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

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

The Prediction of the Area under the Curve and Clearance of Midazolam from Single-Point Plasma Concentration and Urinary Excretion in Healthy Volunteers.

There are large inter- and intra-individual variations in CYP3A4 activity. Midazolam, which is predominantly metabolized to 1'-hydroxymidazolam and 4-hydroxymidazolam by CYP3A4, is considered an effective probe for CYP3A4. To determine the area under the curve (AUC) of midazolam or midazolam clearance for CYP3A4 activity, multiple plasma samples of midazolam are required. This study aimed to evaluate whether measurement of a single plasma concentration or urinary excretion of midazolam could be used to predict the AUC of midazolam in healthy volunteers. We conducted a retrospective analysis of two pharmacokinetic studies. Nineteen volunteers received intravenous (5, 15, and 30 µg/kg) and oral (15, 50, and 100 µg/kg) administration of midazolam on sequential days. The midazolam concentration in plasma and urine was determined by LC-MS/MS. Plasma midazolam concentrations showed a good correlation with the AUC at all blood sampling points after the administrations. The coefficient of determination was highest at 1-2 and 2-4 h after intravenous (>0.96) and oral administration (>0.94), respectively, among all the sampling times. The errors for bias and accuracy of prediction were the lowest at 1.5 and 4 h after intravenous and oral administration, respectively. In case of urinary excretion, a significant positive correlation between midazolam and the AUC was observed only after oral administration. Thus, the AUC of midazolam can be evaluated by blood sampling at 1.5 h after intravenous administration and at 4 h after oral administration.

Hypoalbuminaemia and decreased midazolam clearance in terminally ill adult patients, an inflammatory effect?

AIMS: Midazolam is the drug of choice for palliative sedation and is titrated to achieve the desired level of sedation. Because of large inter-individual variability (IIV), however, the time it takes to achieve adequate sedation varies widely. It would therefore greatly improve clinical care if an individualized dose could be determined beforehand. To find clinically relevant parameters for dose individualization, we performed a pharmacokinetic study on midazolam, 1OH-midazolam (1-OH-M) and 1OH-midazolam-glucuronide (1-OH-MG) in terminally ill patients. METHODS: Using nonlinear mixed effects modelling (NONMEM 7.2), a population pharmacokinetic analysis was conducted with 192 samples from 45 terminally ill patients who received midazolam either orally or subcutaneously. The covariates analysed were patient characteristics, co-medication and blood chemistry levels. RESULTS: The data were accurately described by a one compartment model for midazolam, 1-OH-M and 1-OH-MG. The population mean estimates for midazolam, 1-OH-M and 1-OH-MG clearance were 8.4 l h-1 (RSE 9%, IIV 49%), 45.4 l h-1 (RSE 12%, IIV 60.5%) and 5.1 l h-1 (RSE 11%, IIV 49.9%), respectively. 1-OH-MG clearance was correlated with the estimated glomular filtration rate (eGFR) explaining 28.4% of the IIV in 1-OH-MG clearance. In addition, low albumin levels were associated with decreased midazolam clearance, explaining 18.2% of the IIV. CONCLUSION: Our study indicates albumin levels and eGFR as relevant clinical parameters to optimize midazolam dosing in terminally ill patients. The correlation between low albumin levels and decreased midazolam clearance is probably a result of inflammatory response as high CRP levels were correlated in a similar way.

Autoinhibitory properties of the parent but not of the N-oxide metabolite contribute to infusion rate-dependent voriconazole pharmacokinetics.

