Evaluation of the anaphylactoid activity of a new LHRH antagonist

ORF 23541 [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Pal(3)3,Ser4,Nic-Lys5,D-Nic-Lys 6, Leu7, I-Lys8, Pro9,D-Ala10NH2; "Nal-Lys antagonist"] was identified as a potent LHRH antagonist without significant anaphylactoid activity. It blocked ovulation in proestrus rats when administered subcutaneously with an ED50 of 5.8 micrograms/kg. Much higher doses of ORF 23541 than of other antagonists were required to induce a cutaneous anaphylactoid-like reaction. Intradermal administration of ORF 23541 caused an 8.75 x 8.75 mm wheal response with estimated doses of 10.9 and 13.7 micrograms in rats and guinea pigs, respectively. These doses were at least 10 times greater than that required of other LHRH antagonists for the same response. ORF 23541 also did not alter pulmonary function in guinea pigs or dogs when administered intravenously at doses up to 10 mg. These results indicate that ORF 23541 represents a new generation of LHRH antagonists with an improved safety margin.     

Superiority of an antagonist of the luteinizing hormone releasing hormone with emphasis on arginine in position 8, named Argtide.

In the research for more potent antagonists of the luteinizing hormone releasing hormone (LHRH), 13 new peptides with emphasis on arginine in position 8 were designed, synthesized and tested for anti-ovulatory activity (AOA). Two very potent analogs were achieved. N-Ac-D-3-Qal, D-pClPhe, D-3-Pal, Ser, cis-PzACA1a, D-PicLys, Leu, Arg, Pro, D-AlaNH2 showed 63% AOA at 0.125 microgram and 89% at 0.25 microgram, and an ED50 of 30.8 +/- 0.59 and presently may be the most promising antagonist reported. It is named Argtide. N-Ac-D-3-Qal, D-pClPhe, D-3-Pal, Ser, cis-PzACA1a, D-PicLys, Val, Arg, Pro, D-AlaNH2 showed 18% AOA at 0.125 microgram. Arg8 in antagonists may be significant for receptor binding.     

Benzimidazoles as non-peptide luteinizing hormone-releasing hormone (LHRH) antagonists. Part 3: Discovery of 1-(1H-benzimidazol-5-yl)-3-tert-butylurea derivatives

1-(1H-Benzimidazol-5-yl)-3-tert-butylurea derivatives have been identified as a novel class of non-peptide luteinizing hormone-releasing hormone (LHRH) antagonists. Herein, we disclose the synthesis and structure-activity relationships (SAR) of this class resulting in the identification of compound 12c, with dual functional activity on human and rat receptors (rat LHRH: IC50=120 nM; human LHRH: IC50=18 nM). These SAR studies suggest that 1-(1H-benzimidazol-5-yl)-3-tert-butylurea is a new pharmacophore for small molecule LHRH antagonists.     

Antagonists of the luteinizing hormone releasing hormone (LHRH) with emphasis on the TRP7 of the salmon and chicken II LHRH's

The sequences of four naturally occurring luteinizing hormone releasing hormones (LHRH's) differ only in positions 5, 7 and 8. Salmon and chicken II LHRH's have Trp7; porcine/ovine (P/O) and chicken I LHRH's have Leu7. The receptor for P/O LHRH might effectively bind certain antagonists with Trp7. Thirteen antagonists having Trp7 and eight antagonists with other substitutions in position 7 were synthesized. One of the thirteen antagonists with the natural Trp7, [N-Ac-D-2-Nal1,D-pClPhe2,D-3-Pal3,D-Arg6,Trp7,D- Ala10]-LHRH, not only maintained activity, but had increased potency (ca. 58%; 90% antiovulatory activity/250 ng; rats) in comparison with the companion analog with the natural Leu7 of P/O LHRH. The other twelve Trp7-antagonists had lower potency.     

Comparative studies on the hypotensive effect of LHRH antagonists in anesthetized rats

Certain neuropeptides are known to cause a hypotensive response, thought to be due to mast cell degranulation. The effects of five antagonists of luteinizing hormone-releasing hormone on blood pressure and heart rate were compared in the anesthetized rat. When given intravenously, all five compounds induced hypotensive and bradycardiac effects. The order of potency for these effects was Nal-Arg Antagonist approximately detirelix [( N-Ac-D-Nal(2)1, D-pCl-Phe2,D-Trp3,D-hArg(Et2)6,D-Ala10]LHRH) greater than [N-Ac-D-Nal(2)1, D-pCl-Phe2,D-Pal(3)3,D-hArg(Et2)6,L-hArg (Et2)8,D-Ala10]LHRH (RS-26306) approximately antide greater than [N-Ac-D-Nal(2)1, D-pCl-Phe2,D-Pal(3)3,6, L-hArg(Et2)8,D-Ala10]LHRH (RS-15378) and did not parallel the order of antiovulatory potencies of these compounds. The hypotensive activity of LHRH antagonists, therefore, appeared dissociable from their antiovulatory activity. RS-26306 and RS-15378 appeared to have the greatest therapeutic ratios.     

