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GnRH antagonists: A synopsis
Contraception
46: 109-I
12, 1992
GnRH ANTAGONISTS:
A SYNOPSIS
Rivier JP, Theobald P, Hoeger C, Craig AG, Perrin M, Porter J, Corrigan A, Koerber S, Hagler A*, Vale W and Rivier C The Salk Institute for Biological Studies 10010 N. Torrey Pines Rd., La Jolla, California 92037, USA *BIOSYM Technologies, Inc., 10065 Barnes Canyon Rd. San Diego, California 92121, USA
Since its discovery in 1971 by A. Schally and collaborators (1) approximately three to four thousand analogs of gonadotropin releasing hormone (GNRH) have been synthesized and tested in a variety of assays. It was quickly recognized that GNRH played a major role in triggering the onset of ovulation in the female and spermatogenesis in the male. Therefore, it was proposed that a linear competitive antagonist of GNRH could be used as a contraceptive by preventing the GnRH-stimulated LH surge of proestrus in the female or disrupting spermatogenesis in the male. By 1972, we had shown that designing a pure competitive antagonist of GnRH was definitely possible (2). Fifteen years later, i.e. in 1986, phase I clinical investigations had been carried out with a few potent and long acting antagonists; however, they were either not potent enough or showed some undesirable effects. Structure activity relationship studies (3), mechanistic studies, as well as results from clinical investigations, have been extensively reviewed (4). In this paper we will briefly review our most recent progress in two areas: a) the development of cyclic analogs of GnRH which we believe will ultimately lead to a new generation of orally active analogs, and b) the development of safe, long acting, readily soluble and potent linear GNRH antagonists. Cyclic analogs of GNRH The linear antagonists presently under clinical investigation lack certain appealing characteristics such as high potency, extended duration of action and oral activity. In an attempt to attain the stated goals, we have investigated the conformational constraints compatible with bioactivity using tools such as molecular dynamics, energy minimization, template forcing, NMR (5), peptide synthesis, and in vitro and in vivo bioassays. These concerted efforts led to the design and synthesis of highly conformationally constrained analogs, some with biological potencies equal to that of the corresponding linear analogs (6-11). Whether these compounds, with structural features that may increase their resistance to enzymic degradation and will reduce their apparent size, are the forerunners of a generation of orally active GNRH antagonists, remains to be established.
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Linear analogs of GNRH The design of the “Nal-Glu” antagonist (Ac-DNal-DCpa-DPal-Ser-Arg-D-2-amino-5 oxo-5(4-methoxyphenyl)pentanoic acid-Leu-Arg-Pro-DAla-NH,) (12), its reproducible synthesis in large quantities (13). and its introduction in the clinic was a major breakthrough in allowing the testing of an antagonist to GNRH chronically in the human (see other papers in this issue and references therein). While it is recognized that “Nal-Glu” is relatively safe for use in a clinical setting, it is also apparent that analogs with minimal histamine releasing activity and high inhibitory potency in vivo are ultimately needed for widespread use. One recent approach toward reaching this goal in our laboratory was to attenuate the basicity of various arginine residues of active analogs by the introduction of an electronegative substituent on the Ncnguanidino function. The cyan0 substituent stabilizes the guanidino functional group resulting in a significant lowering of the pK, of the guanidinium cation (the pK, of the guanidinium cation in water is 14.5 while that of the cyanoguanidinium cation is 0.4). The synthesis of GNRH antagonists containing modified No-cyano-No’alkyl or aryl guanidino moieties on homoarginine, arginine or (p-amino)phenylalanine residues (cyanoguanidino peptides) as well as peptides incorporating an amino triazolyl moiety on the distal amino function of lysine, omithine or (p-amino)phenylalanine is the subject of two recent reports (14,15). Most analogs recently developed in our laboratory were tested in the rat antiovulatory assay (AOA) and three in vitro assays: a pituitary cell culture assay, a binding assay to pituitary cell membranes, and a histamine release assay. Selected analogs were tested in the castrated male rat using three modes of injection (subcutaneously at one or three sites and intravenously) in order to compare their duration of action. Of these antagonists, Azaline [Ac-DNal1,DCpa2,DPal3,Lys5(Atz),DLys6(Atz),ILyss,DAla’o]GnRH and Azaline B [Ac-DNal’,DCpa2,DPal3,AphS(Atz),DAph6(Atz),ELys8,DAla’o]-GnRH were found to have in an in vitro histamine release