Metabolism Of Midazolam Research Articles

The potential of enteroids derived from children and adults to study age-dependent differences in intestinal CYP3A4/5 metabolism

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.

Evaluation of the Effects of Extracts Containing Valeriana officinalis and Piper methysticum on the Activities of Cytochrome P450 3A and P-Glycoprotein.

This work investigated interactions ascribed to the administration of phytomedicines containing Valeriana officinalis and Piper methysticum with conventional drugs. The phytomedicines were characterized by HPLC and administered per os to male Wistar rats, either concomitantly or not with the CYP3A substrate midazolam. To distinguish between the presystemic or systemic effect, midazolam was given orally and intravenously. The effects on the P-gp substrate fexofenadine uptake by Caco-2 cells were examined. The valerenic acid content was 1.6 ± 0.1 mg per tablet, whereas kavain was 13.7 ± 0.3 mg/capsule. Valerian and kava-kava extracts increased the maximum plasma concentration (Cmax) of midazolam 2- and 4-fold compared to the control, respectively. The area under the plasma concentrations versus time curve (AUC(0-∞)) was enhanced from 994.3 ± 152.3 ng.h/mL (control) to 3041 ± 398 ng.h/mL (valerian) and 4139 ± 373 ng.h/mL (kava-kava). The half-life of midazolam was not affected. These changes were attributed to the inhibition of midazolam metabolism by the enteric CYP3A since the i. v. pharmacokinetic of midazolam remained unchanged. The kava-kava extract augmented the uptake of fexofenadine by 3.5-fold compared to the control. Although Valeriana increased the uptake of fexofenadine, it was not statistically significant to that of the control (12.5 ± 3.7 ng/mg protein vs. 5.4 ± 0.3 ng/mg protein, respectively). Therefore, phytomedicines containing V.officinalis or P. methysticum inhibited the intestinal metabolism of midazolam in rats. Conversely, the P-gp-mediated transport of fexofenadine was preferably affected by kava-kava.

In vitro demonstration of antedrug mechanism of a pharmacokinetic booster to improve CYP3A4 substrates by CYP3A4-mediated metabolism inhibition

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.

Assessing the role of residue Phe108 of cytochrome P450 3A4 in allosteric effects of midazolam metabolism.

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.

Evaluation of the potential drug-drug interactions of carotegrast methyl with midazolam, prednisolone or atorvastatin in healthy adults.

This study evaluated drug-drug interactions between the CYP3A4 inhibitor carotegrast methyl and the other CYP3A4 substrates, midazolam, atorvastatin and prednisolone. A total of 88 healthy volunteers orally received carotegrast methyl 960 mg 3 times daily for 14 days. A single oral (5 mg) or intravenous (0.017 mg kg-1 ) midazolam, oral (5 mg) prednisolone or oral (10 mg) atorvastatin was administered before, with and after carotegrast methyl treatment. When the 90% confidence interval (CI) for the geometric mean ratios of the pharmacokinetic (PK) parameters with coadministration with carotegrast methyl (Day 14) to those before carotegrast methyl administration was between 0.80 and 1.25, no PK interaction were deemed. The Cmax and AUC0-t of oral midazolam before administration of carotegrast methyl were 30.9 ± 9.8 ng mL-1 and 74.5 ± 21.9 ng h mL-1 , respectively. The geometric mean ratio of the Cmax and AUC0-t of midazolam on Day 14 to those on Day -1 was 1.86 (90% CI, 1.64-2.11) and 3.07 (90% CI, 2.81-3.35), which did not fall within the range of 0.80-1.25, suggesting that carotegrast methyl had a PK interaction with midazolam. Similar PK interactions were found for intravenous midazolam and atorvastatin, but not for prednisolone. The inhibitory effect of carotegrast methyl on CYP3A4-mediated metabolism of midazolam and atorvastatin had almost disappeared by 14 days after the end of administration. Carotegrast methyl was classified as a moderate CYP3A4 inhibitor in humans. Carotegrast methyl might enhance the action of drugs that are metabolized by CYP3A4.

Impact of Heterotropic Allosteric Modulation on the Time-Dependent Inhibition of Cytochrome P450 3A4.

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 µM 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 µM) and CBZ (100 or 1000 µM) 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.

Pregnancy related hormones increase CYP3A mediated buprenorphine metabolism in human hepatocytes: a comparison to CYP3A substrates nifedipine and midazolam.

