Effect of Ketoconazole Treatment on Cortisol Response under Adrenocorticotropin Hormone Stimulation in Dogs

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1 ep ~~~t J Chin Soc Vet Sci 26 (4): , 2000 Effect of Ketoconazole Treatment on Cortisol Response under Adrenocorticotropin Hormone Stimulation in Dogs * Cicero Lee-Tian CHANG, Hang-Poung FUNG, Chi-Liang MAO, Jacky Peng-Wen CHAN, Shih-Te CHUANG, and Feng-Pang CHENG Department of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan 402, ROC (Received: April 28, Accepted: August 16, 2000.) ABSTRACT Ketoconazole (KCl), a clinically used imidazole antifungal agent, also inhibits steroid hormones biosynthesis. This study investigated how different dosages of KCl affect cortisol secretion of adrenal glands and its response towards adrenocorticotropin hormone (ACTH) stimulation in dogs. Two groups of 6 healthy dogs were orally administered with a low dose (10 mg/kg of body weight) and a high dose (30 mg/kg of body weight) of KCl, respectively. Synthetic ACTH (Cortrosyn* ) of 0.25 mg was continuously administered by 1M to each dog. Blood samples were collected from each dog before and 4 hours after KCl administration, and then 2 hours and 4 hours after ACTH injection to determine the level of serum cortisol. Serum concentrations of cortisol were measured using ELISA kit (NEOGEN* ). KCl, particularly the high dose group, significantly inhibited the secretion of cortisol (P<0.005) and decreased the response to ACTH stimulation (P<0.05). However, the increased serum cortisol level at 4 hours after ACTH stimulation was higher than at 2 hours after ACTH stimulation (P<0.05). The experimental results indicated how dosage and time appear to affect KCl treatment on cortisol response to ACTH stimulation in dogs. l ' Chang Cicero LT, Fung HP, Mao CL, Chan Jacky P, Chuang ST, and Cheng FP. Effect of ketoconazole treatment on the response to adrenocorticotropin hormone stimulation in dogs. J Chin Soc Vet Sci 26(4): , Corresponding author TEL: , FAX: , d @mail.nchu.edu.tw ] Keywords: Ketoconazo/e, Cortisol, ACTH, Dogs INTRODUCTION Ketoconazole (KCZ), a synthetic imidazole analog, is widely used against systemic and topical fungal infection [3,16]. It inhibits the synthesis of ergosterol in fungal membranes and of cholesterol in mammalian cells [3,13]. In addition to its antifungal activity, KCZ has been shown to interfere with gonadal [10] and adrenal [7,11] steroid synthesis through inhibition of cytochrome P450-dependent enzymes. Similar endocrinologic effects have been observed in clinically normal dogs [16]. KCZ is a competitive inhibitor of all five cytochrome P450 dependent enzymes, and the effects on 17~-hydroxylase, 17, 20-desmolase and l1~-hydroxylase were greater than 21-hydroxylase and 3~-hydroxysteroid dehydrogenase [3,4,7]. It inhibits adrenal steroidogenesis in the rat [3] and dose-dependently decreases basal and CRF-stimulated ACTH release in primary cultures of rat anterior pituitary cells [13]. In man, studies show dose-dependent inhibition of cortisol and testosterone biosynthesis [3,9].

