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Drug metabolism in the intestinal wall affects bioavailability of orally administered drugs and is influenced by age. Hence, it is important to fully understand the drug metabolizing capacity of the gut to predict systemic exposure. The aim of this study was to investigate the potential of enteroids as a tool to study CYP3A4/5 -mediated metabolism in both children and adults. Bioconversion of midazolam, a CYP3A4/5 model substrate, was studied using enteroid monolayers as well as tissue explants in the Ussing chamber, both derived from pediatric [median (range age): 54 weeks (2 days - 13 years), n = 21] and adult (n = 5) tissue. Caco-2 cellular monolayers were employed as controls. In addition, mRNA expression of CYP3A4 was determined in enteroid monolayers (n = 11), tissue (n = 23) and Caco-2 using RT-qPCR. Midazolam metabolism was successfully detected in all enteroid monolayers, as well as in all tissue explants studied in the Ussing chamber, whereas Caco-2 showed no significant metabolite formation. The extracted fraction of midazolam was similar between enteroid monolayers and tissue. The fraction of midazolam extracted increased with age in enteroid monolayers derived from 0 to 70 week old donors. No statistically significant correlation was observed in tissue likely due to high variability observed and the smaller donor numbers included in the study. At the level of gene expression, CYP3A4 increased with age in tissues (n = 32), while this was not reflected in enteroid monolayers (n = 16). Notably, asymmetric metabolite formation was observed in enteroids and tissue, with higher metabolite formation on the luminal side of the barrier. In summary, we demonstrated that enteroids can be used to measure CYP3A4/5 midazolam metabolism, which we show is similar as observed in fresh isolated tissue. This was the case both in children and adults, indicating the potential of enteroids to predict intestinal metabolism. This study provides promising data to further develop enteroids to study drug metabolism in vitro and potentially predict oral absorption for special populations as an alternative to using fresh tissue., Competing Interests: Declaration of competing interest All authors declared no competing interests for this work., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Hepatic impairment, due to liver cirrhosis, decreases the activity of cytochrome P450 enzymes (CYPs). The use of physiologically based pharmacokinetic (PBPK) modeling to predict this effect for CYP substrates has been well-established, but the effect of cirrhosis on uridine-glucuronosyltransferase (UGT) activities is less studied and few PBPK models have been reported. UGT enzymes are involved in primary N -glucuronidation of midazolam and glucuronidation of 1'-OH-midazolam following CYP3A hydroxylation. In this study, Simcyp was used to establish PBPK models for midazolam, its primary metabolites midazolam- N -glucuronide (UGT1A4) and 1'-OH midazolam (CYP3A4/3A5), and the secondary metabolite 1'-OH-midazolam- O -glucuronide (UGT2B7/2B4), allowing to simulate the impact of liver cirrhosis on the primary and secondary glucuronidation of midazolam. The model was verified in noncirrhotic subjects before extrapolation to cirrhotic patients of Child-Pugh (CP) classes A, B, and C. Our model successfully predicted the exposures of midazolam and its metabolites in noncirrhotic and cirrhotic patients, with 86% of observed plasma concentrations within 5th-95th percentiles of predictions and observed geometrical mean of area under the plasma concentration curve between 0 hours to infinity and maximal plasma concentration within 0.7- to 1.43-fold of predictions. The simulated metabolic ratio defined as the ratio of the glucuronide metabolite AUC over the parent compound AUC (AUC glucuronide /AUC parent , metabolic ratio [MR]), was calculated for midazolam- N -glucuronide to midazolam (indicative of UGT1A4 activity) and decreased by 40% (CP A), 48% (CP B), and 75% (CP C). For 1'-OH-midazolam- O -glucuronide to 1'-OH-midazolam, the MR (indicative of UGT2B7/2B4 activity) dropped by 35% (CP A), 51% (CP B), and 64% (CP C). These predicted MRs were corroborated by the observed data. This work thus increases confidence in Simcyp predictions of the effect of liver cirrhosis on the pharmacokinetics of UGT1A4 and UGT2B7/UGT2B4 substrates. SIGNIFICANCE STATEMENT: This article presents a physiologically based pharmacokinetic model for midazolam and its metabolites and verifies the accurate simulation of pharmacokinetic profiles when using the Simcyp hepatic impairment population models. Exposure changes of midazolam- N -glucuronide and 1'-OH-midazolam- O -glucuronide reflect the impact of decreases in UGT1A4 and UGT2B7/2B4 glucuronidation activity in cirrhotic patients. The approach used in this study may be extended to verify the modeling of other uridine glucuronosyltransferase enzymes affected by liver cirrhosis., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

We previously reported novel benzyl-ether derivatives with an imidazole ring and a hydroxyl group (A-01) or carboxyl group (B-01) and esters (2 esters of A-01, and 7 esters of B-01) as pharmacokinetics (PK) boosters. This study demonstrates how these ester compounds embody the concept of a safe pharmacokinetic booster, with potent and transient inhibition of CYP3A4-mediated drug metabolism. As a model CYP3A4 substrate and CYP3A4 enzyme, midazolam (MDZ) and rat liver microsomes were used. A-01 inhibited MDZ metabolism significantly, while B-01 induced only slight inhibition. Although rat liver microsomes hydrolyzed the ester compounds over time, several ester compounds strongly inhibited MDZ metabolism. Due to the significant activity of A-01, A-01 esters affected MDZ metabolism, irrespective of hydrolysis state. Time-dependent inhibition evaluation indicated that the B-01 ester inhibition is not mechanism-based, as hydrolysis eliminated MDZ metabolism inhibition. We report that the B-01 esters significantly inhibit CYP3A4-mediated drug metabolism, and upon hydrolysis this property is eliminated. In conclusion, B-01 ester compounds may be safe PK boosters with antedrug characteristics., (Copyright © 2024 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

In vitro-in vivo extrapolation (IVIVE) allows prediction of clinical outcomes across populations from in vitro data using specific scalars tailored to the biologic characteristics of each population. This study experimentally determined scalars for patients with varying degrees of nonalcoholic fatty liver disease (NAFLD), ranging from fatty liver to nonalcoholic steatohepatitis (NASH) and cirrhosis. Microsomal, S9, and cytosol fractions were extracted from 36 histologically normal and 66 NAFLD livers (27 nonalcoholic fatty liver [NAFL], 13 NASH, and 26 NASH with cirrhosis). Corrected microsomal protein per gram liver (MPPGL) progressively decreased with disease severity (26.8, 27.4, and 24.3 mg/g in NAFL, NASH, and NASH/cirrhosis, respectively, compared with 35.6 mg/g in normal livers; ANOVA, P < 0.001). Homogenate, S9, and cytosolic protein showed a consistent trend of decline in NASH/cirrhosis relative to normal control (post-hoc t test, P < 0.05). No differences across the groups were observed in homogenate, S9, cytosolic, and microsomal protein content in matched kidney samples. MPPGL-based scalars that combine protein content with liver size revealed that the reduction in MPPGL in NAFL and NASH was compensated by the reported increase in liver size (relative scalar ratios of 0.96 and 0.99, respectively), which was not the case with NASH/cirrhosis (ratio of 0.63), compared with healthy control. Physiologically based pharmacokinetics-informed global sensitivity analysis of the relative contribution of IVIVE scalars (hepatic CYP3A4 abundance, MPPGL, and liver size) to variability in exposure (area under the curve) to three CYP3A substrates (alprazolam, midazolam, and ibrutinib) revealed enzyme abundance as the most significant parameter, followed by MPPGL, whereas liver volume was the least impactful factor. SIGNIFICANCE STATEMENT: Nonalcoholic fatty liver disease-specific scalars necessary for extrapolation from in vitro systems to liver tissue are lacking. These are required in clearance prediction and dose selection in nonalcoholic fatty liver and steatohepatitis populations. Previously reported disease-driven changes have focused on cirrhosis, with no data on the initial stages of liver disease. The authors obtained experimental values for microsomal, cytosolic, and S9 fractions and assessed the relative impact of microsomal scalars on predicted exposure to substrate drugs using physiologically based pharmacokinetics., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