Introduction: Pregnancy increases the clearance of CYP3A4 substrate drugs and pregnancy-related hormones (PRHs) induce hepatic CYP3A4 expression and metabolism. However, it remains unclear to what extent the magnitude of PRH-evoked changes in hepatic CYP3A metabolism varies across multiple substrates. This study quantified the impact of PRHs on CYP3A protein concentrations and buprenorphine metabolism in human hepatocytes, and compared the magnitude of these effects to nifedipine and midazolam metabolism. Methods: Sandwich-cultured human hepatocytes (SCHH) from female donors were exposed to PRHs, administered in combination across a range of physiologically relevant concentrations, for 72h. Absolute protein concentrations of CYP3A4, CYP3A5, and CYP3A7 in SCHH membrane fractions were quantified by nanoLC-MS/MS, and norbuprenorphine (nor-BUP), dehydro-nifedipine (dehydro-NIF), and 1-hydroxy-midazolam (1-OH-MDZ) formation was evaluated. Results: Compared to control, PRH exposure increased CYP3A4, CYP3A7, and total CYP3A protein concentrations, but not CYP3A5 concentrations, and increased nor-BUP, dehydro-NIF, and 1-OH-MDZ formation in a concentration-dependent manner. The formation of nor-BUP, dehydro-NIF, and 1-OH-MDZ each positively correlated with PRH-mediated changes in total CYP3A protein concentrations. The PRH-evoked increase in nor-BUP formation was evident in all donors; however, the PRH induction of dehydro-NIF and 1-OH-MDZ formation was diminished in a hepatocyte donor with high basal CYP3A5 expression. Discussion: These findings demonstrate that PRHs increase buprenorphine, nifedipine, and midazolam metabolism in SCHH via induction of CYP3A4 and total CYP3A protein concentrations, and the magnitude of these effects vary across hepatocyte donors in a substrate-specific manner. These data provide insight into the contribution of PRH induction of CYP3A4 metabolism to increased buprenorphine clearance during pregnancy.

Abstract CT273: Evaluation of the effect of rivoceranib on the pharmacokinetics of cytochrome P450 enzyme substrates in healthy volunteers

Abstract Introduction: Rivoceranib is a selective vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor with potent antitumor activity. Rivoceranib is metabolized in the liver mostly by cytochrome P450 (CYP)3A4/5, with minor contributions from CYP2D6, CYP2C9, and CYP2E1. In vitro and in vivo studies suggest rivoceranib may interact with various CYP substrates, including CYP 1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A. The purpose of study is to evaluate the effect of rivoceranib on the pharmacokinetics (PK) of various CYP substrates. Methods: This study was an open-label, 2-treatment, fixed-sequence drug-drug interaction phase 1 study evaluating the impact of multiple oral doses of 700 mg rivoceranib on the single-dose PK of CYP enzyme substrates administered in a 5+1 probe cocktail (caffeine [CYP1A2], S- and R-warfarin [CYP2C9] + vitamin K, omeprazole [CYP2C19], dextromethorphan [CYP2D6], and midazolam [CYP3A]) in healthy volunteers (N=32). On day 1, volunteers received a single dose of the 5+1 cocktail comprising 200 mg caffeine, 10 mg S- and R-warfarin with 10 mg vitamin K, 40 mg omeprazole, 30 mg dextromethorphan, and 2 mg midazolam. Blood samples were collected predose on day 1 and up to 120 hours post-dose for PK analyses of the substrates in the 5+1 cocktail. On days 6 to 15, volunteers received 700 mg rivoceranib once daily for 10 consecutive days with a single dose of the 5+1 cocktail administered on day 11. Blood samples were collected predose on day 11 and up to 120 hours post cocktail dosing (day 16) for PK analyses. Each dosing was under fasted conditions. There was a washout period of 5 days between Day 1 dosing and the first rivoceranib dose on Day 6; the 2 cocktail dosings were spaced by 10 days. Results: Rivoceranib reduced caffeine AUC0-inf by 15%, and did not change caffeine Cmax, indicating a minimal effect of rivoceranib on the PK of CYP1A2 substrates. S-warfarin and R-warfarin AUC0-inf increased by 68% and 32%, respectively, and Cmax by 19% and 15%, respectively, when co-administered with rivoceranib, indicating rivoceranib weakly inhibits CYP2C9. Rivoceranib appeared to act as a moderate inhibitor of CYP2C19, increasing omeprazole AUC0-inf 3.3-fold and increasing Cmax 2-fold. Dextromethorphan metabolism (CYP2D6) was inhibited, with a 2- to 2.7-fold increase in dextromethorphan exposure. Rivoceranib appeared to moderately inhibit midazolam metabolism by CYP3A4, with 2.4- to 2.8-fold increases in midazolam exposures. Conclusion: In the analysis, the effect of rivoceranib on the PK of CYP1A2 substrates did not appear to be clinically significant. Rivoceranib may inhibit the metabolism of CYP2C9, CYP2C19, CYP2D6, and CYP3A4 substrates, suggesting that dose adjustment of substrates of these CYP isozymes and/or cautiously monitoring patients’ adverse events may be needed when they are co-administered with rivoceranib. Citation Format: Janelle Weyer, Xianzhang Meng, Jennifer Lee, Joseph Reitano, Vinoo Urity, Cheol-Hee Park, Bill Strickland, Jan Van Tornout, Seong Jang. Evaluation of the effect of rivoceranib on the pharmacokinetics of cytochrome P450 enzyme substrates in healthy volunteers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT273.