AIMS: The pharmacokinetics of voriconazole show a nonlinear dose-exposure relationship caused by inhibition of its own CYP3A-dependent metabolism. Because the magnitude of autoinhibition also depends on voriconazole concentrations, infusion rate might modulate voriconazole exposure. The impact of four different infusion rates on voriconazole pharmacokinetics was investigated. METHODS: Twelve healthy participants received 100 mg voriconazole intravenous over 4 h, 400 mg over 6 h, 4 h, and 2 h in a crossover design. Oral midazolam (3 µg) was given at the end of infusion. Blood and urine samples were collected up to 48 h. Voriconazole and its N-oxide metabolite were quantified using high-performance liquid chromatography coupled to tandem mass spectrometry. Midazolam estimated metabolic clearance (eCLmet) was calculated using a limited sampling strategy. Voriconazole-N-oxide inhibition of cytochrome P450 (CYP) isoforms 2C19 and 3A4 were assessed with the P450-Glo luminescence assay. RESULTS: Area under the concentration-time curve for 400 mg intravenous voriconazole was 16% (90% confidence interval: 12-20%) lower when administered over 6 h compared to 2 h infusion. Dose-corrected area under the concentration-time curve for 100 mg over 4 h was 34% lower compared to 400 mg over 4 h. Midazolam eCLmet was 516 ml min-1 (420-640) following 100 mg 4 h-1 voriconazole, 152 ml min-1 (139-166) for 400 mg 6 h-1 , 192 ml min-1 (167-220) for 400 mg 4 h-1 , and 202 ml min-1 (189-217) for 400 mg 2 h-1 . Concentration giving 50% CYP inhibition of voriconazole N-oxide was 146 ± 23 µmol l-1 for CYP3A4, and 40.2 ± 4.2 µmol l-1 for CYP2C19. CONCLUSIONS: Voriconazole pharmacokinetics is modulated by infusion rate, an autoinhibitory contribution voriconazole metabolism by CYP3A and 2C19 and to a lesser extent its main N-oxide metabolite for CYP2C19. To avoid reduced exposure, the infusion rate should be 2 h.

Out of the frying pan, into the fire: a case of heat shock and its fatal complications.

Exertional heat stroke incidence is on the rise and has become the third leading cause of death in high school athletes. It is entirely preventable, yet this is a case of a 15-year-old, 97-kg male football player who presented unresponsive and hyperthermic after an August football practice. His blood pressure was 80/30, and his pulse was 180. He had a rectal temperature of 107.3°F, and upon entering the emergency department, he was rapidly cooled in 40 minutes. As he progressed, he developed metabolic acidosis, elevated liver enzymes, a prolapsed mitral valve with elevated troponin levels, and worsening hypotension even with extracorporeal membrane oxygenation support. After 3 days in the hospital, this young man was pronounced dead as a result of complications from exertional heat stroke. We address not only the complications of his hospital course relative to his positive blood cultures but also the complications that can result from attention-deficit/hyperactivity disorder medication our patient was taking. As the population of young adults becomes more obese and more highly medicated for attention-deficit/hyperactivity disorder, we sought out these growing trends in correlation with the increase in incidence of heat-related illness. We also address the predisposing factors that make young high school athletes more likely to experience heat illness and propose further steps to educate this susceptible population.

Pharmacokinetic and pharmacodynamic alterations in the Roux-en-Y gastric bypass recipients.

OBJECTIVE: We conducted a pharmacokinetic (PK) study and a pharmacodynamic (PD) study to assess whether Roux-en-Y gastric bypass (RYGB) surgery is associated with significant changes to PK and PD of oral medications. BACKGROUND: The effect of RYGB on oral drug disposition is not well understood. METHODS: An oral cocktail of probe drugs for major drug-metabolizing enzymes (caffeine, tolbutamide, omeprazole, dextromethorphan, and oral and intravenous midazolam) was administered to 18 RYGB recipients and 18 controls. Timed blood and urine samples were obtained for PK analyses. Forty mg of oral furosemide was administered to 13 RYGB recipients and 14 controls, and urine and blood samples were collected for assessing furosemidePK, and urine volume and urine sodium excretion for PD analyses. RESULTS: Compared with controls, the RYGB group had significantly lower time to maximum plasma concentration (tmax) for caffeine (0.58 ± 0.5 vs 2.1 ± 2.2 hours, P < 0.0001), tolbutamide (1.4 ± 1.8 vs 2.1 ± 2.2 hours, P = 0.0001), omeprazole (1.1 ± 1.1 vs 4.4 ± 1.3 hours, P < 0.0001), and oral midazolam (0.5 ± 0.2 vs 0.7 ± 0.4 hours, P < 0.01). However, maximum plasma concentration, half-life, area under the curve, and oral bioavailability were not different. Compared with controls, the RYGB group had brisk natriuresis, with significantly lower tmax for urine sodium (1.3 ± 0.5 vs 3.1 ± 2.3 hours, P < 0.02) and correspondingly lower tmax for furosemide (1.8 ± 0.3 vs 4.2 ± 1.2 hours, P = 0.006). However, 6-hour urine sodium and 6-hour urine volume were not different between the two groups. CONCLUSIONS: RYGB recipients have significantly shorter tmax for the studied orally administered medications, but otherwise no other significant changes in PK were reported.

