In this study we have evaluated the reliability of a fluorescence-based method used for rapid identification of irreversible CYP inhibitors (mechanism-based inhibitors). This was accomplished by comparing the time-dependence pattern of IC50 values from fluorometric kinetic measurements. For irreversible CYP inhibitors, IC50 values decreased as incubation proceeded. This was due to progressive inactivation of corresponding enzymes by reactive metabolites generated during the incubation. This change pattern was confirmed using a number of known irreversible CYP inhibitors, including furafylline, midazolam, erythromycin, clarithromycin, oleandomycin, 17alpha-ethynylestradiol and verapamil. The pattern was different in reversible inhibition, depending upon the compounds tested in the fluorometric kinetic assay. For some compounds, such as clotrimazole, IC50 values remained relatively stable, whereas other compounds, such as miconazole, terfenadine and ketoconazole showed a significant increase with incubation time. Monitoring tested compounds by LC-MS/MS during the incubation confirmed that increases of IC50 were probably caused by the loss of inhibitors, resulting from either metabolic degradation, or non-specific binding to microsomal proteins.

1. In vitro studies have been carried out to investigate the metabolic pathways and identify the hepatic cytochrome P450 (CYP) enzymes involved in etoperidone (Et) metabolism. 2. Ten in vitro metabolites were profiled, quantified and tentatively identified after incubation with human hepatic S9 fractions. Et was metabolized via three metabolic pathways: (A) alkyl hydroxylation to form OH-ethyl-Et (M1); (B) phenyl hydroxylation to form OH-phenyl-Et (M2); and (C) N-dealkylation to form 1-m-chlorophenylpiperazine (mCPP, M8) and triazole propyl aldehyde (M6). Six additional metabolites were formed by further metabolism of M1, M2, M6 and M8. 3. Kinetic studies revealed that all metabolic pathways were monophasic, and the pathway leading to the formation of OH-ethyl-Et was the most efficient at eliminating the drug. On incubation with microsomes expressing individual recombinant CYPs, formation rates of M1-3 and M8 were 10-100-fold greater for CYP3A4 than that for other CYP forms. The formation of these metabolites was markedly inhibited by the CYP3A4-specific inhibitor ketoconazole, whereas other CYP-specific inhibitors did not show significant effects. In addition, the production of M1-3 and M8 was strongly correlated with CYP3A4-mediated testosterone 6beta-hydroxylase activities in 13 different human liver microsome samples. 4. Dealkylation of the major metabolite M1 to form mCPP (M8) was also investigated using microsomes containing recombinant CYP enzymes. The rate of conversion of M1 to mCPP by CYP3A4 was 503.0 +/- 3.1 pmole nmole(-1) min(-1). Metabolism of M1 to M8 by other CYP enzymes was insignificant. In addition, this metabolism in human liver microsomes was extensively inhibited by the CYP3A4 inhibitor ketoconazole, but not by other CYP-specific inhibitors. In addition, conversion of M1 to M8 was highly correlated with CYP3A4-mediated testosterone 6beta-hydroxylase activity. 5. The results strongly suggest that CYP3A4 is the predominant enzyme-metabolizing Et in humans.

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A small series of novel, imidazoles 4 have been prepared that exhibit very good binding affinities for the delta and mu opioid receptors (ORs), as well as demonstrate potent agonist functional activity at the delta OR. Representative imidazole 4a (K(i) delta = 0.9 nM; K(i) mu = 55 nM; K(i) kappa = 124 nM; EC(50) delta =13-25 nM) was further profiled for OR related in vivo effects. Compound 4a reduced gastrointestinal (GI) propulsive motility in a dose-dependent and naloxone-reversible manner, based on the results of the mouse glass bead expulsion test (3, 5, and 10 mg/kg, ip) and the mouse fecal pellet output test (1 and 3 mg/kg, ip). Compound 4a showed no analgesic activity as measured by the mouse abdominal irritant test (MAIT) when dosed at 100 mg/kg, sc, but did show significant MAIT activity at doses of both 10 microg (40% inhibition) and 100 microg (100% inhibition) when dosed intracerebroventricularly (icv). Taken together, these in vivo results suggest that 4a acts peripherally when dosed systemically, and that these prototypical compounds may prove promising as medicinal leads for GI indications.