Induction of hepatic CYP3A4 expression by cholesterol and cholic acid: Alterations of gene expression, microsomal activity, and pharmacokinetics.

Human cytochrome P450 3A4 (CYP3A4) is a drug-metabolizing enzyme that is abundantly expressed in the liver and intestine. It is an important issue whether compounds of interest affect the expression of CYP3A4 because more than 30% of commercially available drugs are metabolized by CYP3A4. In this study, we examined the effects of cholesterol and cholic acid on the expression level and activity of CYP3A4 in hCYP3A mice that have a human CYP3A gene cluster and show human-like regulation of the coding genes. A normal diet (ND, CE-2), CE-2 with 1% cholesterol and 0.5% cholic acid (HCD) or CE-2 with 0.5% cholic acid was given to the mice. The plasma concentrations of cholesterol, cholic acid and its metabolites in HCD mice were higher than those in ND mice. In this condition, the expression levels of hepatic CYP3A4 and the hydroxylation activities of triazolam, a typical CYP3A4 substrate, in liver microsomes of HCD mice were higher than those in liver microsomes of ND mice. Furthermore, plasma concentrations of triazolam in HCD mice were lower than those in ND mice. In conclusion, our study suggested that hepatic CYP3A4 expression and activity are influenced by the combination of cholesterol and cholic acid in vivo.

Investigating the Utility of Humanized Pregnane X Receptor-Constitutive Androstane Receptor-CYP3A4/7 Mouse Model to Assess CYP3A-Mediated Induction.

Clinical induction liability is assessed with human hepatocytes. However, underpredictions in the magnitude of clinical induction have been reported. Unfortunately, in vivo studies in animals do not provide additional insight because of species differences in drug metabolizing enzymes and their regulatory pathways. To circumvent this limitation, transgenic animals expressing human orthologs were developed. The aim of this work was to investigate the utility of mouse models expressing human orthologs of pregnane X receptor, constitutive androstane receptor, and CYP3A4/7 (Tg-Composite) in evaluating clinical induction. Rifampin, efavirenz, and pioglitazone, which were employed to represent strong, moderate, and weak inducers, were administered at multiple doses to Tg-Composite animals. In vivo CYP3A activity was monitored by measuring changes in the exposure of the CYP3A probe substrate triazolam. After the in vivo studies, microsomes were prepared from their livers to measure changes of in vitro CYP3A4 activity. In both in vivo and in vitro, distinction of clinic induction was recapitulated as rifampin yielded the greatest inductive effect followed by efavirenz and pioglitazone. Interestingly, with rifampin, in vivo CYP3A activity was approximately 4-fold higher than in vitro activity. Conversely, there was no difference between in vivo and in vitro CYP3A activity with efavirenz. These findings are consistent with the report that, although rifampin exhibits differential inductive effects between the intestines and liver, efavirenz does not. These data highlight the promise of transgenic models, such as Tg-Composite, to complement human hepatocytes to enhance the translatability of clinical induction as well as become a powerful tool to further study mechanisms of drug disposition. SIGNIFICANCE STATEMENT: Underprediction of the magnitude of clinical induction when using human hepatocytes has been reported, and transgenic models may improve clinical translatability. The work presented here showcases the human orthologs of pregnane X receptor, constitutive androstane receptor, and CYP3A4/7 model, which was able to recapitulate the magnitude of clinical induction and to differentiate tissue-dependent induction observed with rifampin but not with efavirenz. These results not only foreshadow the potential application of such transgenic models in assessing clinical induction but also in further investigation of the mechanism of drug disposition.

Kinetic analysis of sequential metabolism of triazolam and its extrapolation to humans using an entero-hepatic two-organ microphysiological system.

