Combined Treatment with the 5Α-Reductase Inhibitor PNU 157706 and the Antiandrogen Flutamide on the Dunning R3327 Prostatic Carcinoma in Rats

Home , Antiandrogen, Turosteride

Endocrine-Related Cancer (1999) 6 429-435 Combined treatment with the 5α-reductase inhibitor PNU 157706 and the antiandrogen flutamide on the Dunning R3327 prostatic carcinoma in rats

T Zaccheo, D Giudici and E di Salle

Department of Pharmacology, Oncology Research, Pharmacia & Upjohn, Nerviano, Italy (Requests for offprints should be addressed to T Zaccheo, Department of Pharmacology, Oncology Research, Pharmacia & Upjohn, Viale Pasteur 10, 20014 Nerviano (MI), Italy)

Abstract The steroid 5α-reductase enzyme catalyzes the conversion of testosterone to the potent androgen 5α-dihydrotestosterone (DHT). PNU 157706, a novel, potent and selective dual 5α-reductase inhibitor, was reported to be effective in inhibiting the growth of established tumors in the Dunning R3327 rat prostatic carcinoma model. We have studied the efficacy of combined treatment with PNU 157706 and the antiandrogen flutamide in this prostatic tumor in rats. Rats with tumor diameters of about 1 cm were treated orally 6 days a week for 9 weeks with PNU 157706 (10 mg/kg per day) alone or in combination with flutamide (1 and 5 mg/kg per day). Animals were killed 24 h after the last treatment and ventral prostates were removed for testosterone and DHT determination. PNU 157706 reduced the growth of established tumors by 36%; flutamide showed a slight effect at 1 mg/kg per day (24% inhibition), while at the dose of 5 mg/kg per day it reduced tumor growth by 48%. The combination of PNU 157706 with the lower dose of flutamide caused an additive tumor growth inhibition (60%) and the combination with the higher dose of flutamide resulted in a better inhibition of tumor growth (68%) than did either treatment alone. Castration resulted in marked tumor growth inhibition (76%). Ventral prostate weight was more markedly reduced by PNU 157706 treatment than by flutamide; combined treatment was as effective as castration. Prostatic DHT content was markedly reduced by PNU 157706 (93%), whereas prostatic testosterone increased (137%). Concomitant treatment with flutamide partially antagonized the testosterone increase induced by PNU 157706 and did not modify the already considerable suppression of DHT. These data show that the inhibitory effects of PNU 157706 and flutamide on Dunning prostatic tumor growth are additive, thus supporting the rationale of this combination therapy in advanced prostate cancer, in order to achieve adequate androgen blockade with minimal side-effects. Endocrine-Related Cancer (1999) 6 429-435

Introduction With the advent of routine prostate specific antigen (PSA) testing (Stamey et al. 1989, Partin et al. 1990) and Androgen deprivation with the combination of luteinizing the consequent early diagnosis of the disease, patients at hormone-releasing hormone analogs and antiandrogens is earlier stages of prostatic cancer may be candidates for the most effective form of treatment for metastatic hormonal therapy. As a result, concerns about the quality carcinoma of the prostate (Labrie et al. 1985, Crawford et of life associated with androgen ablation therapy become al. 1989). However, androgen deprivation therapy has important. side-effects that are the result of castrate levels of serum Testosterone acts in the prostate through the local testosterone, including impotence, loss of libido and formation of its potent androgenic metabolite 5α- decreased muscle mass (Vogelzang & Kennealey 1992, dihydrotestosterone (DHT), synthesized by the enzyme Hsieh & Simons 1993). 5α-reductase (Bruchovsky et al. 1988). Inhibitors of this

