Metabolism Of Midazolam Research Articles

Cannabidiol Inhibits In Vitro Human Liver Microsomal Metabolism of Remdesivir.

Introduction: The year 2020 began with the world being flounced with a wave of novel coronavirus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) disease, named COVID-19. Based on promising pre-clinical and clinical data, remdesivir (RDV) was the first drug to receive FDA approval and so far, it is the most common therapy for treatment of SARS-CoV-2/MERS-CoV. However, following intravenous administration, RDV metabolizes majorly by human liver carboxylesterase 1 (CES1) and marginally by the CYP3A4 enzyme in merely less than an hour. Its resultant active metabolite is a hydrophilic nucleoside with very limited accumulation within lung tissues. Therefore, there is a need to investigate strategies to overcome such premature metabolism issues and improve the antiviral efficacy of RDV at the target site. Objective: Considering the major CES1-mediated metabolism of RDV on systemic administration, we intend to explore the remarkable CES1 plus CYP3A4 inhibitory activity of cannabidiol (CBD) against in vitro microsomal metabolism of RDV to indicate its therapeutic potential as an adjuvant to RDV in the treatment and management of COVID-19. Methods: We investigated the in vitro human liver microsomal metabolism of RDV in the presence of two potential CES1 inhibitors-CBD and nelfinavir, and two standard CYP3A4 inhibitors-ritonavir (RITO) and cyclosporin A. The microsomal metabolism assay was further validated by using a well-characterized CYP3A4-selective substrate, midazolam (MDZ), in the presence of CBD and RITO. Results: Our findings depicted that RDV was rapidly and completely metabolized by human liver microsomes within 60 min. Coincubation with CBD substantially reduced microsomal metabolism of RDV and prolonged its in vitro half-life from 8.93 to 31.07 min. CBD showed significantly higher inhibition of RDV compared with known CES1 and CYP3A4 inhibitors. Inhibition of MDZ metabolism by CBD and RITO further validated the assay. Conclusions: The current study strongly suggests that CBD significantly inhibits human liver microsomal metabolism of RDV and extends its in vitro half-life. Thus, concomitant administration of CBD with RDV intravenous injection could be a promising strategy to prevent premature metabolism in COVID-19 patients.

Lack of inhibitory effect of wood creosote on cytochrome P450‐mediated drug metabolism

ABSTRACTAimWood creosote (WC), an over‐the‐counter (OTC) drug, has been used as an anti‐diarrheal agent for many years; however, its safety in combination with other drugs has not been adequately investigated. The current study was conducted to evaluate whether WC may cause drug interactions through the inhibition of cytochrome P450 (CYP).MethodsThe inhibitory effects of WC on ethoxyresorufin O‐deethylation, paclitaxel 6α‐hydroxylation, S‐warfarin 7‐hydroxylation, S‐mephenytoin 4′‐hydroxylation, bufuralol1′‐hydroxylation, and midazolam (MDZ) 1′‐hydroxylation were investigated using human liver microsomes as the marker activities for CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, respectively. In addition, the effects of single and three‐day multiple treatments of WC (30 mg/kg) on CYP2C‐mediated tolbutamide (TB, 20 mg/kg) and CYP3A‐mediated MDZ (15 mg/kg) metabolism were investigated in rats in vivo.ResultsWC at clinical doses did not show a considerable inhibitory effect on any of the CYP activities examined in the in vitro study. CYP2C9 and CYP2C19 activities were inhibited by high concentrations of the WC (100 μg/mL); however, the IC50 values were approximately 70 times higher than the reported circulating levels of the main ingredients in WC. Furthermore, there were no marked changes in the pharmacokinetic parameters of TB and MDZ (area under the serum concentration–time curve, maximum serum concentrations, and elimination half‐life) after single or three‐day multiple treatments with WC.ConclusionThese results suggest that clinical doses of WC are unlikely to cause drug interactions mediated by the inhibition of CYPs.

Time-dependent inhibition of CYP3A4-mediated midazolam metabolism by macrolide antibiotics in CYP3A4 genetic variants: Comparison with testosterone metabolism.

