Pergamon J. Steroid Biochem. Molec. Biol. Vol. 50, No. 5/6, pp. 267-273, 1994 Copyright ~) 1994 Elsevier Science Ltd 0960-0760(94)00093-X Printed in Great Britain. All rights reserved 0960°0760/94 $7.00 + 0.00 Pharmacology of Novel Steroidal Inhibitors of Cytochrome P45017 (17 -Hydroxylase/C17-20 Lyase) S. E. Barrie, ~* G. A. Potter, 1 P. M. Goddard, 1 B. P. Haynes, 1 M. Dowsett 2 and M. Jarman t 1Drug Development Section, Institute of Cancer Research, Sutton, Surrey SM2 5NG and 2Department of Academic Biochemistry, Royal Marsden Hospital, Fulham Road, London SW3 6JJ, England Medical or surgical castration for the treatment of prostatic cancers prevents androgen production by the testes, but not by the adrenals. Inhibition of the key enzyme for androgen biosynthesis, cytochrome P45017a, could prevent androgen production from both sources. The in vivo effects of 17-(3-pyridyl)androsta-5,16-dien-3fl-ol (CB7598) and 17-(3-pyridyl)androsta-5,16-dien-3-one (CB7627), novel potent steroidal inhibitors of this enzyme, on WHT mice were compared with those of castration and two clinically active compounds, ketoconazole and flutamide. Flutamide and surgical castration caused significant reductions in the weights of the ventral prostate and seminal vesicles. CB7598, in its 3fl-O-acetate form (CB7630), and CB7627 caused significant reductions in the weights of the ventral prostate, seminal vesicles, kidneys and testes when administered once daily for 2 weeks. Ketoeonazole, given on the same schedule, caused no reductions. Plasma testosterone was reduced to ~<0.1 nM by CB7630, despite a 3- to 4-fold increase in the plasma level of luteinizing hormone. Adrenal weights were unchanged following treatment with CB7630 or CB7627 but were markedly increased following ketoconazole, indicating no inhibition of corticosterone production by these steroidal compounds. These results indicate that CB7598, CB7630 or CB7627 may be useful in the treatment of hormone-dependent prostatic cancers. J. Steroid Biochem. Molec. Biol., Vol. 50, No. 5/6, pp. 267-273, 1994 INTRODUCTION Antiendocrine therapy with luteinizing hormone- releasing hormone (LHRH) agonists has become an established alternative to orchidectomy as a means of lowering circulating androgens in patients with cancer of the prostate. This treatment ablates the testicular production of androgens but leaves the adrenal supply of androgens and their precursors un- affected. The long standing premise that this adrenal supply is critical has been tested in several clinical trials, some of which showed a significant increase in survival as a result of combining LHRH agonists with antiandrogens when compared with the use of LHRH agonists alone [1]. An alternative to this combination would be an effective and selective in- hibitor of the key enzyme in the androgen biosynthetic pathway, namely cytochrome P45017 a (17~-hydroxyl- *Correspondence to S. E. Barrie. Received 31 Jan. 1994; accepted 9 May 1994. ase/ C17-20 lyase), which is present in both the testes and the adrenals. Ketoconazole, originally developed as an antifungal agent, potently inhibits this enzyme and has been used clinically in the treatment of hor- mone-dependent prostatic cancers [2]. Unfortunately ketoconazole also inhibits many other cytochrome P450 enzymes [3,4] and was withdrawn from use because it caused liver damage [5, 6]. Several groups are seeking to develop more potent and selective inhibitors [7, 8]. Recently we reported briefly on the in vitro and in vivo results obtained with a novel steroidal compound, 17-(3-pyridyl)androsta-5,16- dien-3fl-ol (CB7598) (Fig. 1), a potent inhibitor (Kiap p < 1 nM for the lyase activity) of the human cytochrome P4501v~ [9]. In this paper the effects of CB7598 in its 3fl-O-acetate form (CB7630), and an equally potent related compound 17-(3- pyridyl)androsta-5,16-dien-3-one (CB7627) (Fig. 1), on the circulating hormone levels and organ weights in mice are described, and compared with those of castration, ketoconazole and flutamide. 267