Downloaded from Bioscientifica.com at 09/28/2021 03:02:34AM via free access Zaccheo et al.: PNU 157706 and flutamide in rat prostatic tumor enzyme provide a novel and selective approach to (22±2 °C) on a circadian rhythm of 12 h of light (0600- androgen deprivation for benign and malignant prostatic 1800 h) and 12 h of darkness. Animal care was in diseases. Both in experimental animals and in humans, accordance with institution guidelines. treatment with such inhibitors results in reduced prostatic DHT without lowering serum testosterone, thus preserv- Prostatic tumor model ing sexual function. However, experimental data in The Dunning R3327 prostatic carcinoma was kindly prostatic tumor therapy indicate that these compounds provided by Dr H Altman, Papanicolau Cancer Research have less effect on tumor than on normal prostate (Brooks Center, University of Miami (FL, USA). The tumor was et al. 1991, Zaccheo et al. 1997), likely because of the maintained by serial transplantation in Copenhagen rats. remaining testosterone which eventually interacts with the The tumor was passed by harvesting fresh tumor, dicing it androgen receptor and stimulates tumor growth. There- with sterile scissors in sterile 0.9% NaCl solution and fore, a combination therapy with a 5α-reductase inhibitor aseptically implanting a single tumor fragment (approxi- and an androgen receptor antagonist to neutralize the mately 3-4 mm in size) subcutaneously in the flank of the remaining testosterone could be of potential value in the recipient animal under mild diethylether anesthesia. therapy of metastatic prostate cancer. PNU 157706 (N-(1,1,1,3,3,3-hexafluorophenylpro- Treatment and experimental setup pyl)-3-oxo-4-aza-5α-androst-1-ene-17β-carboxamide) is a novel dual inhibitor of 5α-reductase. The compound was Tumor-bearing rats with tumor diameters of 0.5-1 cm highly potent in inhibiting human recombinant 5α- were randomly assigned to the different treatment groups. PNU 157706 (synthesized at the Chemistry Department, reductase type I and type II isozymes, showing IC50 values of 3.9 and 1.8 nM, and therefore it was several times more Pharmacia & Upjohn, Italy) and flutamide (kindly supplied by Schering Plough, Kenilworth, NJ, USA), were potent than finasteride (IC50 values of 313 and 11.3 nM), particularly on type I isozyme (di Salle et al. 1998). Tested suspended in 0.5% Methocel (methylcellulose 400; Dow on a crude preparation of rat prostatic 5α-reductase, PNU Chemical, Midland, MI, USA) containing 0.4% Tween 80 (Merck, Darmstadt, Germany) and administered orally in 157706 caused enzyme inhibition with an IC50 value of 34 nM, compared with 58 nM shown by finasteride. In adult a volume of 5 ml/kg, 6 days a week for 9 weeks. Castration rats a single oral dose of 10 mg/kg PNU 157706 caused a was performed on the first treatment day under diethyl- marked and longer lasting reduction of prostatic DHT ether anesthesia. content than did finasteride (at 24 h 89% and 47% Tumor growth was followed by measuring the two inhibition respectively). In prepubertal testosterone- or perpendicular diameters with calipers and tumor weight 2 DHT-implanted castrated rats, PNU 157706, at the oral was calculated according to the formula: d ×D/2, where d dose of 10 mg/kg per day for 7 days, markedly reduced is the minimum and D is the maximum diameter (Geran et ventral prostate weight in testosterone- but not in DHT- al. 1972). The area under the tumor weight to time curve implanted animals, thus showing a lack of antiandrogen (AUC) was calculated by the linear trapezoidal method up activity. After repeated (28 days) oral dosing in rats, the to 9 weeks. Animals were killed 24 h after the last compound was found to be 16-fold more potent than treatment and ventral prostate and seminal vesicles were finasteride in reducing prostate weight. PNU 157706 has removed and weighed. Ventral prostates were immedi- been reported to be effective in inhibiting the growth of the ately frozen on dry ice and stored at -20 °C for androgen androgen-dependent Dunning R3327 prostatic carcinoma hormone assays. in the rat (Zaccheo et al. 1998a). In this study we have investigated the antitumor effect Testosterone and DHT determinations of a combined therapy using PNU 157706 and the Prostatic concentrations of testosterone and DHT were antiandrogen flutamide on the Dunning prostatic tumor measured by specific RIAs, after sample extraction and model; tumor growth rate as well as endocrine organ purification by high-performance liquid chromatography weights and prostatic DHT and testosterone content were (HPLC). Each prostate sample (pool of two to three evaluated. prostates in the castrated group) was thawed and homogenized in 4 ml acetone/acetonitrile mixture (1:1) Materials and methods with a Polytron apparatus. After extraction and centrifu- gation, the organic phase was desiccated, the dried extract was dissolved in 5% methanol aqueous solution and Animals purified on Waters Sep-Pack Plus C18 minicolumns Male Copenhagen rats, weighing approximately 200 g, (Millipore Corporation, Milford, MA, USA), using 60% were supplied by Harlan Nossan Srl (Correzzana, Italy). acetonitrile aqueous solution as the eluting solvent. The Animals were housed in temperature-controlled rooms dried extract was then dissolved in 38% acetonitrile

