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Endocrine Journal 1993, 40 (4), 425-430

An Inhibitory Effect of TZP4238*-on the Growth of -Sensitive Rat Prostatic Tumor (Dunning R3327)

T0M0HIK0 ICHIKAWA, SUSUMUAKIMOTO, ANDJUN SHIMAZAKI Departmentof Urology,School o f Medicine,Chiba University, Chiba260, Japan

Abstract. An inhibitory effect of TZP-4238, a newly synthesized , on the growth of Dunning R3327 rat prostatic tumor was studied and compared with that of (CMA). TZP-4238 markedly suppressed the growth of R3327 tumor. The inhibitory effect of TZP-4238 on the tumor was more potent than that on the . While the inhibitory effect of TZP-4238 on the weight of the ventral prostate was about 6 times as great as that of CMA, the suppressive effect of TZP-4238 on tumor weight was about 40 times as great as that of CMA. Serum levels of and showed no obvious changes after the administration of either TZP-4238 or CMA. The inhibitory effect of TZP-4238 on the growth of R3327 tumor indicated the application of the compound to human prostatic cancer.

Key words: Dunning R3327 prostatic tumor, Antaandrogen, TZP-4238. (Endocrine Journal 40: 425-430, 1993)

IT IS WELL known that human prostatic cancer is [6-7] and antiandrogen [8]. of an androgen-responsive nature. This justifies Antaandrogens are divided into two types; one is the clinical application of endocrine therapy such non-steroidal compounds such as and as , , luteinizing nilutamide, and the other is steroidal ones such as releasing hormone (LH-RH) and anti- acetate and chlormadinone acetate for the effective treatment of the (CMA). The non-steroidal exhibit a disease. While the of these hormonal potent antiandrogenic activity in rat and human, drugs has been recognized, they sometimes cause but these drugs increase blood levels of LH and undesirable side effects. In view of the above, testosterone (T) by blocking , development of a new drug with high efficacy and which is undesirable from a clinical point of view. low side effects has earnestly been desired. In contrast, steroidal antiandrogens do not induce Dunning R3327 rat prostatic tumor (R3327 such an increase on blood levels of LH and T tumor) has been considered to be a suitable animal because of their inhibitory effects on gonado- model of human prostatic cancer because the tropin secretion. TZP-4238 (17a acetoxy-6-chloro- tissue contains adenocarcinoma resembling hu- 2-oxa-4,6-pregnadiene-3,20-dione) is a newly man well-differentiated cancer [1], and the growth synthesized (Fig. 1). TZP- of R3327 tumor is androgen-sensitive [2]. The 4238 is substituted with oxygen in position 2 of the growth of R3327 tumor is suppressed by orchiec- A ring of CMA. Its antiandrogenic activities tomy [3], administration of [4-5], LH-RH against exogenous and endogenous androgens found in animal experiments were 11-13 times Received: January 4, 1993 and 3-5 times more, respectively, than those of Accepted: May 7, 1993 Correspondence to: Dr. Jun SHIMAZAKI, Department of CMA [9]. Urology, School of Medicine, Chiba University, Chuo-ku, Inohana 1-8-1, Chiba 260, Japan. * 17cr-acetoxy-6-chloro-2-oxa-4, 6-pregnadiene-3, 20-dione 426 ICHIKAWA et al.

the following formula: volume = L x W x Hx 0.5236 in L, length; W, width; H, height [10].

Treatment of tumor-bearing rats

Castration was performed via the scrotal route under hexobarbital anesthesia. TZP-4238 and CMA were suspended in aq. dist. containing 2% Tween 80 and administered orally to the rats once a day at daily doses of 2 mg/kg and 10 mg/kg of TZP-4238 or 10 mg/kg and 50 mg/kg of CMA with a volume of 5 ml/kg. Control rats received vehicle. At sacrifice, serum was collected for determination of androgen levels.

