Investigating the Utility of Humanized Pregnane X Receptor-Constitutive Androstane Receptor-CYP3A4/7 Mouse Model to Assess CYP3A-Mediated Induction.

Clinical induction liability is assessed with human hepatocytes. However, underpredictions in the magnitude of clinical induction have been reported. Unfortunately, in vivo studies in animals do not provide additional insight because of species differences in drug metabolizing enzymes and their regulatory pathways. To circumvent this limitation, transgenic animals expressing human orthologs were developed. The aim of this work was to investigate the utility of mouse models expressing human orthologs of pregnane X receptor, constitutive androstane receptor, and CYP3A4/7 (Tg-Composite) in evaluating clinical induction. Rifampin, efavirenz, and pioglitazone, which were employed to represent strong, moderate, and weak inducers, were administered at multiple doses to Tg-Composite animals. In vivo CYP3A activity was monitored by measuring changes in the exposure of the CYP3A probe substrate triazolam. After the in vivo studies, microsomes were prepared from their livers to measure changes of in vitro CYP3A4 activity. In both in vivo and in vitro, distinction of clinic induction was recapitulated as rifampin yielded the greatest inductive effect followed by efavirenz and pioglitazone. Interestingly, with rifampin, in vivo CYP3A activity was approximately 4-fold higher than in vitro activity. Conversely, there was no difference between in vivo and in vitro CYP3A activity with efavirenz. These findings are consistent with the report that, although rifampin exhibits differential inductive effects between the intestines and liver, efavirenz does not. These data highlight the promise of transgenic models, such as Tg-Composite, to complement human hepatocytes to enhance the translatability of clinical induction as well as become a powerful tool to further study mechanisms of drug disposition. SIGNIFICANCE STATEMENT: Underprediction of the magnitude of clinical induction when using human hepatocytes has been reported, and transgenic models may improve clinical translatability. The work presented here showcases the human orthologs of pregnane X receptor, constitutive androstane receptor, and CYP3A4/7 model, which was able to recapitulate the magnitude of clinical induction and to differentiate tissue-dependent induction observed with rifampin but not with efavirenz. These results not only foreshadow the potential application of such transgenic models in assessing clinical induction but also in further investigation of the mechanism of drug disposition.

High-dose green tea polyphenol intake decreases CYP3A expression in a liver-specific manner with increases in blood substrate drug concentrations.

In recent years, the intake of functional foods containing high-doses of green tea polyphenols (GP) has been increasing. In this study, the long-term safety of high-dose GP was assessed from a pharmacokinetic point of view by focusing on the drug-metabolizing enzyme, cytochrome P450 (CYP). Mice were fed a diet containing 3% GP for 4weeks, and the CYP expression levels and activity were determined. The GP-treated group showed a significant decrease in the hepatic CYP3A and an increase in the hepatic CYP2C expression compared with the control group. CYP1A, CYP2D, and CYP2E expression were not different between the GP-treated and the control groups. In the small intestine, there were no differences in the CYP3A protein levels between the groups. The increase in the plasma triazolam concentration in the GP-treated group was observed. Although no changes were found in the hepatic CYP3A levels in mice receiving a diet containing 0.1% GP for 4weeks, a significant decrease was seen in the hepatic CYP3A level in mice receiving a diet containing 3% GP for only 1week. This study revealed that the intake of a high-dose GP results in a liver-specific decrease in the CYP3A expression level. The results also indicated that the effects of GP on CYP3A were not observed following the intake of a low-dose GP. In the future, caution should be taken in cases when functional foods containing a high-dose GP are concomitantly consumed with a CYP3A substrate drug.

Effects of menthol on the pharmacokinetics of triazolam and phenytoin.