The microphysiological system (MPS) is a promising tool for predicting drug disposition in humans, although limited information is available on the quantitative assessment of sequential drug metabolism in MPS and its extrapolation to humans. In the present study, we first constructed a mechanism-based pharmacokinetic model for triazolam (TRZ) and its metabolites in the entero-hepatic two-organ MPS, composed of intestinal Caco-2 and hepatic HepaRG cells, and attempted to extrapolate the kinetic information obtained with the MPS to the plasma concentration profiles in humans. In the two-organ MPS and HepaRG single culture systems, TRZ was found to be metabolized into α- and 4-hydroxytriazolam and their respective glucuronides. All these metabolites were almost completely reduced in the presence of a CYP3A inhibitor, itraconazole, confirming sequential phase I and II metabolism. Both pharmacokinetic model-dependent and -independent analyses were performed, providing consistent results regarding the metabolic activity of TRZ: clearance of glucuronidation metabolites in the two-organ MPS was higher than that in the single culture system. The plasma concentration profile of TRZ and its two hydroxy metabolites in humans was quantitatively simulated based on the pharmacokinetic model, by incorporating several scaling factors representing quantitative gaps between the MPS and humans. Thus, the present study provided the first quantitative extrapolation of sequential drug metabolism in humans by combining MPS and pharmacokinetic modeling.

Effect of pretreatment regimens of 1-aminobenzotriazole on metabolism and gastric emptying of probe compounds in rat.

1-Aminobenzotriazole (ABT) is a mechanism-based inactivator of major cytochrome P450 (CYP) enzymes, which is used in multiple mechanistic studies. The purpose was to evaluate the effect of 2 and 16-h pretreatment regimens of ABT on the exposures of triazolam in rat. Another objective was to evaluate the effect of ABT on gastric emptying of acetaminophen. Plasma area under the curve (AUC) of triazolam was increased by 101-fold and 81-fold for the rats pretreated with ABT at 2 and 16 h, respectively, compared to control rats. Time to reach maximum concentration was 0.3, 4.8 and 3.7 h in control, 2 and 16-h pretreatment animals, respectively. In the case of acetaminophen, where Tmax was not delayed, the mean absorption time (MAT) in control, 2 and 16 h ABT pretreatment groups were 0.3, 4.6 and 2.9 h, respectively, suggesting delayed absorption. This hypothesis was further supported by GastroPlusTM simulation. In summary, extent of triazolam absorption was increased to a similar extent with both 2 and 16 h ABT pretreatment regimens, suggesting that either of the regimen can be used to increase parent exposures in rat. With ABT pretreatment, delayed absorption of triazolam and acetaminophen was observed, as suggested by delay in Tmax and MAT, respectively.

Application of unbound liver-to-plasma concentration ratio to quantitative projection of cytochrome P450-mediated drug-drug interactions using physiologically based pharmacokinetic modelling approach.

1. This study evaluated the prediction accuracy of cytochrome P450 (CYP)-mediated drug-drug interaction (DDI) using minimal physiologically-based pharmacokinetic (PBPK) modelling incorporating the hepatic accumulation factor of an inhibitor (i.e. unbound liver/unbound plasma concentration ratio [Kp,uu,liver]) based on 22 clinical DDI studies. 2. Kp,uu,liver values were estimated using three methods: (1) ratio of cell-to-medium ratio in human cryopreserved hepatocytes (C/Mu) at 37 °C to that on ice (Kp,uu,C/M), (2) multiplication of total liver/unbound plasma concentration ratio (Kp,u,liver) estimated from C/Mu at 37 °C with unbound fraction in human liver homogenate (Kp,uu,cell) and (3) observed Kp,uu,liver in rats after intravenous infusion (Kp,uu,rat). 3. PBPK model using each Kp,uu,liver projected the area under the curve (AUC) increase of substrates more accurately than the model assuming a Kp,uu,liver of 1 for the average fold error and root mean square error did. Particularly, the model with a Kp,uu,liver of 1 underestimated the AUC increase of triazolam following co-administration with CYP3A4 inhibitor itraconazole by five-fold, whereas the AUC increase projected using the model incorporating the Kp,uu,C/M, Kp,uu,cell, or Kp,uu,rat of itraconazole and hydroxyitraconazole was within approximately two-fold of the actual value. 4. The results indicated that incorporating Kp,uu,liver into the PBPK model improved the accuracy of DDI projection.

Comparison of the hepatic metabolism of triazolam in wild-type andCyp3a-knockout mice for understanding CYP3A-mediated metabolism inCYP3A-humanised mice in vivo.

