Inr. 1. Rodicwion Onco/ogv Biol. Phys.. Vol. 8, pp. 585-588 0360-3016/82/03058544$03.00/0

Printed in the U.S.A. All rights reserved Copyright 8 1982 Pergamon Press Ltd.

??Session III-P’rotectors With Radiation

PROTECTION AGAINST CIS-DICHLORODIAMMINE Pt (II) CYTOTOXICITY IN VITRO BY CYSTEAMINE

DEWNIS C. SHRIEVE, A.B. AND JOHN W. HARRIS, PH.D., M.D.

Radiation Oncology Research Laboratory, Department of Radiation Oncology, School of Medicine, University of California, San Francisco, CA 94143

EMT6/SF cells exposed to cis-dichlorodiammine Pt (II) (DDP) for 1 hr in the presence of 10 mM cysteamime (MEA) survived better than cells treated with DDP alone (DMF-3.1). MEA added to cultures ufier removal of DDP also protected, even when the two exposures were separated by as long as 4 hr. Protection by MEA was concentration dependent: no significant protection was observed below 1 mM and maximum protection required 5 mM. When cells were incubated with 10 mM MEA immediately after removal of DDP, survival increased as a function of the length of exposure to MEA, reaching a maximum at 2 hr.

Cis-platinum, Radioprotectors, Chemotherapy drugs, Cell culture.

INTRODUCTION

The radioprotective qu.alities of sulfhydryl-containing compounds have been studied intensely for almost four decades and one such compound, WR-2721 (S-2-(3- aminopropylamino) ethyl phosphorothioic acid), has been introduced into clinical trials in this country and in Japan. WR-2721 also has been reported to protect mouse mar- row colony-forming cells against killing by cyclophos- phamide, nitrogen mustard, cis-dichlorodiammine plati- num (II) (DDP), 5-Fluorouracil and BCNU, with dose modifying factors (DMF’s) ranging from 1.5 to 4.6.” Protection against adria.mycin toxicity by WR-2721 has been found in mouse bone marrow and intestinal crypt cells (T. L. Phillips, oral communication, Sept., 1981). Yuhas and Culo” found that WR-2721 could protect against DDP-induced nephrotoxicity (DMF = 1.7) in rats without reduction in the drug’s antitumor activity. These effects suggest that WIR-2721 may enhance the thera- peutic ratio in clinical chemotherapy.

The mechanism of the protective action remains uncer- tain since WR-2721 h.as both biochemical (thiol) and physiological (cardiovascular and vasoactive) properties in some systems, raising the question as to whether “protection” against drugs in vivo is biochemical, physio- logical (e.g., redistribution of chemotherapeutic drugs) or both. As a baseline approach to answer this question, we undertook to investigate the biochemical aspects of chemoprotection in vit,ro. It was necessary to use the parent compound, @mercaptoethylamine (MEA), be- cause WR-2721 has little or no activity in vitro.S

METHODS AND MATERIALS

EMT6/SF mouse mammary tumor cells were main- tained in exponential growth in Dulbecco’s Modified Eagle Medium (DME) containing 4500 mg/l of glucose and supplemented with fetal calf serum (15%), L-gluta- mine (292 mg/l) and gentamycin (50 pg/ml). Cells grew with a doubling time of 12 hr and were free of detectable mycoplasma contamination as measured by the method of Chen.’

Twenty-four hours prior to experiments, 5 x lo5 cells were plated into 60 mm plastic dishes and allowed to progress into early exponential growth before use. MEA or DDP was dissolved in DME at IO-fold concentrations and added to cells cultures in appropriate dilutions. After drug treatments at 37“C, the drug-containing medium was removed and the cultures were rinsed twice with fresh DME. The cells were trypsinized (0.05% trypsin, 0.02% EDTA, 5-7 min at 37’C), counted and plated for colony formation. Colonies were stained with crystal violet and scored on day 9.

RESULTS

10 mM MEA significantly protected DDP-treated EMT6/SF cells (Fig 1) when present concomitantly with DDP. Exposure to DDP at 16 pg/ml (53pM) reduced the surviving fraction to 0.003, but in the presence of 10 mM MEA this same concentration of DDP reduced survival to only 0.12. Under these conditions a protection factor of 40 was found. Comparison of the slopes of the survival curves

Presented at the Conl’erence on Chemical Modification: Radiation and Cytotoxic Drugs, Key Biscayne, FL, Sept. 17-20, 1981.

Supported by National Cancer Institute Research Grant No. CA-20529-03.

Reprint requests to: Dennis C. Shrieve. Accepted for publication 5 November I98 I.

585

Radiation Oncology 0 Biology 0 Physics March-April 1982, Volume 8, No. 3 and No. 4 586

1.0

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DDP ALpne

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DDP Concmtration IlrMl

Fig. 1. Survival of EMT6/SF cells after exposure (1 hr) to DDP alone (0) or with concomitant exposure to 10 mM MEA (@). Values are the means of 3 experiments + s.e.m.