We have previously shown that menthol attenuates the anticoagulant effect of warfarin by increasing the expression levels of CYP3A and CYP2C in the liver. This study evaluated the effects of menthol on the pharmacokinetics of the CYP3A substrate triazolam and the CYP2C substrate phenytoin. Menthol was orally administered to mice for 7 d. Twenty-four hours after the administration of menthol, triazolam was orally administered, and the plasma concentration was measured. In addition, the CYP3A metabolic activity for triazolam and the CYP3A expression level in the liver were determined. The effects of menthol on the pharmacokinetics of phenytoin were assessed in the same manner. In the menthol-treated group, the area under the blood concentration-time curve (AUC) of triazolam was lower and its clearance was higher compared with the control group. The CYP3A metabolic activity and CYP3A expression level in the liver were significantly increased in the menthol-treated group compared with the control group. Similarly, the AUC of phenytoin was lower and the hepatic CYP2C expression level was higher in the menthol-treated group. Thus, menthol lowered the plasma concentrations of triazolam and phenytoin when concurrently administered. These effects may be attributed to an increased metabolic activity for these drugs due to the increased expression of CYP3A and CYP2C in the liver.

Use of triazolam and alprazolam as premedication for general anesthesia / 대한마취과학회지

BACKGROUND: Triazolam has similar pharmacological properties as other benzodiazepines and is generally used as a sedative to treat insomnia. Alprazolam represents a possible alternative to midazolam for the premedication of surgical patients. The purpose of this study was to evaluate the anxiolytic, sedative, and amnestic properties of triazolam and alprazolam as pre-anesthetic medications. METHODS: Sixty adult patients were randomly allocated to receive oral triazolam 0.25 mg or alprazolam 0.5 mg one hour prior to surgery. A structured assessment interview was performed in the operating room (OR), the recovery room, and the ward. The levels of anxiety and sedation were assessed on a 7-point scale (0 = relaxation to 6 = very severe anxiety) and a 5-point scale (0 = alert to 4 = lack of responsiveness), respectively. The psychomotor performance was estimated using a digit symbol substitution test. As a memory test, we asked the patients the day after the surgery if they remembered being moved from the ward to the OR, and what object we had shown them in the OR. RESULTS: There were no significant differences between the groups with respect to anxiety and sedation. The postoperative interviews showed that 22.2% of the triazolam-treated patients experienced a loss of memory in the OR, against a 0% memory loss in the alprazolam-treated patients. In comparison with alprazolam 0.5 mg, triazolam 0.25 mg produced a higher incidence of amnesia without causing respiratory depression. CONCLUSIONS: Oral triazolam 0.25 mg can be an effective preanesthetic medication for psychomotor performance.

The melatonin receptor agonist is effective for free-running type circadian rhythm sleep disorder: case report on two sighted patients.

Along with urbanization of the living environment, the number of patients with circadian rhythm sleep disorder (CRSD) has been increasing. There are several treatment candidates for CRSD, such as light therapy, drugs (melatonin and vitamin B12), and sleep hygiene education. However, successful treatment method has not been established. In free-running type (FRT) CRSD, the endogenous circadian rhythm cannot be entrained to the 24-h light-dark cycle, resulting in free running on a cycle 0.5-2.5 h longer than the 24-h period. This condition is relatively common in blind individuals and is unusual in sighted individuals. Here we report two sighted patients with FRT, successfully treated with a melatonin receptor agonist, ramelteon. Patient 1 (36-year-old female) had suffered from FRT for nearly 4 months after resigning her job. She was given sleep hygiene education together with ramelteon at first and the free-running cycle stopped after treatment day 15. Triazolam was added from the day 25 to promote earlier sleep onset. And the sleep-wake schedule was normalized by the day 34. Patient 2 (33-year-old male) had suffered from FRT for nearly 8 months after starting to take a leave of absence from his job. He was given sleep hygiene education and was treated with ramelteon and methylcobalamin. His sleep-wake schedule was normalized from the first treatment day. By the combined treatment with ramelteon, both patients have maintained favorable sleep-wake schedules. The agonist action of ramelteon at the melatonin 2 receptor may have primarily contributed to the cessation of the free-running cycle in these patients.

