CYP3A4-mediated Metabolism Research Articles

4β-Hydroxycholesterol as an Endogenous Biomarker for CYP3A Activity: Literature Review and Critical Evaluation.

A number of cytochrome P450 (CYP)3A phenotyping probes have been used to characterize the drug interaction potential of new molecular entities; of these, midazolam has emerged as the gold standard. Recently, plasma 4β-hydroxycholesterol (4β-OHC), the metabolite of CYP3A-mediated cholesterol metabolism, has been championed as an endogenous biomarker for CYP3A, particularly during chronic conditions where CYP3A activity is altered by disease and in long-term treatment studies where midazolam administration is not optimal. Multiple studies in humans have shown that 4β-OHC can qualitatively differentiate among weak, moderate, and potent CYP3A induction when an inducer, typically rifampin, is administered for up to 2weeks. Conversely, longer durations of CYP3A inhibitor administration (≥1month) appear to be necessary to differentiate among weak, moderate, and potent CYP3A inhibitors. A number of studies have reported statistically significant linear relationships between 4β-OHC plasma concentrations (and 4β-OHC:cholesterol ratios) and midazolam clearance. However, sufficiently powered studies assessing the ability of 4β-OHC or 4β-OHC:cholesterol ratios to measure CYP3A activity (ie, predictive performance) have not been conducted to date. Additional limitations associated with 4β-OHC phenotyping include inability to detect acute changes in CYP3A activity, uncertainty with regard to its intestinal formation, ambiguity surrounding the role of CYP3A5 in its metabolism, and lack of clarity regarding the role of transporters in its disposition. As such, the data do not support the use of 4β-OHC or 4β-OHC:cholesterol ratios as an endogenous biomarker for CYP3A activity.

The metabolism and hepatotoxicity of ginkgolic acid (17 : 1) in vitro

Pharmacological activities and adverse side effects of ginkgolic acids (GAs), major components in extracts from the leaves and seed coats of Ginkgo biloba L, have been intensively studied. However, there are few reports on their hepatotoxicity. In the present study, the metabolism and hepatotoxicity of GA (17 : 1), one of the most abundant components of GAs, were investigated. Kinetic analysis indicated that human and rat liver microsomes shared similar metabolic characteristics of GA (17 : 1) in phase I and II metabolisms. The drug-metabolizing enzymes involved in GA (17 : 1) metabolism were human CYP1A2, CYP3A4, UGT1A6, UGT1A9, and UGT2B15, which were confirmed with an inhibition study of human liver microsomes and recombinant enzymes. The MTT assays indicated that the cytotoxicity of GA (17 : 1) in HepG2 cells occurred in a time- and dose-dependent manner. Further investigation showed that GA (17 : 1) had less cytotoxicity in primary rat hepatocytes than in HepG2 cells and that the toxicity was enhanced through CYP1A- and CYP3A-mediated metabolism.

Drug-metabolizing enzymes CYP3A as a link between tacrolimus and vitamin D in renal transplant recipients: is it relevant in clinical practice?

CYP3A enzymes are involved in the metabolism of calcineurin inhibitor tacrolimus as well as vitamin D. In this review, we summarize the clinical aspects of CYP3A-mediated metabolism of tacrolimus and vitamin D with emphasis on the influence of single-nucleotide polymorphisms on tacrolimus disposition. We describe the utility of 4β hydroxycholesterol as a marker of CYP3A activity. Then, we discuss the possible interaction between calcineurin inhibitors and vitamin D in solid organ transplant recipients. Also, we review other mechanisms which may contribute to side effects of calcineurin inhibitors on bone. Lastly, suggestions for future research and clinical perspectives are discussed.

Characterization of Intestinal and Hepatic CYP3A-Mediated Metabolism of Midazolam in Children Using a Physiological Population Pharmacokinetic Modelling Approach