Comparative Study between the 3D‐Liver Spheroid Models Developed from HepG2 and Immortalized Hepatocyte‐Like Cells with Primary Hepatic Stellate Coculture for Drug Metabolism Analysis and Anticancer Drug Screening

AbstractLiver spheroids may be the best alternative models for evaluating efficacy and toxicity of the new anticancer candidates and diagnostics for hepatocellular carcinoma (HCC). Here, novel 3D‐liver spheroid models are constructed from human hepatoma cells (HepG2)/ immortalized human hepatocyte‐like cells (imHCs) with primary hepatic stellate cells (HSCs) coculture using the ultralow attachment technique. Spheroid morphology, HSC distribution, metabolic activity, protein expressions, and drug penetration are evaluated. All developed 3D spheroid models exhibit in spherical shape with narrow size distribution, diameter between 639–743 (HepG2‐10%HSC) and 519–631 (imHC‐10%HSC) µm. Both imHC mono and coculture models significantly express normal liver biomarkers at the higher level than HepG2 models. While 3D‐HepG2 models significantly exhibit HCC biomarkers at the higher level than imHC models. HepG2 and imHC spheroids express basal cytochrom P450 (CYP450) enzymes at different levels depending on cell types, culture period, and ratio of coculture. Their metabolic activities for dextromethorphan (CYP2D6) tolbutamide (CYP2C9) and midazolam (CYP3A4) are routinely evaluated. For midazolam metabolism, imHC models allow the detection of phase II metabolic enzymes (UGT2B4 and UGT2B7). The presence of HSC in HepG2‐HSC model increases biological barrier for doxorubicin (DOX) penetration, and escalates IC50 of DOX from 61.4 to 127.2 µg mL−1.

Sex and Age Influence on Association of CYP450 Polymorphism with Midazolam Levels in Critically Ill Children.

Midazolam is a drug that is metabolized by cytochrome P450 (CYP450) enzymes, particularly CYP3A4 and CYP3A5. The present study aimed to determine the sex and age influence on association of CYP450 polymorphism with midazolam levels in critically ill children. Seventy-two DNA samples were genotyped by real-time PCR. Children ≤ five years of age who carry the rs776746 (T) allele in CYP3A5 gene were associated with lower plasma midazolam levels. The concentration median in patients was 0.0 ng/mL, while in patients with the normal (C) allele, it was 438.17 ng/mL (Q25 135.75-Q75 580.24), p = 0.005. The midazolam plasmatic concentration in female patients with the minor (T) allele was 0.0 ng/mL (Q250.00-Q75204.3), while in patients with the normal (C) allele median it was 459.0 ng/mL (Q25296.9-Q75789.7), p = 0.002. Analysis of the dominant model for the rs2740574 variant in CYP3A4 revealed a median of 0.38 L/kg (Q250.02-Q751.5) for the volume of distribution parameter in female patients with the normal T allele, while female patients with the minor C allele showed a median of 18.1 L/kg (Q257.5-Q7528.7) p = 0.02. Our results suggest an altered midazolam metabolism due to the presence the allelic rs2740574 variants of CYP3A4 and rs776746 of CYP3A5, and also the strong influence of age and sex.

