Diclofenac is eliminated predominantly (ϳ50%) as its 4Ј-hydroxylated metabolite in humans, whereas the acyl glucuronide (AG) pathway appears more important in rats (ϳ50%) and dogs (Ͼ80 -90%). However, previous studies of diclofenac oxidative metabolism in human liver microsomes (HLMs) have yielded pronounced underprediction of human in vivo clearance. We determined the relative quantitative importance of 4Ј-hydroxy and AG pathways of diclofenac metabolism in rat, dog, and human liver microsomes. Microsomal intrinsic clearance values (CL int ϭ V max /K m ) were determined and used to extrapolate the in vivo blood clearance of diclofenac in these species. Clearance of diclofenac was accurately predicted from microsomal data only when both the AG and the 4Ј-hydroxy pathways were considered. However, the fact that the AG pathway in HLMs accounted for ϳ75% of the estimated hepatic CL int of diclofenac is apparently inconsistent with the 4Ј-hydroxy diclofenac excretion data in humans. Interestingly, upon incubation with HLMs, significant oxidative metabolism of diclofenac AG, directly to 4Ј-hydroxy diclofenac AG, was observed. The estimated hepatic CL int of this pathway suggested that a significant fraction of the intrahepatically formed diclofenac AG may be converted to its 4Ј-hydroxy derivative in vivo. Further experiments indicated that this novel oxidative reaction was catalyzed by CYP2C8, as opposed to CYP2C9-catalyzed 4Ј-hydroxylation of diclofenac. These findings may have general implications in the use of total (free ϩ conjugated) oxidative metabolite excretion for determining primary routes of drug clearance and may question the utility of diclofenac as a probe for phenotyping human CYP2C9 activity in vivo via measurement of its pharmacokinetics and total 4Ј-hydroxy diclofenac urinary excretion.In vitro drug metabolism systems, especially liver microsomes, offer tremendous promise as a tool in drug discovery and development to make human pharmacokinetic projections for potential drug candidates (Obach et al., 1997;Obach, 1999). These systems allow for lead selection based on metabolism data in human tissue that seem more relevant to the human in vivo situation than the in vivo animal models. The popularity of liver microsomes, in comparison with other in vitro systems such as hepatocytes and liver slices, stems from the ease of their preparation, use, and long-term storage and viability. However, the use of liver microsomes for extrapolation of in vivo clearance suffers from a number of limitations such as nonspecific binding of compounds to microsomal components, reduced rates of metabolism because of potential product inhibition kinetics, and the difficulties in examining conjugative metabolism (e.g., glucuronidation) in microsomal incubations. These limitations lead to frequent underprediction of in vivo clearance from microsomal metabolism data (Houston and Carlile, 1997;Obach, 1999). There have been only a few attempts to extrapolate in vivo clearance from microsomal metabolism data for compound...

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Recently, it was shown that diclofenac was metabolized in rats to reactive benzoquinone imines via cytochrome P450-catalyzed oxidation. These metabolites also were detected in human hepatocyte cultures in the form of glutathione (GSH) adducts. This report describes the results of further studies aimed at characterizing the human hepatic P450-mediated bioactivation of diclofenac. The reactive metabolites formed in vitro were trapped by GSH and analyzed by LC/MS/MS. Thus, three GSH adducts, namely, 5-hydroxy-4-(glutathion-S-yl)diclofenac (M1), 4'-hydroxy-3'-(glutathion-S-yl)diclofenac (M2), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (M3), were identified in incubations of diclofenac with human liver microsomes in the presence of NADPH and GSH. The formation of the adducts was taken to reflect the intermediacy of the corresponding putative benzoquinone imines. While M2 was the dominant metabolite over a substrate concentration range of 10-50 microM, M1 and M3 became equally important products at >/=100 microM diclofenac. The formation of M2 was inhibited by sulfaphenazole or an anti-P450 2C9 antibody (5-10% of control values). The formation of M1 and M3 was inhibited by troleandomycin, ketoconazole, or an anti-P450 3A4 antibody (30-50% of control values). In studies in which recombinant P450 isoforms were used, M2 was generated only by P450 2C9-catalyzed reaction, while M1 and M3 were produced by P450 3A4-catalyzed reaction. Good correlations were established between the extent of formation of M2 and P450 2C9 activities (r = 0.93, n = 10) and between the extent of formation of M1 and M3 and P450 3A4 activities (r = 0.98, n = 10) in human liver microsomal incubations. Taken together, the data suggest that the biotransformation of diclofenac to M2 is P450 2C9-dependent, whereas metabolism of the drug to M1 and M3 involves mainly P450 3A4. Although P450s 2C9 and 3A4 both catalyze the bioactivation of diclofenac, P450 2C9 is capable of producing the benzoquinone imine intermediate at lower drug concentrations which may be more clinically relevant.

Activation of ␤ 3 adrenergic receptors on the surface of adipocytes leads to increases in intracellular cAMP and stimulation of lipolysis. In brown adipose tissue, this serves to upregulate and activate the mitochondrial uncoupling protein 1, which mediates a proton conductance pathway that uncouples oxidative phosphorylation, leading to a net increase in energy expenditure. While chronic treatment with ␤ 3 agonists in nonprimate species leads to uncoupling protein 1 up-regulation and weight loss, the relevance of this mechanism to energy metabolism in primates, which have much lower levels of brown adipose tissue, has been questioned. With the discovery of L-755,507, a potent and selective partial agonist for both human and rhesus ␤ 3 receptors, we now demonstrate that acute exposure of rhesus monkeys to a ␤ 3 agonist elicits lipolysis and metabolic rate elevation, and that chronic exposure increases uncoupling protein 1 expression in rhesus brown adipose tissue. These data suggest a role for ␤ 3 agonists in the treatment of human obesity.

The regioselective dibenzylphosphorylation of 2 followed by catalytic reduction in the presence of N-methyl-D-glucamine afforded 2-(S)-(1-(R)-(3, 5-bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluoro)phenyl-4-(5-(2- phosphoryl-3-oxo-4H,-1,2,4-triazolo)methylmorpholine, bis(N-methyl-D-glucamine) salt, 11. Incubation of 11 in rat, dog, and human plasma and in human hepatic subcellular fractions in vitro indicated that conversion to 2 would be expected to occur in vivo most readily in humans during hepatic circulation. Conversion of 11 to 2 occurred rapidly in vivo in the rat and dog with the levels of 11 being undetectable within 5 min after 1 and 8 mg/kg doses iv in the rat and within 15 min after 0.5, 2, and 32 mg/kg doses iv in the dog. Compound 11 has a 10-fold lower affinity for the human NK-1 receptor as compared to 2, but it is functionally equivalent to 2 in preclinical models of NK-1-mediated inflammation in the guinea pig and cisplatin-induced emesis in the ferret, indicating that 11 acts as a prodrug of 2. Based in part on these data, 11 was identified as a novel, water-soluble prodrug of the clinical candidate 2 suitable for intravenous administration in humans.