Mutagenic activity and identification of excreted platinum in human and rat urine and rat plasma after administration of cisplatin

Cancer Letters, 18 (1983) 329-338 Elsevier Scientific Publishers Ireland Ltd.

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MUTAGENIC ACTIVITY AND IDENTIFICATION OF EXCRETED PLATINUM IN HUMAN AND RAT URINE AND RAT PLASMA AFTER ADMINISTRATION OF CISPLATIN

R. SAFIRSTEINa, M. DAYE and JOSEPH B. GUTTENPLANb aDivision of Nephrology and Department of Medicine, Mount Sinai School of Medicine, 100th Street and Fifth Avenue, New York, NY 10029 and bDepartment of Biochemistry, New York University Dental Center, New York, NY 10010 (U.S.A.) (Received 11 June 1982) (Revised version received 15 November 1982) (Accepted 30 November 1982)

SUMMARY

Cisplatin and its biotransformation products were analyzed in human and rat urine and in plasma from rats. Analyses were performed using high performance liquid chromatography (HPLC). Microbial mutagenesis assays were performed on effluents from the chromatographic system. After intravenous administration to man (50 mg/m*) and intravenous and intraperitoneal administration to rats (5-10 mg/kg), platinum was excreted in the urine in a form that co-eluted mainly with cisplatin. Unbound drug in the plasma co-eluted with cisplatin. Furthermore excreted platinum exhibited mutagenic and chemical reactivity similar to that of cisplatin. We conclude that the principal form of free platinum circulating in blood and excreted in urine is cisplatin.

INTRODUCTION

The antineoplastic drug, cis-diamminedichloroplatinum II, or cisplatin, is effective against many solid tumors but its use is often limited by its nephrotoxicity. Cisplatin is excreted predominantly via the kidney. While the total amount of the administered drug excreted differs from species to species (50-+0% in rats over the first 24 h and 20-40% in humans over the same time period) the process of renal excretion in animals is a rapid one with as much as 80% of the total excretion achieved in the first hour of its administration [ 1,2]. Cisplatin is extensively bound to plasma proteins with as much as 90% of the drug bound 2 h after an intravenous injection [ 2,3]. The bound portion of the drug is no longer cytotoxic [ 41. These 2 processes, renal excretion and plasma protein binding, lead to very 0304-3835/83/0000-0000/$03.00 0 1983 Elsevier Scientific Publishers Ireland Ltd. - Published and printed in Ireland

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low concentration of the free, and presumably toxic, form of the drug shortly after its injection. This has led to the proposal that the renal toxicity of cisplatin involves the exposure of renal cells to high concentrations of the drug in blood or urine soon after the drug is given [ 51, Consistent with this view is the rapid accumulation of platinum in the kidney, a process which is nearly complete in the first hour after its administration [6,7]. The form cisplatin assumes in the blood or urine during this time, however, is not known. Several studies have attempted to characterize cisplatin in these fluids but the data are conflicting. Both extensive [ 2,8] and limited [9] biotransformation have been reported. Thus far, no studies have reported the biologic activity of the drug excreted in the urine. Because an understanding of the biotransformation of cisplatin in blood and urine would be important in determining the mechanism of its renal toxicity, we have undertaken this study. We report here conditions for the rapid and simple separation by HPLC of cisplatin, its aquated derivatives and one of its metabolites or decomposition products in rat urine and plasma and in human urine. In addition, we report that certain fractions collected from the HPLC of the urines are mutagenic and that the major platinum containing compound co-elutes with cisplatin. MATERIALS

