Triazolam Research Articles

Non-Steroidal Drug Interferences in a Quantitative Multisteroid LC-MS/MS Assay

Screening for possible interferences from steroidal compounds other than the target analytes (endogenous or exogenous) is well established in LC-MS/MS assay development for steroid quantification in a routine clinical setting. However, interferences from non-steroidal substances have, hitherto, not been explored. After screening more than 150 pharmaceuticals and their metabolites by analyzing commercial quality control samples from TDM analysis kits (Recipe, Chromsystems) with a multisteroid LC-MS/MS assay (protein precipitation followed by HybridSPE filtration, biphenyl column, methanol-water gradient with NH4F additive), we can report the finding of two newly discovered potential interferences from non-steroidal drugs. Antidepressant paroxetine (PX) was identified as an interference to 17-hydroxyprogesterone (17P), and α-hydroxytriazolam (α-OH-TZM)-a major metabolite of benzodiazepine triazolam (TZM)-was identified as an interference to aldosterone (ALDO). Despite different elemental and structural compositions and nominal masses, the M+1 isotopologues of PX and α-OH-TZM produced overlapping signals in ion traces monitored for the respective analytes (m/z 331 → 109/97 and 361→315/343, respectively). PX and TZM are frequently prescribed drugs, and their therapeutic ranges are far exceeding the reference ranges of 17P or ALDO (µmol vs nmol); therefore, these interferences should be considered clinically relevant. Striving for faster multi-analyte methods with high sample turnover, especially in the field of steroid quantification, can limit assay selectivity and specificity. Therefore, supported by the findings of this study, screening for potential interferences in multi-analyte LC-MS/MS method development should not cover only substances of the same class but also include a set of common drugs.

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.

Incorporation of five common hypnotics into hair after a single dose and application to a forensic case of drug facilitated crimes

In order to obtain fundamental information on the disposition of hypnotics into hair after a single oral dose the quantitative hair analysis of triazolam (TZ), etizolam (EZ), flunitrazepam (FNZ), nitrazepam (NZ) and zolpidem (ZP) have been performed using a validated LC-MS/MS procedure. Hair specimens (straight, black) were collected from three subjects about one month and three months after a single 0.25 mg dose of TZ, 1 mg of EZ, 2 mg of FNZ, 5 mg of NZ and 10 mg of ZP tartrate. The subjects ingested just one out of five different hypnotics on each day, each of five days in turn. All ingested hypnotics have been detected in hair from each subject both one month and three months after intake, and their concentrations were in the range of 0.023–0.043 pg/hair strand (0.077–0.36 pg/mg) for TZ, 0.11–0.63 pg/hair strand (0.44–5.2 pg/mg) for EZ, 0.14–2.6 pg/hair strand (0.56–22 pg/mg) for FNZ, 0.33–1.7 pg/hair strand (1.3–17 pg/mg) for NZ and 20–40 pg/hair strand (120–270 pg/mg) for ZP. For FNZ and NZ, not only the parent drugs but also their metabolites, 7-amino-FNZ and 7-amino-NZ, were detected in the range of 2.3–9.2 pg/hair strand (9.2–82 pg/mg) and 2.4–9.1 pg/hair strand (8.0–55 pg/mg), respectively. The calculated incorporation ratios into hair against the dose were found to exhibit similarity between the four benzodiazepines. This finding suggests the ability to apply these quantitative data to approximately estimating the amounts of other benzodiazepines, which have similar chemical structures, in hair although it should be noted that the amounts of drugs in hair varies considerably depending on the hair color. On the other hand, the incorporation ratio of ZP showed 15–29 times higher than that of TZ, indicating that lipophilic ZP was more likely to incorporate into hair than benzodiazepines. In addition, the application of the present data to a drug-facilitated sexual assault was shown.

Kinetic analysis of sequential metabolism of triazolam and its extrapolation to humans using an entero-hepatic two-organ microphysiological system.

