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Large interindividual variability has been observed in the metabolism of CYP2C19 substrates in vivo. The study aimed to evaluate sources of this variability in CYP2C19 activity, focusing on CYP2C19 diplotypes and the cytochrome P450 oxidoreductase (POR). CYP2C19 gene analysis was carried out on 347 human liver samples. CYP2C19 activity assayed using human liver microsomes confirmed a significant a priori predicted rank order for (S)-mephenytoin hydroxylase activity of CYP2C19*17/*17 > *1B/*17 > *1B/*1B > *2A/*17 > *1B/*2A > *2A/*2A diplotypes. In a multivariate analysis, the CYP2C19*2A allele and POR protein content were associated with CYP2C19 activity. Further analysis indicated a strong effect of the CYP2C19*2A, but not the *17, allele on both metabolic steps in the conversion of clopidogrel to its active metabolite. The present study demonstrates that interindividual variability in CYP2C19 activity is due to differences in both CYP2C19 protein content associated with gene diplotypes and the POR concentration.The Pharmacogenomics Journal advance online publication, 1 September 2015; doi:10.1038/tpj.2015.58

It was previously shown that CYP3A4 is induced in the human intestinal Caco-2 cell model by treatment with 1alpha,25-dihydroxy vitamin D3 (1,25-D3). We demonstrate the vitamin D analog, 19-nor-1alpha,25-dihydroxy vitamin D2, is also an effective inducer of CYP3A4 in Caco-2 cells, but with half the potency of 1,25-D3. We report that treatment of LS180 cells, a human intestinal cell line, with 1 to 10 nM 1,25-D3 dose dependently increased CYP3A4 protein and CYP3A4 mRNA expression. CYP3A4- and CYP3A23-promoter-Luciferase reporter constructs transiently transfected into LS180 cells were transcriptionally activated in a dose-dependent manner by 1,25-D3, whereas mutation of the nuclear hormone receptor binding motif (ER6) in the CYP3A4 promoter abrogated 1,25-D3 activation of CYP3A4. Although the CYP3A4 ER6 promoter element has been shown to bind the pregnane X receptor (PXR), this receptor does not mediate 1,25-D3 induction of CYP3A4 because a) PXR is not expressed in Caco-2 cells; b) PXR mRNA expression is not induced by 1,25-D3 treatment of LS180 cells; and c) the ligand binding domain of human PXR was not activated by 1,25-D3. 1,25-D3 uses the vitamin D receptor to induce CYP3A4 because a) the vitamin D receptor (VDR)-retinoid X receptor (RXR) heterodimer binds specifically to the CYP3A4 ER6; b) selective mutation of the CYP3A4 ER6 disrupted the binding of VDR-RXR; and c) reporter constructs containing only three copies of the CYP3A4 ER6 linked to a TK-CAT reporter were activated by 1,25-D3 only in cells cotransfected with a human VDR expression plasmid. These data support the hypothesis that 1,25-D3 and VDR induce expression of intestinal CYP3A by binding of the activated VDR-RXR heterodimer to the CYP3A PXR response element and promoting gene transcription.

Under certain culture conditions, exposure of the human colon adenocarcinoma cell line Caco-2 to 1,25-(OH)(2)-D(3) induces expression of CYP3A4 to levels comparable to that in human small intestinal epithelium. To determine whether 1,25-(OH)(2)-D(3) could be used to restore CYP3A expression in other culture models, we examined several cell lines derived from malignancies of human tissues known to express CYP3A enzymes: Hep G2 (liver), LS180 (colon), HPAC (pancreas), Hs746T (stomach). Primary cultures of human hepatocytes from two donors were also examined. 1,25-(OH)(2)-D(3) increased CYP3A catalytic activity in LS180 (15-fold), HPAC (6-fold), and hepatocytes (2- to 3-fold); this was accompanied by induction of CYP3A4 mRNA and CYP3A immunoreactive protein. However, 1,25-(OH)(2)-D(3) had no effect on CYP3A expression in Hs746T or Hep G2. Known ligands for pregnane X receptor (PXR) (rifampin, dexamethasone, and dexamethasone t-butyl acetate) markedly induced CYP3A4 expression in human hepatocytes. In contrast, these ligands had little or no effect on CYP3A4 expression in Caco-2 cells, even at concentrations 1 to 2 orders of magnitude greater than effective concentrations of 1,25-(OH)(2)-D(3) or two other vitamin D receptor (VDR) ligands (25-OH-D(3) and 1-OH-D(3)). The retinoic acid receptor ligand all-trans-retinoic acid augmented the 1,25-(OH)(2)-D(3)-mediated induction of CYP3A4 catalytic activity up to 2-fold in Caco-2 cells, while having no demonstrable effect on levels of CYP3A4 mRNA or protein. The retinoid X receptor ligand 9-cis-retinoic acid appeared to slightly reduce CYP3A4 catalytic activity. We conclude that 1,25-(OH)(2)-D(3) can be used to increase CYP3A4 expression in some, but not all, human cell lines derived from tissues known to express CYP3A enzymes. The mechanisms involved in this induction are unlikely to involve PXR and may involve VDR.

