European Journal of Endocrinology (2005) 153 R7–R10 ISSN 0804-4643

RAPID COMMUNICATION Regulation of CRH-induced secretion of ACTH and corticosterone by SOM230 in rats A P Silva, P Schoeffter, G Weckbecker, C Bruns and H A Schmid Institutes for BioMedical Research, Basel, Switzerland (Correspondence should be addressed to H Schmid; Email: [email protected])

Abstract Objective: Adrenocorticotropic (ACTH)-dependent Cushing’s syndrome is biochemically characterized by increased plasma concentrations of ACTH inducing hypersecretion of . Somatostatin is known to inhibit ACTH secretion, and in vitro data have shown the inhibition of ACTH secretion by agonists activating sst2 and sst5 receptors. The present study aimed to determine the inhibitory effect of the multireceptor ligand SOM230, compared with the sst2-preferring agonist , on corticotropin-releasing hormone (CRH)-stimulated secretion of ACTH and corticoster- one in rats. Methods: Secretion of ACTH and corticosterone was induced by i.v. application of CRH (0.5 mg/kg) in rats pretreated 1 h before by i.v. application of SOM230 (1, 3, or 10 mg/kg), octreotide (10 mg/kg) or NaCl 0.9%. Results: SOM230 (3 and 10 mg/kg) inhibited CRH-induced ACTH release by 45^3% and 51^2%, respectively, and corticosterone release by 43^5% and 27^16%, respectively. 10 mg/kg of octreotide tended to be less potent at inhibiting ACTH release (34^6% inhibition) and did not alter the secretion of corticosterone. Conclusion: SOM230 has a stronger inhibitory effect on ACTH and corticosterone secretion than octreotide in rats. This difference can be explained by its higher affinity to sst1, sst3 and especially sst5 receptors compared with octreotide.

European Journal of Endocrinology 153 R7–R10

Introduction cells (5). Although individual clinical case reports indicate that octreotide was able to restore eucortisolemia in Cushing’s syndrome is characterized by centripetal obes- patients presenting with an ACTH-dependent Cushing’s ity, skin manifestations, hypertension, proximal myopa- syndrome (6), the efficacy of the sst2-prefering agonist, thy, resistance, hyperlipidemia, osteopenia and octreotide, could not be established in clinical trials (7). some psychiatric manifestations. Adrenocorticotropic Most recently, Hofland et al. showed the predominant hormone (ACTH)-dependent forms of the syndrome expression of sst5 receptors in cultured human cortico- include Cushing’s disease, ectopic ACTH-producing troph adenomas (8). In this study, the secretion of ACTH tumors, and corticotropin-releasing hormone (CRH)-pro- from cultured human adenomas was inhibited by ducing tumors. In these three cases, increased plasma SOM230. These in vitro data strongly argue for the useful- concentrations of ACTH induce hypersecretion of cortisol ness of somatostatin analogs with high affinity for sst2 and a bilateral adrenocortical hyperplasia (1). ACTH and sst5 receptors, such as SOM230, to regulate ACTH secretion is physiologically inhibited by somatostatin (2), plasma levels in Cushing’s patients. However, the inhi- a 14 or 28 cyclopeptide involved in the regu- bition of ACTH secretion by somatostatin analogs in vivo lation of hormone and growth factor secretion (3). has not yet been demonstrated. Therefore, the aim of the Somatostatin effects are mediated through five G pro- present study was to investigate the effect of SOM230 on tein-coupled receptors, sst1–sst5, and can be mimicked the stimulated secretion of ACTH and corticosterone, by analogs which have different affinities for the five sst the rodent equivalent of cortisol, in a freely moving rat receptors. Among them are the sst2-prefering agonist model. octreotide, and the multireceptor ligand SOM230 which binds to sst1, sst2, sst3 and sst5 with high affinity, having a 30, 5 and 40 fold higher affinity to sst1, sst3 Materials and methods and sst5 receptors, respectively,compared with octreotide (4). It has been reported that sst2- and sst5-specific ago- The experiments described in this article were per- nists inhibit CRH-induced ACTH release from AtT-20 formed according to the Swiss national animal welfare

q 2005 Society of the European Journal of Endocrinology DOI: 10.1530/eje.1.01998 Online version via www.eje-online.org

