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During the routine validation of a benzodiazepine method (performed on a Liquid Chromatography - Tandem Mass Spectrometer), it was noted that lorazepam, triazolam, and α-hydroxytriazolam showed a quadratic shift/bias in the calibration curve, particularly at high concentrations. The ultimate cause of this bias was determined to be due to the natural presence of chlorine (Cl) isotopes (35Cl and 37Cl) in these benzodiazepines. The presence of the heavy (37Cl) isoforms of Cl resulted in the analyte's mass being the same as the internal standard which, in turn, caused the internal standard to appear "falsely increased", thus skewing the calibration curve. One solution to this potential issue was to take advantage of this natural phenomenon and use the Cl heavy isoforms of the respectively labeled internal standards.

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.

Preparations of plasma-derived small extracellular vesicles (sEVs) were deployed as liquid biopsy to study cytochrome P450 (CYP) 3A4 (CYP3A4) induction following modafinil 400 mg once daily × 14 days (young healthy volunteers, N = 10 subjects). Induction was confirmed using the 4β-hydroxycholesterol-to-cholesterol (4βHC/C) ratio, a plasma CYP3A4/5 biomarker, with a mean 2.1-fold increase (Day 15 vs. Day 1; 90% confidence interval (CI) = 1.8-2.3; P value = 0.0004). Proteomic analysis revealed the induction (mean Day 15 vs. Day 1 fold-increase (90% CI)) of both liver (1.3 (1.1-1.5), P value = 0.014) and nonliver (1.9 (1.6-2.2), P value = 0.04) sEV CYP3A4 protein expression. In CYP3A5 nonexpresser subjects, the baseline (pre-dose) 4βHC/C plasma ratio was more highly correlated with liver sEVs (r = 0.937, P value = 0.001) than nonliver sEVs (r = 0.619, P value = 0.101) CYP3A4 protein expression. When CYP3A5 expressers (CYP3A5*1/*3) were included, the correlation with liver sEVs (r = 0.761, P value = 0.011) and nonliver sEVs (r = 0.391, P value = 0.264) CYP3A4 protein was weaker. Although modafinil-induced changes in plasma 4βHC/C ratio did not correlate with sEVs CYP3A4 protein expression, the individual subject sEVs proteomic data were used successfully to predict victim drug (midazolam, triazolam, dextromethorphan, 17α-ethinylestradiol, and abemaciclib) area under the plasma concentration-time curve (AUC) ratios (AUCRs) following modafinil. Based on the AUCR values, modafinil was classified as a weak to moderate CYP3A4 inducer (vs. rifampicin). For the first time, it was possible to deploy plasma-derived sEVs to study CYP3A4 induction beyond rifampicin, a more potent CYP3A4 inducer.

Background Researchers have long been interested in the potential drug–drug interactions (DDIs) between opioids and benzodiazepines. However, much remains unknown concerning the interactions between these two drug classes. The objective of this work is to study the mechanism underlying the DDIs between opioids and benzodiazepines from the perspective of their pharmacokinetic (PK) interactions. A PK interaction occurs when two drugs are metabolized by the same cytochrome P450 enzymes and is one of the most common reasons for DDIs. Methods We quantitatively predicted the DDIs between three opioids (fentanyl, oxycodone and buprenorphine) and four benzodiazepines (alprazolam, diazepam, midazolam and triazolam) using a physiologically based pharmacokinetic (PBPK) modeling approach. A set of PBPK models was first constructed for these common opioids and benzodiazepines using SimCYP software, and the DDIs between them were then explored at various dosages. Results Our simulation results suggested there were no PK interactions between normal doses of opioids and benzodiazepines; but weak interactions can be expected with the combination of opioids and overdosed benzodiazepines. Particular attention should be given to the combination of fentanyl and overdosed alprazolam since a PK interaction can be observed between them. Conclusion Our results appear to indicate that pharmacodynamics may play a more important role than PKs in causing DDIs between opioids and benzodiazepines. This study also demonstrated that molecular modeling can be a very useful tool to mitigate the problem of “missing metabolic reaction parameters” in PK modeling and simulation. Electronic supplementary material The online version of this article (10.1007/s40268-019-00282-3) contains supplementary material, which is available to authorized users.

