John Humphries

Presence of proline in positions 3 for potent inhibition of the activity of the luteinizing hormone releasing hormone and of ovulation

The effect of various analogs of luteinizing hormone-releasing hormone (LH-RH) on the release of LH and follicle stimulating hormone (FSH) from isolated rat pituitaries was studied in vitro. (D-Phe (2), Pro (3), D-Trp (6))-LH-RH, in a ratio of analog to LH-RH of 50:1, completely inhibited the LH-RH-induced release of LH and FSH. Higher ratios of (D-Phe (2), Leu(3), D-Trp(6))-LH-RH and (D-Phe(2), Leu(3), D-PHe(6)-LH-RH to LH-RH also had a complete inhibitory effect, though they showed no agonistic activity. A single sc injection of the Pro(3)-analog (750 mcg) completely inhibited ovulation in rats, while a dose of 375 mcg inhibited ovulation by 50 percent. Complete inhibition of ovulation was also observed by the Leu(3)-analogs at 750 mcg with D-Phe(6), and at 1.5 mg with D-Trp(6). The in vitro and antiovulatory potency of LH-RH inhibitors was increased by the substitution of proline in position 3 of the inhibitory sequence.

An antiovulatory decapeptide of higher potency which has an L-amino acid (Ac-Pro) in position 1☆

Ac-[Pro1, D-Phe2, D-Trp3, D-Trp6]-LH-RH completely inhibited ovulation in cycling rats at 200μg/rat and is comparable in activity to the corresponding D-<Glu1-analogue. This Ac-Pro1-analogue is the most potent antiovulatory peptide yet known having an L-amino acid residue in position 1. This result shows that for the design of potent inhibitors of ovulation, a D-amino acid residue is not essential in position 1. The corresponding Ac-D-Pro1- and Kic1-analogues completely inhibited ovulation at 750μg/rat, but not at 200μg/rat, and the Cpc1-analogue was inactive at these dosages.

Studies on inhibition of the luteinizing hormone-releasing hormone by an irreversible inhibitor at the receptor site

Abstract [Leu 2 , Leu 3 , D-Ala 6 ]-LHRH is an analog of pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2 (LHRH) and inhibits the release of LH and FSH induced by LHRH. This analog and inhibitor has been modified with the objective of developing an active-site-directed irreversible inhibitor. The modification consisted of replacing 1 with Chl 1 which is the moiety of chlorambucil (a nitrogen mustard). The Chl analog inhibited the release of LH and FSH by LHRH after addition prior to LHRH and after three changes of the incubation medium; in contrast, [Leu 2 , Leu 3 , D-Ala 6 ]-LHRH and [des-His 2 ]-LHRH only inhibit release when added together with LHRH. The Chl analog released LH and FSH but not TSH or GH, indicating that its agonist and antagonist activities could be specific at the receptor site for LHRH.

A new category of ovulation inhibitors linear LH-RH analogues having more than ten residues

Abstract A linear analogue of the luteinizing hormone-releasing hormone, longer than a decapeptide, is described for the first time, which is equivalent in potency to the best known inhibitors of ovulation, and which constitutes an important new lead to the design of inhibitors of even greater potency. At a dosage of 200 μg/rat, the undecapeptide [( 1 , D -Phe 2 , D -Trp 3 , D -Trp 6 ]-LH-RH caused 100% inhibition of ovulation. The related analogues, [( 1 , D -Phe 2 , D -Trp 3 , D -Trp 6 ]-LH-RH and [(Gly-Pro) 1 , D -Phe 2 , D -Trp 3 , D -Trp 6 ]-LH-RH, were less active, in vivo . All of these undecapeptides inhibited the action of 0.6 ng/ml of LH-RH by greater than 50% at the very low level of 10 ng/ml.

On the importance of position one of ovulation inhibitors, as based on studies on [D-Phe2, Pro3, D-Phe6]-LHRH.

Substitution of cyclopentylcarbonyl-(Cpc) for <Glu1 in the effective and potent antiovulatory inhibitor, [D-Phe2, Pro3, D-Phe6]-LHRH (I) retained the in vitro potency. We know of no other inhibitor of the luteinizing hormone releasing hormone (LHRH) with a modification at position 1, which is as potent in vitro. This result agrees with the concept of the role of <Glu for agonist activity in a low energy conformer of LHRH, and underscores the importance of position 1. [Cpc1, D-Phe2, Pro3, D-Phe6]-LHRH did not inhibit ovulation in rats at the same dosage as did I; this result is under study to circumvent. Des-Gly10-[D-Phe2, Pro3, D-Phe6]-LHRH ethylamide and [Glu1, D-Phe2, Pro3, D-Phe6]-LHRH were significantly less active in vitro than I.

Acidic analogs of the luteinizing hormone-releasing hormone and conformational aspects for activity.

[Gly1a]-LHRH acid (<Glu-Gly-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-OH), [Gly2a]-LHRH acid (<Glu-His-Gly-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-OH), and [Tyr3, Trp5]-LHRH acid (<Glu-His-Tyr-Ser-Trp-Gly-Leu-Arg-Pro-Gly-OH), were synthesized; they released LH with potencies of <0.0003, 0.0003, and 0.0003%, respectively, that of LHRH, but did not act as inhibitors up to a 30,000-fold relative dosage. Absence in these analogs of “conformational components” involving a hydrogen bond between the <Glu1 and Ser4 as proposed for LHRH and/or the proposed parallel planarity of the Trp-Tyr aromatic nuclei, and other effects including that of a C-terminal acid, could explain the observed data.

Differentiation of factor C-LHIH and the synthetic contraceptive polypeptide, H-Thr-Pro-Arg-Lys-OH

Summary The contraceptive polypeptide designated by Kent and shown by him to be H-Thr-Pro-Arg-Lys-OH has been synthesized by a solid-phase procedure, and the tetrapeptide was purified and characterized. Factor C-LHIH, partially purified from porcine hypothalami, which inhibits the luteinizing hormone from basal release and from the synthetic luteinizing hormone releasing hormone (LHRH), and the synthetic H-Thr-Pro-Agr-Lys-OH are different by cation exchange and by other high pressure liquid chromatographic techniques. At high dosage, H-Thr-Pro-Arg-Lys-OH released FSH and to a lesser extent LH, but the tetrapeptide showed no antagonist activity against synthetic LHRH, even at very high levels.

Inhibition of preovulatory gonadotropin secretion in the rhesus monkey by [(<Glu-Pro)1,D-Phe2,D-Trp3,6]-LHRH

We report the first example of a complete inhibition of preovulatory gonadotropin secretion resulting from administration of a luteinizing hormone releasing hormone antagonist during a spontaneous menstrual cycle. The antagonist, [(<Glu-Pro)1,D-Phe2,D-Trp3,6]-LHRH, was administered to a rhesus monkey beginning on Day 9 of the menstrual cycle; ovulation did not occur and preovulatory peaks of LH and FSH were not observed despite elevations in serum estradiol-17 beta of sufficient strength and duration to elicit gonadotropin surges. Midcycle gonadotropin surges had already commenced in another monkey, however the antagonist did partially inhibit LH and FSH secretion although ovulation and luteinization were not prevented. Normal hormone secretion patterns and luteal function were observed in another monkey when the antagonist was given after the midcycle FSH and LH peaks had already occurred. These data emphasize the importance of beginning treatment with LHRH antagonists early in the follicular phase of the menstrual cycle.