AND METHODS

Chemicals [ 195mPt] Cisplatin in isotonic saline was made available through Dr. J.D. Hoeschele, at Oak Ridge National Laboratories, Oak Ridge, Tennessee. Non-radioactive cisplatin was obtained through the Drug Synthesis and Chemistry Branch, National Cancer Institute, Bethesda, Maryland. [195mPt]Cisplatin was received with a specific activity of approximately 145 Ci/mol but due to its short half-life (4.02 days), the specific activity declined rapidly. Standards were prepared in the following manner using the method of Johnson et al. [lo]. Cisplatin ( 10e4 M) dissolved in water (pH 3.5) was incubated in the dark at room temperature. Aliquots of the reaction mixture were chromatographed (see below) for up to 72 h after the addition of cisplatin. After 1.75 h a small (15%) portion of the total radioactivity shifts to a later peak. This peak becomes the predominant peak at 24 h but its formation is not complete until 48 h after incubation, when 3 platinum containing peaks (shown in Fig. 1B) are found. The proportions of these peaks are 10: 60: 30, which does not change thereafter. This proportion agrees well with the calculated proportions derived from the rate constants of the 2 aquation reactions [lo]. Confirmation of the identity of the principal peak was derived from potentiometric determination of the chloride ion liberated from 4.6 mM aqueous solution (pH 3.5) of cisplatin after 72 h. Calculations of the liberated chloride released, by assessing the extent of reaction by HPLC, gave a value of 2.8 mM; 2.8 mM Cl- was found by titration. The diaquodiammine platinum derivative was

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prepared by incubating cisplatin ( 10m4M) in water (pH 4.0) for 48 h at room temperature with a molar concentration of AgNOJ equivalent to the total chloride + 2X the cisplatin concentration in the medium [lo]. As was done during the synthesis of the monoaquo monochloro derivative the reaction mixture was analyzed by HPLC at different time periods. After 15 min, nearly complete conversion of the cisplatin to a compound coeluting with the monoaquo derivative was achieved. After 4 h a second smaller peak was formed which eluted after the monoaquo peak. At 24 h this later species became the predominant peak and by 48 h was the sole peak present. Failure to detect chloride after incubation of a 3.8 mM cisplatin with a-fold excess AgNOJ (relative to chloride) and reaction with lo-fold excess of NH,OH (5 min at 100°C) was taken as further evidence for the production of the diaquo derivative [ 111. A glutathione derivative of cisplatin was also prepared by incubating cisplatin (0.1 mM) with 5 mM glutathione in 0.1 M phosphate buffer (pH 7.4) containing 0.02 M NaCl at 37°C for 24 h. No shift in the platinum containing peak from the cisplatin position occurred until overnight incubation. The reaction was judged complete at 24 h since no shift in retention volume of the platilJum-containing peak was observed on subsequent incubation. Cisplatin incubated in the absence of glutathione under these conditions did not show such a pattern. Preparation

of samples

Male Sprague-Dawley rats were treated with 5 mg/kg of cisplatin (1 mg/ ml in 0.9% NaCl, lo- 50 gCi/ml) given intraperitoneally or intravenously. Awake animals were placed in metal metabolic cages and the urine collected for up to 24 h after injections of cisplatin. In experiments using anesthetized animals, urine was collected from a bladder catheter, 5,10,30,60 and 90 min after intravenous injection of cisplatin. Urines were kept on ice and analyzed immediately or frozen after collection, thawed and centrifuged (2000 X g for 10 min) before injection onto the HPLC. Human urine was collected from a patient 6 h after an intravenous bolus injection of 90 mg cisplatin. The patient voided into a container which was refrigerated immediately after each voiding. In order to determine the chemical reactive nature of excreted platinum and compare it to that of cisplatin added to urine in vitro, a rat was anesthetized (inactin, 100 mg/kg body wt.), placed on a heated table and polyethylene tubes placed in the trachea, external jugular vein and bladder. Sixty minutes after the start of a constant infusion of isotonic saline (0.9% NaCl) at 2 ml/h/100 g body wt., urine was collected, under oil, into preweighted plastic test tubes placed on ice. Urine was collected for 60 min before cisplatin, 5 mg/kg (spec. act. 1184.6 cpm/pg cisplatin), was given intravenously over 1 min. Thereafter, urine was collected for 2 h and chloride and platinum concentration determined in each sample. Most (80%) of the platinum to be excreted (59.2% of the total dose)

