Rat Prostatic Weight Regression in Reaction to Ketoconazole

[CANCER RESEARCH 48, 6063-6068, November 1, 1988] Rat Prostatic Weight Regression in Reaction to Ketoconazole, Cyproterone Acetate, and RU 23908 as Adjuncts to a Depot Formulation of Gonadotropin-releasing Hormone Analogue Steven W. J. Lamberts,1 Piet Uitterlinden, and Frank H. de Jong

Department of Medicine, Erasmus University, Rotterdam, The Netherlands

ABSTRACT

The effects of the s.c. administration of a depot formulation of the INTRODUCTION luteinizing hormone-releasing hormone (LHRH) analogue Zoladex were LHRH2 agonists initially stimulate pituitary gonadotropin studied in normal male rats, alone and in combination with three drugs with "antiandrogenic" action (ketoconazole, cyproterone acetate, and RU secretion resulting in an enhanced testosterone secretion, which 23908) on prostatic weight and on circulating hormone levels in order to is eventually followed by a depletion of pituitary LH stores and investigate whether these antiandrogens might prevent the LHRH-A- desensitization of LHRH receptors in the pituitary gland and of LH receptors in the gonads, and "medical" castration (1-4). induced initial increase in these parameters. These effects were compared with those caused by surgical castration. In addition the effects of the For these reasons treatment with LHRH-A results in inhibition antiandrogens on the activity of the hypothalamic-pituitary-adrenal axis of the growth of gonadal steroid-dependent tumors in humans were investigated. and animals (5-9). Several problems remain, however, with The depot LHRH analogue caused an initial increase in ventral pros regard to LHRH analogue treatment of male prostatic cancer tatic weight after 4 days but suppressed the prostatic and testicular patients: (a) the initial LHRH-induced increase in testosterone weights, the pituitary luteinizing hormone (1.11)content, and plasma LH secretion has been shown to cause a "flare-up" of the disease in and testosterone levels after 10 and 17 days. All three antiandrogenic drugs used prevented the initial LHRH analogue-induced rise in prostatic some patients (10, 11); (b) despite adequate suppression of testicular testosterone production during long-term LHRH an weight, while RU 23908 suppressed its weight after only 4 days. After 10 and 17 days cyproterone acetate and RU 23908 had a similar signifi alogue treatment, the remaining androgen production from the cantly greater suppressive effect on prostatic and testicular weights than adrenal cortex may play a role causing tumor progression in the LHRH analogue alone, while the additive inhibitory effect of keto some of these patients (12, 13), while most "antiandrogenic" conazole was smaller. Surgical castration suppressed prostatic weight drugs used to suppress this androgen production cause other significantly more after 4 days, while its effects after 10 and 17 days potential harmful effects on the hypothalamic-pituitary-adrenal were similar to that exerted by the combination of LHRH-A and RU axis in humans (14-17). All three problems cannot be investi 23908. gated properly in the model of the rat because the adrenal gland The antigonadotropic effect of cyproterone acetate and the indirect of this species does not produce androgens. An initial LHRH- gonadotropin-stimulating effects of ketoconazole and RU 23908 were not A-induced increase in prostatic weight and the effects of differ recognized in rats simultaneously treated with the LHRH analogue and did not interfere with the LHRH analogue-induced rapid depletion of the ent antiandrogenic compounds on the activity of the hypothal pituitary LH content and the decrease in circulating LH and testosterone amic-pituitary-adrenal axis in the rat might well be extrapolated levels. to the human situation, however. The LHRH analogue stimulated circulating progesterone and sup In the present study we evaluated the effects of the use of a pressed 17-hydroxyprogesterone levels. Ketoconazole and cyproterone long-acting depot formulation of the LHRH agonist Zoladex acetate caused disorders in the pituitary-adrenal axis via different mech alone and in combination with three differently acting "anti anisms: ketoconazole caused adrenal hypertrophy with normal circulating androgenic" drugs (ketoconazole, cyproterone acetate, and RU corticosterone levels caused by a compensatory increase in ACTH secre 23908) in normal male rats on organ weights, circulating and tion; while cyproterone acetate exerted glucocorticoid-like effects causing pituitary LH levels, circulating and pituitary ACTH concentra a depletion of the pituitary adrenocorticotropic hormone content, adrenal tions, and circulating progesterone and 17-hydroxyprogester atrophy, and lowered corticosterone levels. The addition of RU 23908 did not change the LHRH agonist-induced changes in adrenocortical one, testosterone, and corticosterone levels. The effects of these activity. The adrenal effects probably were the cause of a significant different combinations of these drugs were compared with the delay in body growth in the ketoconazole- and cyproterone-treated rats, effects of surgical castration. in comparison with those treated with the LHRH analogue alone or in combination with RU 23908. This study shows that: (a) the Zoladex depot formulation causes a MATERIALS AND METHODS transient increase in gonadotropin secretion but results in a suppression Animals. Young adult Wistar rats, weighing 200-220 g, were housed of circulating LH and testosterone levels after 10-17 days; (b) addition in a light (14 h/day, lights on at 6 a.m.) and temperature-controlled of ketoconazole, cyproterone acetate, or RU 23908 prevents the initial (22Â°C)environment. They received Purina rat chow and water ad LHRH analogue-induced stimulation of growth of the ventral prostate; (c) both ketoconazole and cyproterone acetate affect pituitary-adrenocor- libitum. Treatments. A group of 18 animals received a single s.c. injection of tical function via different mechanisms; and (antiandrogen RU 23908, together with the LHRH analogue, causes after 10 and 17 days the same "maximal" decrease of the prostatic weight as Ser-(But6AZGly'Â°)LHRH, ICI, Rotterdam, The Netherlands], in which the LHRH analogue is dispersed throughout a matrix of DL-polymer surgical castration without compromising the pituitary-adrenal axis. lactide-glycolide copolymer (18). In addition 3 groups of 18 animals were given injections of 0.9 mg of this LHRH analogue and also Received 1/4/88; revised 6/23/88; accepted 8/3/88. The costs of publication of this article were defrayed in part by the payment received daily i.p. injections of 10 mg/rat ketoconazole (Janssen, of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 2The abbreviations used are: LHRH, luteinizing hormone-releasing hormone; ' To whom requests for reprints should be addressed, at University Hospital LHRH-A, LHRH analogue; LH, luteinizing hormone; ACTH, adrenocorticotro Dijkzigt, 40 Dr. Molewaterplein, 3015 CD Rotterdam, The Netherlands. pic hormone. 6063

