A series of luteinizing hormone-releasing hormone antagonists with new substitutions in position 6 or positions 5 and 6 that included lysine acylated at the epsilon-amino group with different heterocyclic carboxylic acids or amino-substituted heterocyclic carboxylic acids was synthesized. These novel analogs wee synthesized on a solid-phase support via the acylation of lysine residue in otherwise protected resin-bound peptides. All analogs were tested in the rat antiovulatory assay (AOA) and the best of them in in vitro histamine release assay. Introduction of lysine acylated with amino-substituted heterocyclic carboxylic acids yielded several water-soluble antagonists with good therapeutic ratio (high AOA to low histamine releasing activity). The best antagonist in terms of activity, histamine release, and solubility was nictide: NAcDNal-DCpa-DPal-Ser-PicLys-D(6ANic)-Orn- Leu-ILys-Pro-DAlaNH2 (6ANic = 6-aminonicotinoyl).

Peptide-like materials which release thyrotropin (TSH) from the pituitary in vivo and in vitro were obtained from the hypothalamus of the ox, sheep and pig and from human neurohypophysial tissue. Thyrotropic hormone releasing factor (TRF) activity was measured in vitro by the isolated rat pituitary incubation method of Saffran and Schally. TSH secreted into the medium was assayed by the release of 13I I from the thyroid gland of the mouse as described by McKenzie and by the uptake of 131 I by the tadpole's thyroid as described by Bowers. Addition of TRF preparations to the incubation media significantly increased the release of TSH from the pituitaries. TSH release in vitro was related to the dose of TRF preparations from all 4 species. One TRF preparation was active in vitro at a dose of 10 ng. TRF activity was also measured in vivo in the rat. TRF preparations from each species increased plasma TSH levels in thyroidectomized, triiodothyronine-treated rats. These studies support the concept that the hypothalamus takes part in the regulation of TSH release from the anterior pituitary gland. (Endocrinology 77: 609, 1965)

Neonatal rats were injected with L-dopa, 5-hydroxytryptophan (5-HTP) or 6-hydroxy-dopa (6-OH-dopa) and allowed to mature. Eye opening, vaginal opening, endocrine organ weights and pituitary hormone concentration in the serum were measured. Treatment with either the dopamine (DA) precursor or the serotonin (5-HT) precursor caused an acceleration in eye opening, a significant decrease in serum TSH (males) an elevation of serum prolactin (Prl; males), and a lowering of serum growth hormone (females). After chemical sympathectomy (6-OH-dopa) vaginal opening was delayed, thyroid weights were increased and serum TSH decreased (males). It is concluded that during the critical neonatal period overloading either of 2 of the principal monaminergic systems results in similar and persistent alterations in pituitary function. Chemical sympathectomy produced a different pattern of abnormalities.

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A series of reduced-size analogs of LHRH was designed with the length varying from nine to two amino acids. These compounds were tested in vitro for the LH suppression in cultured rat pituitary cells treated with 1 ng of LHRH. The best analogs were also tested in vivo for their antiovulatory activity in rats. It appeared that terminal amino acids as well as the presence of Arg or ILys in the sequence are both crucial for the antagonism. The most potent antagonist in this series was a heptapeptide, AcDNal-Ser-Tyr-DNal-Leu-Arg-ProNHEt, which completely inhibited LH release at the dose 0.1 microgram and inhibited ovulation at 1000 micrograms/rat. For fragments shorter than heptapeptide the inhibition of LH release was observed at the dose 100 micrograms of the analog.