1.0

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Fig. 2. Survival of EMT6/SF cells after exposure to DDP for 1 hr followed by exposure to 10 mM MEA for 1 hr (0) or by incubation in drug-free medium for 1 hr prior to trypsinization (0). Dotted lines are redrawn from Figure 1 for comparison.

for cells treated with DDP alone (D, = 2.1 pg/ml) or with DDP plus MEA (D, = 7.5 pg/ml) reveals a DMF of 3. I.

When cells were treated with DDP for 1 hr, washed, and then exposed to 10 mM MEA for I hr, marked protection of the MEA-treated cells was observed (Fig 2), indicating that MEA protected EMT6/SF cells against DDP even when added after the cytotoxic agent had been removed. Cells that were incubated for 1 hr in drug-free medium after DDP treatment showed somewhat lower survival (D, = 1.5 pg/ml) compared to cells that were assayed for survival immediately after drug treatment (D, = 2.1 pg/ml). Incubation in 10 mM MEA for 1 hr after DDP removal protected by a factor of 2.4 (D, = 3.6

pg/ml). We also studied the ability of MEA to “rescue” cells

after removal of DDP. Cells were treated with 15 pg/ml (50 PM) DDP for 1 hr and then incubated in drug-free medium for various times before a 1 hr exposure to IO mM MEA. As the time between exposure to DDP and MEA increased, the protective effect of MEA progres- sively declined (Fig 3). Whereas simultaneous treatment resulted in a protection factor of 32.5, cells treated with MEA 4 hr after removal of DDP were spared by a factor of only 2.9 (Fig 3). When cells were incubated in drug- free medium after DDP treatment, survival decreased two-fold during the first hour, then remained constant to 5 hr post-treatment.

Protection against DDP cytotoxicity depended on the dose of MEA. Little protection was observed below 1 mM

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Fig. 3. Effect of time between DDP removal and exposure to 10 mM MEA. Cells were exposed to 15 wg/ml(50/1M) DDP for 1 hr and incubated in drug-free medium for times indicated before 1 hr further incubation with (0) or without (0) 10 mM MEA. Protection factor (A) (right-hand scale) is the ratio of the surviving fraction of MEA rescued cells to the surviving fraction of ceils treated with DDP alone.

Protection against cis-Pt (11) by cysteamine 0 DC. SHRIEVE AND J.W. HARRIS 587

MEA, but the protection factor increased sharply from 2.25 at 1 mM to a maximum of 32.0 at 5mM (Fig 4).

The protection by MEA added immediately after treat- ment with 14 rg/ml (471~M) DDP for 1 hr depended on the length of exposure to MEA. Fifteen min exposure to 10 mM MEA increased survival approximately two-fold but the maximum protection factor (10.3) was observed only when exposures were at least 120 min (Fig 5); exposures longer than 180 min caused toxicity.

DIhiCU!SSION Our results with cysteamine and DDP in EMT6/SF

cells are in good agreement with those obtained in L 1210 cells by Zwelling et al.” with thiourea. Cysteamine, like thiourea, reverses the cytotoxic effects of DDP when the sulfhydryl compound is present either concomitantly with the chemotherapy drug or within a few hours of its removal.

DDP reacts with cellular DNA to form interstrand cross-links and these cross-links are believed to be respon- sible for the drug’s antitumor activity.13 Thiourea’,4.‘4 and cysteamine have the unusual property of reversing these interstrand cross-links and restoring the DNA molecule to an apparently undamaged state as determined by cell viability.

We observed no protlection by MEA against BCNU and ADM cytotoxicity in vitro (data not shown), suggest- ing that the modest protection against BCNU toxicity in murine gut (DMF- I .5) seen by Wasserman et ~1.” and that seen against ADM Itoxicity by Phillips (oral commu-

1 I I I I

0.1 0.2 Cl.5 1 2 5

MEA Concentration ImMl

Fig. 4. Protection of EMT6/SF cells against DDP cytotoxicity by MEA as a function of concentration. Cells were exposed to 14rg/ml(47/1M) DDP for one hour with various concentrations of MEA present concomita.ntly.

0 30 60 120 180 TIME lminl

Fig. 5. Protection of EMT6/SF cells against DDP cytotoxicity by MEA as a function of time of exposure to MEA. Cells were exposed to 14 Kg/ml (47rM) DDP for 1 hr followed by incubation with 1OmM MEA for various times after DDP removal.

nication, Sept., 1981) in vivo with WR-2721 may be a result of physiological rather than biochemical effects. The physiological effects of WR-2721 (e.g., altered blood flow), remain to be fully investigated.