Molecular Insights into the Heterotropic Allosteric Mechanism in Cytochrome P450 3A4-Mediated Midazolam Metabolism.

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.

Impact of Inflammation on Midazolam Metabolism in Severe COVID-19 Patients.

Midazolam is a benzodiazepine frequently used for sedation in patients hospitalized in the intensive care unit (ICU) for coronavirus disease 2019 (COVID‐19). This drug is primarily metabolized by cytochrome P450 3A (CYP3A) isoenzymes. Several studies have suggested that inflammation, frequently observed in these patients, could modulate CYP3A activity. The objective of this work was to study the impact of inflammation on midazolam pharmacokinetics in patients with COVID‐19. Forty‐eight patients hospitalized in the ICU for COVID‐19 and treated with midazolam administered by continuous infusion were included in this study. Midazolam and α‐hydroxymidazolam concentrations were measured and patient data, including the use of CYP3A inhibitors, were collected. Total and unbound concentrations of midazolam and α‐hydroxymidazolam were measured in plasma using a validated liquid‐chromatography coupled with mass spectrometry method. Inflammatory condition was evaluated by C‐reactive protein (CRP) level measurement. Both drug concentrations and CRP measurements were performed on 354 plasma samples. CRP elevation was significantly associated with the α‐hydroxymidazolam/midazolam plasma ratio decrease, whether for the unbound fraction or for the total fraction. Conversely, inflammation was not associated with protein binding modifications. Logically, α‐hydroxymidazolam/midazolam plasma ratio was significantly reduced when patients were treated with CYP3A inhibitors. In this study, we showed that inflammation probably reduces the metabolism of midazolam by CYP3A. These results suggest that molecules with narrow therapeutic margins and metabolized by CYP3A should be administrated with care in case of massive inflammatory situations.

The Impact of Suboptimal 25-Hydroxyvitamin D Levels and Cholecalciferol Replacement on the Pharmacokinetics of Oral Midazolam in Control Subjects and Patients With Chronic Kidney Disease.

The aim of this study was to investigate the impact of suboptimal 25-hydroxyvitamin D (25-VitD) and cholecalciferol (VitD3 ) supplementation on the pharmacokinetics of oral midazolam (MDZ) in control subjects and subjects with chronic kidney disease (CKD). Subjects with CKD (n=14) and controls (n=5) with suboptimal 25-VitD levels (<30ng/mL) were enrolled in a 2-phase study. In phase 1 (suboptimal), subjects were administered a single oral dose of VitD3 (5000IU) and MDZ (2mg). In phase 2 (replete) subjects who achieved 25-VitD repletion after receiving up to 16 weeks of daily cholecalciferol were given the identical single oral doses of VitD3 and MDZ as in phase 1. Concentrations of MDZ and metabolites, 1'-hydroxymidazolam (1'-OHMDZ), and 1'-OHMDZ glucuronide (1'-OHMDZ-G) were measured by liquid chromatography-tandem mass spectrometry and pharmacokinetic analysis was performed. Under suboptimal 25-VitD, reductions in MDZ clearance and renal clearance of 47% and 87%, respectively, and a 72% reduction in renal clearance of 1'-OHMDZ-G were observed in CKD vs controls. In phase 1 versus phase 2, MDZ clearance increased in all control subjects, with a median (interquartile range) increase of 10.5 (0.62-16.7)L/h. No changes in MDZ pharmacokinetics were observed in subjects with CKD between phases 1 and 2. The effects of 25-VitD repletion on MDZ disposition was largely observed in subjects without kidney disease. Impaired MDZ metabolism and/or excretion alterations due to CKD in a suboptimal 25-VitD state may not be reversed by cholecalciferol therapy. Suboptimal 25-VitD may augment the reductions in MDZ and 1'-OHMDZ-G clearance values observed in patients with CKD.

