Relationships between Endogenous Plasma Biomarkers of Constitutive Cytochrome P450 3A Activity and Single-Time-Point Oral Midazolam Microdose Phenotype in Healthy Subjects

Due to high basal interindividual variation in cytochrome P450 3A (CYP3A) activity and susceptibility to drug interactions, there has been interest in the application of efficient probe drug phenotyping strategies, as well as endogenous biomarkers for assessment of in vivo CYP3A activity. The biomarkers 4β-hydroxycholesterol (4βHC) and 6β-hydroxycortisol (6βHCL) are sensitive to CYP3A induction and inhibition. However, their utility for the assessment of constitutive CYP3A activity remains uncertain. We investigated whether endogenous plasma biomarkers (4βHC and 6βHCL) are associated with basal CYP3A metabolic activity in healthy subjects assessed by a convenient single-time-point oral midazolam (MDZ) phenotyping strategy. Plasma 4βHC and 6βHCL metabolic ratios (MRs) were analysed in 51 healthy adult participants. CYP3A activity was determined after administration of an oral MDZ microdose (100 μg). Simple linear and multiple linear regression analyses were performed to assess relationships between MDZ oral clearance, biomarkers and subject covariates. Among study subjects, basal MDZ oral clearance, 4βHC and 6βHCL MRs ranged 6.5-, 10- and 13-fold, respectively. Participant age and alcohol consumption were negatively associated with MDZ oral clearance (p = 0.03 and p = 0.045, respectively), while weight and female sex were associated with lower plasma 4βHC MR (p = 0.0003 and p = 0.032, respectively). Neither 4βHC nor 6βHCL MRs were associated with MDZ oral clearance. Plasma 4βHC and 6βHCL MRs do not relate to MDZ single-time-point metabolic phenotype in the assessment of constitutive CYP3A activity among healthy individuals

Clarifying the importance of CYP2C19 and PON1 in the mechanism of clopidogrel bioactivation and in vivo antiplatelet response

AimsIt is thought that clopidogrel bioactivation and antiplatelet response are related to cytochrome P450 2C19 (CYP2C19). However, a recent study challenged this notion by proposing CYP2C19 as wholly irrelevant, while identifying paraoxonase-1 (PON1) and its Q192R polymorphism as the major driver of clopidogrel bioactivation and efficacy. The aim of this study was to systematically elucidate the mechanism and relative contribution of PON1 in comparison to CYP2C19 to clopidogrel bioactivation and antiplatelet response.Methods and resultsFirst, the influence of CYP2C19 and PON1 polymorphisms and plasma paraoxonase activity on clopidogrel active metabolite (H4) levels and antiplatelet response was assessed in a cohort of healthy subjects (n = 21) after administration of a single 75 mg dose of clopidogrel. There was a remarkably good correlation between H4 AUC (0-8 h) and antiplatelet response (r2 = 0.78). Furthermore, CYP2C19 but not PON1 genotype was predictive of H4 levels and antiplatelet response. There was no correlation between plasma paraoxonase activity and H4 levels. Secondly, metabolic profiling of clopidogrel in vitro confirmed the role of CYP2C19 in bioactivating clopidogrel to H4. However, heterologous expression of PON1 in cell-based systems revealed that PON1 cannot generate H4, but mediates the formation of another thiol metabolite, termed Endo. Importantly, Endo plasma levels in humans are nearly 20-fold lower than H4 and was not associated with any antiplatelet response.ConclusionOur results demonstrate that PON1 does not mediate clopidogrel active metabolite formation or antiplatelet action, while CYP2C19 activity and genotype remains a predictor of clopidogrel pharmacokinetics and antiplatelet response. © 2012 The Author