Determination of serum androgen levels The present study was conducted to compare the inhibitory effect of TZP-4238 on the growth of Serum T and dihydrotestosterone (DHT) levels R3327 tumor with that of CMA. were determined by radioimmunoassay [11].

Statistical analysis

Materials and Methods Statistically significant differences between the Animals TZP-4238 or CMA group and the control group were obtained by ANOVA and Dunnet's multiple Copenhagen rats were obtained from National comparison test [12]. The activity of TZP-4238 Cancer Institute (Bethesda, Md, USA) and have compared to that of CMA was determined by the been kept at our laboratory. Ten-week-old male four point assay. rats of this strain, weighing approximately 200 g, were used as host animals for the R3327 tumor. The animals were placed in cages regulated at 22 Results ± 2°C and 55 ± 15% humidity with illumination between 0800 h and 2000 h throughout the An inhibitoryeffect of TZP-4238 and CMA on the experimental period. Commercially available solid growtho f tumorsand weighto f tumor,prostate and testis pellets (CE-2, CLEA Japan Inc.) and tap water in tumor-bearingrats were provided ad libitum. When tumors appeared as subcutaneous Tumor transplantation and determination of tumor nodules 3 months after their implantation, admi- volume nistration of TZP-4238 or CMA was started (indi- cated as 0 on the abscissa in Fig. 2). The growth of The R3327 tumor was obtained from Papanico- the tumors in respective groups after the start of laou Cancer Research Institute and has been the administration is shown in Fig. 2. While the maintained by serial transplantation at our labora- tumors in the rats in the control group increased tory. A piece of tumor (approximately 5 mg wet exponentially, the growth of the tumors was weight) was transplanted subcutaneously into the markedly inhibited in the orchiectomized rats. right flank of the rats with a trocar under ether Growth of the tumors was retarded in the groups anesthesia. Three months after the transplanta- treated with TZP-4238 and CMA. TZP-4238 sup- tion, the tumor appeared as subcutaneous nodules pressed the growth of the tumors more strongly in the animals. The tumor-bearing rats were than CMA, but its effect was not stronger than that divided into six groups. Tumor volume was of orchiectomy. The inhibition of tumor growth calculated by measuring with calipers and using found in the TZP-4238 2 mg/kg-treated group EFFECTS OF TZP-4238 427

Fig. 2. Effects of or daily of TZP-4238 or chlormadinone acetate on growth of 13327 tumor. Treatments of animals in the experimental groups which started 3 months after implantation are as follows: Group 1, Vehicle (0); Group 2, TZP-4238 2 mg/kg/day (El); Group 3, TZP-4238 10 mg/kg/day (•); Group 4, Chlormadinone acetate 10 mg/kg/day (A); Group 5, Chlormadinone acetate 50 mg/kg/day (A); Group 6, Castration (•). Each point indicates the mean ± SEM for 9 animals. The day on which the treatments were started is indicated by 0 on the abscissa in the figure.

showed a tendency to be stronger than in the CMA by comparison with the control, the rate of 50 mg/kg-treated group. inhibition by TZP-4238 was higher for the tumor The relative weights of the tumor, ventral than for the accessory sex organs. The same prostate, seminal vesicle and testis 7 weeks after tendency was found in the rate of inhibition due to start of the administration were compared with castration. There was no significant change in the those of the control animals (Fig. 3). Changes in weight of the testis among the experimental tumor weight in each group were similar to those groups. in tumor volume, as shown in Fig. 2. The inhibi- tory effect of 2 mg/kg of TZP-4238 on the increase Serum T and DHT levels in tumor weight was more potent than that of 50 mg/kg of CMA, so that the inhibitory effect of Table 1 shows serum T and DHT levels 7 weeks TZP-4238 was about 40 times as great as that of after the start of administration. Although the CMA in terms of the molar base. The weight of the orchiectomized group showed a significant de- ventral prostate and seminal vesicle was suppres- crease in the serum T level when compared with sed by TZP-4238, but its inhibitory effect on these that of the control group, neither TZP-4238 nor accessory sex organs was weaker than that exerted CMA had an influence on the serum T level. None on the tumor. The inhibitory effect of TZP-4238 of the groups showed any significant changes in on the ventral prostate was about 6 times as great the serum DHT level. as that of CMA in terms of the molar base. When the inhibitory effect of TZP-4238 on the weight of the tumor and of accessory sex organs was assessed 428 ICHIKAWA et al.