Is pomegranate juice a potential perpetrator of clinical drug-drug interactions? Review of the in vitro, preclinical and clinical evidence.

The area of fruit juice-drug interaction has received wide attention with numerous scientific and clinical investigations performed and reported for scores of drugs metabolized by CYP3A4/CYP2C9. While grapefruit juice has been extensively studied with respect to its drug-drug interaction potential, numerous other fruit juices such as cranberry juice, orange juice, grape juice, pineapple juice and pomegranate juice have also been investigated for its potential to show drug-drug interaction of any clinical relevance. This review focuses on establishing any relevance for clinical drug-drug interaction potential with pomegranate juice, which has been shown to produce therapeutic benefits over a wide range of disease areas. The review collates and evaluates relevant published in vitro, preclinical and clinical evidence of the potential of pomegranate juice to be a perpetrator in drug-drug interactions mediated by CYP3A4 and CYP2C9. In vitro and animal pharmacokinetic data support the possibility of CYP3A4/CYP2C9 inhibition by pomegranate juice; however, the human relevance for drug-drug interaction was not established based on the limited case studies.

In vitro interactions of water-soluble garlic components with human cytochromes p450.

Eight water-soluble components of aged garlic extract were evaluated to assess their potential to inhibit the activity of human cytochrome-P450 (CYP) enzymes. The in vitro model consisted of human liver microsomes with index reactions chosen to profile the activity of the following six CYP isoforms: CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A. With only 2 exceptions, none of the 8 garlic components produced >50% inhibition even at high concentrations (100 micromol/L). S-methyl-L-cysteine and S-allyl-L-cysteine at 100 micromol/L produced modest inhibition of CYP3A, reducing activity to 20-40% of control. However available clinical evidence does not indicate CYP3A inhibition in vivo. The findings suggest that drug interactions involving inhibition of CYP3A enzymes by aged garlic extract are very unlikely.

The use of anxiolytic medications to supplement local anesthesia in the anxious patient.

Local anesthetic failures in dental patients can have many causes, including anatomical variations, technique, and anxiety/fear. By understanding the mechanisms responsible for failed local anesthesia, patients can be treated more comfortably. The interaction of anxiety and fear is discussed. Oral sedation dentistry is highlighted as a way to reduce anxiety/fear and the patient's perception of pain. Profound anesthesia can be accomplished more easily in relaxed patients with diminished or eliminated anxiety/fear.

Sleep-related vagotonic effect of zolpidem in rats.

RATIONALE: Zolpidem is a relatively new nonbenzodiazepine sedative-hypnotic. The effects of zolpidem on autonomic functions remain unclear. OBJECTIVES: The aim of this study was to evaluate the effects of zolpidem on sleep and related cardiac autonomic modulations as compared with triazolam in Wistar-Kyoto rats. METHODS: Continuous power spectral analyses of electroencephalogram (EEG), electromyogram, and heart rate variability were performed on freely moving rats during daytime sleep. The consciousness states were classified into active waking (AW), quiet sleep (QS), and paradoxical sleep (PS). Drugs were administered via gavage and data within 2 h were analyzed. RESULTS: All zolpidem (ZP3, 3 mg/kg; ZP30, 30 mg/kg) and triazolam (TZ0.075, 0.075 mg/kg; TZ0.75, 0.75 mg/kg) groups had longer accumulated QS time and averaged QS duration as compared with the vehicle control. The accumulated QS time and averaged QS duration of ZP3 were similar to those of TZ0.075. Significant suppressions of PS time were noted in all drug groups except ZP3. During QS, ZP3 and ZP30 exhibited significant increases of magnitude and percentage of EEG delta power, whereas TZ0.075 and TZ0.75 did not. Heart period and high-frequency power of heart rate variability increased significantly in ZP3 during all sleep-wake states. Both parameters, however, did not increase but even decreased in ZP30, TZ0.075, and TZ0.75. CONCLUSIONS: Zolpidem not only caused a longer and deeper sleep but also led to an elevated cardiac vagal activity at a specific dose in the rat.