The microphysiological system (MPS) is a promising tool for predicting drug disposition in humans, although limited information is available on the quantitative assessment of sequential drug metabolism in MPS and its extrapolation to humans. In the present study, we first constructed a mechanism-based pharmacokinetic model for triazolam (TRZ) and its metabolites in the entero-hepatic two-organ MPS, composed of intestinal Caco-2 and hepatic HepaRG cells, and attempted to extrapolate the kinetic information obtained with the MPS to the plasma concentration profiles in humans. In the two-organ MPS and HepaRG single culture systems, TRZ was found to be metabolized into α- and 4-hydroxytriazolam and their respective glucuronides. All these metabolites were almost completely reduced in the presence of a CYP3A inhibitor, itraconazole, confirming sequential phase I and II metabolism. Both pharmacokinetic model-dependent and -independent analyses were performed, providing consistent results regarding the metabolic activity of TRZ: clearance of glucuronidation metabolites in the two-organ MPS was higher than that in the single culture system. The plasma concentration profile of TRZ and its two hydroxy metabolites in humans was quantitatively simulated based on the pharmacokinetic model, by incorporating several scaling factors representing quantitative gaps between the MPS and humans. Thus, the present study provided the first quantitative extrapolation of sequential drug metabolism in humans by combining MPS and pharmacokinetic modeling.

Comparison of the hepatic metabolism of triazolam in wild-type andCyp3a-knockout mice for understanding CYP3A-mediated metabolism inCYP3A-humanised mice in vivo

1. To investigate cytochrome P450 3A (CYP3A)-mediated metabolism in vivo, plasma concentrations of triazolam (TRZ) are often monitored as a CYP3A marker in CYP3A-humanised mice. However, it has not been determined whether plasma concentrations of TRZ after intravenous administration can reflect hepatic CYP3A activity in CYP3A-humanised mice.2. Firstly, we investigated the pharmacokinetics of TRZ in wild-type and Cyp3a-knockout (Cyp3a-KO) mice. Plasma concentration profiles of TRZ and α-hydroxy (OH) TRZ were very similar in wild-type and Cyp3a-KO mice. On the other hand, AUC of 4-OH TRZ in Cyp3a-KO mice was significantly lower than that in wild-type mice. Pregnenolone 16α-carbonitrile (PCN) decreased the areas under the plasma concentration-time curves (AUCs) of TRZ and α-OH TRZ in both groups. There was no significant effect of PCN on AUC of 4-OH TRZ in Cyp3a-KO mice.3. Next, we verified that AUC of 4-OH TRZ in CYP3A-humanised mice was higher than that in Cyp3a-KO mice, although the difference was not significant.4. In conclusion, plasma concentrations of 4-OH TRZ, but not those of TRZ and α-OH TRZ, might reflect hepatic CYP3A activity in mice in vivo. These results provide important insights for in vivo studies using a CYP3A-humanised model.

LC-MS determination of triazolam and its hydroxy metabolites in mouse dried blood spots: application to transgenic mouse pharmacokinetic studies.

The objective of this study was to develop a LC-MS/MS method for the simultaneous determination of triazolam (TRZ) and its two hydroxy metabolites in transgenic mouse dried blood spots (DBS) using BALB/c mouse blood as a surrogate biomatrix. The DBS method involved spotting volume of 10 μl using Ahlstrom 226 sample collection cards. A 'whole spot' analysis (6-mm punch) involved extraction of analytes using water and acetonitrile containing an internal standard. DBS samples were analyzed by a validated LC-MS/MS method with a run time of 4 min. This validated LC-MS/MS method using DBS extraction was applied to quantitation of TRZ, α-hydroxytriazolam and 4-hydroxytriazolam in a CYP3A4 transgenic mouse oral pharmacokinetic study of TRZ.

Learning and Memory Deficits in Male Adult Mice Treated with a Benzodiazepine Sleep-Inducing Drug during the Juvenile Period.

Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian central nervous system, is also known to be important for brain development. Therefore, disturbances of GABA receptor (GABA-R) mediated signaling (GABA-R signal) during brain development may influence normal brain maturation and cause late-onset brain malfunctions. In this study, we examined whether the stimulation of the GABA-R signal during brain development induces late-onset adverse effects on the brain in adult male mice. To stimulate the GABA-R signal, we used either the benzodiazepine sleep-inducing drug triazolam (TZ) or the non-benzodiazepine drug zolpidem (ZP). We detected learning and memory deficits in mice treated with TZ during the juvenile period, as seen in the fear conditioning test. On the other hand, ZP administration during the juvenile period had little effect. In addition, decreased protein expression of GluR1 and GluR4, which are excitatory neurotransmitter receptors, was detected in the hippocampi of mice treated with TZ during the juvenile period. We measured mRNA expression of the immediate early genes (IEGs), which are neuronal activity markers, in the hippocampus shortly after the administration of TZ or ZP to juvenile mice. Decreased IEG expression was detected in mice with juvenile TZ administration, but not in mice with juvenile ZP administration. Our findings demonstrate that TZ administration during the juvenile period can induce irreversible learning and memory deficits in adult mice. It may need to take an extra care for the prescription of benzodiazepine sleep-inducing drugs to juveniles because it might cause learning and memory deficits.

Prescription of triazolam for the elderly in Japan:

Prescription of triazolam for the elderly in Japan:

Evaluation of animal models for intestinal first-pass metabolism of drug candidates to be metabolized by CYP3A enzymes via in vivo and in vitro oxidation of midazolam and triazolam

1. To search an appropriate evaluation methodology for the intestinal first-pass metabolism of new drug candidates, grapefruit juice (GFJ)- and vehicle (tap water)-pretreated mice or rats were orally administered midazolam (MDZ) or triazolam (TRZ), and blood levels of the parent compounds and their metabolites were measured by liquid chromatography/MS/MS. A significant effect of GFJ to elevate the blood levels was observed only for TRZ in mice.2. In vitro experiments using mouse, rat and human intestinal and hepatic microsomal fractions demonstrated that GFJ suppressed the intestinal microsomal oxidation of MDZ and especially TRZ. Substrate inhibition by MDZ caused reduction in 1′-hydroxylation but not 4-hydroxylation in both intestinal and hepatic microsomal fractions. The kinetic profiles of MDZ oxidation and the substrate inhibition in mouse intestinal and hepatic microsomal fractions were very similar to those in human microsomes but were different from those in rat microsomes. Furthermore, MDZ caused mechanism-based inactivation of cytochrome P450 3A-dependent TRZ 1′-hydroxylation in mouse, rat and human intestinal microsomes with similar potencies.3. These results are useful information in the analysis of data obtained in mouse and rat for the evaluation of first-pass effects of drug candidates to be metabolized by CYP3A enzymes.

Inhibition of Triazolam Hydroxylation by Ketoconazole, Itraconazole, Hydroxyitraconazole and Fluconazole In‐vitro

The capacity of hydroxyitraconazole, an in-vivo metabolite of the antifungal agent itraconazole, to inhibit human cytochrome P4503A isoforms was evaluated in-vitro using a human microsomal model. Varying concentrations of ketoconazole, itraconazole, hydroxyitraconazole and fluconazole were incubated with human liver microsomes and triazolam (250 μM), an index compound used to profile activity of P4503A isoforms. Formation of α-OH- and 4-OH-triazolam was quantitated by high-performance liquid chromatography. Ketoconazole was the most potent inhibitor of triazolam metabolite formation (mean IC50 0.074–0.076 μM), while fluconazole was less potent (IC50 4.4–4.9 μM). Both itraconazole and hydroxy-itraconazole had similar IC50 values (0.4–0.7 μM), with potency intermediate between ketoconazole and fluconazole. Hydroxyitraconazole may contribute to impaired clearance of P4503A substrates, and consequent pharmacokinetic drug interactions, during treatment with itraconazole in man.

