GYNECOLOGIC

ONCOLOGY

40, 34-37 (1991)

Neutralizing Effect of Sodium Thiosulfate on Antitumor Efficacy of Cisplatin for Human Carcinoma Xenografts in Nude Mice MASAKI INOUE,

M.D., CHIKAKO SHIMIZU, M.D., HIROMU SHIMIZU, M.D., AND OSAMU TANIZAWA, M.D.

Department of Obstetrics and Gynecology,

Osaka University

Medical School, l-l-50

Fukushima Fukushima-ku,

Osaka 553, Japan

Received June 27, 1990

The present study was designed to examine the antitumor efficacy of combination chemotherapy with sodium thiosulfate (STS) and cis-diamminedichloroplatinum (CDDP) using a murine model. Xenograft tumors of the endometrial adenocarcinoma cell line responded well to intraperitoneal chemotherapy with CDDP in nude mice. However, addition of STS to CDDP treatment canceled the antitumor effect of CDDP. Such a finding was obtained in mice injected subcutaneously with STS even 72 hr after treatment with CDDP. On the other hand, alleviation of CDDPinduced side effects by administration of STS was not observed in mice treated by several modalities of combination chemotherapy with STS and CDDP. Thus, addition of STS to CDDP treatment reduced CDDP’s antitumor efficacy with only minimal alleviation of side effects, suggesting that it would provide no benefit in patients treated with CDDP. o WI Academic FWW, IIIC. INTRODUCTION

Although cis-diamminedichloroplatinum (CDDP) is one of the most effective drugs currently available for the treatment of ovarian carcinomas, the dose of CDDP appears to be a critical factor in the successful treatment of patients with refractory ovarian cancers [l-4]. However, the use of high-dose CDDP therapy is impaired by its dose-limiting toxic effects on renal function [1,2,4,5]. Therefore, as a new modality, two-route chemotherapy using the combination of intraperitoneal administration of high-dose CDDP with intravenous or subcutaneous administration of sodium thiosulfate (STS) has been reported to yield favorable results [6-91. As shown in many reports, two-route chemotherapy appears to alleviate nephrotoxicity [6,8,9]. However, it has not been fully clarified whether intravenous administration of STS actually reduces the antitumor efficacy of CDDP. These circumstances prompted us to investigate whether combination chemotherapy of CDDP and STS reduces the antitumor efficacy of CDDP in an animal model. 34 0090-8258/91$1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

MATERIALS

AND METHODS

An endometrial cancer cell line, Ishikawa, was previously established from a well-differentiated adenocarcinoma of the uterine endometrium of a 39-year-old woman [lo]. The cell line was maintained in RPM1 1640 medium (Handai Biken Labs, Osaka, Japan) containing 10% fetal calf serum (FCS), 100 pg/ml streptomycin, and 100 U/ml penicillin. The cells (2 x 10’) were inoculated into the backs of nude mice (BALB/C nu/nu) weighing 18-22 g at 6 to 7 weeks of age. When the tumor had grown to about 100 mm3 in volume, combination chemotherapy with CDDP and STS was started according to the following schedules. In each experiment, chemotherapy was done weekly for 4 weeks. In experiment A, 0.5 ml of saline was injected intraperitoneally. In experiment B, CDDP (5 mg/kg) alone was injected intraperitoneally. In experiment C, CDDP (5 mg/kg) and STS (400 mg/kg) were injected simultaneously into the peritoneal cavity and into the subcutaneous layer of mice, respectively. In experiment D, STS (400 mg/kg) was injected subcutaneously 8 hr after the intraperitoneal administration of CDDP (5 mg/kg). In experiment E, STS (400 mg/kg) was injected subcutaneously 24 hr after the intraperitoneal administration of CDDP (5 mg/kg). In experiment F, STS (400 mg/kg) was injected subcutaneously 72 hr after the intraperitoneal administration of CDDP (5 mg/kg). In experiment G, CDDP (5 mg/kg) was preincubated with human serum in the concentration of 5 pg/ml for 30 hr at 37°C and then injected intraperitoneally. CDDP was provided by Nippon Kayaku Company Ltd. (Tokyo). STS was obtained from Wako Pure Chemical Industries, Ltd. (Osaka). CDDP and STS solutions were each freshly prepared by dissolution in saline. To estimate the antitumor efficacy in each experiment,

