Characterizing pituitary response to a gonadotropinreleasing hormone (GnRH) antagonist in monkeys:

Transcription

1 FERTLTY AND STERLTY Copyright 1987 The American Fertility Society Printed in U.S.A. Characterizing pituitary response to a gonadotropinreleasing hormone (GnRH) antagonist in monkeys: tonic follicle-stimulating hormone/luteinizing hormone secretion versus acute GnRH challenge tests before, during, and after treatment* Claudio F. Chillik, M.D.t:\: Joseph tskovitz, M.D., D.Sc.tll Do Won Hahn, Ph.D.~ John L. McGuire, Ph.D.~ Douglas R. Danforth, Ph.D.t Gary D. Hodgen, Ph.D.tH The Jones nstitute for Reproductive Medicine, Eastern Virginia Medical Schaal, Norfolk, Virginia and Research Laboratories, Ortha Pharmaceutical Corporation, Raritan, New Jersey Pituitary sensitivity to a gonadotropin-releasing hormone (GnRH) challenge test before, during, and after GnRH antagonist administration was compared in four ovariectomized female monkeys receiving GnRH antagonist intramuscularly (1M) at increasing doses of.3, 1., and 3. mg/kg/day over 9 days. Three days before and 3 days after treatment, monkeys received vehicle alone. On experiment days 4,7,1,13, and 16,1 Lg of GnRH was administered intravenously (V) and blood drawn at and 3 minutes. Before treatment, tonic follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were 248 ± 15 and 178 ± 31 ng/ml, respectively; after.3 mg/kg/day of GnRH antagonist, FSH and LH decreased to 3 ± 6 and 41 ± 4 ng/ml, respectively. After treatment with either 1 mg/kg/day or 3 mg/kg/day of GnRH antagonist, both gonadotropins were undetectable in serum. Monkeys with lower initial levels of gonadotropins were suppressed by 48 hours after GnRH antagonist, while those with higher tonic gonadotropins were suppressed 6 days later (FSH: r =.992; LH: r =.833). The data show that initial physiologic status is predictive of the rapidity of the suppression response induced by a GnRH antagonist and that, after achieving pituitary suppression, responsivity to an V GnRH challenge test may be restored before normal tonic FSH/LH secretion is regained. Fertil Steril 48:48, 1987 Previous studies have forecast potential therapeutic uses of gonadotropin-releasing hormone (GnRH) analogs in reproductive endocrinology.1,2 Received February 2,1987; revised and accepted May 14, * Performed in collaboration with Ortho Pharmaceuticals and supported in part by funding from Ortho Pharmaceuticals, Raritan, New Jersey. t The Jones nstitute, Department of Obstetrics and Gynecology, Eastern Virginia Medical School. :j: Recipient of the Rockefeller Foundation postdoctoral fellowship in Population Sciences for Reproductive Biology. Present address: Centro de Estudios Ginecologicos y Reproducion, Buenos Aires, Argentina. Present address: Department of Obstetrics and Gynecology, Rambam Medical Center, Haifa, srael. 48 Chillik et a1. Pituitary response to GnRH antagonist Until now, most human and animal data have been obtained using agonists of GnRH in clinical trials for the study of precocious puberty,3,4 endometriosis, 5 contraception,6,7 prostatic carcinoma, 8 uterine fibroids,9 ovulation induction,lo or in vitro fertilizationy,12 However, the development of useful antagonists of GnRH is now approaching clinical investigation. n some circumstances, these antagonists offer more therapeutic promise than the agonists. 11 Research Laboratories, Ortho Pharmaceutical Corporation. # Reprint requests: Gary D. Hodgen, Ph.D., The Jones nstitute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Lewis Hall, Room 211, Norfolk, Virginia Fertility and Sterility