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Table 1 Effect of 9-week treatment with PNU 157706 or flutamide given alone or in combination, on Dunning R3327 prostatic tumor growth in rats Treatment (mg/kg per day p.o.) Tumor growth Final tumor AUC (0-9 week)° PNU 157706 Flutamide No. of rats (weight in g) (% inhibition) (g x week) (% inhibition) Control 8 18.2±2.9§ — 58.4±10.5 — 10 — 9 13.7±1.0 25 37.1±2.6 36 — 1 9 15.2±2.1 16 44.5±4.9 24 — 5 9 10.5±2.2 42 30.4±5.3 48 10 1 9 7.6±1.3 59 23.4±3.7 60 10 5 9 5.9±1.2 67 18.5±3.1 68 Castration 9 3.0±0.6 83 14.2±2.6 76 ° AUC=area under the tumor weight to time curve, calculated by the linear trapezoidal method from 0 to 9 weeks. § Means±S.E.

aqueous solution and injected into a Hewlett Packard 1050 2.2 ml respectively, then desiccated in a centrifugal HPLC System. Separation was performed using a evaporator and subjected to RIA. Testosterone and DHT 3.9×300 mm Nova-Pack Waters C18 reversed-phase levels in the resuspended samples were estimated, in column (particle size 3 µm), in isocratic conditions, at a duplicate, by using the [3H]testosterone and [3H]DHT column temperature of 40 °C. DHT- and testosterone- RIA kits supplied by bioMérieux (Marcy l’Etoile, France) containing fractions were collected in volumes of 2.7 and and ICN Biomedicals (Costa Mesa, CA, USA)

Figure 1 Effect of 9-week oral treatment with PNU 157706 (10 mg/kg per day) or flutamide (1 and 5 mg/kg per day), given alone or in combination, on tumor growth in the Dunning R3327 prostatic tumor model in rats. Castration was performed on the first treatment day. Groups contained eight to nine rats.

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Table 2 Statistical analysis of final tumor weight (week 9). Results (P values) of multiple comparison test. Groups with a P value ≤0.05 were considered statistically different with the two-tail test PNU 157706 + PNU 157706 + PNU 157706 Flutamide 1 Flutamide 5 Flutamide 1 Flutamide 5 Castration Control 0.00799 0.75077 0.00003 0.00003 0.00003 0.00003 PNU 157706 1.00000 0.46164 0.00003 0.00003 0.00003 Flutamide 1 0.00143 0.00003 0.00003 0.00003 Flutamide 5 0.00003 0.00218 0.00003 PNU 157706 + 1.00000 0.00344 Flutamide 1 PNU 157706 + 0.72941 Flutamide 5 Flutamide 1, dose of 1 mg/kg per day; flutamide 5, dose of 5 mg/kg per day.

respectively. The final sensitivity for testosterone and reduced tumor growth by 48%. Combination of PNU DHT assays were 0.2 and 1 ng/g prostate respectively (for 157706 with the lower dose of flutamide caused an a sample of at least 0.2 g). additive tumor growth inhibition (60%), whereas combination with the higher dose of flutamide resulted in greater Statistical analysis inhibition of tumor growth (68%) than did either treatment The statistical analysis of the effect of the various alone. Statistical analysis of tumor growth over time for all treatments on tumor growth (mean tumor weight from groups showed significant differences between groups week 0 to week 9) was assessed by use of an ANOVA (F=9.3; P<0.001), times (F=207.9; P<0.001) and inter- split-plot design (7 groups×10 observation times). Tukey’s actions (F=9.1; P<0.001). Analysis of final tumor weight HSD test was used for post hoc comparison. The analysis data (Tables 1 and 2) indicated that all treated groups, was performed using the SPSS statistical package. except that treated with 1 mg/kg per day flutamide Statistical significance of data on endocrine organ (P=0.75), were significantly different from the control weight and DHT and testosterone prostatic content was group. Groups treated with the combinations were signifi- measured according to Dunnett’s test. cantly different from those treated with PNU 157706 or flutamide alone. Final tumor weight in the group treated with the combination of PNU 157706 and flutamide at 5 Results mg/kg per day was not different (P=0.73) from that of the castrated group. Effect on tumor growth Figure 1 and Table 1 show the effect of PNU 157706 (10 Effect on endocrine organ weight mg/kg per day) alone or in combination with flutamide (1 Ventral prostates and seminal vesicles of tumor-bearing and 5 mg/kg per day), given orally for 9 weeks, on the rats were excised and weighed when they were killed after growth of the transplanted Dunning tumor. The weekly 9 weeks. The relative weights of these organs are shown recorded tumor weights are reported in Fig. 1. The mean in Fig. 2. PNU 157706 caused a decrease of ventral tumor weight at the beginning of the experiment (week 0) prostate and seminal vesicle weight of 76% and 46% was similar in all groups (P=1.000). The AUC(0-9 weeks) respectively, a greater effect than that observed after values, expressed as g×week, are reported in Table 1. flutamide treatment. In fact flutamide, at the dose of 1 mg/ During the 9-week observation period, the Dunning tumor kg per day, reduced prostate and seminal vesicle weights grew progressively in the control group (AUC, by 23% and 19%, and by 50% and 38% at the dose of 5 mean±S.E.=58.4±10.5), whereas castration resulted in a mg/kg per day. Combined treatment with PNU 157706 marked decrease in tumor growth (76% inhibition), de- and 1 mg/kg per day flutamide caused a reduction of monstrating the androgen responsiveness of the tumor. prostate and seminal vesicle weights of 81% and 57% Treatment with the 5α-reductase inhibitor PNU 157706 respectively, whereas the combination with 5 mg/kg per decreased tumor growth by 36%. Flutamide, at the dose of day flutamide reduced the weight of these organs by 86% 1 mg/kg per day, caused a slight inhibition of tumor and 64% respectively, which was similar to the effect of growth (24%), whereas at the dose of 5 mg/kg per day it castration (92% and 71% reduction).