On the sleep promoting effects of BR-16A: interaction with GABAergic modulators.

Pentobarbitone-induced hypnosis test was used as an animal model to explore the role of BR-16A, a polyherbal formulation in sleep. Pentobarbitone produces quick sleep latency (onset) and prolongation of total sleep time (duration). Sleep latency and total sleep time were used as a parameters for the evaluation. BR-16A potentiated the effect of triazolam (0.1 mg/kg, ip) and alprazolam (0.25 mg/kg, ip). Melatonin (5.0 mg/kg, ip) and zolpidem (0.5 mg/kg, ip) did not produce any significant effect on sleep parameters. However, alprazolam (0.25mg/kg, ip) potentiated the effect of BR-16A (100 mg/ kg, po) in higher dose only. Sleep promoting effect of BR-16A in combination with GABAergic drugs (triazolam and alprazolam,) suggested that these drugs have common mechanism in sleep promoting effect of pentobarbitone and could be used along with other GABAergic hypnotics for the treatment of insomnia. This may reduce the dose of the latter drug(s). BR-16A can be used for the treatment of sleep and sleep-related disorders.

Recent developments in local anesthesia and oral sedation.

This article reviews 3 recent developments in anxiety and pain control with significant potential for altering dental practice. First is the introduction of articaine hydrochloride as an injectable local anesthetic. Although articaine is an amide, its unique structure allows the drug to be quickly metabolized, reducing toxicity associated with repeated injections over time. The second development is the formulation of a lidocaine and prilocaine dental gel for topical anesthesia of the periodontal pocket. This product may significantly reduce the need for anesthetic injections during scaling and root planing. Finally, the use of triazolam as an oral sedative/anxiolytic is reviewed. The recent administration of triazolam in multiple doses has extended the availability of anxiety control to many dental patients, but unknowns about the safety of the technique as practiced by some dentists remains a concern.

Comparative cognitive and psychomotor effects of single doses of Valeriana officianalis and triazolam in healthy volunteers.

OBJECTIVES: To assess the cognitive and psychomotor effects of single oral doses of valerian in healthy volunteers in comparison with a placebo and the hypnotic agent triazolam. METHODS: In a double-blind, placebo-controlled, four-way crossover study nine healthy subjects (5 males, 4 females) received in random order valerian 500 mg, valerian 1000 mg, triazolam 0.25 mg and placebo. Doses were separated by a wash-out period of at least 1 week. Subjects were tested before each dose and at 2, 4 and 8 h after the dose of each compound using the critical flicker fusion (CFF), choice reaction time (CRT), digit symbol substitution test (DSST), symbol search test (SST), digit span test (DST) and visual analogue scales of mood. RESULTS: Repeated measures ANOVA was used to examine the changes in performance on tests over time and significant effects were further analysed using simple main effects analysis with least significant difference corrections. Statistically significant differences were only noted for the cognitive tests: SST (F(3, 8)=3.182, p<0.05) and DSST (F(3, 8)=9.688, p<0.005). In both cases the differences between groups were due to the effects of triazolam. CONCLUSION: These data confirm that at recommended therapeutic doses, triazolam has detrimental effects on cognitive processes in healthy volunteers as found in previous studies. Valerian was without effect on either cognitive or psychomotor performance in healthy volunteers at the doses used in this study. Should the hypnotic activity of valerian be confirmed in randomized double-blind trials it may be a less troublesome alternative to benzodiazepines in the treatment of insomnia.

Bright light treatment for night-time insomnia and daytime sleepiness in elderly people: comparison with a short-acting hypnotic.

