0022-3565/02/3011-95–102$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 301, No. 1 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 4586/967861 JPET 301:95–102, 2002 Printed in U.S.A. Pharmacological Profile of a New, Potent, and Long-Acting Gonadotropin-Releasing Hormone Antagonist: Degarelix PIERRE BROQUA, PIERRE J.-M. RIVIERE, P. MICHAEL CONN, JEAN E. RIVIER, MICHEL L. AUBERT, and JEAN-LOUIS JUNIEN Ferring Research, Division of Biology of Growth and Reproduction, University of Geneva Medical School, Geneva, Switzerland (P.B., M.L.A.); Ferring Research Inc., San Diego, California (P.J.-M.R., J.-L.J.); Oregon Health Sciences University, Beverton, Oregon (P.M.C.); and Salk Institute, La Jolla, California (J.E.R.) Received September 28, 2001; accepted December 6, 2001 This article is available online at http://jpet.aspetjournals.org Downloaded from ABSTRACT We describe the pharmacological profile in rats and monkeys of subcutaneous injection. In comparative experiments, degarelix degarelix (FE200486), a member of a new class of long-acting showed a longer duration of action than the recently developed gonadotropin-releasing hormone (GnRH) antagonists. At single GnRH antagonists abarelix, ganirelix, cetrorelix, and azaline B. subcutaneous injections of 0.3 to 10 ␮g/kg in rats, degarelix The in vivo mechanism of action of degarelix was consistent produced a dose-dependent suppression of the pituitary-go- with competitive antagonism, and the prolonged action of de- jpet.aspetjournals.org nadal axis as revealed by the decrease in plasma luteinizing garelix was paralleled by continued presence of radioimmuno- hormone (LH) and testosterone levels. Duration of LH suppres- assayable degarelix in the general circulation. In contrast to sion increased with the dose: in the rat, significant suppression cetrorelix and similarly to ganirelix and abarelix, degarelix had of LH lasted 1, 2, and 7 days after a single subcutaneous only weak histamine-releasing properties in vitro. These results injection of degarelix at 12.5, 50, or 200 ␮g/kg, respectively. demonstrate that the unique and favorable pharmacological Degarelix fully suppressed plasma LH and testosterone levels properties of degarelix make it an ideal candidate for the man- in the castrated and intact rats as well as in the ovariectomized agement of sex steroid-dependent pathologies requiring long- at ASPET Journals on January 19, 2020 rhesus monkey for more than 40 days after a single 2-mg/kg term inhibition of the gonadotropic axis. The pulsatile secretion of gonadotropin-releasing hormone and/or solubility limitations that affect their clinical usefulness (GnRH) from the hypothalamus and its binding to membrane or even preclude their development as drugs (Bagatell et al., GnRH receptors located on the cell surface of the pituitary 1993, 1995; Hutchison et al., 1999). gonadotropes lead to release of gonadotropins. Subsequently, We have developed a new series of potent and long-acting these hormones, luteinizing hormone (LH) and follicle-stim- competitive antagonists for the GnRH receptor of the general ulating hormone, stimulate steroidogenesis and regulate ga- formula, Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph/Amf (P)-D-4Aph/- metogenesis through activation of cognate receptors in the Amf (Q)-Leu-Ilys-Pro-D-Ala-NH2, showing high water solubility gonads. and low histamine-releasing properties (Jiang et al., 2001). Blockade of the GnRH receptor by GnRH antagonists pro- The aim of the present study was to characterize the in vitro duces a rapid and effective suppression of gonadotropin re- and in vivo pharmacological profile of a selected representa- lease and therefore gonadal steroids secretion in human tive of this series, degarelix (FE200486: Ac-D-2Nal-D-4Cpa-D- males (Jockenho¨vel et al., 1988; Salameh et al., 1991; Baga- 3Pal-Ser-4Aph (L-hydroorotyl)-D-4Aph (carbamoyl)-Leu-Ilys- tell et al., 1993, 1995; Klingmu¨ller et al., 1993). Therefore, Pro-D-Ala-NH2) in comparison with other recently developed such antagonists have been recognized as potential drugs for GnRH antagonists such as abarelix (Cook and Sheridan, the management of sex steroid-dependent pathologies, such as 2000), ganirelix (Gillies et al., 2000), cetrorelix (Reissmann et prostate cancer and endometriosis. These conditions are cur- al., 2000), and azaline B (Rivier et al., 1995b). rently managed with long-acting preparations of GnRH super- agonists that suppress sex steroids through desensitization of the pituitary-gonadal axis (Conn and Crowley, 1991). Despite Materials and Methods the advantages of GnRH antagonists over agonists in suppress- Animals. Male Sprague-Dawley rats, intact or castrated, were ing serum gonadal steroids, many of the peptidic GnRH antag- purchased from Iffa-Credo (L’Arbresle, France) at 6 to 8 weeks of age onists investigated so far show histamine-releasing properties and housed in a temperature-, humidity-, and light-controlled room ABBREVIATIONS: GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; Aph, aminophenylalanine; Amf, aminomethylphenylalanine; RIA, radioimmunoassay; ANOVA, analysis of variance. 