Releasing Hormone Agonist and Chlormadinone Acetate on Rat Prostate Weight and Plasma Testosterone Levels

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Prostate Cancer and Prostatic Diseases (2003) 6, 66–72 ß 2003 Nature Publishing Group All rights reserved 1365–7852/03 $15.00 www.nature.com/pcan Effects of combination therapy with a luteinizing hormone- releasing hormone agonist and chlormadinone acetate on rat prostate weight and plasma testosterone levels

K Gotanda1*, A Shinbo1, M Okada1, Y Nakano1, H Kobayashi1, T Sasaki1, M Hagiwara1 & H Akaza2 1Pharmacological Research Department, Teikoku Hormone Mfg Co. Ltd, Kawasaki, Japan; 2Department of Urology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Japan

We investigated whether the combination of chlormadinone acetate (CMA) and a luteinizing hormone releasing hormone (LH-RH) agonist, leuprorelin acetate (leuprorelin), more markedly decreased ventral prostate and seminal vesicle weights and plasma sex hormone levels in male rats. Four weeks after administration of 0.28, 0.84 or 2.8 mg/kg of leuprorelin, ventral prostate weights significantly decreased (53.8, 54.4 and 64.1%) and the plasma testosterone levels significantly lowered, but not dose-dependently. After repetitive admin- istrations of 3 and 30 mg/kg/day of CMA, the rates of ventral prostatic atrophy were 37.1 and 65.9%, respectively. Although there was no change in the plasma testosterone level at 3 mg/kg, 30 mg/kg of CMA significantly decreased the level. A combination of leuprorelin (0.28 mg/kg) and CMA (3 or 30 mg/kg) more potently induced ventral prostatic and seminal vesicle atrophy than leuprorelin alone. Furthermore, a combination of leuprorelin and CMA (30 mg/kg) more markedly decreased the plasma testosterone level. According to the pharmacokinetic data for CMA in male rats, the doses of CMA correspond to the clinical dose. These findings suggest that combination therapy with an LH-RH agonist and CMA is more useful than therapy with the agonist alone in the treatment of prostate cancer. Prostate Cancer and Prostatic Diseases (2003) 6, 66–72. doi:10.1038/sj.pcan.4500617

Keywords: chlormadinone acetate; LH-RH agonist; testosterone; prostate cancer; combination therapy

Introduction Chlormadinone acetate (CMA) is one of the steroidal anti-androgen agents used in CAB therapy as well as Endocrine therapy is the most frequently employed monotherapy for prostate cancer in Japan.4,8 Steroidal treatment for prostate cancer, and luteinizing hormone anti-androgen agents have been known to possess both releasing hormone (LH-RH) agonists alone or in combi- actions of antagonizing androgen to bind its receptor and nation with anti-androgen agents are commonly lowering the plasma androgen levels. Moreover, CMA prescribed.1–7 This combination is sometimes referred to reduces the secretion of androgen not only from the as combined androgen blockade (CAB) therapy. gonads but also from the adrenal. A recent study has reported that CAB therapy with CMA and an LH-RH agonist achieved better clinical results than LH-RH agonist monotherapy for prostate cancer.4 However, it is still *Correspondence: K Gotanda, Teikoku Hormone Mfg Co. Ltd, 1604 unknown whether the better clinical result with CMA is Shimosakunobe, Takatsu-ku, Kawasaki 213-8522, Japan. due to the action of reducing the secretion of androgen Received 22 November 2001; revised 21 June 2002 from the gonads or adrenal. Synergistic effect of CMA and LH-RH agonist K Gotanda et al 67 In this study, we examined whether the combination of used to measure not only the plasma testosterone level an LH-RH agonist and CMA more potently decreased but also the levels of two gonadotropins (luteinizing accessory genital organ weights as well as plasma hor- hormone (LH) and follicle stimulating hormone (FSH)). mone levels. We also investigated whether the above- mentioned clinical results were supported at the basic experimental level. To avoid interference by adrenal Measurement of plasma testosterone, LH, androgen, we used male rats in which the secretion of FSH and CMA levels testosterone occurs exclusively in the testis. Plasma samples were isolated from the whole blood samples obtained. Plasma testosterone levels were measured by liquid chromatography – tandem mass spectrometry Methods (LC-MS/MS),9 because LC-MS/MS is more precise and reliable than the conventional radioimmunoassay (RIA) Animals method. Plasma LH and FSH levels were measured by RIA. Eleven-week-old male Sprague – Dawley rats (Charles The lower detection limits of plasma testosterone, LH and River Japan) were used. All experiments were conducted FSH were 0.05, 0.8 and 1.6 ng/ml, respectively. in accordance with the Guideline for Animal Experiments (Japanese Association for Laboratory Animal Science, 1987). As a negative and a positive control, a vehicle-treated group and a castration group were established, respectively. The number of animals per group was eight. Body weight was measured the day before the initial administration. Grouping was conducted so that there was no difference in mean body weight. In the castration group, bilateral orchiectomy was performed under anesthesia with ketamine (50 – 80 mg/kg i.p.). Four weeks after the ﬁrst administration of drugs to be tested, blood was collected under ether anesthesia after body weight was measured. Blood samples were used to measure the plasma testosterone levels. Then, the ventral prostate and seminal vesicle were extirpated, and wet weights were measured.