PurposeChanges in drug absorption and first-pass metabolism have been reported throughout the pediatric age range. Our aim is to characterize both intestinal and hepatic CYP3A-mediated metabolism of midazolam in children in order to predict first-pass and systemic metabolism of CYP3A substrates.MethodsPharmacokinetic (PK) data of midazolam and 1-OH-midazolam from 264 post-operative children 1–18 years of age after oral administration were analyzed using a physiological population PK modelling approach. In the model, consisting of physiological compartments representing the gastro-intestinal tract and liver,intrinsic intestinal and hepatic clearances were estimated to derive values for bioavailability and plasma clearance.ResultsThe whole-organ intrinsic clearance in the gut wall and liver were found to increase with body weight, with a 105 (95% confidence interval (CI): 5–405) times lower intrinsic gut wall clearance than the intrinsic hepatic clearance (i.e. 5.08 L/h (relative standard error (RSE) 10%) versus 527 L/h (RSE 7%) for a 16 kg individual, respectively). When expressed per gram of organ, intrinsic clearance increases with increasing body weight in the gut wall, but decreases in the liver, indicating that CYP3A-mediated intrinsic clearance and local bioavailability in the gut wall and liver do not change with age in parallel. The resulting total bioavailability was found to be age-independent with a median of 20.8% in children (95%CI: 3.8–50.0%).ConclusionIn conclusion, the intrinsic CYP3A-mediated gut wall clearance is substantially lower than the intrinsic hepatic CYP3A-mediated clearance in children from 1 to 18 years of age, and contributes less to the overall first-pass metabolism compared to adults.

Inflammation down-regulates CYP3A4-catalysed drug metabolism in hemodialysis patients

BackgroundRecent studies indicate that inflammation may also affect CYP3A4 activity. Associations of CYP3A4-mediated metabolism of quinine, with inflammatory biomarkers were investigated in patients undergoing maintenance hemodialysis (HD).MethodsA single dose of 100 mg quinine was given to 44 HD patients and the plasma concentration of quinine and its metabolite 3-OH-quinine were measured 12 h after drug intake. The ratios of quinine/3-OH-quinine and 4β-OH-cholesterol/cholesterol were used as markers of CYP3A4 activity. Inflammatory biomarkers, high-sensitive CRP (hsCRP), pentraxin 3 (PTX3) and orosomucoid were followed during 4 weeks prior to quinine administration.ResultsThe quinine/3-OH-quinine ratio correlated with median concentrations of hsCRP (Rho = 0.48; p = 0.001) and orosomucoid (Rho = 0.44; p = 0.003), and also with interleukin-6 at 12 h after drug intake (Rho = 0.43; P = 0.004) but not PTX3. In multivariate regression analysis, the correlation between CYP3A4 activity and median hsCRP remained borderline significant (p = 0.05). 4β-OH-cholesterol/cholesterol ratio correlated with quinine/3-OH-quinine (p = 0.008), but not with any of the inflammation markers.ConclusionsThe association between CYP3A4 activity and inflammatory biomarkers suggest that the activity of CYP3A4 is reduced by inflammation in HD patients. Further studies are needed to confirm this finding and to assess to what extent magnitude and duration of inflammation as well as the microbiota affect drug metabolism.

Nutritional Status Differentially Alters Cytochrome P450 3A4 (CYP3A4) and Uridine 5'-Diphospho-Glucuronosyltransferase (UGT) Mediated Drug Metabolism: Effect of Short-Term Fasting and High Fat Diet on Midazolam Metabolism.

Background and ObjectivesPrevious studies have shown that nutritional status can alter drug metabolism which may result in treatment failure or untoward side effects. This study assesses the effect of two nutritional conditions, short-term fasting, and a short-term high fat diet (HFD) on cytochrome P450 3A4 (CYP3A4) and uridine 5′-diphospho-glucuronosyltransferase (UGT) mediated drug metabolism by studying the pharmacokinetics of midazolam and its main metabolites.MethodsIn a randomized-controlled cross-over trial, nine healthy subjects received a single intravenous administration of 0.015 mg/kg midazolam after: (1) an overnight fast (control); (2) 36 h of fasting; and (3) an overnight fast after 3 days of a HFD consisting of 500 ml of cream supplemented to their regular diet. Pharmacokinetic parameters were analyzed simultaneously using non-linear mixed-effects modeling.ResultsShort-term fasting increased CYP3A4-mediated midazolam clearance by 12% (p < 0.01) and decreased UGT-mediated metabolism apparent 1-OH-midazolam clearance by 13% (p < 0.01) by decreasing the ratio of clearance and the fraction metabolite formed (ΔCL1-OH-MDZ/f1-OH-MDZ). Furthermore, short-term fasting decreased apparent clearance of 1-OH-midazolam-O-glucuronide (CL1-OH-MDZ-glucuronide/(f1-OH-MDZ-glucuronide × f1-OH-MDZ)) by 20% (p < 0.01). The HFD did not affect systemic clearance of midazolam or metabolites.ConclusionsShort-term fasting differentially alters midazolam metabolism by increasing CYP3A4-mediated metabolism but by decreasing UGT-mediated metabolism. In contrast, a short-term HFD did not affect systemic clearance of midazolam.