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`268 S.E. Barrie et al. EXPERIMENTAL CB7598, CB7630 and CB7627 were prepared from dehydroepiandrosterone where the key step involved a novel palladium catalysed cross-coupling of a steroidal enol triflate with a pyridyl borane [10, 11]. Ketocona- zole was a gift from Janssen Pharmaceuticals (Beerse, Belgium) and flutamide was a gift from Kirby-Warwick Pharmaceuticals Ltd, (Bury St Edmunds, England) The WHT mice came from the Gray Lab (Mount Vernon Hospital, Mddx, England). Adrenal cell assay Guinea pig adrenal cells were obtained and assayed for the ACTH stimulated production of androstene- dione and cortisol as described previously [12]. Radioimmunoassays Testosterone was analysed using the DPC total tes- tosterone coat-a-count kit. The detection limit was 0.1 nM and the within and between assay CVs were 8.0 and 12.9%, respectively at an assay value of 2.5 nM. The assay for luteinizing hormone (LH) used the Amersham International rat LH[12sI] assay system employing a magnetic separation system which cross- reacts with mouse LH. The detection limit was 0.4 ng/ml and the within and between assay CVs were 6.5 and 6.604, respectively at an assay value of 4.5 ng/ml. chromatography. Plasma samples (250 pl) were mixed with acetonitrile (375pl) and centrifuged. Aliquots (100-200 #1) of the supernatant so obtained were in- jected onto a 15cm Spherisorb $3PC18 cartridge column (4.6mm i.d.) with an eluent of aceto- nitrile-50 mM ammonium acetate buffer (60:40, v/v) flowing at 1.5 ml/min. Eluates were detected using a Perkin-Elmer LC-240 fluorescence detector set up with a excitation wavelength of 262 nm and an emission wavelength of 353 nm. The detection limit of the assay was 20 ng/ml (~ 50 nM) for both compounds. Adult male WHT mice were castrated via the scrotal route while under pentobarbital anaesthesia. The sham operated group underwent the same surgical pro- cedures as the castrates except that the testes were not II No CB7598 I In vivo studies Adult male WHT mice (12 weeks old) were housed under conditions of 12 h light-dark and were allowed free access to food (SDS expanded rodent diet) and water. CB7598 was used in its 3fl-O-acetate form (CB7630) as this was easier to formulate than the free 3/3-hydroxy compound. CB7630, CB7627 and ketoconazole were prepared in 5 % benzyl alcohol, 95 % safflower oil. The preparations were given intraperitoneally, 5 ml/kg, to the mice once daily for 14 days. Flutamide was pre- pared in 5°; dimethylsulphoxide, 95% arachis oil and was given subcutaneously, 5 ml/kg, following the same schedule. The doses given ranged from 0.02 to 0.5 mmol/kg/day. On day 15, the animals were anaes- thetized with fluothane, the blood collected by cardiac puncture into heparinized tubes, and the organs of interest dissected out and weighed. After weighing, the organs from the animals treated with CB7627 were stored in modified methacarn for histological examin- ation. The plasma was separated by centrifugation and stored at - 20°C. For the pharmacokinetic experiment, a single in- traperitoneal dose of 0.5 mmol/kg CB7630 was given to the mice which were sacrificed at various times up to 24 h afterwards, and the blood taken by cardiac punc- ture as above. CB7598 and CB7630 plasma concen- trations were determined by high pressure liquid /. Ae CB7630 I ° I I Fig. 1. Structures of the compounds CB7598, the 3-O-acetate form CB7630, and CB7627
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`Pharmacology of P45013~ Steroidal Inhibitors 269 Table 1. The effects of castration on the organ weights of WHT mice Treatment/ Seminal time Adrenals Prostate vesicles Testes Kidneys Untreated 3.9 ± 0.1 9.5 ± 0.4 186 ± 6 137 ± 2 651 ± 26 (n = 21) (n = 21) (n = 21) (n = 21) (n = 8) 3.4±0.1 11.1_+0.5 170±10 124±5 ND 3.9±0.1 9.5±0.5 168±6 126±6 ND 3.3±0.1 7.1±0.5 167_+_8 137±2 571±23 Sham 1 wk (n = 7) 2wk (n = 8) 4wk (n = 8) Castrates 1 wk (n = 8) 2wk (n = 8) 4wk 3.6±0.1 4.2±0.2* 27±2" ND 3.9±0.1 2.5±0.2* 16±1" ND 4.1±0.1 1.9±0.1" 15±1" 413±13" The results are given in mg as means + SE, with the number of animals in parentheses. *P < 0.01 for the differences between the castrate groups and the sham groups; ND, not determined. removed. After 1, 2, or 4 weeks the animals were sacrificed and the organs removed and weighed. Statistical significance was assessed by analysis of variance, and subsequent multiple range testing used the least significant difference method using the Sta- graphics 5 computer program. RESULTS ACTH stimulated the production of androstene- dione and cortisol by guinea pig adrenal cells in vitro by 1703 and 38nM, respectively after 3 h. CB7598, CB7627 and ketoconazole were able to inhibit this, achieving almost total suppression (< 10% stimulated production) at 10 s M. This concentration was much higher than the K,s for the human cytochrome P45017 ~ enzyme as measured in vitro, and may refect the effects of protein binding and membrane transport as well as differences in the enzyme structure and environment. A similar concentration of ketoconazole was required to inhibit corticosterone synthesis in rat adrenal cells [4]. The effect of castration on the organ weights of the WHT mice is shown in Table 1. There were marked decreases in the sizes of the ventral prostate, seminal vesicles and kidneys to 20, 8, and 63% of untreated controls, respectively. The effect on the seminal ves- icles was greater than on the prostate at all times. The maximum effect was seen after approx. 