Effects of hypothermia on the disposition of morphine, midazolam, fentanyl, and propofol in intensive care unit patients.

Therapeutic hypothermia (TH) may induce pharmacokinetic changes that may affect the level of sedation. We have compared the disposition of morphine, midazolam, fentanyl, and propofol in TH with normothermia in man. Fourteen patients treated with TH following cardiac arrest (33-34°C) were compared with eight matched critically ill patients (36-38°C). Continuous infusions of morphine and midazolam were stopped and replaced with infusions of fentanyl and propofol to describe elimination and start of infusion pharmacokinetics, respectively. Serial serum and urine samples were collected for 6-8 hours for validated quantification and subsequent pharmacokinetic analysis. During TH, morphine elimination half-life (t(1/2)) was significantly higher, while total clearance (CL(tot)) was significantly lower (median [semi-interquartile range (s-iqr)]): t(1/2), 266 (43) versus 168 (11) minutes, P < 0.01; CL(tot), 1201 (283) versus 1687 (200) ml/min, P < 0.01. No significant differences were seen for midazolam. CL(tot) of fentanyl and propofol was significantly lower in hypothermic patients [median (s-iqr)]: fentanyl, 726 (230) versus 1331 (678) ml/min, P < 0.05; propofol, 2046 (305) versus 2665 (223) ml/min, P < 0.05. Compared with the matched, normothermic intensive care unit patients, t(1/2) of morphine was significantly higher during TH. CL(tot) was lower during TH for morphine, fentanyl, and propofol but not for midazolam. Reducing the infusion rates of morphine, fentanyl, and propofol during TH is encouraged.

A fast and simple method for the simultaneous analysis of midazolam, 1-hydroxymidazolam, 4-hydroxymidazolam and 1-hydroxymidazolam glucuronide in human serum, plasma and urine.

For the quantification of the sedative and anesthetic drug midazolam and its main (active) metabolites 1-hydroxymidazolam, 4-hydroxymidazolam and 1-hydroxymidazolam glucuronide in human serum, human EDTA plasma, human heparin plasma and human urine a single accurate method by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) has been developed. Protein precipitation as sample preparation, without the need of a time-consuming deglucuronidation step for the quantification of 1-hydroxymidazolam glucuronide, resulted in a simple and rapid assay suitable for clinical practice with a total runtime of only 1.1  min. The four components and the isotope-labeled internal standards were separated on a C18 column and detection was performed with a triple-stage quadrupole mass spectrometer operating in positive ionization mode. The method was validated based on the "Guidance for Industry Bioanalytical Method Validation" (Food and Drug Administration, FDA) and the "Guideline on bioanalytical method validation" of the European Medicines Agency (EMA). Linearity was proven over the ranges of 5-1500 µg/L for midazolam, 1-hydroxymidazolam and 4-hydroxymidazolam and 25-5000 µg/L for 1-hydroxymidazolam glucuronide, using a sample volume of 100 µL. Matrix comparison indicated that the assay is also applicable to other human matrices like EDTA and heparin plasma and urine. Stability experiments showed good results for the stability of midazolam, 1-hydroxymidazolam and 1-hydroxymidazolam glucuronide in serum, EDTA and heparin plasma and urine stored for 7 days under different conditions. At room temperature, 4-hydroxymidazo-lam is stable for 7 days in EDTA plasma, but stable for only 3 days in serum and heparin plasma and less than 24 h in urine. All four compounds were found to be stable in serum, EDTA plasma, heparin plasma and urine for 7 days after sample preparation and for 3 freeze-thaw cycles. The assay has been applied in therapeutic drug monitoring of midazolam for (pediatric) intensive care patients.

Urinary metabolic markers reflect on hepatic, not intestinal, CYP3A activity in healthy subjects.