Benzimidazole derivatives as novel nonpeptide luteinizing hormone-releasing hormone (LHRH) antagonists. Part 1: Benzimidazole-5-sulfonamides

A new class of benzimidazole-5-sulfonamides has been identified as nonpeptide luteinizing hormone-releasing hormone (LHRH) antagonists. Initial structure-activity relationships are presented resulting in compounds 19 and 28 with submicromolar dual functional activity on human and rat receptors.     

Properties of LHRH release from a hypothalamic synaptosomal fraction of estrogen-primed ovariectomized rats

The release of luteinizing hormone-releasing hormone (LHRH) from mitochondrial-synaptosomal fractions (P₂) of basomedial hypothalamus was examined under various conditions. Less than 3% of the LHRH in P₂ suspensions was released under control conditions while the addition of 60 mM KCl or NaCl effected an 8-fold increase in LHRH as measured by radioimmunoassay. Equiosmolar sucrose effected only a 1.8-fold increase in LHRH release. The stimulatory effects of both Na⁺ and K⁺ were significantly inhibited by Mn²⁺ or La³⁺. Two forms of released LHRH were observed, one soluble and the other particulate. Soluble LHRH release was effected by hypertonic sucrose or 60 mM KCl and was not inhibited by Ca²⁺ antagonists. The release of particulate LHRH was unaffected by hypertonic sucrose, was stimulated 10-fold by 60 mM KCl, and was abolished with Ca²⁺ antagonists. These results suggest that the released soluble LHRH results from nonspecific leakage while the release of particulate LHRH reflects a Ca²⁺-dependent secretory process.     

Elimination of antibacterial activities of non-peptide luteinizing hormone-releasing hormone (LHRH) antagonists derived from erythromycin A

Antibacterial SAR for a series of macrolides derived from erythromycin A that are potent LHRH antagonists was developed in an attempt to eliminate the antibiotic activities of these compounds. Increasing the size of the alkyl substituents on the desosamine 3'-amine resulted in potent LHRH antagonists that were inactive against staphylococcal bacteria strains, and were significantly (>10-fold) less active against streptococcal bacteria strains. Complete elimination of antibacterial activities could be achieved by replacement of one or both methyl groups on the 3'-amine with a large alkyl substituent.     

A new class of potent nonpeptide luteinizing hormone-releasing hormone (LHRH) antagonists: design and synthesis of 2-phenylimidazo[1,2-a]pyrimidin-5-ones

The design and synthesis of a new class of nonpeptide luteinizing hormone-releasing hormone (LHRH) receptor antagonists, the 2-phenylimidazo[1,2-a]pyrimidin-5-ones, is reported. Among compounds described in this study, we identified the potent antagonist 15b with nanomolar in vitro functional antagonism. The result might suggest that the heterocyclic 5-6-ring system possessing a pendant phenyl group attached to the five-membered ring is the important structural feature for a scaffold of small molecule LHRH antagonists.     

Epostane and indomethacin actions on ovarian kallikrein and plasminogen activator activities during ovulation in the gonadotropin-primed immature rat.

Kallikrein and plasminogen activator (PA) are serine proteases that have been implicated in the ovulatory process. Epostane and indomethacin are anti-ovulatory agents that inhibit steroid and eicosanoid synthesis, respectively. This study examines the effects of these two anti-ovulatory agents on ovarian kallikrein and PA activities during ovulation. The proteases were assayed by their actions on chromogenic peptide substrates S-2266 and S-2251, respectively. The ovulatory process was induced in 25-day-old Wistar rats by giving them hCG (10 IU, s.c.) 2 days after the animals had been primed with eCG (10 IU, s.c.). Control animals ovulated approximately 60-70 ova/rat, with the first ova appearing in the oviducts at 10-12 h after hCG administration, and this was the same time ovarian kallikrein and PA activities reached a peak. When doses of epostane ranging from 0.1-5.0 mg/rat or doses of indomethacin ranging from 0.03 to 3.16 mg/rat were administered s.c. at 3 h after hCG, the two drugs inhibited ovulation and ovarian kallikrein and PA activities in a dose-dependent manner. However, the anti-ovulatory action of the two drugs was more closely correlated with suppression of kallikrein activity than with PA activity. Treatment of the animals with exogenous progesterone reversed the inhibitory action of epostane, but not of indomethacin. The results suggest that the increase in ovarian progesterone at the time of ovulation may influence ovarian kallikrein and PA activities.     

