Midazolam Plasma Research Articles

Quantitation of drugs via molecularly imprinted polymer solid phase extraction and electrospray ionization mass spectrometry: benzodiazepines in human plasma

The association of solid phase extraction with molecularly imprinted polymers (MIP) and electrospray ionization mass spectrometry (ESI-MS) is applied to the direct extraction and quantitation of benzodiazepines in human plasma. The target analytes are sequestered by MIP and directly analyzed by ESI-MS. Due to the MIP highly selective extraction, ionic suppression during ESI is minimized; hence no separation is necessary prior to ESI-MS, which greatly increases analytical speed. Benzodiazepines (medazepam, nitrazepam, diazepam, chlordiazepoxide, clonazepam and midazolam) in human plasma were chosen as a proof-of-principle case of drug analyses by MIP-ESI-MS in a complex matrix. MIP-ESI-MS displayed good figures of merits for medazepam, nitrazepam, diazepam, chlordiazepoxide and midazolam, with analytical calibration curves ranging from 10 to 250 μg L(-1) (r > 0.98) with limit of quantification <10 μg L(-1) and acceptable within-day and between-day precision and accuracy.

Assessment of oral midazolam limited sampling strategies to predict area under the concentration time curve (AUC) during cytochrome P450 (CYP) 3A baseline, inhibition and induction or activation

A previous study reported a 2- and 3-timepoint limited sampling strategy (LSS) model accurately predicted oral midazolam area under the concentration time curve (AUC), and thus cytochrome P450 (CYP) 3A activity. This study evaluated whether the LSS models predict midazolam AUC during CYP3A baseline, inhibition and induction/activation. Plasma midazolam concentrations from 106 healthy adults from 6 published studies were obtained where oral midazolam was co-administered alone or with ketoconazole, double-strength grapefruit juice, Ginkgo biloba extract, pleconaril, or rifampin. Observed and predicted midazolam AUCs were determined. Bias and precision of the LSS models were determined. Contrasting results were observed for the 2- and 3-timepoint LSS models in accurately predicting midazolam AUC during baseline CYP3A conditions. With the exception of 1 study (single dose, double-strength grapefruit juice), the 2- and 3-timepoint LSS models did not accurately predict midazolam AUC during conditions of CYP3A inhibition and induction/activation. The previously reported 2- and 3-timepoint oral midazolam LSS models are not applicable to the evaluated conditions of CYP3A baseline, inhibition, and induction/ activation.

Evaluation of the pharmacokinetic interaction of midazolam with ursodeoxycholic acid, ketoconazole and dexamethasone by brain benzodiazepine receptor occupancy

To clarify whether alterations in midazolam pharmacokinetics resulting from changes in cytochrome P450 3A (CYP3A) activity lead to changes in its pharmacodynamic effects, benzodiazepine receptor occupancy was measured in the brain of rats after oral administration of midazolam. Receptor occupancy was measured by radioligand binding assay in rats pretreated with ursodeoxycholic acid (UDCA), ketoconazole and dexamethasone, and the plasma concentration of midazolam was simultaneously determined. There was a significant increase in the apparent dissociation constant and decrease in the maximum number of binding sites for specific [(3) H]flunitrazepam binding after oral administration of midazolam at pharmacologically relevant doses, suggesting that midazolam binds significantly to brain benzodiazepine receptors. Pretreatment with UDCA significantly enhanced the binding. This correlated well with significant enhancement by UDCA of the plasma midazolam concentration. The brain benzodiazepine receptor binding of oral midazolam was significantly enhanced by pretreatment with ketoconazole, a potent inhibitor of CYP3A, whereas it was significantly reduced by treatment with dexamethasone, an inducer of this enzyme. These effects paralleled changes in the plasma concentration of midazolam. The results indicate that pharmacokinetic changes such as altered CYP3A activity significantly influence the pharmacodynamic effect of midazolam by affecting occupancy of benzodiazepine receptors in the brain. They also suggest in-vivo or ex-vivo time-dependent measurements of receptor occupancy by radioligand binding assay to be a tool for elucidating the pharmacokinetic interaction of benzodiazepines with other agents in pre-clinical and clinical evaluations.