PHARMACOGENETIC ASPECTS OF THE USE OF PERIOPERATIVE DRUGS IN PEDIATRICS. Review

Relevance. It is known that in children the frequency of perioperative critical events due to the introduction of drugs is more than 5%. For example fentanyl, which is used as an adjunct to surgical anesthesia, is a major factor in increasing the number of deaths from opioid overdoses. Among the areas of reducing the frequency of severe adverse reactions to perioperative drugs - taking into account the pharmacogenetic variations of the patient.&#x0D; Objective is to consider current data on pharmacogenetic aspects of pharmacokinetics and pharmacodynamics of perioperative drugs.&#x0D; Methods. Analysis of the data presented in PubMed by keywords "pharmacogenetics", "general anesthetics", "analgesics", "muscle relaxants", "children". Search depth – 7 years (2014-2020), with a retrospective deepening of some positions until 2002.&#x0D; Results. The pharmacogenetic aspects of the pharmacokinetics of perioperative drugs are related to the genes of the enzymes that metabolize them and their transporters. Current data on the prevalence of polymorphic alleles of CYP2C9 genes (provides metabolism of nonsteroidal anti-inflammatory and anticonvulsant drugs) and CYP2D6 (metabolizes opioids, antidepressants, antiemetics) in Europe and, in particular, in Ukraine were presented. Thus, the inactive allele CYP2C19 * 2 was found in 13%, while the allele of increased activity CYP2C19 * 17 - in 25% of the population of Ukraine; allele with lost CYP2D6 function (CYP2D6 * 4) - in 18.6% of Ukrainians. Homozygotes with CYP2C9 * 3 polymorphism metabolize nonsteroidal anti-inflammatory drugs much more slowly than wild-type carriers, which may lead to their accumulation and side effects. The analgesic effect of codeine occurs only after it is metabolized in the liver by CYP2D6 to morphine. Respiratory depression, apnea and death may occur in patients with excessive metabolic rate even after a single dose of codeine; however, was noted the ineffectiveness of ondansetron due to a decrease in its concentration in the blood in thesepatients. Concomitant use of midazolam and fentanyl may prolong the effect of fentanyl by competing metabolism of midazolam by the hepatic enzyme CYP3A4, especially in patients with low metabolism. Plasma butyrylcholinesterase deficiency reduces succinylcholine inactivation in 1 in 1,500 people. Changing the genotype of uridine diphosphate glycosyltransferase causes increased glucuronidation of morphine, which may lead to a decrease in its effectiveness. As an example of pharmacogenetic changes in pharmacodynamics can be considered malignant hyperthermia, which occurs due to exposure to volatile anesthetics and depolarizing muscle relaxants in mutations in the RYR1 gene. Carriers of the minor allele (G) of the 5HT2A rs6313 gene require less propofol and 40% less time to start induction of anesthesia.&#x0D; Conclusions. The efficacy and safety of perioperative drugs are largely related to the pharmacogenetic aspects of their pharmacokinetics, especially mutations in genes of enzymes of the metabolism. Mutations in the genes of volatile anesthetic receptors and depolarizing muscle relaxants when their used cause a risk of malignant hyperthermia. The application of pharmacogenetics is the need to titrate drugs that have significant variability of action depending on the genotype. It is advisable to avoid the use of drugs with a high risk of pharmacogenetic reactions, if other alternative drugs are available.

Short-term administration of Polypodium leucotomos extract does not inhibit CYP3A4-mediated metabolism of midazolam in healthy subjects: an open-label, two-period, fixed-sequence study.

The extract of Polypodium leucotomos is used as a dietary supplement for its ultraviolet radiation-protective properties. Polypodium leucotomos extract reportedly inhibits CYP3A, which is important for drug metabolism in vitro in human microsomes and in vivo in rats. Inthis study, we explored the inhibitory effect of the P. leucotomos extract on CYP3A4-mediated midazolam metabolism in humans. This open-label, two-period, fixed-sequence study was performed on six healthy, Japanese, male volunteers. During period 1 (control), midazolam (1 mg) was orally administered. After a wash-out period of at least 5 days, period 2 was initiated. Subjects ingested P. leucotomos extract (240 mg) once in the morning and once at noon on the day before midazolam administration, and once the next morning (thrice overall). Midazolam was administered as in period 1. Blood samples were regularly collected for 8 hours after drug administration, and serum midazolam concentration was determined by ultra-fast liquid chromatography-tandem mass spectrometry. The pharmacokinetic parameters of midazolam were calculated and compared between the two periods. The area under the concentration-time curve was 19.18 ± 3.65 ng h/ml, maximum serum concentration was 7.81 ± 1.25 ng/ml, and half-life was 2.32 ± 0.35 hours during period 2. These parameters did not differ from those recorded in period 1 (area under the concentration-time curve: 18.74 ± 2.97 ng h/ml, maximum serum concentration: 8.78 ± 1.67 ng/ml, half-life: 2.52 ± 0.52 h). Therefore, short-term oral administration of P. leucotomos extract did not cause food-drug interactions mediated by CYP3A4 inhibition in humans.

