displacement of either ligand in vitro. The platinum-amino acid complexes accumulated gradually throughout the 24-h incubation. The free trans-DACH carrier ligand also accumulated to a level approaching 20% of filterable counts during the 24-h incubation, probably due to trans-labilization of the carrier ligand by sulfur-containing nucleophiles. A combination of reverse phase high performance liquid chromatography and a DNA binding assay was used to identify and quantitate the reactive biotransformation products. As expected from previous studies (Mauldin et al., Cancer Res., 46: 2876, 1986), the PtCl₂(trans-DACH)-treated cells had approximately 3 times more reactive platinum biotransformation product at early times, but the levels of reactive biotransformation product fell much more rapidly than in Pt(mal)(trans-DACH)-treated cells. In the PtCl₂(trans-DACH)-treated cells, the major reactive biotransformation product was the aquachloro species at all time points tested. In Pt(mal)(trans-DACH)-treated cells, however, one or more additional reactive biotransformation products were found to accumulate at later times. We believe that these techniques are applicable to detailed biotransformation studies of a wide range of platinum compounds.

¹ This research was supported in part by USPHS Grant CA 34082 awarded by the National Cancer Institute. S. K. M. was supported by a predoctoral traineeship (National Research Service Award 5T32 ES-07126) from the National Institute of Environmental Health Sciences.

² Present address: Molecular and Cell Biology Program, Pennsylvania State University, State College, PA 16802.

³ To whom requests for reprints should be addressed.

Received 5/20/87. Revised 11/ 3/87. Revised 3/ 4/88. Revised 5/17/88. Accepted 5/26/88.

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