1. To investigate cytochrome P450 3A (CYP3A)-mediated metabolism in vivo, plasma concentrations of triazolam (TRZ) are often monitored as a CYP3A marker in CYP3A-humanised mice. However, it has not been determined whether plasma concentrations of TRZ after intravenous administration can reflect hepatic CYP3A activity in CYP3A-humanised mice. 2. Firstly, we investigated the pharmacokinetics of TRZ in wild-type and Cyp3a-knockout (Cyp3a-KO) mice. Plasma concentration profiles of TRZ and α-hydroxy (OH) TRZ were very similar in wild-type and Cyp3a-KO mice. On the other hand, AUC of 4-OH TRZ in Cyp3a-KO mice was significantly lower than that in wild-type mice. Pregnenolone 16α-carbonitrile (PCN) decreased the areas under the plasma concentration-time curves (AUCs) of TRZ and α-OH TRZ in both groups. There was no significant effect of PCN on AUC of 4-OH TRZ in Cyp3a-KO mice. 3. Next, we verified that AUC of 4-OH TRZ in CYP3A-humanised mice was higher than that in Cyp3a-KO mice, although the difference was not significant. 4. In conclusion, plasma concentrations of 4-OH TRZ, but not those of TRZ and α-OH TRZ, might reflect hepatic CYP3A activity in mice in vivo. These results provide important insights for in vivo studies using a CYP3A-humanised model.

LC-MS determination of triazolam and its hydroxy metabolites in mouse dried blood spots: application to transgenic mouse pharmacokinetic studies.

AIM: The objective of this study was to develop a LC-MS/MS method for the simultaneous determination of triazolam (TRZ) and its two hydroxy metabolites in transgenic mouse dried blood spots (DBS) using BALB/c mouse blood as a surrogate biomatrix. METHODOLOGY/RESULTS: The DBS method involved spotting volume of 10 µl using Ahlstrom 226 sample collection cards. A 'whole spot' analysis (6-mm punch) involved extraction of analytes using water and acetonitrile containing an internal standard. DBS samples were analyzed by a validated LC-MS/MS method with a run time of 4 min. CONCLUSION: This validated LC-MS/MS method using DBS extraction was applied to quantitation of TRZ, α-hydroxytriazolam and 4-hydroxytriazolam in a CYP3A4 transgenic mouse oral pharmacokinetic study of TRZ.

Prediction of drug interaction between oral adsorbent AST-120 and concomitant drugs based on the in vitro dissolution and in vivo absorption behavior of the drugs.

PURPOSE: AST-120 is used to decrease the abundance of serum uremic toxins in treatment of chronic kidney disease; however, it could also adsorb concomitantly administered drugs. This study aimed to develop a prediction method for drug interaction between AST-120 and concomitantly administered drugs based on in vitro dissolution and in vivo absorption behavior. METHODS: Sixty-eight drugs were selected for the analysis. For each drug, theoretical dissolution (R d) and absorption (R a) rates at estimated dosing intervals (1, 30, 60, 90, 120, and 240 min) were calculated using the Noyes-Whitney formula and compartment analysis, respectively. The optimal thresholds for R d and R a (R dth and R ath) were estimated by comparing the results with those of previous drug interaction studies for six drugs. Four drug interaction risk categories for 68 drugs at each dose interval were defined according to the indices of dissolution and absorption against their thresholds. RESULTS: The in vitro dissolution and in vivo absorption behavior of the selected drugs were well fitted to the Noyes-Whitney formula and one- or two-compartment models. The optimal R dth and R ath that gave the highest value of consistency with the equivalence of drug interaction studies were 90 and 30 %, respectively. As the dosing intervals were lengthened, the number of drugs classified into the low-risk categories increased. CONCLUSION: A new drug interaction prediction method based on the pharmacokinetic parameters of drugs was developed. The new model is useful for estimating the risk of drug interaction in clinical practice when AST-120 is used in combination with other drugs.

Effect of buffer conditions on CYP2C8-mediated paclitaxel 6α-hydroxylation and CYP3A4-mediated triazolam α- and 4-hydroxylation by human liver microsomes.