Intestinal cytochrome P450 3A (CYP3A) plays an important role in oral drug metabolism, but only endogenous metabolic markers for measuring hepatic CYP3A activity were identified. Our study evaluated whether hepatic CYP3A markers reflected intestinal CYP3A activity. An open-label, three-period, six-treatment, one-sequence clinical trial was performed in 16 healthy Korean males. In the control phase, all subjects received a single dose of intravenous (IV) and oral midazolam (1 mg and 5 mg, respectively). Clarithromycin (500 mg) was administered twice daily for 4 days to inhibit hepatic and intestinal CYP3A, and 500 mL of grapefruit juice was given to inhibit intestinal CYP3A. Clarithromycin significantly inhibited total CYP3A activity, and the clearance of IV and apparent clearance of oral midazolam decreased by 0.15- and 0.32-fold, respectively. Grapefruit juice only reduced the apparent clearance of oral midazolam by 0.84-fold, which indicates a slight inhibition of intestinal CYP3A activity. Urinary markers, including 6ß-OH-cortisol/cortisol and 6ß-OH-cortisone/cortisone, were significantly decreased 0.5-fold after clarithromycin administration but not after grapefruit juice. The fold changes in 6ß-OH-cortisol/cortisol and 6ß-OH-cortisone/cortisone did not correlate to changes in intestinal availability but did correlate to hepatic availability. In conclusion, endogenous metabolic markers are only useful to measure hepatic, but not intestinal, CYP3A activity.

Ontogeny of midazolam glucuronidation in preterm infants.

PURPOSE: In preterm infants, the biotransformation of midazolam (M) to 1-OH-midazolam (OHM) by cytochrome P450 3A4 (CYP3A4) is developmentally immature, but it is currently unknown whether the glucuronidation of OHM to 1-OH-midazolam glucuronide (OHMG) is also decreased. The aim of our study was to investigate the urinary excretion of midazolam and its metabolites OHM and OHMG in preterm neonates following the intravenous (IV) or oral (PO) administration of a single M dose. METHODS: Preterm infants (post-natal age 3-13 days, gestational age 26-34 4/7 weeks) scheduled to undergo a stressful procedure received a 30-min IV infusion (n = 15) or a PO bolus dose (n= 7) of 0.1 mg/kg midazolam. The percentage of midazolam dose excreted in the urine as M, OHM and OHMG up to 6 h post-dose was determined. RESULTS: The median percentage of the midazolam dose excreted as M, OHM and OHMG in the urine during the 6-h interval after the IV infusion was 0.44% (range 0.02-1.39%), 0.04% (0.01-0.13%) and 1.57% (0.36-7.7%), respectively. After administration of the PO bolus dose, the median percentage of M, OHM and OHMG excreted in the urine was 0.11% (0.02-0.59%), 0.02% (0.00-0.10%) and 1.69% (0.58-7.31%), respectively. The proportion of the IV midazolam dose excreted as OHMG increased significantly with postconceptional age (r = 0.73, p < 0.05). CONCLUSION: The glucuronidation of OHM appears immature in preterm infants less than 2 weeks of age. The observed increase in urinary excretion of OHMG with postconceptional age likely reflects the combined maturation of glucuronidation and renal function.

Proof of Concept: First Pediatric [14 C]microtracer Study to Create Metabolite Profiles of Midazolam.

Growth and development affect drug-metabolizing enzyme activity thus could alter the metabolic profile of a drug. Traditional studies to create metabolite profiles and study the routes of excretion are unethical in children due to the high radioactive burden. To overcome this challenge, we aimed to show the feasibility of an absorption, distribution, metabolism, and excretion (ADME) study using a [14 C]midazolam microtracer as proof of concept in children. Twelve stable, critically ill children received an oral [14 C]midazolam microtracer (20 ng/kg; 60 Bq/kg) while receiving intravenous therapeutic midazolam. Blood was sampled up to 24 hours after dosing. A time-averaged plasma pool per patient was prepared reflecting the mean area under the curve plasma level, and subsequently one pool for each age group (0-1 month, 1-6 months, 0.5-2 years, and 2-6 years). For each pool [14 C]levels were quantified by accelerator mass spectrometry, and metabolites identified by high resolution mass spectrometry. Urine and feces (n = 4) were collected up to 72 hours. The approach resulted in sufficient sensitivity to quantify individual metabolites in chromatograms. [14 C]1-OH-midazolam-glucuronide was most abundant in all but one age group, followed by unchanged [14 C]midazolam and [14 C]1-OH-midazolam. The small proportion of unspecified metabolites most probably includes [14 C]midazolam-glucuronide and [14 C]4-OH-midazolam. Excretion was mainly in urine; the total recovery in urine and feces was 77-94%. This first pediatric pilot study makes clear that using a [14 C]midazolam microtracer is feasible and safe to generate metabolite profiles and study recovery in children. This approach is promising for first-in-child studies to delineate age-related variation in drug metabolite profiles.

