Adv. Contracept. 1 (1985) 109-117
Comparison of the subcutaneous and intranasal administration of an LH-RH antagonist ([N-Ac-D-p-C1-Phel'2,D-Trp3,D Arg6,D-Alal°]-LH-RH) in the rhesus monkey R. H. ASCH*, J. P. BALMACEDA*, M. NEVES DE CASTRO* and A. V. SCHALLYt
* Department of Obstetrics and Gynecology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78284, USA t Tulane University School of Medicine, V.A. Medical Center, New Orleans, LA 70146, USA
Abstract Although luteinizing hormone-releasing hormone (LH-RH) agonists have been administered successfully by other than systemic routes (oral, intranasal (i.n.) and vaginal), there is no evidence that inhibitory analogues may be used in any form other than injectable. In the present study, we compared the effect of two routes of administration: (1) subcutaneous (s.c.), 0.5, 0.2 and l m g ; and (2) i.n., 0.2, 1 and 5 mg of an LH-RH antagonist, ORG 30276 ([N-Ac-D-p-C1-Phel,2,D-Trp3,D-Arg6,D-Ala~°]-LH-RH) on gonadotropin levels in oophorectomized monkeys. One hour after s.c. administration, FSH and LH values exhibited a dose-dependent fall that lasted for up to 12 h. After s.c. administration, the maximum inhibition of serum FSH and LH was 29 and 41% (0.2mg dose) and 41 and 58% (1 mg dose), respectively. After i.n. administration, maximum inhibition of serum FSH and LH was 19 and 40% (lmg) and 32 and 53% (5mg), respectively. These decreases were dose-related and lasted for up to 12 h. Analysis of the data revealed that the bioavailability of the i.n. route versus the s.c. route ranged from 16 to 26%. This high effectiveness of the i.n. route in terms of bioavailability is markedly greater than that previously reported for LH-RH agonists (1%) and is probably due to a resistance to enzymatic hydrolysis in the nasal mucosa. These results show for the first time that antagonists of LH-RH can be administered by routes other than parenteral, increasing their potential clinical use in conditions in which inhibition of gonadotropins is desired, as in contraception and in therapy for endometriosis, precocious puberty, and hormone-dependent neoplasms. 109
Advances in Contraception. ISSN 0267-4874. Copyright © MTP Press Ltd., Falcon House, Lancaster, UK. Printed in The Netherlands.
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Introduction Luteinizing hormone-releasing hormone (LH-RH) and its agonistic analogues have been shown to be effective when administered by different routes, i.e. parenteral, intranasal (i.n.), oral, vaginal, and rectal [1-5]. Although almost all modes have proven adequate for effective administration of LH-RH or LH-RH agonists, those modes other than the parenteral require considerably larger doses of the decapeptide or its derivatives to achieve a comparable response [5-9]. The most intensively studied route of administration of LH-RH agonists, other than the injectable, has been the i.n., due to its easy applicability and lack of major side-effects. Recently, however, Tolis et al. [6] reported that even large i.n. doses of an LH-RH agonist are not able to induce complete pituitary and gonadal sensitization, as compared to the effects induced by parenteral administration of the same compound. Unfortunately, no studies are available yet on the i.n. absorption of LH-RH antagonists, despite their widespread use in research in recent years in humans and in non-human primates, as well as in smaller laboratory animals [10-14]. The purpose of the present study was to compare the effects of a potent LH-RH inhibitory analogue, ([N-Ac-D-p-C1-Phel,2,D-Trp3,D-Arg6,D-Alal°]-LH-RH) ORG 30276, administered subcutaneously (s.c.) or i.n., on serum levels of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in castrated female rhesus monkeys.