Physiologically Based Predictions of the Impact of Inhibition of Intestinal and Hepatic Metabolism on Human Pharmacokinetics of CYP3A Substrates

The first objective of the present study was to predict the pharmacokinetics of selected CYP3A substrates administered at a single oral dose to human. The second objective was to predict pharmacokinetics of the selected drugs in presence of inhibitors of the intestinal and/or hepatic CYP3A activity. We developed a whole-body physiologically based pharmacokinetics (WB-PBPK) model accounting for presystemic elimination of midazolam (MDZ), alprazolam (APZ), triazolam (TRZ), and simvastatin (SMV). The model also accounted for concomitant administration of the above-mentioned drugs with CYP3A inhibitors, namely ketoconazole (KTZ), itraconazole (ITZ), diltiazem (DTZ), saquinavir (SQV), and a furanocoumarin contained in grape-fruit juice (GFJ), namely 6′,7′-dihydroxybergamottin (DHB). Model predictions were compared to published clinical data. An uncertainty analysis was performed to account for the variability and uncertainty of model parameters when predicting the model outcomes. We also briefly report on the results of our efforts to develop a global sensitivity analysis and its application to the current WB-PBPK model. Considering the current criterion for a successful prediction, judged satisfied once the clinical data are captured within the 5th and 95th percentiles of the predicted concentration–time profiles, a successful prediction has been obtained for a single oral administration of MDZ and SMV. For APZ and TRZ, however, a slight deviation toward the 95th percentile was observed especially for Cmax but, overall, the in vivo profiles were well captured by the PBPK model. Moreover, the impact of DHB-mediated inhibition on the extent of intestinal pre-systemic elimination of MDZ and SMV has been accurately predicted by the proposed PBPK model. For concomitant administrations of MDZ and ITZ, APZ and KTZ, as well as SMV and DTZ, the in vivo concentration–time profiles were accurately captured by the model. A slight deviation was observed for SMV when coadministered with ITZ, whereas more important deviations have been obtained between the model predictions and in vivo concentration–time profiles of MDZ coadministered with SQV. The same observation was made for TRZ when administered with KTZ. Most of the pharmacokinetic parameters predicted by the PBPK model were successfully predicted within a two-fold error range either in the absence or presence of metabolism-based inhibition. Overall, the present study demonstrated the ability of the PBPK model to predict DDI of CYP3A substrates with promising accuracy. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:486–514, 2010

Separate evaluation of intestinal and hepatic metabolism of three benzodiazepines in rats with cannulated portal and jugular veins: comparison with the profile in non-cannulated mice

Pharmacokinetic analyses of three kinds of benzodiazepines—midazolam (MDZ), triazolam (TRZ) and alprezolam (APZ)—were performed in rats with cannulated portal and jugular veins. Each drug was administered to the double-cannulated rats, and pharmacokinetic data for the parent drugs and their 1′- and 4-hydroxylated metabolites were compared with those obtained in non-cannulated mice.In bioavailability, the drugs ranked APZ >> TRZ = MDZ in rats, and APZ > TRZ >> MDZ in mice, with the values for MDZ remarkably different between rats and mice (19% in rats versus 2.3% in mice). In contrast, hepatic availability (Fh) was similar (APZ > TRZ > MDZ) in both species. Highly significant relationships were found between the ratio of the area under the plasma concentration–time curve (AUC) for the parent drugs in portal blood (AUCpor) to that in systemic blood (AUCsys) and Fh in rats and mice.The double-cannulated rat is useful for estimating the hepatic availability of drug candidates by determining the AUC values for the parent drugs in portal and systemic blood samples.