The CYTONOX trial.

INTRODUCTION: In Denmark, it is estimated that 3-5% of children are obese. Obesity is associated with pathophysiological alterations that may lead to alterations in the pharmacokinetics of drugs. In adults, obesity was found to influence important drug-metabolising enzyme pathways. The impact of obesity-related alterations on drug metabolism and its consequences for drug dosing remains largely unknown in both children and adults. An altered drug metabolism may contribute significantly to therapeutic failure or toxicity. The aim of this trial is to investigate the in vivo activity of CYP3A4, CYP2E1 and CYP1A2 substrates in obese versus non-obese children. METHODS: The CYTONOX trial is an open-label explorative pharmacokinetic trial. We intend to include 50 obese and 50 non-obese children. The primary end points are: in vivo clearance of CYP3A4, CYP2E1 and CYP1A2 substrates, which will be defined by using well-tested probes; midazolam, chlorzoxazone and caffeine. Each of the probes will be administered as a single dose. Subsequently, blood and urine samples will be collected at pre-specified times. CONCLUSION: The aim of the CYTONOX trial is to investigate the in vivo activity of CYP3A4, CYP2E1 and CYP1A2 in obese and non-obese children. The results are expected to be used in the future as a basis for drug dosing recommendations in obese children. FUNDING: The study was funded by the Danish Regions' "Medicinpuljen". The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. TRIAL REGISTRATION: EudraCT: 2014-004554-34.

Use of midazolam urinary metabolic ratios for cytochrome P450 3A (CYP3A) phenotyping.

Midazolam (MDZ) total clearance (ClT) is widely used for cytochrome P450 3A (CYP3A) phenotyping, but requires up to eight blood samples. This study was conducted to compare the use of midazolam ClT to use of a midazolam urinary metabolic ratio for CYP3A phenotyping. Ten male and 10 female subjects received i.v. midazolam 0.025 mg/kg eight times over a 4-month period at approximately 2-week intervals. The first six phenotyping measures were used to estimate baseline CYP3A activity, then subjects received the moderate CYP3A inhibitor fluvoxamine 150 mg/day for the last 4 weeks (two phenotyping visits) of the study. Serial blood samples were obtained for calculation of ClT. Urine was collected for 6 h following each midazolam dose. Midazolam, 1'-hydroxymidazolam (1-OHMDZ), and 4-hydroxymidazolam were measured in plasma and urine by liquid chromatography with tandem mass spectrometry (LC/MS/MS). Analysis of 148 samples from 20 subjects revealed a weak overall correlation between the urinary ratio of 1-OHMDZ/MDZ to midazolam ClT of r(s) = 0.372 (P = 0.0001). There was no correlation when examining either baseline samples or fluvoxamine-inhibited samples alone (r(s) = 0.101, P = 0.289 and r(s) = 0.266, P = 0.123, respectively). The median (range) urinary ratio decreased significantly with fluvoxamine [219 (141-409) versus 127 (50-464); P = 0.005] and to a similar extent to the midazolam ClT (-33.6% versus -42.4%, respectively; P > 0.05). Median urinary recovery of the i.v. midazolam dose varied between 1.4% and 53% and was significantly lower in samples collected while patients were receiving fluvoxamine (34.3% versus 23.1%; P= 0.0004). Based on these results, although this midazolam urinary ratio was not very reflective of baseline CYP3A activity, it may be a useful indicator of CYP3A inhibition.

The effect of hormone replacement therapy on CYP3A activity.

BACKGROUND: The effect of menopause and hormone replacement therapy on hepatic and intestinal wall CYP3A activity is poorly defined. This study was therefore designed to determine the effect of menopause and estrogen replacement therapy on hepatic and intestinal CYP3A activity with a specific CYP3A substrate, midazolam. METHODS: Twelve young women (27 +/- 5 years), 10 elderly women receiving estrogen replacement therapy (71 +/- 6 years), and 14 elderly women not receiving estrogen replacement therapy (71 +/- 5 years) received simultaneous intravenous (0.05 mg/kg over 30 minutes) and oral (3 to 4 mg of a stable isotope, 15N3-midazolam) doses of midazolam. Serum and urine samples were assayed for midazolam, 15N3-midazolam, and metabolites by use of gas chromatography-mass spectrometry. RESULTS: No significant (P > .05) differences were observed in systemic clearance and oral clearance between the three groups. Likewise, no differences were observed in oral, hepatic, or intestinal availability. A significant correlation was observed between oral and intestinal availability and not hepatic availability. CONCLUSION: Neither menopause nor menopause with estrogen replacement therapy altered intestinal or hepatic CYP3A activity relative to that in a control group of young women.