95 96 Broqua et al. and were given free access to food and water. Experimental protocols tion-response curves. These parameters were obtained by Hill equa- concerning the use of laboratory animals were reviewed by the Uni- tion curve fitting. versity of Geneva School of Medicine Ethical Committee for Animal Drugs. Degarelix and the reference GnRH antagonists abarelix, Experimentation and approved by the State of Geneva Veterinary azaline B, cetrorelix, ganirelix, and Nal-Glu were synthesized at Office. Ovariectomized female rhesus monkeys 8 to 10 years of age Ferring Research Inc. (San Diego, CA) according to published proto- were housed in individual cages at the Oregon Regional Primate cols. For in vivo studies, all antagonists were dissolved in a 5% Research Center. The animals were fed Purina Lab Diet (high- mannitol solution. For in vitro studies, all antagonists were dissolved protein monkey diet #5047; Purina, St. Louis, MO) and received in water (histamine release assay). approximately 125 to 175 g twice daily at around 8:00 AM and 3:00 Statistical Analysis. In the rat studies, untransformed hormone PM and fruit two or three times per week. Animals had free access to data were analyzed by Kruskal-Wallis one-way ANOVA on ranks or water. Experimental protocols concerning the use of laboratory ani- by a two-way repeated measures ANOVA (comparison between du- mals followed the guidelines for animal experimentation in Oregon ration of action after subcutaneous or intravenous administration). Regional Primate Research Center. After a significant ANOVA, statistical analysis continued by either Experimental Procedure. Rats weighed 200 to 300 g at the Student-Newman-Keuls or Dunn’s multiple comparison procedures. initiation of the study. Antagonists were injected either subcutane- For the dose-response studies with degarelix and abarelix on testos- ously in the scapular region or intravenously using a jugular cathe- terone, a Dunnett’s multiple comparison procedure versus vehicle ter. After jugular catheter implantation, rats were allowed to recover mean was performed. Differences between organ weights were mea- for at least 24 h in individual cages with food and water available ad sured by the Student’s t test or by the Mann-Whitney rank sum test ␮ when normality test failed. In the monkey studies, untransformed libitum. Blood sampling (200–250 l) was performed through the Downloaded from jugular catheter or at the tail tip and blood was collected in hepa- hormone data were analyzed by one-way repeated measures analysis rinized tubes (30 IU/ml). Plasma was extracted by centrifugation at of variance followed by Tukey’s multiple comparisons versus pre- 3000 rpm for 10 min then stored at Ϫ20°C until determination of LH treatment mean, or when normality or equal variance test failed by or testosterone. Friedman’s repeated measures analysis of variance on ranks fol- Rhesus monkeys had a range of weight during study of 5.0 to 6.7 lowed by Dunnett’s multiple comparison procedure versus pretreat- ment mean. Differences were considered statistically significant kg. Antagonists were injected subcutaneously in the right hip. Blood when p was smaller than 0.05. All statistics were performed using samples of 2.5 ml were collected from the saphenous vein, into jpet.aspetjournals.org SigmaStat 2.0 software (Jandel Scientific, San Rafael, CA). unheparinized Vacutainer tubes, placed on wet ice immediately after collection, and then refrigerated for storage and separation. The samples were spun the next day in a refrigerated centrifuge at 8°Cat Results 2500 rpm for 15 min. Suppression of Plasma LH Levels in Castrated Male Determination of LH Levels. LH levels in plasma samples were determined by standard RIA techniques with reagents prepared by Rat by Degarelix. When injected s.c. at doses ranging from ␮ Dr. A. F. Parlow (Pituitary Hormone and Antisera Center, Harbor- 0.3 to 10 g/kg, degarelix produced a dose-dependent and UCLA Medical Center, Torrance, CA) and provided by the National reversible decrease in plasma LH levels with a minimal at ASPET Journals on January 19, 2020 Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, effective dose of 3 ␮g/kg. At 10 ␮g/kg, degarelix produced a MD), and a commercially obtained secondary antiserum. National significant suppression of plasma LH up to 12 h postinjection Institute of Diabetes and Digestive and Kidney Diseases anti-rat LH (Fig. 1). S11 sera were used. Values are expressed in terms of RP-3 reference Comparison of Duration of Action of Degarelix and standard. For each experiment, all plasma samples (vehicle control Abarelix after Subcutaneous Administration in Cas- and tested antagonists) were measured in the same RIA. Monkey trated Rat. Degarelix and abarelix were given at doses of serum LH was determined by RIA with standard LH, LH antisera, 12.5, 50, and 200 ␮g/kg, and plasma samples were collected and iodination grade LH obtained from Dr.