Drugs As an LH-RH agonist, a leuprorelin acetate (leuprorelin) microcapsule sustained-release preparation (Leuplin1) was used. CMA was synthesized at Teikoku Hormone Mfg Co. Ltd. Leuprorelin was suspended in the accompanying sus- pension to prepare a concentration of 1.875 mg/ml. CMA was suspended in 2% Tween 80/physiological saline to prepare concentrations of 0.6 and 6 mg/ml.

Dose-dependency of leuprorelin and CMA We investigated doses of 0.28, 0.84 and 2.8 mg/kg of leuprorelin, which was subcutaneously administered once to the back of rats. We also investigated doses of 3 and 30 mg/kg of CMA. Three or 30 mg/kg of CMA was subcutaneously administered to the back once a day for 4 weeks. Six hours after the ﬁnal administration, rats were sacriﬁced.

Combination with leuprorelin and CMA We compared the effects of 0.28 mg/kg of leuprorelin Figure 1 Effects of leuprorelin acetate on ventral prostate weight (upper panel) and seminal vesicle weight (lower panel) in rats. Data were obtained alone with a combination of leuprorelin and either 3 or 4 weeks after the administration. Each column represents the mean Æ s.d. of 30 mg/kg of CMA. Six hours after the ﬁnal administra- eight rats. *P < 0.05, vs vehicle-control (two-sample t-test). {P < 0.05, vs tion of CMA, rats were sacriﬁced. Blood samples were vehicle-control (Dunnett’s test).

Prostate Cancer and Prostatic Diseases Synergistic effect of CMA and LH-RH agonist K Gotanda et al 68 To evaluate whether the doses of CMA investigated in vesicle weights were reduced significantly (Figure 1). In this study were appropriate, 3 mg/kg of CMA was sub- comparison to the vehicle-treated group, the rates of cutaneously administered once, and plasma CMA levels prostatic atrophy were 53.8, 54.4 and 64.1% in the three were measured. At 1, 2, 3, 6, 8 and 24 h after CMA dose groups, respectively. The rates of seminal vesicle administration, blood was collected under ether anesthe- atrophy were 55.3, 57.4 and 61.1%, respectively. After the sia. At each time point, plasma CMA levels were three doses of leuprorelin, the plasma testosterone levels measured by gas chromatography – mass spectrometry were reduced significantly (Figure 2). (GC/MS) in three animals. The lower detection limit of There was no significant difference among the three plasma CMA was 0.2 ng/ml. dose groups in these leuprorelin-related actions. In addition, even at the highest dose (2.8 mg/kg) tested in this study, these leuprorelin-related effects were less Data analysis marked than the castration-related effects. Data are expressed as the mean Æ s.d. or mean alone. Wet organ weights are expressed as corrected values (mg/100 g body weight). When each plasma concentration was below the lower detection limit, the value was taken as one-half of the individual limit. Significance was tested as follows: when values were compared between two groups, the two-sample t-test was used. When values were compared among three or more groups, one-sided Dunnett’s test was used (SAS system Release 6.12). P < 0.05 was regarded as significant.