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was excreted in the first 30 min. Platinum and chloride concentration in this 30-min urine sample was 0.86 mM and 139 mM, respectively. Cisplatin was added (final concentration 0.86 mM) to a urine sample excreted before the rat received cisplatin. The 2 urines, labeled experimental and control, were used in the following experiment: 0.1 ml of each urine was injected onto the HPLC system and eluded with water (pH 3.5) at a flow rate of 1 ml/min; 0.3-ml fractions were collected from the column and chloride and platinum determined in each fraction. The water elution enabled separation of platinum from chloride (and other urinary components) as most of the chloride eluted before cisplatin. AgN03 (lo-fold excess) was added to the fractions containing the highest content of platinum (fraction 13 for both control and experimental urines) and the mixture brought to a final volume of 1 ml with Hz0 (pH 3.5). The final concentration of platinum in control and experimental urine was 3.5 X 10V5 M respectively. This mixture was incubated for 3 h at 40°C filtered and 0.2 ml re-injected and analyzed by HPLC. Plasma samples were obtained 2 h after the start of a constant infusion of cisplatin (2.5 mg/kg/h) at 10 pCi/h from 2 rats. The rats were exsanguinated through a carotid artery catheter, and the blood spun in a cold centrifuge to separate serum from cells. The serum was ultrafiltered through a UM 10 filter in the cold and the ultrafiltrate injected onto the column. In 2 additional rats, cisplatin (5 mg/kg) was given intravenously and the rat killed 1 h later. Ultrafiltrate of serum was obtained as above and the ultrafiltrate injected onto the column. HPLC was performed on a system constructed from the following components: pump, Eldex B-100s; injector, Altex Model 210; and a Whatman Partisil-10 SCX 4 mm (i.d.) X 25 cm, strong cationic exchange column. The column was eluted with 0.012 M or 0.03 M ammonium formate or 0.03 M sodium acetate buffer (pH 3.5) at a flow rate of 1 ml/min. Fractions were collected every 0.4 min and counted in 4.5 ml of Budget-Solv (Research Products International Corp., Elk Grove, IL). Recoveries of injected radioactivity were 90-100% in urine and plasma ultrafiltrates and 60--80% for the aquated species of cisplatin. Atomic

absorption

An Instrumentation Laboratory model 951 Atomic Absorption Spectrometer was used which was equipped with a controlled temperature atomizer (Model 555), deuterium background corrector, and real-time video display screen. The hollow-cathode platinum lamp was operated at a current of 10 mA and the atomic absorption spectrometer was used to monitor the platinum line at 265.9 mm with a slit band width of 0.5 nm. Deuterium background correction was used in all analyses. Other conditions were similar to those described by Pera and Harder [ 121. Mutagenesis

Bacterial mutagenesis was assayed by the standard Ames test using Sal-

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monella typhimurium TA100 (kindly supplied by B. Ames, Berkeley, CA). Fractions from the chromatographic system were collected into sterile tubes, combined with top agar and bacteria and overlayed onto minimal plates. An equal portion of each fraction was combined with diluted bacteria (5 X lo6 dilution) and plated onto histidine-containing minimal agar to determine toxicity. Toxic fractions were diluted, and mutagenedis reassayed under conditions of minimal (10%) toxicity. The mutagenic activity of cisplatin varied somewhat from day to day as is typical in Ames assays, with an average value of 120 f 40 (S.E.) revertants/nmol platinum. RESULTS

Cisplatin eluted with a retention volume of 3.6 ml (Fig. lA), and its mono and diaquo derivatives eluted with retention volumes of 7.6 ml and 9.6 ml, respectively (Fig. 1B and C). The larger retention volumes of the aquated derivatives were presumably due to the greater binding affinities of these positively charged molecules with the cationic exchange resin. As the retention volume of cisplatin was relatively small, another platinum species was prepared (see Materials and Methods) and analyzed by HPLC to determine if it would separate from cisplatin. This derivative of cisplatin (presumably the sulfhydryl bound glutathione derivative) eluted at 2.7 ml (results not shown) and was readily separated from cisplatin. Urines from 8 of the rats treated with cisplatin showed a major peak corresponding in retention volume to cisplatin (Fig. 2A and B). Similar elution profiles were obtained in urines collected from 3 min to 24 h after injection. Addition of AgN03 to these platinum containing eluates resulted in a shift in the elution profile to a later peak corresponding to the diaquo derivative (Fig. 4A -C). Cisplatin added to control urine eluted in an identical fashion when treated with AgNO,. Some urines contained a minor peak (15% of total Pt recovered) whose retention time corresponded closely to the monoaquomonochloro derivative. When the HPLC eluant was analyzed by atomic absorption, profiles were obtained which were nearly identical with those obtained by gamma counting (Fig. 2A). Fractions from the eluted urine were assayed for mutagenic activity and mutagenesis compared to platinum content (Fig. 3A). The fractions with the highest platinum content exhibited the highest mutagenic activity. The mutagenic activity per nmol of platinum (119 revertants/nmol platinum) in the first peak (the sum of fractions 9,lO and 11) was similar to that obtained for cisplatin determined on the same day (156 revertants/ nmol platinum). The later peak, presumably an aquated species of platinum, was also mutagenic. The mutagenic activity in the later peak was more apparent when larger samples were plated. For example, when 3 times as much sample was plated, the mutagenic activity in fraction 17 was 150 revertants per plate above background. The elution profile of human urine from a patient treated with cisplatin is shown in Fig. 3B. The platinum content in aliquots of fractions collected