ventral prostate weight Beerse, Belgium), 20 mg/rat cyproterone acetate (Schering, Berlin, a of control Federal Republic of Germany), or 5 mg/rat RU 23908 (Roussel, Paris, France). In addition a control group of 18 rats and a group of 24 rats which had been castrated on day 0 received daily placebo injections. The 3 latter drugs were injected in 0.5 ml 1% gelatin (0.9% NaCl). On the morning of the last day of treatment, all animals were sacrificed by decapitation 30-60 min after the last injection. The organs were rapidly removed, deposited on ice, and weighed. Trunk blood was collected for plasma hormone determination (see below). The pituitary glands were <5 LHRH homogenized in 0. l N HC1 for determination of the LH and ACTH contents. Groups of 36 rats (6 rats of each of the treatment groups) were LHRH â€¢ketoconazole decapitated after 4, 10, and 17 days of treatment. From the first group LHRH * cyproterone acetate LHRH â€¢RU 23908 which was killed on day 4, additional tail blood was collected on day 2. surgical castration The dosages of the antiandrogenic compounds used in this study were chosen on the basis of data published in the literature. The dose of 10 mg/rat of ketoconazole i.p. (40 mg/kg/day) was chosen on the Fig. 1. Effect of s.c. administration of the LHRH analogue Zoladex alone or basis of studies by English et al. (19) and Trachtenberg et al. (20) who in combination with ketoconazole, cyproterone acetate, or RU 23908 and of surgical castration on the weight of the ventral prostate as percentage of the showed a maximal inhibitory effect on testosterone levels with 20 mg/ weight of the control rats. N = 6 animals/group; mean Â±SE + M (bars). kg twice daily s.c. and 10 mg/kg once daily i.p., respectively. The dose of 20 mg/rat cyproterone acetate (80 mg/kg/day) is maximal with regard to its antiandrogenic effect as shown in several studies reviewed LHRH agonist with RU 23908 significantly reduced prostate by Neumann (16). The dose of 5 mg/rat RU 23908 (20 mg/kg/day) weight after 4 days (P < 0.01 versus control and versus LHRH- was chosen on the basis of studies by Moguilewsky et al. (21) who A alone). The combined administration of the LHRH agonist showed that this is the maximal antiandrogenic dose in normal male and ketoconazole resulted after 17 days in a further decrease of rats. the prostatic weight than after LHRH-A alone (P < 0.01), while Hormone Determinations. Rat LH in the plasma and pituitary ex tracts was measured by a double antibody radioimmunoassay using both cyproterone acetate and RU 23908 had after 10 days National Institute of Arthritis and Metabolic Disease rat LH 1-1 for already caused a larger decrease of the weight of the prostate iodination and National Institute of Arthritis and Metabolic Disease in comparison with that caused by the LHRH agonist alone rat LH RP-1 as standard. The antiserum was obtained by immunization (P < 0.01 in both instances). After 17 days the combination of of rabbits with NIH-LH S 17, as described previously (22). The intraas- cyproterone acetate or RU 23908 with the LHRH agonist say variation was 16.3%, while the interassay variation mounted to caused no further reduction of the prostatic weight. At 10 and 10.8%. 