Cytochrome P450 3A4 Expression and Regulation in Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is defined as lipid accumulation within hepatocytes (steatosis) in the absence of excess alcohol consumption. It is the most common liver disease in the western world, affecting one third of the general adult population with particularly high prevalence in obesity and type 2 diabetes. NAFLD is a disease continuum originating with simple hepatic steatosis that can progress to non-alcoholic steatohepatitis (NASH) with fibrosis and potentially cirrhosis, which places patients at risk for hepatocellular carcinoma. Unfortunately, there are not yet specific pharmacologic agents to treat NAFLD and so its management involves treatment of comorbidities, but there are significant efforts to develop new drugs to reverse disease course and prevent progression. While the liver is the major organ of drug elimination, little is known regarding the effect of NAFLD on hepatic drug metabolism in humans. The most important drug metabolizing enzyme is cytochrome P450 (CYP) 3A4, which metabolizes medications widely prescribed in NAFLD patients including those to treat hypertension, type 2 diabetes and dyslipidemia. We tested the hypothesis that CYP3A4 expression and activity are altered in NAFLD. In the first study, we evaluated the use of predictive endogenous plasma biomarkers of drug metabolic function against the gold-standard midazolam pharmacokinetic phenotyping approach for the assessment of constitutive CYP3A activity in healthy subjects. There was a lack of association between levels of the CYP3A derived metabolites 4β-hydroxycholesterol and 6β-hydroxycortisol with midazolam pharmacokinetics indicating that these biomarkers have limited utility within the context of relatively narrow enzyme activity variability in healthy individuals. CYP3A activity was next examined in patients with biopsy-proven NAFLD in comparison to healthy control subjects using midazolam pharmacokinetic phenotyping and biomarker approaches. We demonstrated, for the first time, that in vivo CYP3A activity is reduced in patients with simple steatosis or NASH and that fibrosis was also associated with lower enzyme function. Likewise, experimental NAFLD in mice and cultured hepatoma cells was associated with lower CYP3A4 expression. In the third study, mouse and cell models of NAFLD were used to define a novel regulatory pathway involved in the observed reduction of CYP3A4 expression and activity. We found that NAFLD causes hepatic upregulation of the metabolic hormone, fibroblast growth factor 21 (FGF21), stimulating a canonical cellular phospho-signaling pathway. FGF21 acted on the liver to decrease the nuclear localization and activity of the pregnane X receptor, a key transcriptional regulator of CYP3A4 gene expression. These findings provide novel insights to altered drug metabolism in NAFLD and a mechanistic basis for studies aimed to optimize pharmacotherapy and drug development for this common liver disease

A fibroblast growth factor 21-pregnane X receptor pathway downregulates hepatic CYP3A4 in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) alters drug response. We previously reported that NAFLD is associated with reduced in vivo CYP3A drug-metabolism activity and hepatic CYP3A4 expression in humans as well as mouse and human hepatoma models of the disease. Here, we investigated the role of the lipidand glucose-modulating hormone fibroblast growth factor 21 (FGF21) in the molecular mechanism regulating CYP3A4 expression in NAFLD. In human subjects, mouse and cellular NAFLD models with lower CYP3A4 expression, circulating FGF21, or hepatic FGF21 mRNA levels were elevated. Administration of recombinant FGF21 or transient hepatic overexpression of FGF21 resulted in reduced liver CYP3A4 luciferase reporter activity in mice and decreased CYP3A4 mRNA expression and activity in cultured Huh7 hepatoma cells. Blocking canonical FGF21 signaling by pharmacological inhibition of MEK1 kinase in Huh7 cells caused de-repression of CYP3A4 mRNA expression with FGF21 treatment. Mice with high-fat diet-induced simple hepatic steatosis and lipid-loaded Huh7 cells had reduced nuclear localization of the pregnane X receptor (PXR), a key transcriptional regulator of CYP3A4. Furthermore, decreased nuclear PXR was observed in mouse liver and Huh7 cells after FGF21 treatment or FGF21 overexpression. Decreased PXR binding to the CYP3A4 proximal promoter was found in FGF21-treated Huh7 cells. An FGF21-PXR signaling pathway may be involved in decreased hepatic CYP3A4 metabolic activity in NAFLD