Zwitterionic uracil derivatives as potent GnRH receptor antagonists with improved pharmaceutical properties.
A novel series of potent zwitterionic uracil GnRH antagonists were discovered that showed reduced liability for CYP3A4 enzyme inhibition.
Non-peptide gonadotropin-releasing hormone receptor antagonists.
Introduction Gonadotropin-releasing hormone (GnRH) is the principal neuroendocrine regulator of the reproductive system in humans. It is a linear decapeptide, pGlu-His-Trp-Ser-Tyr-Gly-Leu-ArgPro-Gly-NH2, that was first isolated from porcine and ovine hypothalamii. This peptide and related isoforms have subsequently been identified in a wide range of species, indicating that core features of the sequence have been conserved over ∼600 million years of chordate evolution. The GnRH peptide is made by neurons in the hypothalamus and secreted in pulses approximately hourly into the portal blood supplying the pituitary (Figure 1). There, it stimulates secretion of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), into the general circulation. The gonadotropins in turn act at the gonads to support spermatogenesis and synthesis of testosterone in the male and follicular development and production of estrogen and progesterone in the female. Gonadal steroids in turn feed back to regulate the hypothalamus and pituitary. The actions of GnRH are mediated by the GnRH receptor (GnRH-R). It is a member of the rhodopsin family of seventransmembrane receptors and was first cloned from the mouse and subsequently from a variety of other species, including human. It differs from other class A GPCRs by the absence of a C-terminal tail, which is a predominate site of regulation by various kinases in other family members. Binding of GnRH induces a conformational change in the receptor, which in turn activates the GTPases GRq and GR11 resulting in G-protein activation, which stimulates activity of phospholipase C resulting in phosphatidyl inositol turnover, intracellular calcium release, and gonadotropin secretion. Continuous agonist administration causes an initial stimulatory effect, followed by desensitization of the pituitary and eventually down-regulation of gonadotropin secretion over the course of 1-2 weeks. This pituitary down-regulation leads to a profound suppression of the reproductive endocrine axis and can be exploited therapeutically to produce medical gonadectomy which reduces circulating sex steroids to levels equivalent to surgical castration. On the basis of this somewhat paradoxical mechanism, several GnRH agonist peptides such as leuprolide, goserelin, and triptorelin are now commercially available in long acting injectable depot formulations for gonadal suppression. They have found widespread utility for a range of steroid hormone dependent diseases and assisted reproductive therapy as outlined in Table 1. Early studies in the structure–activity relationships of GnRH peptides identified His as critical for receptor activation. Substitutions with D-amino acids at this position and optimization of multiple additional residues in the peptide led to the identification of potent antagonists. These efforts eventually led to multiple peptides that have been evaluated in clinical studies. In contrast to the initial stimulation caused by agonists, antagonists immediately inhibit pituitary gonadotropin secretion. Thus, in patients with advanced prostate cancer, antagonists avoid the initial “flare” in testosterone produced by agonists and result in a more rapid reduction in testosterone. Analogously in women, peptide antagonists reduce uterine fibroid volume in 2-4 weeks, more rapidly than is observed with agonist therapy. Antagonists also require fewer injec* To whom correspondence should be addressed. Phone: 858-617-7740. Fax: 858-617-7696. E-mail: sstruthers@neurocrine.com. a Abbreviations: AUC, area under the curve; b.i.d., twice daily dosing; Cmax, highest concentration; E2, estradiol; ECL, extracellular loop; FSH, follicle-stimulating hormone; GnRH, gonadotropin-releasing hormone; GnRH-R, gonadotropin-releasing hormone receptor; GPCR, G-proteincoupled receptor; GTPase, guanosine triphosphatase; HPG, hypothalamicpituitary–gonadal; im, intramuscular; ip, intraperitoneal; IVF, in vitro fertilization; LH, luteinizing hormone; NTD, N-terminal domain; sc, subcutaneous; TM, transmembrane.  Copyright 2008 by the American Chemical Society
Coexistence of Virulence and Antibiotic Resistance Genes in Pasteurella multocida Isolated from Diseased Rabbits
Pasteurella multocida (P. multocida)is considered a predominant pathogenic bacterial agent causing respiratory manifestations (snuffles) in rabbits with considerable economic losses and unfavorable prognosis in Egypt. A few recent P. multocida strains exhibit resistance to the most commonly used antibiotics in the veterinary field. Therefore, the present study was carried out to investigate the prevalence of both virulence and antibiotic resistance genes among P. multocida isolated from diseased rabbits in Sharkia Governorate, Egypt. Only 10 out of 100 tested rabbits` nasal swabs were finally confirmed positive for P. multocida of serogroup A by polymerase chain reaction (PCR). Antibiotic susceptibility testing of the recovered isolates revealed that they were all multidrug resistant (MDR) with a predominance of resistance to amoxicillin, ampicillin, amoxicillin/clavulanic acid, neomycin and tetracycline (100% each), followed by kanamycin and streptomycin (90% each). All the recovered isolates were further subjected to PCR screening of some common virulence and antibiotic resistance genes of interest . With the exception of toxA gene, the other virulence associated genes (ptfA, Omp87 and nanB) were found among all the examined isolates. Totally, all MDR P. multocida isolates contained at least one antibiotic resistance gene with aphA1 being the most prevalent (100%), followed by ermX gene (40%). Antibiotic resistance genotyping demonstrated the presence of multiple antibiotic resistance genes among majority of the isolates (40%) with only one isolate harboring 4 genes encoding identical resistance phenotypes. Evidentially, all MDR P. multocida isolates possessed at least 3 virulence genes accompanied by the attendance of antibiotic resistance genes. These findings evidenced that rabbits are potential sources of pathogenic P. multocida strains harboring virulence and antibiotic resistance genes. Therefore, it is evident that there is an urgent need for the judicious use of antibiotics in rabbits` treatment systems to successfully mitigate the propagation of drug resistance across P. multocida species.