Acetaminophen (APAP), a widely used analgesic and antipyretic agent, can cause acute hepatic necrosis in both humans and experimental animals when consumed in large doses. It is generally accepted that N-acetyl-p-benzoquinone imine (NAPQI) is the toxic, reactive intermediate whose formation from APAP is mediated by cytochrome P450. Several forms of P450 in humans, including 2E1, 1A2, 2A6, 3A4, have been shown to catalyze the oxidation of APAP to NAPQI. We now present evidence which demonstrates that human cytochrome P450 2D6 (CYP2D6) is also involved in the bioactivation of APAP. The formation of NAPQI from APAP by cDNA-expressed CYP2D6 was examined. K(m) and V(max) values were 1.76 mM and 3.02 nmol/min/nmol of P450, respectively, such that the efficiency of CYP2D6 in the conversion of APAP to NAPQI is approximately one-third of that of CYP2E1. The contribution of CYP2D6 to the total formation of NAPQI from APAP (1 mM) in human liver was investigated using quinidine (1 microm) as a CYP2D6-specific inhibitor, and varied from 4.5 to 22.4% among 10 livers, with an average at 12.6%. The correlation between the contribution of CYP2D6 to NAPQI formation in human liver microsomes and the CYP2D6 activity probed by the O-demethylation of dextromethorphan was studied, and found to be strong (r(2) = 0.85), and significant (P <.0001). Our findings indicate that CYP2D6, one of the major P450 isoforms in humans and also one of the pharmacogenetically important isoforms, may contribute significantly to the formation of the cytotoxic metabolite NAPQI, especially in CYP2D6 ultra-rapid and extensive metabolizers and at toxic doses of APAP when plasma APAP concentrations reach 2 mM or more.

To determine the level of FMO1 protein present in human liver tissues, a monospecific antibody was prepared and a sensitive Western blotting procedure with enhanced chemiluminescence detection was developed. Human FMO1, purified from insect cells expressing the recombinant protein, was used as a protein standard for absolute quantification. The average concentrations of FMO1 in microsomes prepared from human liver, kidney, intestine, and fetal liver were found to be <1, 47 +/- 9, 2.9 +/- 1.9, and 14.4 +/- 3.5 pmol/mg, respectively. Quantitation in intestinal microsomes was complicated by variable degrees of proteolytic degradation of FMO1, not seen in microsomes prepared from liver or kidney. Recombinant human FMO1 and detergent-solubilized human duodenal microsomes both metabolized p-tolyl methyl sulfide stereoselectively to the (R)-sulfoxide, indicating the expression of functional FMO1 in human intestine. The relatively high levels of immunoquantifiable FMO1 in human kidney and fetal liver complement our previous catalytic studies in these tissues, which also demonstrated preferential (R)-p-tolyl methyl sulfoxide formation. These data demonstrate a profound ontogenic change in expression of hepatic FMO1 in humans, such that in adult life FMO1 is exclusively an extrahepatic drug-metabolizing enzyme. The marked expression levels of FMO1 found in human kidney coupled to the high catalytic activity of this isoform toward a diverse array of sulfides and tertiary amines suggest the possibility that human renal FMO1 is a significant contributor to the metabolic clearance of drugs and other xenobiotics bearing these functionalities.