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Discussion Although hormonal treatment of advanced prostatic cancer has relatively small effects on patient survival, it is an important development in palliative care, improving the patient's quality of life. Since the initial report by Huggins & Hodges (1941), the hormonal therapy of advanced or metastatic prostate cancer has traditionally consisted of surgical or chemical castration in order to block testicular androgens and therefore to lower circulating testosterone. An important consideration in the endocrine therapy of prostate cancer is that both testes and adrenals provide almost equal amount of the androgens responsible for cancer growth (Labrie et al. 1993b). In fact, in humans, unlike other species (e.g. rats), the adrenals secrete a large amount of the inactive androgen precursor dehydroepiandrosterone, which is converted into DHT within the prostate. Therefore, castration (surgical or medical) causes 90-95% reduction in serum testosterone concentration, although a much smaller effect is seen on the intraprostatic concentration of DHT, the potent metabolite of testosterone, that remains at about 40% of that measured in intact men (Geller et al. 1984, Labrie et al. 1993b). More recently, combined therapy involving surgical or medical castration plus an antiandrogen to block the remaining adrenal androgens has been tried in an attempt to achieve total androgen blockade (Labrie et al. 1993a). However, such a systemic reduction Figure 2 Effect of 9-week oral treatment with PNU 157706 (10 in the circulating testosterone level has several side- mg/kg per day) or flutamide (1 and 5 mg/kg per day), given effects, including sterility, impotence, loss of libido, hot alone or in combination, on the relative weight of ventral flushes and decreased muscle mass (Vogelzang & prostate and seminal vesicles in rats bearing the Dunning Kennealey 1992, Hsieh & Simons 1993). Inhibitors of 5α- R3327 prostatic carcinoma. Bars represent means±S.E. (eight to nine animals per group). **P<0.01 vs controls by Dunnett's reductase provide a novel and selective approach to test. androgen deprivation. Treatment with such inhibitors results in reduced prostatic DHT and consequently in Effect on prostatic DHT and testosterone involution of prostate size (Schroder 1994). Oral treatment for 9 weeks with PNU 157706 resulted in Two 5α-reductase isozymes with different charac- a marked decrease (93%) in prostatic DHT content, teristics have been described: type I and II (Andersson & measured 24 h after the last dose (Fig. 3). As expected for Russell 1990). While type II is mainly localized in the 5α-reductase inhibitors, the prostatic testosterone content prostate, type I is predominant in the peripheral tissues, increased in PNU 157706-treated animals (137%). Flut- mainly the skin and the liver. As regards the tumor 5α- amide caused a reduction of DHT (48% and 50%) and an reductase enzyme, it has been reported that both isozymes increase in testosterone content (126% and 33%) at the are expressed in human prostatic carcinoma (Silver et al. doses of 1 and 5 mg/kg per day respectively. 1994, Bonkhoff et al. 1996). No data are so far available Combined treatment with PNU 157706 and flutamide on the characteristics of the enzyme in the Dunning at 1 or 5 mg/kg per day was as effective as PNU 157706 prostatic tumor, but both isoforms of 5α-reductase are alone in reducing DHT content of the prostate (92% or expected to be expressed in this prostatic tumor tissue, as 93% vs 93%) but was more effective than flutamide alone reported for the normal rat prostate (Normington & (48% or 50%). The prostatic testosterone content Russell 1992). increased to a lesser extent in the groups treated with the PNU 157706, a dual 5α-reductase inhibitor (di Salle et combination (69% or 72%) in comparison with the PNU al. 1998), has previously been reported to be effective, as 157706-treated group (137%). Castration markedly re- a single agent, in reducing Dunning prostatic tumor duced prostatic DHT (97%) and testosterone (88%) growth (Zaccheo et al. 1998a). However, its effect was content. found to be weaker than that caused by castration, likely