The well-stirred model (WSM) incorporating the fraction of unbound drug (f u ) to account for the effect of plasma binding on intrinsic clearance has been widely used for predicting hepatic clearance under the assumption that drug protein binding reaches equilibrium instantaneously. Our theoretical analysis reveals that the effect of protein binding on intrinsic clearance is better accounted for with the dynamic free fraction (f D ), a measure of drug protein binding affinity, which leads to a putative dynamic well-stirred model (dWSM) without the instantaneous equilibrium assumption. Using recombinant CYP3A4 as the in vitro clearance system, we demonstrate that the binding effect of albumin on the intrinsic clearance of both highly bound midazolam and highly free verapamil is fully corrected by their corresponding f D values, respectively. On the other hand, f u only corrects the binding effect of albumin on the intrinsic clearance of verapamil, and yields severe over-correction of the intrinsic clearance of midazolam. The results suggest that the traditional WSM is suitable for highly free drugs like verapamil but not necessarily for highly bound drugs such as midazolam due to the violation of the instantaneous equilibrium assumption or under-estimating the true free drug concentration. In comparison, the dWSM incorporating f D holds true as long as drug elimination follows steady-state kinetics, and hence, it is more broadly applicable to drugs with different protein binding characteristics. Here we demonstrate with 36 diverse drugs, that the dWSM significantly improves the accuracy of predicting human hepatic clearance and liver extraction ratio from in vitro microsomal clearance data, highlighting the importance of drug plasma protein binding kinetics in addressing the under-prediction of hepatic clearance by the WSM., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of more drugs in clinical use than any other xenobiotic-metabolizing enzyme. CYP3A4-mediated drug metabolism is usually allosterically modulated by substrate concentration (homotropic allostery) and other drugs (heterotropic allostery), exhibiting unusual kinetic profiles and regiospecific metabolism. Recent studies suggest that residue Phe108 (F108) of CYP3A4 may have an important role in drug metabolism. In this work, residue mutations were coupled with well-tempered metadynamics simulations to assess the importance of F108 in the allosteric effects of midazolam metabolism. Comparing the simulation results of the wild-type and mutation systems, we identify that the π-π interaction and steric effect between the F108 side chain and midazolam is favorable for the stable binding of substrate in the active site. F108 also plays an important role in the transition of substrate binding mode, which mainly induces the transition of substrate binding mode by forming π-π interactions with multiple aromatic rings of the substrate. Moreover, the side chain of F108 is closely related to the radius and depth of the 2a and 2f channels, and F108 may further regulate drug metabolism by affecting the pathway, orientation, or time of substrate entry into the CYP3A4 active site or product egress from the active site. Altogether, we suggest that F108 affects drug metabolism and regulatory mechanisms by affecting substrate binding stability, binding mode transition, and channel characteristics of CYP3A4. Our findings could promote the understanding of complicated allosteric mechanisms in CYP3A4-mediated drug metabolism, and the knowledge could be used for drug development and disease treatment.

In the early stages of drug discovery, adequate evaluation of the potential drug-drug interactions (DDIs) of drug candidates is important. Several CYP3A activators are known to lead to underestimation of DDIs. These compounds affect midazolam 1'-hydroxylation but not midazolam 4-hydroxylation.We used both metabolic reactions of midazolam to evaluate the activation and inhibition of CYP3A activators simultaneously. For our CYP inhibition assay using cocktail probe substrates, simultaneous liquid chromatography-tandem mass spectrometry monitoring of 1'-hydroxymidazolam and 4-hydroxymidazolam for CYP3A was established in addition to monitoring of 4-hydroxydiclofenac and 1'-hydroxybufuralol for CYP2C9 and CYP2D6.The results of our cocktail inhibition assay were well correlated with those of a single inhibition assay, as were the estimated inhibition parameters for typical CYP3A inhibitors. In our assay, a proprietary compound that activated midazolam 1'-hydroxylation and tended to inhibit 4-hydroxylation was evaluated along with known CYP3A activators. All compounds were well characterised by comparison of the results of midazolam 1'- and 4-hydroxylation.In conclusion, our CYP cocktail inhibition assay can detect CYP3A activation and assess the direct and time-dependent inhibition potentials for CYP3A, CYP2C9, and CYP2D6. This method is expected to be very efficient in the early stages of drug discovery.

Cytochrome P450 3A (CYP3A) participates in the metabolism of more than 30% of clinical drugs. The vast intra- and inter-individual variations in CYP3A activity pose great challenges to drug development and personalized medicine. It has been disclosed that human CYP3A4 and CYP3A7 are exclusively responsible for the tertiary oxidations of deoxycholic acid (DCA) and glycodeoxycholic acid (GDCA) regioselectivity at C-1 β and C-5 β This work aimed to compare the 1 β - and 5 β -hydroxylation of DCA and GDCA as potential in vitro CYP3A index reactions in both human liver microsomes and recombinant P450 enzymes. The results demonstrated that the metabolic activity of DCA 1 β - and 5 β -hydroxylation was 5-10 times higher than that of GDCA, suggesting that 1 β -hydroxyglycodeoxycholic acid and 5 β -hydroxyglycodeoxycholic acid may originate from DCA oxidation followed by conjugation in humans. Metabolic phenotyping data revealed that DCA 1 β -hydroxylation, DCA 5 β -hydroxylation, and GDCA 5 β -hydroxylation were predominantly catalyzed by CYP3A4 (>80%), while GDCA 1 β -hydroxylation had approximately equal contributions from CYP3A4 (41%) and 3A7 (58%). Robust Pearson correlation was established for the intrinsic clearance of DCA 1 β - and 5 β -hydroxylation with midazolam (MDZ) 1'- and 4-hydroxylation in fourteen single donor microsomes. Although DCA 5 β -hydroxylation exhibited a stronger correlation with MDZ oxidation, DCA 1 β -hydroxylation exhibited higher reactivity than DCA 5 β -hydroxylation. It is therefore suggested that DCA 1 β - and 5 β -hydroxylations may serve as alternatives to T 6 β -hydroxylation as in vitro CYP3A index reactions. SIGNIFICANCE STATEMENT: The oxidation of DCA and GDCA is primarily catalyzed by CYP3A4 and CYP3A7. This work compared the 1β- and 5β-hydroxylation of DCA and GDCA as in vitro index reactions to assess CYP3A activities. It was disclosed that the metabolic activity of DCA 1β- and 5β-hydroxylation was 5-10 times higher than that of GDCA. Although DCA 1β-hydroxylation exhibited higher metabolic activity than DCA 5β-hydroxylation, DCA 5β-hydroxylation demonstrated stronger correlation with MDZ oxidation than DCA 1β-hydroxylation in individual liver microsomes., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

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.

Graphical abstract Abstract Human CYP3A4 (Cyp3a11 in mice) is one of the most important enzymes for drug metabolism and detoxification. Here, we aimed to investigate a potential role for E4bp4 in regulation of Cyp3a11 expression and activity. The regulatory effects of E4bp4 on Cyp3a11 enzyme were assessed using E4bp4 −/− mice and Hepa-1c1c7 cells. The mRNA and protein levels were quantified using qPCR and Western blotting, respectively. In vitro microsomal Cyp3a11 activity was probed using its specific substrates midazolam and testosterone. Pharmacokinetic studies were performed with wild-type and E4bp4 −/− mice after midazolam administration. Global deletion of E4bp4 led to significant upregulation of Cyp3a11 mRNA and protein in major metabolic organs (i.e., the liver, kidney and small intestine). E4bp4 ablation also caused an increased microsomal Cyp3a11 activity consistent with the enzyme's expression change. Overexpression of E4bp4 in Hepa-1c1c7 cells resulted in reduced levels of Cyp3a11 mRNA and protein, whereas E4bp4 knockdown caused upregulation of Cyp3a11 expression. In addition, the systemic exposure of midazolam was lowered in E4bp4 −/− mice compared with wild-type mice. This was accompanied by enhanced formation of its metabolite 1′-hydroxymidazolam. Furthermore, luciferase reporter and mobility shift assays revealed that E4bp4 repressed Cyp3a11 transcription via direct binding to C-site (−1539/−1529 bp) in the promoter region. In conclusion, E4bp4 negatively regulates Cyp3a11 expression, thereby impacting drug metabolism and pharmacokinetics. [ABSTRACT FROM AUTHOR]

The current study was designed to investigate the influence of allosteric effectors on the metabolism of the prototypical cytochrome P450 (CYP) 3A4 substrate midazolam (MDZ), and on the determination in vitro time-dependent inhibition (TDI) of CYP3A4 using human liver microsomes (HLM). As the concentration of midazolam increased to 250 µ M in HLMs, homotropic cooperativity resulted in a decrease in the 1'-hydroxymidazolam to 4-hydroxymidazolam ratio to a maximum of 1.1. The presence of varying concentrations of testosterone, progesterone (PGS), or carbamazepine (CBZ) in HLMs with MDZ could recapitulate the effect of homotropic cooperativity such that the formation rates of the 1'hydroxymidazolam and 4-hydroxymidazolam were equal even at low concentrations of MDZ. The presence of PGS (10 or 100 µ M) and CBZ (100 or 1000 µ M) in in vitro TDI determination of four known CYP3A4 time-dependent inactivators (clarithromycin, troleandomycin, mibefradil, raloxifene) simultaneously decreased potency and inactivation rate constant, resulting in fold changes in inactivation efficiency on average of 1.6-fold and 13-fold for the low and high concentrations of allosteric modulator tested, respectively. The formation of a metabolic-intermediate complex (MIC) for clarithromycin and troleandomycin decreased in the presence of the allosteric modulators in a concentration-dependent manner, reaching a new steady state formation that could not be overcome with increased incubation time. Maximum reduction of the MIC formed by clarithromycin was up to ∼91%, while troleandomycin MIC decreased up to ∼31%. These findings suggest that the absence of endogenous allosteric modulators may contribute to the poor translation of HLM-based drug-drug interaction predictions. SIGNIFICANCE STATEMENT: The reported overprediction of in vitro human liver microsome time-dependent inhibition of CYP3A4 and observed drug interactions in vivo remains an issue in drug development. We provide characterization of allosteric modulators on the CYP3A4 metabolism of the prototypical substrate midazolam, demonstrating the ability of the modulators to recapitulate the homotropic cooperativity of midazolam. Furthermore, we demonstrate that allosteric heterotropic cooperativity of CYP3A4 can impact the time-dependent inhibition kinetics of known mechanisms-based inhibitors, providing a potential mechanism to explain the overprediction., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)

