Midazolam Hydroxylation Research Articles

High-throughput assay to simultaneously evaluate activation of CYP3A and the direct and time-dependent inhibition of CYP3A, CYP2C9, and CYP2D6 using liquid chromatography-tandem mass spectrometry

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.

The influence of temperature on the metabolic activity of CYP2C9, CYP2C19, and CYP3A4 genetic variants in vitro

1. Temperature is considered to affect the activity of drug-metabolizing enzymes; however, no previous studies have compared temperature dependency among cytochrome P450 genetic variants. This study aimed to analyze warfarin 7-hydroxylation by CYP2C9 variants; omeprazole 5-hydroxylation by CYP2C19 variants; and midazolam 1-hydroxylation by CYP3A4 variants at 34 °C, 37 °C, and 40 °C. 2. Compared with that seen at 37 °C, the intrinsic clearance rates (V max/K m) of CYP2C9.1 and .2 were decreased (76 ∼ 82%), while that of CYP2C9.3 was unchanged at 34 °C. At 40 °C, CYP2C9.1, .2, and .3 exhibited increased (121%), unchanged and decreased (87%) intrinsic clearance rates, respectively. At 34 °C, the clearance rates of CYP2C19.1A and .10 were decreased (71 ∼ 86%), that of CYP2C19.1B was unchanged, and those of CYP2C19.8 and .23 were increased (130 ∼ 134%). At 40 °C, the clearance rates of CYP2C19.1A, .1B, .10, and .23 remained unaffected, while that of CYP2C19.8 was decreased (74%). At 34 °C, the clearance rates of CYP3A4.1 and .16 were decreased (79 ∼ 84%), those of CYP3A4.2 and .7 were unchanged, and that of CYP3A4.18 was slightly increased (112%). At 40 °C, the clearance rate of CYP3A4.1 remained unaffected, while those of CYP3A4.2, .7, .16, and .18 were decreased (58 ∼ 82%). 3. These findings may be clinically useful for dose optimization in patients with hypothermia or hyperthermia.

Danazol increases the oral bioavailability of midazolam by inactivation of hepatic and intestinal CYP3A in rats

Danazol (DNZ) is a synthetic androgen derivative used for the treatment of intractable hematological disorders. In this study, we investigated the effects of DNZ on CYP3A activity in hepatic and small intestinal microsomes and the pharmacokinetics of midazolam (MDZ), a typical substrate for CYP3A, in rats. MDZ 4-hydroxylation activities in hepatic and small intestinal microsomes significantly decreased 24 h after DNZ (100 mg/kg, i.p.) treatment. Time-dependent inactivation of MDZ 4-hydroxylation activities was noted when microsomes were pre-incubated with DNZ in the presence of a NADPH-generating system. The Western blot analysis indicated that the decrease observed in enzyme activity was not due to changes in the protein expression of CYP3A. In contrast to the intravenous administration, serum MDZ concentrations in DNZ-treated rats were markedly higher than those in control rats when administered orally. DNZ treatment increased MDZ oral bioavailability by approximately 2.5-folds. We herein demonstrated that DNZ increased the bioavailability of orally administered MDZ through irreversible inactivation of hepatic and intestinal CYP3A in rats.

Differential expression and activities of cytochrome P450 3A in the rat brain microsomes and mitochondria.

Midazolam (MDZ), a benzodiazepine derivative, is metabolized to 1'- and 4-hydroxylated metabolites (1'-OH-MDZ and 4-OH-MDZ, respectively) by cytochrome P450 3A (CYP3A). The purpose of this study was to investigate the CYP3A-mediated hydroxylation of MDZ in the rat brain mitochondria (MT). Brain microsomes (MC) and MT fractions were prepared from rats (n = 8) using differential and density gradient centrifugations, and the purity of the fractions was evaluated using VDAC1 and calreticulin as markers of MT and MC, respectively. The formation rates of 1'-OH-MDZ and 4-OH-MDZ in the rat brain MC and MT samples were determined using an LC-MS/MS method after validation. Subsequently, Michaelis-Menten kinetics of 1'- and 4-hydroxylation of MDZ were estimated. Western blot (WB) analysis was used to determine the protein expression of CYP3A in the rat brain MC and MT. The MC fractions had 5.93% ± 3.01% mitochondrial impurity, and the MT fractions had 19.3% ± 7.8% microsomal impurity (mean ± SD). The maximum velocity (Vmax ) values of the formation of the hydroxylated metabolites in the brain MT were 2.4-9-fold higher than those in MC. Further, the Vmax values of 4-OH-MDZ in both MC and MT fractions were substantially higher than those of 1'-OH-MDZ. The WB analysis showed that the intensity of the CYP3A immunoreactive band in MT was more than twofold higher than that in MC. It is concluded that compared with MC, rat brain MT contains substantial CYP3A, which may affect the pharmacology or toxicology of centrally acting xenobiotic and endogenous substrates of this enzyme.