assay, an EC,, of approximately 50 pg/mL, which is 30 to 40 times that of “Nal-Glu” [(atz)=Ne-5’-(3-amino-lH-1,2,4-triazole)]. Of these two analogs, the most interesting seems at present to be Azaline B which was found to be long acting after intravenous administration in the castrated male rat, in contradt to “Nal-Glu”, Azaline, Antide [AcDNal’,DCpa2,DPal3,Lys5(Nic),DLys6(Nic),DLys*,DAla1~-GnRH (16), and other similar analogs while remaining readily soluble in aqueous solutions. Taking all results into consideration, we have selected Azaline B for further development. Azaline B is extremely long acting, resistant to degradation in the blood stream, relatively hydrophilic compared to Antide or “Nal-Glu” and significantly less potent than “Nal-Glu” in its ability to release histamine while being essentially equipotent to “Nal-Glu” in an acute assay such as the AOA. Conclusion Considerable progess has been achieved in the development of GNRH antagonists and several may be good candidates as therapeutic agents or for the control of fertility. Of all the analogs recently tested by us in a variety of assays, Azaline B seems to have the best solubility characteristics while maintaining unusually long half life in vivo even after iv injection. It is also one of the safest and most potent analogs on the basis of animal testing to be presently considered for further clinical development. It is clearly more hydrophilic than “Nal-Glu”, Antide (16) and SB-75 and releases less histamine than ganirelix or cetrorelix (see other contributions in this issue). In the castrated male rat, after intravenous injection, it is clearly longer acting than any of the other analogs presently designed for human investigations.
Contraception
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Acknowledgements This work was supported by MH under Contract NOl-HD-9-2903 and the Hearst Foundation. We acknowledge the outstanding technical contributions of R. Kaiser, D. Pantoja, Y. Haas and R. Chavarin, and the assistance of R. Hensley in manuscript preparation. References 1. Matsuo H, Baba Y, Nair R M, Arimura A, Schally AV. Structure of the porcine LH- and FSH-releasing hormone. 1. The proposed amino acid sequence. Biochem Biophys Res Commun, 1971;43:1334-1339. 2. Vale W, Grant G, Rivier J, Monohan M, Amoss M, Blackwell R, Burgus R, Guillemin R. Synthetic polypeptide antagonists of the hypothalamic luteinizing hormone releasing factor. Science, 1972;176:933-934. 3. Karten MJ, Rivier JE. Gonadotropin-releasing hormone analog design. Structure-function studies toward the development of agonists and antagonists: rationale and perspective. Endocrine Rev, 1986;7:44-66. 4. Endocrine Reviews Vol. 7, 1986 5. Struthers RS, Tanaka G, Koerber SC, Solmajer T, Baniak EL, Gierasch LM, Vale W, Rivier J, Hagler AT. Design of biologically active, conformationally constrained GNRH antagtonists. Proteins: Structure, Function, and Genetics, 1990;8:295-304. 6. Rivier J, Kupryszewski G, Varga J, Porter J, Rivier C, Pen-in M, Hagler A, Struthers S, Corrigan A, Vale W. Design of potent cyclic gonadotropin releasing hon-none (GNRH) antagonists. J Med Chem, 1988;31:677-682. 7. Rivier J, Kupryszewski G, Theobald P, Hoeger C, Porter J, Penin M, Corrigan A, Strutliers S, Koerber S, Hagler A, Vale W, Rivier C. Two distinct approaches for the design of potent GNRH antagonists. In Proc. of the 2nd Int’l. Symp. on GNRH Analogues in Cancer and Human Reproduction; Lunenfeld, B., Eds.; Parthenon Publishing, Camforth, Lancaster, UK, 1991; pp in press. 8. Rivier J, Koerber S, Rivier C, Porter J, Hagler A. Design, computer derived structure and biological activity of three bicyclic gonadotropin releasing hormone (G&H) antagonists. In Current Research in Protein Chemistry; Villafranca, J., Eds.; Academic Press, 1990; pp 273-28 1. 9. Rivier J, Koerber S, Rivier C, Hagler A, Perrin M, Gierasch L, Corrigan A, Porter J, Vale W. Design, physics-chemical characterization and biological activity of gonadotropin releasing hormone (GMH) antagonists as potential contraceptives. In Proc. of Int’l. Symp. on Frontiers in Reproduction Research: The role of Growth Factors, Oncogenes, Receptors and Gonadal Polypeptides; Chen, H.-C., Eds., 1988; pp in press. 10. Rivier J, Varga J, Porter J, Perrin M, Haas Y, Corrigan A, Rivier C, Vale W, Struthers S, Hagler A. Potent conformationally constrained analogs of GNRH. In Proc. of the Ninth American Peptide Symposium; Deber, C. M., Hruby, V. J.Kopple, K. D., Eds.; Pierce Chemical Company, Rockford, EL, 1986; pp 541-544. 11. Rivier JE, Rivier C, Vale W, Koerber S, Corrigan A, Porter J, Gierasch LM, Hagler AT. Bicyclic gonadotropin releasing hormone (GNRH) antagonists. In Peptides, Chemistry, Structure and Biology; Rivier, J. E.Marshall, G. R., Eds.; Escom Science Publishers, Lciden, The Netherlands, 1990; pp 33-37.