Objectives: The present study aimed to evaluate the effects of CYP3A4 genetic variation on the kinetics of mechanism-based inhibition (MBI) of both inhibitors using midazolam as a substrate for comparison with our previous study, as midazolam and testosterone have different binding sites. Background: The genetic variation of cytochrome P450 (CYP) 3A4 affects MBI, expressed as the maximum inactivation rate constant (k inact,max) and the inhibitor concentration required to achieve half-maximal inactivation (K I). We previously showed, using testosterone as a substrate, that the MBI kinetics of erythromycin and clarithromycin differ among CYP3A4 variants. Materials and methods: Midazolam 1’-hydroxylation inactivation profiles of erythromycin and clarithromycin were assessed using recombinant CYP3A4.1, .2, .7, .16, and .18 expressed in Escherichia coli. MBI parameters were calculated from changes in the inactivation rate constant (Δk obs) by the inhibitors. Results: Both inhibitors increased Δk obs value in a concentration- and preincubation time-dependent manner, and MBI kinetics differed among variants. Trends of differences in MBI parameters among variants were similar to those assessed using testosterone as a substrate; K I decreased for CYP3A4.7, and k inact,max decreased for CYP3A4.2, .7, and .16. Conclusion: The genetic variation of recombinant CYP3A4 affects the MBI profile of CYP3A4 by erythromycin and clarithromycin, while the influence of genetic variation was similarly observed regardless of substrates. Our findings are of clinical relevance because the residual enzyme activity of CYP3A4 in the presence of inhibitor was estimated to vary among genetic variants.

Complex Cytochrome P450 Kinetics Due to Multisubstrate Binding and Sequential Metabolism. Part 2. Modeling of Experimental Data.

Three CYP3A4 substrates, midazolam, ticlopidine, and diazepam, display non-Michaelis-Menten kinetics, form multiple primary metabolites, and are sequentially metabolized to secondary metabolites. We generated saturation curves for these compounds and analyzed the resulting datasets using a number of single-substrate and multisubstrate binding models. These models were parameterized using rate equations and numerical solutions of the ordinary differential equations. Multisubstrate binding models provided results superior to single-substrate models, and simultaneous modeling of multiple metabolites provided better results than fitting the individual datasets independently. Although midazolam datasets could be represented using standard two-substrate models, more complex models that include explicit enzyme-product complexes were needed to model the datasets for ticlopidine and diazepam. In vivo clearance predictions improved markedly with the use of in vitro parameters from the complex models versus the Michaelis-Menten equation. The results highlight the need to use sufficiently complex kinetic schemes instead of the Michaelis-Menten equation to generate accurate kinetic parameters. SIGNIFICANCE STATEMENT: The metabolism of midazolam, ticlopidine, and diazepam by CYP3A4 results in multiple metabolites and sequential metabolism. This study evaluates the use of rate equations and numerical methods to characterize the in vitro enzyme kinetics. Use of complex cytochrome P450 kinetic models is necessary to obtain accurate parameter estimates for predicting in vivo disposition.

UPLC-MS/MS analysis of the Michaelis-Menten kinetics of CYP3A-mediated midazolam 1′- and 4-hydroxylation in rat brain microsomes

Midazolam (MDZ) is a short-acting benzodiazepine with rapid onset of action, which is metabolized by CYP3A isoenzymes to two hydroxylated metabolites, 1′-hydroxymidazolam and 4-hydroxymidazolam. The drug is also commonly used as a marker of CYP3A activity in the liver microsomes. However, the kinetics of CYP3A-mediated hydroxylation of MDZ in the brain, which contains much lower CYP content than the liver, have not been reported. In this study, UPLC-MS/MS and metabolic incubation methods were developed and validated for simultaneous measurement of low concentrations of both hydroxylated metabolites of MDZ in brain microsomes. Different concentrations of MDZ (1–500 µM) were incubated with rat brain microsomes (6.25 µg) and NADPH over a period of 10 min. After precipitation of the microsomal proteins with acetonitrile, which contained individual isotope-labeled internal standards for each metabolite, the analytes were separated on a C18 UPLC column and detected by a tandem mass spectrometer. Accurate quantitation of MDZ metabolism in the brain microsomes presented several challenges unique to this tissue, which were resolved. The optimized method showed validation results in accordance with the FDA acceptance criteria, with a linearity ranging from 1 to 100 nM and a lower limit of quantitation of 0.4 pg on the column for each of the two metabolites. The method was successfully used to determine the Michaelis-Menten (MM) kinetics of MDZ 1′- and 4-hydroxylase activities in rat brain microsomes (n = 5) for the first time. The 4-hydroxylated metabolite had 2.4 fold higher maximum velocity (p < 0.01) and 1.9 fold higher (p < 0.05) MM constant values than the 1′-hydroxylated metabolite. However, intrinsic clearance values of the two metabolites were similar. The optimized analytical and metabolic incubation methods reported here may be used to study the effects of various pathophysiological and pharmacological factors on the CYP3A-mediated metabolism of MDZ in the brain.

Homotropic Cooperativity of Midazolam Metabolism by Cytochrome P450 3A4: Insight from Computational Studies.