2 weeks, and a 2-week dosing schedule with the various drugs was therefore followed. The results of treating the mice with CB7630 and CB7627 are given in Figs 2 and 3 and the results obtained with ketoconazole and flutamide, agents that have been used clinically to treat hormone dependent prostatic cancer, are given in Figs 4 and 5. The mice did not lose weight or appear unwell with any of the treatments, and there were no drug related features seen at post-mortem except in the case of ketoconazole at the highest dose when the livers looked brown (no histology was carried out). CB7630 and CB7627 caused a marked suppression in the weights of several androgen-dependent organs in a dose-dependent way. The reductions in the seminal vesicle weights (maximum reductions 87 and 85%, respectively) were more marked than that in the ventral prostate weights (maximum reductions 48 and 51%, respectively), as had been the case following castration. The kidneys were also reduced in weight (by 37 and 28%) as were the testes (by 62 and 58%). Flutamide, in contrast, produced equal reductions in the prostate and seminal vesicles (54 and 60%, respectively), and no change in the testicular weight (kidney weights were not measured). Ketoconazole did not produce any change in the weights of these organs. After the experiment with CB7627, the testes were histologically examined. There was a thinning of the germinal epithelium with a reduced number of mature forms. There was no obvious reduction in Leydig cell numbers, although this was difficult to estimate in the presence of marked changes in the seminiferous tubules. There were no obvious toxic effects of the drug and all the changes were consistent with a reduction in testosterone. Following treatment of the mice with ketoconazole, there was a marked increase in adrenal weight, while treatment with CB7630, CB7627, or flutamide caused no change in the weight of the adrenals. The plasma testosterone and LH levels 24 h after the end of treatment are given in Table 2. The level of testosterone in control animals varied considerably, and there was a dramatic reduction in the levels in the vehicle controls probably related to stress. Comparing the vehicle controls with the drug treated groups, there was a decrease in circulating testosterone after keto-
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`125
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`vehicle only
`[] 0.02 mmol/kglday
`[] 0.1 mmol/kglday
`[] 0.5 mmol/kglday
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`.. 100
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`................. i T \1 ~¢ ventral prostate \1 seminal vesicles X adrenals b* testes -Z r_ ~ xSt¢ ~* Xx \\X ',~'× kidneys Fig. 2. Effects of 14 days treatment with CB7630 on the organ weights of WHT mice. Results are expressed as % of the untreated controls for which the values were ventral prostate 9.5 + 0.3, seminal vesicles 182 + 6, adrenals 4.2 + 0.1, testes 144 + 2, kidneys 677 + 17 mg (n = 20). *P < 0.01 for the difference from the vehicle controls. conazole, but this was not statistically significant, and was not associated with a rise in plasma LH. However, there was a marked dose-dependent decrease in testos- terone to almost undetecteble levels following CB7630 and this was maintained despite the increased levels of LH which would normally stimulate the testicular production of the androgen. Following CB7627 there was also a dose-dependent decrease in plasma testoster- one but not to such low levels as following CB7630. This was not statistically significant as the variability of the vehicle control group was so great. The pharmacokinetics of CB7598 following the intraperitoneal administration of CB7630, the acetyl- ated form of CB7598, are shown in Fig. 6. CB7630 was detected in only 2 of the 25 mice, indicating rapid de-acetylation to CB7598. The peak plasma concentration of CB7598 of approx. 400ng/ml (1.0 #M) was seen after 6 h with the level dropping to 1/3 after 8 h. The concentration at 24 h was similar to that at 8h, possibly due to a depot effect from the route of administration, and/or enterohepatic recir- culation.
