ABSTRACT:Glucuronidation is a listed clearance mechanism for 1 in 10 of the top 200 prescribed drugs. The objective of this article is to encourage those studying ligand interactions with UDP-glucuronosyltransferases (UGTs) to adequately consider the potential consequences of in vitro UGT inhibition in humans. Spurred on by interest in developing potent and selective inhibitors for improved confidence around UGT reaction phenotyping, and the increased availability of recombinant forms of human UGTs, several recent studies have reported in vitro inhibition of UGT enzymes. In some cases, the observed potency of UGT inhibitors in vitro has been interpreted as having potential relevance in humans via pharmacokinetic drug-drug interactions. Although there are reported examples of clinically relevant drug-drug interactions for UGT substrates, exposure increases of the aglycone are rarely greater than 100% in the presence of an inhibitor relative to its absence (i.e., AUC i /AUC <2). This small magnitude in change is in contrast to drugs primarily cleared by cytochrome P450 enzymes, where exposures have been reported to increase as much as 35-fold on coadministration with an inhibitor (e.g., ketoconazole inhibition of CYP3A4-catalyzed terfenadine metabolism). In this article the evidence for purported clinical relevance of potent in vitro inhibition of UGT enzymes will be assessed, taking the following into account: in vitro data on the enzymology of glucuronide formation from aglycone, pharmacokinetic principles based on empirical data for inhibition of metabolism, and clinical data on the pharmacokinetic drug-drug interactions of drugs primarily cleared by glucuronidation.

1 Several selective 5-HT reuptake inhibitors (SSRIs) are inhibitors of the genetically polymorphic drug metabolizing enzyme, CYP2D6. We studied the interaction of venlafaxine, a new SSRI, with CYP2D6 in human liver microsomes. 2 Venlafaxine was a less potent inhibitor of this enzyme activity in vitro than other SSRIs tested. The average apparent K i values determined using CY P2D6-dependent dextromethorphan 0-demethylation were: 33, 52 and 22 PM for rac-venlafaxine, R( +)-venlafaxine and S (-)-venlafaxine, respectively, us 0.065 to 1.8 p~ for paroxetine, fluoxetine, norfluoxetine, fluvoxamine and sertraline. 3 Microsomes from human livers ( n = 3 ) and from yeast transformed with an expression plasmid containing human CYP2D6 cDNA catalyzed the 0-demethylation of venlafaxine, which is the major metabolic pathway in uiuo. Intrinsic metabolic clearance values ( Vmax/Km) indicated that S( -)-venlafaxine was cleared preferentially via this pathway. 4 In microsomes from CYP2D6-deficient livers (n = 2), Vmax/Km of O-demethylation of venlafaxine was one to two orders of magnitude lower and was similar to the rate of N-demethylation. 5 Studies with chemical probes which preferentially inhibit P450 isoforms suggested that CYP3A3/4 is involved in venlafaxine N-demethylation. 6 These in vitro findings predict phenotypic differences in the kinetics of venlafaxine in vivo, although the clinical importance of this is unclear as 0-demethylvenlafaxine is pharmacologically similar to the parent drug. The findings also predict relatively limited pharmacokinetic interaction between venlafaxine and other CYP2D6 substrates.

This promotor region polymorphism does not appear to play a major role in determining constitutive CYP3A4 expression.

Cytochrome P450s play a central role in the metabolism and disposition of an extremely wide range of drugs and chemical carcinogens. Individual differences in the expression of these enzymes may be an important determinant in susceptibility to adverse drug reactions, chemical toxins and mutagens. In this paper, we have measured the relative levels of expression of cytochrome P450 isoenzymes from eight gene families or subfamilies in a panel of twelve human liver samples in order to determine the individuality in their expression and whether any forms are co-regulated. Isoenzymes were identified in most cases on Western blots based on the mobility of authentic recombinant human cytochrome P450 standards. The levels of the following P450 proteins correlated with each other: CYP2A6, CYP2B6 and a protein from the CYP2C gene subfamily, CYP2E1 and a member of the CYP2A gene subfamily, CYP2C8, CYP3A3/A4 and total cytochrome P450 content. Also, the levels of two proteins in the CYP4A gene subfamily were highly correlated. These correlations are consistent with the relative regulation of members of these gene families in rats or mice. In addition, the level of expression of specific isoenzymes has also been compared with the rate of metabolism of a panel of drugs, carcinogens and model P450 substrates. These latter studies demonstrate and confirm that the correlations obtained in this manner represent a powerful approach towards the assignment of the metabolism of substrates by specific human P450 isoenzymes.