Background Previous studies have investigated α1GABAA and α5GABAA receptor mechanisms in the behavioral effects of ethanol (EtOH) in monkeys. However, genetic studies in humans and preclinical studies with mutant mice suggest a role for α2GABAA and/or α3GABAA receptors in the effects of EtOH. The development of novel positive allosteric modulators (PAMs) with functional selectivity (i.e., selective efficacy) at α2GABAA and α3GABAA receptors allows for probing of these subtypes in preclinical models of the discriminative stimulus and reinforcing effects of EtOH in rhesus macaques. Methods In discrimination studies, subjects were trained to discriminate EtOH (2 g/kg, intragastrically) from water under a fixed-ratio (FR) schedule of food delivery. In oral self-administration studies, subjects were trained to self-administer EtOH (2% w/v) or sucrose (0.3 to 1% w/v) under an FR schedule of solution availability. Results In discrimination studies, functionally selective PAMs at α2GABAA and α3GABAA (HZ-166) or α3GABAA (YT-III-31) receptors substituted fully (maximum percentage of EtOH-lever responding ≥80%) for the discriminative stimulus effects of EtOH without altering response rates. Full substitution for EtOH also was engendered by a nonselective PAM (triazolam), an α5GABAA -preferring PAM (QH-ii-066) and a PAM at α2GABAA , α3GABAA , and α5GABAA receptors (L-838417). A partial (MRK-696) or an α1GABAA -preferring (zolpidem) PAM only engendered partial substitution (i.e., ~50 to 60% EtOH-lever responding). In self-administration studies, pretreatments with the functionally selective PAMs at α2GABAA and α3GABAA (XHe-II-053 and HZ-166) or α3GABAA (YT-III-31 and YT-III-271) receptors increased EtOH, but not sucrose, drinking at doses that had few, or no, observable sedative-motor effects. Conclusions Our results confirm prior findings regarding the respective roles of α1GABAA and α5GABAA receptors in the discriminative stimulus effects of EtOH and, further, suggest a key facilitatory role for α3GABAA and potentially α2GABAA receptors in several abuse-related effects of EtOH in monkeys. Moreover, they reveal a potential role for these latter subtypes in EtOH's sedative effects.

1. This study evaluated the prediction accuracy of cytochrome P450 (CYP)-mediated drug-drug interaction (DDI) using minimal physiologically-based pharmacokinetic (PBPK) modelling incorporating the hepatic accumulation factor of an inhibitor (i.e. unbound liver/unbound plasma concentration ratio [Kp,uu,liver]) based on 22 clinical DDI studies. 2. Kp,uu,liver values were estimated using three methods: (1) ratio of cell-to-medium ratio in human cryopreserved hepatocytes (C/Mu) at 37 °C to that on ice (Kp,uu,C/M), (2) multiplication of total liver/unbound plasma concentration ratio (Kp,u,liver) estimated from C/Mu at 37 °C with unbound fraction in human liver homogenate (Kp,uu,cell) and (3) observed Kp,uu,liver in rats after intravenous infusion (Kp,uu,rat). 3. PBPK model using each Kp,uu,liver projected the area under the curve (AUC) increase of substrates more accurately than the model assuming a Kp,uu,liver of 1 for the average fold error and root mean square error did. Particularly, the model with a Kp,uu,liver of 1 underestimated the AUC increase of triazolam following co-administration with CYP3A4 inhibitor itraconazole by five-fold, whereas the AUC increase projected using the model incorporating the Kp,uu,C/M, Kp,uu,cell, or Kp,uu,rat of itraconazole and hydroxyitraconazole was within approximately two-fold of the actual value. 4. The results indicated that incorporating Kp,uu,liver into the PBPK model improved the accuracy of DDI projection.