1. Buffer conditions in in vitro metabolism studies using human liver microsomes (HLM) have been reported to affect the metabolic activities of several cytochrome P450 (CYP) isozymes in different ways, although there are no reports about the dependence of CYP2C8 activity on buffer conditions. 2. The present study investigated the effect of buffer components (phosphate or Tris-HCl) and their concentration (10-200 mM) on the CYP2C8 and CYP3A4 activities of HLM, using paclitaxel and triazolam, respectively, as marker substrates. 3. The Km (or S50) and Vmax values for both paclitaxel 6α-hydroxylation and triazolam α- and 4-hydroxylation, estimated by fitting analyses based on the Michaelis-Menten or Hill equation, greatly depended on the buffer components and their concentration. 4. The CLint values in phosphate buffer were 1.2-3.0-fold (paclitaxel) or 3.1-6.4-fold (triazolam) higher than in Tris-HCl buffer at 50-100 mM. These values also depended on the buffer concentration, with a maximum 2.3-fold difference observed between 50 and 100 mM which are both commonly used in drug metabolism studies. 5. These findings suggest the necessity for optimization of the buffer conditions in the quantitative evaluation of metabolic clearances, such as in vitro-in vivo extrapolation and also estimating the contribution of a particular enzyme in drug metabolism.

Residual effects of zolpidem, triazolam, rilmazafone and placebo in healthy elderly subjects: a randomized double-blind study.

With current hypnotic agents, next-day residual effects are a common problem. The purpose of the present study was to evaluate the residual effects of the commercially available hypnotics - zolpidem, triazolam, and rilmazafone - on the physical and cognitive functions of healthy elderly people in the early morning and the day following drug administration. In this study, the next-day residual effects of zolpidem, triazolam, and rilmazafone, following bedtime dosing in elderly subjects, were evaluated. Women (n = 11) and men (n = 2) aged 60-70 years received a single dose (at 23:00) of one of these, zolpidem 5 mg, triazolam 0.125 mg, rilmazafone 1 mg and placebo in a randomized, double-blind, crossover design. Measures of objective parameters and psychomotor performances (Timed up and Go test, Functional Reach Test, body sway test, critical flicker fusion test, simple discrimination reaction test, short-term memory test) and subjective ratings were obtained at 04:00, 07:00, and the next time of the day. All hypnotics were generally well tolerated; there were no serious adverse side effects and no subjects discontinued the evaluations. Compared to placebo, zolpidem and rilmazafone had good results on the Functional Reach Test. Although subjective assessments tended to be poor in the early morning, rilmazafone significantly improved the body sway test in the other hypnotics. A single dose of zolpidem 5 mg and triazolam 0.125 mg did not have any next-day residual effects on healthy elderly subjects. Residual effects appeared to be related to the compound's half-life and the dose used. Rilmazafone 1 mg exhibited steadiness in static and dynamic balance and seemed to be more favorable for the elderly with early morning awakening.

Effects of menthol on the pharmacokinetics of triazolam and phenytoin.

We have previously shown that menthol attenuates the anticoagulant effect of warfarin by increasing the expression levels of CYP3A and CYP2C in the liver. This study evaluated the effects of menthol on the pharmacokinetics of the CYP3A substrate triazolam and the CYP2C substrate phenytoin. Menthol was orally administered to mice for 7 d. Twenty-four hours after the administration of menthol, triazolam was orally administered, and the plasma concentration was measured. In addition, the CYP3A metabolic activity for triazolam and the CYP3A expression level in the liver were determined. The effects of menthol on the pharmacokinetics of phenytoin were assessed in the same manner. In the menthol-treated group, the area under the blood concentration-time curve (AUC) of triazolam was lower and its clearance was higher compared with the control group. The CYP3A metabolic activity and CYP3A expression level in the liver were significantly increased in the menthol-treated group compared with the control group. Similarly, the AUC of phenytoin was lower and the hepatic CYP2C expression level was higher in the menthol-treated group. Thus, menthol lowered the plasma concentrations of triazolam and phenytoin when concurrently administered. These effects may be attributed to an increased metabolic activity for these drugs due to the increased expression of CYP3A and CYP2C in the liver.

Cytochrome b5 is a major determinant of human cytochrome P450 CYP2D6 and CYP3A4 activity in vivo.