First-Pass CYP3A-Mediated Metabolism of Midazolam in the Gut Wall and Liver in Preterm Neonates.

To predict first‐pass and systemic cytochrome P450 (CYP) 3A‐mediated metabolism of midazolam in preterm neonates, a physiological population pharmacokinetic model was developed describing intestinal and hepatic midazolam clearance in preterm infants. On the basis of midazolam and 1‐OH‐midazolam concentrations from 37 preterm neonates (gestational age 26–34 weeks) receiving midazolam orally and/or via a 30‐minute intravenous infusion, intrinsic clearance in the gut wall and liver were found to be very low, with lower values in the gut wall (0.0196 and 6.7 L/h, respectively). This results in a highly variable and high total oral bioavailability of 92.1% (range, 67–95%) in preterm neonates, whereas this is around 30% in adults. This approach in which intestinal and hepatic clearance were separately estimated shows that the high bioavailability in preterm neonates is explained by, likely age‐related, low CYP3A activity in the liver and even lower CYP3A activity in the gut wall.

Application of Physiologically Based Pharmacokinetic Modeling in Understanding Bosutinib Drug-Drug Interactions: Importance of Intestinal P-Glycoprotein

Bosutinib is an orally available Src/Abl tyrosine kinase inhibitor indicated for the treatment of patients with Ph+ chronic myelogenous leukemia at a clinically recommended dose of 500 mg once daily. Clinical results indicated that increases in bosutinib oral exposures were supraproportional at the lower doses (50-200 mg) and approximately dose-proportional at the higher doses (200-600 mg). Bosutinib is a substrate of CYP3A4 and P-glycoprotein and exhibits pH-dependent solubility with moderate intestinal permeability. These findings led us to investigate the factors influencing the underlying pharmacokinetic mechanisms of bosutinib with physiologically based pharmacokinetic (PBPK) models. Our primary objectives were to: 1) refine the previously developed bosutinib PBPK model on the basis of the latest oral bioavailability data and 2) verify the refined PBPK model with P-glycoprotein kinetics on the basis of the bosutinib drug-drug interaction (DDI) results with ketoconazole and rifampin. Additionally, the verified PBPK model was applied to predict bosutinib DDIs with dual CYP3A/P-glycoprotein inhibitors. The results indicated that 1) the refined PBPK model adequately described the observed plasma concentration-time profiles of bosutinib and 2) the verified PBPK model reasonably predicted the effects of ketoconazole and rifampin on bosutinib exposures by accounting for intestinal P-glycoprotein inhibition/induction. These results suggested that bosutinib DDI mechanism could involve not only CYP3A4-mediated metabolism but also P-glycoprotein-mediated efflux on absorption. In summary, P-glycoprotein kinetics could constitute an element in the PBPK models critical to understanding the pharmacokinetic mechanism of dual CYP3A/P-glycoprotein substrates, such as bosutinib, that exhibit nonlinear pharmacokinetics owing largely to a saturation of intestinal P-glycoprotein-mediated efflux.

Kinetics of Letrozole Metabolism by Cytochrome P450 3A4: Modulation and Fitting to Non‐Michaelis Menten Models