In order to develop improved anxiolytic drugs, 8-substituted analogs of triazolam were synthesized in an effort to discover compounds with selectivity for α2/α3 subunit-containing GABAA subtypes. Two compounds in this series, XLi-JY-DMH (6-(2-chlorophenyl)-8-ethynyl-1-methyl-4H-benzo [f][1,2,4]triazolo[4,3-a][1,4]diazepine) and SH-TRI-108 [(E)-8-ethynyl-1-methyl-6-(pyridin-2-yl)-4H-benzo [f][1,2,4]triazolo[4,3-a][1,4]diazepine], were evaluated for in vitro and in vivo properties associated with GABAA subtype-selective ligands. In radioligand binding assays conducted in transfected HEK cells containing rat αXβ3γ2 subtypes (X = 1,2,3,5), no evidence of selectivity was obtained, although differences in potency relative to triazolam were observed overall (triazolam > XLi-JY-DMH > SH-TRI-108). In studies with rat αXβ3γ2 subtypes (X = 1,2,3,5) using patch-clamp electrophysiology, no differences in maximal potentiation of GABA-mediated Cl− current was obtained across subtypes for any compound. However, SH-TRI-108 demonstrated a 25-fold difference in functional potency between α1β3γ2 vs. α2β3γ2 subtypes. We evaluated the extent to which this potency difference translated into behavioral pharmacological differences in monkeys. In a rhesus monkey conflict model of anxiolytic-like effects, triazolam, XLi-JY-DMH, and SH-TR-108 increased rates of responding attenuated by shock (anti-conflict effect) but also attenuated non-suppressed responding. In a squirrel monkey observation procedure, both analogs engendered a profile of sedative-motor effects similar to that reported previously for triazolam. In molecular docking studies, we found that the interactions of the 8-ethynyl triazolobenzodiazepines with the C-loop of the α1GABAA site was stronger than that of imidazodiazepines XHe-II-053 and HZ-166, which may account for the non-sedating yet anxiolytic profile of these latter compounds when evaluated in previous studies.

The degradation behavior of eight benzodiazepines (BZPs): alprazolam, etizolam, diazepam, triazolam, nitrazepam (NZP), flunitrazepam (FNZ), bromazepam, and lorazepam, in artificial gastric juice was monitored by a LC/photodiode array detector (PDA) to estimate their pharmacokinetics in the stomach. For drugs that were degradable, such physicochemical parameters as reaction rate constant were measured to evaluate the effect of storage conditions on drug degradability, such as whether the degradation proceeds faster by increasing storage temperature, or whether the degradation reaction is reversible by adjusting pH. As a result, it was confirmed that although the eight BZPs degraded in artificial gastric juice, most of them could be restored when pH was increased, and the restoration rates differed depending on the pH and the type of BZP. As for NZP, an Arrhenius plot was drawn to obtain the physicochemical parameters, such as activation energy and activation entropy involved in the degradation reaction, and the reaction kinetics was discussed. In addition, two substances were confirmed as the degradation products of NZP in artificial gastric juice: one was a reversible degradation product (A) (intermediate) and the other was an irreversible degradation product (B) (final degradation product). The intermediate was identified as 2-amino-N-(2-benzoyl-4-nitrophenyl)-acetamide, and the final degradation product was 2-amino-5-nitrobenzophenone. Therefore, when detecting NZP in human stomach contents, such as during judicial dissection, it would be prudent to target NZP as well as the intermediate (A) and the final degradation product (B).

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.