Night-time bright light (BL) treatment and triazolam (0.125 mg/day) were given to three healthy elderly people in a cross-over design. They kept a daytime sleepiness test and a sleep log, and their wrist-activity was monitored simultaneously. Subjectively, BL increased daytime sleepiness and naps, and decreased night-time sleep. Triazolam decreased daytime sleepiness and naps, and increased night-time sleep. Actigraphic night-time sleep and naps on the first day were similar to these results. However, on the fourth day night-time insomnia induced by BL had recovered, and naps were shorter than the baseline. Triazolam increased actigraphic naps as the days passed.

Overnight effects of triazolam on cognitive function: an event-related potentials study.

The effects of triazolam on cognitive function and vigilance on the morning following a nocturnal administration were investigated using event-related potentials (ERP) measurement and a sleep latency test (SLT). We previously reported a significant reducing effect on target N1 amplitude on the morning following triazolam administration, suggesting a residual effect of triazolam. In order to demonstrate, which aspect of cognitive function alteration caused the reducing effect on N1 amplitude, we added the ignore condition for ERP measurement, which enabled us to separate mismatch negativity (MMN) from other subcomponents overlapping N1. As a result, MMN was attenuated and sleep latency was shortened on the morning following triazolam administration. Two possibilities were suggested for the mechanism of MMN attenuation. One is GABAergic activation caused by the residual effect of triazolam per se, and the other is the lowered vigilance level demonstrated in the SLT. Further studies are necessary to determine whether this alteration in physiological bases underlying mismatch detection is specific to triazolam and/or other benzodiazepines or related to nonspecific vigilance level.

Differential catalytic properties in metabolism of endogenous and exogenous substrates among CYP3A enzymes expressed in COS-7 cells.

The catalytic properties of CYP3A7 in the metabolism of endogenous and exogenous substrates were compared with those of CYP3A4 and CYP3A5 using COS-7 expressing enzymes. The highest activities of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone 3-sulfate (DHEA-S) 16alpha-hydroxylase were observed in COS-7 cells expressing CYP3A7. In contrast, the activity of testosterone 6beta-hydroxylase of CYP3A7 expressed in COS-7 cells was much less than that of CYP3A4 expressed in COS-7 cells. The rate of carbamazepine 10, 11-epoxidation was the greatest in COS-7 cells expressing CYP3A4, followed by CYP3A5 and CYP3A7. On the other hand, the formation of reductive metabolite of zonisamide was the highest in COS-7 cells expressing CYP3A4, followed by CYP3A7 and CYP3A5. Furthermore, the addition of triazolam resulted in a decrease in 6beta-hydroxylation catalyzed by CYP3A7, but not by CYP3A4, whereas the pretreatment of microsomes with triacetyloleandomycin (TAO) resulted in a decrease in the reaction catalyzed by CYP3A4, but not by CYP3A7. Together with these results, it was suggested that CYP3A7 exerts differential catalytic properties not only in metabolism of endogenous substrates but also in drug metabolism compared to CYP3A4 and CYP3A5.

[Jet lag syndrome].

Jet lag is a travel-induced circadian rhythm disorders. Symptoms of jet lag include difficulty sleeping at the new sleep time, daytime sleepiness and fatigue, and impaired performance. Treatment of jet lag includes both behavioral and pharmacological component. A short-half-life benzodiazepine hypnotic for several nights at the new sleep time have been recommended to decrease jet lag symptoms. Melatonin also can alleviate jet lag and bright light exposure is a useful countermeasure for jet lag.

Can we mix behavioral therapy with hypnotics when treating insomniacs?