MDMA alters the response of the mammalian circadian clock in hamsters: Effects on re-entrainment and triazolam-induced phase shifts

Serotonin (5-hydroxytryptamine or 5-HT) is a neurotransmitter that is involved in a wide range of behavioural and physiological processes. Previous work has indicated that serotonin is important in the regulation of the circadian clock, which is located in the suprachiasmatic nuclei (SCN) of the hypothalamus. 3,4-methylenedioxymethamphetamine (MDMA or ‘Ecstasy’), which is widely used as a recreational drug of abuse, is a serotonin neurotoxin in animals and non-human primates. Previous work has shown that MDMA exposure can alter circadian clock function both in vitro and in vivo. Evidence shows that 5-HT may have a modulatory role in the regulation of the circadian clock by non-photic stimuli, such as the benzodiazepine triazolam (TRZ). Triazolam is a short-acting benzodiazepine that results in phase advances of the wheel running activity in hamsters when administered during the mid-subjective day. In the present study, male Syrian hamsters treated with TRZ (5 mg/kg) at ZT6 significantly phase advanced their clock. Treatment with MDMA significantly diminished the TRZ induced phase shift in hamsters. Previous evidence shows the involvement of 5-HT in the re-synchronisation of the endogenous clock to a new shifted light–dark cycle. Untreated animals were successfully entrained to a new, 6 h advanced light–dark cycle within an average of 4.5 ± 0.1 days. Following treatment with MDMA, these animals took an average of 8.3 ± 0.1 days to re-entrain to a shifted environmental cycle. Immunohistochemical analysis revealed that animals treated with MDMA showed reduced serotonin staining, as evidenced by a decrease in innervation density in the SCN. No significant differences were found in cell counts within the raphe nuclei. These results demonstrate the importance of the serotonergic system in the modulation of photic and non-photic responses of the circadian pacemaker.

Inhibitory Action of 5-HT1A Agonist MKC-242 on Triazolam-Induced Phase Advances in Hamster Circadian Activity Rhythms

Mammalian circadian rhythms can be entrained by photic and non-photic stimuli. Although we know that non-photic entrainment interferes with photic entrainment signals, there is no information about whether photic entrainment interferes with non-photic entraining signals. We examined whether triazolam (TRZ), a non-photic form of entrainment, could be attenuated by pre-treatment with (S)-5-[3-[(1,4-benzodioxan-2-ylmethyl)amino]propoxy]-1,3-benzodioxole hydrochloride (MKC-242), a photic enhancing drug. We found that TRZ-induced phase advances in hamster behavioral circadian rhythms and the increase of locomotor activity were both significantly attenuated by MKC-242. Thus, in hamsters, the photic enhancing drug MKC-242 seemed to hinder benzodiazepine-induced non-photic entrainment.

Role of CYP3A enzymes in the biotransformation of triazolam in rat liver

1. Triazolam (TRZ) has been used extensively in rat to evaluate its benzodiazepine agonist central nervous system effects. However, the pharmacokinetics of TRZ in the male rat are not well understood.2. To characterize further TRZ biotransformation across species, the NADPH-dependent biotransformation of TRZ was examined in rat and human liver microsomes. The role of specific cytochrome P450s (CYPs) in the biotransformation of TRZ in the rat were also determined using both rat cDNA-expressed CYPs and chemical and antibody inhibition techniques.3. The formation of TRZ's primary hydroxylated products, α-OH- and 4-OH-TRZ, was consistent with a single-enzyme Michaelis–Menten model in humans.4. Although 4-OH-TRZ formation in the male rat liver was also approximated by a single-enzyme model, a second low-affinity component was identified as contributing to α-OH-TRZ formation in the rat.5. The Km values for the primary metabolic pathway differed between the two species. However, the net intrinsic clearances were similar for the rat and human.6. As observed previously for humans, chemical and antibody inhibition studies suggested that CYP3A enzymes contribute significantly to TRZ hydroxylation in the rat. This finding was further supported by the use of rat cDNA-expressed CYPs.7. The male rat might serve as a useful model for evaluating mechanisms regulating TRZ metabolism in vivo.

Effect of age on in vitro triazolam biotransformation in male human liver microsomes.