17 days the prostatic weight was significantly lower in the ACTH was measured by direct radioimmunoassay in unextracted LHRH-A plus cyproterone acetate and RU 23908-treated rats plasma and in pituitary homogenates using a commercial kit supplied than in the animals treated with the combination of LHRH-A by Byk-Mallinckrodt (Dietzenbach, Federal Republic of Germany). The and ketoconazole (P < 0.05 in both instances). Actual weights intraassay variability of plasma ACTH determinations was 12%, and of the anterior prostate at the end of the 17-day experiment are of that of pituitary extracts was 6.8%. All samples were measured in duplicate within the same assay. shown in Fig. 2. Four days after surgical castration the weight Plasma testosterone was estimated as described by Verjans et al. of the ventral prostate had already decreased by 49% (P< 0.01 (23), and corticosterone was measured by radioimmunoassay in ethyl versus control), which was a greater decrease than that observed acetate extracts of the plasma, using the antiserum described by van by treatment with LHRH-A plus RU 23908 (P< 0.01). At days Zon et al. (24). This antiserum was a gift from Dr. T. Benraad, 10 and 17, however, the effects of surgical castration and the Nijmegen, The Netherlands. The intraassay variations for the testos treatment with LHRH-A plus RU 23908 were similar but in terone and corticosterone assays were 8.1 and 5.0% and the interassay all instances more inhibitory than that exerted by LHRH-A variations were 11.8 and 9.3%, respectively. Plasma progesterone and 17-hydroxyprogesterone concentrations were also measured by radio with or without ketoconazole and/or cyproterone acetate (P < immunoassay, using the procedures described earlier (25). The antisera 0.01 in all instances). used were raised in the department of Biochemistry, Division of Chem Changes of testicular weight differed from those of the pros ical Endocrinology, Erasmus University (Rotterdam, The Netherlands), tatic weight in that no initial increase was noticed after a 4-day and their specificity was described earlier (25, 26). Intraassay variations exposure to the LHRH agonist (Fig. 3). A significant decrease for the progesterone and 17-hydroxyprogesterone assays were 11.2 and of the testicular weight (P < 0.01) was observed 10 days after 12.7%, respectively, while the interassay variations were 13.7 and the administration of the depot formulation of the LHRH 11.8%, respectively. analogue, while there was a further decrease after 17 days of Statistics. All results have been expressed as means Â±SEM. Statis the analogue (P < 0.05). Combined administration of the ana tical evaluation was performed by analysis of variance. logue with ketoconazole initially did not further decrease testic ular weight, but it enhanced the effect of the LHRH analogue RESULTS after 17 days significantly (P< 0.05). Combined administration of the analogue with both cyproterone acetate and RU 23908 Administration of the depot formulation of the LHRH ago suppressed testicular weight after 4 days (P < 0.05 versus nist Zoladex s.c. resulted in a statistically significant increase control in both instances), while both combinations similarly of the weight of the ventral phosphate after 4 days (+27%; P < further suppressed testicular weights after both 10 and 17 days 0.01 versus control), which reversed to a decrease of 44% after in comparison with those measured after the LHRH analogue 10 and 17 days (P < 0.01 versus control in both instances; Fig. alone or after the combination of LHRH-A and ketoconazole 1). Simultaneous administration of the agonist and ketocona (in all cases P < 0.01 versus LHRH-A alone or in combination zole, cyproterone acetate, or RU 23908 in all instances pre with ketoconazole). The actual weight of the testes at the end vented the initial LHRH agonist-induced increase in the ventral of the 17-day experiment are shown in Fig. 2. prostatic weight after 4 days. Combined administration of the LHRH agonist administration resulted after 17 days in a 6064