Variable blood plasma concentration is an issue for many drugs, including letrozole, which can contribute to toxicity or sub‐effective dosing levels. There is evidence that letrozole is primarily metabolized by CYP2A6 however, CYP2A6 genetic variation only explains a portion (about 30%) of the variability in letrozole plasma concentrations. We hypothesized that CYP3A4 may play a larger role in letrozole metabolism than the current evidence suggests due to the influence of multiple ligand binding and the observation of non‐Michaelis‐Menten kinetic plots. The kinetics of letrozole metabolism by CYP3A4 were analyzed in the presence and absence of ketoconazole, a CYP3A4 modulator, and fitted to multiple ligand binding functions as well as the Michaelis‐Menten model. Further, the time‐dependent inhibition parameters of CYP2A6‐mediated letrozole metabolism were measured in the presence of the CYP2A6‐specific inactivator, trans‐cinnamic aldehyde (tCA). While the Eadie‐Hofstee plot for CYP3A4‐mediated letrozole metabolism was ambiguous, curve fitting showed the Michaelis‐Menten model appropriately characterized metabolism in the absence of ketoconazole (KM: 142+/−23.0 μM, Vmax: 13.0+/−1.44 pmol/nmol/min), suggesting a single ligand bound to the enzyme. A biphasic model most closely modeled letrozole metabolism in the presence of ketoconazole (KM1: 4.71+/−4.55 μM KM2: 513+/−164 μM, Vmax1:0.525+/−0.392 Vmax2: 30.9+/−7.60 pmol/nmol/min) and this was also reflected in the Eadie‐Hofstee plot, which was clearly biphasic; this supports a mechanism involving multiple‐ligand binding. TDI analysis of tCA revealed less effective inhibition of CYP2A6‐mediated letrozole metabolism by tCA (KI: 136 μM, kinact: 0.019 min−1) as compared to CYP2A6‐mediated coumarin metabolism. Together, these results suggest that CYP3A4 may contribute to variability in letrozole metabolism especially in the presence of an effector molecule (i.e., ketoconazole) since the value of KM1 is much closer to the letrozole plasma concentrations observed clinically than the KM value observed in the absence of an effector.Support or Funding InformationFunding from National Institute on Drug AbuseThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Vinca Alkaloid Pharmacokinetics in the Context of a Physiologically‐Based Murine Model

Vinca alkaloids are a class of molecule clinically used as cytotoxic chemotherapeutics, and exert their activity through mitotic disruption by preventing the polymerization of microtubules. While successfully used to treat a variety of cancer types, patients receiving these treatments remain at risk of toxic side‐effects due to significant variability observed in drug disposition. The objective of this study was to characterize the primary factors driving vinca alkaloid pharmacokinetics (PK), and develop a model capable of predicting PK based on alterations in these drivers. Physiologic sources of variability are known to include a wide range of reported values for CYP3A‐mediated metabolism and plasma protein binding, as well as a strong correlation between tissue uptake and tissue tubulin content. Additionally, vinca alkaloids are known substrates of ABCB1 (MDR1, PGP), which mediates drug efflux from tissues. To investigate dependence on these factors we initially developed a physiologically‐based pharmacokinetic (PBPK) model for vinblastine (VBL) dosed IV at 2 mg/kg in MDR1a −/− (KO) and MDR1a +/+ (WT) mice. An increase in VBL exposure was observed in all KO tissues compared to WT, particularly in brain which accumulated roughly 10‐fold more VBL over 6 hours. This is of particular importance as neurotoxicity is the most commonly reported dose‐limiting toxicity for vinca alkaloids. Additionally, excretion through biliary and renal routes were 6‐fold and 10‐fold lower, respectively, for both VBL and the active metabolite, desacetylVBL. Using known physiological and biochemical parameters the PBPK model was developed and validated for key organs involved in distribution, metabolism, and excretion of VBL in the MDR1a WT and KO mice. Through adjustment of relevant biochemical parameters the model was validated for vincristine (VCR) PK in MDR1a WT mice receiving the same 2mg/kg IV bolus dose, and used to simulate what VCR PK might look like in an MDR1a KO scenario. The validated PBPK model for VBL and VCR allows for investigation of PK based on alterations in measurable, physiologically‐relevant driving factors in murine models. Due to the pharmacologic similarities between molecules within the vinca alkaloid class, biochemical parameters can easily be adjusted to predict PK of vindesine and vinorelbine as well. Value of this model lies in its ability predict exposure in pre‐clinical studies involving vinca alkaloids, and provide insight into how exposure would be altered in combination therapy or gene knockout settings.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Midazolam Hydroxylation Assay as a Marker for Endogenous CYP3A4‐mediated Metabolism: Metabolic Interaction with Natural Health Supplements