Results Dose-dependency of leuprorelin In comparison to the vehicle-treated group, the rates of atrophy of prostate and seminal vesicle in the castration group were 91.4 and 76.5%, respectively (Figure 1). The plasma testosterone level in the castration group was less than the lower detection limit, 0.05 ng/ml (Figure 2). After administration of 0.28, 0.84 and 2.8 mg/kg of leuprorelin, ventral prostate weights and seminal

Figure 2 Effects of leuprorelin acetate on plasma testosterone concentra- Figure 3 Effects of chlormadinone acetate (CMA) on ventral prostate tion in rats. Data were obtained 4 weeks after the administration. Each weight (upper panel) and seminal vesicle weight (lower panel) in rats. Each column represents the mean Æ s.d. of eight rats. *P < 0.05, vs vehicle-control column represents the mean Æ s.d. of eight rats. *P < 0.05, vs vehicle- (two-sample t-test). {P < 0.05, vs vehicle-control (Dunnett’s test). control (two-sample t-test). {P < 0.05, vs vehicle-control (Dunnett’s test).

Prostate Cancer and Prostatic Diseases Synergistic effect of CMA and LH-RH agonist K Gotanda et al 69 Dose-dependency of CMA been reported that plasma LH-lowering effects do not normally appear in rats, dissimilarly form humans. After repetitive subcutaneous administration of 3 and Moreover, plasma LH levels in rats may be less sensitive 30 mg/kg of CMA for 4 weeks, ventral prostate weights to change than FSH. and seminal vesicle weights were reduced significantly and dose-dependently (Figure 3). In comparison to vehicle-treated group, the rates of prostatic atrophy were Plasma CMA level after a single dose of CMA 37.1 and 65.9%, respectively. The rates of seminal vesicle atrophy were 27.9 and 71.5%, respectively. The plasma The maximal CMA concentration (Cmax 25.8 ng/ml) was testosterone level in the 30 mg/kg group was significantly obtained 1 h after the administration. The area under the lower than the vehicle-treated group. However, there was no significant change in the 3 mg/kg group (Figure 4).

Combination with leuprorelin and CMA The rates of prostatic atrophy by the combination of 3 or 30 mg/kg of CMA with 0.28 mg/kg of leuprorelin, compared with the vehicle-treated group, were 60.0 and 69.3%, respectively. These effects were significantly more potent than that of the rats given 0.28 mg/kg of leuprorelin alone (37.7%). The similar results were obtained in the seminal vesicle weights (Figure 5). A combination of 30 mg/kg of CMA with leuprorelin decreased the plasma testosterone level to 0.08 Æ 0.06 ng/ml. This value was significantly lower than that in the group treated with leuprorelin alone (0.44 Æ 0.30 ng/ml). On the other hand, the testosterone-lowering effects of leuprorelin were not influenced by a combination with 3 mg/kg of CMA (Figure 6). Administration of 0.28 mg/kg of leuprorelin alone significantly decreased the plasma FSH level. This effect was significantly potentiated when leuprorelin was combined with 3 or 30 mg/kg of CMA (Figure 6). However, FSH levels in rats seem to decrease less than those in humans. There was no change in plasma LH levels in any groups (data not shown). Generally, it has

Figure 4 Effects of chlormadinone acetate (CMA) on plasma testosterone Figure 5 Effects of leuprorelin acetate in combination with chlormadinone concentration in rats. Each column represents the mean Æ s.d. of eight rats. acetate (CMA) on ventral prostate weight (upper panel) and seminal vesicle *P < 0.05, vs vehicle-control (two-sample t-test). {P < 0.05, vs vehicle- weight (lower panel) in rats. Each column represents the mean Æ s.d. of eight control (Dunnett’s test). rats. {P < 0.05, vs Leuprorelin acetate monotreatment (Dunnett’s test).

