Structure-activity studies on the N-terminal region of growth hormone releasing factor.

In previous reports illustrating the effects of conformational restriction of the N-terminal region of human pancreatic growth hormone releasing factor, we demonstrated that D-amino acid substitutions in either of positions 1, 2, or 3 resulted in greatly increased growth hormone releasing activity both in vivo and in vitro. The most active compound, [D-Ala-2]GRF(1-29)NH2, was 51 times more active than the parent 29 amino acid peptide in the sodium pentobarbital anesthetized rat. These observations have now been extended to analogues containing multiple D-amino acid replacements in these three positions. Once again, peptides with superagonist potencies ranging from 1200% to 3800% were obtained after solid-phase synthesis and purification by medium-pressure reverse-phase liquid chromatography. In addition, it was found that [D-Asn-8]- and [D-Ala-4]GRF(1-29)NH2 were, respectively, 2.43 and 1.1 times more active than GRF(1-29)NH2 itself. In contrast, [D-Phe-6] and [D-Thr-7] analogues were virtually inactive. Chou-Fasman structural predictions suggest that the first three residues of the peptide assume no fixed type of conformation but that a reverse turn could be present between residues 6 and 10. Attempts are made to rationalize the biological results with these calculations. The effects of other side chains on the D-amino acid in position 2 were also investigated. Both the Ac-[D-Phe-2]- and Ac-[D-Arg-2]peptides had very low activity. Several of the inactive peptides were tested as possible antagonists of GRF; however, none was able to block the stimulatory effects of GRF(1-29)NH2 after combined administration.

Synthesis and biological activity of LH-RH antagonists modified in position 1.

As part of our studies on the design of more potent antagonists of the LH-RH (luteinizing hormone-releasing hormone) decapeptide, twelve new highly soluble D-Arg6-analogs have been synthesized. These peptides contain modifications in position 1 and are typified by the general formula (N-acetyl-X1, D-p-Cl-Phe2, D-Trp3, D-Arg6, D-Ala10) LH-RH. We have found that a lyophilic, aromatic substituent is required in position 1 in order to elicit antiovulatory activity at a dose as low as 3 micrograms. The larger the hydrophobic amino acid (X: p-Br-Phe, beta-Nal-2) in position 1, the higher is the antiovulatory activity that can be attained. Analogs with non-aromatic or hydrophilic amino acids (X: Gly, Leu, Arg, His, Glu) in position 1 generally have much lower activities in this series of LH-RH antagonists.

Peptide antagonists of LH-RH: large increases in antiovulatory activities produced by basic D-amino acids in the six position.

It has been assumed, usually with good reason, that D-amino acids with large aromatic side-chains must be present in position 6 of both LH-RH superagonists and antagonists for the highest levels of biological activity to be reached. However, using one of a recent generation of potent lH-RH inhibitory analogs as a model, we have found that the insertion of D-lysine or, better still, D-arginine in this position results in greater antiovulatory activity in the rat over corresponding D-phenylalanine6- and D-tryptophan6-analogs. For instance, [Ac-D-p-Cl-Phe1,2,D-Trp3,D-Arg6,D-Ala10]-LH-RH exhibits antiovulatory activity at a dose of 750 ng per animal and appears to be the first competitive antagonist with activity in the nanogram region in vivo. This effect seems to be highly dependent on the degree of basicity of the amino acid side-chain since D-amino acids with neutral or acidic groups produced far less active compounds.

Solid-phase synthesis and biological activities of gastrointestinal hormones: secretin and motilin.

Many successful solid-phase syntheses of peptide chains in the region of 20-40 amino acid residues have now been routinely reported. Utilizing standard solid-phase synthetic methodologies but, particularly, new and powerful purification techniques we have been developing rapid and efficient preparative routes for the numerous neuro-gastrointestinal peptides. In the present study, secretin and motilin were obtained in 16% and 10% yields, respectively, after simplified two-step purification of hydrogen fluoride-cleaved peptides by gel filtration followed by preparation high performance liquid chromatography. Peptides were essentially homogeneous by TLC and analytical high performance liquid chromatography. Secretin was found to have full biological activity when tested against a standard sample of natural material for effects on pancreatic secretion in the dog. Motilin exhibited full biological activity on interdigestive motility in the dog. Secretin has been reported to undergo rearrangement with loss of bioactivity during purification and prolonged storage. We observed no obvious problems during our abbreviated purification schedule and have found no loss of purity of peptide which has been kept for 6 months as power lyophilized from dilute acetic acid.