OBJECTIVE The contribution ratios (CR) of metabolic enzymes to the systemic clearance of a drug can be estimated from in vitro studies. Another feasible approach is to calculate them based on the increase in the area under the time-concentration curve (AUC) caused by the co-administration of a potent and selective inhibitor in a clinical drug-drug interaction (DDI) study. However, some factors, such as the inhibitory potency of the inhibitor and the inhibition of first-pass metabolism, might affect the estimation of CR based on clinical DDI studies. We aimed to validate the accuracy of the DDI-based estimation of CR using an in silico approach. MATERIALS AND METHODS An in silico DDI study was conducted using a population-based physiological pharmacokinetic simulator to estimate the CR of cytochrome P450 (CYP)3A4 for zolpidem, sildenafil, omeprazole, triazolam, and repaglinide. The ratio of the AUC value seen in the presence of an inhibitor (ketoconazole or itraconazole) to that observed in the absence of the inhibitor (AUC ratio) was also calculated. The CR for CYP3A4 obtained using the simulator (CRdef) were compared with those calculated from the AUC ratio (CRest). RESULTS When ketoconazole was used, good correlations between the CRest and CRdef were obtained for all examined substrates (inconsistencies were seen in < 10% of subjects). CR estimates derived from the AUC ratio were found to be accurate. Some underestimation was observed, possibly due to incomplete inhibition, and some overestimation caused by extensive first-pass metabolism was noted. CONCLUSION This study verified that CR obtained from AUC ratios in DDI studies are quite reliable.

It is important to predict drug-drug interactions via inhibition of intestinal cytochrome P450 3A (CYP3A) which is a determinant of bioavailability of orally administered CYP3A substrates. However, inhibitory effects of macrolide antibiotics on CYP3A-mediated metabolism are not entirely identical between humans and rodents.We investigated the effects of macrolide antibiotics, clarithromycin and erythromycin, on in vitro and in vivo metabolism of triazolam, a CYP3A substrate, in CYP3A-humanised mice generated by using a mouse artificial chromosome vector carrying a human CYP3A gene.Metabolic activities of triazolam were inhibited by macrolide antibiotics in liver and intestine microsomes of CYP3A-humanised mice.The area under the plasma concentration-time curve ratios of 4-hydroxytriazolam to triazolam after oral dosing of triazolam were significantly decreased by multiple administration of macrolide antibiotics. The plasma concentrations ratios of α-hydroxytriazolam and 4-hydroxytriazolam to triazolam in portal blood were significantly decreased by multiple administration of clarithromycin in CYP3A-humanised mice.These results suggest that intestinal CYP3A activity was inhibited by macrolide antibiotics in CYP3A-humanised mice in vitro and in vivo. The plasma concentrations of triazolam and its metabolites in the portal blood of CYP3A-humanised mice would be useful for direct evaluation of intestinal CYP3A-mediated drug-drug interactions. It is important to predict drug-drug interactions via inhibition of intestinal cytochrome P450 3A (CYP3A) which is a determinant of bioavailability of orally administered CYP3A substrates. However, inhibitory effects of macrolide antibiotics on CYP3A-mediated metabolism are not entirely identical between humans and rodents. We investigated the effects of macrolide antibiotics, clarithromycin and erythromycin, on in vitro and in vivo metabolism of triazolam, a CYP3A substrate, in CYP3A-humanised mice generated by using a mouse artificial chromosome vector carrying a human CYP3A gene. Metabolic activities of triazolam were inhibited by macrolide antibiotics in liver and intestine microsomes of CYP3A-humanised mice. The area under the plasma concentration-time curve ratios of 4-hydroxytriazolam to triazolam after oral dosing of triazolam were significantly decreased by multiple administration of macrolide antibiotics. The plasma concentrations ratios of α-hydroxytriazolam and 4-hydroxytriazolam to triazolam in portal blood were significantly decreased by multiple administration of clarithromycin in CYP3A-humanised mice. These results suggest that intestinal CYP3A activity was inhibited by macrolide antibiotics in CYP3A-humanised mice in vitro and in vivo. The plasma concentrations of triazolam and its metabolites in the portal blood of CYP3A-humanised mice would be useful for direct evaluation of intestinal CYP3A-mediated drug-drug interactions.