Background and Objective: The impact of liver cirrhosis on the activity of UDP-glucuronosyltransferases (UGTs) is currently not well characterized. We investigated the glucuronidation capacity and glucuronide accumulation in patients with liver cirrhosis., Methods: We administered the Basel phenotyping cocktail (caffeine, efavirenz, flurbiprofen, omeprazole, metoprolol, midazolam) to patients with liver cirrhosis (n = 16 Child A, n = 15 Child B, n = 5 Child C) and n = 12 control subjects and obtained pharmacokinetic profiles of substrates and primary metabolites and their glucuronides., Results: Caffeine and its metabolite paraxanthine were only slightly glucuronidated. The metabolic ratio (AUC glucuronide /AUC parent , MR) was not affected for caffeine but decreased by 60% for paraxanthine glucuronide formation in Child C patients. Efavirenz was not glucuronidated whereas 8-hydroxyefavirenz was efficiently glucuronidated. The MR of 8-hydroxyefavirenz-glucuronide formation increased three-fold in Child C patients and was negatively correlated with the glomerular filtration rate. Flurbiprofen and omeprazole were not glucuronidated. 4-Hydroxyflurbiprofen and 5-hydroxyomeprazole were both glucuronidated but the corresponding MRs for glucuronide formation were not affected by liver cirrhosis. Metoprolol, but not α-hydroxymetoprolol, was glucuronidated, and the MR for metoprolol-glucuronide formation dropped by 60% in Child C patients. Both midazolam and its metabolite 1'-hydroxymidazolam underwent glucuronidation, and the corresponding MRs for glucuronide formation dropped by approximately 80% in Child C patients. No relevant glucuronide accumulation occurred in patients with liver cirrhosis., Conclusions: Detailed analysis revealed that liver cirrhosis may affect the activity of UGTs of the UGT1A and UGT2B subfamilies according to liver function. Clinically significant glucuronide accumulation did not occur in the population investigated., Clinical Trial Registration: NCT03337945., (© 2023. The Author(s).)

Drug oxidations are mediated mainly by cytochromes P450 (P450s or CYPs). CYP3As are an important P450 subfamily and include liver-specific CYP3A12 and intestine-specific CYP3A98 in dogs. Individual differences in drug oxidation activities were investigated, including correlations with immunoreactive CYP3A protein intensities and CYP3A mRNA expression levels in livers.Pooled and individual dog liver microsomes showed activities towards nifedipine, midazolam, alprazolam, and estradiol, but the levels of catalytic activities varied approximately twofold among the individual dogs. One dog harboured a CYP1A2 variant causing protein deletion but showed higher activities than the other dogs towards nifedipine oxidation, midazolam 1'-hydroxylation, alprazolam 4-hydroxylation, estradiol 16α-hydroxylation activities, and caffeine C 8 -hydroxylation; the latter is used as a reference reaction for CYP1A.In individual dog liver microsomes, the intensities of the immunochemical bands with anti-human CYP3A4 and anti-rat CYP3A2 antibodies along with CYP3A12 and CYP3A26 mRNA expression levels showed good correlations ( p  < 0.05) with nifedipine oxidation, midazolam 1'- and 4-hydroxylation, alprazolam 1'- and 4-hydroxylation, and estradiol 16α-hydroxylation activities.These results suggest that the oxidation activities of dog liver microsomes towards nifedipine and other typical CYP3A-catalyzed drugs exhibit approximately twofold individual differences and were predominantly mediated by liver-specific CYP3A12 in the dogs.

In this study, we investigated the effect of type 1 diabetes mellitus on the modulation of the activities of CYP450s in dynamics by a UHPLC-MS/MS method. The diabetic rat model was constructed by an intraperitoneal single injection of streptozotocin. Fasting blood glucose levels > 16.7 mmol/L were considered as diabetic. The rats were given a cocktail of four probe drugs (10 mg/kg phenacetin, 1 mg/kg tolbutamide, 10 mg/kg metoprolol, and 10 mg/kg midazolam) by oral administration for the pharmacokinetic study. Thereafter, the metabolic ratio (MR) of the metabolites to probe substrates were determined. The results indicated that two weeks after diabetes was induced, diabetes increased the MRs of acetaminophen/phenacetin (CYP1A2) and 4-hydroxyl tolbutamide/tolbutamide (CYP2C9); however, it decreased the MRs of α-hydroxy metoprolol/metoprolol (CYP2D6) and 1-hydroxy midazolam/midazolam (CYP3A4). Two months after diabetes was induced, diabetes increased the MRs of acetaminophen/phenacetin and 4-hydroxyl tolbutamide/tolbutamide. The MR of α-hydroxy metoprolol/metoprolol was decreased and the MR of 1-hydroxy midazolam/midazolam was increased but the difference was not significant. According to the results, CYP1A2 and CYP2C9 activities were enhanced in the diabetic rats. and CYP2D6 activity was inhibited in a short period of diabetes; however, the decrease in CYP2D6 activity was not significant in the long period. CYP3A4 activity was decreased in a short period of diabetes and increased in a long period of diabetes but was not significant in the two periods. This study suggests the activity change rule of the CYP450 enzyme system in diabetes mellitus, which can provide a reference for precise clinical medication., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Cytochrome P450 3A4 (CYP3A4) is the main P450 enzyme for drug metabolism and drug-drug interactions (DDIs), as it is involved in the metabolic process of approximately 50% of drugs. A detailed mechanistic elucidation of DDIs mediated by CYP3A4 is commonly believed to be critical for drug optimization and rational use. Here, two typical probes, midazolam (MDZ, substrate) and testosterone (TST, allosteric effector), are used to investigate the molecular mechanism of CYP3A4-mediated heterotropic allosteric interactions, through conventional molecular dynamics (cMD) and well-tempered metadynamics (WT-MTD) simulations. Distance monitoring shows that TST can stably bind in two potential peripheral sites (Site 1 and Site 2) of CYP3A4. The binding of TST at these two sites can induce conformational changes in CYP3A4 flexible loops on the basis of conformational analysis, thereby promoting the transition of the MDZ binding mode and affecting the ratio of MDZ metabolites. According to the results of the residue interaction network, multiple allosteric communication pathways are identified that can provide vivid and applicable insights into the heterotropic allostery of TST on MDZ metabolism. Comparing the regulatory effects and the communication pathways, the allosteric effect caused by TST binding in Site 2 seems to be more pronounced than in Site 1. Our findings could provide a deeper understanding of CYP3A4-mediated heterotropic allostery at the atomic level and would be helpful for rational drug use as well as the design of new allosteric modulators.

Human cytochrome P450 CYP3A4 is involved in the processing of more than 35% of current pharmaceuticals and therefore is responsible for multiple drug-drug interactions (DDI). In order to develop a method for the detection and prediction of the possible involvement of new drug candidates in CYP3A4-mediated DDI, we evaluated the application of midazolam (MDZ) as a probe substrate. MDZ is hydroxylated by CYP3A4 in two positions: 1-hydroxy MDZ formed at lower substrate concentrations, and up to 35% of 4-hydroxy MDZ at high concentrations. The ratio of the formation rates of these two products (the site of metabolism ratio, SOM) was used as a measure of allosteric heterotropic interactions caused by effector molecules using CYP3A4 incorporated in lipid nanodiscs. The extent of the changes in the SOM in the presence of effectors is determined by chemical structure and is concentration-dependent. MD simulations of CYP3A4 in the lipid bilayer suggest that experimental results can be explained by the movement of the F-F' loop and concomitant changes in the shape and volume of the substrate-binding pocket. As a result of PGS binding at the allosteric site, several residues directly contacting MDZ move away from the substrate molecule, enabling the repositioning of the latter for minor product formation.