Quantification of midazolam, morphine and metabolites in plasma using 96‐well solid‐phase extraction and ultra‐performance liquid chromatography–tandem mass spectrometry

Currently, pharmacokinetic-pharmacodynamic studies of sedatives and analgesics are performed in neonates and children to find suitable dose regimens. As a result, sensitive assays using only small volumes of blood are necessary to determine drug and metabolite concentrations. We developed an ultra-performance liquid chromatographic method with tandem mass spectrometry detection for quantification of midazolam, 1-hydroxymidazolam, hydroxymidazolamglucuronide, morphine, morphine-3-glucuronide and morphine-6-glucuronide in 100 microL of plasma. Cleanup consisted of 96 wells micro-solid phase extraction, before reversed-phase chromatographic separation (ultra-performance liquid chromatography) and selective detection using electrospray ionization tandem mass spectrometry. Separate solid-phase extraction methods were necessary to quantify morphine, midazolam and their metabolites because of each group's physicochemical properties. Standard curves were linear over a large dynamic range with adequate limits of quantitation. Intra- and interrun accuracy and precision were within 85-115% (of nominal concentration using a fresh calibration curve) and 15% (coefficient of variation, CV) respectively. Recoveries were >80% for all analytes, with interbatch CVs (as a measure of matrix effects) of less than 15% over six batches of plasma. Stability in plasma and extracts was sufficient, allowing large autosampler loads. Runtime was 3.00 min per sample for each method. The combination of 96-well micro-SPE and UPLC-MS/MS allows reliable quantification of morphine, midazolam and their major metabolites in 100 microL of plasma.

Propofol Reduces the Distribution and Clearance of Midazolam

Midazolam, at sedative levels, increases blood propofol concentrations by 25%. We evaluated the reverse interaction and determined the influence of propofol on the pharmacokinetics of midazolam. Eight healthy male volunteers were studied on 2 occasions in a random crossover manner. During session A, volunteers received midazolam 0.035 to 0.05 mg x kg(-1) IV for 1 minute followed by an infusion of 0.035 to 0.05 mg x kg(-1) x h(-1) for 59 minutes. During session B, in addition to this midazolam infusion scheme, a target-controlled infusion of propofol (constant C(T): 0.6 or 1.0 microg x mL(-1)) was given from 15 minutes before the start until 6 hours after termination of the midazolam infusion. Arterial blood samples for propofol and midazolam concentration analysis were taken until 6 hours after termination of the midazolam infusion. Nonlinear mixed-effect models examining the influence of propofol and hemodynamic variables on midazolam pharmacokinetics were constructed using Akaike's information-theoretic criterion for model selection. In the presence of a mean blood propofol concentration of 1.2 microg x mL(-1), the plasma midazolam concentration was increased by 26.9% + or - 9.4% compared with midazolam given as a single drug. Propofol (C(blood): 1.2 microg x mL(-1)) reduced midazolam central volume of distribution from 5.37 to 2.98 L, elimination clearance from 0.39 to 0.31 L x min(-1), and rapid distribution clearance from 2.77 to 2.11 L x min(-1). Inclusion of heart rate further improved the pharmacokinetic model of midazolam. Propofol reduces the distribution and clearance of midazolam in a concentration-dependent manner. In addition, inclusion of heart rate as a covariate improved the pharmacokinetic model of midazolam predominantly through a reduction in the intraindividual variability.

Pharmacokinetics (PK) of PF-562271, a focal adhesion kinase (FAK) inhibitor, and its effect on CYP3A in patients with advanced nonhematologic malignancies.