Inhibitory effects of traditional Chinese medicine colquhounia root tablet on the pharmacokinetics of tacrolimus in rats

Ethnopharmacological relevanceTacrolimus (TAC) was widely used in various renal diseases while high recurrence rate and high expense restricted its applications. Traditional herbal medicine has become increasingly popular as an adjuvant therapy to minimize the adverse effects of TAC. Colquhounia root tablet (CRT), a prescribed drug prepared from the water extract of the peeled root of Tripterygium hypoglaucum (H. Lév.) Hutch., showed excellent anti-inflammatory, analgesic and immunosuppressive pharmacological properties. TAC used in combination with CRT was substantially more efficacious and safer than the monotherapy for the treatment of nephrotic syndrome. However, studies on their herb-drug interaction were scanty. Aim of the studyThe study was proposed to examine the effect of CRT on the pharmacokinetics of TAC in rats and identify the key natural constituents in CRT that affected the metabolism of TAC. Materials and methodsTAC was orally and intravenously administered to rats alone or in combination with CRT and the pharmacokinetic parameters of TAC were compared. After pretreatment with CRT for 15 d, the expressions of the drug-metabolizing enzymes (DMEs), drug transporters (DTs) and nuclear receptors (NRs) were determined by polymerase chain reaction and western blotting and compared with the control group. The hepatic microsomal incubation system was employed to confirm the inhibitory effects of CRT and its major components on rat cytochrome P450 (CYP) 3A2. The roles of the primary components in the regulation of human CYP3A4 and mouse P-gp activities were evaluated by using docking analysis. ResultsThe blood concentrations of TAC were significantly increased in a dose- and pretreatment time-dependent manner after combined administration of CRT. The maximal effect was found at 300 mg/kg (43.70 ± 8.77 ng/mL and 141.45 ± 21.58 h·ng/mL) in a single dose run and the pharmacokinetic parameters gradually returned to the normal levels at 24 h interval of long-term CRT pretreatment. In contrast, CRT had no effect on the pharmacokinetics of intravenous TAC. Further study indicated that the mRNA and protein expressions of DMEs and DTs, such as CYP3A1, CYP3A2, P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 in rat intestine and liver were down-regulated, whereas the expressions of NRs like constitutive androstane receptor and pregnane X receptor were up-regulated after multiple oral doses of CRT. Molecular docking showed the binding potency of five CRT major constituents with both human CYP3A4 and mouse P-gp. Celastrol, wilforgine and wilforine were the strongest inhibitors towards midazolam metabolism in rat liver microsomes, with the 50% inhibition concentrations being at 8.33 μM, 22.18 μM and 22.22 μM, respectively. ConclusionsOur results revealed that co-dosing of CRT could lead to a significant increase in blood concentration of TAC and this effect could be ascribed to the resultant co-regulation of DMEs, DTs and NRs. Our study provided an experimental basis for the combination use of CRT and TAC in clinical practice.

Extending the viability of human precision-cut intestinal slice model for drug metabolism studies

Human Precision-cut intestinal slices (hPCIS) are used to study intestinal physiology, pathophysiology, drug efficacy, toxicology, kinetics, and metabolism. However, the use of this ex vivo model is restricted to approximately a 24 h timeframe because of declining viability of the hPCIS during traditional culture. We hypothesized that we could extend the hPCIS viability by using organoid medium. Therefore, we cultured hPCIS for up to 72 h in organoid media [expansion medium (Emed) and differentiation medium (Dmed)]. After incubation, we assessed culture-induced changes on viability markers, specific cell type markers and we assessed the metabolic activity of enterocytes by measuring midazolam metabolite formation. We show that the adenosine triphosphate (ATP)/protein ratio of Emed-cultured hPCIS and morphology of both Emed- and Dmed-cultured hPCIS was improved compared to WME-cultured hPCIS. Emed-cultured hPCIS showed an increased expression of proliferation and stem cell markers, whereas Dmed-cultured hPCIS showed an increased expression of proliferation and enterocyte markers, along with increased midazolam metabolism. Using the Emed, the viability of hPCIS could be extended for up to 72 h, and proliferating stem cells remained preserved. Using Dmed, hPCS also remained viable for up to 72 h, and specifically rescued the metabolizing enterocytes during culture. In conclusion, by using two different organoid culture media, we could extend the hPCIS viability for up to 72 h of incubation and specifically steer stem cells or enterocytes towards their original function, metabolism, and proliferation, potentially allowing pharmacokinetic and toxicology studies beyond the 24 h timeframe.