Fig. 3. Effects of castration or daily oral administration of TZP-4238 or chlormadinone acetate on the weights of the R3327 tumor, ventral prostate, seminal vesicle and testis. Tumor- bearing animals indicated in Fig. 2 were sacrificed 7 weeks after the start of treatment and the weights of the respective organs were measured. The weights of the seminal vesicle and testis are totalled for both organs. Numbers on the abscissa in this figure indicate the experimental groups described in the legend to Fig. 2. Data are shown as the mean ± SEM as a percentage (9 animals) to the control group (9 animals, Group 1). Actual weights of the organs in the control group were as follows: tumor, 20.4 ± 4.3 g; ventral prostate, 112.1 ± 10.3 mg/100 g b.w; seminal vesicle, 272.6 ± 3.3 mg/100 g b.w; testis, 809.1 ± 16.2 mg/100 g b.w. Significantly different from control group: *, P<0.05; **, P<0.01; ***, P<0.001.

Table 1. Serum androgen levels

inhibitory action on the growth of the tumor. An Discussion inhibitory effect of TZP-4238 and CMA on the There are many reports indicating that the growth of tumors and on the weight of tumors and growth of R3327 tumor is suppressed by castration accessory sex organs was not associated with or antiandrogens such as flutamide [3, 8]. The decrease in either testis weight or the serum present study also showed that TZP-4238 and androgen level (Fig. 3, Table 1). Possible participa- CMA, potent steroidal antiandrogens, have an tion of the central in the secretion EFFECTS OF TZP-4238 429

of pituitary and testicular androgen in the tumor. is usually regarded as the main cause of a decrease It is generally known that the mitosis index is in testis weight and the serum androgen level [13]. different in normal and tumorous tissues. An It is, however, reasonable to suppose that the inhibitory effect of antiandrogens including TZP- inhibitory effects of the two compounds are 4238 and of orchiectomy on dividing cells seems mainly attributable to their direct action on the more potent than that on non-dividing cells. The prostate and testis rather than due to a decrease in difference between the rate of inhibition in the the pituitary gonadotropin and testicular tumor and accessory sex organs found in TZP- androgen secretion. This is supported by reports 4238 can be explained by the different population stating that TZP-4238 [14] and CMA [15] competi- of dividing cells in the respective tissues in addi- tively inhibit the binding of tion to the differences in delivery and with androgenic ligands in the rat prostate. in the drugs. In fact it was reported that the tumor Because the inhibitory effect of TZP-4238 on and prostate responded differently to the adminis- the rat prostate was 3-5 times as potent as that of tration of T [16]. CMA [9], the authors compared the pharmacolo- The growth of the R3327 tumor in the animals gical action of 2 and 10 mg/kg of TZP-4238 and was markedly inhibited by the oral administration that of 10 and 50 mg/kg of CMA in the present of TZP-4238. The results indicate a possible study. The suppressive action of TZP-4238 on the application of the antiandrogen for the treatment weight of ventral prostate was 6 times as great as of prostatic cancer. that of CMA in terms of the molar base, but its suppression of tumor weight was about 40 times as

great as that of CMA. It is obvious that the Acknowledgments inhibitory effect of TZP-4238 on the growth of the tumor is very powerful. Differences between the The authors are grateful to Mr. M. Mieda, inhibitory effects of TZP-4238 and CMA on tumor Teikoku Hormone Mfg. Co., Ltd. for cooperation growth may be attributable, at least in part, to the in this study, and to Teikoku Hormone Mfg. Co., uptake, retention and metabolism of these Ltd. for providing TZP-4238 and CMA.