This study asks whether insomniacs undergoing behavioral training need to do so while totally free of hypnotics. Twenty-six insomniacs participated in the study, which included extensive monitoring of sleep both in the laboratory and at home. All subjects received six sessions of training in sleep hygiene and relaxation. About half the subjects were also given a hypnotic for occasional use; the others were asked to abstain from all hypnotics. Follow-up was performed immediately after treatment and again 10 months later. When compared with a waiting list, 6 hours of behavioral therapy improved insomniacs' sleep latency and sleep efficiency immediately after treatment, whether or not hypnotics were given concomitantly. Immediately following the 6 hours of therapy, those who had combined pharmacotherapy and behavior therapy improved about the same as those who had received behavior therapy alone. However, on the 10-month follow-up, those who had learned sleep hygiene and relaxation without the help of occasional hypnotics had more sleep and a better sleep efficiency than those who had been allowed an occasional hypnotic. We conclude that when teaching sleep hygiene and behavioral therapy to insomniacs, it might be advantageous to disallow the concomitant use of hypnotics as needed.

Role of GABA-A and mitochondrial diazepam binding inhibitor receptors in the anti-stress activity of neurosteroids in mice.

Neuroactive steroidal modulation of immobilization-stress and possible involvement of GABA-A and mitochondrial diazepam binding inhibitor (DBI) receptors (MDR) has been investigated in mice. Immobilization of mice for 2 h induced intense antinociception, anxiety state, and associated with a fall in adrenal ascorbic acid levels. Pretreatment with high dose of progesterone (10 mg/kg), a precursor of neurosteroids, significantly decreased the stress-induced antinociception, anxiety and fall in adrenal ascorbic acid, while low doses (1 and 5 mg/kg) or hydrocortisone (10 and 100 mg/kg) were ineffective. In contrast, progesterone (1 mg/kg, for 9 days) produced a significant antistress effect, which was blocked by GABA-A antagonists picrotoxin (1 mg/kg) and bicuculline (1 mg/kg), but not by flumazenil (2 mg/kg), a specific benzodiazepine (BZD) antagonist. 4'-chlordiazepam (0.1 and 0.25 mg/kg), a specific high affinity MDR agonist, produced significant anti-stress effect in a flumazenil-insensitive manner, but was blocked by pretreatment with PK11195 (1.5 mg/kg), a selective partial agonist of MDR, and with bicuculline (1 mg/kg), a potent GABA-A receptor antagonist. At higher doses, progesterone and 4'-chlordiazepam which are effective in immobilization stress also reduced locomotion. However, lower doses of progesterone (6.5 mg/kg) neither affected locomotion, nor produced any motor toxicity on rota-rod test. At the lower doses, the MDR ligand 4'-chlordiazepam (50 micrograms/kg) decreased locomotor activity, without altering motor toxicity on rota-rod test. Further, the per se effects of these treatments on unstressed mice were not significantly different from those of untreated controls, except for plus-maze test. The antistress profile of progesterone may be attributed to the in vivo production of neurosteroid allopregnanolone, thus resembled that of BZDs. Furthermore, the antistress actions are flumazenil-resistant, reaffirming that there may be an increase in the levels of pregnane neurosteroids in vivo, which may act on a specific allosteric site on GABA-A receptors distinct from BZD site. Because 4'-chlordiazepam binds to MDRs and stimulate mitochondrial neurosteroidogenesis, the anti-stress effects of 4'-chlordiazepam may be imputed to its MDR-induced neurosteroids, which then act on GABA-A receptors. These data suggest a pivotal role for GABA-A and mitochondrial DBI receptors in the antistress actions of neurosteroids and reinforces their ameliorative effect in physiological stress.

A multicenter study of sleep-wake rhythm disorders: therapeutic effects of vitamin B12, bright light therapy, chronotherapy and hypnotics.

One hundred and six subjects with primary sleep-wake rhythm disorders [13 non-24 hour sleep-wake syndrome (non-24), 76 delayed sleep phase syndrome (DSPS), 11 irregular sleep-wake pattern (irregular) and six long sleepers] were treated with vitamin B12, bright light, chronotherapy and/or hypnotics. These therapies caused moderate or remarkable improvement in 32% of the non-24, 42% of DSPS, 45% of irregular and 67% of long sleepers. A lack of adequate sleep, unpleasant feelings at waking and daytime drowsiness were also improved in DSPS.