The cytochrome P450-dependent mono-oxygenase system is responsible for the metabolism and disposition of chemopreventive agents, chemical toxins and carcinogens, and >80% of therapeutic drugs. Cytochrome P450 (P450) activity is regulated transcriptionally and by the rate of electron transfer from P450 reductase. In vitro studies have demonstrated that cytochrome b5 (Cyb5) also modulates P450 function. We recently showed that hepatic deletion of Cyb5 in the mouse (HBN) markedly alters in vivo drug pharmacokinetics; a key outstanding question is whether Cyb5 modulates the activity of the major human P450s in drug disposition in vivo. To address this, we crossed mice humanized for CYP2D6 or CYP3A4 with mice carrying a hepatic Cyb5 deletion. In vitro triazolam 4-hydroxylation (probe reaction for CYP3A4) was reduced by >50% in hepatic microsomes from CYP3A4-HBN mice compared with controls. Similar reductions in debrisoquine 4-hydroxylation and metoprolol α-hydroxylation were observed using CYP2D6-HBN microsomes, indicating a significant role for Cyb5 in the activity of both enzymes. This effect was confirmed by the concentration-dependent restoration of CYP3A4-mediated triazolam turnover and CYP2D6-mediated bufuralol and debrisoquine turnover on addition of Escherichia coli membranes containing recombinant Cyb5. In vivo, the peak plasma concentration and area under the concentration time curve from 0 to 8 hours (AUC0-8 h) of triazolam were increased 4- and 5.7-fold, respectively, in CYP3A4-HBN mice. Similarly, the pharmacokinetics of bufuralol and debrisoquine were significantly altered in CYP2D6-HBN mice, the AUC0-8 h being increased ∼1.5-fold and clearance decreased by 40-60%. These data demonstrate that Cyb5 can be a major determinant of CYP3A4 and CYP2D6 activity in vivo, with a potential impact on the metabolism, efficacy, and side effects of numerous therapeutic drugs.

Physiologically based pharmacokinetic modeling of CYP3A4 induction by rifampicin in human: influence of time between substrate and inducer administration.

The induction of cytochrome P450 enzymes (CYPs) is an important source of drug-drug interaction (DDI) and can result in pronounced changes in pharmacokinetics (PK). Rifampicin (RIF) is a potent inducer of CYP3A4 and also acts as a competitive inhibitor which can partially mask the induction. The objective of this study was to determine a clinical DDI study design for RIF resulting in maximum CYP3A4 induction. A physiologically based pharmacokinetic (PBPK) model was developed to project the dynamics and magnitude of CYP3A4 induction in vivo from in vitro data generated with primary human hepatocytes. The interaction model included both inductive and inhibitory effects of RIF on CYP3A4 in gut and liver and accounting for the observed RIF auto-induction. The model has been verified for 4 CYP3A4 substrates: midazolam, triazolam, alfentanil and nifedipine using plasma concentration data from 20 clinical study designs with intravenous (n=7) and oral (n=13) administrations. Finally, the influence of the time between RIF and substrate administration was explored for the interaction between midazolam and RIF. The model integrating in vitro induction parameters correctly predicted intravenous induction but underestimated oral induction with 30% of simulated concentrations more than 2-fold higher than of observed data. The use of a 1.6-fold higher value for the maximum induction effect (Emax) improved significantly the accuracy and precision of oral induction with 82% of simulated concentrations and all predicted PK parameters within 2-fold of observed data. Our simulations suggested that a concomitant administration of RIF and midazolam resulted in significant competitive inhibition limited to intestinal enzyme. Accordingly, a maximum induction effect could be achieved with a RIF pretreatment of 600 mg/day during 5 days and a substrate administration at least 2 h after the last RIF dose. A period of 2 weeks after RIF removal was found sufficient to allow return to baseline levels of enzyme.

Hepatic early inflammation induces downregulation of hepatic cytochrome P450 expression and metabolic activity in the dextran sulfate sodium-induced murine colitis.

Ulcerative colitis (UC) patients may have increased concentrations of drugs in their blood. We hypothesized that this response is mainly due to a decrease in the expression and activity of the drug-metabolizing enzyme, cytochrome P450 (CYP), in the liver. In this study, we have tried to demonstrate the hypothesis. UC was induced in mice by treatment with dextran sulfate sodium (DSS) solution. The mRNA and protein expression levels of CYP, inflammatory cytokine levels, and the metabolic activity of CYP3A in the liver were measured. The nuclear translocations of nuclear factor kappa B (NF-κB), pregnane X receptor (PXR), and constitutive androstane receptor (CAR) were analyzed. The levels of hepatic inflammatory cytokines increased in the DSS-treated group. The hepatic mRNA and protein expression of CYP (CYP1A, CYP2C, CYP2D, CYP2E, and CYP3A) and the CYP3A metabolic activity significantly decreased compared to the control group. Hepatic NF-κB nuclear translocation significantly increased in the DSS-treated group. In contrast, the nuclear translocations of PXR and CAR were decreased. Lipopolysaccharides from inflammatory sites in the colon induce hepatic inflammation in DSS-induced murine colitis. This inflammation then causes an increase in the nuclear translocation of hepatic NF-κB and a decrease in the nuclear translocation of PXR and CAR, resulting in the decreased expression and activities of CYP. The results of this study indicated that at the onset of UC, the decreased activity of hepatic CYP causes an increase in the concentrations of drugs in the blood, leading to an increase in the incidence of adverse reactions.