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The efficacy of this combined treatment has been demonstrated in other preclinical studies. Experimental studies in rats have shown that flutamide and the 5α- reductase inhibitor N,N-diethyl-4-methyl-3-oxo-4-aza- 5α−androstane−17β−carboxamide were more effective in combination than when given alone in reducing andros- tenedione-induced prostatic growth in castrated rats (Labrie et al. 1991). In addition, flutamide and the 5α- reductase inhibitor, finasteride, in combination caused a significant decrease in rat ventral prostate size compared with either drug alone (Fleshner & Trachtenberg 1992). In mice bearing the androgen-sensitive Shionogi mammary carcinoma, flutamide and finasteride had an additive inhibitory effect on tumor growth and on ventral prostate weight (Chen et al. 1996). Further, we have previously reported that PNU 156765, a 5α-reductase inhibitor structurally related to PNU 157706, showed an additive antitumor effect when given in combination with flutamide in the Dunning prostatic tumor model (Zaccheo et al. 1998b). Early results in patients with prostatic carcinoma have shown that combined therapy with flutamide and finasteride was effective in reducing PSA levels and in maintaining sexual function in most men (Fleshner & Fair 1996). A potent, dual inhibitor of both type I and II 5α- reductases could better suppress circulating DHT and provide more efficient treatment for prostate cancer than a Figure 3 Effect of 9-week oral treatment with PNU 157706 (10 type II inhibitor like finasteride (Span et al. 1999). PNU mg/kg per day) or flutamide (1 and 5 mg/kg per day), given 157706 represents an improvement in the field of 5α- alone or in combination, on prostatic DHT and testosterone reductase inhibitors on account of its in vitro potency on content in rats bearing the Dunning R3327 prostatic carcinoma. both human isozymes and its extraordinary potency in Animals were killed 24 h after the last dose. Bars represent means±S.E. (eight to nine animals per group). **P<0.01 vs reducing prostate weight in the rat (di Salle et al. 1998). controls by Dunnett's test. The experimental data presented in this paper indicate that a combination of a dual inhibitor of 5α-reductase (like because of the remaining tumor testosterone, at variance PNU 157706) and an antiandrogen could represent a with castration. Therefore, in this study we investigated further improvement in endocrine therapy of prostate the effect of combination therapy using PNU 157706 and cancer. In fact, such a combined treatment could have a the androgen receptor antagonist flutamide (Labrie et al. significant impact on the management of advanced 1993a). In fact the PNU 157706-induced increase in tissue prostate cancer, as it should maintain sexual function and testosterone level could be neutralized by flutamide at the provide ‘androgen blockade’. The possible role of androgen receptor level. In addition, the PNU 157706- hormonal therapy in early stage prostate cancer has not induced decrease in DHT could give flutamide a further been explored systematically in clinical studies. However, competitive advantage over DHT for binding to the the possibility that very early lesions could be more androgen receptor. The present data show that the androgen-dependent and more sensitive to DHT with- inhibitory effects of PNU 157706 and flutamide on drawal supports the use of a 5α-reductase inhibitor alone Dunning prostatic tumor growth are additive, thus in prevention studies (Ford et al. 1994) or in combination supporting the rationale of this combination therapy. In with an antiandrogen in early stage prostate cancer. fact, treatment with PNU 157706 alone caused 36% tumor growth inhibition; flutamide alone, at the doses of 1 or 5 mg/kg per day, caused 24% and 48% inhibition of tumor Acknowledgements growth respectively. When these two doses of flutamide were combined with PNU 157706, tumor growth The authors would like to thank Professor Martino inhibition reached 60% and 68%. Recchia for his assistance in the statistical analysis.

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