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The two native forms of gonadotropin-releasing hormones (GnRH) present in goldfish, salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II), stimulate gonadotropin-II (GTH-II) and growth hormone (GH) release both in vivo and in vitro. In our previous study using perifused goldfish pituitary fragments, many mammalian GnRH antagonists, especially those with D-Arg6, showed weak to strong stimulation of GTH-II and GH release. In the present study, the dose-related stimulation of GTH-II and GH release by [Ac-D(2)-Nal1, 4Cl-D-Phe2, D-Trp3, D-Arg6, Trp7, D-Ala10] mGnRH (analog J) and [Ac-D(2)-Nal1, 4Cl-D-Phe2, D-Trp3, D-hArg(Et2)6, D-Ala10] mGnRH (analog K) was demonstrated; the stimulatory potency of both analogs was significantly lower than that of native sGnRH. In the presence of analogs J and K, cGnRH-II stimulated GTH-II release was significantly suppressed. Further, GTH-II and GH stimulation by 2 microM of analog K was significantly suppressed by a 'true' GnRH antagonist, [Ac-delta 3-Pro1, 4FD-Phe2, D-Trp3,6] mGnRH (analog E). These results indicate that analogs J and K increase GTH-II and GH release in goldfish by acting on GnRH receptors on gonadotrophs and somatotrophs. Since analog K, having [D-hArg(Et2)6], strongly stimulated GTH-II release, the potency of [D-hArg(Et2)6] or [D-hArg(CH2CF3)2(6)] substituted analogs to stimulate GTH-II and GH release from the perifused goldfish pituitary fragments was tested. Among the peptides tested, [D-hArg(Et2)6, Pro9-NHEt] sGnRH had a higher potency in stimulating GTH-II release than any other analog tested in the present or in previous studies. For stimulation of GH release, [D-hArg(Et2)6, Pro9-NHEt] sGnRH and [D-Arg6, Pro9-NHEt] sGnRH were the most potent analogs tested; analogs of mGnRH were less potent than sGnRH, indicating the importance of Trp7, Leu8 residues in the native peptide. These results suggest the importance of [D-Arg6] or alkylated [D-Arg6] in determining the intrinsic activity and potency of GnRH peptides in goldfish.

A new series of unnatural amino acids has been prepared and incorporated into antagonistic analogues of luteinizing hormone-releasing hormone (LH-RH), on the basis of the hypothesis that stabilization of a proposed phospholipid membrane interaction might yield analogues with high potency and a prolonged duration of action. Thus a series of NG,NG'-dialkyl-D-homoarginine analogues [H-D-hArg(R2)-OH; R = Me, Et, Pr, i-Pr, Bu, hexyl, cyclohexyl, (Et, Me2NPr)] was conveniently prepared by semisynthesis from D-Lys using the appropriate dialkylcarbodiimide. A number of the analogues that were prepared by using these new amino acid analogues exhibited very high potency and a prolonged duration of action. One of the most potent members of the series, [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Trp3,D-hArg(Et2)6,D-Ala10 ]LH-RH (detirelix), had an ED50 of 0.7 microgram in the rat antiovulatory assay when administered at noon on proestrus and only 2.5 micrograms when administered 24 h earlier, at noon on diestrus II. This antagonist is undergoing detailed biological and clinical evaluation.