Downloaded from Bioscientifica.com at 09/25/2021 12:07:14AM via free access R8 A P Silva and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2005) 153 requirements (Eidgeno¨ssische Tierversuchsbewilligung, commercially available kits: (125I) ACTH RIA kit (Dia- Switzerland). Sorin, Stillwater, Minnesota); (125I)corticosterone RIA (ICN Biomedicals GmbH, Eschwege, Germany). Compounds and formulations SOM230 acetate was dissolved in sterile water, and Statistical analysis stock solutions stored frozen at 220 8C. On the day of Results are expressed as mean^S.E.M. Data obtained at experiment, the stock solutions were diluted with each time point were compared between groups using 0.9% sterile saline to the final concentrations as indi- the non-parametric Kruskal–Wallis test and a Dunn’s cated (pH 5–6). SMS 201-995 acetate (octreotide) multiple comparison test, with the program GraphPad was prepared in the same way as SOM230. CRH Prism, version 4.02. (human/rat, Bachem AG, Bubendorf, Switzerland) was dissolved in water, and stocks stored frozen at 220 8C. Results

Animals and treatments The basal plasma ACTH concentration in rats was 46^3 pg/ml (mean^S.E.M., n ¼ 6). After i.v. injection Adult male Sprague-Dawley rats (Ico:OFA-SD, Iffa- of CRH (0.5 mg/kg), ACTH levels increased up to Credo, Lyon, France) were housed in groups of five 229^52 pg/ml within 20 min (Fig. 1A). Similarly, rats per cage under controlled light (12 h light:12 h corticosterone levels increased from 60^26 to dark) and temperature (22 8C). The rats were fed with 374^24 ng/ml (Fig. 1B). Before stimulation with water and laboratory food ad libitum. Animals were weighed on the day of surgery. The rats were allowed to adjust to the light/dark cycle for 1 week prior to the experiment. They were used with a body weight of 275–336 g. On the day before the experiment (day 0), rats were anesthetized by inhalation of isoflurane 5% in oxygen/air and kept under anesthesia with iso- flurane 2% in oxygen/air. The right femoral vein and artery were catheterized in order to perform i.v. injec- tions and blood sampling, respectively. After surgery, rats were placed individually in cages, and the arterial catheter was connected to a pump with constant infu- sion of NaCl 0.9% containing heparin 10 IU/ml. On day 1, animals were pretreated i.v. with either SOM230 (1, 3or10mg/kg), octreotide (10 mg/kg) or saline (NaCl 0.9%) for 1 h. Then they were injected i.v. with CRH 0.5 mg/kg to stimulate the secretion of ACTH and corti- costerone. The injection of CRH was considered as the time point 0 min. Blood samples were collected at time points (—60, 0, 10, 15, 20, 120 min, in pre- chilled Eppendorf tubes containing EDTA (WED-19, Milian SA, Geneva, Switzerland) and aprotinin (100 U/ml, Calbiochem, Merck KGaA, Darmstadt, Germany). The plasma was separated by centrifugation and frozen until measurement of hormone levels. Catheterized, non-anaesthetized rats were chosen in order to avoid the effect of acute stress on ACTH levels caused by handling and blood sampling. Also, the experiment was performed one day after surgery to allow the rats to recover from surgery, and to prevent Figure 1 CRH-induced increase in rat plasma ACTH (A) and corti- costerone (B). Rats were pretreated 1 h before with SOM230 any influence of the anesthetics used, i.e. isoflurane. 1 mg/kg X,3mg/kg O,or10mg/kg V (red), octreotide 10 mg/kg V (blue) or NaCl 0.9% B. ACTH release was stimulated by i.v. injection of CRH (0.5 mg/kg). Blood samples were taken at time Analytical methodology points 260, 0, 10, 15, 20 and 120 min. ACTH and corticosterone Plasma concentrations of ACTH and corticosterone concentrations were determined by RIA in rat plasma. Results are expressed as mean^S.E.M. of data from seven rats for SOM230 were determined according to the manufacturer’s and octreotide, and eight rats for vehicle group. * P , 0.05, instructions, by radioimmunoassay (RIA) using ** P , 0.01 vs (NaCl þ CRH) group.