Is pomegranate juice a potential perpetrator of clinical drug-drug interactions? Review of the in vitro, preclinical and clinical evidence.

The area of fruit juice-drug interaction has received wide attention with numerous scientific and clinical investigations performed and reported for scores of drugs metabolized by CYP3A4/CYP2C9. While grapefruit juice has been extensively studied with respect to its drug-drug interaction potential, numerous other fruit juices such as cranberry juice, orange juice, grape juice, pineapple juice and pomegranate juice have also been investigated for its potential to show drug-drug interaction of any clinical relevance. This review focuses on establishing any relevance for clinical drug-drug interaction potential with pomegranate juice, which has been shown to produce therapeutic benefits over a wide range of disease areas. The review collates and evaluates relevant published in vitro, preclinical and clinical evidence of the potential of pomegranate juice to be a perpetrator in drug-drug interactions mediated by CYP3A4 and CYP2C9. In vitro and animal pharmacokinetic data support the possibility of CYP3A4/CYP2C9 inhibition by pomegranate juice; however, the human relevance for drug-drug interaction was not established based on the limited case studies.

Different diets cause alterations in the enteric environment and trigger changes in the expression of hepatic cytochrome P450 3A, a drug-metabolizing enzyme.

Changes in the expression level and activity of cytochrome P450 (CYP) in the liver are caused by various factors and affect the pharmacokinetics of drugs. The purpose of this study was to determine whether the expression of CYP3A is affected by a high-fat diet. In addition, we examined whether the type of diet given to mice could produce changes in the expression level and activity of CYP3A. Mice were fed a purified diet containing 10 kcal% lard (control group) or 60 kcal% lard (HF group) or regular mouse chow containing 13 kcal% of fat (MF group) for 4 weeks. No significant differences were observed in the hepatic CYP3A protein expression level between the HF group and the control group. The CYP3A protein expression in the MF group was significantly higher than that observed in the control group. In the MF group, the area under the curve (AUC) of intraperitoneally administered triazolam was lower. Because lithocholic acid (LCA) is known to increase hepatic CYP3A expression, the levels of Clostridium sordellii and LCA in the feces were measured. In the MF group, the levels of Clostridium sordellii and LCA were higher. It has been demonstrated that a high-fat diet does not cause any changes in hepatic CYP3A expression. In addition, the different diets caused alterations in the enteric environment, which triggered changes in CYP3A expression. Therefore, it is necessary to carefully consider the type of feed while performing animal experiments to evaluate the pharmacokinetics of drugs.

Evaluation of animal models for intestinal first-pass metabolism of drug candidates to be metabolized by CYP3A enzymes via in vivo and in vitro oxidation of midazolam and triazolam.

1. To search an appropriate evaluation methodology for the intestinal first-pass metabolism of new drug candidates, grapefruit juice (GFJ)- and vehicle (tap water)-pretreated mice or rats were orally administered midazolam (MDZ) or triazolam (TRZ), and blood levels of the parent compounds and their metabolites were measured by liquid chromatography/MS/MS. A significant effect of GFJ to elevate the blood levels was observed only for TRZ in mice. 2. In vitro experiments using mouse, rat and human intestinal and hepatic microsomal fractions demonstrated that GFJ suppressed the intestinal microsomal oxidation of MDZ and especially TRZ. Substrate inhibition by MDZ caused reduction in 1'-hydroxylation but not 4-hydroxylation in both intestinal and hepatic microsomal fractions. The kinetic profiles of MDZ oxidation and the substrate inhibition in mouse intestinal and hepatic microsomal fractions were very similar to those in human microsomes but were different from those in rat microsomes. Furthermore, MDZ caused mechanism-based inactivation of cytochrome P450 3A-dependent TRZ 1'-hydroxylation in mouse, rat and human intestinal microsomes with similar potencies. 3. These results are useful information in the analysis of data obtained in mouse and rat for the evaluation of first-pass effects of drug candidates to be metabolized by CYP3A enzymes.