Human cytochrome P450 3A4 (CYP3A4) is responsible for the metabolism of ∼50% clinically used drugs. Midazolam (MDZ) is a commonly used sedative drug and serves as a marker substrate for the CYP3A4 activity assessment. MDZ is metabolized by CYP3A4 to two hydroxylation products, 1′-OH-MDZ and 4-OH-MDZ. It has been reported that the ratio of 1′-OH-MDZ and 4-OH-MDZ is dependent on the MDZ concentration, which reflects the homotropic cooperative behavior in MDZ metabolism by CYP3A4. Here, we used quantum chemistry (QC), molecular docking, conventional molecular dynamics (cMD), and Gaussian accelerated molecular dynamics (GaMD) approaches to investigate the mechanism of the interactions between CYP3A4 and MDZ. QC calculations suggest that C1′ is less reactive for hydroxylation than C4, which is a pro-chirality carbon. However, the 4-OH-MDZ product is likely to be racemic due to the chirality inversion in the rebound step. The MD simulation results indicate that MDZ at the peripheral allosteric site is not stable and the binding modes of the MDZ molecules at the productive site are in line with the experimental observations.

Hepatic Cytochrome P450 Abundance and Activity in the Developing and Adult Göttingen Minipig: Pivotal Data for PBPK Modeling.

The Göttingen Minipig is gaining ground as nonrodent species in safety testing of drugs for pediatric indications. Due to developmental changes in pharmacokinetics and pharmacodynamics, physiologically based pharmacokinetic (PBPK) models are built to better predict drug exposure in children and to aid species selection for nonclinical safety studies. These PBPK models require high quality physiological and ADME data such as protein abundance of drug metabolizing enzymes. These data are available for man and rat, but scarce for the Göttingen Minipig. The aim of this study was to assess hepatic cytochrome P450 (CYP) protein abundance in the developing Göttingen Minipig by using mass spectrometry. In addition, sex-related differences in CYP protein abundance and correlation of CYP enzyme activity with CYP protein abundance were assessed. The following age groups were included: gestational day (GD) 84–86 (n = 8), GD 108 (n = 6), postnatal day (PND) 1 (n = 8), PND 3 (n = 8), PND 7 (n = 8), PND 28 (n = 8) and adult (n = 8). Liver microsomes were extracted and protein abundance was compared to that in adult animals. Next, the CYP protein abundance was correlated to CYP enzyme activity in the same biological samples. In general, CYP protein abundance gradually increased during development. However, we observed a stable protein expression over time for CYP4A24 and CYP20A1 and for CYP51A1, a high protein expression during the fetal stages was followed by a decrease during the first month of life and an increase toward adulthood. Sex-related differences were observed for CYP4V2_2a and CYP20A1 at PND 1 with highest expression in females for both isoforms. In the adult samples, sex-related differences were detected for CYP1A1, CYP1A2, CYP2A19, CYP2E1_2, CYP3A22, CYP4V2_2a and CYP4V2_2b with higher values in female compared to male Göttingen Minipigs. The correlation analysis between CYP protein abundance and CYP enzyme activity showed that CYP3A22 protein abundance correlated clearly with the metabolism of midazolam at PND 7. These data are remarkably comparable to human data and provide a valuable step forward in the construction of a neonatal and juvenile Göttingen Minipig PBPK model.

Tertiary Oxidation of Deoxycholate Is Predictive of CYP3A Activity in Dogs.