Ovulation inhibition in the pregnant mare's serum gonadotropin-treated immature rat: a bioassay for luteinizing hormone-releasing hormone antagonists

A convenient method for evaluating the biological activity of luteinizing hormone-releasing hormone (LHRH) antagonists was devised. Pregnant mare's serum gonadotropin (PMSG) treatment of immature rats is known to stimulate follicular growth and estrogen production, that in turn stimulates the release of LHRH which triggers an ovulatory discharge of luteinizing hormone (LH) from the pituitary. The present bioassay of the antagonists is based on the inhibition of ovulation in the PMSG-treated rats. Twenty-eight-day-old Sprague Dawley rats maintained under a light period of 12 h/day (lights on at 0630 h) were given 10 IU of PMSG s.c. at 0930 h. On Day 30 of age the antagonist was given s.c. at 1430 h. The rats were killed on the following morning and the oviducts examined for the presence of ova. In addition, the antagonists were compared in their ability to inhibit serum testosterone levels in adult male rats. In the PMSG-treated rats the order of ovulation-inhibiting potency of the following antagonists was: [Ac-D-NAL(2)1,4FD-Phe2,D-Trp3,D-Arg6]-LHRH (LHRH-1) greater than [Ac-delta 3 Pro1,4FD-Phe2,D-NAL(2)3.6]-LHRH (LHRH-2) greater than [Ac-delta 3 Pro1,4FD-Phe2,D-Trp3,6]-LHRH (LHRH-3). The order of potency was confirmed by their antitesticular effects in adult male rats.     

Human chorionic gonadotropin and luteinizing hormone-releasing hormone reverse the blockade of ovulation in pregnant mare's serum gonadotropin-primed immature rats by the anti-androgenic drug, hydroxyflutamide

The present study was designed to examine mechanism(s) of the anti-ovulatory action of the anti-androgen, hydroxyflutamide (OH-F). Prepubertal rats were treated with 4 IU pregnant mare's serum gonadotropin (PMSG) (day -2) to induce first estrus and ovulation. They received OH-F in sesame oil or oil alone at 08:00 and 20:00 h on day 0 (the day of proestrus) and ovulations were assessed on the morning of day 1. Eighty-three percent of control animals ovulated with a mean of 7.7 +/- 1.1 corpora lutea per rat. Hydroxyflutamide blocked ovulation in all but 2 of the 12 rats receiving this drug alone. All of OH-F treated rats that received 5 and 25 IU human chorionic gonadotropin (hCG) ovulated with means +/- SEM of 9.1 +/- 0.1 and 7.3 +/- 1.4 corpora lutea per rat, respectively. The dose of 0.2 IU hCG was essentially ineffective, while the effect of 1.0 IU hCG was intermediate. At the dose of 20 ng and above (100 and 500 ng) luteining hormone-releasing hormone (LHRH) completely overcame the ovulation blockade in the OH-F treated animals, while a 4-ng dose was ineffective. At 18:00 h on the day of proestrus, serum LH levels in control animals were 17.56 +/- 2.60 ng/mL, which were 920% above basal levels (1.90 +/- 0.13) indicating a spontaneous LH surge. This surge was suppressed in OH-F treated rats. Injection of LHRH, at the dose of 20 ng and above, reinstated the LH release in OH-F treated animals. Thus, the anti-androgen, OH-F, inhibits ovulation in PMSG-treated immature rats through its interference with the preovulatory LH surge; the inhibition can be reversed by hCG or LHRH. Hydroxyflutamide does not appear to interfere at the level of the pituitary, but may have direct action at the hypothalamic and (or) extrahypothalamic sites involved in the generation of positive feedback signals that control LH release.     