Hyperinflammation Reduces Midazolam Metabolism in Critically Ill Adults with COVID-19.

Background and ObjectiveMany patients treated for COVID-19 related acute respiratory distress syndrome in the intensive care unit are sedated with the benzodiazepine midazolam. Midazolam undergoes extensive metabolism by CYP3A enzymes, which may be inhibited by hyperinflammation. Therefore, an exaggerated proinflammatory response, as often observed in COVID-19, may decrease midazolam clearance. To develop a population pharmacokinetic model for midazolam in adult intensive care unit patients infected with COVID-19 and to assess the effect of inflammation, reflected by IL-6, on the pharmacokinetics of midazolam.MethodsMidazolam blood samples were collected once a week between March 31 and April 30 2020. Patients were excluded if they concomitantly received CYP3A4 inhibitors, CYP3A4 inducers and/or continuous renal replacement therapy. Midazolam and metabolites were analyzed with an ultra-performance liquid chromatography–tandem mass spectrometry method. A population pharmacokinetic model was developed, using nonlinear mixed effects modelling. IL-6 and CRP, markers of inflammation, were analyzed as covariates.ResultsThe data were described by a one-compartment model for midazolam and the metabolites 1-OH-midazolam and 1-OH-midazolam-glucuronide. The population mean estimate for midazolam clearance was 6.7 L/h (4.8–8.5 L/h). Midazolam clearance was reduced by increased IL-6 and IL-6 explained more of the variability within our patients than CRP. The midazolam clearance was reduced by 24% (6.7–5.1 L/h) when IL-6 increases from population median 116 to 300 pg/mL.ConclusionsInflammation, reflected by high IL-6, reduces midazolam clearance in critically ill patients with COVID-19. This knowledge may help avoid oversedation, but further research is warranted.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40262-022-01122-5.

Investigation of the molecular and mechanistic basis for the regioselective metabolism of midazolam by cytochrome P450 3A4.

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.

Inhibition of CYP3A-mediated Midazolam Metabolism by Kaempferia Parviflora.

Kaempferia parviflora (KP) extract has recently attracted attention in Japan as a dietary supplement; however, there is little information regarding food-drug interactions (FDIs). The current study was conducted to clarify the FDI of KP extract via inhibition of cytochrome P450 3A (CYP3A), a typical drug-metabolizing enzyme. The inhibitory effects of KP extract and its main ingredients, 5,7-dimethoxyflavone (5,7-DMF) and 3,5,7,3’,4’-pentamethoxyflavone (3,5,7,3’,4’-PMF), on CYP3A-mediated midazolam 1’-hydroxylation (MDZ 1’-OH) activity were investigated in human liver microsomes. In addition, the effect of a single oral treatment with KP extract (135 mg/kg) on oral MDZ (15 mg/kg) metabolism was investigated in rats. Serum MDZ concentration was analyzed and pharmacokinetic parameters were compared with the control group. KP extract competitively inhibited MDZ 1’-OH activity with an inhibition constant value of 78.14 µg/ml, which was lower than the estimated concentration in the small intestine after ingestion. Furthermore, KP extract, 5,7-DMF, and 3,5,7,3’,4’-PMF inhibited the activity in a time-, NADPH-, and concentration-dependent manner. In vivo study showed that administration of KP extract to rats 2 h before MDZ significantly increased the area under the serum concentration-time curve and the maximum concentration of MDZ significantly by 2.3- and 1.9- fold, respectively (p < 0.05). Conversely, administration of MDZ 18 h after KP extract treatment displayed a weaker effect. These results suggest that KP extract competitively inhibits CYP3A-mediated MDZ metabolism, and that this inhibition may be time-dependent but not irreversible. This work suggests an FDI through CYP3A inhibition by KP extract.