Central nervous system (CNS) dysfunction induced by sepsis and pathogenic microbial infections is reported to be closely associated with increased permeability of the blood-brain barrier (BBB), which is mainly mediated by the stimulation of lipopolysaccharide (LPS) on inflammatory signaling. Midazolam is a novel sedative acting on the benzodiazepine receptor, which is recently reported to exert a neuroprotective effect by inhibiting inflammation. The present study will explore the potential repair capacity of Midazolam on LPS-induced damage to the BBB. The in vivo mice model was established by intraperitoneal injection of LPS, while the in vitro model was constructed by stimulating endothelial cells utilizing LPS. We found that the increased malondialdehyde (MDA) level and reduced superoxide dismutase (SOD) activity in the brain cortices, promoted serum concentration of inflammatory factors, and elevated BBB permeability were found in the LPS group, all of which were dramatically reversed by 1 mg/kg and 2 mg/kg Midazolam. Interestingly, Midazolam increased the expression of the tight junction protein zonula occludens-1 (ZO-1). In LPS-challenged in vitro human brain microvascular endothelial cells (HBMECs), the increased concentration of inflammatory factors, reduced trans-endothelial electrical resistance (TEER) level, elevated relative value of trans-endothelial permeability, and downregulated ZO-1 were observed, all of which were pronouncedly alleviated by Midazolam, accompanied by the inhibition on the Ras homolog family member A/ Rho-kinase 2 (RhoA/ROCK-2) pathway. Furthermore, the regulatory effects of Midazolam on ZO-1 expression and the endothelial monolayer permeability in LPS-challenged HBMECs were abolished by the overexpression of RhoA. Collectively, our data imply that Midazolam ameliorated the impairment of the BBB against LPS by regulating the RhoA/ROCK2 pathway., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Cytochrome P450 3A (CYP3A), the most important class of drug-metabolizing enzymes, participates in the metabolism of half of clinically used drugs. The CYP3A index reactions of dogs, one of the most widely used preclinical nonrodent species, are still poorly understood. This work evaluated the activity and selectivity of 10 CYP3A index reactions, including midazolam (MDZ) 1'- and 4-hydroxylation, alprazolam (APZ) and triazolam (TRZ) α - and 4-hydroxylation, testosterone (T) 6 β -hydroxylation, lithocholate (LCA) 6 α -hydroxylation, deoxycholate (DCA) 1 β - and 5 β -hydroxylation, with quantitative reaction phenotyping and kinetic analysis in human and canine recombinant CYP enzymes (rCYPs). In human studies, all reactions are reconfirmed as mixed index reactions of CYP3A with minor contributions from non-CYP3A isoforms. In canine studies, all reactions are also primarily catalyzed by CYP3A12 with lower contributions from CYP3A26. However, the canine CYP2B11 appreciably contributes to the hydroxylation of benzodiazepines except for APZ 4-hydroxylation. The canine CYP3A isoforms have lower activity than human isoforms toward T 6 β -hydroxylation and LCA 6 α -hydroxylation and both substrates undergo non-CYP3A catalyzed side reactions. DCA 1 β - and 5 β -hydroxylation are validated as the CYP3A index reactions in both humans and dogs with limited non-CYP3A contributions and side reactions. In conclusion, this work provides a comprehensive overview for the selectivity and activity of in vitro CYP3A index reactions in humans and dogs. The validated CYP3A index reactions between humans and dogs may benefit future practices in drug metabolism and drug interaction studies. SIGNIFICANCE STATEMENT: Dogs are one of the most important nonrodent animals with limited studies of cytochrome P450 enzymes than humans. This work provides the most comprehensive quantitative data to date for the selectivity and activity of CYP3A index reactions in humans and dogs. The canine CYP2B11 was found to appreciably contribute to hydroxylation of midazolam, alprazolam and triazolam, the well-known probes for human CYP3A. Deoxycholate 1 β - and 5 β -hydroxylation are validated as the CYP3A index reactions in both humans and dogs., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Acetaminophen (APAP)-induced liver injury (AILI) is the leading cause of acute liver failure in the United States, but its impact on metabolism, therapeutic efficacy, and adverse drug reactions (ADRs) of co- and/or subsequent administered drugs are not fully investigated. The current work explored this field with a focus on the AILI-mediated alterations of cytochrome P450-mediated drug metabolism. Various levels of liver injury were induced in mice by treatment with APAP at 0, 200, 400, and 600 mg/kg. Severity of liver damage was determined at 24, 48, 72, and 96 hours by plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), microRNA miR122, and tissue staining. The expression and activities of CYP3A11, 1A2, 2B10, 2C29, and 2E1 were measured. Sedation efficacy and ADRs of midazolam, a CYP3A substrate, were monitored after APAP treatment. ALT, AST, and miR122 increased at 24 hours after APAP treatment with all APAP doses, whereas only groups treated with 200 and 400 mg/kg recovered back to normal levels at 72 and 96 hours. The expression and activity of the cytochromes P450 significantly decreased at 24 hours with all APAP doses but only recovered back to normal at 72 and 96 hours with 200 and 400, but not 600, mg/kg of APAP. The alterations of cytochrome P450 activities resulted in altered sedation efficacy and ADRs of midazolam, which were corrected by dose justification of midazolam. Overall, this work illustrated a low cytochrome P450 expression window after AILI, which can decrease drug metabolism and negatively impact drug efficacy and ADRs. SIGNIFICANCE STATEMENT: The data generated in the mouse model demonstrated that expression and activities of cytochrome P450 enzymes and correlated drug efficacy and ADRs are altered during the time course of liver repair and regeneration after liver is injured by treatment with APAP. Dose justifications based on predicted changes of cytochrome P450 activities can achieve desired therapeutic efficacy and avoid ADRs. The generated data provide fundamental knowledge for translational research to drug treatment for patients during liver recovery and regeneration who have experienced AILI., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Abstract: Shenmai injection (SMI), one of the most popular herbal preparations, is widely used for the treatment of coronary atherosclerotic cardiopathy and viral myocarditis. The purpose of this study was to investigate the effect of Shenmai injection (SMI) on the CYP3A-mediated metabolism of midazolam (MDZ). The present study demonstrated that SMI could significantly inhibit MDZ 4-hydroxylation but activate its 1′-hydroxylation in human liver microsomes (HLMs), rat liver microsomes (RLM) and recombinant human CYP3A4 and CYP3A5. The opposing effect of SMI was characterized by the kinetic change of increasing V max/K m for MDZ 1′-hydroxylation and decreasing V max/K m for MDZ 4-hydroxylation in HLM and RLM. The presence of SMI enhanced the inhibition of ketoconazole on MDZ 4-hydroxylation but weakened or reversed its inhibition on MDZ 1′-hydroxylation in HLM. After single or multiple pretreatment with SMI, the ratios of AUC4-OH MDZ/AUCMDZ in rats were significantly decreased, while the ratios of AUC1′-OH MDZ/AUCMDZ were increased. Among the major components in SMI, total ginsenoside (TG), ophiopogon total saponins (OTS), ophiopogon total flavone (OTF), ginsenoside Rd, ophiopogonin D and ophiopogonone A exhibited significant inhibition on both 4-hydroxylation and 1′-hydroxylation of MDZ in HLM and RLM, while no activation on MDZ metabolism was observed in the presence of these major constituents alone or together. To further explore the responsible components, 3mL of SMI was loaded on a solid phase extraction (SPE) C18 cartridge and then separated by different concentrations of methanol. The fractions eluted with 60% and 90% methanol both showed significant activation on MDZ 1′-hydroxylation in HLM, but the fraction eluted with 30% methanol had no such effect. The results indicated that the activation of SMI on MDZ 1′-hydroxylation might be mainly resulted from the lipid-soluble components in SMI. [Copyright &y& Elsevier]

Critical illness and associated sequelae can cause severe metabolic disturbances. The effects these have on hepatic drug metabolism are poorly understood. In vivo, enzyme specific drug probes are used to measure changes in hepatic drug metabolism but they require multiple blood sampling and are time consuming. We suggest that a single measurement, 4 h after intravenous administration of midazolam is a reliable indicator of integral plasma midazolam exposure or area under the curve (AUC) in critically ill patients. We also explore the hypothesis that acute kidney injury (AKI) directly impairs hepatic metabolism of drugs in critically ill patients. A prospective study in 20 critically ill patients who were not taking specific enzyme inhibitors or inducers or benzodiazepines. Correlation between 4 h midazolam concentration and AUC was calculated. We also assessed the difference in metabolism between the patients with normal renal function and those with AKI. Four hour midazolam concentration correlated with AUC r = 0.956 ( p < 0.0001). In addition, the 4 h midazolam concentration was greater in critically ill patients with AKI than those with normal renal function p = 0.023. A single-time-point determination of plasma midazolam concentration is a reliable predictor of integral plasma midazolam exposure in critically ill patients. This tool can now be used to assess the effects of critical illness on hepatic drug metabolism. Using this method, we suggest that AKI reduces the hepatic metabolism of midazolam in critically ill patients. [ABSTRACT FROM AUTHOR]

Cytochrome P450 3A4 (CYP3A4) is the most important P450 enzyme for drug metabolism and drug-drug interaction, due to it being responsible for the biotransformation of approximately 50% of clinically used drugs. Advance knowledge of the molecular and mechanistic basis of CYP3A4 regioselective metabolism is beneficial for understanding the production of metabolites, and may allow personalized metabolic pathways or designing pathway-specific therapeutics. In this work, we focus on investigating the ligand-receptor interactions, substrate conformational transition, and key factors regulating the specificity of metabolic pathways using midazolam (MDZ) as a probe. Here, three types of substrate-binding conformations related to the diversity of MDZ metabolites are identified. The results also suggest that an allosteric site for MDZ is located near the F'-helix, A-anchor, and C-terminal loop of CYP3A4. The presence of an effector in the allosteric site can accelerate the conformational transition of the substrate via modulating a "sandwich" structure, and may affect the proportion of metabolites at high substrate concentration. We hope that the results can improve the understanding of the CYP3A4 structure and function, and provide a new perspective for drug development.