We studied age-related changes in enzyme kinetic parameters in human liver microsomes (HLMs) in vitro, using triazolam (TRZ), an index of CYP3A activity. HLMs were prepared from male livers from four age groups, n = 5 per group: A (14-20 years), B (21-40 years), C (41-60 years), and D (61-72 years). Mean V(max) values in groups B and C for both 1-hydroxytriazolam (1-OH-TRZ) and 4-hydroxy-triazolam (4-OH-TRZ) formation were significantly greater as compared with groups A and D individually, as well as the net intrinsic clearance (sum of the two pathways). The mean net intrinsic clearance (Cl(int)) values were 25.2, 89.8, 78, and 20.6 nl/min/mg protein in A, B, C, and D, respectively. TRZ Cl(int) correlated well with total CYP3A content (r(s) = 0.84; P < 0.0001). Testosterone (TST) inhibited 1-OH-TRZ formation and activated 4-OH-TRZ formation in all age groups, with no significant differences among the groups; this suggests that the drug-drug interaction potential using TRZ and TST as index CYP3A substrates may not change with age. Reduced V(max) and Cl(int) for TRZ hydroxylation and CYP3A protein in livers from elderly men suggest reduced CYP3A gene expression in this group.

Behavioral effects of acute and chronic triazolam treatments in albino rats

Previous behavioral studies on triazolam (TZ), which are small in number, could only speculate about tolerance to the anxiolytic effect of TZ, as the experiments did not cover sufficient time (of 4 to 7 days) for tolerance to develop. Therefore longer time for chronic TZ administration is used. We investigated the effects of TZ on motor activity and exploratory behavior using plus maze and open field. Three experiments were conducted. In the first, five groups of rats were acutely treated with different doses of TZ (0.25 mg/kg-4.0 mg/kg). In the second set of experiments, rats were treated chronically with a single daily dose of TZ (started with 0.25 mg/kg and increased by time to 1.0 mg/kg) for 5 weeks (representing clinical use). In the third, rats were treated chronically with three daily doses of TZ (started with 0.25 mg/kg and increased by time to 0.5 mg/kg) for 20 days (mimicking drug abuse). Acute TZ administration produced dose dependent anxiolytic effects and a decrease in motor activity with higher doses. Chronically treated rats, either once daily or three times daily doses, showed tolerance to both anxiolytic and sedative effects of TZ. It may be concluded that tolerance to the anxiolytic and sedative effects of TZ would develop after chronic administration either with clinical use or its abuse.

IN VITRO METABOLISM OF MIDAZOLAM, TRIAZOLAM, NIFEDIPINE, AND TESTOSTERONE BY HUMAN LIVER MICROSOMES AND RECOMBINANT CYTOCHROMES P450: ROLE OF CYP3A4 AND CYP3A5

Midazolam, triazolam (TRZ), testosterone, and nifedipine have all been widely used as probes for in vitro metabolism of CYP3A. We used these four substrates to assess the contributions of CYP3A4 and CYP3A5 to in vitro biotransformation in human liver microsomes (HLMs) and in recombinant enzymes. Recombinant CYP3A4 and CYP3A5 (rCYP3A4 and rCYP3A5) both produced 1-OH and 4-OH metabolites from midazolam and triazolam, 6 beta-hydroxytestosterone from testosterone, and oxidized nifedipine from nifedipine. Overall, the metabolic activity of CYP3A5 was less than that of CYP3A4. Ketoconazole potently inhibited midazolam, triazolam, testosterone, and nifedipine metabolite formation in HLMs and in rCYP3A4. The inhibitory potency of ketoconazole in rCYP3A5 was about 5- to 19-fold less than rCYP3A4 for all four substrates. In testosterone interaction studies, testosterone inhibited 1-OH-TRZ formation, but significantly activated 4-OH-TRZ formation in HLMs and rCYP3A4 but not in rCYP3A5. Oxidized nifedipine formation was inhibited by testosterone in rCYP3A4. However, in rCYP3A5, testosterone slightly activated oxidized nifedipine formation at lower concentrations, followed by inhibition. Thus, CYP3A4 and CYP3A5 both contribute to midazolam, triazolam, testosterone, and nifedipine biotransformation in HLMs, with CYP3A5 being metabolically less active than CYP3A4 in general. Because the inhibitory potency of ketoconazole in rCYP3A5 is substantially less than in rCYP3A4 and HLMs, CYP3A5 is probably less important than CYP3A4 in drug-drug interactions involving ketoconazole and CYP3A substrates.