Oral first-pass elimination of midazolam involves both gastrointestinal and hepatic CYP3A-mediated metabolism.

OBJECTIVE: To determine in humans the relative roles of intestinal and hepatic metabolism in the oral first-pass elimination of a CYP3A substrate using midazolam as a model compound. METHODS: Midazolam was administered intravenously (1 mg) or orally (2 mg) to 20 healthy young subjects (10 men and 10 women) in a random fashion, and the disposition of the drug and its 1'-hydroxy metabolite were determined. In separate in vitro studies, the CYP3A-mediated formation of 1'-hydroxymidazolam by human hepatic and intestinal microsomes was investigated. RESULTS: No gender-related differences were noted in either the systemic (370 +/- 114 ml/min [mean +/- SD]) or oral (1413 +/- 807 ml/min) clearance values of midazolam. Despite complete oral absorption, measured oral bioavailability was on average about 50% less than that predicted on the assumption that only the liver contributed to first-pass metabolism. Pharmacokinetic estimation of the intestinal component indicated an extraction ratio (0.43 +/- 0.24) that was similar to that of the liver (0.44 +/- 0.14). 1'-Hydroxymidazolam was extensively but variably formed in vitro by both hepatic and intestinal microsomes and, although the intrinsic clearance (V(max)/Km) was higher in the liver preparations (540 +/- 747 versus 135 +/- 92 microliters/min/mg protein), this difference was not statistically significant. CONCLUSIONS: These results show that the small intestine can be a major site for presystemic, CYP3A-mediated metabolism after oral administration. Moreover, it appears that this represents a true first-pass effect. In addition, intestinal and hepatic metabolism may be important factors in interindividual variability in disposition after oral administration of midazolam and similar CYP3A substrates. Finally, intestinal localization of CYP3A may be significant in metabolism-based drug-drug interactions.

The contribution of intestinal and hepatic CYP3A to the interaction between midazolam and clarithromycin.

OBJECTIVE: To assess the relative contribution of intestinal and hepatic CYP3A inhibition to the interaction between the prototypic CYP3A substrates midazolam and clarithromycin. METHODS: On day 1, 16 volunteers (eight men and eight women; age range, 20 to 40 years; weight range, 45 to 100 kg) received simultaneous doses of midazolam intravenously (0.05 mg/kg over 30 minutes) and orally (4 mg of a stable isotope, 15N3-midazolam). Starting on day 2, 500 mg clarithromycin was administered orally twice daily for 7 days. On day 8, intravenous and oral doses of midazolam were administered 2 hours after the final clarithromycin dose. Blood and urine samples were assayed for midazolam, 15N3-midazolam, and metabolites by gas chromatography-mass spectrometry. RESULTS: There was no significant (p > 0.05) difference in the urinary excretion of 1'-hydroxymidazolam after intravenous and oral dosing on day 1 or day 8, indicating that the oral dose was completely absorbed into the gut wall. The oral clearance of midazolam was found to be significantly greater in female subjects (1.9 +/- 1.0 versus 1.0 +/- 0.3 L/hr/kg; p < 0.05) than in male subjects but not systemic clearance (0.35 +/- 0.1 versus 0.44 +/- 0.1 L/hr/kg). For women not receiving oral contraceptives (n = 6) a significant gender-related difference was observed for systemic and oral clearance and for area under the curve and elimination half-life after oral administration. A significant (p < 0.05) reduction in the systemic clearance of midazolam from 28 +/- 9 L/hr to 10 +/- 3 L/hr occurred after clarithromycin administration. Oral midazolam availability was significantly increased from 0.31 +/- 0.1 to 0.75 +/- 0.2 after clarithromycin dosing. Likewise, intestinal and oral availability were significantly increased from 0.42 +/- 0.2 to 0.83 +/- 0.2 and from 0.74 +/- 0.1 to 0.90 +/- 0.04, respectively. A significant correlation was observed between intestinal and oral availability (n = 32, r = 0.98, p < 0.05). After clarithromycin administration, a significant correlation was observed between the initial hepatic or intestinal availability and the relative increase in hepatic or intestinal availability, respectively. Female subjects exhibited a greater extent of interaction after oral and intravenous dosing than male subjects (p < 0.05). CONCLUSION: These data indicate that in addition to the liver, the intestine is a major site of the interaction between oral midazolam and clarithromycin. Interindividual variability in first-pass extraction of high-affinity CYP3A substrates such as midazolam is primarily a function of intestinal enzyme activity.