Background: Hypnotics, such as benzodiazepines and nonbenzodiazepines, have been used to aid sleep and treat insomnia in older adults. However, there is limited information for the comparative efficacy and safety of different hypnotics for insomnia in older adults. Methods: EMBASE, PubMed, ClinicalTrials.gov, and ProQuest Dissertations and Theses A&I databases were searched. We included randomised controlled trials comparing hypnotics with either another hypnotic or placebo for insomnia treatment in elderly people. A multivariate random effect network meta-analysis in a frequentist framework was used for estimations of treatment effects and safety. Findings: Twenty-two articles with 5172 older adults were included. Eszopiclone was ranked as the best therapy for prolonging objective total sleep time (28·60 mins), and low-dose doxepin appeared to be the optimal treatment for increasing subjective total sleep time (28·19 mins) and objective sleep efficiency (6.8%) compared with control. Zaleplon was the most effective therapy in reducing objective and subjective sleep onset latency (-21·63 mins and -15·92 mins) compared with control. The risk of overall adverse events was highest with triazolam use (odd ratio, 1·89, 95% confidence interval 1·10 to 3·25) compared to placebo. Interpretation: Considering unclear risk of bias and the potential adverse effects of benzodiazepines and nonbenzodiazepines, we suggest that low-dose doxepin can be the optimal pharmacotherapy for the improvements of sleep duration and sleep efficiency. Future RCTs investigating the treatment effects of hypnotics, particularly low-dose doxepin, on insomnia in older adults are warranted. Funding Statement: HYC acknowledges the support of the Taiwan Ministry of Science and Technology (MOST 106-2314-B-038 -058 -MY3). YKT was supported by the Ministry of Science and Technology, Taiwan (MOST 106-2314-B-002 -098 -MY3). Declaration of Interests: All authors declared no conflicts of interest in this work. Ethics Approval Statement: The study is registered with PROSPERO, number CRD42016046301.

Retention of the ability to wake from sleep in response to dangerous situations is an ideal characteristic of safe hypnotics. We studied the effects of a dual orexin receptor antagonist-22 (DORA-22) and the GABA-A receptor modulator, triazolam, on the ability to wake in response to aversive stimuli. We examined four modalities of sensory inputs, namely, auditory (ultrasonic sound), vestibular (trembling), olfactory (predator odor), and autonomic (hypoxia) stimuli. When the mice fell asleep, one of the four stimuli was applied for 30 s. In the case of auditory stimulation, latency to arousal following vehicle, DORA-22, and triazolam administration was 3.0 (2.0–3.8), 3.5 (2.0–6.5), and 161 (117–267) s (median and 25–75 percentile in the parentheses, n = 8), respectively. Latency to return to sleep after arousal was 148 (95–183), 70 (43–98), and 60 (52–69) s, respectively. Similar results were obtained for vestibular and olfactory stimulation. During the hypoxic stimulation, latencies for arousal and returning to sleep were not significantly different among the groups. The findings of this study are consistent with the distinct mechanisms of these sleep promoting therapies; GABA-A receptor activation by triazolam is thought to induce widespread central nervous system (CNS) suppression while DORA-22 more specifically targets sleep/wake pathways through orexin receptor antagonism. These data support the notion that DORA-22 preserves the ability to wake in response to aversive and consciousness-inducing sensory stimuli, regardless of modality, while remaining effective in the absence of threat. This study provides a unique and important safety evaluation of the potential for certain hypnotics., Shouhei Iwakawa, Yuichi Kanmura and Tomoyuki KuwakiOrexin Receptor Blockade-Induced Sleep Preserves the Ability to Wake in the Presence of Threat in MiceFrontiers in Behavioral Neuroscience, 2019https://doi.org/10.3389/fnbeh.2018.00327