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`0 k~X ~X ventral .-. ..... -i ........ prostate % seminal vesicles \ adrenals
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`testes kidneys Fig. 3. Effects of 14 days treatment with CB7627 on the organ weights of WHT mice. Results are expressed as % untreated controls (values given in Fig. 2). *P < 0.01 for the difference from the vehicle controls.
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`270 S.E. Barrie et al.
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`[] vehicle only
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`\N ventral prostate -7,- seminal vesicles testes ........... i- \ kidne rs Fig. 4. Effects of 14 days treatment with ketoconazole on the organ weights of WHT mice. Results are expressed as % untreated controls (values given in Fig. 2). *P < 0.01 for the difference from the vehicle controls. DISCUSSION Plasma levels of testosterone and LH are very vari- able in mice [13] and can be affected by stress of varying types [14], making the detection of significant drug induced changes difficult. Both the variability and the effect of stress are illustrated by the data here for the control and the vehicle control groups. Following ketoconazole, the level of circulating testosterone was reduced but this was not associated with an increase in plasma LH, nor with regression of the androgen-de- pendent organs. These results suggest that the testos- terone suppression may have been of insufficient duration to cause any regression of the androgen- dependent organs and so might have been stress in- duced. On the other hand, following CB7630, acting as a prodrug form of CB7598, there was a marked drop in the circulating level of testosterone which was main- tained despite the reflex rise in LH, which in turn would have stimulated testosterone biosynthesis. This indicates that CB7630 produced potent inhibition of testosterone production in vivo. The significant re- gression of several androgen-dependent organs could have resulted from this reduced level of circulating 0 N
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`\X i ] \M ) ! ......... 3 ..... \N x NN_ \x Nx \X \ \ \ ventral seminal adrenals testes prostate vesicles Fig. 5. Effects of 14 days treatment with flutamide on the organ weights of WHT mice. Results are expressed as % untreated controls (values given in Fig. 2). *P < 0.01 for the difference from the vehicle controls.
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`Pharmacology of P45017 ~ Steroidal Inhibitors 271
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`272 S.E. Barrie et al. Table 2. The levels of plasma testosterone and LH following drug treatments Testosterone LH (nM) (ng/ml) Ketoconazole Control 17.3 + 7.1 a 0.66 + 0.05 Vehicle 1.3 + 0.4 <0.4 0.1 mmol/kg/day 0.9 + 0.2 <0.4 0.225 mmol/kg/day 0.7 + 0.15 0.75 ± 0.02 0.5 mmol/kg/day 0.4 + 0.1 0.76 + 0.03 CB7630 Control 9.8 ± 5.6 0.63 + 0.16 Vehicle 2.5 + 1.2 0.80 + 0.09 0.02 mmol/kg/day 2.7 + 0.5 3.4 + 0.5* 0.1 mmol/kg/day 0.2 + 0.1" 2.55 + 0.45* 0.5 mmol/kg/day 0.1 + 0.0" 2.25 + 0.67* CB7627 Control 27.8 + 11.4 ND Vehicle 11.0 ± 5.6 ND 0.02 mmol/kg/day 4.5 + 0.3 ND 0.1 mmol/kg/day 3.5 + 1.1 ND 0.5 mmol/kg/day 0.4 _+ 0.1 ND The results are given as mean + SE with 5 animals per group. aDue to the large variation in the testosterone values in the untreated control group, the analysis of variance was carried out without including this group. *P < 0.05 for difference from the vehicle only group; ND, not determined. testosterone, although an additional direct effect of the compound cannot be excluded. CB7627 also induced a marked drop in plasma testosterone, although this was not to as low a level as CB7630. However, the effects on the target organs were significant indicating that this compound is also active by inhibiting testosterone biosynthesis and/or acting directly as an antiandrogen. The reduction in testicular weight following treat- ment with CB7630 or CB7627 was surprising as no
