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1.
Pharmacogenomics J ; 16(4): 375-87, 2016 08.
Article in English | MEDLINE | ID: mdl-26323597

ABSTRACT

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.


Subject(s)
Cytochrome P-450 CYP2C19/genetics , Cytochrome P-450 CYP2C19/metabolism , Cytochrome P-450 Enzyme System/metabolism , Liver/enzymology , Mephenytoin/metabolism , Pharmacogenomic Variants/genetics , Ticlopidine/analogs & derivatives , Activation, Metabolic , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Clopidogrel , Female , Gene Expression Regulation, Enzymologic , Gene Frequency , Genotype , Humans , Hydroxylation , Infant , Infant, Newborn , Kinetics , Linear Models , Male , Microsomes, Liver/enzymology , Middle Aged , Multivariate Analysis , Oxidation-Reduction , Phenotype , Substrate Specificity , Ticlopidine/metabolism , Young Adult
2.
Clin Pharmacol Ther ; 92(6): 737-45, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23073208

ABSTRACT

We evaluated the hypothesis that cytochrome P450 3A5 (CYP3A5) expression can affect intrarenal tacrolimus accumulation. Tacrolimus was administered orally to 24 healthy volunteers who were selected on the basis of their CYP3A5 genotype. As compared with CYP3A5 nonexpressors, expressors had a 1.6-fold higher oral tacrolimus clearance and 2.0- to 2.7-fold higher metabolite/parent area under the curve (AUC) ratios for 31-desmethyl tacrolimus (31-DMT), 12-hydroxy tacrolimus, and 13-desmethyl tacrolimus (13-DMT). In addition, the apparent urinary tacrolimus clearance was 36% lower in CYP3A5 expressors as compared with nonexpressors. To explore the mechanism behind this observation, we developed a semiphysiological model of renal tacrolimus disposition and predicted that tacrolimus exposure in the renal epithelium of CYP3A5 expressors is 53% of that for CYP3A5 nonexpressors, when normalized to blood AUC. These data suggest that, at steady state, intrarenal accumulation of tacrolimus and its primary metabolites will depend on the CYP3A5 genotype of the liver and kidneys. This may contribute to interpatient differences in the risk of tacrolimus-induced nephrotoxicity.


Subject(s)
Cytochrome P-450 CYP3A/genetics , Immunosuppressive Agents/pharmacokinetics , Tacrolimus/pharmacokinetics , Adult , Algorithms , Area Under Curve , Biotransformation , DNA/genetics , Female , Genetic Variation , Genotype , Half-Life , Heterozygote , Humans , Immunosuppressive Agents/urine , Kidney/metabolism , Male , Models, Genetic , Polymerase Chain Reaction , Polymorphism, Single Nucleotide , Tacrolimus/urine
3.
J Pharmacol Exp Ther ; 289(2): 1134-42, 1999 May.
Article in English | MEDLINE | ID: mdl-10215697

ABSTRACT

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.


Subject(s)
Calcitriol/pharmacology , Midazolam/metabolism , Biotransformation , Blood Proteins/metabolism , Blotting, Western , Caco-2 Cells , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/metabolism , Electric Conductivity , Extracellular Space/metabolism , Humans , Kinetics , Midazolam/analogs & derivatives , Midazolam/blood , Midazolam/pharmacokinetics , Mixed Function Oxygenases/metabolism , Permeability , Reproducibility of Results
4.
J Pharmacol Exp Ther ; 289(2): 1143-50, 1999 May.
Article in English | MEDLINE | ID: mdl-10215698

ABSTRACT

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.


Subject(s)
Extracellular Matrix Proteins/metabolism , Midazolam/metabolism , Algorithms , Caco-2 Cells , Electric Conductivity , Extracellular Space/metabolism , Humans , Hydroxylation , Midazolam/analogs & derivatives , Midazolam/pharmacokinetics , Protein Binding , Serum Albumin/metabolism
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