LH-RH antagonists: further analogs with ring-substituted aromatic residues.

Although the systematic substitution of benzene and other aromatic ring systems with various atoms and groups has been a standard approach in conventional pharmaceutical research, it has only recently received the attention it deserves in peptide research. The observation that D-p-Cl-Phe inserted in position 2 of certain LH-RH (luteinizing hormone-releasing hormone) analogs results in large improvements in antagonist activity led us to examine the effect of this and other substituents on position 1 and 2 D-phenylalanyl analog side chains. Analogs containing two D-p-Cl-phenylalanines were found to be particularly powerful competitive inhibitors when assayed in cycling rat for blockade of ovulation. Analogs with non-aromatic amino acids in the first position exhibited much lower activities.

Suppression of LH-RH-induced ovulation in hamsters and rats by synthetic analogues of LH-RH.

Peptide antagonists of LH-RH, [D-Phe2, D-Phe6]-LH-RH, [D-Phe2,D-Leu6]-LH-RH, [D-Phe2, D-Ala6]-LH-RH, [D-Phe2, D-Phe3,D-Phe6]-LH-RH, [DesHis2, D-Leu6]-LH-RH, and [DesHis2, D-Phe6]-LH-RH, were examined for their ability to suppress LH-RH-induced ovulation in phenobarbital-blocked hamsters and Nembutal-blocked rats. All of these peptides, with the exception of [DesHis2, Phe6]- LH-RH suppressed ovulation to various degrees, but alos exhibited various degrees of agonistic activities. Complete suppression of ovulation was achieved in rats with 3 mg [D-Phe2,Phe3,-D-Phe6]-LH-RH given in 4 divided doses at 30-min intervals starting 2 hr before the LH-RH injection. It was found that LH levels had to be lower than 5 ng/ml (control: 20-9 ng/ml) to suppress ovulation. However, the extent of suppression of ovulationdid not correlate with the serum LH levels less than 5 ng/ml. The incidence of ovulation induced by the intrinsic LH-RH activity of some of these analogues was similar to or greater than that resulting from administration of LH-RH plus analogue, suggesting that the activity of LH-RH itself was eliminated by pretreatment with the analogues.

Antagonistic activity of analogs of luteinizing hormone-releasing hormone (LH-RH) in vitro.

The ability of eighteen analogs of LH-RH to inhibit LH-RH-induced LH release was tested in primary cultures of rat anterior pituitary cells. [Des-His2]LH-RH, [Des-His2, D-Leu6]LH-RH and [Des-His2, D-Phe6]LH-RH inhibited 50% of LH-RH-induced LH release at molar ratios (MR50S) of 3000, 500 and 60, respectively, while [D-Phe2]LH-RH, [D-Phe2, D-Leu6]LH-RH and [D-Phe2, D-Phe6]LH-RH had similar effects at MR50S of 1000, 150 and 25, respectively. This indicates that substitution of D-phenylalanine for histidine at position 2 of LH-RH leads to compounds approximately 3-fold more potent than the corresponding [Des-His2]-analogs. [D-Phe2, D-Phe6]LH-RH, the most potent antagonist tested has however a slight agonistic activity (0.003% that of LH-RH itself). [D-Phe2, D-Phe6, Phe7]LH-RH, [D-Phe2, Phe3, D-Phe6]-LH-RH and [D-Phe2, Phe5, D-Phe6]LH-RH inhibit 50% of LH-RH action at MR50S of 400, 100 and 75, respectively. All of the analogs mentioned in the last group have LH-releasing activities below 1/100,000 that of LH-RH itself.

Prolonged anti-luteinizing hormone/follicle-stimulating hormone-releasing activities of some synthetic antagonists of luteinizing hormone-releasing hormone.

A number of synthetic analogs of luteinizing hormone-releasing hormone (LH-RH) were screened for their in vivo antigonadotropin-releasing activities using a four-point bioassay test in immature male rats. Of the peptides tested, the most effective were those containing D-phenylalanine in position 2 of the decapeptide chain in conjunction with D-leucine or, preferably, D-phenylalanine in position 6. Several inhibitory peptides that were found to be very potent were reassayed and compared for duration of inhibition in immature male rats, whereupon the D-Phe2 analogs were found to be particularly long-acting. The most active and persistent peptide of the series, [D-Phe2, D-Phe6]-LH-RH, was able to inhibit the response to exogenous LH-RH for up to 6 hours after its injection.