Deoxycholic acid (DCA, 3α, 12α-dihydroxy-5β-cholan-24-oic acid) is the major circulating secondary bile acid, which is synthesized by gut flora in the lower gut and selectively oxidized by CYP3A into tertiary metabolites, including 1β,3α,12α-trihydroxy-5β-cholan-24-oic acid (DCA-1β-ol) and 3α,5β,12α-trihydroxy-5β-cholan-24-oic acid (DCA-5β-ol) in humans. Since DCA has the similar exogenous nature and disposition mechanisms as xenobiotics, this work aimed to investigate whether the tertiary oxidations of DCA are predictive of in vivo CYP3A activities in beagle dogs. In vitro metabolism of midazolam (MDZ) and DCA in recombinant canine CYP1A1, 1A2, 2B11, 2C21, 2C41, 2D15, 3A12, and 3A26 enzymes clarified that CYP3A12 was primarily responsible for either the oxidation elimination of MDZ or the regioselective oxidation metabolism of DCA into DCA-1β-ol and DCA-5β-ol in dog liver microsomes. Six male dogs completed the CYP3A intervention studies including phases of baseline, inhibition (ketoconazole treatments), recovery, and induction (rifampicin treatments). The oral MDZ clearance after a single dose was determined on the last day of the baseline, inhibition, and induction phases, and subjected to correlation analysis with the tertiary oxidation ratios of DCA detected in serum and urine samples. The results confirmed that the predosing serum ratios of DCA oxidation, DCA-5β-ol/DCA, and DCA-1β-ol/DCA were significantly and positively correlated both intraindividually and interindividually with oral MDZ clearance. It was therefore concluded that the tertiary oxidation of DCA is predictive of CYP3A activity in beagle dogs. Clinical transitional studies following the preclinical evidence are promising to provide novel biomarkers of the enterohepatic CYP3A activities. SIGNIFICANCE STATEMENT: Drug development, clinical pharmacology, and therapeutics are under insistent demands of endogenous CYP3A biomarkers that avoid unnecessary drug exposure and invasive sampling. This work has provided the first proof-of-concept preclinical evidence that the CYP3A catalyzed tertiary oxidation of deoxycholate, the major circulating secondary bile acid synthesized in the lower gut by bacteria, may be developed as novel in vivo biomarkers of the enterohepatic CYP3A activities.

Midazolam and hydroxymidazolam plasma concentrations can be monitored with selected biochemical and physiological parameters of palliative care patients

Rationale & objectiveMidazolam is one of top three drugs used in palliative care. Its use increases in the last days of hospice patients’ lives while safe dosage can be challenging. Equations currently used to estimate glomerular filtration rate, e.g: the Cockroft-Gault (eGFRCR) and the Modification of Diet in Renal Disease (eGFRMDRD) ones, do not generate precise calculations, especially in palliative patients exhibiting variations in body parameters. Our aim was to seek new relationships between mean midazolam (Mavg) and alfahydroxymidazolam (OH-Mavg) concentrations in plasma, and selected biochemical and physiological parameters of palliative patients, to enable optimal midazolam pharmacotherapy. Study design, participants and interventionsThe pilot study included 11 Caucasians, aged 42–95, with advanced cancer disease, receiving midazolam in a hospice in-patient unit. We tested correlations among Mavg, BMI, eGFRMDRD, midazolam clearance (CL), OH-Mavg, bilirubin (Bil) and blood creatinine concentration (Cr). F test and leave-one out (LOO) validation was applied to verify the correlations’ significance and predictive ability. ResultsWe found ten statistically significant (p < 0.05) correlations related to midazolam pharmacokinetics and physiological factors. We formulated two equations with high degree of predictive ability, based on the eGFRMDRD→CL and the (Bil + BMI × Ln(Cr))→Mavg-(OH-Mavg) correlations.The limitations of the study mainly revolve around its pilot nature and the need to continue testing the results on a bigger population.No funding to disclose. ConclusionsThe significance of correlations corresponding to the arithmetic expressions confirms that Bil, BMI, Ln(Cr) analyzed simultaneously report a series of processes on which midazolam metabolism depends. Two of ten correlations proposed came close to meet all LOO validation criteria. Current findings can help optimize midazolam treatment in palliative therapy.

Effect of ceritinib on the pharmacokinetics of coadministered CYP3A and 2C9 substrates: a phase I, multicenter, drug\u2013drug interaction study in patients with ALK\u2009+\u2009advanced tumors

PurposeCeritinib is an ALK receptor tyrosine kinase inhibitor approved as first- and second-line treatment in adult patients with ALK + metastatic non-small cell lung cancer (NSCLC). The study investigated the drug–drug interaction (DDI) potential of ceritinib when coadministered with midazolam and warfarin as probe substrates for CYP3A and CYP2C9 activity, respectively.MethodsThis was a phase I, multicenter, open-label, single sequence, crossover DDI study in 33 adult patients with ALK + NSCLC or other advanced tumors. A single dose of a cocktail consisting of midazolam and warfarin was administered with and without concomitant administration of ceritinib. The primary objective was to evaluate the pharmacokinetics of midazolam and warfarin. Secondary objectives included pharmacokinetics, safety, tolerability, overall response rate (ORR), and duration of response (DOR) of ceritinib 750 mg once daily.ResultsCeritinib inhibited CYP3A-mediated metabolism of midazolam, resulting in a markedly increased AUC (geometric mean ratio [90% confidence interval]) by 5.4-fold (4.6, 6.3). Ceritinib also led to an increase in the AUC of S-warfarin by 54% (36%, 75%). The pharmacokinetics and safety profile of ceritinib in this study are consistent with previous reports and no new safety signals were reported. Among the 19 patients with NSCLC, efficacy (ORR: 42.1% and DCR: 63.2%) was similar to that reported previously in studies of pretreated patients with ALK + NSCLC.ConclusionCeritinib is a strong CYP3A inhibitor and a weak CYP2C9 inhibitor. These findings should be reflected as actionable clinical recommendations in the prescribing information for ceritinib with regards to concomitant medications whose pharmacokinetics may be altered by ceritinib.