Study of Nocturnal Sleep and the Carryover Effects of Triazolam and Brotizolam Using Neurophysiological and Subjective Methods

In the present study, the effects of short-acting benzodiazepines on nocturnal sleep and the carryover effects of these drugs were studied. The study involved 10 young, healthy male subjects who had given their written informed consent to participate. Either a placebo (PLA), 0.125 mg triazolam (TRZ), 0.25 mg TRZ or 0.25 mg brotizolam (BRZ) was administered to the subjects in a double-blind crossover design by randomized allocation with a single oral administration at 23.00 h. A polysomnography (PSG) was recorded for each subject from 23.00 to 07.00 h the following day. Then, the Stanford Sleepiness Scale (SSS) and Kwansei Gakuin Sleepiness Scale (KSS) were checked between 07.55 and 08.00 h, and the sleep latency test (SLT) was performed between 08.00 and 08.20 h. Event-related potentials (ERPs) were then recorded with an oddball paradigm; the reaction time (RT) was measured simultaneously. According to the PSG, treatment with 0.25 mg TRZ resulted in a statistically significant increase in the percentage of stage 2 sleep (p < 0.05) and a reduction in the percentage of rapid eye movement sleep (p < 0.05) compared with PLA. None of the drugs had any effect on the percentage of slow-wave sleep compared with PLA. With regard to carryover effects, although none of the drugs had any effect on SSS, KSS, RT or ERPs, BRZ did cause a statistically significant decrease in sleep latency (p < 0.05) compared with PLA. TRZ (0.125 and 0.25 mg) and 0.25 mg BRZ exerted different effects on SLT. We suggest that these different effects are attributable to differences in the half-life of these hypnotics.

Semi-micro column HPLC of triazolam in rat plasma and brain microdialysate and its application to drug interaction study with itraconazole

Semi-micro column high-performance liquid chromatographic method with ultraviolet detection for the determination of triazolam (TZ) in rat plasma and brain microdialysate is described. The separation was achieved on a 250×1.5 mm, i.d. C 18 column and the column effluent was monitored at 222 nm. The detection limits at a signal-to-noise ratio of 3 obtained using spiked plasma and artificial cerebrospinal fluid were 2.1 and 0.7 ng/ml, respectively. The method was applied to drug–drug interaction study of TZ with itraconazole (ITZ). The peak concentration ( C max) and the area under the curve (AUC) of TZ in brain microdialysate after simultaneous administration of TZ (2.5 mg/kg, intravenously (i.v.)) and ITZ (25 mg/kg, p.o.) to rats increased 3.4-folds ( P<0.001) and 2.9-folds ( P<0.001), respectively, compared to those of TZ alone. Also, the AUC of TZ in plasma increased 2.6-folds and remarkable delay in its elimination half-life ( t 1/2) was observed. The concentrations of TZ in brain microdialysate and plasma were also measured after single administration of TZ (2.5 mg/kg, i.v.) to rats pretreated with daily administration of ITZ (25 mg/kg, p.o.) once a day for a week. There was no significant difference in TZ's C max in both ITZ treatments ( P>0.2) however its t 1/2 after the daily pretreatment with ITZ was significantly increased ( P<0.05). In plasma, the AUC of TZ after daily pretreatment of ITZ was lower than the single combined treatment, but significantly different from TZ's AUC in the absence of ITZ ( P<0.05). As a result, single simultaneous administration of TZ with ITZ and single administration of TZ after daily pretreatment with ITZ to rats, ITZ seriously interfered with the pharmacokinetic parameters of TZ in plasma and brain micodialysate.