2553 Background: FAK transduces signaling from integrins and growth factors to modulate tumor cell invasion, proliferation, and survival. PF-562271, a potent FAK inhibitor, is under development as an anticancer agent. Preclinical studies demonstrated that PF-562271 is an inhibitor of its primary metabolizing enzyme, CYP3A. PF-562271 PK and its effect on CYP3A were investigated in a dose-escalation trial in patients with nonhematologic malignancies. Methods: Escalating doses of PF-562271 were administered orally to a total of 67 patients twice a day (BID) or once a day (QD), with or without food. Serial serum samples were collected after the first dose and at steady state to determine PK of PF-562271. To evaluate the effect of PF-562271 on CYP3A activity, plasma PK of orally administered midazolam were determined prior to PF-562271 administration and after repeated PF-562271 dosing to steady state in 8 patients who received the recommended phase 2 dose of 125 mg BID. Serum PF-562271 and plasma midazolam concentrations were determined using validated HPLC-MS/MS methods. The concentration-time data for PF-562271 and midazolam were analyzed by noncompartmental methods. Results: After a single oral dose, PF-562271 was readily absorbed with the maximum serum concentration achieved in 0.5 to 6 hours. The increases in systemic exposure (AUCinf) were dose proportional over the 5 to 25 mg range, but greater than dose proportional from 35 to 225 mg. After multiple oral doses, PF-562271 demonstrated nonlinear accumulation in exposure; the mean ratio of steady state AUC during a dose interval to AUCinf after the first dose ranged from 1.3 to 5.3, indicating time-dependent PK. Similar PK parameters for PF-562271 were observed at comparable doses given with or without food. When co-administered, PF-562271 (125 mg BID) increased total midazolam plasma exposure approximately 5.9-fold. Conclusions: PF-562271 displayed time- and dose-dependent nonlinear PK, and is a potent CYP3A inhibitor. The nonlinear PK of PF-562271 likely resulted from auto-inhibition of CYP3A. In the limited number of subjects evaluated, food did not substantially alter the PK of PF-562271. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Pfizer Pfizer

High performance liquid chromatographic assay for the simultaneous determination of midazolam and ketoconazole in plasma

A high performance liquid chromatographic (HPLC) assay was developed for the simultaneous quantitation of midazolam (MDZ) and ketoconazole (KTZ) in plasma. MDZ, KTZ and diazepam (internal standard) were extracted from 100 μL or 500 μL plasma from rat or human, respectively, using liquid–liquid extraction with diethyl ether in the presence of 0.1N NaOH. After vortexing, centrifugation and freezing, the organic layer was transferred to clean tubes and evaporated. The dried residue was reconstituted in mobile phase and injected into the HPLC through a C18 column. The mobile phase consisted of acetonitrile:15 mM potassium dihydrogen orthophosphate (45:55, v/v), pumped at 1 mL/min and measured at λ = 220 nm. The method was tested in a pharmacokinetic study involving orally dosed KTZ 40 mg/kg in 1% methylcellulose followed by intravenous dosing of 5 mg/kg MDZ to rats 1.5 h latter. The components eluted within 10 min and were baseline resolved with no interferences from endogenous substances in plasma. The calibration curves were linear ( r 2 = 0.999) over the range of 25–25,000 and 5–10,000 ng/mL of KTZ and MDZ in rat and human plasma, respectively. The intraday and interday CV% were <15% and <6% for KTZ and <7% and <4% for MDZ and the mean error was <13% for both drugs in rat plasma. In human plasma the intraday CV% and % error of the mean were <11% and <10% for KTZ, respectively; both values were <13% for MDZ. The validated lower limit of quantitation was 25 and 5 ng/mL for both drugs based on 100 μL rat plasma and 500 μL human plasma, respectively. In rats, plasma concentrations of MDZ and KTZ were simultaneously measured up to 8 and 9.5 h, respectively. In conclusion, the assay was shown to be rapid, sensitive and appropriate for use in drug–drug interaction studies involving MDZ and KTZ in rat, and potentially in humans.

Effect of glycyrrhizin on the activity of CYP3A enzyme in humans

Glycyrrhizin is a major ingredient of licorice which is widely used in the treatment of various diseases such as chronic hepatitis. Licorice or glycyrrhizin has been shown to alter the activity of CYP3A in rodents. The influence of glycyrrhizin on CYP3A has not been elucidated in humans. To investigate the effects of repeated glycyrrhizin ingestion on the oral pharmacokinetics of midazolam, a probe drug for CYP3A activity in humans. Sixteen healthy adult male subjects were enrolled in a two-phase randomized crossover design. In each phase the volunteers received placebo or glycyrrhizin for 14 days. On the 15th day, midazolam was administered and blood samples were obtained to determine midazolam plasma concentrations. Bioequivalence was assessed by determining geometric mean ratios (GMRs) and 90% confidence intervals (90% CI). The geometric mean (geometric coefficient of variation) for the AUC(0-infinity) of midazolam in the placebo group was 196.4 ng x h/ml (30.3%) and after glycyrrhizin treatment, 151.3 ng x h/ml (34.7%). The GMRs and 90% CI for AUC(0-infinity) and Cmax of midazolam in the presence/ absence of glycyrrhizin were 0.77 (0.70, 0.89) and 0.83 (0.74, 1.01), respectively. The 90% CI for AUC(0-infinity) and Cmax for the GMR of glycyrrhizin over placebo were both out of the no-effect boundaries of 0.80-1.25. Administration of glycyrrhizin resulted in a modest induction of CYP3A that was clinically relevant according to the bioequivalence analysis.

