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...

Cleft lip and cleft palate (CLP) are common disorders that occur either as part of a syndrome, where structures other than the lip and palate are affected, or in the absence of other anomalies. Van der Woude syndrome (VWS) and popliteal pterygium syndrome (PPS) are autosomal dominant disorders characterized by combinations of cleft lip, CLP, lip pits, skin-folds, syndactyly and oral adhesions which arise as the result of mutations in interferon regulatory factor 6 (IRF6). IRF6 belongs to a family of transcription factors that share a highly conserved N-terminal, DNA-binding domain and a less well-conserved protein-binding domain. To date, mutation analyses have suggested a broad genotype–phenotype correlation in which missense and nonsense mutations occurring throughout IRF6 may cause VWS; in contrast, PPS-causing mutations are highly associated with the DNA-binding domain, and appear to preferentially affect residues that are predicted to interact directly with the DNA. Nevertheless, this genotype–phenotype correlation is based on the analysis of structural models rather than on the investigation of the DNA-binding properties of IRF6. Moreover, the effects of mutations in the protein interaction domain have not been analysed. In the current investigation, we have determined the sequence to which IRF6 binds and used this sequence to analyse the effect of VWS- and PPS-associated mutations in the DNA-binding domain of IRF6. In addition, we have demonstrated that IRF6 functions as a co-operative transcriptional activator and that mutations in the protein interaction domain of IRF6 disrupt this activity.

This article presents the features and structures of protein crystals naturally grown in vivo within developing embryos of the only known viviparous cockroach, D. punctata. This study reveals the heterogeneous nature of the crystalline protein with respect to amino-acid sequence, glycosylation and bound fatty acid at atomic resolution.