Development of a method to determine cytochrome P450 1A2, 2C9, 2D6 and 3A4 activity sheep hepatic microsomes

IntroductionEx vivo studies of human fetal hepatic drug metabolism are uncommon as it requires access to functional liver tissue and therefore raises practical and ethical concerns. Large animal models provide an alternative opportunity to study changes in cytochrome P450 (CYP) activity in the mother and fetus during pregnancy. We aimed to develop methods to determine the activity of CYP1A2, CYP2C9, CYP2D6 and CYP3A4 in sheep hepatic microsomes. MethodsWe identified optimal conditions to determine the activity of CYP1A2 (using the probe drug phenacetin), CYP2C9 (diclofenac), CYP2D6 (dextromethorphan) and CYP3A4 (midazolam) by varying techniques for microsome extraction, probe drug concentration, incubation time and microsome concentration. The specificity of each probe drug was assessed by determining the rate of metabolism when specific CYP enzyme inhibitors were included in the reaction. ResultsThe optimum incubation time and probe drug concentration was six hours with 5 μM phenacetin (CYP1A2), four hours with 10 μM diclofenac (CYP2C9), 30 min with 1 μM of midazolam (CYP3A4) and 10 min with 1 μM dextromethorphan (CYP2D6). For both CYP2D6 and CYP3A4 reactions required 20 μg of microsomal protein, whereas for CYP1A2 and CYP2C9, reactions required 40 μg of microsomal protein. Metabolism of phenacetin, dextromethorphan and midazolam was reduced by specific enzyme inhibitors, but the specific CYP2C9 inhibitor sulfaphenazole did not substantially inhibit diclofenac metabolism. DiscussionThis study identifies the optimal conditions for determining CYP activity in maternal sheep hepatic microsomes. In doing so, we have developed a standardised protocol for assessment of microsomal activity of CYP3A4, CYP1A2 and CYP2D6, but we were unable to optimise conditions for assessment of CYP2C9. This approach can be applied to investigate the impact of pregnancy complications on maternal and fetal hepatic drug metabolism.

PBPK modeling of CYP3A and P-gp substrates to predict drug-drug interactions in patients undergoing Roux-en-Y gastric bypass surgery.

Roux-en-Y gastric bypass surgery (RYGBS) is an effective surgical intervention to reduce mortality in morbidly obese patients. Following RYGBS, the disposition of drugs may be affected by anatomical alterations and changes in intestinal and hepatic drug metabolizing enzyme activity. The aim of this study was to better understand the drug-drug interaction (DDI) potential of CYP3A and P-gp inhibitors. The impacts of RYGBS on the absorption and metabolism of midazolam, acetaminophen, digoxin, and their major metabolites were simulated using physiologically-based pharmacokinetic (PBPK) modeling. PBPK models for verapamil and posaconazole were built to evaluate CYP3A- and P-gp-mediated DDIs pre- and post-RYGBS. The simulations suggest that for highly soluble drugs, such as verapamil, the predicted bioavailability was comparable pre- and post-RYGBS. For verapamil inhibition, RYGBS did not affect the fold-change of the predicted inhibited-to-control plasma AUC ratio or predicted inhibited-to-control peak plasma concentration ratio for either midazolam or digoxin. In contrast, the predicted bioavailability of posaconazole, a poorly soluble drug, decreased from 12% pre-RYGBS to 5% post-RYGBS. Compared to control, the predicted posaconazole-inhibited midazolam plasma AUC increased by 2.0-fold pre-RYGBS, but only increased by 1.6-fold post-RYGBS. A similar trend was predicted for pre- and post-RYGBS inhibited-to-control midazolam peak plasma concentration ratios (2.0- and 1.6-fold, respectively) following posaconazole inhibition. Absorption of highly soluble drugs was more rapid post-RYGBS, resulting in higher predicted midazolam peak plasma concentrations, which was further increased following inhibition by verapamil or posaconazole. To reduce the risk of a drug-drug interaction in patients post-RYGBS, the dose or frequency of object drugs may need to be decreased when administered with highly soluble inhibitor drugs, especially if toxicities are associated with plasma peak concentrations.