Disulfiram (DSF) is a mechanism-based inhibitor of cytochrome P-450 2E1 (CYP2E1), resulting in loss of CYP2E1 protein and activity, which may be useful in preventing CYP2E1-mediated xenobiotic toxicity. The duration of inhibition after a single DSF dose is, however, unknown. The purpose of this investigation was to determine this duration, and CYP2E1 formation and degradation rates, in humans. Oral chlorzoxazone (CLZ) was used as the selective in vivo probe for CYP2E1. Healthy subjects received CLZ to determine baseline CYP2E1 activity (CLZ plasma clearance and 6-hydroxychlorzoxazone fractional metabolic clearance). One week later, DSF (500 mg orally) was administered at bedtime, and CLZ administered the following morning and 3, 6, 8, 10, and 13 days after DSF. A terminal DSF metabolite, 2-thiothiazolidine-4 carboxylic acid, was also measured in each 24-h urine sample. The mean CLZ clearance and 6-hydroxychlorzoxazone fractional metabolic clearance on the first day declined to 10.2 and 5.5% of baseline values, indicating rapid and profound CYP2E1 inhibition. CYP2E1 activity returned to half that of control on day 3, and to baseline values on day 8. Assuming zero-order synthesis and first-order degradation, the in vivo CYP2E1 synthesis rate and degradation half-life was estimated to be 11 +/- 5 nmol/h and 50 +/- 19 h, respectively. Significant amounts of 2-thiothiazolidine-4 carboxylic acid were present only on day 1, suggesting that the return of in vivo CYP2E1 activity was not caused by inhibitor washout, but by enzyme resynthesis. Results regarding CYP2E1 disposition may be useful for modeling the effects of CYP2E1 inducers and inhibitors. For prevention of CYP2E1-mediated bioactivation, depending on protoxicant disposition, a second DSF dose might be necessary to completely prevent toxicity.

It has been suggested that the binding of a drug to plasma proteins will influence the intestinal extraction efficiency when drug is delivered to the mucosal epithelium via either the gut lumen or vasculature. We evaluated this hypothesis using cytochrome P-450 (CYP)3A4-expressing Caco-2 monolayers as a model for the intestinal epithelial barrier and midazolam as a CYP3A-specific enzyme probe. The rate of 1'-hydroxylation was measured following apical or basolateral midazolam administration to monolayers incubated in the presence or absence of 4 g/dl of human serum albumin (HSA) in the basolateral compartment medium. The midazolam-free fraction in culture medium containing HSA was 3.3%. Inclusion of HSA in the basolateral medium decreased peak intracellular midazolam accumulation after an apical midazolam dose (3 microM) by 35% and reduced the 1'-hydroxymidazolam formation rate by approximately 20%. Because of the accelerated diffusion of midazolam through the cell monolayer and into the basolateral compartment, there was a 61% reduction in the first-pass metabolic extraction ratio: 13.3 +/- 0. 12% for control versus 5.2 +/- 1% with HSA. Compared with control, addition of HSA resulted in a 91% decrease in the peak intracellular midazolam level and a 86% decrease in the rate of 1'-hydroxylation after the administration of midazolam into basolateral medium. These findings suggest that, in vivo, binding of a drug to plasma proteins will impact both first-pass and systemic intestinal midazolam extraction efficiency. Furthermore, the effect will be more pronounced for a drug that is delivered to mucosal enterocytes by way of arterial blood, compared with oral drug delivery.