Molecular dynamics simulation and in vitro evaluation of herb-drug interactions involving dietary polyphenols and CDK inhibitors in breast cancer chemotherapy.

Dietary polyphenols such as quercetin and curcumin have been extensively administered to patients with cancer in the form of herbal supplements. They may have a synergistic anticancer effect; however, a risk of pharmacokinetic interactions with selective CDK-4/6 inhibitors that are metabolized by the CYP3A4 enzyme exists. Considering these pharmacokinetic aspects, the current study examined the effects of curcumin and quercetin on human CYP3A4 to ascertain CYP3A4-mediated herb-drug interactions with CDK inhibitors. In this study, using in silico methods and CYP3A4 inhibition kinetics in human liver microsomes and recombinant CYP3A4 enzymes, the effects of concentration-dependent inhibition of CYP3A4 by quercetin and curcumin on CDK inhibitors metabolism were examined. Based on our in-silico docking findings, curcumin and quercetin were considerably bound to CYP3A4 protein and displace CDK inhibitors from the CYP3A4 substrate binding domain. The IC50 values of curcumin and quercetin were 16.10 and 0.05 μM, respectively, for CYP3A4-mediated 1'-hydroxylation of midazolam. The dietary polyphenols prolonged the in vitro half-life of palbociclib and ribociclib by 6.4-fold and decreased their intrinsic microsomal clearance by approximately 4.6 times. Our findings indicate that curcumin and quercetin effectively cause herb-drug interactions and should be cautiously used to avoid therapeutic failure.

Reevaluate In Vitro CYP3A Index Reactions of Benzodiazepines and Steroids between Humans and Dogs.

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) 6β-hydroxylation, lithocholate (LCA) 6α-hydroxylation, deoxycholate (DCA) 1β- and 5β-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 6β-hydroxylation and LCA 6α-hydroxylation and both substrates undergo non-CYP3A catalyzed side reactions. DCA 1β- and 5β-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 1β- and 5β-hydroxylation are validated as the CYP3A index reactions in both humans and dogs.

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

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

Midazolam oxidation in cattle liver microsomes: The role of cytochrome P450 3A.

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 Vmax and Km were 0.67nmol/min/mg protein and 6.16μM for 4-OHMDZ, and 0.06nmol/min/mg protein and 10.08μM 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.

Modulation of Major Human Liver Microsomal Cytochromes P450 by Component Alkaloids of Goldenseal: Time-Dependent Inhibition and Allosteric Effects.