Analogs of luteinizing hormone-releasing hormone with increased biological activity produced by D-amino acid substitutions in position 6.

The incorporation of simple d-amino acids in place of glycine in position 6 of the LH-RH decapeptide produces analogs which have far greater gonadotropin-releasing activities in vivo and in vitro than the natural hormone. An investigation of the structural features of the d-amino acids responsible for this phenomenon suggests that an increase in the lipophilic character and perhaps the size and aromaticity of the side chain coincides with an increase in biological activity. This is demonstrated by the LH-releasing activities of the following series of peptides which were assayed over a period of 6 h in immature male rats: [d-Glu(6)]-,1.8;[d-Ala(6)]-,7.0;[d-Leu(6)]-,9,0;[d-Phe(6)]-,10;[d-Trp(6)]-LH-RH, 13 times more active than LH-RH itself. In contrast to previous results with [d-Ala(6)]-and [d-Leu(6)]-LH-RH, where the substitution of an ethylamide group for the glycine amide at the C-terminus produces large increases in LH/FSH releasing activity, the ethylamide derivatives of [d-Phe(6)]-and [d-Trp(6)]-LH-RH were actually less potent than their parent peptides. [(N-Me-d-Ala)(6)]-LH-RH was found to be approximately 70 times less active than [d-Ala(6)]-LH-RH which indicates that disruption of a preferred receptor-site binding conformation might be brought about by methylation of the amide linkage in this position.

Inhibition of luteinizing hormone release by analogs of luteinizing hormone-releasing hormone (LHRH) in vitro.

Sixteen synthetic analogs of LH-releasing hormone (LHRH) were tested for their ability to inhibit the stimulation of LH release induced by 3 X 10(-9)M LHRH in anterior pituitary cells in monolayer culture. Half-maximal inhibition of LHRH-induced LH release was obtained with 7 analogs at concentrations which ranged from 3 X 10(-6)M to 3 X 10(-5)M. None of these seven analogs had significant LH-releasing activity at concentrations up to 10-5M. Nine analogs had no detectable antagonistic activity when tested in up to a 3000-fold molar ratio of analog to LHRH.

Inhibitory activity of analogues of luteinizing hormone-releasing hormone (LH-RH) in vitro and in vivo.

Improved inhibitors of LH-RH are those which, beside removal of the histidine residue at position 2 of LH-RH, include replacement of glycine at position 6 by a D-amino acid. A still better modification is replacement of the histidine residue at position 2 by D-phenylalanine. As examples, when tested in pituitary cells in culture, [Des-His2]LH-RH, [Des-His2, D-Leu6]LH-RH, [Des-His2, D-Phe6]-LH-RH, [D-Phe2]LH-RH, [D-Phe2, D-Leu6]LH-RH and [D-Phe2, D-Phe6]LH-RH inhibit 50% of LH release induced by LH-RH at molar ratios (MR50S) of 3000, 500, 60, 1000, 150 and 25, respectively. [D-Phe2, D-Phe6, D-Phe7]LH-RH, [D-Phe2, Phe3, D-Phe6]LH-RH and [D-Phe2, Phe5, D-Phe6]LH-RH have MR50 values of respectively 400, 100, and 75. When evaluated in vivo, some of the mentioned structural modifications permit inhibition of LH-RH action at molar ratios lower than observed in vitro. At a 500 molar ratio, [D-Phe2, Phe5, D-Phe6]-LH-RH inhibits the plasma LH rise induced by LH-RH by 75% up to 5 h after its injection. When administered at 12.00 hours at the dose of 2 mg, this analogue inhibits the spontaneous pro-oestrus LH surge and ovulation by 85 and 75%, respectively.

Comparison of the anti-LH/FSH-RH and anti-ovulatory activities of (D-Phe2, D-Leu6)-LH-RH and (D-Phe2, D-Ala6)-LH-RH.

The anti-LH/FSH-RH and antiovulatory activities of [D-Phe2, D-Leu6]-LH-RH and [D-Phe2, D-Ala6]-LH-RH were compared in rats. Both peptides inhibited the LH and FSH release induced by LH-RH in immature male rats, but, 4 hr after the injection, [D-Phe2, D-Leu6]-LH-RH still suppressed the LH and FSH release whereas the [D-Phe2, D-Ala6]-LH-RH did not. When the peptides were administered in equal doses on the afternoon of the day of proestrus in 4-day cycling rats, [D-Phe2, D-Leu6]-LH-RH more completely inhibited the ovulation occurring on the following morning than [D-Phe2, D-Ala6]-LH-RH. Thus, the incorporation of D-Leucine into position six of the decapeptide chain gives a more potent inhibitor than that resulting from the insertion of D-Alanine.