Effects of Dexmedetomidine on the Pharmacokinetics of Dezocine, Midazolam and Its Metabolite 1-Hydroxymidazolam in Beagles by UPLC-MS/MS.

ObjectiveWe developed and validated a sensitive and reliable UPLC-MS/MS method for simultaneous determination of dezocine (DEZ), midazolam (MDZ) and its metabolite 1-hydroxymidazolam (1-OH-MDZ) in beagle plasma and investigated the effect of dexmedetomidine (DEX) on the pharmacokinetics of DEZ, MDZ and 1-OH-MDZ in beagles.Materials and MethodsDiazepam was used as the internal standard (IS); the three analytes and IS were extracted by acetonitrile precipitation and separated on an Acquity UPLC BEH C18 column using acetonitrile-0.1% formic acid as mobile phase in gradient mode. In positive ion mode, the three analytes and IS were monitored by multiple reaction monitoring (MRM). Six beagles were designed as a double cycle self-control experiment with 0.15 mg/kg in the first cycle (Group A). After a 1-week washout period, the same six beagles were slowly injected intravenously with 2 µg/kg DEX in the second cycle (Group B), with continuous injection for 7 days. On the seventh day, 0.5 hr after intravenous injection of 2 µg/kg DEX, the six beagles were intramuscularly given with DEZ 0.33 mg/kg and MDZ 0.15 mg/kg.ResultsUnder the conditions of this experiment, this method exhibited a good linearity for each analyte. The accuracy and precision were all within the acceptable limits in the bioanalytical method, and the results of recovery, matrix effect and stability have also met the requirements.ConclusionThe developed UPLC-MS/MS method for simultaneous determination of DEZ, MDZ and 1-OH-MDZ in beagles plasma was accurate, reproducible, specific, and suitable. DEX could inhibit the metabolism of DEZ and MDZ and increase the exposure of DEZ and MDZ in beagles. Therefore, the change of therapeutic effect and the occurrence of adverse reactions caused by drug–drug interaction should be paid attention to when the drugs were used in combination.

A Novel Study Design Using Continuous Intravenous and Intraduodenal Infusions of Midazolam and Voriconazole for Mechanistic Quantitative Assessment of Hepatic and Intestinal CYP3A Inhibition.

The extent of a drug-drug interaction (DDI) mediated by cytochrome P450 (CYP) 3A inhibitors is highly variable during a dosing interval, as it depends on the temporal course of victim and perpetrator drug concentrations at intestinal and hepatic CYP3A expression sites. Capturing the time course of inhibition is therefore difficult using standard DDI studies assessing changes in area under the curve; thus, a novel design was developed. In a 4-period changeover pilot study, 6 healthy men received intraduodenal or intravenous infusions of the CYP3A substrate midazolam (MDZ) at a rate of 0.26 mg/h for 24 hours. This was combined with intraduodenal or intravenous infusion of the CYP3A inhibitor voriconazole (VRZ), administered at rates of 7.5 mg/h from 8 to 16 hours and of 15 mg/h from 16 to 24 hours, after starting midazolam administration. Plasma and urine concentrations of VRZ, MDZ, and its major metabolites were quantified by liquid chromatography-tandem mass spectrometry and analyzed by semiphysiological population pharmacokinetic nonlinear mixed-effects modeling. A model including mechanism-based inactivation of the metabolizing enzymes (maximum inactivation rate constant kinact , 2.83 h-1 ; dissociation rate constant , 9.33 μM) described the pharmacokinetics of VRZ well. By introducing competitive inhibition by VRZ on primary and secondary MDZ metabolism, concentration-time profiles, MDZ and its metabolites were captured appropriately. The model provides estimates of local concentrations of substrate and inhibitor at the major CYP3A expression sites and thus of the respective dynamic extent of inhibition. A combination of intravenous and intraduodenal infusions of inhibitors and substrates has the potential to provide a more accurate assessment of DDIs occurring in both gut wall and liver.

Inhibitory Effect of Lygodium Root on the Cytochrome P450 3A Enzyme in vitro and in vivo.