Botanical and other natural products (NPs) are often coconsumed with prescription medications, presenting a risk for cytochrome P450 (P450)-mediated NP-drug interactions. The NP goldenseal (Hydrastis canadensis) has exhibited antimicrobial activities in vitro attributed to isoquinoline alkaloids contained in the plant, primarily berberine, (-)-β-hydrastine, and to a lesser extent, hydrastinine. These alkaloids contain methylenedioxyphenyl rings, structural alerts with potential to inactivate P450s through formation of metabolic intermediate complexes. Time-dependent inhibition experiments were conducted to evaluate their ability to inhibit major P450 activities in human liver microsomes by using a cocktail of isozyme-specific substrate probes. Berberine inhibited CYP2D6 (dextromethorphan O-demethylation; K I = 2.7 μM, kinact = 0.065 minute-1) and CYP3A4/5 (midazolam 1'-hydroxylation; K I = 14.8 μM, kinact = 0.019 minute-1); (-)-β-hydrastine inhibited CYP2C9 (diclofenac 4'-hydroxylation; K I = 49 μM, kinact = 0.036 minute-1), CYP2D6 (K I > 250 μM, kinact > 0.06 minute-1), and CYP3A4/5 (K I = 28 μM, kinact = 0.056 minute-1); and hydrastinine inhibited CYP2D6 (K I = 37 μM, kinact = 0.049 minute-1) activity. Berberine additionally exhibited allosteric effects on midazolam hydroxylation, showing both positive and negative heterotropic cooperativity. Experiments with recombinant isozymes showed that berberine activated midazolam 1'-hydroxylation by CYP3A5, lowering K m(app), but showed mixed inhibition and negative cooperativity toward this reaction when catalyzed by CYP3A4. Berberine inactivated CYP3A4 at a much faster rate than CYP3A5 and was a noncompetitive inhibitor of midazolam 4-hydroxylation by CYP3A4 but a strong mixed inhibitor of the CYP3A5 catalyzed reaction. These complex kinetics should be considered when extrapolating the risk for NP-drug interactions involving goldenseal. SIGNIFICANCE STATEMENT: Robust kinetic parameters were determined for the reversible and time-dependent inhibition of CYP2C9, CYP2D6, and CYP3A4/5 activities in human liver microsomes by major component isoquinoline alkaloids contained in the botanical natural product goldenseal. The alkaloid berberine also exhibited opposing, isozyme-specific allosteric effects on midazolam hydroxylation mediated by recombinant CYP3A4 (inhibition) and CYP3A5 (activation). These data will inform the development of a physiologically based pharmacokinetic model that can be used to predict potential clinically relevant goldenseal-drug interactions.

Evaluation of Erythromycin as a Tool to Assess CYP3A Contribution of Low Clearance Compounds in a Long-Term Hepatocyte Culture

Long-term hepatocyte culture systems such as HepatoPac are well suited to evaluate the metabolic turnover of low clearance (CL) drugs because of their sustained metabolic capacity and longer-term viability. Erythromycin (ERY), a moderate, mechanism-based inhibitor of CYP3A, was evaluated as a tool in the HepatoPac model to assess contribution of CYP3A to the clearance of drug candidates. ERY inhibited CYP3A activity by 58% and 80% at 3 and 10 μM, respectively, for up to 72 hours. At 30 µM, ERY inhibited midazolam hydroxylation by >85% for the entire 144-hour duration of the incubation. Alprazolam CLint was inhibited 58% by 3 μM of ERY, 75% by 15 μM of ERY, 89% by 30 μM of ERY, and 94% by 60 μM of ERY. ERY (30 μM) did not markedly affect CLint of substrates for several other major cytochrome P450 isoforms evaluated and did not markedly inhibit uridine diphosphoglucuronosyl transferase (UGT) isoforms 1A1, 1A3, 1A4, 1A6, 1A9, 2B7, or 2B15 as assessed using recombinant UGTs. ERY only mildly increased CYP3A4 gene expression by 2.1-fold (14% of rifampicin induction) at 120 µM, indicating that at effective concentrations for inhibition of CYP3A activity (30-60 µM), arylhydrocarbon receptor, constitutive androstane receptor, and pregnane-X-receptor activation are not likely to markedly increase levels of other drug-metabolizing enzymes or transporters. ERY at concentrations up to 60 µM was not toxic for up to 6 days of incubation. Use of ERY to selectively inhibit CYP3A in high-functioning, long-term hepatocyte models such as HepatoPac can be a valuable strategy to evaluate the contribution of CYP3A metabolism to the overall clearance of slowly metabolized drug candidates. SIGNIFICANCE STATEMENT: This work describes the use of erythromycin as a selective inhibitor of CYP3A to assess the contribution of CYP3A in the metabolism of compounds using long-term hepatocyte cultures.