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Aims In order to anticipate drug-interactions of potential clinical significance the ability of the novel antidepressant, venlafaxine, to inhibit CYP2D6 dependent imipramine and desipramine 2-hydroxylation was investigated in human liver microsomes. The data obtained were compared with the selective serotonin re-uptake inhibitors, fluoxetine, sertraline, fluvoxamine and paroxetine. Venlafaxine's potential to inhibit several other major P450s was also studied (CYP3A4, CYP2D6, CYP1A2). Methods K i values for venlafaxine, paroxetine, fluoxetine, fluvoxamine and sertraline as inhibitors of imipramine and desipramine 2-hydroxylation were determined from Dixon plots of control and inhibited rate data in human hepatic microsomal incubations. The inhibitory effect of imipramine and desipramine on liver microsomal CYP2D6 dependent venlafaxine O-demethylation was determined similarly. Venlafaxine's IC 50 values for CYP3A4, CYP1A2 CYP2C9 were determined based on inhibition of probe substrate activities (testosterone 6b-hydroxylation, ethoxyresorufin O-dealkylase and tolbutamide 4-hydroxylation, respectively). Results Fluoxetine, paroxetine, and fluvoxamine were potent inhibitors of imipramine 2-hydroxylase activity (K i values of 1.6±0.8, 3.2±0.8 and 8.0±4.3 mm, respectively; mean±s.d., n=3), while sertraline was less inhibitory (K i of 24.7±8.9 mm). Fluoxetine also markedly inhibited desipramine 2-hydroxylation with a K i of 1.3±0.5 mm. Venlafaxine was less potent an inhibitor of imipramine 2-hydroxylation (K i of 41.0±9.5 mm) than the SSRIs that were studied. Imipramine and desipramine gave marked inhibition of CYP2D6 dependent venlafaxine O-demethylase activity (K i values of 3.9±1.7 and 1.7±0.9 mm, respectively). Venlafaxine did not inhibit ethoxyresorufin O-dealkylase (CYP1A2), tolbutamide 4-hydroxylase (CYP2C9) or testosterone 6b-hydroxylase (CYP3A4) activities at concentrations of up to 1 mm. Conclusions It is concluded that venlafaxine has a low potential to inhibit the metabolism of substrates for CYP2D6 such as imipramine and desipramine compared with several of the most widely used SSRIs, as well as the metabolism of substrates for several of the other major human hepatic P450s.

For the pharmaceutical industry, one of the challenges in evaluating the risk of future compound attrition at the discovery stage is the successful prediction of the major routes of clearance in humans. For compounds cleared by metabolism, such information will help to avoid the development of compounds that will exhibit large interpatient differences in pharmacokinetics via 1). routes of metabolism catalyzed by functionally polymorphic enzymes and/or 2). clinically significant metabolic drug-drug interactions, in the later stages of development. The degree of intersubject variability that is acceptable for a drug candidate is uncertain in the discovery stage where knowledge of other important factors is limited or unavailable (i.e. therapeutic index, pharmacodynamic variability, etc). Reaction phenotyping is the semi-quantitative in vitro estimation of the relative contributions of specific drug-metabolizing enzymes to the metabolism of a test compound. However, reaction phenotyping in the discovery stage of drug development is complicated by the absence of radiolabelled parent compound or metabolite bioanalytical standards relative to later stages of development. In this commentary, some of the approaches, based on published data, which can be taken to overcome these challenges are discussed. In addition, knowledge of the molecular structure (i.e. specific chemical substituents), physicochemical properties, and routes of clearance in animals can all help in making a successful prediction for the routes of clearance in humans. In combination, the objective of these studies should be to reduce to a minimum the risk of finding significant inter-patient differences in pharmacokinetics at a later stage in development due to significant metabolism by polymorphic enzymes or drug-drug interactions. Consequently, this data should be used to avoid costly late stage attrition.

The study of the method in the determination of the isoleucine/leucine ratio in-the leucine fraction from the hydrolysates of casein, insulin and edestin gave results which agree with those obtained by microbiological methods except in the case of edestin where there is a very large discrepancy' We are grateful to. Prof. A. C. Chibnall, at whose instigatien this investigation was undertaken, for his interest and helpfil advice. We are indebted to the Medical Research Council (S.E.D.) and to the Agricultural Research Council (G.R.T.) for grants during the tenures of which this investigation was undertaken.