2 248 Effect of Ketoconazole Treatment in Dogs This leads to the therapeutic use of KCZ for patients with Cushing's syndrome [3,4] and advanced prostate cancer [3,6,14]. In man, cortisol responsiveness to ACTH is depressed for up to 8 hours after a single KCZ dose, but returns to normal within the next 8 hours [11]. The purpose of this study was to evaluate the effect of KCZ treatment, using different dosages, on cortisol response under ACTH stimulation in dogs. MATERIALS AND METHODS Experimental dogs Twelve adult dogs, weighing 12 to 25 kg, were physically examined twice and clinically evaluated with CBC, serum biochemical values (AST, ALT, ALP, BUN, creatinine, glucose, total protein, and albumin), urinalysis, heart worm disease assay and fecal specimens (floatation examination for parasites). The dogs also showed normal cortisol response to ACTH stimulation (basal cortisol: 17.7 ± 9.2 ng/ml; post ACTH cortisol: ± 23.5 ng/ml) (Table 1). They were quarantined at least two weeks for observation and acclimation prior to this study. Serum biochemical analysis Serum biochemical values of AST, ALT, ALP, BUN, creatinine, glucose, total protein and albumin were analyzed using an automatic analyzer (Express plus, C1BA CORNING Diagnostics Corp., USA). KCZ treatment and hormone assay Twelve dogs were separated into two dose groups, with 6 dogs per group. The high dose group was administered with 30 mg/kg of KCZ per body weight, while the low dose group was administered with 10 mg/kg of KCZ per body weight. Synthetic ACTH (Cortrosyn" Depot, N. V. Organon/Oss, Holland) of 0.25 mg was continuously administered 1M to each dog. Blood samples were collected from each dog before and 4 hours after KCZ administration, and 2 hours and 4 hours after ACTH injection. Serum concentrations of cortisol were determined by a commercial ELISA kit (Cortisol ELISA kit instructions, NEOGEN' CORPORATION, USA). One hundred microliter of canine serum was pipetted into a glass test tube (10 X 75 mm) and 1 ml of ethyl ether was added. The tube was vortexed for 30 seconds and the aqueous and organic phases were allowed to separate, after which the aqueous layer was frozen and the solvent layer (organic phase) was poured into another test tube. The solvent was gently evaporated with a stream of nitrogen, and the residue was dissolved in 100 fll diluted extraction buffer. Fifty fll diluted extract was vortexed and assayed in duplicates. The plate was read in a microplate reader at 650 nrn, The obtained values of cortisol multiplied by 100 to produce a final ng/ml concentration. Regarding possible cross-reactivity of related steroid hormones in these assays was provided by the manufacturer's information. Statistical analysis Paired Student's t-test was used for a comparison of individual set of data (mean ± SD, n = 6). RESULTS Serum biochemical analysis The values of AST, ALT, ALP, BUN, creatinine, glucose, total protein and albumin were in the normal range for dog (data not shown). Serum cortisol assay KCZ, especially the highdose group, significantly inhibited the secretion of cortisol (P < a.005) and decreased the response to ACTH stimulation (P < O.05). time factor, In regard to the with both high and low KCZ doses, the increased serum cortisol level at 4 hours after ACTH stimulation is higher than at 2 hours after ACTH stimulation (P < O.05). All serum cortisol values of post-acth stimulation, recombined with both dosages of KCZ, are higher than the values of KCZ administration alone. In addition, the high dose group's serum cortisol value of 2 hours post ACTH stimulation is lower than the basal value (P <0.05). Meanwhile, the high-dose group's serum cortisol value of 4 hours post-acth stimulation is not significantly higher than the basal value (P >