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12. Rivier J, Porter J, Rivier C, Perrin M, Corrigan A, Hook WA, Siraganian RP, Vale WW. New effective gonadotropin releasing hormone antagonists with minimal potency for histamine release in vitro. J Med Chem, 1986;29:1846-1851. 13. Hoeger C, Porter J, Boublik J, Rivier J. Preparative-scale synthesis and reversephase purification of a gonadotropin-releasing hormone antagonist. J Chromatog, 1989;404:307-310. 14. Theobald, P.; Porter, J.; Hoeger, C.; Rivier, J. A general method for incorporation of modified N(O-cyanoguanidino moieties on selected amino functions during SPPS. JACS, 1990; 112:9624-9626. 15. Theobald P, Porter J, Rivier C, Corrigan A, Perrin, M, Vale W, Rivier J. Novel gonadotropin releasing hormone antagonist: Peptides incorporating modified N(O-cyanoguanidino moieties. J Med Chem, 1991;34:2395-2402. 16. Ljungqvist A. Feng D-M, Tang P-F L, Kubota M, Okamoto T, Zhang Y, Bowers CY, Hook WA, Folkers K. Design, synthesis and bioassays of antagonists of LHRH which have high antiovulatory activity and release negligible histamine. Biochem Bioshys Res Commun, 1987; 148:849-856.
46: 109-I
12, 1992
GnRH ANTAGONISTS:
A SYNOPSIS
Rivier JP, Theobald P, Hoeger C, Craig AG, Perrin M, Porter J, Corrigan A, Koerber S, Hagler A*, Vale W and Rivier C The Salk Institute for Biological Studies 10010 N. Torrey Pines Rd., La Jolla, California 92037, USA *BIOSYM Technologies, Inc., 10065 Barnes Canyon Rd. San Diego, California 92121, USA
Since its discovery in 1971 by A. Schally and collaborators (1) approximately three to four thousand analogs of gonadotropin releasing hormone (GNRH) have been synthesized and tested in a variety of assays. It was quickly recognized that GNRH played a major role in triggering the onset of ovulation in the female and spermatogenesis in the male. Therefore, it was proposed that a linear competitive antagonist of GNRH could be used as a contraceptive by preventing the GnRH-stimulated LH surge of proestrus in the female or disrupting spermatogenesis in the male. By 1972, we had shown that designing a pure competitive antagonist of GnRH was definitely possible (2). Fifteen years later, i.e. in 1986, phase I clinical investigations had been carried out with a few potent and long acting antagonists; however, they were either not potent enough or showed some undesirable effects. Structure activity relationship studies (3), mechanistic studies, as well as results from clinical investigations, have been extensively reviewed (4). In this paper we will briefly review our most recent progress in two areas: a) the development of cyclic analogs of GnRH which we believe will ultimately lead to a new generation of orally active analogs, and b) the development of safe, long acting, readily soluble and potent linear GNRH antagonists. Cyclic analogs of GNRH The linear antagonists presently under clinical investigation lack certain appealing characteristics such as high potency, extended duration of action and oral activity. In an attempt to attain the stated goals, we have investigated the conformational constraints compatible with bioactivity using tools such as molecular dynamics, energy minimization, template forcing, NMR (5), peptide synthesis, and in vitro and in vivo bioassays. These concerted efforts led to the design and synthesis of highly conformationally constrained analogs, some with biological potencies equal to that of the corresponding linear analogs (6-11). Whether these compounds, with structural features that may increase their resistance to enzymic degradation and will reduce their apparent size, are the forerunners of a generation of orally active GNRH antagonists, remains to be established.
