Effects of Carbenoxolone on the Canine Pituitary-Adrenal Axis

RESEARCH ARTICLE Effects of Carbenoxolone on the Canine Pituitary-Adrenal Axis Takahiro Teshima*, Hirotaka Matsumoto, Tomoko Okusa, Yumi Nakamura, Hidekazu Koyama Division of Therapeutic Science I, Department of Veterinary Clinical Medicine, School of Veterinary Medicine, Faculty of Veterinary Science, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musashinoshi, Tokyo 180-8602, Japan * Abstract a11111 OPEN ACCESS Citation: Teshima T, Matsumoto H, Okusa T, Nakamura Y, Koyama H (2015) Effects of Carbenoxolone on the Canine Pituitary-Adrenal Axis. PLoS ONE 10(8): e0135516. doi:10.1371/journal. pone.0135516 Editor: Peyman Björklund, Uppsala University, SWEDEN Received: February 18, 2015 Accepted: June 13, 2015 Published: August 11, 2015 Copyright: © 2015 Teshima et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Cushing’s disease caused by pituitary corticotroph adenoma is a common endocrine disease in dogs. A characteristic biochemical feature of corticotroph adenomas is their relative resistance to suppressive negative feedback by glucocorticoids. The abnormal expression of 11beta-hydroxysteroid dehydrogenase (11HSD), which is a cortisol metabolic enzyme, is found in human and murine corticotroph adenomas. Our recent studies demonstrated that canine corticotroph adenomas also have abnormal expression of 11HSD. 11HSD has two isoforms in dogs, 11HSD type1 (HSD11B1), which converts cortisone into active cortisol, and 11HSD type2 (HSD11B2), which converts cortisol into inactive cortisone. It has been suggested that glucocorticoid resistance in corticotroph tumors is related to the overexpression of HSD11B2. Therefore it was our aim to investigate the effects of carbenoxolone (CBX), an 11HSD inhibitor, on the healthy dog’s pituitary-adrenal axis. Dogs were administered 50 mg/kg of CBX twice each day for 15 days. During CBX administration, no adverse effects were observed in any dogs. The plasma adrenocorticotropic hormone (ACTH), and serum cortisol and cortisone concentrations were significantly lower at day 7 and 15 following corticotropin releasing hormone stimulation. After completion of CBX administration, the HSD11B1 mRNA expression was higher, and HSD11B2 mRNA expression was significantly lower in the pituitaries. Moreover, proopiomelanocortin mRNA expression was lower, and the ratio of ACTH-positive cells in the anterior pituitary was also significantly lower after CBX treatment. In adrenal glands treated with CBX, HSD11B1 and HSD11B2 mRNA expression were both lower compared to normal canine adrenal glands. The results of this study suggested that CBX inhibits ACTH secretion from pituitary due to altered 11HSD expressions, and is potentially useful for the treatment of canine Cushing’s disease. Data Availability Statement: All relevant data are within the paper and its Supporting Information file. Funding: TT received Japan Society for the Promotion of Science KAKENHI Grant Number 24780315, http://www.jsps.go.jp/english/index.html. Introduction Competing Interests: The authors have declared that no competing interests exist. Corticotroph adenoma is the most common cause of canine Cushing’s disease, and the treatment options for dogs with Cushing’s disease are pituitary resection by hypophysectomy, PLOS ONE | DOI:10.1371/journal.pone.0135516 August 11, 2015 1 / 11 CBX Inhibits ACTH Secretion in Dogs radiotherapy, and medical management [1–3]. In humans, the typical treatment of Cushing’s disease is surgical resection of the pituitary tumor [4,5]. However, in veterinary medicine, the most common treatment is medical management, whereby trilostane and mitotane are most often used for the treatment [6,7]. These drugs can decrease circulating cortisol levels by inhibiting steroid synthesis (trilostane) or inducting adrenal gland necrosis (mitotane). The utilization and efficacy of these drugs has been well documented [8–12]. However, there are no reports that trilostane nor mitotane has curative effects on corticotroph adenoma. Moreover, use of these drugs may lead to the development of Nelson’s syndrome, as a consequence of suppressing cortisol negative feedback [13,14]. Our previous study found that pituitary size gradually enlarged and circulating adrenocorticotropic hormone concentrations increased via inhibited cortisol secretion after trilostane treatment in healthy dogs [15]. Recently, new drugs such as pasireotide and cabergoline, which are targeted at decreasing ACTH secretion from corticotroph tumors, have been studied for possible use in the management of human Cushing’s disease [16–19]. However, there is little research to support the direct targeting of canine corticotroph adenoma [20–22]. The candidates for the therapeutic agent of canine Cushing’s disease such as retinoic acid, pasireotide, and gefitinib, which are also targeted at decreasing ACTH secretion from corticotroph tumors. Retinoic acid and pasireotide have been reported that decreasing circulating ACTH concentrations and tumors size using dogs with Cushing’s disease [20,21]. Glucocorticoid resistance, which is a characteristic of corticotroph tumors, is partially caused by abnormal expression of 11-beta hydroxysteroid dehydrogenase (11HSD) [23,24]. 11HSD has two isoforms in humans, 11HSD type 1 (HSD11B1), which catalyzes the conversion of cortisone into active cortisol, and 11HSD type 2 (HSD11B2), which catalyzes the conversion of cortisol into inactive cortisone. Expression of both HSD11B1 and HSD11B2 have been documented in healthy dogs [25], and abnormal HSD11B1 and HSD11B2 expression patterns were found in canine corticotroph adenomas [26]. These findings are similar to those found in human and murine corticotroph adenomas [23,24,27]. A previous study using murine corticotroph tumor cells found that carbenoxolone (CBX), an 11HSD inhibitor, improved the negative feedback effect of glucocorticoids and enhanced apoptosis under existing cortisol levels [24]. However, the effect of CBX in dogs has not been studied. We aimed to investigate the effect of CBX on the pituitary–adrenal axis in healthy dogs. Materials and Methods Animals Thirteen healthy Beagles (ORIENTAL YEAST, Tokyo, Japan) were randomly assigned to a control group or carbenoxolone administration group (CBX group). All dogs were male aged 1 to 3 years old (mean: 2.0 years old), and 8.9 to 12.4 kg body weight (mean: 10.2 kg). The control group contained six dogs, with a mean age of 2.3 years and a mean weight of 10.1 kg. The CBX group had seven dogs, with a mean age of 1.9 years and a mean weight of 10.5 kg. All dogs were individually housed at the same laboratory animal unit in separate pens (1.1x0.9 m) in temperature-controlled rooms with a 12-h light: 12-h dark cycle, with be provided small blanket and free acces (...truncated)