3 Cicero Lee-Tian CHANG, et al 249 Table 1. Serum levels of cortisol before and 2 hours after ACTH (0.25 mg/dog) stimulation. (n= 12) Dog number Determinant Basal cortisol (ng/ml) Post-ACTH Cortisol (ng/ml) a. mean±sd,b. P<O.OOI Average value' 17.7±9.2 b ± L Table 2. Basal and post-acth stimulation serum cortisol levels in dogs given KCZ. Dosage group Dog No. Basal Serum cortisol levels (ng/ml) 4 hours after KCZ treatment post-acth 2 hours 4 hours Low (10 mg/kg BW) Mean±SD (n=6) 9.5 ± 1.3'b 5.5 ± 1.0' 36.6 ± 8.2'" 54.4 ± 15.1",g High (30 mg/kg BW) Mean±SD (n=6) 23.9 ± 10. 3'ef 5.3 ± 0.8' 16.4 ± 8.3 de 28.2 ± dfg a. d. g. P<O.005,b. P<O.OOl,c. e. P<O.05,f. P>O ) (Table 2). DISCUSSIONS KCZ is an orally active imidazole derivative widely used clinically as antifungal agent [1,2,4]. KCZ has been used as a palliative treatment of Cushing's syndrome, due to its ability to lower cortisol production [1]. Because gastric acid may enhance the absorption of KCZ [15], it was administered immediately before feeding in the present study, however, food may also deleteriously effect KCZ absorption [8]. KCZ showed competitive inhibition of five cytochrome P450 enzymes, and the effects on 17~-hydroxylase, 17, 20-desmolase and l ljl-hydroxylase were greater than 21-hydroxylase and 3~-hydroxysteroid dehydrogenase [3,4,7]. Side chain cleavage of cholesterol and l1~-hydroxylation of deoxycorticosterone in the adrenal gland were inhibited by KCZ [5]. In vivo, the administration of low doses of KCZ leads to a significant reduction in serum androgen level while at higher doses castration levels are achieved and cortisol secretion is suppressed [ 4]. Thus, both KCZ dosages of the present study, especially the higher dose, revealed the sig-

4 250 Effect of Ketoconazole Treatment in Dogs nificant inhibitory effect of cortisol secretion (P < 0.005) (Table 2). At higher doses (400 and 600 mg for human), the cortisol response to exogenous ACTH was significantly blunted 4 hours after a single dose [11]. This inhibition persisted for 8 hours but was absent by 16 hours. Our data suggested that KCZ at both doses have the same effect of cortisol response to exogenous ACTH stimulation, especially as seen in the value at 2 hours post-acth stimulation (P < o.05). Its value was even lower than the basal one. At 4 hours post-acth stimulation, the value was not significantly higher (P>0.05) (Table 2). The serum half-life of KCZ was 2.7 hours in the dog [16]. The high-dose KCZ may have a longer retention time. Apparently, the high-dose KCZ can last a longer inhibitory effect on the cortisol response under ACTH stimulation in our study (P < 0.005). In regard to time factor, the increased serum cortisol levels at 4 hours post-acth stimulation was higher than at 2 hours post-acth stimulation (P < 0.05) (Table 2). The increased cortisol level after KCZ treatment and ACTH stimulation was lower than the result without KCZ treatment (Table 1-2). KCZ inhibits the corticotropin releasing hormone (CRH) stimulated ACTH release from rat anterior pituitary fragments in a dose dependent fashion. The effects of KCZ on ACTH release and biosynthesis seems to be mediated by cyclic AMP (camp) since KCZ inhibits basal and CRH stimulated camp release and content within the same dose range [12, 13]. The lower cortisol response to exogenous ACTH stimulation under KCZ treatment, especially the high-dose group, was due to a direct inhibitory effect on ACTH synthesis and release. It resulted from a longer retention time of high dose KCZ to decrease more serum cortisol secretion. Simultaneously, there is no combined effect of endogenous and exogenous ACTH stimulation. Thus, a mild cortisol response to ACTH stimulation is observed within the high-dose KCZ group (Table 2). The data in the