Copyright 0 1992 Butterworth-Heinemann
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Linear analogs of GNRH The design of the “Nal-Glu” antagonist (Ac-DNal-DCpa-DPal-Ser-Arg-D-2-amino-5 oxo-5(4-methoxyphenyl)pentanoic acid-Leu-Arg-Pro-DAla-NH,) (12), its reproducible synthesis in large quantities (13). and its introduction in the clinic was a major breakthrough in allowing the testing of an antagonist to GNRH chronically in the human (see other papers in this issue and references therein). While it is recognized that “Nal-Glu” is relatively safe for use in a clinical setting, it is also apparent that analogs with minimal histamine releasing activity and high inhibitory potency in vivo are ultimately needed for widespread use. One recent approach toward reaching this goal in our laboratory was to attenuate the basicity of various arginine residues of active analogs by the introduction of an electronegative substituent on the Ncnguanidino function. The cyan0 substituent stabilizes the guanidino functional group resulting in a significant lowering of the pK, of the guanidinium cation (the pK, of the guanidinium cation in water is 14.5 while that of the cyanoguanidinium cation is 0.4). The synthesis of GNRH antagonists containing modified No-cyano-No’alkyl or aryl guanidino moieties on homoarginine, arginine or (p-amino)phenylalanine residues (cyanoguanidino peptides) as well as peptides incorporating an amino triazolyl moiety on the distal amino function of lysine, omithine or (p-amino)phenylalanine is the subject of two recent reports (14,15). Most analogs recently developed in our laboratory were tested in the rat antiovulatory assay (AOA) and three in vitro assays: a pituitary cell culture assay, a binding assay to pituitary cell membranes, and a histamine release assay. Selected analogs were tested in the castrated male rat using three modes of injection (subcutaneously at one or three sites and intravenously) in order to compare their duration of action. Of these antagonists, Azaline [Ac-DNal1,DCpa2,DPal3,Lys5(Atz),DLys6(Atz),ILyss,DAla’o]GnRH and Azaline B [Ac-DNal’,DCpa2,DPal3,AphS(Atz),DAph6(Atz),ELys8,DAla’o]-GnRH were found to have in an in vitro histamine release assay, an EC,, of approximately 50 pg/mL, which is 30 to 40 times that of “Nal-Glu” [(atz)=Ne-5’-(3-amino-lH-1,2,4-triazole)]. Of these two analogs, the most interesting seems at present to be Azaline B which was found to be long acting after intravenous administration in the castrated male rat, in contradt to “Nal-Glu”, Azaline, Antide [AcDNal’,DCpa2,DPal3,Lys5(Nic),DLys6(Nic),DLys*,DAla1~-GnRH (16), and other similar analogs while remaining readily soluble in aqueous solutions. Taking all results into consideration, we have selected Azaline B for further development. Azaline B is extremely long acting, resistant to degradation in the blood stream, relatively hydrophilic compared to Antide or “Nal-Glu” and significantly less potent than “Nal-Glu” in its ability to release histamine while being essentially equipotent to “Nal-Glu” in an acute assay such as the AOA. Conclusion Considerable progess has been achieved in the development of GNRH antagonists and several may be good candidates as therapeutic agents or for the control of fertility. Of all the analogs recently tested by us in a variety of assays, Azaline B seems to have the best solubility characteristics while maintaining unusually long half life in vivo even after iv injection. It is also one of the safest and most potent analogs on the basis of animal testing to be presently considered for further clinical development. It is clearly more hydrophilic than “Nal-Glu”, Antide (16) and SB-75 and releases less histamine than ganirelix or cetrorelix (see other contributions in this issue). In the castrated male rat, after intravenous injection, it is clearly longer acting than any of the other analogs presently designed for human investigations.