Antagonists of luteinizing hormone releasing hormone with novel unnatural amino acids at position six

Five new antagonists of luteinizing hormone releasing hormone (LHRH) containing novel unnatural amino acids at position six are reported. They are very effective in the rat antiovulatory assay. Using saline as vehicle, antagonist-[N-Ac-D-2-Nal1, D-4-Cl-Phe2, D-3-Pal3, Arg5, D-A26, D-Ala10]-LHRH inhibited ovulation completely at 1 micrograms/rat and three of the other antagonists showed some antiovulatory activity at 0.5 micrograms/rat.     

Peptidase activity in the hypothalamus of the rat: utilization of leucine-p-nitroanilide to monitor the activity degrading luteinizing hormone releasing hormone

Previous studies by other investigators have shown that luteinizing hormone releasing hormone (LHRH) and amino acid derivatives of p-nitroanilide are probably degraded by a common enzymatic activity; however, most of these studies are inferential in that they are largely based upon kinetic inhibition data derived from relatively crude tissue preparations. The purpose of this work was to determine whether the synthetic substrate leucine-p- nitroanilide (Leu-p-NA) and LHRH were degraded by the same peptidase activity. Supernatants (10,000 X g) from homogenates of rat hypothalami were eluted from Sephadex G-200, and the resultant fractions were assayed for degrading activity toward LHRH and Leu-p-NA. Radioimmunoassay (RIA) indicated that loss of immunologically active LHRH occurred in the same fractions in which maximal Leu-p-NA degrading activity eluted. Kinetically, exogenous LHRH inhibited degradation of Leu-p-NA in a concentration-dependent manner. When fractions evidencing Leu-p-NA degrading activity were incubated with 125I-LHRH, polyacrylamide gel electrophoresis (PAGE) indicated a time-dependent loss of LHRH with the concomitant production of a radioactive peptide fragment. High-performance liquid chromatography (HPLC) analysis of unlabeled LHRH incubations revealed, within the Leu-p-NA degrading fractions, the formation of two peptide fragments. These studies have further substantiated the likelihood that LHRH and Leu-p-NA are degraded by a common enzyme activity as indicated not only by kinetic inhibition data, but also by cofractionation of activity toward both substrates and by two analytical methods capable of detecting LHRH fragmentation (PAGE and HPLC).     

Effects of LHRH on copulatory behavior and locomotor activity in sexually inexperienced male rats

To determine whether luteinizing hormone-releasing hormone (LHRH) contributes to the sexual behavior and locomotor activity of sexually inexperienced male rats, subcutaneous injections of LHRH (500 ng) were given to males. The males showed significant facilitation of a few aspects of sexual behavior 2h after LHRH injection, compared with saline-injected controls. However, the locomotor activity of males injected with LHRH showed no significant change. These results may indicate that LHRH mechanisms play a direct role in the normal regulation of sexual arousal of male behavior in rats.     

New antagonists of LHRH. II. Inhibition and potentiation of LHRH by closely related analogues

Modifications of the previously described LHRH antagonists, [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Trp3, D-Cit6, D-Ala10]LHRH and the corresponding D-Hci6 analogue, have been made to alter the hydrophobicity of the N-terminal acetyl-tripeptide portion. Substitution of D-Trp3 with the less hydrophobic D-Pal(3) had only marginal effects on the antagonistic activities and receptor binding potencies of the D-Cit/D-Hci6 analogues, but it appeared to further improve the toxicity lowering effect of D-Cit/D-Hci6 substitution. Antagonists containing D-Pal(3)3 and D-Cit/D-Hci6 residues, i.e. [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Cit6, D-Ala10]LHRH (SB-75) and [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Hci6, D-Ala10]LHRH (SB-88), were completely free of the toxic effects, such as cyanosis and respiratory depression leading to death, which have been observed in rats with the D-Trp3, D-Arg6 antagonist and related antagonists. Replacement of the N-acetyl group with the hydrophilic carbamoyl group caused a slight decrease in antagonistic activities, particularly in vitro. Introduction of urethane type acyl group such as methoxycarbonyl (Moc) or t-butoxycarbonyl (Boc) led to analogues that showed LHRH-potentiating effect. The increase in potency induced by these analogues, e.g. [Moc-D-Nal(2)1, D-Phe(4Cl)2, D-Trp3, D-Cit6, D-Ala10]LHRH and [Boc-D-Phe1, D-Phe(4Cl)2, D-Pal(3)3, D-Cit6, D-Ala10]LHRH, was 170-260% and persisted for more than 2 h when studied in a superfused rat pituitary system.     