Long-acting luteinizing homone-releasing hormone (LH-RH) analogs in the treatment of female infertility.

Induction of ovulation in 20 infertile women was attempted by the use of two synthetic analogs of LH-RH, D-ALA6-Des-Gly10-ethylamide and D-Leu6-Des-Gly10-ethylamide. Ovulation was obtained in five out of the 16 in whom D-Leu6-Des-Gly10-LH-RH ethylamide was administered intramuscularly. Two of the five women received the LH-RH analog additioned of polysaturated gelatines as carrier medium. None of these women got pregnant. It is concluded that better therapeutic results might be expected when a suitable LH-RH vehicle allowing a gradual release of LH-RH analog is obtained.

Synthesis and biological properties of the 2-L-beta-(pyrazolyl-1)alanine analogs of luteinizing hormone-releasing hormone and thyrotropin-releasing hormone.

The luteinizing hormone-releasing hormone (LH-RH) analog, less thanGlu-Pyr(1)Ala-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, and the thyrotropin-releasing hormone (TRH) analog, less thanGlu-Pyr(1)Ala-Pro-NH2, were synthesized by azide couplings of the dipeptide hydrazide, less thanGlu-Pyr(1)Ala-NHNH2, to the C-terminal octapeptide of LH-RH and to proline amide, respectively. In an ovariectomized, steroid-blocked rat assay, the LH-RH analog was found to have only 1% of the LH-releasing activity of the natural hormone. The TRH analog was 1.5 times more effective than TRH itself in releasing TSH in vivo from the anterior pituitary of mice. This peptide is one of two synthetic peptides so far discovered which are more potent than TRH.

Increased and prolonged LH-RH/FSH-RH activity of synthetic (D-Ala6, Des-Gly-NH210)-LH-RH-ethylamide in normal women.

A synthetic LH-RH analogue (D-Ala6, Des-Gly-NH210)-LH-RH ethylamide exhibited an increased and prolonged LH-RH/FSH-RH activity in normal women. The integrated amounts of LH and FSH levels for this LH-RH analogue were about nine and five times greater than for the same doses of synthetic LH-RH. It is expected that this synthetic LH-RH analogue might yield more positive results than with LH-RH when used in infertility problems.

Inhibitory activity of four analogs of luteinizing hormone-releasing hormone in vivo.

Four analogs of luteinizing hormone-releasing hormone (LH-RH), [des-His2,D-Ala6]-LH-RH, [des-His2,D-Ala6, des-Gly-NH2(10)1-LH-RH ethylamide, [des-His2,D-Leu6]-LH-RH, and [D-Phe2,D-Leu6]-LH-RH, at 300-fold molar ratios (analog/LH-RH) led to an almost complete inhibition of LH response to LH-RH in anesthetized 4-day cycling rats on the afternoon of proestrus. At a 75-fold molar ratio, [des-His2,D-Ala6]-LH-RH still inhibited the LH-RH-induced LH release by 50%. The ethylamide substitution at the COOH terminus of [des-His2,D-Ala6]-LH-RH did not significantly improve the inhibitory activity of the molecule.

Gonadotropin-releasing activity of two highly active and long-acting analogs of luteinizing hormone-releasing hormone after subcutaneous, intravaginal, and oral administration.

The gonadotropin-releasing activities of two synthetic analogs of luteinizing hormone-releasing hormone (LH-RH), D-Ala6-des-Gly10-LH-RH ethylamide and D-Leu6-des-Gly10-LH-RH ethylamide were evaluated in immature female rats after subcutaneous, intravaginal, and oral administration. Maximal peaks of serum LH and follicle-stimulating hormone (FSH) levels after administration of both analogs by any of the three routes were obtained at 2 hours. Therefore, serum gonadotropin levels declined slowly, so that at 6 hours LH levels had returned to base line values, whereas FSH levels remained elevated for up to 10 hours. The integrated serum gaondotropin levels after LH-RH and both analogs over a 10-hour period indicated that D-Leu6-des-Gly10-LH-RH EA and D-Ala6-des-Gly10-LH-RH EA released more LH and FSH than did the LH-RH decapeptide. The intense and long-acting properties of these analogs in releasing LH and FSH suggest the possibility that they may be more useful therapeutically than LH-RH.