`i 18 i 24 Fig. 6. Pharmacokinetics of CB7598 following intraperiton- neal administration of 0.5 mmol/kg CB7630. The data from 5 mice per time point are expressed as mean + SE. changes had been reported with ketoconazole, and antiandrogens like flutamide had no effect [15] as found here. Reductions in testicular weight have been docu- mented following LHRH analogues [15, 16], but in these situations there was low LH in contrast to the situation following CB7630. The histology indicated that the changes were consistent with low tissue testos- terone, which would result from the effective inhibition of the biosynthetic pathway by the test compounds. The pharmacokinetic study showed that the de- acetylated drug (CB7598) persisted at quite high levels (~0.3 #M) at 24 h. This may be related to the route of administration or may be the result of enterohepatic recirculation increasing the effective half-life consider- ably. This persistence of CB7598 would have been very important in maintaining adequate enzyme inhibition throughout the experiment. Ketoconazole has been shown to reduce circulating testosterone levels in rats and cause regression of the ventral prostate and Dunning R3327H tumours [17 18]. In those exper- iments it was given twice daily and this schedule difference may explain the lack of effect on the andro- gen-dependent organs observed here, despite a higher dose of the drug being given. However there were effects on the liver and adrenals indicating unwanted side-effects. Ketoconazole is known to inhibit other cytochrome P450s in addition to the P45017~ [3, 4] and was withdrawn from cancer therapy because of hepato- toxicity [5, 6] which was probably a result of these additional unwanted enzyme inhibitions. 17e-Hydroxylation is an essential step in the biosyn- thesis of androstenedione and cortisol, and inhibition of this step should inhibit the production of both steroids. This is borne out by the results with the guinea pig adrenal cells. In mice, corticosterone, and not cortisol, is the predominant glucocorticoid, and the biosynthesis of corticosterone does not require the cytochrome P450~v~ enzyme. Therefore, a selective inhibitor should have no effect on adrenal function in mice. Inhibition of corticosterone production by, for example, amino- glutethimide inhibiting the cytochrome P450~cc enzyme results in a marked increase in adrenal weight [19, 20]. Ketoconazole inhibits several steps in this pathway [3,4] and, as shown here, also results in a marked increase in adrenal weight. The lack of effect on the mouse adrenals following CB7630 and CB7627 indi- cates that these compounds do not significantly inhibit other steps in the biosynthetic pathway, and therefore that they are more selective for the target enzyme. For an inhibitor of cytochrome P450~7~ (17:~-hy- droxylase/C17-20 lyase) to be effective in the treatment of hormone-dependent cancer of the prostate, it needs to be potent, selective and to have favourable pharma- cokinetics and metabolic profile. The compound 17fl- (cyclopropylamino)-androst-5-en-3flol has been shown to be an irreversible inhibitor of the target enzyme [7], but is considerably less potent than CB7598 or CB7627. No information was given as to its in vivo
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`Pharmacology of P45017 , Steroidal Inhibitors 273 activity. Another compound, 4-pregnen-3-one-20/3- carboxaldehyde, has been developed to exploit this target in combination with 5~-reductase [8]. It was highly potent against the human 5g-reductase in vitro with Ki = 16 nM but in contrast poorly active against the rat cytochrome P45017~ enzyme giving Ki values in the/~M range. In vivo repeated administration of the compound produced a very modest drop (50%) in plasma testosterone, and no details were given as to any effect on the androgen-dependent organs of the animals. In conclusion, the steroidal compounds CB7630, acting as a prodrug form of CB7598, and CB7627 are potent inhibitors of the human target enzyme, while not inhibiting several other P450 enzymes, and show significant activity in vivo, making them worthy of further study as potential agents for the treatment of hormone-dependent prostate cancer. Acknowledgements--This work was supported by grants from the Cancer Research Campaign and Medical Research Council. Finan- cial support (to S.E.B. and G.A.P.) from the British Technology Group is also gratefully acknowledged. We also thank Dr K. MacLennan for carrying out the histological examinations, and M. G. Rowlands, J. Houghton, M, Valenti, and N. King for their valuable technical help. REFERENCES 1. Crawford E. D., Eisenberger M. A., McLeod D. G., Spaulding J. T., Benson R., Dorr F. A., Blumenstein B. A., Davies M. A. and Goodman P. J.: A controlled trial of leuprolide with and without flutamide in prostatic carcinoma. New Engl. J. Med. 321 (1989) 419-424. 