Control of luteinizing hormone-releasing hormone pulse generation in nonhuman primates

Summary 1. The pulsatile release of luteinizing hormone-releasing hormone (LHRH) is critical for reproductive function. However, the exact mechanism of LHRH pulse generation is unclear. The purpose of this article is to review the current knowledge on LHRH pulse generation and to discuss a series of studies in our laboratory.2. Using push-pull perfusion in the stalk-median eminence of the rhesus monkey several important facts have been revealed. There is evidence indicating that LHRH neurons themselves have endogenous pulse-generating mechanisms but that the pulsatility of LHRH release is also modulated by input from neuropeptide Y (NPY) and norepinephrine (NE) neurons. The release of NPY and NE is pulsatile, with their pulses preceding or occurring simultaneously with LHRH pulses, and the neuroligands NPY and NE and their agonists stimulate LHRH pulses, while the antagonists of the ligands suppress LHRH pulses.3. The pulsatile release of LHRH increases during the estrogen-induced LH surge as well as the progesterone-induced LH surge. These increases are partly due to the stimulatory effects of estrogen and progesterone on NPY neurons.4. An increase in pulsatile LHRH release occurs at the onset of puberty. This pubertal increase in LHRH release appears to be due to the removal of tonic inhibition from aminobutyric acid (GABA) neurons and a subsequent increase in the inputs of NPY and NE neurons to LHRH neurons.5. There are indications that additional neuromodulators are involved in the control of the LHRH pulse generation and that glia may play a role in coordinating pulses of the release of LHRH and neuromodulators.6. It is concluded that the mechanism generating LHRH pulses appears to comprise highly complex cellular elements in the hypothalamus. The study of neuronal and nonneuronal elements of LHRH pulse generation may serve as a model to study the oscillatory behavior of neurosecretion.     

Luteinizing hormone secretion from the quail pituitary in vitro

An enzymatically dispersed pituitary preparation from Japanese quail (Coturnix coturnix) was used to study the dynamics of gonadotropin release. After an 18-h incubation, the cells were challenged with different luteinizing hormone-releasing hormones (LHRH) for 90 min. Using pituitary cells from mature males, mammalian and chicken LHRH I (Gln8-LHRH) had approximately equal luteinizing hormone (LH)-releasing activity whereas chicken LHRH II (His5, Trp7, Tyr8-LHRH) was 8-9 times more potent. The LHRH agonist (Trp6, Pro9-NEt-LHRH) had 15 times greater potency than chicken LHRH I. Pre-incubation with an LHRH antagonist (D-Phe2, D-Trp6-LHRH) significantly suppressed LH release. Acid extracts of median eminence released LH from pituitary cells, extracts from short-day and long-day males had equal activity, while tissue extracts from castrated males had significantly greater LH-releasing activity. Pituitary cells from sexually immature males released LH in response to chicken LHRH I in a similar profile to cells from mature males. These data indicate that the quail LHRH receptor in the male recognizes several different molecular species of LHRH and the response to LHRH is comparable between short- and long-day males. Pituitary cells from ovulating females were variably sensitive to LHRH peptides, possibly due to changes in pituitary sensitivity during the ovulatory cycle. Pituitary cells from immature females did not release LH in response to chicken LHRH I. However, pituitary cells from immature females photostimulated for 1 wk displayed a response to chicken LHRH I and II similar to that of pituitary cells from males.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic stability of long-acting luteinizing hormone-releasing hormone antagonists

Long-acting luteinizing hormone-releasing hormone (LHRH) antagonists designed to be protease resistant consisted of a series of novel decapeptides structurally similar to LHRH. The aim of this study was to evaluate the in vitro metabolic stability of the LHRH decapeptides using pancreatin and homogenates models and identify the metabolites in rat liver homogenate for the purpose of illustrating the metabolic features of the decapeptides. The major metabolites in rat liver homogenate were identified by LC-ESI-MS(n). The half-lives of the 11 LHRH decapeptides were from 44 to 330?min in the pancreatin model. The half-lives of the five decapeptides in rat liver, kidney and lung homogenates were between 8 and 462?min. The most stable decapeptides were the LY616 and LY608 peptides with half-lives of 36?min in liver homogenate. Two major cleavage sites were found by analysing the metabolites of the LY618 peptide in rat liver homogenate, between the Pal(3)-Ser(4) and the Leu(7)-Ilys(8) peptide bonds. The major metabolites were produced via cleavages of peptide bonds at these sites, and further metabolic reactions such as hydroxylation, oxidative dechlorination, alcohol dehydration and isopropyl dealkylation were also observed.