BackgroundEndogenous substances (endobiotics) such as vitamin D3 and estrogen are metabolized by cytochrome P450 3A4 (CYP3A4). However, measurement of endobiotic metabolites is often technology‐limited and expensive. Because of structural similarity of midazolam with vitamin D3, midazolam metabolic profile could present valuable information in identifying potential interaction of drugs or natural health supplements (NHS) with endogenous substances. Polyphenols (quercetin, raspberry ketone, gallic acid, rutin), furanocoumarin (imperatorin) and terpenoids (ursolic acid) are the biologically active human dietary constituents from grapes, berry fruits, pomegranate, and olive oil, but are also widely ingested as over‐the‐counter natural health supplements (NHS) in United States. NHS have the ability to inhibit CYP3A4‐mediated metabolism, possibly causing the plasma concentrations of the midazolam as well as vitamin D3 to increase. The objective of the present study was to develop a simple and effective metabolism assay to measure CYP3A4‐mediated midazolam hydroxylation and to screen certain NHS (polyphenols, furanocoumarin, terpenoids) for their potential to cause CYP3A4 related herb‐endobiotic interactions in humans.MethodsThe hydroxylation of midazolam to 1′‐hydroxymidazolam assay was developed and optimized. Reaction mixtures containing potassium phosphate buffer (pH 7.4), recombinant CYP3A4 protein (30 pmol/ml), NADPH‐regenerating system, and midazolam (5 uM or 2 uM) were incubated for 15 min. Substrate, metabolites and internal standard were analyzed with a reverse phase assay by a Shimadzu Prominence high‐performance liquid chromatography (HPLC) coupled to a photodiode array detector set at 220 nm using a C18 column. SigmaPlot enzyme kinetics module (version 13; Systat Software, Inc., San Jose, CA) and GraphPad Prism software (GraphPad Software Inc., San Diego, CA) were used to determine the kinetics parameters and inhibitory constants, respectively.ResultsThe Km and Vmax values for rCYP3A4‐mediated 1′‐hydroxymidazolam formation were 1.9±0.3 uM and 75.4±3.7 pmol/min/mg, respectively. Imperatorin inhibited 1′‐hydroxymidazolam formation to undetectable levels at both 2 uM and 5 uM concentrations of midazolam, whereas quercetin inhibited the formation by 31–44%. The IC50 values of imperatorin for 1′‐hydroxymidazolam and 4‐hydroxymidazolam were 7.7 uM and 2.6 uM, respectively. Ketoconazole, a known potent inhibitor of CYP3A4, yielded IC50 concentrations of 0.16–0.20 uM for midazolam hydroxylation.ConclusionsA midazolam hydroxylation assay was developed to screen potential metabolic interactions. Imperatorin was identified as an inhibitor of rCYP3A4. Quercetin showed low rCYP3A4 inhibition potential. This research further shows that NHS studied here can potentially inhibit one of the most ubiquitous enzymes responsible for inactivation of endobiotics and may provide additional health benefits. Vitamin D3 is metabolized by CYP3A4 and midazolam could potentially be used as a marker for interaction of commonly used NHS with vitamin D3. The cost‐effective HPLC method developed in this study provides a useful tool in predicting NHS‐endobiotic interactions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterisation of the absorption, distribution, metabolism, excretion and mass balance of doravirine, a non-nucleoside reverse transcriptase inhibitor in humans

Absorption, distribution, metabolism and elimination of doravirine (MK-1439), a novel non-nucleoside reverse transcriptase inhibitor, were investigated. Two clinical trials were conducted in healthy subjects: an oral single dose [14 C]doravirine (350 mg, ∼200 µCi) trial (n = 6) and an intravenous (IV) single-dose doravirine (100 µg) trial (n = 12). In vitro metabolism, protein binding, apparent permeability and P-glycoprotein (P-gp) transport studies were conducted to complement the clinical trials.Following oral [14 C]doravirine administration, all of the administered dose was recovered. The absorbed dose was eliminated primarily via metabolism. An oxidative metabolite (M9) was the predominant metabolite in excreta and was the primary circulating metabolite (12.9% of circulating radioactivity). Following IV administration, doravirine clearance and volume of distribution were 3.73 L/h (95% confidence intervals (CI) 3.09, 4.49) and 60.5 L (95% CI 53.7, 68.4), respectively.In vitro, doravirine is not highly bound to plasma proteins (unbound fraction 0.24) and has good passive permeability. The metabolite M9 was generated by cytochrome P450 3A (CYP3A)4/5-mediated oxidation. Doravirine was a P-gp substrate but P-gp efflux is not expected to play a significant role in limiting doravirine absorption or to be involved in the elimination of doravirine.In conclusion, doravirine is a low clearance drug, primarily eliminated by CYP3A-mediated metabolism.

Antiretroviral Drug Metabolism in Humanized PXR-CAR-CYP3A-NOG Mice.