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from HPLC were analyzed by atomic absorption. The major peak eluted with the same retention volume as cisplatin. Analysis of the HPLC fractions in the mutagenesis assay system again showed a close relationship between mutagenic activity and platinum content in the HPLC fractions. There was @

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Fig. 1. HPLC of [ 195mPtlcisplatin (A) and aquated derivatives of cisplatin (B and C). Chromatography was performed using a partisil SCX/lO column (4 mm i.d., 25 cm length) with a mobile phase consisting of 0.03 M sodium acetate buffer (pH 3.5) at a flow rate of 1 .O ml/min. Fractions were collected at 0.4-min intervals. (A) Chromatograph of [195mPt]cisplatin in 0.9% NaCl (spec. act. 0.25 &i/mg) was injected. (B) Chromatograph of a solution of cisplatin ( 10m4 M) in water incubated for 48 h at 23°C. [195mPt]cisplatin in 0.9% NaCl was eluted from column with water (pH 4.0) and collected on ice. This was done to separate cisplatin from NaCl in the solvent. Fractions free of chloride were added to the cisplatin solution above to achieve a final concentration of 10e4 M, and incubated; 75 ~1 of this sample was injected. The 3 species present are cisplatin (PtAm,Cl,), monoaquomonochlorodiammine platinum (PtAm, ClH,O+ ), and diaquodiammine platinum (PtAm,Aq,S+). (C) Chromatograph of a cisplatin solution ( 10F4 M) incubated with AgNO, for 48 h at room temperature. 20 ~1 injected and eluted as above. Fig. 2. HPLC of rat urine collected 3 h after administration of 5 mg/kg of cisplatin (i.p.). Chromatographic conditions were the same as described in Fig. 1 except 0.03 M NH,COOH was used. Fractions were analyzed by atomic absorption (as described in Methods) and gamma counting. 10 ~1 of urine containing 433 nmol/ml of Pt was chromatographed.

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Fig. 3. HPLC of rat (A) and human (B) urine and a comparison of platinum concentration obtained by atomic absorption with mutagenic activity in each fraction. HPLC and platinum analyses were carried out as described in Fig. 2 and in Methods. Mutagenesis assays are described in Methods. 100 ~1 of rat urine containing 155.8 gg/ml(519 nmol/ml) of Pt was injected onto the column and 50 ~1 of each fraction (larger volumes were toxic) was used for the mutagenesis assay. For the human urine 500 ~1 of urine containing 13.9 pg/ml(46 nmoI/ml) platinum was injected and 50 ~1 of each fraction was used for the mutagenesis assay. In order to depict the results as actual observed revertant numbers and not derived numbers, the values given in Fig. 3 are taken directly from each plate and are not corrected to give the induced revertant numbers in the entire fractions. Fig. 4. HPLC of rat urine. (A) Excreted platinum in urine. (B) Excreted platinum in urine after addition of AgNO, (lo-fold platinum concentration) and incubation at 40°C for 3 h. (C) Added cisplatin in urine after incubation with AgNO, as above. See Methods for details.

a close correspondence, as well, between the mutagenic activity of this early peak (60 revertants/nmol platinum) and cisplatin (78 revertants/nmol cisplatin) determined on the same day. A similar chromatographic profile was obtained in ultrafiltrates of serum (Fig. 5). Again, as in the urine, the major peak corresponded in retention time to cisplatin with a minor secondary peak eluting later. A similar pattern of elution was observed in blood samples taken 1 h after intraperitoneal injection of the drug.