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CRH, rats were pretreated 1 h before with either sst5 in corticotrophs (9, 10). Most recently, an elegant SOM230 (1, 3, or 10 mg/kg, i.v.), octreotide in vitro study by Hofland et al. has shown that SOM230 (10 mg/kg, i.v.) or saline. SOM230 at 1 mg/kg did not inhibits ACTH secretion from primary cultures of alter CRH-induced ACTH and corticosterone secretion. human corticotroph adenomas, whereas octreotide Pretreatment of rats with SOM230 at 3 and 10 mg/kg was not able to inhibit ACTH secretion in most cases resulted in a 56^12% and a 70^5% (P ¼ 0.011) (8). The difference in activity of the two somatostatin decrease in plasma ACTH levels, respectively, compared analogs was associated with a predominant expression with saline-treated control animals at time point of sst5 receptor mRNA in these cells. The stronger effect 20 min (Fig. 1A). Octreotide at 10 mg/kg also inhibited of SOM230 compared with octreotide observed in the CRH-induced ACTH release (55^6% decrease), but present study may be explained by the binding proper- tended to be less potent than SOM230, although the ties of the two compounds to sst receptors. Indeed, difference between both compounds was not signifi- SOM230 binds with sub-nanomolar affinity to sst2 cant. Accordingly, the calculation of the area under and sst5 receptors (IC50 values of 1^0.1 nM and the curve (AUC from time—60 min to time 120 min) 0.16^0.01 nM, respectively (4)), whereas octreotide showed a dose-dependent inhibition of CRH-induced has a preferential affinity for sst2 receptors compared ACTH secretion by SOM230, with 45^3% inhibition with sst5 (IC50 values of 0.38^0.08 nM compared at 3 mg/kg and a 51^2% inhibition at 10 mg/kg with 6.3^1 nM (4)). The differences are even more pro- (P ¼ 0.015). Octreotide at 10 mg/kg did not signifi- nounced with regard to their functional activity, as cantly inhibit ACTH secretion (34^6% decrease, reported for the inhibition of forskolin-induced intra- P . 0.05). SOM230 also inhibited CRH-induced corti- cellular cAMP accumulation (11). Clinical studies costerone secretion (Fig. 1B). At 3 mg/kg SOM230, have shown that in most cases, octreotide fails to nor- the corticosterone levels were reduced by 43^5% malize ACTH and cortisol levels in Cushing’s disease (P ¼ 0.069 for the Kruskal–Wallis test, P , 0.05 for patients (7). However, octreotide could reduce ACTH Dunn’s multiple comparison test), and at 10 mg/kg plasma levels in patients with Nelson’s syndrome SOM230 the corticosterone levels were reduced by (12). This difference of efficacy may be explained by a 27^16%, as calculated from the AUC values. However, receptor subtype specific downregulation of sst recep- the reduction did not reach statistical significance tors by the high cortisol plasma levels observed in (Table 1). Octreotide did not alter CRH-induced corti- Cushing’s patients, as recently suggested (8). According costerone secretion (Fig. 1B, Table 1). to this hypothesis, recent in vitro studies have shown that the cortisol analog dexamethasone was able to abolish the effect of octreotide on ACTH secretion, but Discussion did not affect the inhibition produced by SOM230. This difference was associated with a down-regulation This study demonstrates the inhibition of CRH-induced of sst2 and no change in sst5 receptor expression (8, secretion of both ACTH and corticosterone by the mul- 13). In the present study, CRH-induced corticosterone tireceptor ligand SOM230 in vivo in rats. Octreotide did increase was inhibited by SOM230, but not by octreo- not inhibit CRH-induced corticosterone secretion, and tide. This may suggest a direct effect of SOM230 on tended to be less efficacious than SOM230 for the inhi- adrenals through a receptor other than sst2. Indeed, bition of ACTH release. The effect observed for SOM230 the expression of sst receptors has been reported in on ACTH secretion was expected since it had previously adrenal tumors (14) and in the rat been described that sst2- and sst5-specific agonists inhi- (15). mRNA for sst1, sst2, sst4 and sst5 was detected bit CRH-induced ACTH release from the mouse pitu- by RT-PCR with a predominant amount of sst2, and itary cell line AtT-20 (5). In addition, sst receptor in situ hybridization showed major localization of sst2 expression studies on tissue and purified receptor mRNA in the adrenal cortex, in the zona pituitary cells showed the expression of both sst2 and glomerulosa, but also some labeling in the external

Table 1 Area under curve (AUC) calculated from the data represented in Fig. 1. Results are expressed as mean^S.E.M., n ¼ 7 for SOM230 and octreotide, and n=8 for vehicle.

AUC

Group ACTH (ng/min/ml21) Corticosterone (mg/min/ml21)

NaCl 0.9% þ CRH 0.5 mg/kg 20.18^3.20 36.87^4.37 SOM230 1 mg/kg þ CRH 0.5 mg/kg 23.23^7.23 30.41^4.21 SOM230 3 mg/kg 1 CRH 0.5 mg/kg 11.00 6 1.46* 21.14 6 1.80* SOM230 10 mg/kg 1 CRH 0.5 mg/kg 9.86 6 1.03* 26.85^5.68 Octreotide 10 mg/kg þ CRH 0.5 mg/kg 13.24^2.89 33.56^5.62

* P , 0.05 vs (NaClþCRH) group.