No evidence of a genetic polymorphism in the oxidative metabolism of midazolam.

The benzodiazepine midazolam is rapidly eliminated by oxidative metabolism. In young healthy volunteers elimination half-life (t1/2) is about 2.4 hours. A recent study showed a prolonged t1/2 from 8 to 22 hours in 6.5% of surgical patients, and a genetic polymorphism of midazolam's metabolism has been suggested. Therefore, we measured in 168 surgical patients the elimination of midazolam and its major hydroxylated metabolite (alpha-OH-midazolam) in blood and urine. Co-medication, disease status, smoking habits and alcohol intake were recorded; normal liver and kidney functions were assessed by routine laboratory tests. Midazolam was administered intravenously (0.1 to 0.2 mg/kg) for the induction of anaesthesia. Blood was drawn 1.5, 3, 4.5 and 6 hours after application and urine was collected for 6 hours. Plasma protein binding of midazolam was determined by equilibrium dialysis. Midazolam and alpha-OH-midazolam were measured in plasma by specific gas-liquid chromatography and in urine by high performance liquid chromatography. Data for the dose-corrected area under plasma-level curve of midazolam (AUC-midazolam/dose: 1.23 +/- 961 x 10(5) h/ml; mean +/- SD) and for the metabolic plasma ratio (AUC of alpha-OH-midazolam/AUC-midazolam: 0.52 +/- 0.28) demonstrated a log-normal distribution. Likewise, the percentage of the unbound fraction of midazolam in plasma (5.0 +/- 2.4%), urinary excretion of alpha-OH-midazolam (55.9 +/- 22.7% of dose) and the values for t1/2 (2.9 +/- 1.1 hours) did indicate a unimodal distribution. Age, comedication and smoking habits did not affect the disposition of midazolam. However, patients with regular intake of alcohol had a higher (p less than 0.05) metabolic ratio. Only in 3 patients could a prolonged t1/2 of midazolam from 7.5 to 10.2 hours be detected, but plasma levels and urinary excretion of alpha-OH-midazolam in those individuals were found to be normal. Therefore it is very unlikely that the oxidative metabolism of midazolam exhibits a genetic polymorphism.

Urinary screening for midazolam and its major metabolites with the Abbott ADx and TDx analyzers and the EMIT d.a.u. benzodiazepine assay with confirmation by GC/MS.

Midazolam is a short-acting 1,4-imidazole benzodiazepine with sedative-hypnotic, anxiolytic, and amnestic properties. It is administered orally for sleeping disorders and intravenously for sedation during surgery. This drug has a short half-life (1.5-3.5 h), with less than 1% of a midazolam dose being excreted unchanged. The major urinary metabolite is alpha-hydroxy midazolam glucuronide. The objective of this study was to characterize the reactivity of midazolam and its two major metabolites in the EMIT d.a.u. benzodiazepine assay and in the Abbott TDx and ADx urine benzodiazepine assays. Midazolam and alpha-OH midazolam gave an equivalent response to the EMIT low calibrator at 200 ng/mL. On both Abbott analyzers, midazolam and alpha-OH midazolam gave an equivalent net polarization at 500 ng/mL to the Abbott low control. All three screening assays were positive in all of 21 random urine specimens collected from midazolam-treated patients. Confirmation testing was performed by analyzing for alpha-OH midazolam after enzymatic hydrolysis and formation of a TMS derivative for GC/MS. All urine specimens were confirmed positive for alpha-OH midazolam. In conclusion, all three immunoassay screening assays are acceptable for detecting the presence of midazolam metabolites in urine.

High-throughput assay for simultaneous quantification of the plasma concentrations of morphine, fentanyl, midazolam and their major metabolites using automated SPE coupled to LC-MS/MS.