References 1. Smolev JK, Heston WDW, Scott WW, Coffey DS analogs as adjuncts to agonists of luteinizing (1977) Characterization of the Dunning R-3327-H hormone releasing hormone in the treatment of prostatic adenocarcinoma: An appropriate animal experimental . Proc Natl Acad Sci model for prostatic cancer. Cancer Treat Rep 61: USA 84: 7275-7279. 273-287. 7. Ichikawa T, Akimoto S, Shimazaki J (1988) Effect 2. Isaacs JT, Coffey DS (1981) Adaptation versus of leuprolide on growth of rat prostatic tumor selection as the mechanism responsible for the (R3327) and weight of male accessory sex organs. relapse of prostatic cancer to androgen ablation EndocrinolJapon 35: 181-187. therapy as studied in the Dunning R-3327-H 8. Redding TW, Schally AV (1985) Investigation of adenocarcinoma. Cancer Res 41: 5070-5075. the combination of the agonist D-Trp-6-LHRH 3. Trachtenberg J (1987) Experimental treatment of and the antiandrogen flutamide in the treatment prostatic cancer by intermittent hormonal therapy. of Dunning R-3327-H prostate cancer model. J Urol 137: 785-788. Prostate 6: 219-232. 4. Daehlin L, Bergh A, Damber JE (1987) Direct 9. Mieda M, Oota Y, Saito T, Takahashi H, Shimaza- effects of oestradiol on growth and morphology of wa E, Miyasaka K. submitted. the Dunning R-3327-H prostatic carcinoma. Urol 10. Janik P, Briand P, Hartmann NR (1975) The effect Res 15: 169-172. of estrone-progesterone treatment on prolif- 5. Furuya Y, Sato N, Watanabe Y, Shimazaki J (1991) eration kinetics of hormone-dependent GR mouse Effects of estrogen on growth of androgen- mammary tumors. Cancer Res 35: 3698-3704. responsive rat prostatic tumor (R3327). Endocrinol 11. Hosaka M, Oshima H, Nankin H, Troen P (1978) Japon 38: 67-73. Studies of the human testis. XI Leydig cell clusters 6. Schally AV, Redding TW (1987) Somatostatin and levels of intratesticular testosterone and 20cr 430 ICHIKAWA et al.

dihydroprogesterone. J Clin Endocrinol Metab 47: compounds on the in vitro formation of androgen- 1164-1167. receptor complex. Folia Endocrinol Japon 67: 12. Sakuma A (1977) Statistical Method in Pharma- 1197-1210 (In Japanese). cometrics. Tokyo Univ. Press, 150-241 (In 15. Kodama T, Wakisaka M, Shimazaki J (1980) Japanese). Binding of chlormadinone acetate to cytosol from 13. Ito Y, Kurosawa I, Yamanaka H, Koya A, Imai K, human benign prostatic hypertrophy. Endocrinol Kosaku N, Shida K (1977) The mechanism of Japon 27: 53-57. antiandrogenic action of chlormadinone acetate. 16. Brooks JR, Berman C, Nguyen H, Prahalada S, Jpn J Urol 68: 537-552 (In Japanese). Primka RL, Rasmusson GH, Slater EE (1991) 14. Imai K, Watanabe K, Takahashi 0, Nakata S, Effect of castration, DES, flutamide, and the Nakazawa Y, Yamanaka H (1991) The effects of 5a-reductase inhibitor, MK-906, on the growth of anti-androgen TZP4238 (17a acetoxy-6-chloro-2- the Dunning rat prostatic carcinoma, R3327. Pros- oxapregna-4,6-diene-3,20-dione) and its related tate 18: 215-227.