Materials and methods Chronically oophorectomized rhesus monkeys were used in this study. The animals were individually caged and housed in a constantly controlled environment - temperature (23+1°C), humidity (50%), and light:dark photoperiod (06.00-20.00h). Other details on the housing, feeding and general husbandry practices have been described elsewhere [14]. Animals were divided into six groups of five each according to type of treatment. The LH-RH antagonist, ORG 30276, was administered in a single s.c. dose (total volume 0.5 ml = 0.2 ml propylene glycol + 0.3 ml saline solution) to groups 1-3 in doses of 50, 200 and 1000/~g, respectively. ORG 30276 was administered by nasal drops (total volume 0.4ml= 0.16ml propylene glycol + 0.24ml saline solution, 0.2ml in each nostril) in doses of 0.2, 1 and 5mg to animals of groups 4-6, respectively. ORG 30276 solution was prepared at 37°C and administered to the animals within 5 rain. Blood (3 ml) was drawn from the femoral or saphenous veins of animals lightly sedated with ketamine HC1 (5-7mgkg -~) (Vetalar@; Parke-Davis, Morris Plains, NJ) according to the following schedule: 1. every day at 08.00 for the 5 days preceding the administration of ORG 30276 in order to determine baseline levels of gonadotropins; 2. immediately before (-30 min and 0 h) the administration of ORG 30276 (08.00); 3. at 1, 2, 4, 6, 8, 12, 33, 48, 57, 72 and 96 h after administration of the drug. Serum was obtained after centrifugation of blood and stored at -20°C until
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assayed for FSH and LH by radioimmunoassay, as described previously [13]. All samples were assayed in duplicate within the same assay for each hormone. Sensitivity was 1500 and 210ng/ml for FSH and LH, respectively. Intra-assay coefficients of variation at 70% maximum binding were 2.8 and 5.3% for FSH and LH, respectively. Concentrations of gonadotropins were averaged at each time-point following the administration of ORG 30276 and compared to the baseline levels. Statistical analysis was performed using multiple analysis of variance. Differences were considered significant when p values were <0.05. Special attention was given to possible side-effects or anaphylactic reactions following the administration of ORG 30276.
Results Figure 1 shows the effects on serum FSH levels of different doses of ORG 30276 administered s.c. or i.n., respectively. The lowest doses (s.c., 0.05 rag; i.n., 0.2 rag) had no demonstrable effect on circulating FSH concentrations. The s.c. doses of 0.2 and I m g induced rapid decreases in FSH levels as early as I h after administration. This effect was evident for as long as 12 h. Maximum inhibition was 29 and 41% for the 0.2 and l m g doses, respectively. Intranasal administration of 1 and 5 mg of the LH-RH antagonist also caused a rapid fall of FSH levels, noticeable in the first blood sample (1 h) after drug administration. The inhibition of FSH lasted for 6 and 12h and maximum reduction was 19 and 40% for the 1 and 5mg i.n. doses, respectively. Figure 2 shows the effects on LH levels of the different doses of ORG 30276 administered s.c. or i.n., respectively. The lowest doses (s.c., 0.05 mg; i.n., 0.2 rag) had no demonstrable effect on circulating LH concentrations. As early as l h after the LH-RH antagonist injection, the s.c. doses of 0.2 and l m g induced rapid decreases of LH levels that lasted for 8 and 12 h, respectively. Maximum inhibition was 41 and 58%, respectively, for 0.2 and lmg. Intranasal administration of ORG 30276 produced, as early as l h after administration, suppression of LH levels that lasted for 8-12 h (1 and 5 mg, respectively). Maximum inhibition observed after the i.n. administration of I or 5 mg of ORG 30276 was 32 and 53%, respectively.
Discussion The results of this study clearly show that the LH-RH inhibitory analogue, ([N-Ac-D-p-C1-Phel,2,D-Trp3,D-Arg6,D-Alal°]-LH-RH) ORG 30276, is effective in suppressing FSH and LH levels when administered by the i.n. route to oophorectomized rhesus monkeys. Previous studies have shown that LH-RH agonists are active when used by different routes of administration, including intranasal, for contraception [15,16], treatment of endometriosis [17], precocious puberty [18,19] and prostatic carcinoma
112
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[8,20]. Although the i.n. form of administration is relatively simple, it is necessary to give considerably larger doses of LH-RH agonists by this route as compared with parenteral administration [6]. In previous studies, the amount of LH-RH
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agonist necessary to induce the same degree of inhibition of FSH, LH and gonadal steroids has been s h o w n to be about 100 times greater for i.n. than for parenteral administration [6, 9, 21]; thus, the bioavailability of the nasal route compared to
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the parenteral route is about 1%. The results of the present study indicate a dosecalculated nasal bioavailability of 16-26% compared to the s.c. route, with a similar duration of effect on serum gonadotropin levels. The reason(s) for the increased bioavailability of ORG 30276 after intranasal administration, as compared to that of LH-RH agonists, is, at present, incompletely understood. It is likely that the substitution of five D-amino acids into the LH-RH sequence results in a potent LH-RH antagonist with high resistance to enzymatic hydrolysis in the nasal mucosa [22]. Studies designed to enhance the bioavailability of i.n.-administered LH-RH antagonists b y inhibiting their enzymatic hydrolysis are n o w in progress in our laboratories. The lipophilic character of this antagonist, as compared to the LH-RH agonists, m a y also facilitate the nasal absorption. In summary, this study shows for the first time that the nasal route offers a useful alternative to the parenteral route for the administration of LH-RH antagonists. The possible clinical implications of the administration of LH-RH antagonists b y routes other than injectable are obvious, because of the practicality of the intranasal route. Another advantage of the antagonists, as compared to the LH-RH agonists, is that they do not produce an initial pituitary and gonadal stimulation. The clinical use of intranasally administered LH-RH antagonists is indicated in cases in which gonadotropin or sex h o r m o n e suppression is desired, as in contraception in both sexes and in the treatment of endometriosis, precocious p u b e r t y and sex h o r m o n e d e p e n d e n t neoplasms.