Simultaneous determination of tolbutamide, omeprazole, midazolam and dextromethorphan in human plasma by LC–MS/MS—A high throughput approach to evaluate drug–drug interactions

Drug–drug interactions involving cytochrome P450 (CYP450s) are an important factor for evaluation of a new chemical entity (NCE) in drug development. To evaluate the potential inhibitory effects of a NCE on the pharmacokinetics of a cocktail of representative probes of CYP enzymes (midazolam for CYP3A4, tolbutamide for CYP2C9, omeprazole for CYP2C19 and dextromethorphan for CYP2D6) and the safety and tolerability of the NCE in the presence of probe substrates, a high throughput liquid chromatography/tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of tolbutamide, omeprazole, midazolam and dextromethorphan in human plasma using tolbutamide-d 9, midazolam-d 4, (±)-omeprazole-d 3, and dextromethorphan-d 3 as the internal standards (ISs). Human plasma samples of 50 μL were extracted by a simple protein-precipitation procedure and analyzed using a high performance liquid chromatography electrospray tandem mass spectrometer system. Reversed-phase HPLC separation was achieved with a Hypersil GOLD AQ column (50 mm × 4.6 mm, 5 μm). MS/MS detection was set at mass transitions of 271 → 172 m/ z for tolbutamide, 346 → 198 m/ z for omeprazole, 326 → 291 m/ z for midazolam, 272 → 171 m/ z for dextromethorphan, 280 → 172 m/ z for tolbutamide-d 9 (IS), 349 → 198 m/ z for (±)-omeprazole-d 3 (IS), 330 → 295 m/ z for midazolam-d 4 (IS), and 275 → 171 m/ z for dextromethorphan-d 3 (IS) in positive mode. The high throughput LC–MS/MS method was validated for accuracy, precision, sensitivity, stability, recovery, matrix effects, and calibration range. Acceptable intra-run and inter-run assay precision (<10%) and accuracy (<10%) were achieved over a linear range of 50–50,000 ng/mL for tolbutamide, 1–1000 ng/mL for omeprazole, 0.1–100 ng/mL for midazolam and 0.05–50 ng/mL for dextromethorphan in human plasma. Method robustness was demonstrated by the 100% pass rate of 24 incurred sample analysis runs and all of the 50 clinical study samples used for incurred sample reproducibility (ISR) test having met the acceptance criterion (%Diff within 20%). The overall ISR results for all compounds showed that over 95% of the samples had a %Diff of less than 10%. The method is simple, rapid and rugged, and has been applied successfully to sample analysis in support of a drug–drug interaction study.

Variability in drug metabolizing enzyme activity in HIV-infected patients

To evaluate variability in cytochrome P450 (CYP) 1A2, CYP2D6, CYP3A, N-acetyltransferase 2 (NAT2), and xanthine oxidase (XO) activity in HIV-infected patients and compare this with data from uninfected, healthy volunteers. Ten HIV-infected men and seven women on medication affecting CYP enzyme activity were phenotyped four times over 2 months using caffeine, dextromethorphan, and midazolam. Urinary caffeine and dextromethorphan metabolite ratios were used to phenotype CYP1A2, NAT2, XO, and CYP2D6 activity and midazolam plasma clearance was used to phenotype CYP3A activity. Plasma and urine samples were analyzed by validated LC/UV or LC/MS methods for midazolam, caffeine, and dextromethorphan. Noncompartmental pharmacokinetics and nonparametric statistical analyses were performed, and the data compared with those of healthy volunteer historic controls. Compared with age and sex-matched healthy volunteers, HIV-infected subjects had 18% lower hepatic CYP3A4 activity, 90% lower CYP2D6 activity, 53% lower NAT2 activity, and 22% higher XO activity. No significant difference was found in CYP1A2 activity. Additionally, 25% genotype-phenotype discordance in CYP2D6 activity was noted in HIV-infected subjects. Intraindividual variability in enzyme activity increased by 42-62% in HIV-infected patients for CYP1A2, NAT2, and XO, and decreased by 33% for CYP2D6. Interindividual variability in enzyme activity increased by 27-63% in HIV-infected subjects for CYP2D6, CYP1A2, and XO, and decreased by 38% for NAT2. Higher plasma TNFalpha concentrations correlated with lower CYP2D6 and CYP3A4 activity. Infection with HIV or stage of HIV infection may alter Phase I and II drug metabolizing enzyme activity. HIV infection was related to an increase in variability of these drug-metabolizing enzymes. Altered metabolism may be a consequence of immune activation and cytokine exposure.