PurposeThe aim of the present study was to investigate the interactions of the main components of Lygodium root (ie, p-coumaric acid, acacetin, apigenin, buddleoside and Diosmetin-7-O-β-D-glucopyranoside) with cytochrome P450 3A enzyme activity both in vitro and in vivo.MethodsIn vitro inhibition of drugs was assessed by incubating rat liver microsomes (RLMs) with a typical P450 3A enzyme substrate, midazolam, to determine their 50% inhibitory concentration (IC50) values. For the in vivo study, healthy male Sprague Dawley rats were consecutively administered acacetin or apigenin for 7 days at the dosage of 5 mg/kg after being randomly divided into 3 groups: Group A (control group), Group B (acacetin group) and Group C (apigenin group).ResultsAmong the five main components of Lygodium root, only acacetin and apigenin showed inhibitory effects on the cytochrome P450 3A enzyme in vitro. The IC50 values of acacetin and apigenin were 58.46 μM and 8.20 μM, respectively. Additionally, the in vivo analysis results revealed that acacetin and apigenin could systemically inhibit midazolam metabolism in rats. The Tmax, AUC(0-t) and Cmax of midazolam in group B and group C were significantly increased (P<0.05), accompanied by a significant decrease in Vz/F and CLz/F (P<0.05).ConclusionAcacetin and apigenin could inhibit the activity of the cytochrome P450 3A enzyme in vitro and in vivo, indicating that herbal drug interactions might occur when taking Lygodium root and midazolam synchronously.

Effects of Vitamin D Receptor, Cytochrome P450 3A, and Cytochrome P450 Oxidoreductase Genetic Polymorphisms on the Pharmacokinetics of Remimazolam in Healthy Chinese Volunteers.

Remimazolam is a new ultra-short-acting benzodiazepine used to induce and maintain anesthesia and procedural sedation. Its compound structure is similar to midazolam's. Midazolam metabolism might be affected by vitamin D receptor (VDR), cytochrome P450 3A, and cytochrome P450 oxidoreductase genetic polymorphisms. This study investigated the effects of VDR, cytochrome P450 3A, and cytochrome P450 oxidoreductase genetic polymorphisms on the pharmacokinetics of remimazolam in healthy Chinese volunteers after a single intravenous injection of remimazolam besylate. Blood samples were collected from subjects (n = 62) at scheduled time intervals before and after injection. High-performance liquid chromatography-tandem mass spectrometrywas used to quantify plasma remimazolam and RF7054 (its inactive carboxylic acid metabolite) concentrations. The relationship between plasma remimazolam concentration, pharmacokinetic parameters, and polymorphic alleles was assessed for each subject. The rs4516035 allele affected the elimination half-life of RF7054 (P = .043), while the rs1544410 allele affected the dose-normalized maximum observed plasma concentration (Cmax /D) of remimazolam (P = .025) in 46 volunteers. Results showed that VDR genetic polymorphisms might affect the pharmacokinetics of remimazolam in the Chinese population.

Numerical method analysis of the activation and time‐dependent inhibition of midazolam metabolism by ticlopidine

Drug metabolism plays an important role in the pharmacokinetic and pharmacodynamic properties of drug candidates. In most cases, drug metabolizing enzymes (DMEs) display non‐Michalis‐Menten or atypical kinetics. Atypical kinetics includes activation, autoactivation, partial inhibition, substrate inhibition, and biphasic saturation curves. These kinetics make it difficult to obtain in vitro kinetic constants to be used for in vivo predictions of drug‐drug interactions. Numerical methods offer a unique tool to estimate improved kinetic constants. In this study, we use ordinary differential equations to model the activation and time‐dependent inactivation of midazolam (MDZ) hydroxylation by ticlopidine. Ticlopidine (TCP) is the first thioenopyridine‐based prodrug developed to inhibit platelet aggregation. Time‐dependent inactivation (TDI) assays showed both activation and partial inactivation of midazolam hydroxylation. Co‐incubation of MDZ with different TCP concentrations (0–250 μM) activated the formation of 1‐hydroxymidazolam, increasing kcat by 30%. Additional experiments are currently underway to model the observed activation and time‐dependent inactivation of CYP‐mediated metabolism of MDZ by TCP.Support or Funding InformationFunding: NIGMS grants R01GM114369 and R01GM104178.

Vitamin D3 Is Transformed into 1,25(OH)2D3 by Triggering CYP3A11(CYP3A4) Activity and Hydrolyzing Midazolam.