A rapid LC-MS/MS assay method for simultaneous quantification of morphine, fentanyl, midazolam and their major metabolites: morphine-3-ß-D-glucuronide (M3G), morphine-6-ß-D-glucuronide (M6G), norfentanyl, 1'-hydroxymidazolam (1-OH-MDZ) and 4-hydroxymidazolam (4-OH-MDZ) in samples of human plasma has been developed and validated. Robotic on-line solid phase extraction (SPE) instrumentation was used to elute the eight analytes of interest from polymeric SPE cartridges to which had been added aliquots (150 µL) of human plasma and aliquots (150 µL) of a mixture of two internal standards, viz. morphine-d3 (200 ng/mL) and 1'-hydroxymidazolam-d5 (50 ng/mL) in 50 mM ammonium acetate buffer (pH 9.25). Cartridges were washed using 10% methanol in ammonium acetate buffer, pH 9.25 (1 mL, 2 mL/min) before elution with mobile phase comprising 0.1% formic acid in water (A) and acetonitrile (B) with a flow rate of 0.6 mL/min using an 11.5 min run time. The analytes were separated on a C18 X-Terra® analytical column. The linear concentration ranges were 0.5-100 ng/mL for fentanyl, norfentanyl and midazolam; 1-200 ng/mL for 4-hydroxymidazolam, 2.5-500 ng/mL for 1'-hydroxymidazolam and 3.5-700 ng/mL for morphine, M3G, and M6G. The method showed acceptable within-run and between-run precision (relative standard deviation (RSD) and accuracy <20%) for quality control (QC) samples spiked at concentrations of 80% and 50% of the ULOQ, 3 times higher than the LLOQ, and also at the LLOQ. Furthermore, analytes were stable in samples (after mixing with internal standard) for at least 48 h in the autosampler (except for 4-hydroxymidazolam which decreased by 22% after 24 h), 5 h at room temperature and after three cycles of freeze and thaw. No autosampler carry-over was observed and the absolute recovery (the area ratio of analyte in plasma relative to that in ammonium acetate buffer 50 mM, pH 9.25) was in the range 40% (midazolam) to 110% (morphine). The assay was applied successfully to the measurement of the analytes of interest in plasma samples from patients on extracorporeal membrane oxygenation (ECMO).

Effect of 2 weeks' consumption of pomegranate juice on the pharmacokinetics of a single dose of midazolam: an open-label, randomized, single-center, 2-period crossover study in healthy Japanese volunteers.

BACKGROUND: It has been reported that pomegranate juice significantly increased the AUC of orally administered carbamazepine in rats, which suggests that pomegranate may inhibit the cytochrome P450 3A (CYP3A)-mediated carbamazepine metabolism. OBJECTIVE: The aim of the present study was to clarify the effect of repeated consumption of pomegranate juice on CYP3A activity by assessing the pharmacokinetics of midazolam, a typical CYP3A probe drug, and its metabolites in healthy volunteers. METHODS: An open-label, randomized, single-center, 2-period crossover study was conducted on healthy Japanese volunteers. Each subject received 200 mL of pomegranate juice twice daily for 2 weeks. On day 14, they were administered 15 µg/kg midazolam orally with either pomegranate juice or water. Plasma concentrations and urinary excretions of midazolam, 1'-hydroxymidazolam, and 4-hydroxymidazolam were determined up to 24 hours using LC/MS/MS and analyzed by a noncompartmental method. RESULTS: Sixteen subjects (11 men and 5 women) were enrolled and completed the study. The mean (SD) age was 24.1 (4.8) years (range 22-40), mean body weight was 62.9 (8.8) kg (range 45.6-79.9). Differences in the mean AUC(0-∞) were 12.7 (4.4) and 14.2 (6.6) ng/mL/h in pomegranate juice and control groups, respectively (geometric mean ratio: 1.02 [95% CI, 0.95-1.09]; P = 0.40). Differences in C(max) for midazolam did not reach the level of statistical significance (5.1 [1.7] vs 5.0 [2.0] ng/mL, geometric mean ratio: 0.95 [95% CI, 0.79-1.11]; P = 0.68). Excretions of 1'-hydroxymidazolam (P = 0.34) and 4-hydroxymidazolam (P = 0.32) were not significantly altered by ingestion of pomegranate juice. CONCLUSION: In this small Japanese adult volunteer population receiving single subtherapeutic doses of midazolam, 2 weeks' consumption of pomegranate juice did not significantly alter the pharmacokinetic profile of midazolam compared with that of the control. Protocol identifier: UMIN000004459.