Effects of Thioacetamide—Induced Cirrhosis, after a Drug Washout Period, on the Michaelis–Menten Kinetics of Major Cytochrome P450 Enzymes in the Rat Liver Microsomes

PurposeThioacetamide (TAA) is used to induce liver cirrhosis in rodents. However, the drug itself directly inhibits cytochrome P450 (CYP450) enzymes. The purpose of this study was to determine the effects of TAA‐induced liver cirrhosis, after a washout period to eliminate TAA, on the Michaelis‐Menten (MM) kinetics and protein contents of the major CYP450 enzymes CYP2D, CYP2E1, and CYP3A in rat liver microsomes.MethodsAdult, male Sprague‐Dawley rats were treated for 10 weeks with either TAA (200 mg/kg ip, 3 days per week) or vehicle (n = 7/each group). After a 10‐day washout period, animals were euthanized, and the liver was collected after cardiac perfusion with cold saline. Liver microsomes (MC) were prepared by differential centrifugation. The activities of the liver microsomal CYP2D (dextromethorphan O‐demethylation), CYP2E1 (chlorzoxazone 6‐hydroxylation) and CYP3A (midazolam 1′‐hydroxylation) were estimated by measurement of the metabolites by LC‐MS/MS. The microsomal CYP450 reductase (CPR) activity was analyzed spectrophotometry. Furthermore, the protein expressions of the isoenzymes were determined using Western blot analysis. The MM kinetic parameters maximum velocity (Vmax) and MM constant (Km) were determined using nonlinear regression analysis. Unpaired t‐test was used for statistical analysis of data, with a p value of &lt; 0.05 as significant.ResultsWhereas the MM kinetics of dextromethorphan demethylation (CYP2D) were best fit by a two‐enzyme equation, those of chlorzoxazone hydroxylation (CYP2E1) and midazolam 1′‐hydroxylation (CYP3A) were best fit by a single‐enzyme equation. Cirrhosis resulted in significant reductions in the Vmax values for all the tested microsomal activities. The microsomal high‐ and low‐capacity Vmax values for CYP2D demethylase activity were significantly reduced by 74% (p value &lt; 0.0001) and 51% (p &lt; 0.05), respectively, in the TAA group. This was associated with a significant (p &lt; 0.001) reduction (71%) in the protein contents of CYP2D in the TAA group. However, Km values were not affected by cirrhosis. For CYP2E1, cirrhosis caused a significant reduction (52%) in the Vmax value (p &lt; 0.0001) for the chlorzoxazone hydroxylation activity. However, the Km values and protein contents of CYP2E1 remained unchanged. The Vmax and Km values of microsomal CYP3A hydroxylase activity, resulting in 1′‐hydroxylation of midazolam, were significantly reduced by 71% (p &lt; 0.001) and 46% (p &lt; 0.05), respectively, which was associated with a 30% reduction (p &lt; 0.05) in the protein contents of CYP3A. Cirrhotic animals also showed a 31% decline (p &lt; 0.05) in the microsomal CPR activity.ConclusionsTAA‐induced cirrhosis in rats resulted in a decreased liver microsomal activity of CPR and major CYP450 enzymes (CYP2D, CYP2E1, and CYP3A). Except for CYP2E1, the decreased isoenzyme activities were also associated with decreases in the protein concentrations of the isoenzymes. However, the cirrhosis‐induced decrease in the CYP2E1 activity, which was in the absence of a change in its protein levels, is possibly due to a decrease in the microsomal CPR activity.Support or Funding InformationFunding: Chapman University School of Pharmacy

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

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

Janus kinase-dependent regulation of drug detoxifying protein expression by interleukin-22 in human hepatic cells