ti MU ni,r weight of control

_ o IS ^ ,SÂ¿"i CSX ta. t" O)Â¿ pituitary c: ^ID_i^oj 31 >- 13 weight (mg)S9-8-7-i-Tr Â«u_L__ju _jo;

LHRH â€¢cyproterone acetate J^Tf T LHRH â€¢RU 23908

T T T 'r T i0Â£X1:*Â° TT Fig. 3. Effect of s.c. administration of the LHRH analogue Zoladex alone or 80-, in combination with ketoconazole, cyproterone acetate, or RU 23908 on the adrenals weight of the testis as a percentage of the weight of the control rats. N = animals (mg) 60- per group; mean + SEM (bars).

140- Table 1 Effect of s.c. administration of the LHRH analogue Zoladex alone or in combination with ketoconazole, cyproterone acetate, and RU 23908 and of 20- surgical castration on plasma LH concentrations, the pituitary LH content, and plasma testosterone concentrations of normal male rats The animals were decapitated and trunk blood was collected on days 4, 10, and 17, 30-60 min after the last injection of placebo or LHRH-A alone or in combination with ketoconazole, cyproterone acetate, or RU 23908, while tail prostate blood was collected on day 2 from the animals killed on day 4, 6 animals/group; (mg) mean Â±SEM. (ng/ml)DayControl Plasma LH 200- 427 1126 Â± Â±4 Â± LHRH-A 301224Â±38* 46 Â±12 16Â±1 11 Â±3Â» 100- LHRH-A + ketoconazole -t-24Â« 55 Â±6 13Â±3 8Â±2* LHRH-A -1-cyproterone acetate 271 + 35" 41 Â±5 13Â±321 6Â±72* LHRH-A + RU 2390846 217+Â±28IS"Day37 Â±66Day1022Â±2Day Â±72* Surgical castration 449 Â±49Â°312 Â±19Â°707Â±97Â°694 Â±102Â° LH content (Â»ig/pituitarygland) testes 2Control Day 17150 4 Day 10 Day (9) 3- 188Â±12 128 Â±16 148 Â± LHRH-ALHRH-A 5Â°'c1Â±4Â° 6 Â±1Â°'* 5 Â± ketoconazoleLHRH-A+ O.S"-'61 Â±3Â° 6 Â±1Â° 5 Â± + cyproterone acetate Â±3Â° 7 Â±1Â° 5 Â±0.5" LHRH-A + RU 23908 8 Â±2Â° 5 Â±1Â° 5 Â±5Â°'c Surgical castrationDay 136 Â±7 170 Â±19 256 Â±41* Plasma testosterone (nmol/liter) Day 2 Day 4 Day 10 Day 17 Control 24.8 Â±1.4 31+8 15 + 6 28 Â±3 LHRH-A 29. l Â±3.8 18 + 3 4 Â±0.5Â° LHRH-A + ketoconazole 21.1 Â±2.4 22 Â±2 6 Â±1Â° l Â±{"â€¢" LHRH-A + cyproterone acetate 23.9 Â±4.4 14 Â±2 5 Â±0.4Â° 1 Â±0.1"'d LHRH-A -t-RU 23908 23 Â±7.3 23 + 4 4 Â±0.4Â° l Â±0.1Â°'Â°' Fig. 2. Effect of s.c. administration of the LHRH analogue Zoladex alone or Surgical castration Not det.' Not det. Not det. Not det. in combination with ketoconazole, cyproterone acetate, or RU 23908 and of Â°P< 0.01 versus control. surgical castration on the weight of the anterior pituitary gland, of both adrenals, of the ventral prostate, and the testes after 17 days of treatment. N = 6 animals/ *P < 0.05 versus control. c P < 0.05 versus day 4. group; mean + SEM (ears). Ã¡P< 0.01 versus day 10. ' Not det., not detectable. significant decrease of the anterior pituitary weight of the animals (P < 0.01), which was prevented by the simultaneous alone). After the combination of the agonist and RU 23908 the administration of ketoconazole, cyproterone acetate, and RU weight gain was 37 Â±6 g, which is not significantly different 23908 (Fig. 2). LHRH agonist administration did not affect from the results in control and LHRH agonist-treated rats. the adrenal weight. The combination of the analogue with Plasma LH concentrations were highly stimulated on day 2 ketoconazole significantly increased and the combination with after the injection of the depot preparation of the LHRH cyproterone acetate significantly decreased adrenal weights, agonist (P < 0.01 versus control). This increase was not signif however (Fig. 2). icantly affected by the simultaneous administration of the three During the 17-day experiment control rats gained 59 Â±5 g antiandrogenic drugs and was similar to that observed after while the weights of the LHRH agonist-treated animals in surgical castration (Table 1). After 4 and 10 days the stimula creased by 48 Â±9g (P not significant), and that of the surgically tory effect of the agonist on LH secretion was no longer evident, castrated rats increased by 42 Â±8 g (P not significant). The while after 17 days circulating LH levels were significantly LHRH agonist plus ketoconazole- and the LHRH agonist plus suppressed to a similar extent in the LHRH analogue and cyproterone acetate-treated rats gained only 15 Â±9 and 8 Â±7 the three LHRH analogue plus ketoconazole-, cyproterone g, respectively (in both instances P < 0.01 versus LHRH-A acetate-, and RU 23908-treated groups of rats (P < 0.05 versus 6065