Prostate Cancer and Prostatic Diseases Synergistic effect of CMA and LH-RH agonist K Gotanda et al 70

Figure 7 Plasma concentration of chlormadinone acetate (CMA) after single subcutaneous administration of 3 mg/kg of CMA in rats. Each point represents the mean of three rats.

with LH-RH agonists and anti-androgen agents has commonly been administered to improve clinical results.1 – 7 CAB therapy aims at not only eliminating testis-derived testosterone by LH-RH agonists but also blocking the action of adrenal androgen by anti-androgen agents, since it is thought that adrenal androgens are also impor- tant to the active androgen, dihydrotestosterone, in the prostate in men, 10,11 unlike in rats where the secretion of androgens occurs exclusively in the gonads. However, LH-RH agonists may be unable to sufﬁciently lower plasma testosterone levels, according to the report by Oefelein and colleagues.12 They have reported that the plasma testosterone level was still higher than a tentative castration level of 0.5 ng/ml in 5% of patients with prostate cancer treated with an LH-RH agonist monotherapy, and such percentage of patients increased to 13% if the tentative castration level was 0.2 ng/ml.12 Among anti-androgen agents, steroidal compounds can lower the plasma testosterone level by a central negative feedback mechanism and antagonize androgen to bind its receptor. In Japan, CMA, one of the steroidal anti-androgen agents, had been the only available anti-androgen agent for prostate cancer until a non-steroidal anti-androgen agent, ﬂutamide, was launched. Moreover, CMA has been known to reduce the secretion of adrenal androgen by inhibiting the secretion of adrenocorticotropic hormone Figure 6 Effects of leuprorelin acetate in combination with chlormadinone from the pituitary, as well as the secretion of gonadal acetate (CMA) on plasma testosterone (upper panel) and plasma follicle androgen. A recent clinical study has reported that combi- stimulating hormone (FSH) (lower panel) concentrations in rats. Each nation therapy with CMA and an LH-RH agonist showed { column represents the mean Æ s.d. of eight rats. P < 0.05, vs Leuprorelin better long-term results than the LH-RH agonist mono- acetate monotreatment (Dunnett’s test). therapy.4 These better long-term results are probably due to the synergistic effect of CMA on the plasma testosterone- lowering effect of the LH-RH agonist. Therefore, we concentration – time curve until 24 h after administration investigated whether CMA potentiated the plasma (AUC0 – 24hr) was approximately 225 ng h/ml (Figure 7). testosterone-lowering effect of the LH-RH agonists in rats, and whether the above-mentioned clinical results and the hypothesis about the mechanism of better clinical results were supported at the basic experimental level. Discussion We used leuprorelin as an LH-RH agonist in this study. The clinical dose of leuprorelin in the treatment of pros- Currently, LH-RH agonists are mainly prescribed for tate cancer is 3.75 mg at 4 week intervals, in Asia includ- endocrine therapy for prostate cancer, and CAB therapy ing Japan and in Europe. Based on body surface area in