2 GnRH antagonists are analogs of GnRH having a high affinity for the pituitary GnRH receptor, but with no intrinsic biologic activity to stimulate follicle-stimulating hormone/luteinizing hormone (FSH/LH) secretion.13 Persistent administration of a GnRH antagonist suppresses basal levels of gonadotropins, creating a reversible "medical hypophysectomy."14 Not only do GnRH antagonists almost immediately suppress basal gonadotropin secretion, but they also blunt the response of the pituitary to an estrogen (positive feedback) challenge test. S-S We designed the following experiment in order to evaluate the following: (1) dose-response characteristics of a newer generation GnRH antagonist; (2) correlation between initial basal (tonic) gonadotropin levels and days needed to achieve pituitary suppression; and (3) pituitary response to a GnRH challenge test before, during, and after GnRH antagonist administration. MATERALS AND METHODS Four ovariectomized (8 weeks or more) female rhesus monkeys (Macaca mulatta) were assigned to this study. Animal husbandry, blood collection techniques, and assays have been described previously.19 Reagents for FSH/LH assays were obtained from the Hormone Distribution Office (NAMDD, Bethesda, MD). Coefficients of variation for within and between assays were 7.1 and 11.8%, respectively. Drug Administration The GnRH antagonist used in this study ([Ac D2NAU, 4ClDPhe2, D3PaP, Arg S, DGlu 6 (AA), DAlalO]-GnRH) was synthesized by The Clayton Foundation Laboratories for Peptide Biology (The Salk nstitute,2 La Jolla, CA). The antagonist was dissolved in sesame oil and administered intramuscularly (1M). Figure 1 illustrates the experimental protocol. During the first 3 days, 2 ml of vehicle (sesame oil only) was given 1M (control). From days 4 to 6, monkeys received.3 mg/kg/day of the GnRH antagonist; the dose was increased to 1. mg/kg/day from days 7 to 9, and to 3. mg/kg/day from days 1 to 12. Finally, vehicle only was given again from days 13 to 15 during post-treatment recovery from GnRH antagonist administration. GnRH Challenge Test ntravenous (V) GnRH (1 f.lg) was given as a bolus injection via a catheter inserted into the sa- ~ o Acute GnRH Challenge Tests 3 o (1lJg iv) 2.5 o 5.5 Pre -Treatment l' Vehicle Low Dose Mid Dose High Dose j Post-Treatment Vehicle Day of Experiment Figure 1 Experimental protocol. Four ovariectomized female monkeys received GnRH antagonist 1M over 9 days in increasing doses of.3, 1., and 3. mg/kg/day. GnRH (1!'g/V) was administered on experiment days 4, 7, 1, 13, and 16; blood samples were drawn at and +3 minutes. phenous vein. The GnRH challenge test was performed on days 4 (control), 7, 1, 13 (treatment), and 16 (recovery). On these occasions, GnRH was injected 6 minutes before administration of the GnRH antagonist. Serum Collection Blood was drawn daily on day 1 until experiment day 16 from the femoral vein before the injection of GnRH antagonist, as well as at and +3 minutes relative to GnRH administration. Therefore, the levels of LH and FSH reported for a certain day reflect the effect(s) of the antagonist given on the previous day(s), as well as the acute effects of GnRH challenge tests given intermittently. LH and FSH levels are expressed in ng/ml of the mean ± standard error of the mean ex ± SEM). Statistical differences were measured through analysis of variance and correlation coefficients for changes in tonic gonadotropin levels in serum and the response to GnRH challenge tests.21 RESULTS Tonic Gonadotropin Profiles nitial levels of plasma gonadotropins were in the ovariectomized range (FSH, 248 ± 15 ng/ml; LH, 178 ± 31 ng/ml). After 3 days of vehicle administration, there were no meaningful changes in the tonic levels of gonadotropins. However, within 24 hours after administration of.3 mg/kg of the Chillik et al. Pituitary response to GnRH antagonist 481