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1988 American Association for Cancer Research. OPTIMAL MEDICAL CASTRATION IN MALE RATS control in all instances). In contrast LH levels remained ele not cause cross-reactivity in any of the other radioimmuno- vated after surgical castration. The pituitary content of LH was assays used in this study. already significantly depleted on day 4 after LHRH agonist administration but was not affected by simultaneous adminis DISCUSSION tration of any of the three antiandrogenic drugs (Table 1). During further exposure to the LHRH analogue a further In the present study we evaluated whether a potential draw nonsignificant decrease of the LH content of the pituitary gland back of the treatment of men with metastatic prostatic cancer was noted on day 17, which again was not affected by the with LHRH analogues (an initial flare-up of the disease) might simultaneous administration of ketoconazole, cyproterone ace be prevented by the use of a depot formulation of a LHRH tate, or RU 23908. The LH content of the pituitary gland of analogue (Zoladex) alone or in combination with a drug which surgically castrated rats remained unchanged (days 4 and 10) either may suppress androgen production further (ketocona or increased (after 17 days) in the presence of greatly increased zole) or has antiandrogenic properties at the androgen receptor circulating LH levels (Table 1). level (cyproterone acetate, RU 23908). These effects were com Plasma testosterone concentrations had become undetectable pared with that of surgical castration. In addition we evaluated in surgically castrated animals from 2 days onward. They were the effect of these three antiandrogenic compounds on the not affected yet by medical treatment on days 2 and 4 but were pituitary-adrenal axis. significantly suppressed on day 10 (P < 0.01 versus control) The depot formulation in which the LHRH analogue Zoladex and even further on day 17 (P < 0.01 versus levels on day 10). is dispersed slowly degrades after s.c. injection and the analogue Again the combination of the LHRH analogue with any of the is continuously released into the systemic circulation for 21-28 three drugs mentioned above did not cause further suppressed days. Previous studies involving twice daily administration of testosterone levels, in comparison with the results after admin LHRH agonists in rats for several weeks showed incomplete istration of the LHRH analogue alone (Table 1). inhibition of testicular androgen production in most studies Finally, the effect of the LHRH analogue alone or in com (27, 28), resulting in regression of the ventral prostate to a bination with ketoconazole, cyproterone acetate, or RU 23908 lesser degree as reached after castration (28). Most injections was measured on the plasma concentrations and the pituitary of LHRH analogues even after 15-28 days of administration content of ACTH and on the circulating corticosterone, pro stimulated circulating LH and follicle-stimulating hormone gesterone, and 17-hydroxyprogesterone levels (Table 2). The levels (7, 27-29), in some instances by an ensuing pulse of combination of the LHRH analogue with ketoconazole resulted testosterone (19). The hypothalamic-pituitary-testicular axis of in higher circulating ACTH levels (P < 0.05 versus control), rats seems less sensitive to the inhibitory effects of LHRH without affecting the pituitary ACTH content and the corticos analogues than that of humans. Most reports regarding a lack terone levels. Cyproterone acetate, however, significantly sup of LH and testosterone suppression in men seem to reflect pressed the ACTH content of the pituitary gland (P < 0.05 different doses and routes of administration of the analogues versus control) and the corticosterone levels (P < 0.01 versus which resulted in insufficient absorption or circulating levels control), without affecting plasma ACTH concentrations. too low to achieve a therapeutic effect (30, 31). After implan The administration of the LHRH analogue resulted after 17 tation of the depot formulation of Zoladex in normal male rats, days in a significant increase in progesterone (P < 0.05 versus we observed only a transient stimulation of LH secretion on control) and a significant decrease in 17-hydroxyprogesterone day 2. No stimulation was observed after 4 and 10 days, while levels (P < 0.05 versus control; Table 2). The combined admin plasma LH concentrations were significantly suppressed after istration of the LHRH agonist with RU 23908 did not change 17 days. Testosterone levels remained unchanged on days 2 and these LHRH agonist-induced changes. Ketoconazole and cy 4 and were suppressed on days 10 and 17 after the injection of proterone acetate, however, prevented both the LHRH agonist- the depot LHRH analogue. In other studies it was shown that induced increase in progesterone (P < 0.05 versus LHRH-A the effect of the Zoladex depot preparation in rat lingers after alone in both instances), while cyproterone acetate prevented 21-28 days (32). Our data in the rat point to a considerable the LHRH agonist-induced decrease in 17-hydroxyprogester advantage of the use of such a long-acting depot formulation one (P < 0.05 versus LHRH agonist). The cyproterone acetate- over chronic twice daily s.c. administration of the LHRH mediated changes in the circulating progesterone and 17-hy analogue. However, the early effects of surgical castration still droxyprogesterone levels, however, are probably influenced by remain more profound. the fact that cyproterone acetate cross-reacted in the radio- Ketoconazole is an antifungal imidazole derivative which has immunoassay of these two hormones, especially 17-hydroxy been shown to inhibit androgen production via an inhibition of progesterone. Cyproterone acetate and the other drugs used did 17,20-lyase activity in the testis and the adrenal cortex, while