Trans-chromosomic mice containing a human CYP3A cluster for prediction of xenobiotic metabolism in humans.

Human CYP3A is the most abundant P450 isozyme present in the human liver and small intestine, and metabolizes around 50% of medical drugs on the market. The human CYP3A subfamily comprises four members (CYP3A4, CYP3A5, CYP3A7, CYP3A43) encoded on human chromosome 7. However, transgenic mouse lines carrying the entire human CYP3A cluster have not been constructed because of limitations in conventional cloning techniques. Here, we show that the introduction of a human artificial chromosome (HAC) containing the entire genomic human CYP3A locus recapitulates tissue- and stage-specific expression of human CYP3A genes and xenobiotic metabolism in mice. About 700 kb of the entire CYP3A genomic segment was cloned into a HAC (CYP3A-HAC), and trans-chromosomic (Tc) mice carrying a single copy of germline-transmittable CYP3A-HAC were generated via a chromosome-engineering technique. The tissue- and stage-specific expression profiles of CYP3A genes were consistent with those seen in humans. We further generated mice carrying the CYP3A-HAC in the background homozygous for targeted deletion of most endogenous Cyp3a genes. In this mouse strain with 'fully humanized' CYP3A genes, the kinetics of triazolam metabolism, CYP3A-mediated mechanism-based inactivation effects and formation of fetal-specific metabolites of dehydroepiandrosterone observed in humans were well reproduced. Thus, these mice are likely to be valuable in evaluating novel drugs metabolized by CYP3A enzymes and in studying the regulation of human CYP3A gene expression. Furthermore, this system can also be used for generating Tc mice carrying other human metabolic genes.

Consumption of a high-fat diet during pregnancy decreases the activity of cytochrome P450 3a in the livers of offspring.

Recent studies have reported that a high-fat diet during pregnancy exerts various effects on the foetus and newborn. The purpose of this study was to clarify the effects of a high-fat diet during pregnancy on the activity of hepatic cytochrome P450 (Cyp) 3a in offspring in mice. The protein expression level and activity of Cyp3a in the livers of 6-week-old mice born to mothers that were given a high-fat diet during pregnancy (HF group) decreased significantly compared with the Control group. Triazolam, which is a substrate of Cyp3a, was intraperitoneally administered to the mice in the HF group. Compared with the Control group, an increase in the area under the plasma concentration-time curve and a decrease in total clearance were observed in the HF group. The hepatic constitutive androstane receptor (CAR) mRNA expression level in the HF group was significantly lower than that in the Control group. An increase in phosphorylation of extracellular signal-regulated kinase (ERK) was also observed in the HF group. The results of this study revealed that a high-fat diet during pregnancy causes an increase in ERK phosphorylation and a decrease in the expression level of CAR in the livers of offspring, which leads to decreased Cyp3a expression and activity. The results suggest that individual differences in pharmacokinetics may not only be expressed by genetic predisposition but also by a mother's living environment during pregnancy.

Effects of dosing interval on the pharmacokinetic and pharmacodynamic interactions between the oral adsorbent AST-120 and triazolam in humans.

OBJECTIVE: The objective of this study was to evaluate the pharmacokinetic and pharmacodynamic interactions between the oral adsorbent AST-120 and triazolam. METHODS: In this randomized, cross-over study, 12 healthy volunteers received a single oral dose of triazolam 0.25 mg alone or with AST-120 2 g given 0, 30 or 60 min before triazolam administration. RESULTS: The area under the plasma triazolam concentration-time curve (AUC(0-∞)) significantly decreased with simultaneous AST-120 + triazolam (alone vs simultaneous: 10.9 ± 6.0 vs 6.4 ± 2.6 ng·h/mL, p = 0.003). Triazolam-induced impairment in psychomotor performance assessed by the digit symbol substitution test was significantly attenuated when AST-120 was administered simultaneously. No significant changes in pharmacokinetic and pharmacodynamic parameters were observed when AST-120 was given 30 or 60 min before triazolam administration. CONCLUSIONS: Administering AST-120 simultaneously with triazolam affects the pharmacokinetics and pharmacodynamics of triazolam. Dosing AST-120 at least 30 min before triazolam administration may avoid these interactions.