Givosiran (trade name GIVLAARI) is a small interfering ribonucleic acid that targets hepatic delta-aminolevulinic acid synthase 1 (ALAS1) messenger RNA for degradation through RNA interference (RNAi) that has been approved for the treatment of acute hepatic porphyria (AHP). RNAi therapeutics, such as givosiran, have a low liability for drug-drug interactions (DDIs) because they are not metabolized by cytochrome 450 (CYP) enzymes, and do not directly inhibit or induce CYP enzymes in the liver. The pharmacodynamic effect of givosiran (lowering of hepatic ALAS1, the first and rate limiting enzyme in the heme biosynthesis pathway) presents a unique scenario where givosiran could potentially impact heme-dependent activities in the liver, such as CYP enzyme activity. This study assessed the impact of givosiran on the pharmacokinetics of substrates of 5 major CYP450 enzymes in subjects with acute intermittent porphyria (AIP), the most common type of AHP, by using the validated "Inje cocktail," comprised of caffeine (CYP1A2), losartan (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), and midazolam (CYP3A4). We show that givosiran treatment had a differential inhibitory effect on CYP450 enzymes in the liver, resulting in a moderate reduction in activity of CYP1A2 and CYP2D6, a minor effect on CYP3A4 and CYP2C19, and a similar weak effect on CYP2C9. To date, this is the first study evaluating the DDI for an oligonucleotide therapeutic and highlights an atypical drug interaction due to the pharmacological effect of givosiran. The results of this study suggest that givosiran does not have a large effect on heme-dependent CYP enzyme activity in the liver., (© 2021 Alnylam Pharmaceuticals. Clinical Pharmacology & Therapeutics. © 2021 American Society for Clinical Pharmacology and Therapeutics.)

Aim: In critically ill mechanically ventilated children, midazolam is used first line for sedation, however its exact sedative effects have been difficult to quantify. In this analysis, we use parametric time-to-event (PTTE) analysis to quantify the effects of midazolam in critically ill children., Methods: In the PTTE analysis, data was analyzed from a published study in mechanically ventilated children in which blinded midazolam or placebo infusions were administered during a sedation interruption phase until, based on COMFORT-B and NISS scores, patients became undersedated and unblinded midazolam was restarted. Using NONMEM® v.7.4.3., restart of unblinded midazolam was analysed as event. Patients in the trial were divided into internal and external validation cohorts prior to analysis., Results: Data contained 138 events from 79 individuals (37 blinded midazolam; 42 blinded placebo). In the PTTE model, the baseline hazard was best described by a constant function. Midazolam reduced the hazard for restart of unblinded midazolam due to undersedation by 51%. In the blinded midazolam group, time to midazolam restart was 26 h versus 58 h in patients with low versus high disease severity upon admission (PRISM II < 10 versus > 21), respectively. For blinded placebo, these times were 14 h and 33 h, respectively. The model performed well in an external validation with 42 individuals., Conclusion: The PTTE analysis effectively quantified the effect of midazolam in prolonging sedation and also the influence of disease severity on sedation in mechanically ventilated critically ill children, and provides a valuable tool to quantify the effect of sedatives. Clinical trial number and registry URL: Netherlands Trial Register, Trial NL1913 (NTR2030), date registered 28 September 2009 https://www.trialregister.nl/trial/1913 ., (© 2021. The Author(s).)

Mounting evidence has revealed that despite the high degree of sequence homology between cytochrome P450 3A isoforms (i.e., CYP3A4 and CYP3A5), they have the propensities to exhibit vastly different irreversible and reversible interactions with a single substrate. We have previously established that benzbromarone (BBR), a potent uricosuric agent used in the management of gout, irreversibly inhibits CYP3A4 via mechanism-based inactivation (MBI). However, it remains unelucidated if CYP3A5-its highly homologous counterpart-is susceptible to inactivation by BBR. Using three structurally distinct probe substrates, we consistently demonstrated that MBI was not elicited in CYP3A5 by BBR. Our in silico covalent docking models and molecular dynamics simulations suggested that disparities in the susceptibilities toward MBI could be attributed to the specific effects of BBR covalent adducts on the F-F' loop. Serendipitously, we also discovered that BBR reversibly activated CYP3A5-mediated rivaroxaban hydroxylation wherein apparent V max increased and K m decreased with increasing BBR concentration. Fitting data to the two-site model yielded interaction factors α and β of 0.44 and 5.88, respectively, thereby confirming heterotropic activation of CYP3A5 by BBR. Furthermore, heteroactivation was suppressed by the CYP3A inhibitor ketoconazole in a concentration-dependent manner and decreased with increasing preincubation time, implying that activation was incited via binding of parent BBR molecule within the enzymatic active site. Finally, noncovalent docking revealed that CYP3A5 can more favorably accommodate both BBR and rivaroxaban in concert as compared with CYP3A4, which further substantiated our experimental observations. SIGNIFICANCE STATEMENT: Although it has been previously demonstrated that benzbromarone (BBR) inactivates CYP3A4, it remains uninterrogated whether it also elicits mechanism-based inactivation in CYP3A5, which shares ∼85% sequence similarity with CYP3A4. This study reported that BBR exhibited differential irreversible and reversible interactions with both CYP3A isoforms and further unraveled the molecular determinants underpinning their diverging interactions. These data offer important insight into differential kinetic behavior of CYP3A4 and CYP3A5, which potentially contributes to interindividual variabilities in drug disposition., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

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 C 18 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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Deoxycholic acid (DCA, 3 α , 12 α -dihydroxy-5 β -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 1 β ,3 α ,12 α -trihydroxy-5 β -cholan-24-oic acid (DCA-1 β -ol) and 3 α ,5 β ,12 α -trihydroxy-5 β -cholan-24-oic acid (DCA-5 β -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-1 β -ol and DCA-5 β -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-5 β -ol/DCA, and DCA-1 β -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., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

CYP3A4 is among the most abundant liver and intestinal drug-metabolizing cytochrome P450 enzymes, contributing to the metabolism of more than 30% of clinically used drugs. Therefore, interindividual variability in CYP3A4 activity is a frequent cause of reduced drug efficacy and adverse effects. In this study, we characterized wild-type CYP3A4 and 40 CYP3A4 variants, including 11 new variants, detected among 4773 Japanese individuals by assessing CYP3A4 enzymatic activities for two representative substrates (midazolam and testosterone). The reduced carbon monoxide-difference spectra of wild-type CYP3A4 and 31 CYP3A4 variants produced with our established mammalian cell expression system were determined by measuring the increase in maximum absorption at 450 nm after carbon monoxide treatment. The kinetic parameters of midazolam and testosterone hydroxylation by wild-type CYP3A4 and 29 CYP3A4 variants ( K m , k cat , and catalytic efficiency) were determined, and the causes of their kinetic differences were evaluated by three-dimensional structural modeling. Our findings offer insight into the mechanism underlying interindividual differences in CYP3A4-dependent drug metabolism. Moreover, our results provide guidance for improving drug administration protocols by considering the information on CYP3A4 genetic polymorphisms. SIGNIFICANCE STATEMENT: CYP3A4 metabolizes more than 30% of clinically used drugs. Interindividual differences in drug efficacy and adverse-effect rates have been linked to ethnicity-specific differences in CYP3A4 gene variants in Asian populations, including Japanese individuals, indicating the presence of CYP3A4 polymorphisms resulting in the increased expression of loss-of-function variants. This study detected alterations in CYP3A4 activity due to amino acid substitutions by assessing the enzymatic activities of coding variants for two representative CYP3A4 substrates., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