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DISCUSSION

A previous report has indicated that cisplatin is predominantly excreted unchanged in human urine for up to 6 h after an intravenous bolus of the drug [9]. However, other reports [ 2,8] presented evidence of extensive biotransformation of excreted cisplatin. This finding may have been a result of aquation and possible subsequent reaction of excreted cisplatin during the analytic procedure, a problem recognized by the authors [2]. Our results provide strong evidence that cisplatin is excreted largely unchanged in human and rat urine. The evidence is based not only on HPLC retention volume, but also the mutagenic potency of the platinumcontaining peak and its chemical reactivity. The potency of a compound in the Ames test is a characteristic property of the compound tested. The potency of mutagens varies by some 106-fold [13] and, for a group of about 20 liganded platinum II compounds with a wide potency distribution, potency may vary by at least 104-fold [ 14-161 Platinum eluted from urine had the same potency as the platinum in cisplatin. Such concurrence in potency is unlikely to be fortuitous. Also, AgNO, added to the HPLC eluates of urine, converted both excreted platinum and cisplatin added to normal rat urine to the same product. Taken together the data indicate that excreted platinum is predominantly in the form of cisplatin. While in a few samples a smaller peak eluted later than

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cisplatin, this species cannot account for more than 20% of the excreted drug over 24 h. This is true because recoveries of injected platinum from the HPLC system was 95.0 f 1.6 (n = 7) (X f S.E.) and this species never exceeded 15% of the injected drug. Thus, the similarity in chromatographic, mutagenic and chemical reactive nature of cisplatin and excreted platinum provides further evidence that cisplatin is excreted unchanged by the kidney. The results indicate that free cisplatin is relatively stable in the extracellular fluids examined. This is consistent with the known dependence of cisplatin aquation on chloride concentration. Thus the substitution of water ligands for chloride, a process which has been shown to accelerate the rate of reaction of platinum species with DNA [lo] and increase its toxicity [ 171, is inhibited in the presence of chloride at a concentration as low as 17 mM [ 181. This aquation of cisplatin is almost completely inhibited by chloride concentrations of 77 mM or above [l&-20]. Thus, at the high chloride concentration of blood and urine, it is not surprising that the chloride ligands on cisplatin would not be appreciably dissociated. The binding of cisplatin with plasma proteins, on the other hand, is not inhibited by chloride and presumably involves a different mechanism [ 21. Such a reaction may involve the rather strong electrophiles on proteins, such as -SMe and imidazole-N groups [ 211 and may operate at the cellular level as well. In summary, our studies show that the principal species of platinum to which renal cells are exposed is cisplatin. The mutagenicity of excreted cisplatin may pose a potential risk of genotoxic effects in the urinary tracts of patients treated with cisplatin. ACKNOWLEDGEMENTS

We wish to thank Dr. Fred S. Wright, Yale University School of Medicine, West Haven VA Hospital who made available the use of the atomic absorption spectrometer and Dr. Howard Bruckner, Mount Sinai School of Medicine, for providing patient’s urine. We wish to thank Ms. Lilia Shoichet for her expert technical assistance and Ms. Ricky Herskovitz and Ms. Helen Phillips for preparation of the manuscript. This investigation was supported by PHS grant number lROlCA/AM28683-OlAl awarded by the National Cancer Institute, DHHS and Grant number K-064 awarded by the Health Research Council of New York. REFERENCES 1 Safirstein, R., Daye, M., Miller, P. and Guttenplan, J. (1981) Renal disposition and metabolism of liganed platinum: Implications to toxicity. Fed. Proc., 40, 651A. 2 DeConti, R.C., Toftness, B.R., Lange, R.C. et al. (1973) Clinical and pharmacological studies with cis-diamminedichloroplatinum (II). Cancer Res., 33, 13101315. 3 Himmelstein, K.J., Patton, T.F., Belt, R.J. et al. (1981) Clinical kinetics of intact cisplatin and some related species. Clin. Pharmacol. Ther., 29, 658-664.