3 GnRH antagonist, there was a striking decrease in serum levels of FSH (78 ± 24) and LH (69 ± 18). After 3 days of GnRH antagonist treatment at.3 mg/kg, tonic FSH concentrations in serum decreased to near minimal detectable limits of 3 ± 7 and LH to 41 ± 4 ng/ml (Fig. 2). After 3 days of treatment with 1 mg/kg/day of the GnRH antagonist, LH and FSH levels were suppressed below assay detection limits in all monkeys (FSH < 18; LH < 35 ng/ml). Figure 2 illustrates this decrease of serum gonadotropins during the study; the decrease was highly significant for both FSH and LH (P <.5). As expected, when the dose of antagonist was increased to 3 mg/kg/day, gonadotropins remained suppressed. During the recovery interval (days 14 to 16), despite administration of vehicle alone, tonic serum gonadotropin levels in all monkeys remained undetectable. The time needed to achieve suppression was not the same in all monkeys. Whereas some needed only 2 days of treatment to achieve full suppression, others required as many as 6 days of treatment with the GnRH antagonist in order to reach a "medical hypophysectomy" state. Table 1 demonstrates a correlation between the initial tonic levels of gonadotropins and the days of antagonist administration necessary to achieve suppression. That is, monkeys whose pituitaries quickly submitted to GnRH antagonist suppression had begun with lower tonic levels of FSH and LH in serum compared with females having higher initial levels of pituitary gonadotropins. Accordingly, there was a high correlation (r =.992) between initial FSH levels and the interval of treatment necessary to 25 2 : 15 OJ c 1 5 Pre-Treatment ljehic;1e Only Low Dose Mid Dose High Dose Post-Treatment 1.3 mg!kg~ 1 mgikg/day : 3 mg/kg/day: Vehicle Only, i, O" -O~~-o---t " 1\ '\.-efshlp<o LH p<o.31 Table 1 Correlation Between nitial FSH and LH Levels in Serum and Days of Treatment Necessary to Achieve Maximal Suppression Days until Monkey FSH/LH below no. nitial FSH detection limits nitial LH ng/ml FSHlLH ng/ml Sl / /3 14 U / H 44 6/5 26 Correlation FSH: r =.992 LH: r =.839 (P <.8) (P>.5) achieve maximal suppression (P <.1). Although there was also a strong correlation (r =.839) between tonic LH levels and days of treatment until full suppression, this was not significant (P>.5). Response to GnRH Challenge Test After 3 days of treatment with.3 mg/kg of GnRH antagonist, bolus administration of 1 J.l.g of GnRH V elicited the release of gonadotropins not dissimilar from responses under pretreatment conditions (Fig. 3). Upon increasing the dose of GnRH antagonist to 1 mg/kg, although tonic secretion of LH and FSH was markedly suppressed, acute responses to the GnRH challenge were again similar to pretreatment tests. Despite 9 days of treatment with increasing doses of the GnRH antagonist and with gonadotropins consistently below detection limits in all monkeys, the GnRH challenge still elicited a small release of FSH (P <.1; Fig. 3a); indeed, the LH secretory response was only slightly inhibited (P <.5; Fig. 3b). nterestingly, as soon as 3 days after post-treatment recovery, although tonic gonadotropins remained suppressed, there was partial restoration of FSH secretory response to the GnRH challenge test, while acute LH release was already fully recovered. mportantly, we found no histamine release effects either locally or systemically during 9 days of GnRH antagonist therapy at doses up to 3. mg/kg/day. This dose is up to lo-fold more than that required to achieve a persistent state of "medical hypophysectomy." O~~~~~~~~-L~~~~~ Day of Experiment Figure 2 Tonic serum concentrations of FSH and LH before, during, and after GnRH antagonist treatment. Each point represents the mean of four animals. The decrease in serum FSH and LH throughout the study was significant (P <.5). 482 Chillik et al. Pituitary response to GnRH antagonist DSCUSSON Our data indicate that, within 24 hours after a single dose of.3 mg/kg of GnRH antagonist, profound suppression of FSH and LH concentrations in peripheral serum was evident. Depending on the Fertility and Sterility