Vitamin E Supplementation and Hepatic Drug Metabolism in Humans

Meta-analyses studies suggest that high-dose vitamin E may be associated with increased mortality in some populations. Vitamin E may increase the production of CYP3A4 in the liver, and this could lead to an increase in drug metabolism, potentially lowering the efficacy of therapeutic drugs. We hypothesized that upregulation of CYP3A4 by alpha-tocopherol (alpha-TOH) would decrease the plasma concentration of the CYP3A4 substrate midazolam. Baseline metabolism of midazolam (1 mg intravenously) was determined in 12 healthy subjects before randomization into 2 groups of 6 to receive either RRR-alpha-TOH (750 IU/d) or placebo for 3 weeks. At completion, subjects were given an additional 1 mg intravenous bolus of midazolam. Plasma midazolam, 1-hydroxy-midazolam, and urinary alpha-TOH metabolite excretion were measured using gas chromatography mass spectrometry. Serum alpha-TOH was measured using high performance liquid chromatography with electrochemical detection. Serum alpha-TOH increased by 100% (P = 0.002) and urinary alpha-TOH metabolite excretion increased 20-fold in the treatment group versus placebo (P = 0.001). There was no effect on the area under time curve of midazolam in subjects taking alpha-TOH compared with placebo. These findings do not support the hypothesis that alpha-TOH supplementation interferes with hepatic CYP3A4-mediated drug metabolism.

A PBPK model for midazolam in four avian species

A physiologically based pharmacokinetic (PBPK) model was developed for midazolam in the chicken and extended to three other species. Physiological parameters included organ weights obtained from 10 birds of each species and blood flows obtained from the literature. Partition coefficients for midazolam in tissues vs. plasma were estimated from drug residue data obtained at slaughter. The avian models include separate compartments for venous plasma, liver, kidney, muscle, fat and all other tissues. An estimate of total body clearance from an earlier in vitro study was used as a starting value in the model, assuming almost complete removal of the parent compound by liver metabolism. The model was optimized for the chicken with plasma and tissue data from a pharmacokinetic study after intravenous midazolam (5 mg/kg) dose. To determine which parameters had the most influence on the goodness of fit, a sensitivity analysis was performed. The optimized chicken model was then modified for the turkey, pheasant and quail. The models were validated with midazolam plasma and tissue residue data in the turkey, pheasant and quail. The PBPK models in the turkey, pheasant and quail provided good predictions of the observed tissue residues in each species, in particular for liver and kidney.

Limited sampling strategy in rats to predict the inhibited activities of hepatic CYP3A

The present study was undertaken in order to evaluate feasibility of a limited sampling strategy (LSS) to predict the systemic clearance of midazolam (MDZ), which is a hepatic CYP3A activity phenotyping probe. Groups of rats pretreated with or without serial doses of ketoconazole, which is a selective inhibitor on CYP3A, were used as training set. Linear regression analysis and a Jack-knife validation procedure were performed based on plasma MDZ concentrations at specific time points after sublingual vein injection of MDZ to establish the most informative LSS equations for accurately estimating the clearance of MDZ. Another group of rats in the same setting was used as the validation set to confirm the individual values of estimated clearance (Clest) that were derived from the predictive equations developed in the training set. LSS that were derived from one, two or three sampling times, namely 90 min, 60-90 min, 30-60-90 min and 30-60-120 min, gave the best correlation and acceptable errors between the values of observed clearance (Clobs) and Clest and were chosen to evaluate hepatic CYP3A activity. Our results supported the hypothesis that using limited plasma sampling is simpler than the usual method of estimating CYP3A phenotyping by predicting the systemic clearance of MDZ when the hepatic activity of CYP3A is reduced in the rat. This experimental design offers opportunities to reduce animal use in the study of drug metabolism.