NEUTRALIZATION

35

OF CDDP EFFECT BY Na THIOSULFATE

5-

O-

-5

1

IO-

15-

FIG. 1. Tumor growth curves of xenografts in nude mice which were treated by the following modalities of chemotherapy. In each experiment, chemotherapy was done weekly for 4 weeks. Experiment A: 0.5 ml of saline was injected intraperitoneally. Experiment B: CDDP (5 mg/kg) alone was injected intraperitoneally. Experiment C: CDDP (5 mg/kg) and STS (400 mg/kg) were simultaneously injected into the peritoneal cavity and in the subcutaneous layer of mice, respectively. Experiment D: STS (400 mg/kg) was injected subcutaneously 8 hr after the intraperitoneal administration of CDDP (5 mg/kg). Experiment E: STS (400 mg/kg) was injected subcutaneously 24 hr after the intraperitoneal administration of CDDP (5 mg/kg). Experiment F: STS (400 mg/kg) was injected subcutaneously 72 hr after the intraperitoneal administration of CDDP (5 mg/kg).

the tumor volume was calculated weekly according to the following formula: tumor volume = (the longest diameter) x (the shortest diameter)* 2

Chemotherapes

0

I

2

Experiments A:B:t--, C:M D:A--=4 E.W F.

3

4

5

6

7

M

8 Weeks

FIG 3. Changes in average body weight of mice treated by the modalities of chemotherapy described in the legend to Fig. 1.

In addition, to evaluate the adverse effects, the body weight of each mouse was determined weekly. Statistical differences in mean tumor volume and body weight (indicated as mean -+ SD) between experiments were calculated by Fisher’s exact test and Student’s twotailed t test. A P value less than 0.5% was considered significant. Each experiment consisted of five mice. The percentage of body weight loss compared with initial body weight was assessedfor each group. RESULTS

The present results of tests of the antitumor efficacy of combination chemotherapy with STS and CDDP are summarized in Figs. 1 and 2. Growth of the inoculated tumors was significantly retarded by the intraperitoneal administration of CDDP (P < 0.01). However, such tumor growth retardation was not observed in mice treated with STS simultaneously or 8 hr after the injection of CDDP (not significant; among experiments A, C, D, and E). Moreover, coadministration of STS even 72 hr after the injection of CDDP also reduced the antitumor efficacy of CDDP, although a slight retardation of tumor growth was observed (P < 0.05; at 2-5 weeks after treatment between experiments A and F) (Fig. 1). Interestingly, 0 I 2 3 4 5 0 I 8 Weeks potent tumor growth retardation was also observed in FIG. 2. Tumor growth curves of xenografts in nude mice which mice treated with CDDP preincubated in serum (P < were treated by the following modalities of chemotherapy. Experiment 0.01) (Fig. 2). A: 0.5 ml of saline was injected intraperitoneally. Experiment B: CDDP The adverse effects of the combination chemotherapy (5 mg/kg) dissolved in 0.5 ml saline was injected intraperitoneally. Experiment G: CDDP (5 mg/kg) incubated in serum for 30 hr at 37°C with STS and CDDP are summarized in Figs. 3 and 4. (protein-bound CDDP) was injected intraperitoneally. The administration of CDDP induced weight loss up to

36

INOUE ET AL.