Interleukin (IL)-22 is a cytokine up-regulated in inflammatory situations and known to exert various hepatic effects. The potential impact of IL-22 towards liver drug detoxifying proteins remains nevertheless unknown, but may be important to determine owing to the well-established alterations of liver detoxification occuring during inflammation. The present study was therefore designed to analyze the effects of IL-22 towards drug metabolizing enzyme and drug transporter expression and activity in cultured human hepatic cells. Exposure of differentiated hepatoma HepaRG cells or primary human hepatocytes to 10 ng/mL IL-22 was found to repress mRNA expression of cytochrome P-450 (CYP) 1A2, CYP3A4, CYP2B6 and CYP2C9 and of the sinusoidal sodium-taurocholate co-transporting polypeptide (NTCP); such IL-22 effects were concentration-dependent for CYP3A4 (IC50 = 1.7 ng/mL), CYP2B6 (IC50 = 0.9 ng/mL) and NTCP (IC50 = 1.8 ng/mL). Activity of CYP1A2 (phenacetin O-deethylation), CYP3A4 (midazolam hydroxylation) and CYP2B6 (bupropion hydroxylation), as well as that of NTCP (taurocholate uptake) were concomitantly decreased in IL-22-treated HepaRG cells; by contrast, activity of organic anion transporter polypeptides (OATPs) (estrone-3-sulfate uptake) and of organic cation transporter (OCT) 1 (tetra-ethylammonium uptake) remained unchanged. IL-22 was next found to activate the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) 3 pathway, whose inhibition by the JAK inhibitor ruxolitinib fully prevented the IL-22-mediated CYP3A4, CYP2B6 and NTCP repression in HepaRG cells. This JAK-dependent down-regulation of hepatic drug detoxifying proteins, notably of CYPs, by IL-22 may contribute to alteration of pharmacokinetics in patients suffering from acute and chronic inflammatory diseases and may be the source of drug-drug interactions.

Effects of several organophosphates on hepatic cytochrome P450 activities in rats.

Four commonly used organophosphates (fenitrothion, dichlorvos, chlorpyrifos, and trichlorfon) were orally administered to male Sprague-Dawley rats for five days in order to explore their effects on the activities of liver cytochrome P450 (CYP). In addition, Michaelis-Menten kinetics of the metabolic reactions catalyzed by liver CYPs were analyzed following the addition of these compounds to the assay system to examine their potential inhibitory effects on liver CYPs activities. These reactions included ethoxyresorufin O-deethylation, midazolam 4-hydroxylation, tolbutamide hydroxylation, and bufuralol 1’-hydroxylation for CYP1A, 3A, 2C, and 2D activities, respectively. Total CYP content was also examined after oral administration of each organophosphate. Results revealed that oral giving of fenitrothion inhibited significantly CYP1A and 3A activities while elevated activity of CYP2C. Fenitrothion is a potent inhibitor for CYP1A and 2C with Ki values of 0.42 and 36.1 µM, respectively but had a weak inhibitory effect on CYP2D and 3A with Ki values of 290 and 226 µM, respectively. Chlorpyrifos is a potent inhibitor of CYP1A with Ki 0.24 µM and moderately inhibited CYP2C or 3A with Ki values of 84.8 and 77.7 µM, respectively. On the other hand, dichlorvos and trichlorfon caused extremely low or negligible inhibition of different CYP activities. From these results, it is concluded that both fenitrothion and chlorpyrifos may increase the toxicity of chemicals in environmental living organisms through their potent inhibitory effects on these CYP activities, but dichlorvos and trichlorfon may not.

Effect of Pomelo Juice on the Pharmacokinetics of Simvastatin, CYP3A2 Activity and Mdr1a, Mdr1b and Slc21a5 Expressions in Rats

Background: Food-drug interaction can decrease drug effectiveness or increase risk of drug toxicity. Simvastatin is widely used for treatment of hypercholesterolemia and hypertriglyceridemia. Therefore, this study aimed to investigate the effects of pomelo juice on the pharmacokinetics of simvastatin, CYP3a2 activity and Mdr1a, Mdr1b and Slc21a5 expressions in rats. Methods: Rats were divided into 4 groups including (i) control, (ii) pomelo that received pomelo juice orally twice daily for 7 days, (iii) simvastatin that received simvastatin on day 8, and (iv) simvastatin + pomelo juice. Plasma concentrations of simvastatin and simvastatin acid were analyzed using LC-MS/MS. Hepatic CYP3a2 activity was evaluated using midazolam hydroxylation assay. The expressions of hepatic and intestinal Mdr1a, Mdr1b and Slc21a5 were measured using the real-time RT-PCR. Results: Oral administration of pomelo juice for 7 days altered pharmacokinetic profiles of simvastatin and its primary active metabolite, simvastatin acid, in rats. Real-time RT-PCR analysis revealed that pomelo juice significantly suppressed the expression of intestinal Mdr1a and Mdr1b and hepatic Slc21a5. Rat hepatic CYP3a2 catalytic activity was also inhibited following pomelo juice administration. Conclusion: The results of this study suggested that there was a risk of potential drug interaction associated with inhibition of drug transporters and CYP3A caused by pomelo juice.

