Extrapolation of Diclofenac Clearance from in Vitro Microsomal Metabolism Data: Role of Acyl Glucuronidation and Sequential Oxidative Metabolism of the Acyl Glucuronide

Abstract

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...

In Vitro Metabolism of 2-[6-(4-Chlorophenyl)-2,2-dimethyl-7-phenyl-2,3-dihydro-1H-pyrrolizin-5-yl] Acetic Acid (Licofelone, ML3000), an Inhibitor of Cyclooxygenase-1 and -2 and 5-Lipoxygenase

“…CYP2C8, which was identified as the only P450 isoform involved in M1 hydroxylation, was previously reported as the enzyme responsible for hydroxylation of diclofenac acyl glucuronide (Kumar et al, 2002) and estradiol-17␤-glucuronide (Delaforge et al, 2005). Despite the similarity of the elimination pathway of diclofenac and licofelone, the results published by Kumar et al (2002) show an important difference between these two drugs.…”

“…Despite the similarity of the elimination pathway of diclofenac and licofelone, the results published by Kumar et al (2002) show an important difference between these two drugs. In incubations with microsomes, a rapid degradation of diclofenac acyl glucuronide was observed, irrespective of the presence or absence of NADPH.…”

“…In the present article, results from in vitro metabolism studies are presented that show that in humans hydroxylation of the glucuronide M1 represents the pivotal step in the biosynthesis of M2. Although the cytochrome P450 (P450)-dependent hydroxylation of glucuronides has been described in the literature (Kumar et al, 2002;Delaforge et al, 2005), the formation of M2 represents a unique example as the systemic exposure of humans to this major metabolite is based on the glucuronidation of the parent drug followed by hydroxylation of the glucuronide.…”

Prediction of Drug Clearance by Glucuronidation from in Vitro Data: Use of Combined Cytochrome P450 and UDP-Glucuronosyltransferase Cofactors in Alamethicin-Activated Human Liver Microsomes

“…UGTs are primarily involved in conjugation of metabolites from oxidation reactions. However, if a suitable electrophilic acceptor group is present, UGTs can also conjugate drugs directly without any prior oxidation step, as seen in the case of buprenorphine (Picard et al, 2005) and diclofenac (Kumar et al, 2002).…”

Plasma Stability-Dependent Circulation of Acyl Glucuronide Metabolites in Humans: How Circulating Metabolite Profiles of Muraglitazar and Peliglitazar Can Lead to Misleading Risk Assessment

“…1-O-␤-Acyl glucuronides, which are formed by UDP-glucuronosyltransferase (UGT) enzymes as the original isomer, can undergo a number of reactions including hydrolysis, rearrangement via acyl migration (Stachulski et al, 2006;Zhang et al, 2006;Xue et al, 2008), further metabolism (Kumar et al, 2002;Kochansky et al, 2005;Ogilvie et al, 2006), and reaction with nucleophiles (Sallustio and Foster, 1995;Akira et al, 2002;Bailey and Dickinson, 2003). The chemical reactivity of acyl glucuronides proceeds via two distinct pathways (Bailey and Dickinson, 2003;Stachulski et al, 2006;Corcoran et al, 2001): 1) direct displacement of the acyl residue with a nucleophile to produce an aglycon (hydrolysis product) and an acylated nucleophile; and 2) alternatively, migration of the acyl group around the sugar ring to yield 2-, 3-, and 4-acyl isomers.…”