BackgroundVitamin D3 (VD3) is a commonly used supplement in clinical practice. Cytochrome P450 3A11 (CYP3A11) is the most important monomeric enzyme involved in metabolism of drugs. This study aimed to investigate effects of vitamin D3 (VD3) on CYP3A11 activity.Material/MethodsForty male Sprague-Dawley (SD) rats were randomly divided a Control group (peanut oil 0.1 ml/kg/d), a Low-VD3 group (100 IU/kg/d), a Medium-VD3 group (400 IU/kg/d), and a High-VD3 (1600 IU/kg/d) group. Blood samples were collected from the jugular vein after midazolam (MDZ) administration. CYP3A11 expressions in liver and colon were detected by Western blotting and immunohistochemistry (IHC) assay. The concentration of serum 25(OH)D3 and serum 1,25(OH)2D3 were evaluated using ELISA. Effects of different dosages of vitamin D3 on metabolism of MDZ were evaluated using high-performance liquid chromatography (HPLC).ResultsVitamin D3 significantly enhanced serum 25(OH)D3 and 1,25(OH)2D3 levels in rats compared to Control rats (p<0.05). Expressions of hepatic CYP3A11 were more than 10-fold higher in rats treated with vitamin D3 compared to Control rats (p<0.05). Expressions of colon CYP3A11 were 5-fold higher than in Control rats (p<0.05). CYP3A11 expressions in vitamin D3-treated groups were significantly higher compared to the Control group (p<0.05). MDZ levels were significantly higher in vitamin D3-treated rats compared to that in Control rats (p<0.05). Concentrations of serum MDZ at every sampling point were remarkably lower in the vitamin D3-treated rats than in Control rats (p<0.05).ConclusionsVitamin D3 was transformed into 1,25(OH)2D3 by triggering CYP3A11 and CYP3A11 activity and by hydrolyzing MDZ.

Effects of dexamethasone to reverse decreased hepatic midazolam metabolism in rats with acute renal failure

The inductive effects of dexamethasone on hepatic midazolam metabolism were examined in Wistar rats with acute renal failure (ARF) to clarify whether the ARF-related decrease in the hepatic expression of drug-metabolizing enzymes is caused by an impairment in the translation/polypeptide formation process.ARF was induced with intramuscular glycerol injection. Dexamethasone was orally administered. Pooled liver microsomes from five rats were prepared with ultracentrifugation for each of four groups, namely, control and ARF rats, control rats with dexamethasone treatment and ARF rats with dexamethasone treatment.Hepatic drug-metabolizing activity was examined in an incubation study with the microsomes, where midazolam was employed as a substrate of cytochrome P450 (CYP) 3A enzymes. The hepatic protein and mRNA expressions of CYP3A23/3A1 and 3A2 enzymes were also evaluated.With dexamethasone treatment, the hepatic metabolic rate of midazolam increased 1.4 times in control rats, while it increased 19.6 times in ARF rats, reflecting the greater induction of hepatic protein expressions of CYP3A enzymes in ARF rats than in control rats.The hepatic protein expression process for CYP3A23/3A1 and 3A2 responds well to dexamethasone treatment in ARF rats, indicating that the translation/polypeptide formation process is not impaired in the presence of ARF.

Indinavir Increases Midazolam N-Glucuronidation in Humans: Identification of an Alternate CYP3A Inhibitor Using an In Vitro to In Vivo Approach.

Midazolam is a widely used index substrate for assessing effects of xenobiotics on CYP3A activity. A previous study involving human hepatocytes showed the primary route of midazolam metabolism, 1'-hydroxylation, shifted to N-glucuronidation in the presence of the CYP3A inhibitor ketoconazole, which may lead to an overprediction of the magnitude of a xenobiotic-midazolam interaction. Because ketoconazole is no longer recommended as a clinical CYP3A inhibitor, indinavir was selected as an alternate CYP3A inhibitor to evaluate the contribution of the N-glucuronidation pathway to midazolam metabolism. The effects of indinavir on midazolam 1'-hydroxylation and N-glucuronidation were first characterized in human-derived in vitro systems. Compared with vehicle, indinavir (10 μM) inhibited midazolam 1'-hydroxylation by recombinant CYP3A4, human liver microsomes, and high-CYP3A activity cryopreserved human hepatocytes by ≥70%; the IC50 obtained with hepatocytes (2.7 μM) was within reported human unbound indinavir Cmax (≤5 μM). Midazolam N-glucuronidation in hepatocytes increased in the presence of indinavir in both a concentration-dependent (1-33 μM) and time-dependent (0-4 hours) manner (by up to 2.5-fold), prompting assessment in human volunteers (n = 8). As predicted by these in vitro data, indinavir was a strong inhibitor of the 1'-hydroxylation pathway, decreasing the 1'-hydroxymidazolam/midazolam area under the plasma concentration versus time curve (AUC)0-12h ratio by 80%. Although not statistically significant, the midazolam N-glucuronide/midazolam AUC0-12h ratio increased by 40%, suggesting a shift to the N-glucuronidation pathway. The amount of midazolam N-glucuronide recovered in urine increased 4-fold but remained <10% of the oral midazolam dose (2.5 mg). A powered clinical study would clarify whether N-glucuronidation should be considered when assessing the magnitude of a xenobiotic-midazolam interaction.