Heterotropic Cooperativity for CYP3A4‐Mediated 1′‐Hydroxylation of Midazolam by Berberine: An In Silico Modeling and Simulation Study

ObjectiveCytochrome P450 (CYP) 3A4, a membrane‐associated enzyme located primarily in the liver and small intestine, is involved in the oxidation of &gt;50% of marketed drugs. Due to its broad substrate specificity and voluminous binding pocket, CYP3A4 can bind two or more drugs simultaneously, leading to non‐hyperbolic kinetics and heterotropic cooperativity, resulting in activation or inhibition of substrate metabolism. In vitro experiments involving human liver and intestinal microsomes suggested that a major constituent of goldenseal, berberine, both activates and inhibits CYP3A‐mediated 1′‐hydroxylation of midazolam at low (≤5 μM) and high (≥100 μM) concentrations, respectively. Molecular dynamics (MD) simulations were conducted to investigate the structural basis for the observed dual cooperativity effects of berberine on midazolam 1′‐hydroxylation. Similarities in structure and membrane partitioning between berberine and progesterone suggested the progesterone site as a potential allosteric site.MethodsAll‐atom MD simulations using explicit water were conducted using a midazolam‐bound crystal structure of CYP3A4 (PDB ID 5TE8) anchored in a 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine (POPC) bilayer with its modeled transmembrane helix. Two additional systems were modeled: (1) berberine docked (using MOE software) in the progesterone binding allosteric site, topologically distinct from the midazolam binding site, and (2) berberine placed at its favorable bilayer depth, near the progesterone binding site. Each all‐atom MD simulation was conducted for 250 ns using NAMD2.12 software and CHARMM36 and CGenFF force fields.ResultsAnalyses of trajectories from all three MD simulations indicated that the conformational changes associated with the dynamics of the CYP3A4‐midazolam complex in which berberine was docked to the peripheral progesterone‐binding site were distinct from that shown in the other two simulations. Berberine caused notable conformational changes in the F‐F′ loop, increasing immobilization of midazolam (decrease in root‐mean‐square‐deviation) within the binding site, as well as decreasing the distance between the heme iron and C1 of midazolam, possibly resulting in activation (positive allosteric modulation). Additionally, a berberine molecule docked in the vicinity of the progesterone site entered the substrate binding site, altering the interaction of the midazolam molecule with the binding site residues to modulate metabolism. Berberine may also block the dissociation of 1′‐hydroxymidazolam from the binding site, causing inhibition (negative cooperativity).ConclusionComputational results provide a structural basis for the concentration‐dependent heterotropic cooperativity of CYP3A4 consistent with both activation and inhibition of midazolam 1′‐hydroxylation.Support or Funding InformationNational Institutes of Health ‐ R15GM13129 and U54AT008909This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nonalcoholic Fatty Liver Disease and Diabetes Are Associated with Decreased CYP3A4 Protein Expression and Activity in Human Liver.