Mixed-Effects Modeling of the Influence of Midazolam on Propofol Pharmacokinetics

The combined administration of anesthetics has been associated with pharmacokinetic interactions that induce concentration changes of up to 30%. Midazolam is often used as a preoperative sedative in advance of a propofol-based anesthetic. In this study, we identified the influence of midazolam on the pharmacokinetics of propofol. Eight healthy male volunteers were studied on two occasions in a random crossover manner. During Session A, volunteers received propofol 1 mg/kg in 1 min followed by an infusion of 2.5 mg x kg(-1) x h(-1) for 59 min. During Session B, in addition to this propofol infusion scheme, a target-controlled infusion of midazolam (constant C(t): 125 ng/mL) was given from 15 min before the start until 6 h after termination of the propofol infusion. Arterial blood samples for blood propofol and plasma midazolam concentration analysis were taken until 6 h after termination of the propofol infusion. Nonlinear mixed-effects models examining the influence of midazolam and hemodynamic variables on propofol pharmacokinetics were constructed using Akaike criterion for model selection. In the presence of midazolam (C(blood): 224.8 +/- 41.6 ng/mL), the blood propofol concentration increased by 25.1% +/- 13.3% compared with when propofol was given as single drug. Midazolam (C(blood): 225 ng/mL) reduced propofol Cl(1) from 1.94 to 1.61 L/min, Cl(2) from 2.86 to 1.52 L/min, and Cl(3) from 0.95 to 0.73 L/min. Inclusion of mean arterial blood pressure further improved the propofol pharmacokinetic model. Midazolam reduces the metabolic and rapid and slow distribution clearances of propofol. In addition, a reduction in mean arterial blood pressure is associated with propofol pharmacokinetic alterations that increase the blood propofol concentration.

Effects of Schisandra sphenanthera extract on the pharmacokinetics of midazolam in healthy volunteers

To assess the effect of Schisandra sphenanthera extract (SchE) on the pharmacokinetics of midazolam, a probe drug of CYP3A, and its metabolite 1'-hydroxy midazolam in healthy volunteers. Twelve healthy male volunteers were orally treated with SchE, three capsules twice daily for 7 days. Pharmacokinetic investigations of oral midazolam administration at 15 mg were performed both before and at the end of the SchE treatment period. The plasma midazolam and 1'-hydroxy midazolam concentrations were determined by high-performance liquid chromatography-tandem mass spectrometry. Estimated pharmacokinetic parameters before and with SchE were calculated with noncompartmental techniques. Following administration of SchE, the average increases (%) of individual increases in AUC, AUMC and C(max) of midazolam were 119.4% [95% confidence interval (CI) 83.9, 155.0], 183.4% (95% CI 120.5, 246.2) and 85.6% (95% CI 14.4, 156.9), respectively (P < 0.01 or 0.05). On average, there was a 133.3% (95% CI 8.9, 257.7) increase in midazolam t(max) (P < 0.01). The average decrease (%) in CL/F was 52.1% (95% CI 44.9, 59.4) (P < 0.01). No significant changes were seen in midazolam half-life. After co-administration of SchE, the average increase (%) in t(max) of 1'-hydroxy midazolam was 150.0% (95% CI 22.2, 277.8) (P < 0.05). No significant differences were observed in the other pharmacokinetic parameters of 1'-hydroxy midazolam. SchE can markedly increase the oral bioavailability of midazolam in healthy volunteers. SchE is an inhibitor of CYP3A and has a high susceptibility to alter the disposition of drugs metabolized by CYP3A.

Lack of effect of subject posture on intravenous midazolam clearance: implications for hepatic cytochrome P450 3A phenotyping