4 ~ Ol c 15 (/) LL ~ g' ' 1 5 o a b Tre~~~ent 3 mg 1 mg 3. mg T r:~~nt Control Control Daily Dose of GnRH Antagonist Time in minutes Do mins!i!l3 mins after GnRH bolus Figure 3 Serum concentrations offsh (a) and LH (b) before and 3 minutes after a GnRH challenge test (1 /Lg/V). Each bar represents the mean gonadotropin level ± SEM, n = 4. initial tonic levels of LH and FSH, gonadotropins were suppressed below detectable limits after 2 to 6 days of GnRH antagonist administration. Tonic FSH/LH levels remained suppressed during the study interval, even 3 days into the post-treatment recovery interval. These data demonstrate that (Ac-D2NAU, 4ClDPhe2, D3PaP, Arg5, DGlu6[AA], DAlalO)-GnRH is a potent antagonist of GnRH, and that, with at least some doses, it has a duration of action of several days. GnRH antagonists have significant therapeutic advantages over the agonists in that they almost immediately inhibit gonadotropin secretion without inducing an initial rise in gonadotropins, as is characteristic of GnRH agonists during the first 2 to 3 weeks of treatment.22 There have been relatively few clinical trials using GnRH antagonists because of the lack of a potent GnRH antagonist without side effects. One of the main side effects of GnRH antagonists was reported by Schmidt et al.,23 who demonstrated that administration of cer- tain antagonists of GnRH to rats produced transient edema of the face and extremities, including a cutaneous anaphylactoid-like reaction.24 This side effect was due to release of histamine by the drug. Among the most potent analogs associated with this histamine release phenomenon are those having structures that include a D-arginine at position 6. The antagonist used in the present study has a D-arginine substitution in position 5 with a D-glutamate in position 6. Although our observations were not specifically directed at this issue, the combination of structural features avoided discernible histamine release in monkeys. We found a strong correlation between tonic levels of gonadotropins and the days of GnRH antagonist treatment necessary to achieve full suppression. Using a different GnRH antagonist, Cetel et al.25 reported that the net decrement in gonadotropin concentrations was correlated with tonic values of LH and FSH in women. Thus, a higher dynamic state of pituitary gonadotropin secretion portends greater relative resistance to suppression by the GnRH antagonist in women and monkeys. Therefore, when evaluating the potency of a GnRH antagonist, it may be important to take into consideration the endocrine milieu of the individual. Although we were able to decrease the levels of gonadotropins within 24 hours after administration of a single dose (.3 mg/kg) of GnRH antagonist and were able to achieve undetectable levels of LH and FSH after 2 to 6 days of treatment in all monkeys (perhaps due to increased doses), even 9 days of GnRH antagonist treatment did not fully block FSH or LH secretion during a GnRH challenge test. ndeed, LH secretory responses were hardly affected. This agrees with a recent study done by Kenigsberg and Hodgen,16 but is in contrast to a study done by Balmaceda et al. 18 in which bolus injections of GnRH 18 hours after administration of GnRH antagonist elicited a blunted release of gonadotropins. A similar response was seen in adult male monkeys by other authorsy mportantly, we performed the GnRH challenge test 23 hours after GnRH antagonist administration. Thus, the pituitary may have had enough time to "recover" from the acute effects of the antagonist in that antagonist occupation of the GnRH receptor is diminished and/or circulating GnRH antagonist levels are decreased. That Balmaceda et al. 18 observed a blunted response to GnRH 18 hours after antagonist treatment agrees with this hypothesis, and suggests that the pituitary is indeed recovering from the acute antagonist effect. Chillik et al. Pituitary response to GnRH antagonist 483