Nonalcoholic fatty liver disease (NAFLD) is a major cause of chronic liver disease in the Western population. We investigated the association of nonalcoholic fatty liver disease (NAFLD) and diabetes mellitus on CYP3A4 activity in human liver tissue from brain dead donors ( n = 74). Histopathologically graded livers were grouped into normal ( n = 24), nonalcoholic fatty liver (NAFL, n = 26), and nonalcoholic steatohepatitis (NASH, n = 24) categories. The rate of conversion of midazolam to its 1-hydroxy metabolite was used to assess in vitro CYP3A4 activity in human liver microsomes (HLM). A proteomics approach was utilized to quantify the protein expression of CYP3A4 and related enzymes. Moreover, a physiologically based pharmacokinetic (PBPK) model was developed to allow prediction of midazolam concentration in NAFL and NASH livers. CYP3A4 activity in NAFL and NASH was 1.9- and 3.1-fold ( p < 0.05) lower than normal donors, respectively. Intrinsic clearance (CLint) was 2.7- ( p < 0.05) and 4.1-fold ( p < 0.01) lower in donors with NAFL and NASH, respectively. CYP3A4 protein expression was significantly lower in NAFL and NASH donors ( p < 0.05) and accounted for significant midazolam hydroxylation variability in a multiple linear regression analysis (β = 0.869, r2 = 0.762, P < 0.01). Diabetes was also associated with decreased CYP3A4 activity and protein expression. Both midazolam CLint and CYP3A4 protein abundance decreased significantly with increase in hepatic fat accumulation. Age and gender did not exhibit any significant association with the observed alterations. Predicted midazolam exposure was 1.7- and 2.3-fold higher for NAFL and NASH, respectively, which may result in a longer period of sedation in these disease-states. Data suggests that NAFLD and diabetes are associated with the decreased hepatic CYP3A4 activity. Thus, further evaluation of clinical consequences of these findings on the efficacy and safety of CYP3A4 substrates is warranted.

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

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

In vitro analysis of itraconazole cis-diastereoisomers inhibition of nine cytochrome P450 enzymes: stereoselective inhibition of CYP3A

1. Itraconazole (ITZ), an antifungal azole derivate is a chiral drug that consists of four cis-diastereoisomers ((+)-2R,4S,2′R-ITZ-A; (+)-2R,4S,2′S-ITZ-B; (–)-2S,4R,2′S-ITZ-C and (–)-2S,4R,2′R-ITZ-D) which may differ in their pharmacokinetics and pharmacodynamics.2. As ITZ is known as a CYP3A4 inhibitor causing severe drug–drug interaction, the inhibitory potencies of its individual optical isomers towards nine drug-metabolising cytochrome P450 (including CYP3A, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1), were investigated.3. All ITZ diastereoisomers dose-dependently inhibited CYP3A activity in both used assays, midazolam and testosterone hydroxylation. The Ki values were assessed: for testosterone ITZ-A/0.085 µM; ITZ-B/0.91 µM, ITZ-C/0.20 µM and ITZ-D/0.022 µM; for midazolam ITZ-A/0.44 µM; ITZ-B/0.48 µM, ITZ-C/1.56 µM and ITZ-D/3.48 µM. The enzyme activity of CYP2C19 was moderately inhibited (IC50 30–53 µM), but in this case without large differences between the individual optical isomers.4. The significant differences between diastereoisomers were presented. Antifungal potency of ITZ stereoisomers also differs so the potential enantiopure preparations of ITZ was not of interest.

Influence of Amlodipine Enantiomers on Human Microsomal Cytochromes P450: Stereoselective Time-Dependent Inhibition of CYP3A Enzyme Activity.

Amlodipine (AML) is available as a racemate, i.e., a mixture of R- and S-enantiomers. Its inhibitory potency towards nine cytochromes P450 (CYP) was studied to evaluate the drug–drug interactions between the enantiomers. Enzyme inhibition was evaluated using specific CYP substrates in human liver microsomes. With CYP3A, both enantiomers exhibited reversible and time-dependent inhibition. S-AML was a stronger reversible inhibitor of midazolam hydroxylation: the Ki values of S- and R-AML were 8.95 µM, 14.85 µM, respectively. Computational docking confirmed that the enantiomers interact differently with CYP3A: the binding free energy of S-AML in the active site was greater than that for R-AML (−7.6 vs. −6.7 kcal/mol). Conversely, R-AML exhibited more potent time-dependent inhibition of CYP3A activity (KI 8.22 µM, Kinact 0.065 min−1) than S-AML (KI 14.06 µM, Kinact 0.041 min−1). R-AML was also a significantly more potent inhibitor of CYP2C9 (Ki 12.11 µM/S-AML 21.45 µM) and CYP2C19 (Ki 5.97 µM/S-AML 7.22 μM. In conclusion, results indicate that clinical use of S-AML has an advantage not only because of greater pharmacological effect, but also because of fewer side effects and drug–drug interactions with cytochrome P450 substrates due to absence of R-AML.