Table 2 Effect of the s.c. administration of the LHRH analogue Zoladex alone or in combination with ketoconazole, cyproterone acetate, or RU 23908 on plasma ACTH, corticosterone, progesterone, and 17-hydroxy-progesterone levels and the pituitary ACTH content of normal male rats The animals were decapitated and trunk blood was collected on day 17, 30-60 min after the last injection of placebo, ketoconazole, cyproterone acetate, or RU 23908. Six animals/group; mean Â±SEM. content 17-hydroxy ACTH ofACTH(ng/gland)88 corticosterone progesterone progesterone (ng/liter)112Â± (nmol/liter)166 (nmol/liter)1.0 (nmol/liter)2.4 Control 12 Â±3 Â±46 + 0.2 Â±0.3 LHRH A 271 Â±92 76 Â±6 418Â± 115 4.9 Â±2.1Â° 1.3 Â±0.2Â° LHRH-A ketoconazole 337 Â±85" 93 Â±5 278 Â±70 2.2 Â±1.3* 1.2 Â±0.1Â° LHRH A + cyproterone acetate 142 Â±36 60Â±3Â°72 54 Â±5C 2.1 Â±0.6* 3.3 Â±0.4* LHRH-A+ + RU 23908Plasma 173 Â±66Pituitary Â±4Plasma 308 Â±136Plasma 5.2 Â±2.7Â°Plasma 1.5 Â±0.1Â° Â°P< 0.05 versus control. * P< 0.05 versus LHRH alone. f /><0.01 versus control.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1988 American Association for Cancer Research. OPTIMAL MEDICAL CASTRATION IN MALE RATS at higher concentrations it also suppresses glucocorticoid pro been observed in humans. They became manifest during high- duction (14). dose cyproterone acetate therapy of children with precocious Ketoconazole administered in combination with the LHRH puberty, but no signs of adrenal insufficiency have been reported agonist was shown in this and an earlier investigation (19) to in adult men (17, 39). prevent the initial LHRH agonist-induced increase of prestaiic Finally the effects of the combination of the LHRH agonist weight, while after 17 days of treatment this combination of with a "pure" antiandrogen were investigated. In this study we drugs induced a significantly greater reduction of the testicular used RU 23908, a compound which is from a pharmacological and prostatic weights than the LHRH agonist alone. The ke- viewpoint quite similar to other compounds like flutamide (40). toconazole-induced acute blockade of 17,20-lyase activity and Antiandrogens of this type specifically block the action of the ensuing blockade in androgen production result in normal androgens at all target sites. Blockade of the pituitary androgen animals in a compensatory increase in gonadotropin secretion, receptors interferes with the feedback mechanism which con which in humans has been suggested to be a good test of the trols testosterone secretion (see Refs. 21 and 41). Consequently secretory reserve of LH and follicle-stimulating hormone re these compounds increase testicular androgen production, lease (33). In theory this ketoconazole-induced blockade of the which in turn counteracts the antiandrogen effect in the periph feedback regulation of gonadotropin secretion might have in eral organs like the prostate (41). In the case of RU 23908 the terfered with the desensitizing effect of the LHRH analogue at antiandrogenic activity of the compound on the prostate indeed pituitary levels. However, such an interference was not ob is fully manifest in castrated animals only but is weakened in served. Ketoconazole treatment resulted in an increase in the intact animals by the compensatory increase of testosterone size of the adrenal gland and of the anterior pituitary in com secretion which follows from the interference with the hypothal- parison with those observed after LHRH agonist administra amic pituitary-go nada I axis (21, 41). tion alone. This points to blocking effects of ketoconazole on In the present study it was shown that RU 23908 not only enzyme activities in the adrenal gland which is further sup prevented the initial LHRH agonist-induced increase in pros ported by the increased circulating ACTH levels and the in tatic weight but even suppressed it after only 4 days. Similar creased pituitary ACTH content. These changes probably re observations were reported previously (21). The combination flect the ketoconazole-induced partial inhibition of corticoster of the LHRH agonist and RU 23908 caused after 10 and 17 one biosynthesis which was compensated for by a rise in ACTH days a similar further decrease in testicular and prostatic weight synthesis and release and which indeed resulted in normal as observed after the combination with cyproterone acetate. circulating corticosterone levels (15). LHRH analogues induce