Acknowledgements This study was supported in part by NIH Grant SP30 HD10202 (Radioimmunoassay Core); by NIH Biomedical Research Support Grant RRO5654; by Grant 820-1973 from the Ford Foundation; and by the Program for Applied Research on Fertility Regulation (PARFR-347), Northwestern University, under a co-operative agreement with the United States Agency for International Development (AID) (DPE-0546-A-00-1003-00). We are grateful to Dr Evert de Jager of Organon, OSS, Holland for his kindness in supplying the LH-RH antagonist, ORG 30276. We also wish to thank Wendy Anderson for her technical assistance and Kim Francis and Gretta Small for their help in the preparation of the manuscript. The views expressed by the authors do not necessarily reflect the views of AID.
References 1. Nishi, N., Arimura, A., Coy, D. H., Vilchez-Martin@z, J. A. and Schally, A. V. (1975). The effect of oral and vaginal administration of synthetic LH-RH and [D-Ala6,DesGlyI°NH2]-LH-RH ethylamide on serum LH levels in ovariectomized, steroid blocked rats (38678). Proc. Soc. Exp. Biol. Med., 148, 1009 2. DeLaCruz, A., DeLaCruz, K. G., Arimura, A., Coy, D. H., Vilchez-Martinez, J. A., Coy, E. J. and Schally, A. V. (1975). Gonadotropin-releasing activity of two highly active and long-acting analogs of luteinizing hormone-releasing hormone after subcutaneous, intravaginal, and oral administration. Fertil. Steril., 26, 894 3. Saito, M., Kumasaki, T., Yaoi, Y., Nishi, N., Arimura, A., Coy, D. H. and Schally, A. V. (1977). Stimulation of luteinizing hormone (LH) and follicle-stimulating hormone by [D-Leu6, Des-Glyl°-NH2]-LH-releasing hormone ethylamide after subcutaneous, intravaginal, and intrarectal administration to women. Fertil. Steril., 28, 240
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4. Gonzalez-Barcena, D., Kastin, A. J., Schalch, D. S., Coy, D. H. and Schally, A. V. (1976). Prolonged elevation of luteinizing hormone (LH) after intranasal administration of an analog of LH-releasing hormone. Fertil. Steril., 27, 1246 5. Gonzalez-Barcena, D., Kastin, A. J., Miller, M. CI III, Schalch, D. S., Coy, D. H., Schally, A. V. and Escalante-Herrera, A. (1975). Stimulation of luteinising hormone (LH) release after oral administration of an analogue of LH releasing hormone. Lancet, 2, 1126 6. Tolis, G., Faure, N., Koutsilieris, M., Lemay, A., Klioze, S., Yakabow, A. and Fazekas, A . T . A . (1983). Suppression of testicular steroidogenesis by the GnRH agonistic analogue buserelin (HOE-766) in patients with prostatic cancer: Studies in relation to dose and route of administration. J. Steroid Biochem., 19, 995 7. Faure, N., Lemay, A., B61anger, A. and Labrie, F. (1981). Inhibition of androgen biosynthesis in the human male by chronic administration of [D-Ser(TBU)6-des-Gly-NH21°] LH-RH ethylamide (buserelin). In: LH-RH Peptides as Female and Male Contraceptives, G: I. Zatuchni, J. D. Shelton and J. J. Sciarra, eds., Harper & Row, Philadelphia, p. 307 8. Tolis, G., Ackman, D., Stellos, A., Mehta, A., Labrie, F., Fazekas, A. T., Comaru:Schally, A. M. and Schally, A. V. (1982). Tumor growth inhibition in patients with prostatic carcinoma treated with luteinizing hormone-releasing hormone agonists. Proc. Natl. Acad. Sci. USA, 79, 1658 9. Sandow, Y., Clayton, R. N. and Kuhl, H. (1981). Pharmacology of LH-RH and its analogues. In: Endocrinology of Human Infertility - New Aspects, P. G. Crosignani and R. L. Rubin, eds., Academic Press, London, p. 221 10. Gonzalez-Barcena, D., Kastin, A. J., Coy, D. H., Nikolics, K. and