338 Holdener, E.E., Park, C.H., Belt, R.J. et al. (1978) Effect of mannitol and human plasma on cytotoxicity of cis-dichlorodiammineplatinum. Clin. Res., 26, 436A. Guarino, A.M., Miller, D.S., Arnold, S.T., Pritchard, J.B. et al. (1979) Platinate toxicity: Past, present, and prospects. Cancer Treat. Rep., 63, 1475-1483. Litterst, C., Torres, I.J. and Guarino, A.M. (1979) Plasma levels and organ distribution of platinum in the rat, dog, and dog fish shark following single intravenous administration of cis-dichlorodiammine platinum (II). J. Clin. Hematol. Oncol., 7,169-179. Subcellular localization of I Choie, D.D., DelCamp, A.A. and Guarino, A.M. (1980) cis-dichlorodiammineplatinum (II) in rat kidney and liver. Toxicol. Appl. Pharmacol., 55, 245-252. Reactions of cisplatin with human plasma and 8 Repta, A.L. and Long, D.F. (1982) plasma fractions. In: Cisplatin: Current Status and New Developments, pp. 285304. Editors: A.W. Prestayko, S.T. Crooke and S.K. Carter. Academic Press, New York. 9 LeRoy, A.F., Wehling, M., Gormley, P. et al. (1980) Quantitative changes in cisdichlorodiammineplatinum (II) speciation in excreted urine with time after intravenous infusion in man: methods of analysis, preliminary studies, and clinical results. Cancer Treat. Rep., 64, 123-132. N.P., Hoeschele, J.D. and Rahn, R.O. (1980) Kinetic analysis of the in10 Johnson, vitro binding of radioactive cis- and trans-dichlorodiammineplatinum (II) to DNA. Chem.-Biol. Interact., 30, 151-169. Chlorotriammineplatinum (II) ion. Acid 11 Aprile, F. and Martin, Jr., D.S. (1962) hydrolysis and isotopic exchange of chloride ligand. Inorg. Chem., 1, 551-557. Analysis of platinum in biological material by 12 Pera, M.F. and Harder, H.C. (1977) flameless atomic absorption spectrometry. Clin. Chem., 23, 1245-1249. E. and Ames, B.N. (1975) Detection of carcino13 McCann, J., Choie, E., Yamasaki, gens as mutagens in the Salmonella/microsome test: assay of three hundred chemicals. Proc. Natl. Acad. Sci. U.S.A., 72, 5135-5139. W.R., Miller, E.C. and Miller, J.A. (1979) Carcinogenicity of antitumor 14 Leopold, cis-platinum (II) coordination complexes in the mouse and rat. Cancer Res., 39, 913-918. P., Macquet, J.-P., Butour, J.-L. and Pasletti, C. (1977) Relative efficiencies 15 Lecointe, of a series of square-planar platinum (II) compounds on Salmonella mutagenesis. Mutat. Res., 48, 139-144. P., Macquet, J.-P. and Butour, J.-L. (1979) Correlation between the tox16 Lecointe, icity of platinum drugs to L1210 Leukemia cells and their mutagenic properties. Biochem. Biophys. Res. Commun. 90, 209-213. 17 Litterst, C.L. (1981) Alterations in the toxicity of cis-dichlorodiammineplatinum II and in tissue localization of platinum as a function of NaCl concentration in the vehicle of administration. Toxicol. Appl. Pharmacol., 61, 99-108. 18 Hincal, A.A., Long, D.F. and Repta, A.J. (1979) Cisplatin stability in aqueous parenteral vehicles. J. Parenteral Drug Assoc., 33, 107-116. Cis-dichlorodiammineplatinum (II) Acid 19 Reishus, J.W. and Martin, Jr., D.S. (1961) hydrolysis and isotopic exchange of the chloride ligands. J. Am. Chem. Sot., 83, 2457-2462. D.C., Hiranaka, P.K. and Gallelli, J.F. (1979) Stability of 20 Greene, R.F., Chatterji, cisplatin in aqueous solution. Am. J. Hosp. Pharm., 36, 38-43. 21 Cleare, M.J. (1977) Some aspects of platinum complex chemistry and their relation to antitumor activity. J. Clin. Hematol. Oncol., 7, 1-21.