2. Trachtenberg J.: Ketoconazole therapy in advanced prostatic cancer. J. Urol. 132 (1984) 61-63. 3. Nagai K., Miyamori I., Ikeda M., Koshida H., Takeda R., Suhara K. and Katagiri M.: Effect of ketoconazole (an imidazole antimycotic agent) and other inhibitors of steroidogenesis on cytochrome P450-catalysed reactions. J. Steroid Biochem. 24 (1986) 321-323. 4. Loose D. S., Kan P. B., Hirst M. A., Marcus R. A. and Feldman D.: Ketoconazole blocks adrenal steroidogenesis by inhibiting cytochrome P450-dependent enzymes. J. Clin. Invest. 71 (1983) 1495-1499. 5. Lake-Bakaar G., Scheuer P. J. and Sherlock S.: Hepatic reac- tions associated with ketoconazole in the United Kingdom. Br. Med. J. 294 (1987) 419-422. 6. Moffat L. E. F., Kirk D., Tolley D. A., Smith M. F. and Beastall G.: Ketoconazole as primary treatment of prostatic cancer. Br. J. Urol. 61 (1988) 439-440. 7. Angelastro M. R., Laughlin M. E., Schatzman G. L., Bey P. and Blohm T. R.: 17/3-(cyclopropylamino)-andros-5-en-3/3-ol, a se- lective mechanism based inhibitor of cytochrome P45017 , (steroid 17~-hydroxylase/C17-20 lyase). Biochem. Biophys, Res. Commun. 162 (1989) 1571-1577. 8. Li J., Li Y., Son C., Banks P. and Brodie A.: 4-Pregnene-3-one- 20/3-carboxaldehyde: a potent inhibitor of 17e~-hydroxylase/C 17- 20 lyase and of 5~ -reductase. J. Steroid Biochem. Molec. Biol. 42 (1992) 313-320. 9. Barrie S. E., Potter G. A., Jarman M. and Dowsett M.: Highly potent inhibitors of human cytochrome P-450(17~) : Activity in vitro and in vivo. Br. J. Cancer (Suppl. XX) 67 (1993) 75. 10. Potter G. A. and Barrie S. E.: Discovery of highly potent enzyme inhibitors with potential for the treatment of prostate cancer; the important dual role of transition metal chemistry in both drug design and synthesis. In Organometallic Reagents in Organic synthesis (Edited by J. H. Bateson and M. B. Mitchell). Academic Press, London (1994) p. 235 11. Barrie S. E., Jarman M. and Potter G. A.: UK Patent Appli- cation GB 2,265,624A (1993). 12. Newton C. J., Mehta A. and Dowsett M.: The effect of the aromatase inhibitor, rogletimide (pyridoglutethirnide), on guinea pig adrenal cell steroidogenesis and placental microsomal aroma- tase activity: Comparison with aminoglutethimide and CGS16949A. J. Steroid Biochem. Molec. Biol. 39 (1991) 723-727. 13. Barke A., Steele R. E., Musto N. and Caldwell B. V.: Fluctu- ations in plasma testosterone levels in adult male rats and mice. Endocrinology 92 (1973) 1223-1228. 14. Knol B. W.: Stress and the endocrine hypothalamus-pituitary- testis system: a review. Vet. Quart. 13 (1991) 104-114. 15. Chandolia R. K., Weinbauer G. F., Simoni M., Behre H. M. and Nieschlag E.: Comparative effects of chronic administration of the non-steroidal antiandrogens flutamide and Casodex on the reproductive system of the adult male rat. Acta Endocr. (Copen- hagen) 125 (1991) 547-555. 16. Redding T. W. and Schally A. V.: Inhibition of the pituitary- gonadal axis in nude male mice by continuous administration of LHRH agonists and antagonists. J. Endocr. 126 (1990) 309-315. 17. Trachtenberg J.: The effects of ketoconazole on testosterone production and normal and malignant androgen dependent tis- sues of the adult rat. J. Urol. 132 (1984) 599-601. 18. English H. F., Santner S. J., Levine H. B. and Santen R. J.: Inhibition of testosterone production with ketoconazole alone and in combination with a gonadotropin releasing hormone analogue in the rat. Cancer Res. 46 (1986) 38-42. 19. Malendowicz L. K.: Comparative studies on the effects of aminoglutethimide on hamster and rat adrenal cortex. Exp. Toxic. Path. 44 (1992) 81-85. 20. Ricciardi M. P., Pellegrini A., Grieco M., Marroni P. and Materazzi F.: Effects of stress on adrenal steroidogenesis block- ade by aminoglutethimide. In Vivo 6 (1992) 103-106.
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