Schally, A. V. (1977). Suppression of gonadotrophin release in man by an inhibitory analogue of LHreleasing hormone. Lancet, 2, 997 11. Asch, R. H., Balrnaceda, J. P., Eddy, C. A., Siler-Khodr, T., Coy, D. H. and Schally, A. V. (1981). Inhibition of the postcastration rise of luteinizing hormone and folliclestimulating hormone in female rhesus monkeys (Macaca mulatta) by the administration of a luteinizing hormone-releasing hormone inhibitory analog ([N-Ac-D-Trp1,3,Dp-C1-Phe2,D-Phe6,D-Alal°]-LH-RH). Fertil. Steril., 36, 388 12. Balmaceda, J. P., Borghi, M. R., Burgos, L., Pauerstein, C. J., Schally, A. V. and Asch, R. H. (1984). The effects of chronic administration of LH-RH agonists and antagonists on the menstrual cycle and endometrium of the rhesus monkey. Contraception, 29, 83 13. Asch, R. H., Balmaceda, J. P., Borghi, M. R., Niesvizky, R., Coy, D. H. and Schally, A. V. (1983). Suppression of the positive feedback of estradiol benzoate on gonadotropin secretion by an inhibitory analog of luteinizing hormone-releasing hormone (LRH) in oophorectomized rhesus monkeys: Evidence for a necessary synergism between LRH and estrogens. J. Clin. Endocrinol. Metab., 57, 367 14. Balmaceda, J. P., Schally, A. V., Coy, D. and Asch, R. H. (1981). The effects of an LH-RH antagonist ([N-Ac-D-Trpl,3,D-p-CI-Phe2,D-Phe6,D-Alal°]-LH-RH) during the preovulatory period of the rhesus monkey. Contraception, 24, 275 15. Nillius, S. J., Bergquist, C. and Wide, L. (1978). Inhibition of ovulation in women by chronic treatment with a stimulatory LRH analogue- a new approach to birth control? Contraception, 17, 537 16. Schmidt-Gollwitzer, M., Hardt, W., Schmidt-Gollwitzer, K., von der Ohe, M. and Nevinny-Stickel, J. (1981). Influence of the LH-RH analogue buserelin on cyclic ovarian function and on endometrium. A new approach to fertility control? Contraception, 23, 187 17. Meldrum, D. R., Chang, R. J., Lu, J., Vale, W., Rivier, J. and Judd, H. (1982). 'Medical oophorectomy' using a long acting GnRH agonist - a possible new approach to the treatment of endometriosis. J. Clin. Endocrinol. Metab., 54, 1081 18. Laron, A., Kauli, R., Ben Zeev, Z., Comaru-Schally, A. M. and Schally, A. (1981). D-Trp6analogue of luteinizing hormone-releasing hormone in combination with cyproterone acetate to treat precocious puberty. Lancet, 2, 955 19. Crowley, W. F., Comite, F., Vale, W., Rivier, J., Loriaux, D. L. and Cutler, G. B. (1981). Therapeutic use of pituitary desensitization with a long acting LH-RH agonist: A
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potential new treatment for idiopathic precocious puberty. J. Clin. Endocrinol. Metab., 52, 370 20. Borgmann, Y., Hardt, W., Schmidt-Gollwitzer, M., Adenauer, H. and Nagel, R. (1982). Sustained suppression of testosterone production by the luteinizing hormone-releasing hormone agonist buserelin in patients with advanced prostate carcinoma. A new therapeutic approach? Lancet, 1, 1097 21. Wass, J. A. H., Besser, G. M., Gomez-Pan, A., Scanlon, M. F., Hall, R., Kastin, A. J., Coy, D. H. and Schally, A. V. (1979). Comparison of long-acting analogues of luteinizing hormone releasing hormone in man. Clin. Endocrinol., 10, 419 22. Schally, A. V. (1983). Current status of antagonistic analogs of LH-RH as a contraceptive method in the female. In: Research Frontiers in Fertility Regulation, Vol. 2, no. 5. Program for Applied Research on Fertility Regulation, Northwestern University, Chicago
MS received 15 March 85. Accepted for publication 3 April 85.