Cytochrome P-450 (CYP) 3A4 accounts for approximately 50% of all P-450s found in the small intestine (Paine et al., 1997) and contributes to the extensive and variable first-pass extraction of drugs such as cyclosporine and saquinavir. We recently demonstrated that CYP3A4 expression in a differentiated Caco-2 subclone is increased when cell monolayers are treated with 1alpha,25-dihydroxy-vitamin-D3 (Schmiedlin-Ren et al., 1997). This improved metabolic capacity permits the in vitro modeling of first-pass intestinal metabolic kinetics. Midazolam (MDZ) 1'-hydroxylation was used as a specific probe for CYP3A-mediated metabolism in modified Caco-2 monolayers. Caco-2 cells were grown to confluence on laminin-coated culture inserts, and then for two additional weeks in the presence of 1alpha,25-dihydroxy vitamin-D3. Cell monolayers were subsequently exposed to MDZ for varying lengths of time and concentrations. The amount of MDZ in the monolayer increased rapidly after apical drug administration, reaching a pseudo steady state within 6 min. The cellular uptake rate was considerably slower after a basolateral dose. By either route of administration, the rate of 1'-hydroxymidazolam formation was stable and linear for 2 h. Under basolateral sink conditions and low apical MDZ dosing concentration (1-8 microM), the first-pass extraction ratio was found to be approximately 15%. Higher dosing concentrations led to saturation of the hydroxylation reaction and reduction in the extraction ratio. The modified Caco-2 cell monolayer is an excellent model for studying drug absorption and first-pass intestinal metabolic kinetic processes. In this system, the selective CYP3A probe MDZ was rapidly absorbed, yet extensively metabolized, as is observed in vivo.

Mexiletine, lidocaine, and tocainide are class IB antiarrhythmic drugs that are used for the treatment of ventricular arrhythmias and are known to inhibit drug metabolism. The objectives of this study were to characterize the inhibitory effects of mexiletine, lidocaine, and tocainide on cytochrome P-450 1A2 (CYP1A2) activity in human liver microsomes and to evaluate their relative inhibitory potencies by using a molecular model of this P-450 isozyme. The inhibitory effect of mexiletine, lidocaine, and tocainide on cytochrome CYP1A2 in human liver microsomes was examined with methoxyresorufin O-demethylase activity as an index of the catalytic activity of this P-450 isozyme. The kinetic inhibition types and Ki values were determined by Lineweaver-Burk plots and Dixon plots, respectively. Molecular modeling was used to assess the interaction of these agents with the CYP1A2 active site. Methoxyresorufin O-demethylase activity was inhibited 67 +/- 8%, 20 +/- 5%, and 7 +/- 4% by 2 mM mexiletine, lidocaine, and tocainide, respectively. Mexiletine and lidocaine exhibited competitive inhibition with Ki values of 0.28 +/- 0.12 mM and 1.54 +/- 0.74 mM, respectively, whereas the inhibition type of tocainide could not be determined because of its weak potency. A charge interaction between mexiletine and the Asp313 side chain in the CYP1A2 active site was found, and varying degrees of hydrogen bond formation between these three compounds and the CYP1A2 active site were observed. The in vitro inhibitory potencies in human liver microsomes (mexiletine > lidocaine > tocainide) are consistent with the structural interactions found in a molecular model of the active site of CYP1A2.

The purpose of this work was to evaluate the effect of mutual unbound inhibitor and unbound enzyme depletion on the potency of three antifungal cytochrome P-450 (CYP)3A inhibitors with over 1000-fold range in enzyme affinity. Incubations were performed with human liver microsomal protein concentrations that varied from 25 to 1000 microg/ml. The effect of each inhibitor was evaluated using midazolam as a CYP3A probe. Clotrimazole was found to be a tight binding inhibitor of CYP3A with a Ki of 250 pM. Analysis of percent inhibition data by stepwise linear regression for the matrix of inhibitor and enzyme concentrations used showed that protein concentrations predicted the percent inhibition by clotrimazole (r2 = 0.60, p <.001). When clotrimazole concentrations were added to the model, the r2 improved to 0.81, p =.003. Clotrimazole concentrations alone were not a significant predictor of percent inhibition (r2 = 0. 21, p =.08). For ketoconazole, protein concentrations provided a weak prediction of the percent inhibition (r2 = 0.39, p =.006). Conversely, ketoconazole concentrations alone were a good predictor of percent inhibition (r2 = 0.55, p <.001). In contrast to results with clotrimazole and ketoconazole, percent inhibition by fluconazole was not dependent on protein concentrations (r2 = 0.06, p =.39). We conclude that microsomal inhibitory potency can be affected by incubation conditions that deplete the unbound concentration of inhibitor available to the enzyme. This may introduce serious error into a quantitative prediction of an in vivo drug-drug interaction based on an in vitro derived Ki value.