Antiretroviral drug (ARV) metabolism is linked largely to hepatic cytochrome P450 activity. One ARV drug class known to be metabolized by intestinal and hepatic CYP3A are the protease inhibitors (PIs). Plasma drug concentrations are boosted by CYP3A inhibitors such as cobisistat and ritonavir (RTV). Studies of such drug-drug interactions are limited since the enzyme pathways are human specific. While immune-deficient mice reconstituted with human cells are an excellent model to study ARVs during human immunodeficiency virus type 1 (HIV-1) infection, they cannot reflect human drug metabolism. Thus, we created a mouse strain with the human pregnane X receptor, constitutive androstane receptor, and CYP3A4/7 genes on a NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac background (hCYP3A-NOG) and used them to evaluate the impact of human CYP3A metabolism on ARV pharmacokinetics. In proof-of-concept studies we used nanoformulated atazanavir (nanoATV) with or without RTV. NOG and hCYP3A-NOG mice were treated weekly with 50 mg/kg nanoATV alone or boosted with nanoformulated ritonavir (nanoATV/r). Plasma was collected weekly and liver was collected at 28 days post-treatment. Plasma and liver atazanavir (ATV) concentrations in nanoATV/r-treated hCYP3A-NOG mice were 2- to 4-fold higher than in replicate NOG mice. RTV enhanced plasma and liver ATV concentrations 3-fold in hCYP3A-NOG mice and 1.7-fold in NOG mice. The results indicate that human CYP3A-mediated drug metabolism is reduced compared with mouse and that RTV differentially affects human gene activity. These differences can affect responses to PIs in humanized mouse models of HIV-1 infection. Importantly, hCYP3A-NOG mice reconstituted with human immune cells can be used for bench-to-bedside translation.

Dicloxacillin induces CYP2C19, CYP2C9 and CYP3A4 in vivo and in vitro.

The aim of this study was to study potential cytochrome P450 (CYP) induction by dicloxacillin. We performed an open-label, randomized, two-phase, five-drug clinical pharmacokinetic cocktail crossover study in 12 healthy men with and without pretreatment with 1g dicloxacillin three times daily for 10days. Plasma and urine were collected over 24h and the concentration of all five drugs and their primary metabolites was determined using a liquid chromatography coupled to triple quadrupole mass spectrometry method. Cryopreserved primary human hepatocytes were exposed to dicloxacillin for 48h and changes in gene expression and the activity of CYP3A4, CYP2C9, CYP2B6 and CYP1A2 were investigated. The activation of nuclear receptors by dicloxacillin was assessed using luciferase assays. A total of 10days of treatment with dicloxacillin resulted in a clinically and statistically significant reduction in the area under the plasma concentration-time curve from 0 to 24h for omeprazole (CYP2C19) {geometric mean ratio [GMR] [95% confidence interval (CI)]: 0.33 [0.24, 0.45]}, tolbutamide (CYP2C9) [GMR (95% CI): 0.73 (0.65, 0.81)] and midazolam (CYP3A4) [GMR (95% CI): 0.54 (0.41, 0.72)]. Additionally, other relevant pharmacokinetic parameters were affected, indicating the induction of CYP2C- and CYP3A4-mediated metabolism by dicloxacillin. Investigations in primary hepatocytes showed a statistically significant dose-dependent increase in CYP expression and activity by dicloxacillin, caused by activation of the pregnane X receptor. Dicloxacillin is an inducer of CYP2C- and CYP3A-mediated drug metabolism, and we recommend caution when prescribing dicloxacillin to users of drugs with a narrow therapeutic window.

Effects of Cilostazol on the Pharmacokinetics of Nifedipine After Oral and Intravenous Administration in Rats

The purpose of this study was to investigate the effects of cilostazol on the bioavailability and pharmacokinetics of nifedipine and its main metabolite, dehydronifedipine, in rats. The pharmacokinetic parameters of nifedipine and dehydronifedipine were determined following oral and intravenous administration of nifedipine (1.5 and 6.0 mg ・ kg-1) in rats. Cilostazol inhibited CYP3A4 enzyme activity at a 50% inhibitory concentration (IC50) of 4.1 μM. The areas under the plasma concentration–time curve (AUC 0-∞) and the peak concentration (C max) of nifedipine were significantly increased, respectively, in the presence of cilostazol compared to that in the control. The total body clearance (CL/F) was significantly decreased by cilostazol. Consequently, the absolute bioavailability (AB) of nifedipine with cilostazol was significantly higher than that in the control. The metabolite to parent AUC ratio (MR) in the presence of cilostazol was significantly decreased compared to that in the control. The AUC 0-∞ of intravenous nifedipine was significantly increased with cilostazol compared to that in the control. The increased bioavailability of nifedipine in rats can be mainly due to the inhibition of CYP3A4-mediated metabolism in the small intestine and/or liver by cilostazol. In addition, the reduction of CL/F of nifedipine by cilostazol may also be a factor.