We investigate the effect of the alcohol-induced increase in the content of CYP2E1 in human liver microsomes (HLM) on the function of CYP3A4. Membrane incorporation of the purified CYP2E1 into HLM considerably increases the rate of metabolism of 7-benzyloxyquinoline (BQ) and attenuates the homotropic cooperativity observed with this CYP3A4-specific substrate. It also eliminates the activating effect of α-naphthoflavone (ANF) seen in some HLM samples. To probe the physiological relevance of these effects, we compared three pooled preparations of HLM from normal donors (HLM-N) with a pooled preparation from ten heavy alcohol consumers (HLM-A). The composition of the P450 pool in all samples was characterized by the mass-spectrometric determination of 11 cytochrome P450 species. The fractional content of CYP2E1 in HLM-A was from 2.0 to 3.4 times higher than in HLM-N. In contrast, the content of CYP3A4 in HLM-A was the lowest among all samples. Despite that, HLM-A exhibited a much higher metabolism rate and a lower homotropic cooperativity with BQ, similar to CYP2E1-enriched HLM-N. To substantiate the involvement of interactions between CYP2E1 and CYP3A4 in these effects, we probed hetero-association of these proteins in CYP3A4-containing Supersomes™ with a technique employing CYP2E1 labeled with BODIPY-618 maleimide. These experiments evinced the interactions between the two enzymes and revealed an inhibitory effect of ANF on their association. Our results demonstrate that the functional properties of CYP3A4 are fundamentally dependent on the composition of the cytochrome P450 ensemble and suggest a possible impact of chronic alcohol exposure on the pharmacokinetics of drugs metabolized by CYP3A4., (Copyright © 2020 Elsevier Inc. All rights reserved.)

ANS-6637, a pro-drug of GS-548351, is a selective, reversible inhibitor of aldehyde dehydrogenase isoform 2 under development as an anticraving agent for the treatment of substance use disorders. In vitro testing indicates that GS-548351 is an inhibitor and inducer of cytochrome P450 family 3, subfamily A (CYP3A). In this phase 1 single-center, open-label, fixed-sequence drug-drug interaction study we assessed the impact of steady-state GS-548351 on single-dose pharmacokinetics of midazolam, an index substrate for CYP3A. Twelve healthy volunteers received 600 mg of ANS-6637 by mouth daily from study days 3 to 8 and a single 5-mg oral dose of midazolam on days 1 and 8. Pharmacokinetic samples were collected over 24 hours on days 1 and 8, then analyzed using liquid chromatography-tandem mass spectrometry. The prespecified no-effect range for the 90% confidence interval (CI) of the geometric mean ratio (GMR) of midazolam coadministered with ANS-6637 (day 8) compared with midazolam alone (day 1) was 0.7-1.43. There was an increase in midazolam AUC 0-∞ (GMR [90%CI]) that was within the no-effect range (1.26 [1.12-1.425]) and an increase in midazolam C max that was outside the range (1.22 [1.03-1.45]). The AUC 0-∞ (1.08 [0.91-1.27]) and C max (0.95 [0.75-1.2]) of 1-hydroxymidazolam, the primary metabolite of midazolam, were also within the no-effect range. A single grade 3 adverse event (alanine aminotransferase elevation) was identified and resolved following discontinuation of the study drug. Overall, multidose ANS-6637 was well tolerated and did not alter the PK of midazolam beyond a small increase in AUC 0-∞ that is unlikely to be clinically significant., (© 2020, The American College of Clinical Pharmacology.)

In humans, the cytochrome P450 3A (CYP3A) subfamily is involved in midazolam (MDZ) biotransformation into 1'- and 4-hydroxy metabolites, and the former serves as a probe for CYP3A catalytic activity. In veterinary species is still crucial to identify enzyme- and species-specific CYP substrates; thus, the aim of this study was to characterize MDZ oxidation in cattle liver. A HPLC-UV method was used to measure 1'- and 4-hydroxy MDZ (1'- and 4-OHMDZ, respectively) formation in cattle liver microsomes and assess the role of CYP3A by an immunoinhibition study. Moreover, MDZ hydroxylation was evaluated in 300 cattle liver samples and results were correlated with testosterone hydroxylation. Formation of both metabolites conformed to a single-enzyme Michaelis-Menten kinetics. Values of V max and K m were 0.67 nmol/min/mg protein and 6.16 μM for 4-OHMDZ, and 0.06 nmol/min/mg protein and 10.08 μM for 1'-OHMDZ. An anti-rat CYP3A1 polyclonal antibody inhibited up to 50% and 94% 1'- and 4-OHMDZ formation, respectively. MDZ oxidation in liver microsomes was poorly correlated with testosterone hydroxylation. In conclusion, cattle metabolized MDZ to 1'-OHMDZ and 4-OHMDZ. The immunoinhibition results indicated a major contribution of CYP3As to 4-OHMDZ formation and the involvement of other CYPs in 1'-OHMDZ production, paving the way for further investigations., (© 2020 John Wiley & Sons Ltd.)

Hemoglobin-vesicles (Hb-V) are being developed as red blood cell (RBC) substitutes. In this study, we report on quantitative and qualitative alterations of hepatic cytochrome P450 (CYPs) and the pharmacokinetics of CYP-metabolizing drugs, with a focus on four CYP isoforms (CYP1A2, CYP2C11, CYP2E1 and CYP3A2), after Hb-V resuscitation from a massive hemorrhage. The results of proteome analysis and western blot data indicate that resuscitation with both Hb-V and packed RBC (PRBC) resulted in a decrease in the protein levels of CYPs. Along with a decrease in the protein expression of CYPs, pharmacokinetic studies showed that the elimination of CYP-metabolizing drugs was prolonged in the Hb-V and PRBC resuscitation groups. It is also noteworthy that the CYP-metabolizing drugs in the Hb-V resuscitation group was retained for a longer period compared to the PRBC resuscitation group, and this is attributed to the CYP isoforms having a lower metabolic activity in the Hb-V resuscitation group than that for the PRBC resuscitation group. These findings suggest that resuscitation with Hb-V after a massive hemorrhage has a slight but not clinically significant effect on drug metabolism via CYPs in the liver due to decreased protein levels and the metabolic activity with respect to the CYPs., Competing Interests: Declaration of competing interest All of authors have disclosed no conflicts of interest., (Copyright © 2020 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Commercial formulations of 29 commonly used herbal supplements (HSs) and grapefruit juice were evaluated for drug interaction potential via quantification of their CYP3A inhibitory potential in two in vitro experimental models of human small intestine, cryopreserved human intestinal mucosa (CHIM), and cryopreserved human enterocytes (CHEs). Two CYP3A substrates were used-in the studies with CHIM, CYP3A activity was quantified via liquid chromatography tandem mass spectrometry quantification of midazolam 1'-hydroxylation, whereas in CHE, luciferin-IPA metabolism to luciferin was quantified by luminescence. Upon treatment of CHIM with the estimated lumen concentration of the HS upon each oral administration (manufacturers' recommended dosage dissolved in 200 ml of culture medium), >80% CYP3A inhibition was observed for green tea extract, St. John's wort, valerian root, horehound, and grapefruit juice. Less than 50% inhibition was observed for fenugreek, aloe vera, guarana, soy isoflavone, maca, echinacea, spirulina, evening primrose, milk thistle, cranberry, red yeast rice, rhodiola, ginkgo biloba, turmeric, curcumin, white kidney bean, garlic, cinnamon, saw palmetto berries, panax ginseng, black elderberry, wheat grass juice, flaxseed oil, black cohosh, and ginger root. The results were confirmed in a a dose-response study with HSs obtained from three suppliers for the four inhibitory HSs (green tea extract, horehound, St. John's wort, valerian root) and three representative noninhibitory HSs (black cohosh, black elderberry, echinacea). Similar results were obtained with the inhibitory HSs in CHE. The results illustrate that CHIM and CHE represent physiologically relevant in vitro experimental models for the evaluation of drug interaction potential of herbal supplements. Based on the results, green tea extract, horehound, St. John's wort, and valerian root may cause drug interactions with orally administered drugs that are CYP3A substrates, as was observed for grapefruit juice. SIGNIFICANCE STATEMENT: In vitro evaluation of 29 popular herbal supplements in cryopreserved human intestinal mucosa identified green tea extract, horehound, St. John's wort, and valerian root to have CYP3A inhibitory potential similar to that for grapefruit juice, suggesting their potential to have clinically significant pharmacokinetic interaction with orally administered drugs that are CYP3A substrates. The results suggest that cryopreserved human intestinal mucosa can be used for in vitro evaluation of drug interactions involving enteric drug metabolism., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

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 k inact , 2.83 h -1 ; dissociation rate constant K I , 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., (© 2020 The Authors. The Journal of Clinical Pharmacology published by Wiley Periodicals LLC on behalf of American College of Clinical Pharmacology.)