Prostate Cancer and Prostatic Diseases Synergistic effect of CMA and LH-RH agonist K Gotanda et al 71 humans weighing 60 kg (1.62 m2) and rats weighing 100 g The effect obtained by combination with 0.28 mg/kg of (0.017 m2), the dose of 3.75 mg was calculated into leuprorelin and 30 mg/kg of CMA was more potent than approximately 0.4 mg/kg at 4 week intervals as the that by the dose escalating up to 10-fold (2.8 mg/kg) in corresponding dose for rats. In this study, we investigated leuprorelin monotreatment. This result suggests that the the dose-dependency of leuprorelin within a range mechanism of action differs between leuprorelin and including the calculated dose, that is, at 0.28, 0.84 and CMA. The mechanism of LH-RH agonists to lower 2.8 mg/kg. Four weeks after a single-dose subcutaneous plasma testosterone levels is thought to down-regulate administration of leuprorelin, prostate weight and the LH-RH receptors in the pituitary. Dissimilarly, a negative plasma testosterone levels in rats decreased significantly, feedback mechanism in the hypothalamus and pituitary even at the minimal dose investigated (0.28 mg/kg). seems to be involved in the plasma testosterone-lowering However, the actions of leuprorelin at higher doses effect of the steroidal anti-androgen agents, including were not enhanced, and did not reach the level in CMA. With regard to the negative feedback mechanism castrated rats. This result is consistent with results in the hypothalamus, Skinner et al reported that proges- reported previously, investigating the dose-dependency terone powerfully inhibits the secretion of LH-RH from of leuprorelin.13 the hypothalamus via a negative feedback mechanism at Repetitive subcutaneous daily administration of 3 and its own receptor in the hypothalamus.15 In this respect, 30 mg/kg of CMA for 4 weeks decreased dose-dependently CMA also probably inhibits LH-RH secretion from the prostate and seminal vesicle weights in rats. Dissimilarly hypothalamus, since CMA possesses progesterone to accessory genital organ weights, 3 mg/kg of CMA did mimetic action. Thus, it is suggested that CMA exhibits not influence the plasma testosterone levels, although negative feedback action in the hypothalamus as well as 30 mg/kg of CMA markedly decreased those levels. At in the pituitary and that such action is reflected in the the two doses of CMA investigated in this study, the rates results in the present study with rats and the better long- of prostatic atrophy were near to 50%, and the dose term results by CAB therapy using CMA in clinical. This ranges included the threshold of the testosterone- feature is not shared by non-steroidal anti-androgen lowering effect. These doses exhibit moderate effects agents, flutamide, bicalutamide, etc, and may be a and may not be excessive. To examine the relation major advantage in CAB therapy with LH-RH agonists. between the doses administered to rats and the clinical Furthermore, agents lowering the plasma testosterone dose, furthermore, we measured plasma CMA levels in levels may also be useful for preventing a worsening of rats after the administration of 3 mg/kg of CMA. As a symptoms caused by transient increases in testosterone, result, the AUC0 – 24hr for the plasma CMA level was that is, flare-up, after the initial administration of LH-RH approximately 225 ng h/ml. In humans, it was reported agonists. In clinical practice, it has been reported that CMA that the AUC0 – 24hr for the plasma CMA level was administered prior to leuprorelin inhibited a transient approximately 200 ng h/ml after oral administration of a increase in the secretion of LH in patients with prostate 14 16 25 mg tablet. Therefore, the AUC0 – 24hr after adminis- cancer in comparison with patients not given CMA, and tration of 3 and 30 mg/kg of CMA to rats may be one- that CAB therapy with CMA was found effective in 17,18 third and 3-fold of the AUC0 – 24hr in humans after oral inhibiting flare-up related to LH-RH agonists. administration of 100 mg, clinical dose for prostate cancer. Although this estimation should be verified further, doses of 3 and 30 mg/kg of CMA to rats may correspond to the clinical dose for prostate cancer. Conclusion Based on the results regarding the dose-dependency of leuprorelin and CMA, and pharmacokinetic profile of CMA, The steroidal anti-androgen agent, CMA, may have the we investigated and effects of combination of 0.28 mg/kg advantage of lowering the plasma testosterone level via of leuprorelin with 3 or 30 mg/kg of CMA in rats. central negative feedback mechanisms. The results of this Similarly to the effects after administration of CMA simple experiment using rats may show one of the alone, 3 mg/kg of CMA with leuprorelin did not influence mechanisms by which combination therapy with an LH- the plasma testosterone-lowering effect, but potentiated RH agonist and CMA achieves better clinical results than the prostatic and seminal vesicle atrophy by leuprorelin treatment with the agonist alone. CMA may be a useful alone. 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