Predicting CYP3A-mediated midazolam metabolism in critically ill neonates, infants, children and adults with inflammation and organ failure.

AimsInflammation and organ failure have been reported to have an impact on cytochrome P450 (CYP) 3A‐mediated clearance of midazolam in critically ill children. Our aim was to evaluate a previously developed population pharmacokinetic model both in critically ill children and other populations, in order to allow the model to be used to guide dosing in clinical practice.MethodsThe model was evaluated externally in 136 individuals, including (pre)term neonates, infants, children and adults (body weight 0.77–90 kg, C‐reactive protein level 0.1–341 mg l–1 and 0–4 failing organs) using graphical and numerical diagnostics.ResultsThe pharmacokinetic model predicted midazolam clearance and plasma concentrations without bias in postoperative or critically ill paediatric patients and term neonates [median prediction error (MPE) <30%]. Using the model for extrapolation resulted in well‐predicted clearance values in critically ill and healthy adults (MPE <30%), while clearance in preterm neonates was over predicted (MPE >180%).ConclusionThe recently published pharmacokinetic model for midazolam, quantifying the influence of maturation, inflammation and organ failure in children, yields unbiased clearance predictions and can therefore be used for dosing instructions in term neonates, children and adults with varying levels of critical illness, including healthy adults, but not for extrapolation to preterm neonates.

Pregnane X receptor and constitutive androstane receptor modulate differently CYP3A-mediated metabolism in early- and late-stage cholestasis.

AIMTo ascertain whether cholestasis affects the expression of two CYP3A isoforms (CYP3A1 and CYP3A2) and of pregnane X receptor (PXR) and constitutive androstane receptor (CAR).METHODSCholestasis was induced by bile duct ligation in 16 male Wistar rats; whereas 8 sham-operated rats were used as controls. Severity of cholestasis was assessed on histological examination of liver sections, and serum concentrations of albumin, AST, ALT, GGT, ALPK and bilirubin. Gene and protein expressions of PXR, CAR, CYP3A1 and CYP3A2 were assessed by means of qRT-PCR and Western blot, respectively. Alterations in CYP3A activity were measured by calculating the kinetic parameters of 4-OH and 1’-OH-midazolam hydroxylation, marker reactions for CYP3A enzymes.RESULTSThe mRNA and protein expression of CYP3A1 increased significantly in mild cholestasis (P < 0.01). At variance, mRNA and protein expression of CYP3A2 didn’t change in mild cholestasis, whereas the expression and activity of both CYP3A1 and CYP3A2 decreased dramatically when cholestasis became severe. Consistently with these observations, the nuclear expression of both PXR and CAR, which was measured because they both translocate into the cell nucleus after their activation, virtually disappeared in the late stage of cholestatic injury, after an initial increase. These results indicate that early- and late-stage cholestasis affects CYP3A-mediated drug metabolism differently, probably as consequence of the different activation of PXR and CAR.CONCLUSIONEarly- and late-stage cholestasis affects CYP3A-mediated drug metabolism differently. PXR and CAR might be targeted therapeutically to promote CYP3A-mediated liver detoxification.

Modulation of CYP3A enzyme activity by diosmin and its consequence on carbamazepine pharmacokinetics in rats.

Diosmin is a widely used flavonoid for the treatment of varicose veins and hemorrhoids. Epileptic patients with hemorrhoids and varicose veins may use diosmin along with carbamazepine (CBZ) therapy, which leads to pharmacokinetic interaction between diosmin and CBZ. Therefore, the present study was performed to evaluate the effect of diosmin on the pharmacokinetics of CBZ in rats. Diosmin-mediated altered CYP3A enzyme activity in human and rat liver microsomes was examined using CYP3A dependent erythromycin N-demethylase assay. Further, an in vivo pharmacokinetic study of oral administered CBZ in rats with and without diosmin pretreatment was performed. The CYP3A enzyme activity in human and rat liver microsomes was significantly (p < 0.05) decreased by diosmin when compared to control. Pretreatment with diosmin significantly (p < 0.05) enhanced maximum plasma concentration (C max), area under the curve (AUC), and half life (t 1/2), while significantly (p < 0.05) decreased elimination rate constant (k el) and apparent oral clearance (CL/F) of CBZ as compared to control rats. On the other hand, C max, AUC, and t 1/2 of carbamazepine 10, 11-epoxide (CBZE) were significantly (p < 0.05) decreased after diosmin pretreatment. Furthermore, diosmin pretreatment significantly (p < 0.05) decreased metabolic ratios of C max and AUC when compared to control, suggesting reduced formation of CBZ to CBZE. The results suggest that diosmin pretreatment might have inhibited CYP3A-mediated metabolism of CBZ. Accordingly, caution should be taken when diosmin is used in combination with therapeutic drugs metabolized by CYP3A enzyme in addition to CBZ.