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Downloaded from Bioscientifica.com at 09/25/2021 12:07:14AM via free access R10 A P Silva and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2005) 153 part of zona fasciculata (15). Therefore, a direct effect adrenocorticotropin secretion from AtT-20 cells. Neuroendocrin- of somatostatin analogs on corticosterone secretion ology 2002 75 339–346. 6 Uwaifo GI, Koch CA, Hirshberg B, Chen CC, Hartzband P, cannot be excluded at present. However, the fact that Nieman LK & Pacak K. Is there a therapeutic role for octreotide SOM230 has a relatively stronger inhibitory effect on in patients with ectopic Cushing’s syndrome? Journal of Endocrino- CRH-stimulated ACTH secretion than on corticosterone logical Investigation 2003 26 710–717. secretion is in line with a primary action on the pitu- 7 de Herder WW & Lamberts SW. Is there a role for somatostatin and its analogs in Cushing’s syndrome? Metabolism 1996 45 itary. These are the first preclinical data comparing 83–85. the effects of SOM230 and octreotide on ACTH 8 Hofland LJ, van der Hoek J, Feelders R, van Aken MO, van secretion in vivo. The present study, together with the Koetsveld PM, Waaijers M, Sprij-Mooij D, Bruns C, in vitro data reported by others (8) support the idea Weckbecker G, de Herder WW, Beckers A & Lamberts SWJ. The that SOM230 may have potential for the regulation of multi-ligand somatostatin analogue SOM230 inhibits ACTH secretion by cultured human corticotroph adenomas via somato- ACTH plasma levels. This would be of major interest statin receptor type 5. European Journal of Endocrinology 2005 for the treatment of ACTH-dependent Cushing’s syn- 152 643–652. drome, which includes Cushing’s disease, ectopic 9 Day R, Dong W, Panetta R, Kraicer J, Greenwood MT & Patel YC. ACTH-producing tumors, and CRH-producing tumors, Expression of mRNA for (sstr) types 2 and 5 in individual rat pituitary cells. A double labeling in situ hybridiz- as well as Nelson’s syndrome, where the elevated ation analysis. Endocrinology 1995 136 5232–5235. secretion of ACTH needs to be controlled. Further 10 Mezey E, Hunyady B, Mitra S, Hayes E, Liu Q, Schaeffer J & studies in animal models showing increased or Schonbrunn A. Cell specific expression of the sst2A and sst5 decreased levels of ACTH and/or corticosterone might somatostatin receptors in the rat . Endocrinology help to further strengthen this concept. 1998 139 414–419. 11 Schmid HA & Schoeffter P. Functional activity of the multiligand analog SOM230 at human recombinant somatostatin receptor subtypes supports its usefulness in neuroendocrine tumors. Acknowledgements 2004 80 47–50. 12 de Herder WW & Lamberts SW. Octapeptide somatostatin- We would like to thank Ionel Bobirnac for his skillful analogue therapy of Cushing’s syndrome. Postgraduate Medicine technical assistance. Journal 1999 75 65–66. 13 van der Hoek J, Waaijers M, van Koetsveld PM, Sprij-Mooij D, Feelders RA, Schmid HA, Schoeffter P, Hoyer D, Cervia D, References Taylor JE, Culler MD, Lamberts SW & Hofland LJ. Distinct func- tional properties of native somatostatin receptor subtype 5 com- 1 Miyachi Y. Pathophysiology and diagnosis of Cushing’s syndrome. pared with subtype 2 in the regulation of ACTH release by Biomedicine and Pharmacotherapy 2000 54 113s–117s. corticotroph tumour cells. American Journal of Physiology-Endo- 2 Engler D, Redei E & Kola I. The corticotropin-release inhibitory crinology and Metabolism 2005 289 278–287. factor hypothesis: a review of the evidence for the existence of 14 Unger N, Serdiuk I, Sheu SY, Walz MK, Schulz S, Schmid KW, inhibitory as well as stimulatory hypophysiotropic regulation of Mann K & Petersenn S. Immunohistochemical determination of adrenocorticotropin secretion and biosynthesis. Endocrine Reviews somatostatin receptor subtypes 1, 2A, 3, 4, and 5 in various adre- 1999 20 460–500. nal tumors. Endocrine Research 2004 30 931–934. 3 Gillies G. Somatostatin: the neuroendocrine story. Trends in Phar- 15 O’Carroll AM. Localization of messenger ribonucleic acids for macological Sciences 1997 18 87–95. somatostatin receptor subtypes (sstr1-5) in the rat adrenal 4 Bruns C, Lewis I, Briner U, Meno-Tetang G & Weckbecker G. gland. Journal of Histochemistry and Cytochemistry 2003 51 SOM230: a novel somatostatin peptidomimetic with broad 55–60. somatotropin release inhibiting factor (SRIF) receptor binding and a unique antisecretory profile. European Journal of Endocrin- ology 2002 146 707–716. 5 Strowski MZ, Dashkevicz MP, Parmar RM, Wilkinson H, Kohler M, Schaeffer JM & Blake AD. Somatostatin receptor subtypes Received 12 April 2005 2 and 5 inhibit corticotropin-releasing hormone-stimulated Accepted 5 July 2005

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