tually, some investigators observed a reduction in both CDDP-induced toxicity and the antitumor activity of CDDP in mice bearing murine and human tumors [ 11,121. Thus, the question of whether STS reduces the antitumor efficacy of CDDP in clinical use persists. In the present study, addition of STS to CDDP treatment, even after an interval of 72 hr, completely reduced the antitumor efficacy of CDDP as well as CDDP-induced side effects. This finding suggests that the duration of contact as well as the dose of CDDP is another important factor in killing tumor cells. Curiously, protein-bound CDDP, which has shown no cytotoxity in vitro [14], demonstrated an antitumor effect against the endometrial cancer cell line inoculated into nude mice. Similar observations were also reported by other investigators [15]. Chmotheraples The mechanism of in vivo antitumor activity of proteinbound CDDP is not clear. It is speculated that proteinbound CDDP can gradually be converted to free CDDP FIG. 4. Changes in average body weight of mice treated with free as the binding of CDDP with serum protein loosens and CDDP or protein-bound CDDP. Experiments are described in the leg- gain cytotoxicity. Another possible mechanism is recomend to Fig. 1 and Fig. 2. position of antitumor active platinum complex within cancer cells after the pinocytotic uptake of protein-bound 15% at maximum. The coadministration of STS with platinum. Intraperitoneal administration of CDDP has been faCDDP alleviated this side effect to various degrees acvored among some clinical oncologists because a high cording to the various modalities. However, no statistical concentration of the drug can be achieved in the perisignificance was observed except for in the group treated toneal cavity [16-N]. Actually, several reports demonby stimultaneous administration of both STS and CDDP strate the effectiveness of intraperitoneal chemotherapy (P < 0.05; experiment C versus experiment B). Adminfor patients with small-volume disease [19-211. However, istration of STS 72 hr after injection of CDDP showed the results in patients with bulky disease have been disno alleviating effect (Fig. 3). Protein-bound CDDP also appointing [20,21]. Penetration of drugs from the periinduced weight loss to the same degree as did free CDDP toneal cavity into the tumor is restricted to around five (not significant; experiment B versus experiment G) (Fig. cell layers [22] and tumor cells implanted on the peri4). toneum are fed mainly from the surrounding circulation, not from ascites. The main source of antitumor activity DISCUSSION of intraperitoneally administered CDDP can be considRecent in vitro and in vivo studies have demonstrated ered to be free CDDP which has escaped from the perthat the cytotoxic effects of CDDP are canceled by ad- itoneal cavity and entered the systemic circulation. Thereministration of high-dose STS [5-9,11-131. It was spec- fore, we doubt the actual clinical value of intraperitoneal ulated that STS combines directly with CDDP to form a chemotherapy for disseminated ovarian cancers, compared to intravenous administration. In addition, we complex no longer capable of reacting with nucleophilic sites of other molecules. Accordingly, two-route chemo- worry whether addition of STS to intraperitoneal CDDP therapy using CDDP and STS was designed in which the treatment will eliminate the antitumor efficacy, as shown cancer cells come under direct attack by high doses in the present study. However, to draw a definitive conof CDDP administered intraperitoneally, while CDDP clusion regarding this issue, randomized clinical trials with entering the circulation from the peritoneal cavity may and without STS must be performed. be neutralized by STS administered intravenously or subcutaneously [6-9,131. Most investigators who have reREFERENCES ported the superiority of this new combination modality, compared with single treatment with CDDP, in humans 1. Young, R. C., Von Hoff, D. D., and Gormely, P. cis-Dichloroand animal models have shown only the neutralization of diammineplatinum(I1) for the treatment of advanced ovarian canside effects but have not fully mentioned the therapeutic cer, Cancer Treat. Rep. 63, 1539-1.544 (1979). efficacy of this therapy, although a few reports have 2. Frei, E., III, and Ganellos, G. P. Dose: A critical factor in cancer shown prolongation of the life span of mice [7,9]. Acchemotherauv. . I Amer. J. Med. 69, 585-594 (1980).

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