In vitro - in vivo extrapolation (IVIVE) using human liver microsomes has been widely used to predict metabolic clearance, but some of the factors used in the process of prediction show variability for the same compound: notably, microsomal intrinsic clearance values corrected by the unbound fraction (CL int, u ), physiological parameters used for scale-up, and the source of in vivo clearance data.The purpose of this study was to assess the correlation between in vitro and in vivo CL int with a focus on factors showing variability using four cytochrome P450 (CYP)3A substrates.We surveyed in vivo clearance values in literature and also determined the microsomal CL int, u values. A scaling factor (SF direct ) was defined as in vivo CL int divided by the microsomal CL int, u , which ranged from 1190 to 2310 (mg protein per kg body weight). The application of a mean SF direct of 1600 (mg protein per kg body weight) and further normalization by the microsomal CL int, u values of midazolam, the most commonly used substrate, resulted in improved prediction accuracy for CL int, u values from various microsomal batches.The results suggest the normalization of variability might be useful for predicting the in vivo CL int .

Drug-drug interactions (DDI) have been examined for various drugs for oral use, but less for non-oral applications. This study provides DDI prediction methods for non-orally administered CYP3A4 substrates based on clinical DDI data of oral dosages. Gut availability (F g ) and fraction contribution of CYP3A4 to hepatic intrinsic clearance (fm CYP3A4 ) were predicted by AUC ratio (AUCR) in oral DDI study with/without grapefruit juice, and alteration in intrinsic clearances with/without ketoconazole, respectively. AUCRs of non-orally administered CYP3A4 substrates with/without inhibitors or inducers were predicted with the estimated F g , fm CYP3A4 and changes in liver CYP3A4 activities with inhibitors/inducers predicted using Simcyp library. DDIs of intravenously administered midazolam and alfentanil with CYP3A4 inhibitors/inducers could be predicted well by this method with predicted AUCRs within ±64% of observed values. Moreover, maximum DDIs with strong CYP3A4 inducers could be predicted by comparing hepatic clearance with hepatic blood flow, as hepatic blood flow indicates the possible maximum hepatic clearance after strong enzyme induction. Predicted AUCRs of midazolam, alfentanil and R- and S-verapamil were less than, but not far from observed ratios, suggesting good conservative prediction. These methods were applied to blonanserin transdermal patch, suggesting much smaller interaction with CYP3A4 inhibitors/inducers compared to oral dosage of blonanserin., Competing Interests: Declaration of competing interest The authors declared no conflict of interst. All authors are or were employees of Sumitomo Dainippon Pharma Co., Ltd., (Copyright © 2020 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Objective: We 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 Methods: Diazepam 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., Results: Under 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., Conclusion: The 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., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Zhou et al.)

GABA A receptors (GABA A R) are inhibitory ion channels ubiquitously expressed in the central nervous system and play critical roles in brain development and function. Benzodiazepines are positive allosteric modulators of GABA A R, enhancing channel opening frequency when GABA is bound to the receptor. Midazolam is a commonly used benzodiazepine. It is frequently used for premature infants, but the long-term consequences of its use in this patient population are not well established. Here, we studied the acute effects of midazolam on immature synapses. Using a rodent organotypic hippocampal slice preparation, we evaluated how midazolam affects inhibitory synaptic transmission onto CA1 pyramidal neurons. We found that 1 μM midazolam enhances evoked inhibitory post synaptic currents (eIPSCs) at a holding potential of -60 mV. Similarly, 1 μM midazolam enhances miniature IPSCs (mIPSCs) in CA1 pyramidal neurons at holding potentials of -60 mV and -30 mV. At depolarized holding potentials, however, midazolam no longer enhances mIPSCs. Depolarization of the postsynaptic cell by itself increases mIPSC decay, which occludes the allosteric effects of midazolam. These results provide insight into how a benzodiazepine and membrane voltage may modulate GABA A R function in developing circuits., (Copyright © 2020. Published by Elsevier B.V.)

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.

Measuring in vivo changes in the drug metabolizing activity of cytochrome P450 (CYP) enzymes is critical to understanding and assessing drug-drug, drug-diet and drug-disease interactions. The sensitivity and specificity of ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) makes it an ideal tool for analyzing drugs and their metabolites in biological matrices, and has demonstrated utility in CYP phenotyping across varied applications. Published CYP phenotyping cocktail assays often require large plasma sample volumes (0.5-1 mL), have relatively low sensitivity and multi-step complex sample preparation and extraction procedures. Further, variability exists in the way that recovery and matrix effects are investigated and reported, and some studies fail to report these data altogether. Therefore, the aim of this study was to develop, validate and optimize a simplified assay for the probe drugs caffeine (metabolized by CYP1A2), omeprazole (CYP2C19), losartan (CYP2C9), dextromethorphan (CYP2D6), midazolam (CYP3A4) and their respective enzyme-specific metabolites in small volumes (100 μL) of human plasma, that addresses the issues noted. Analyte extraction involved protein precipitation with acetonitrile and solid-phase extraction (SPE). Samples were analyzed using an Agilent 1290 infinity LC system in tandem with 6460A triple quadrupole mass spectrometers. The assay met FDA guideline-recommended requirements for specificity, sensitivity (analyte LLOQs 0.78-23.4 ng/mL), accuracy (intra-day RE% nominal concentration 90.7-110.2%; inter-day RE% 87.0-110.5%) and precision (intra-day analyte RSD% 0.46-11.4%; inter-day RSD% 1.36-11.2%). Recovery and matrix effects were thoroughly investigated and excluded as potential interferers with assay performance. This assay has been used successfully to phenotype CYP activity in a human clinical trial participant. Importantly, the authors provide a contemporary commentary on commonly found issues in the CYP phenotyping cocktail assay literature, and make recommendations concerning best-practice approaches and the standardization of data reporting in this area., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: To estimate the pharmacokinetics of midazolam and 1-hydroxymidazolam after midazolam administration as an intravenous bolus in sevoflurane-anesthetized cats., Study Design: Prospective pharmacokinetic study., Animals: A group of six healthy adult, female domestic cats., Methods: Anesthesia was induced and maintained with sevoflurane. After 30 minutes of anesthetic equilibration, cats were administered midazolam (0.3 mg kg -1 ) over 15 seconds. Venous blood was collected at 0, 1, 2, 4, 8, 15, 30, 45, 90, 180 and 360 minutes after administration. Plasma concentrations for midazolam and 1-hydroxymidazolam were measured using high-pressure liquid chromatography. The heart rate (HR), respiratory rate (f R ), rectal temperature, noninvasive mean arterial pressure (MAP) and end-tidal carbon dioxide (Pe'CO 2 ) were recorded at 5 minute intervals. Population compartment models were fitted to the time-plasma midazolam and 1-hydroxymidazolam concentrations using nonlinear mixed effect modeling., Results: The pharmacokinetic model was fitted to the data from five cats, as 1-hydroxymidazolam was not detected in one cat. A five-compartment model best fitted the data. Typical values (% interindividual variability where estimated) for the volumes of distribution for midazolam (three compartments) and hydroxymidazolam (two compartments) were 117 (14), 286 (10), 705 (14), 53 (36) and 334 mL kg -1 , respectively. Midazolam clearance to 1-hydroxymidazolam, midazolam fast and slow intercompartmental clearances, 1-hydroxymidazolam clearance and 1-hydroxymidazolam intercompartment clearance were 18.3, 63.5 (15), 22.1 (8), 1.7 (67) and 3.8 mL minute -1 kg -1 , respectively. No significant changes in HR, MAP, f R or Pe'CO 2 were observed following midazolam administration., Conclusion and Clinical Relevance: In sevoflurane-anesthetized cats, a five-compartment model best fitted the midazolam pharamacokinetic profile. There was a high interindividual variability in the plasma 1-hydroxymidazolam concentrations, and this metabolite had a low clearance and persisted in the plasma for longer than the parent drug. Midazolam administration did not result in clinically significant changes in physiologic variables., (Copyright © 2019 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by Elsevier Ltd. All rights reserved.)

Midazolam, a widely used anesthetic, inhibits proliferation of neural stem cells (NSCs) and induces neuroapoptosis in neonates. Dexmedetomidine, an effective auxiliary medicine in clinical anesthesia, protects the developing brain against volatile anesthetic-induced neuroapoptosis. Whether dexmedetomidine protects against neurogenesis damage induced by midazolam remains unknown. This study aims to clarify the protective effect of dexmedetomidine on midazolam-induced neurogenesis damage and explore its potential mechanism. Postnatal 7-day-old Sprague-Dawley (SD) rats and cultured NSCs were treated with either normal saline, midazolam, or dexmedetomidine combined with midazolam. The rats were sacrificed at 1, 3, and 7 days after treatment. Cell proliferation was assessed by 5-bromodeoxyurdine (BrdU) incorporation. Cell viability was determined using MTT assay. Cell differentiation and apoptosis were detected by immunofluorescent staining and terminal dUTP nick-end labeling (TUNEL), respectively. The protein levels of p-JNK, p-P38, and cleaved caspase-3 were quantified using Western blotting. Midazolam decreased cell proliferation and increased cell apoptosis in the subventricular zone (SVZ), the subgranular zone (SGZ) of the hippocampus, and cultured NSCs. Moreover, midazolam decreased cell viability and increased the expression of p-JNK and p-P38 in cultured NSCs. Co-treatment with dexmedetomidine attenuated midazolam-induced changes in cell proliferation, viability, apoptosis, and protein expression of p-JNK and p-P38 in cultured NSCs. Midazolam and dexmedetomidine did not affect the differentiation of the cultured NSCs. These results indicate that dexmedetomidine alleviated midazolam-induced neurogenesis damage via JNK and P38 MAPK pathways.