The RU 23908-induced reduction of the feedback of testoster increases in plasma progesterone and decreases in 17-hydrox- one on pituitary gonadotropin secretion did not interfere with yprogesterone levels (see Refs. 34 and 35). Ketoconazole pre the LHRH agonist-induced early depletion of the pituitary LH vented the LHRH agonist-induced increase of progesterone content or with the course of plasma LH concentrations. In secretion, probably via an additional inhibitory effect in the addition there was no interference observed of RU 23908 with testes or in the adrenal gland at a "high" step in steroidogenesis, adrenocortical function, neither with regard to adrenal gland presumably the side chain cleavage of cholesterol (14, 36). weights, plasma and pituitary ACTH, and corticosterone levels However, the absence of suppressed corticosterone levels does nor with regard to the LHRH agonist-induced changes in not support the latter mechanism of action of ketoconazole. circulating progesterone and 17-hydroxyprogesterone levels. Cyoproterone acetate has powerful inhibitory effects on an In conclusion we showed in this study that the new depot drogen action while it also inhibits gonadotropin release (16). formulation of the LHRH analogue used rapidly induces a The drug can be classified as a partial agonist/antagonist, decrease in circulating testosterone levels and eventually signif because it was shown to have slight androgenic activity in icantly suppressed LH levels. This contrasts to earlier studies castrated animals (37). In addition it has progestational and in rats with intermittent LHRH agonist injections in which this glucocorticoid activities (16). In the present study cyproterone goal was not reached. In parallel recent studies in humans also acetate was shown to prevent the LHRH-induced initial in showed this long-term (21-28 days) effectivity of the depot crease of the prostatic weight, while it significantly further Zoladex preparation without the often described tendency of decreased the testicular and prostatic weights after 10 and 17 the gonadotropins or of testosterone to escape during intermit days. The antigonadotropic properties of the compound did not tent (p.o.) administration of LHRH agonists (42). All three help to prevent the LHRH agonist-induced initial marked in "antiandrogenic" compounds used prevented the LHRH ago crease of the plasma LH levels after 2 days, and it also did not nist-induced initial increase of the prostatic weight. However, increase the speed at which the circulating LH concentrations there were profound differences in the mechanism of action of and the pituitary LH content were suppressed. Cyproterone the drugs used. It is obvious from our study that the first choice acetate administration resulted in a significant decrease of the of treatment of metastatic prostatic cancer in humans should circulating corticosterone levels, of the ACTH content of the be the combination of a LHRH agonist with a "pure" antian pituitary gland, and of the adrenal gland weight, effects prob drogen like RU 23908, because this combination not only ably mediated via the glucocorticoid-like effects of the com prevents the initial increase of prostatic weight observed after pound (17, 38). Its discrepant effects on circulating progester the administration of the LHRH agonist but also actually one and 17-hydroxyprogesterone levels are difficult to interpret decreases the prostatic weight after only 4 days. No interaction because of cross-reactivity of the drug in the radioimmuno- of RU 23908 administration with the desensitizing effect of the assays used. We suggest that the decrease in plasma progester LHRH-A on pituitary gonadotropins was observed, while no one levels in comparison with the effect of LHRH-A alone action on the pituitary-adrenocortical axis occurred. The drug might be caused by the glucocorticoid-like suppressive effects was well tolerated as evidenced by the increase in body weight of cyproterone acetate on the adrenal gland, while the unex which was similar with that observed after LHRH agonist pected high 17-hydroxyprogesterone levels are probably caused administration alone. by the cross-reactivity of the drug in the radioimmunoassay. Our study showed that surgical castration induced a signifi The glucocorticoid-like effects of cyproterone acetate have also cantly more rapid decrease in the weight of the ventral prostate 6067