Contraception
111
Acknowledgements This work was supported by MH under Contract NOl-HD-9-2903 and the Hearst Foundation. We acknowledge the outstanding technical contributions of R. Kaiser, D. Pantoja, Y. Haas and R. Chavarin, and the assistance of R. Hensley in manuscript preparation. References 1. Matsuo H, Baba Y, Nair R M, Arimura A, Schally AV. Structure of the porcine LH- and FSH-releasing hormone. 1. The proposed amino acid sequence. Biochem Biophys Res Commun, 1971;43:1334-1339. 2. Vale W, Grant G, Rivier J, Monohan M, Amoss M, Blackwell R, Burgus R, Guillemin R. Synthetic polypeptide antagonists of the hypothalamic luteinizing hormone releasing factor. Science, 1972;176:933-934. 3. Karten MJ, Rivier JE. Gonadotropin-releasing hormone analog design. Structure-function studies toward the development of agonists and antagonists: rationale and perspective. Endocrine Rev, 1986;7:44-66. 4. Endocrine Reviews Vol. 7, 1986 5. Struthers RS, Tanaka G, Koerber SC, Solmajer T, Baniak EL, Gierasch LM, Vale W, Rivier J, Hagler AT. Design of biologically active, conformationally constrained GNRH antagtonists. Proteins: Structure, Function, and Genetics, 1990;8:295-304. 6. Rivier J, Kupryszewski G, Varga J, Porter J, Rivier C, Pen-in M, Hagler A, Struthers S, Corrigan A, Vale W. Design of potent cyclic gonadotropin releasing hon-none (GNRH) antagonists. J Med Chem, 1988;31:677-682. 7. Rivier J, Kupryszewski G, Theobald P, Hoeger C, Porter J, Penin M, Corrigan A, Strutliers S, Koerber S, Hagler A, Vale W, Rivier C. Two distinct approaches for the design of potent GNRH antagonists. In Proc. of the 2nd Int’l. Symp. on GNRH Analogues in Cancer and Human Reproduction; Lunenfeld, B., Eds.; Parthenon Publishing, Camforth, Lancaster, UK, 1991; pp in press. 8. Rivier J, Koerber S, Rivier C, Porter J, Hagler A. Design, computer derived structure and biological activity of three bicyclic gonadotropin releasing hormone (G&H) antagonists. In Current Research in Protein Chemistry; Villafranca, J., Eds.; Academic Press, 1990; pp 273-28 1. 9. Rivier J, Koerber S, Rivier C, Hagler A, Perrin M, Gierasch L, Corrigan A, Porter J, Vale W. Design, physics-chemical characterization and biological activity of gonadotropin releasing hormone (GMH) antagonists as potential contraceptives. In Proc. of Int’l. Symp. on Frontiers in Reproduction Research: The role of Growth Factors, Oncogenes, Receptors and Gonadal Polypeptides; Chen, H.-C., Eds., 1988; pp in press. 10. Rivier J, Varga J, Porter J, Perrin M, Haas Y, Corrigan A, Rivier C, Vale W, Struthers S, Hagler A. Potent conformationally constrained analogs of GNRH. In Proc. of the Ninth American Peptide Symposium; Deber, C. M., Hruby, V. J.Kopple, K. D., Eds.; Pierce Chemical Company, Rockford, EL, 1986; pp 541-544. 11. Rivier JE, Rivier C, Vale W, Koerber S, Corrigan A, Porter J, Gierasch LM, Hagler AT. Bicyclic gonadotropin releasing hormone (GNRH) antagonists. In Peptides, Chemistry, Structure and Biology; Rivier, J. E.Marshall, G. R., Eds.; Escom Science Publishers, Lciden, The Netherlands, 1990; pp 33-37.
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12. Rivier J, Porter J, Rivier C, Perrin M, Corrigan A, Hook WA, Siraganian RP, Vale WW. New effective gonadotropin releasing hormone antagonists with minimal potency for histamine release in vitro. J Med Chem, 1986;29:1846-1851. 13. Hoeger C, Porter J, Boublik J, Rivier J. Preparative-scale synthesis and reversephase purification of a gonadotropin-releasing hormone antagonist. J Chromatog, 1989;404:307-310. 14. Theobald, P.; Porter, J.; Hoeger, C.; Rivier, J. A general method for incorporation of modified N(O-cyanoguanidino moieties on selected amino functions during SPPS. JACS, 1990; 112:9624-9626. 15. Theobald P, Porter J, Rivier C, Corrigan A, Perrin, M, Vale W, Rivier J. Novel gonadotropin releasing hormone antagonist: Peptides incorporating modified N(O-cyanoguanidino moieties. J Med Chem, 1991;34:2395-2402. 16. Ljungqvist A. Feng D-M, Tang P-F L, Kubota M, Okamoto T, Zhang Y, Bowers CY, Hook WA, Folkers K. Design, synthesis and bioassays of antagonists of LHRH which have high antiovulatory activity and release negligible histamine. Biochem Bioshys Res Commun, 1987; 148:849-856.