5 Because some recovery of pituitary FSH/LH secretion in response to the acute GnRH challenge test was seen post-treatment, we postulated that low doses of GnRH antagonist may suppress tonic gonadotropin secretion, while the synthesis and storage of LH and FSH are replenished and sustained concurrently. This would allow for maintenance of the ready releasable pool of gonadotropins, as when high doses of GnRH (1 J.tg V) are given. Administration of high doses of GnRH antagonist may not only suppress the secretion of basal gonadotropins, but also block the synthesis and storage of gonadotropins in the anterior pituitary. This scenario may not be immediately evident until depletion of stored pools of FSH and LH are revealed by serial GnRH challenge tests. Alternatively, the GnRH antagonist we used may have an extended duration of action on the pituitary gonadotropins. n summary, although all monkeys were completely suppressed by treatment day 6, pituitary response to GnRH was retained, albeit diminished. After 3 days post-treatment, although tonic gonadotropins remained suppressed, pituitary release of FSH and particularly LH in response to a GnRH challenge test had increased when compared with the tests administered during antagonist treatment. None of the monkeys showed either local or general evidence of histamine release side effects. We conclude that (1) the GnRH antagonist used in this study is potent and without discernible side effects; (2) tonic gonadotropins (particularly FSH) are predictive of the treatment time needed to achieve full pituitary suppression of tonic gonadotropin secretion; (3) normal pituitary response to a GnRH challenge test is retained for several days, even after obtaining full suppression of tonic gonadotropins via a GnRH antagonist; and (4) recovery of pituitary secretory capacity in response to a GnRH bolus V is manifest before restoration of tonic levels of serum gonadotropins. This GnRH antagonist, as well as others having similar structure-activity correlations without significant histamine release effects, deserve consideration for pursuit of clinical trials. The rapidity of action of antagonists of GnRH and their absence of interim stimulatory effects (such as agonists manifest) may allow greater practical utility for both therapeutic and diagnostic applications. Acknowledgments. We appreciate the superb technical assistance of Ms. Lynn Sharpe and the timely efforts of Ms. Dara Leary in the preparation of this manuscript. REFERENCES 1. Yen SCC: Clinical applications of gonadotropin-releasing hormone and gonadotropin releasing hormone analogs. Fertil Steril 39:257, Hodgen GD: Releasing hormones as diagnostic and therapeutic agents. Fertil Steril 39:592, Comite F, Cutler GB, Rivier J, Vale W, Loriaux DL, Crowley WF: Short-term treatment of idiopathic precocious puberty with a long-acting analogue of luteinizing-releasing hormone. N Engl J Med 35:1546, Luder AS, Holland FJ, Costigan DC, Jenner MR, Wielgosz G, Fazeekas ATA: ntranasal and subcutaneous treatment of central precocious puberty in both sexes with a long-acting analog of luteinizing hormone-releasing hormone. J Clin Endocrinol Metab 58:966, Lemay A, Maheux R, Faure N, Jean C, Fazekas ATA: Reversible hypogonadism induced by a luteinizing hormonereleasing hormone (LH-RH) agonist (Buserelin) as a new therapeutic approach for endometriosis. Fertil Steri41:863, Nillius SJ, Gudmundsson J, Bergquist C: A new superagonist of GnRH for inhibition of ovulation in women. Ups J Med Sci 89:147, Linde R, Doelle