present study (Table 1-2) reveals the apparent difference between dosage and time on the effects of KCZ treatment on cortisol response under ACTH stimulation in dogs. ACKNOWLEDGEMENTS This study was supported in parts by grants from the Council of Agriculture, Taiwan. REFERENCES 1. Boscaro M. Sonino N, Rampazzo A, Menegus AM. Mantero F. ACTH response to corticotropin releasing hormone in Cushing's disease before and after ketoconazole: in vivo and in vitro studies. Hor Metab Res Suppl 16: 60-62, Burrin JM, Yeo TH, Ashby MJ, Bloom SR. Effect of ketoconazole on adrenocorticotrophic hormone secretion in vitro and in vivo. J Endocrinol 108: 37-41, Couch RM, Muller J, Perry YS, Winter JSD. Kinetic analysis of inhibition of human adrenal steroidogenesis by ketoconazole. J Clin Endocrinol Metab 65: , Engelhardt D, Webber MM, Miksch T, Abedinpour F, Jaspers C. The influence of ketoconazole on human adrenal steroidogenesis: incubation studies with tissue slices. Clin Endocrinol 35: , Higashi Y. Masako 0, Keiko S, Hiroshi I. Ketoconazole as a possible universal inhibitor of cytochrome P-450 dependent enzymes: its mode of inhibition. Endocrinol Japan 34: , Jack G. Nonsurgical treatment of prostatic hyperplasia. Cancer Suppl 70: , Loose DS, Kan PB. Hirst MA, Marcus RA, Feldman D. Ketoconazole blocks adrenal steroidogenesis by inhibiting cytochrome P45O-dependent enzymes. J Clin Invest 71: , Mannisto PT, Mantyla R, Mykanea S. Impairing effect of food in ketoconazole absorption. Antimicrob Agents Chemother 21: , Pont A, Graybill JR, Craven PC, Galgiani IN, Dismukes WE. Reitz RE, Stevens DA. High-dose ketoconazole therapy and adrenal and testicular function in humans. Arch Intern Med 144: , Pont A, Williams PL, Azhar DA. Ketoconazole blocks testosterone synthesis. Arch Intern Med 142: , Pont A, Williams PL, Loose DS, Feldman D, Reitz RE, Bochra C, Stevens DA. Ketoconazole blocks adrenal steroid synthesis. Annals Intern Med 97' , Stalla GK, Stalla J, Loeffler JP, von Werder K, MOiler OA. Pharmacological modulation of CRH-stimulated ACTH secretion by ketoconazole. Horm Metab Res Suppl 16: 31-36, Stalla GK, Stalla J, von Werder K, MOiler OA, Gerzer R,

5 Cicero Lee-Tian CHANG, et al 251 Hallt V, Jakobs KH. Nitroimidazole derivatives inhibit anterior pituitary cell function apparently by a direct effect on the catalytic subunit of the adenylate cyclase holoenzyme. Endocrinol 125' , Trump DL, Havlin KH, Messing EM, Cummings KB, Lange PH, Jordan VC. High-dose ketoconazole in advanced hormone-refractory prostate cancer: endocrinologic and clinical effects. J Clin Oncol 7: , Van Der Moor JWM, Kenning JJ, Scheijgrond HW. The influence of gastric acidity on the bio-availability of ketoconazole. J Antimicrob Chemother 6: , Willard MD, Nachreiner R, McDonald R, Roudebush P. Ketoconazole induced changes in selected canine hormone concentrations. Am J Vet Res 47: , 1986.

6 252 Effect of Ketoconazole Treatment in Dogs Ketoconazole 於犬隻血清氫基皮質輛生合成對親 腎上腺皮質激素刺激反應之作用 勢張力夭馮翰鵬茅繼良陳鵬文莊士德鄭豐邦 國立中興大學獸醫學系臺中市 收稿日期 : 89 年 4 月 28 日 接受日期 : 89 年 8 月 1 6 日 ) ~it 要 K e t o co n a zo l e (KCZ) 為臨床上 imidazole 類衍生物之抗徽菌劑, 亦為類固醇荷爾蒙生合成之抑制劑 本研究之目的在於瞭解不同劑量之 KCZ 對犬隻腎上腺氫基皮質觀生成及對親腎上腺皮質激素刺激反應之影響 以高劑量 ( 30 mg/kg of body weight) 及低劑量 (10 mg/kg of body weight) 之 KCZ 分別投予各六隻對親腎上腺皮質激素刺激正常反應之犬隻, 之後再以 mg 合成的親腎上腺皮質激素 (Cortrosyn") 肌肉注射犬隻, 而分別於 KCZ 投藥前 投藥後四小時及親腎上腺皮質激素注射後二及四小時抽血分離血清檢體 以 E LI SA 組 ( N EOG E N " ) 檢測氫基皮質關血清濃度 KCZ 可顯著地抑制氫基皮質間的分泌 ( P < O. 5 ), 尤其是高劑量組, 另外, KCZ 亦可使犬隻對親腎上腺皮質激素刺激反應顯著地降低 ( P < ), 尤其是高劑量組 至於在時間方面, 親腎上腺皮質激素刺激後四小時的氫基皮質關上升的數值比二小時後為高 (P<0.05) 結果顯示 KCZ 的劑量及時間上對親腎上腺皮質激素刺激反應之抑制具有顯著的差異性 [ 勢張力天 馮翰鵬 茅繼良 陳鵬文 莊士德 鄭豐邦 o Ketoconazole 於犬隻血清氫基皮質明生合成對親腎上腺皮質激素刺激反應之作用 中華獸醫誌 26 (4): , 聯絡人 TE L: , FAX: , d @mail.nchu.edu.tw] illiffj: K etoconazo 侶, /M6&NJIiJ, 1J!'I!...t 嚴 '&ff, 嚴索, ;f