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1988 American Association for Cancer Research. OPTIMAL MEDICAL CASTRATION IN MALE RATS than the combination of LHRH-A and RU 23908, but this Inhibitors of Hormone Secretion and Action, pp. 132-160. London: Bailliere Tindall, 1987. difference had disappeared after 10 and 17 days. No data are 17. Girard, J., Baumann, J. B., Buhler, V., Zuppinger, K., Haas, H. G., StÃ¤up, available with regard to a longer follow-up. It is possible that J. J., and Wyss, H. I. Cyproterone acetate and ACTH adrenal function. J. Clin. Endocrinol. Metab., 47: 581-586, 1978. after a longer period of time ventral prostate weight would have 18. Hutchinson, F. G., and Furr, B. J. A. Biodegradable polymers for the been identically suppressed with all therapeutic regimens used. sustained release of peptides. Biochem. Soc. Trans., 13: 520-523, 1985. 19. English, H. F., Santner, S. J., Levine, H. B., and Santen, R. J. Inhibition of A shortcoming of the present study is that the additive testosterone production with ketoconazole alone and in combination with a inhibitory effects of the three antiandrogenic drugs after 10 and gonadotropin releasing hormone analogue in the rat. Cancer Res., 46: 38- 17 days on the LHRH analogue-induced decrease in the pros- 42, 1986. 20. Trachtenberg, J. The effects of ketoconazole on testosterone production and tatic weight might not pertain to men, inasmuch as in patients normal and malignant androgen dependent tissues of the adult rat. J. l rol.. a complete suppression of LH and testosterone levels to those 132: 599-601, 1984. 21. Moguilewsky, M., Fiet, J., Tournemine, C., and Raynaud, J.-P. Pharmacol observed after surgical castration is more easily reached during ogy of an antiandrogen, anandron, used as adjuvant therapy in the treatment LHRH analogue therapy. of prostate cancer. J. Steroid Biochem., 24: 139-146, 1986. 22. 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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1988 American Association for Cancer Research. Rat Prostatic Weight Regression in Reaction to Ketoconazole, Cyproterone Acetate, and RU 23908 as Adjuncts to a Depot Formulation of Gonadotropin-releasing Hormone Analogue

Steven W. J. Lamberts, Piet Uitterlinden and Frank H. de Jong

Cancer Res 1988;48:6063-6068.

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