No abstract

A series of antagonists of gonadotropin-releasing hormone (GnRH) of the general formula Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph/4Amf(P)-D4Aph/D4Amf(Q)-Leu-ILys-Pro-DAla-NH2 was synthesized, characterized, and screened for duration of inhibition of luteinizing hormone release in a castrated male rat assay. Selected analogues were tested in a reporter gene assay (IC50 and pA2) and an in vitro histamine release assay. P and Q contain urea/carbamoyl functionalities designed to increase potential intra- and intermolecular hydrogen bonding opportunities for structural stabilization and peptide/receptor interactions, respectively. These substitutions resulted in analogues with increased hydrophilicity and a lesser propensity to form gels in aqueous solution than azaline B [Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(Atz)-D4Aph(Atz)-Leu-ILys-Pro-DAla-NH2 with Atz = 3'-amino-1H-1',2',4'-triazol-5'-yl, 5], and in some cases they resulted in a significant increase in duration of action after subcutaneous (s.c.) administration. Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(L-hydroorotyl)-D4Aph(carbamoyl)-Leu-ILys-Pro-DAla-NH2 (acetate salt is FE200486) (31) and eight other congeners (20, 35, 37, 39, 41, 45-47) were identified that exhibited significantly longer duration of action than acyline [Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(Ac)-D4Aph(Ac)-Leu-ILys-Pro-DAla-NH2] (6) when administered subcutaneously in castrated male rats at a dose of 50 microg in 100 microL of phosphate buffer. No correlation was found between retention times on a C18 reverse phase column using a triethylammonium phosphate buffer at pH 7.0 (a measure of hydrophilicity) or affinity in an in vitro human GnRH report gene assay (pA2) and duration of action. FE200486 was selected for preclinical studies, and some of its properties were compared to those of other clinical candidates. In the intact rat, ganirelix, abarelix, azaline B, and FE200486 inhibited plasma testosterone for 1, 1, 14, and 57 days, respectively, at 2 mg/kg s.c. in 5% mannitol (injection volume = 20 microL). Based on the information that 31, 33, 35 and 37 were significantly shorter acting than acyline or azaline B after intravenous administration (100 microg/rat), we surmised that the very long duration of action of the related FE200486 (for example) was likely due to unique physicochemical properties such as solubility in aqueous milieu, comparatively low propensity to form gels, and ability to diffuse at high concentrations in a manner similar to that described for slow release formulations of peptides. Indeed, in rats injected s.c. with FE200486 (2 mg/kg), plasmatic concentrations of FE200486 remained above 5 ng/mL until day 41, and the time after which they dropped below 3 ng/mL and plasma LH levels started rising until full recovery was reached at day 84 with levels of FE200486 hovering around 1 ng/mL. Additionally, FE200486 was less potent at releasing histamine from isolated rat mast cells than any of the GnRH antagonists presently described in preclinical reports.

No abstract

Experiments were performed in 2 volunteers to define the biotransformation and physiological properties of norursodeoxycholic acid (norUDCA), the C 23 (C 24 -nor) homolog of UDCA. To complement the in vivo studies, the biotransformation of norUDCA ex vivo using precision-cut human liver slices was also characterized. In the human studies, both a tracer dose given intravenously and a physiological dose (7.9 mmol, 3.0 g) given orally were excreted equally in bile and urine. By chromatography and mass spectrometry, the dominant biotransformation product of norUDCA in bile and urine was the C-23 ester glucuronide. Little N-acyl amidation (with glycine or taurine) occurred. The oral dose induced a sustained bicarbonate-rich hypercholeresis, with total bile flow averaging 20 L/kg/min, a rate extrapolating to 2 L/d. The increased bile flow was attributed to cholehepatic shunting of norUDCA as well to the lack of micelles in bile. Phospholipid and cholesterol secretion relative to bile acid secretion decreased during secretion of norUDCA and its metabolites, presumably also because of the absence of micelles in canalicular bile. When incubated with human liver slices, norUDCA was glucuronidated, whereas UDCA was conjugated with glycine or taurine. In conclusion, in humans, norUDCA is glucuronidated rather than amidated. In humans, but not animals, there is considerable renal elimination of the C-23 ester glucuronide, the dominant metabolite. NorUDCA ingestion induces a bicarbonaterich hypercholeresis and evokes less phospholipid and cholesterol secretion into bile than UDCA. Molecules that undergo cholehepatic shunting should be powerful choleretics in humans. (HEPATOLOGY 2005;42:1391-1398