Intravenous (i.v.) midazolam is used to phenotype hepatic cytochrome P450 (CYP) 3A [1]. Midazolam clearance (CL) is believed to correlate with the intrinsic hepatic CL (CLint) of midazolam and thus acts as a surrogate for hepatic CYP3A activity. Midazolam is a moderate extraction ratio drug [2]. Based on the well-stirred model of hepatic clearance [3], midazolam CL depends on CLintand hepatic blood flow. Subject posture alters hepatic blood flow, is often not controlled during blood sample collection [1], and impacts pharmacokinetic studies [4]. The magnitude of posture-induced blood flow changes has been seen in a study of six subjects. Plasma midazolam CL decreased 48% in an ambulant (sitting/walking) posture compared with a supine posture [5] and suggests that changes in midazolam CL may be dependent on hepatic blood flow and not exclusively CYP3A activity. This study evaluated the effect of subject posture on midazolam CL to confirm previous findings. We quantified posture-induced hepatic blood flow changes with i.v. indocyanine green as a biomarker. Results are published elsewhere [6]. The Institutional Review Board of Bassett Healthcare (Cooperstown, NY, USA) approved the study. Written informed consent was obtained. Healthy adults 18–55 years old who were not on any medications were enrolled. Subjects were randomized to a 7-h supine or ambulant posture. A minimum of 7 days passed between study phases. Subjects maintained strict horizontal bed rest during supine posture. During ambulant posture, subjects were seated for the first 3 h and then allowed to stand, walk, or sit. Sessions began between 06.00 h and 07.00 h to minimize temporal variation [5]. Subjects fasted (except water) overnight and for the duration of each posture. To stabilize hepatic blood flow, subjects were in the assigned posture for 1 h prior to midazolam 0.025 mg kg−1 i.v. (midazolam hydrochloride, 1 mg ml−1 injection, North Chicago, IL, USA) administration. Eight blood samples were obtained over 6 h. Samples were centrifuged and stored at −80°C. Plasma concentrations were analysed by liquid chromatography/mass spectrometry/mass spectrometry as described elsewhere [7]. The linear range of the assay was 0.50–100 ng ml−1, with intra- and interday coefficients of variation of ≤7.9% at 0.75, 7.5 and 75 ng ml−1. Midazolam pharmacokinetics was determined by noncompartmental analysis (WinNonlin v3.1; Pharsight, Cary, NC, USA). Sample size calculations were based on previously published data [5, 8–10]. Using an α= 0.05 and β= 0.20, a sample size of 12 would detect a 25% difference in midazolam CL. Bioequivalence testing was used by calculating the least squares geometric mean ratios (LS-GMR; CLambulant/CLsupine) and 90% confidence intervals (CI). Bioequivalence was concluded if the 90% CIs were within the 80–125% interval. Thirteen (seven female) subjects completed both postures. Plasma midazolam concentrations vs. time are shown in Figure 1. Bioequivalence was observed for the geometric mean midazolam CLs in the supine and ambulant phases (595.6 vs. 565.1 ml min−1; LS-GMR 0.95, 90% CI 0.88, 1.02). Intersubject variation of midazolam CL was small, varying 1.7- and two-fold in the supine and ambulant postures, respectively. The volume of distribution (Vd) decreased 18% in the ambulant posture. The difference in Vd may be due to changes in tissue binding, but for CYP3A phenotyping in healthy adults this change is unlikely to be of clinical significance. Figure 1 Mean ± SD midazolam plasma concentrations vs. time in the ambulant (closed squares) and supine (open circles) postures (n= 13) Studies have observed posture-induced changes in hepatic blood flow of 17–37% for several medications [11, 12]. In the current study, subject posture did not affect midazolam CL. In contrast to the study of Klotz et al. [5], the current study used a smaller midazolam i.v. dose (0.025 vs. 0.075 mg kg−1), had a larger sample size (13 vs. 6), and had subjects remain fasting during the entire sampling period. Food intake increases hepatic blood flow and lasts up to 190 min [13]. We speculate that the small sample size and food intake may have affected midazolam CL in the Klotz et al. study [5]. Neither study reported the exact amount of time during the ambulant posture that subjects were in a sitting, walking and standing posture, and potential differences between subjects and studies may have existed in the amount of time spent in a specific ambulant position. Phenotyping studies using i.v. midazolam as a hepatic CYP3A probe do not need to control for subject posture during blood sample collection. These findings are not applicable when using oral midazolam to phenotype intestinal and hepatic CYP3A activity, as i.v. midazolam exclusively measures hepatic CYP3A.

Using midazolam to monitor changes in hepatic drug metabolism in critically ill patients

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.