The major limiting toxicities of DDP are to the diges- tive and hematopoietic systems and particularly to the kidney.3*6.7V* Nephrotoxicity may be reduced by intensive hydration and administration of diuretics,6 but this approach may be contraindicated in some cases (e.g., congestive heart failure). It is, therefore, of interest to investigate alternate means of preventing DDP toxicity.

On the basis of the pharmacokinetic data available for WR-2721” and the in vitro results reported here with cysteamine (MEA), it seems reasonable to suggest that administration of WR-2721 within an hour after DDP may protect normal tissue while not interfering with tumor cell kill. Since 70-90s of DDP is cleared from plasma 15 min after i.v. injection in humans,’ administra- tion of WR-2721 at that time would avoid any significant inactivation of the chemotherapy drug in the blood yet give a maximum area under the concentration ver~tl~ time curve for WR-2721 in normal tissues while DDP was present. The latter point is particularly pertinent since our results indicate that protection against DDP toxicity by MEA is a function of both concentration and duration of exposure (Fig 4 & 5).

WR-2721 is now being tested in clinical trials. Optimal design of these trials requires an understanding of the potential interaction of all agents available for clinical use: radiation, chemotherapy drugs, radiation sensitizers and protectors. Our results suggest that the combination of DDP and WR-2721 might result in an enhanced therapeutic ratio. WR-2721 may also be a useful “res- cue” agent in case of accidental overdose of DDP. Further studies of the mechanism by which DDP exerts its cytotoxic effects, of the manner in which thiol compounds decrease this and of the pharmacokinetics of these com- pounds are clearly indicated.

588 Radiation Oncology 0 Biology 0 Physics March-April 1982, Volume 8, No. 3 and No. 4

REFERENCES I. Burchenal, J.H., Kalaher, K., Dew, K., Lokys, L., Gale, G.:

Studies of cross-resistance, synergistic combinations and blocking of activity of platinum derivatives. Biochimie 60: 96l-965,1978.

2. Chen, T.R.: In situ detection of mycoplasma contamination in cell culture by fluorescent Hoechst 33258 stain. Exp. Cell Res. 104: 255-262, 1977.

3. DeConti, R.C., Toftness, B.R., Langle, R.C., Creasey, W.A.: Clinical and pharmacological studies with cis-diam- minedichloroplatinum (II). Cancer Res. 33: I3 IO-I 3 15, 1973.

4. Filipski, J., Kohn, K.W., Prather, R., Bonner, W.M.: Thiourea reverses cross-links and restores biological activity in DNA treated with dichloro-diamminoplatinum (II). Science204: 181-183, 1979.

5. Harris, J.W., Phillips, T.L.: Radiobiological and biochemi- cal studies of thiophosphate radioprotective compounds related to cysteamine. Radiat. Res. 46: 362-379, 197 I.

6. Hayes, D.M., Cvitkovic, E., Golbey, R.B., Scheiner, E., Helson, L., Krakoff, I.H.: High dose cis-platinum diam- mine-dichloride. Amelioration of renal toxicity by mannitol diuresis. Cancer 39: 1372-l 38 I, 1977.

7. Madias, N.E., Harrington, J.T.: Platinum nephotoxicity. Am. J. Med. 65: 307-3 14, 1978.

8. Stark, J.J., Howell, S.B.: Nephrotoxicity of cis-platinum

(II) dichlorcdiammine. Clin. Pharmacol. Ther. 23: 461- 466, 1978.

9. Taylor, D.M.: The pharmacokinetics of cis-diamminodi- chloro-platinum (II) in animals and man: Relation to treatment scheduling. Biochimie 60: 949-956, 1978.

10. Wasserman, T.H., Phillips, T.L., Ross, G., Kane, L.S.: Differential protection against cytotoxic chemotherapeutic effects on bone marrow CFUs by WR-2721. Cancer Clin. Trials 4: 3-6, 198 I.

I I. Yuhas, J.M.: Active versus passive absorption kinetics as the basis for selective protection of normal tissues by S-2-(3-amino proplyamino)-ethyl-phosphorothioic acid. Cuncer Res. 40: I5 19-l 524, 1980.

12. Yuhas, J.M., Culo, F.: Selective inhibition of the nephrotox- icity of cis-dichlorodiammineplatinum (II) by WR-272 I without altering its antitumor properties. Cancer Treat. Rep. 63: 1439-1444, 1979.

13. Zwelling, L.A., Filipski, J., Kohn, K.W.: Effect of thiourea on survival and DNA cross-link formation in cells treated with platinum (II) complexes, L-phenylalanine mustard, and bis(2-chloroethyl)methylamine. Cancer Res. 39: 4989- 4995, 1979.

14. Zwelling, L.A., Kohn, K.W.: Mechanism of action of cis-dichlorodiammineplatinum (II). Cancer Treat. Rep. 63: l439-1444,1979.