Resum4 Bien que des agonistes de l'hormone de lib6ration de la lut6inostimuline (LH-RH) aient 6t6 administr6s avec succ6s par des voies autres que syst6miques (orale, intranasale (i.n.) et vaginale), il n'existe aucune preuve que des analogues inhibiteurs puissent 6tre utilis6s sous une forme autre qu'injectable. Dans la pr6sente 6tude, nous avons compar6 les effets de deux voies d'administration (1. sous-cutan4e (s.c.) (0,5, 0,2 et lmg) et 2. i.n. (0,2, 1 et 5 mg)) d'un antagoniste de la LH-RH, le ORG30276 ((N-Ac-D-p-C1-Phel,2,D-Trp3,D-Arg6,D Alal°)-LH-RH) sur les taux de gonadotropine chez des singes oophorectomis6s. Une heure apr6s administration s.c., les valeur de la FSH et de la LH pr6sentaient une baisse d6pendant de la dose, qui durait jusqu'a 12 heures. Apr6s administration s.c., l'inhibition maxima de la FSH et de la LH du s6rum 6tait de 29% et 41% (dose 0,2mg) et de 41% et 58% (dose lmg), respectivement. Apr6s administration i.n., l'inhibition maxima de la FSH et de la LH du s6rum 6tait de 19% et 40% (ling) et de 32% et 53% (5mg), respectivement. Ces diminutions 6taient li6es a la dose et duraient jusqu'a 12h. L'analyse des r6sultats a r6v616 que la biodisponibilit6 de la voie i.n. par rapport ~ la voie s.c. allait de 16% a 26%. Cette efficacit6 61ev6e de la voie i.n. exprim6e par la biodisponibilit6 est nettement plus 61ev6e que celle pr6c6demment indiqu6e pour les agonistes de la LH-RH (1%) et elle est probablement dfie a une r6sistance ~ l'hydrolyse enzymatique dans la muqueuse nasale. Ces r6sultats montrent pour la premi6re fois que des antagonistes de la LH-RH peuvent &re administr6s par des voies autres que parent6rales, ce qui am61iore leur potentiel d'utilisation dinique dans des conditions dans lesquelles une inhibition de gonadtropines est d6sir6e, comme dans la contraception et le traitement de l'endom6triose, de la pubert6 pr6coce et des n6oplasmes hormonod6pendants.
Resumen Aunque los agonistas de la hormona liberadora de hormona luteinizante (LHRH) en han sido utilizados exitosamente de otras maneras, ademfis de sist6micas (tales como oral, intranasal (i.n.) y vaginal), no existe evidencia que anfilogos inhibitorios puedan ser usados en otra forma que la inyectable. En el presente estudio hemos comparado el efecto en dos formas de administraci6n: (1) subcutfinea (s.c.) (0,5, 0,2 y lmg); y (2) i.n. (0,2, i y 5 mg) de un antagonista a LHRH, el ORG30276 ([N-Ac-D-p-C1-Phea,R,D-Trp 3, D-Arg6, D-Alal°]-LHRH) en los niveles de gonadotropinas en monas ooforectomizadas. Una hora despu6s de la
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administraci6n subcutfinea, los valores de la hormona foliculo estimulante (FSH) y de la hormona luteinizante (LH), exhibieron una caida dependiente del dosaje que dur6 hasta 12 horas. Luego de la administraci6n s.c. la mfixima inhibici6n de FSH y de LH en suero, fu6 respectivamente 29% y 41% (dosis de 0,2mg) y 41% y 58% (dosis de ling). Luego de una administraci6n i.n., la m~xima inhibici6n de FSH y de LH en suero fu6 respectivamente 19% y 40% ( l m g ) y 32% y 53% (5rag). Estas disminuciones estuvieron relacionadas con el dosaje y duraron hasta 12 horas. E1 an~lisis de los datos revel6 que la disponibilidad biol6gica de la administraci6n i.n. versus la s.c. vari6 de 16% a 26%. Esta efectividad alta de la administraci6n i.n. en t6rminos de disponibilidad es acentuadamente mayor que la previamente notada para los agonistas LHRH (1%) y posiblemente sea debido a la resistencia a hidr61isis enzim~tica en la mucosa nasal. Estos resultados indican por primera vez que los antagonistas de la LHRH p u e d e n ser administrados por rutas distintas a la parenteral, aumentando su potencial uso cl~nico en condiciones en las que se desee la inhibici6n de las gonadotropinas, tal como en la anticoncepci6n y e n el tratamiento de la endometriosis, pubertad precoz y neoplasmas horrnono-dependientes.