Pharmacogenomics of CYP2A6, CYP2B6, CYP2C19, CYP2D6, CYP3A4, CYP3A5 and MDR1 in Vietnam

The aim of this study was to obtain pharmacogenetic data in a Vietnamese population on genes coding for proteins involved in the elimination of drugs currently used for the treatment of malaria and human immunodeficiency virus/acquired immunodeficiency syndrome. The main polymorphisms on the cytochrome P450 (CYP) genes, CYP2A6, CYP2B6, CYP2C19, CYP2D6, CYP3A4 and CYP3A5, and the multi-drug resistance 1 gene (MDR1) were genotyped in 78 healthy Vietnamese subjects. Pharmacokinetic metrics were available for CYP2A6 (coumarin), CYP2C19 (mephenytoin), CYP2D6 (metoprolol) and CYP3As (midazolam), allowing correlations with the determined genotype. In the CYP2 family, we detected alleles CYP2A6*4 (12%) and *5 (15%); CYP2B6*4 (8%), *6 (27%); CYP2C19*2 (31%) and *3 (6%); CYP2D6*4, *5, *10 (1, 8 and 44%, respectively). In the CYP3A family, CYP3A4*1B was detected at a low frequency (2%), whereas CYP3A5 *3 was detected at a frequency of 67%. The MDR1 3435T allele was present with a prevalence of 40%. Allele proportions in our cohort were compared with those reported for other Asian populations. CYP2C19 genotypes were associated to the S-4'-OH-mephenytoin/S-mephenytoin ratio quantified in plasma 4 h after intake of 100 mg mephenytoin. While CYP2D6 genotypes were partially reflected by the alpha-OH-metroprolol/metoprolol ratio in plasma 4 h after dosing, no correlation existed between midazolam plasma concentrations 4 h post-dose and CYP3A genotypes. The Vietnamese subjects of our study cohort presented allele prevalences in drug-metabolising enzymes that were generally comparable with those reported in other Asian populations. Deviations were found for CYP2A6*4 compared to a Chinese population (12 vs. 5%, respectively; P = 0.023), CYP2A6*5 compared with a Korean population (15 vs. <1%, respectively; P < 0.0001), a Malaysian population (1%; P < 0.0001) and a Chinese population (1%; P < 0.0001); CYP2B6*6 compared with a Korean population (27 vs. 12%; P = 0.002) and a Japanese population (16%; P = 0.021). Pharmacokinetic metrics versus genotype analysis reinforces the view that the predictive value of certain globally common variants (e.g. CYP2D6 single nucleotide polymorphisms) should be evaluated in a population-specific manner.

Research Highlights

Research Highlights

Pharmacokinetics of intravenous and oral midazolam in plasma and saliva in humans: usefulness of saliva as matrix for CYP3A phenotyping

To compare midazolam kinetics between plasma and saliva and to find out whether saliva is suitable for CYP3A phenotyping. This was a two way cross-over study in eight subjects treated with 2 mg midazolam IV or 7.5 mg orally under basal conditions and after CYP3A induction with rifampicin. Under basal conditions and IV administration, midazolam and 1'-hydroxymidazolam (plasma, saliva), 4-hydroxymidazolam and 1'-hydroxymidazolam-glucuronide (plasma) were detectable. After rifampicin, the AUC of midazolam [mean differences plasma 53.7 (95% CI 4.6, 102.9) and saliva 0.83 (95% CI 0.52, 1.14) ng ml(-1) h] and 1'-hydroxymidazolam [mean difference plasma 11.8 (95% CI 7.9 , 15.7) ng ml(-1) h] had decreased significantly. There was a significant correlation between the midazolam concentrations in plasma and saliva (basal conditions: r = 0.864, P < 0.0001; after rifampicin: r = 0.842, P < 0.0001). After oral administration and basal conditions, midazolam, 1'-hydroxymidazolam and 4-hydroxymidazolam were detectable in plasma and saliva. After treatment with rifampicin, the AUC of midazolam [mean difference plasma 104.5 (95% CI 74.1, 134.9) ng ml(-1) h] and 1'-hydroxymidazolam [mean differences plasma 51.9 (95% CI 34.8, 69.1) and saliva 2.3 (95% CI 1.9, 2.7) ng ml(-1) h] had decreased significantly. The parameters separating best between basal conditions and post-rifampicin were: (1'-hydroxymidazolam + 1'-hydroxymidazolam-glucuronide)/midazolam at 20-30 min (plasma) and the AUC of midazolam (saliva) after IV, and the AUC of midazolam (plasma) and of 1'-hydroxymidazolam (plasma and saliva) after oral administration. Saliva appears to be a suitable matrix for non-invasive CYP3A phenotyping using midazolam as a probe drug, but sensitive analytical methods are required.