Promiscuous and allosteric drug interactions with cytochrome P450 3A4 (CYP3A4) are ubiquitous but incompletely understood at the molecular level. A classic allosteric CYP3A4 drug interaction includes the benzodiazepine midazolam (MDZ). MDZ exhibits homotropic and heterotropic allostery when metabolized to 1'-hydroxy and 4-hydroxy metabolites in varying ratios. The combination of hydrogen-deuterium exchange mass spectrometry (HDX-MS) and Gaussian accelerated molecular dynamics (GaMD) simulations of CYP3A4 in lipid nanodiscs and in a lipid bilayer, respectively, reveals MDZ-dependent changes in dynamics in a membrane environment. The F-, G-, and intervening helices, as well as the loop preceding the β1-sheets, display the largest observed changes in HDX. The GaMD suggests a potential allosteric binding site for MDZ in the F'- and G'-regions, which undergo significant increases in HDX at near-saturating MDZ concentrations. The HDX-MS and GaMD results confirm that changes in dynamics are most significant near the developing consensus allosteric site, and these changes are distinct from those observed previously with the nonallosteric inhibitor ketoconazole. The results suggest that the allosteric MDZ remains mobile in its binding site at the Phe-cluster. The results further suggest that this binding site remains dynamic or changes the depth of insertion in the membrane.

Ketamine (Ket) and midazolam (MDZ) are commonly administered drugs in the intensive care setting for analgesia and sedation. Ket and MDZ are metabolized to dehydro-norketamine (DHNK), nor-ketamine (NK) and 1-hydroxy midazolam (1HMDZ). Limited studies evaluating their pharmacokinetics exists in patients on extracorporeal membrane oxygenation (ECMO) therapy. Therefore, we developed a quantitative, high-performance liquid chromatography-mass spectrometry (with single ion monitoring) method to simultaneously detect Ket, MDZ and their (DHNK, NK and 1HMDZ) metabolites in human plasma. Considerable sensitivity was obtained for the analytes using a C 18 HILIC column operated by a high-performance liquid chromatography system coupled with a Thermo Exactive Orbitrap mass spectrometer. Calibration curves were developed for analyte molecules (n = 5) in the presence of carbamazepine (CBZ) as an internal standard. The lower limits of quantitation (LLOQ) for Ket and MDZ were 20 and 10 ng/mL, respectively with the LLOQ for DHNK, NK and 1HMDZ at 470, 320 and 150 ng/ml. Moreover, the percent coefficient of variance and precision for inter- and intra-day runs were within the standards set forth by the ICH and FDA guidelines. This method is sensitive and has been successfully applied to an ongoing pharmacokinetic study in patients on ECMO therapy., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

Background: Calycosin (CAL), a type of O-methylated isoflavone extracted from the herb Astralagusmembranaceus (AM), is a bioactive chemical with antioxidative, antiphlogistic and antineoplastic activities commonly used in traditional alternative Chinese medicine. AM has been shown to confer health benefits as an adjuvant in the treatment of a variety of diseases., Aim: The main objective of this study was to determine whether CAL influences the cytochrome P450 (CYP450) system involved in drug metabolism., Methods: Midazolam, tolbutamide, omeprazole, metoprolol and phenacetin were selected as probe drugs. Rats were randomly divided into three groups, specifically, 5% Carboxymethyl cellulose (CMC) for 8 days (Control), 5% CMC for 7 days + CAL for 1 day (single CAL) and CAL for 8 days (conc CAL), and metabolism of the five probe drugs evaluated using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)., Results: No significant differences were observed for omeprazole and midazolam, compared to the control group. T max and t 1/2 values of only one probe drug, phenacetin, in the conc CAL group were significantly different from those of the control group ( T max h: 0.50±0.00 vs 0.23±0.15; control vs conc CAL). C max of tolbutamide was decreased about two-fold in the conc CAL treatment group (conc vs control: 219.48 vs 429.56, P <0.001)., Conclusion: Calycosin inhibits the catalytic activities of CYP1A2, CYP2D6 and CYP2C9. Accordingly, we recommend caution, particularly when combining CAL as a modality therapy with drugs metabolized by CYP1A2, CYP2D6 and CYP2C9, to reduce the potential risks of drug accumulation or ineffective treatment., Competing Interests: The authors have no conflicts of interest to declare., (© 2020 Wu et al.)

BACKGROUND Vitamin 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 AND METHODS Forty 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). RESULTS Vitamin 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). CONCLUSIONS Vitamin D3 was transformed into 1,25(OH)₂D₃ by triggering CYP3A11 and CYP3A11 activity and by hydrolyzing MDZ.

Midazolam is a benzodiazepine with sedative, muscle relaxant, anxiolytic, and anticonvulsant effects. Twelve ball pythons (Python regius) were used in a parallel study evaluating the pharmacokinetics of 1 mg/kg midazolam following a single intracardiac (IC) or intramuscular (IM) administration. Blood was collected from a central venous catheter placed 7 days prior, or by cardiocentesis, at 15 time points starting just prior to and up to 72 hr after drug administration. Plasma concentrations of midazolam and 1-hydroxymidazolam were determined by the use of high-performance liquid chromatography tandem-mass spectrometry and pharmacokinetic parameters were estimated using noncompartmental analysis. The mean ± SD terminal half-lives of IC and IM midazolam were 12.04 ± 3.25 hr and 16.54 ± 7.10 hr, respectively. The area under the concentration-time curve extrapolated to infinity, clearance, and apparent volume of distribution in steady-state of IC midazolam were 19,112.3 ± 3,095.9 ng*hr/ml, 0.053 ± 0.008 L hr -1  kg -1 , and 0.865 ± 0.289 L/kg, respectively. The bioavailability of IM midazolam was estimated at 89%. Maximum plasma concentrations following an IM administration were reached 2.33 ± 0.98 hr and 24.00 ± 14.12 hr postinjection for midazolam and 1-hydroxymidazolam, respectively, and 22.33 ± 20.26 hr postinjection for 1-hydroxymidazolam following IC administration., (© 2019 John Wiley & Sons Ltd.)

Transdermal fentanyl is widely used to control pain in cancer patients. The high pharmacokinetic variability of fentanyl is assumed to be due to cytochrome P450 3A-mediated (CYP3A) N-dealkylation to norfentanyl in humans. However, recently published clinical studies question the importance of the described metabolic pathway. In this small study in palliative cancer patients under real-life clinical conditions, the influence of CYP3A on fentanyl variability was investigated. In addition to the determination of midazolam plasma concentration to reveal CYP3A activity, plasma concentrations of fentanyl and its metabolite, norfentanyl, were measured in identical blood samples of 20 patients who participated in an ongoing trial and had been on transdermal fentanyl. Fentanyl, norfentanyl, midazolam, and 1'-OH-midazolam were quantified by liquid chromatography/tandem mass spectrometry. Plasma concentrations of fentanyl and norfentanyl exhibited a large variability. Mean estimated total clearance of fentanyl and mean metabolic clearance of midazolam (as a marker of CYP3A activity) were 75.5 and 36.3 L/h. Both clearances showed a weak correlation and hence a minimal influence of CYP3A on fentanyl elimination.

Inhibitors of acetylcholinesterase (AChE), which have an important role in the prevention of excessive AChE activity and β-amyloid (Aβ) formation are widely used in the symptomatic treatment of Alzheimer's disease (AD). The inhibitory effect of anesthetic agents on AChE was determined by several approaches, including binding mechanisms, molecular docking and kinetic analysis. Inhibitory effect of intravenous anesthetics on AChE as in vitro and in vivo have been discovered. The midazolam, propofol and thiopental have shown competitive inhibition type (midazolam > propofol > thiopental) and Ki values were found to be 3.96.0 ± 0.1, 5.75 ± 0.12 and 29.65 ± 2.04 µM, respectively. The thiopental and midazolam showed inhibition effect on AChE in vitro, whereas they showed activation effect in vivo when they are combined together. The order of binding of the drugs to the active site of the 4M0E receptor was found to be midazolam > propofol > thiopental. This study on anesthetic agents that are now widely used in surgical applications, have provided a molecular basis for investigating the drug-enzyme interactions mechanism. In addition, the study is important in understanding the molecular mechanism of inhibitors that are effective in the treatment of AD.