O-33 Feasibility of a paediatric micro-dose study of [14c]midazolam to study the ontogeny of cyp3a-mediated drug metabolism

Background Microdose studies present an interesting innovation to study age-related changes in drug metab-olism in young children. To further delineate maturation of intestinal and hepatic CYP3A activity, in this pilot study we aimed to study the feasibility of an oral [14C]midazol-am (MDZ) microdosing study in children. Methods Children admitted to the paediatric intensive care unit were eligible to receive a single oral [14C]MDZ microdose when they received IV midazolam for thera-peutic reasons and had an arterial line in place enabling blood sampling. Blood samples were taken up to 24 hours after dose administration. Plasma concentrations of [14C] MDZ and the metabolite [14C]OH-MDZ were determined by accelator mass spectrometry (AMS). Pharmacokinetic (PK) parameters were estimated using non-compartmen-tal PK models with PKSolver software (Microsoft Excel). Results Of 139 eligible patients, 125 were excluded and informed consent was obtained from parents of nine chil-dren [median age 3.3 months (range 12 days – 4.2 years)] who received a midazolam microdose (19.3 [18.7–21.3] ng/kg; 58 [56-64] Bq/kg). [14C]MDZ and [14C]1-OH-MDZ were detectable at expected concentrations: plasma [14C]MDZ AUC0-∞ was 49.9 (4.0–107.7) ng/L*h, Cmax was 7.5 (1.5–22.2) ng/L, Tmax was 0.5 (0.3–3.1) hour, T0.5 was 4.6 (1.1–14.0) hour, CL/F was 0.4 (0.2–5.3) L/h/kg and Vss/F was 3.1 (1.7–10.7) L/kg. Plasma [14C]1-OH-MDZ AUC0-∞ was 7.8 (1.3–28.3) ng/L*h and CL/F was 2.4 (0.7–14.6) L/h/kg. Plasma Cmax of [14C]MDZ normalised to a dose of 0.1 mg/kg was 39.9 (7.0–114.9) ng/ml. Conclusion We demonstrate the feasibility of an oral [14C]MDZ microdose to study MDZ and 1-OHMDZ dis-position in young infants and children with AMS. This method can be used to study developmental changes in intestinal and hepatic CYP3A activity.

Characterization of Maternal and Fetal CYP3A-Mediated Progesterone Metabolism

ABSTRACTIntroduction: Progesterone is critical for maintaining pregnancy and onset of labor. We evaluated CYP450-mediated progesterone meta-bolism, specifically the contribution of CYP3A isoforms. Materials and Methods: In vitro progesterone metabolism was characterized in human liver microsomes (HLMs) with and without selective cytochrome P450 inhibitors and in recombinant CYP3A4, CYP3A5, and CYP3A7. 6β-hydroxyprogesterone (6β-OHP) and 16α-hydroxyprogesterone (16α-OHP) metabolites were quantified by HPLC/UV and fit to the Michaelis–Menten equation to determine Km and Vmax. The effect of CYP3A5 expression on progesterone clearance was determined by in vitro in vivo extrapolation. Results: Ketoconazole inhibited formation of both 6β-OHP and 16α-OHP more than 95%. 6β-OHP and 16α-OHP were both produced by CYP3A4 (2.3 and 1.3 µL/min/pmol, respectively) to a greater extent than by CYP3A5 (0.09 and 0.003 µL/min/pmol) and CYP3A7 (0.004 and 0.003 µL/min/pmol). Conclusions: Maternal clearance of progesterone by hepatic CYP450's is driven primarily by CYP3A4, with limited contributions from CYP3A5 and CYP3A7.