GC, Alexander N, Kirchner F, Vale W, Rivier J, Rabin D: Reversible inhibition of testicular steroidogenesis and spermatogenesis by a potent gonadotropin-releasing hormone agonist in normal men: an approach toward the development of a male contraceptive. N Engl J Med 35:663, Labrie F, Dupont A, Belanger A, Lacourciere Y, Raynaud JP, Husson JM, Gareau J, Fazekas ATA, Sandow J, Monfette G, Girard JG, Emond J, Houle JG: New approach in the treatment of prostate cancer: complete instead of only partial withdrawal of androgens. Prostate 4:579, Filicori M, Hall DA, Loughlin JS, Rivier J, Vale W, Crowley WF: A conservative approach to the management of uterine leiomyoma: pituitary desensitization by a luteinizing hormone-releasing hormone analogue. Am J Obstet Gynecol 147:726, Kenigsberg D, Littman BA, Williams RF, Hodgen GD: Medical hypophysectomy.. Variability of ovarian response to gonadotropin therapy. Fertil Steri42:116, Craft L, Porter RN, Smith B, Jacobs HS: Ovulation induction in the poor responder (Abstr). Presented at the Fourth World Conference on n Vitro Fertilization, Melbourne, Australia, Wildt L, Diedrich K, Van der Ven H, Al Hasani S, Hubner H, Klasen R: Ovarian hyperstimulation for in vitro fertilization controlled by GnRH agonist administered in combination with human menopausal gonadotropins. Hum Reprod 1:15, Schally A V, Arimura A, Coy DH: Recent approaches to fertility control based on derivatives of LH-RH. Vitam Horm 38:257, Kenigsberg D, Littman BA, Hodgen GD: Medical hypophysectomy.. Dose-response using a gonadotropin-releasing hormone antagonist. Fertil Steril 42:112, Norman RL, Rivier J, Vale W, Spies HG: nhibition of estradiol-induced gonadotropin release in ovariectomized rhesus macaques by a gonadotropin-releasing hormone antagonist. Fertil Steril 45:288, Chillik et al. Pituitary response to GnRH antagonist Fertility and Sterility

6 16. Kenigsberg D, Hodgen GD: Ovulation-inhibition by administration of weekly gonadotropin-releasing hormone antagonist. J Clin Endocrinol Metab 62:734, Bint Akhtar F, Weinbauer GF, Nieschlag E: Acute and chronic effects of a gonadotropin-releasing hormone antagonist on pituitary and testicular function in monkeys. J Endocrinol 14:345, Balmaceda JP, Coy DH, Schally AV, Asch RH: Temporal changes in FSH and LH concentrations following the administration of a potent LH-RH inhibitory analogue ([N Ac-D-Trpl.3, D-p-C-Phe 2, D-Phe 6, D-AlalOJ-LH-RH) to oophorectomized rhesus monkeys. Acta Eur Fertil 14:249, Goodman AL, Descalzi CD, Johnson DK, Hodgen GD: Composite pattern of circulating LH, FSH, estradiol and progesterone during the menstrual cycle in cynomolgus monkeys. Proc Soc Exp Bio Med 155:479, Rivier JE, Porter J, Rivier CL, Perrin M, Corrigan A, Hook W A, Siraganian RP, Vale WW: New effective gonadotropin releasing hormone antagonists with minimal potency for histamine release in vitro. J Med Chem. n press 21. Dixon WJ, Massey F: ntroduction to Statistical Analysis. New York, McGraw-Hill, Schriock ED, Monroe SE, Martin MC, Henzel MR, Jaffe RB: Effect on corpus luteum function of luteal phase administration of a potent gonadotropin-releasing hormone analog (nafareline). Fertil Steril43:844, Schmidt F, Sundaram K, Thau RB, Bardin CW: [Ac-D Nal(2)" 4FD-Phe 2, D-Trp3, D-Arg26J-LHRH, a potent antagonist of LHRH, produces transient edema and behavioral changes in rats. Contraception 29:283, Hahn DW, McGuire JL, Vale WW, Rivier J: Reproductive/ endocrine and anaphylactoid properties of an LHRH antagonist, ORF 1826 [Ac-D-Nal(2)" 4FDPhe 2, D-Trp3-D ArtJ-GnRH. Life 37:55, Cetel NS, Rivier J, Vale W, Yen SSC: The dynamics of gonadotropin inhibition in women induced by an antagonist analog of gonadotropin-releasing hormone. J Clin Endocrinol Metab 57:62, 1983 Chillik et a. Pituitary response to GnRH antagonist 485