Study Objectives This study investigated the association between ambient nighttime temperature and sleep problems assessed by the prescription dose of sleeping pills in South Korean adults. Methods We used the 2002–2015 National Health Insurance Service-National Sample Cohort. A total of 711,079 adults who were 20 years old or older were included, wherein 42,858 adults (~6%) had been prescribed hypnotic medications including zolpidem (N05CF02) and triazolam (N05CD05). Ambient temperature data was calculated as the mean highest temperature of nighttime (23:00–07:00) for every month from January to December. We combined the drug-prescribed date with the administrative districts-level daily nighttime temperature between 2002 and 2015. Results We found that a non-linear, U-shaped relationship between nighttime temperature and hypnotic medication prescription. With an increase per 1°C temperature or an increase in a square per 1°C, the prescription dose of sleeping pills was significantly increased (both p < 0.05). At each 5°C nighttime temperature, subjects belonging to low (≤0°C and 0–5°C) or high (20–25°C and ≥25°C) temperature categories had significantly higher doses of sleeping pills than those at the reference temperature (10–15°C). Changes in nighttime temperature had a significant non-linear effect on the prescribed dosage of hypnotic medications for both adults (p < 0.0001) and the elderly (p = 0.0006). Conclusion We found that either a high or low nighttime temperature was significantly associated with a high daily dose of hypnotic medications in the Korean population.

Benzodiazepines are a commonly prescribed class of drugs that have the potential for abuse. The Palm Beach County Sheriff’s Office received drug seizure submissions that included novel and/or nonroutine benzodiazepines of increasing prevalence from 2017 to 2019. This prompted the development of a method of analysis for these compounds in biological specimens. The method tests for 16 novel and nonroutine benzodiazepines and suvorexant in whole blood by liquid chromatography–tandem mass spectrometry (LC–MS-MS). The target analytes included bromazepam, clobazam, clonazolam, clotiazepam, diclazepam, estazolam, etizolam, flualprazolam, flubromazepam, flubromazolam, loprazolam, lormetazepam, phenazepam, prazepam, suvorexant, tetrazepam and triazolam. The method uses 200 µL of sample, protein precipitation and an instrument run-time of 8 min. The limit of detection was either 1 or 5 ng/mL and the limit of quantitation was either 5 or 25 ng/mL depending on the analyte. The method was validated for quantitative analysis for 15 out of the 17 analytes. Flubromazepam and prazepam were validated for qualitative identification only. A quadratic calibration model (r2 > 0.990) with 1/x weighting was used for all analytes for quantitative analysis. The calibration range was either 5–100 or 25–500 ng/mL depending on the analyte. The coefficient of variation of replicate analyses was within 14% and bias was within ±14%. The method provides a sensitive, efficient and robust procedure for the quantitation and/or qualitative identification of select novel and nonroutine benzodiazepines and suvorexant using LC–MS-MS and a sample volume of 200 µL.

Estazolam (Z1) and related derivatives, adinazolam (Z2), alprazolam (Z3), 4-hydroxyalprazolam (Z4) and triazolam (Z5) have been studied by using various computational tools to analyze their geometry and spectral characteristics. The compounds were found to interact with graphene monolayer results shows that there is enhancement in various physico-chemical descriptors and surface enhanced Raman spectra (SERS). The various reactive descriptors obtained from the FMO analysis predict the reactive nature of the compound. The various lone pair/sigma to pi conjugation was analyzed using NBO formalism, which provides valuable information about intra molecular electron transfer which is vital in predicting the inherent stability of the molecule. Simulated electronic spectra using TD-DFT and CAM-B3LYP functional are discussed in detail with respect to electronic transitions and light harvesting efficiency. Suitability of candidates as a photo sensitizer in dye sensitized solar cells was studied and 4-Hydroxyalprazolam is identified as a suitable candidate. Nucleophilic and electrophilic regions of the molecules are identified using MESP, which adds to the reactivity information. It can be seen that the highest interaction energy has been obtained in the case of the Z5-graphene system, while the lowest interaction energy has been obtained in the case of the Z1-graphene system. Docking indicates that the ligands adsorbed over graphene also form stable complexes with the receptors as indicated by the high binding affinity energy values.

Flualprazolam is a novel designer benzodiazepine, structurally related to alprazolam, flubromazolam and triazolam. In the last couple of years, it has been frequently detected in seizures and in forensic cases in Sweden and Finland. However, there is a lack of published blood concentrations for the drug, which presents difficulties when assessing its relevance for the cause of death. A quantitative method for the determination of flualprazolam in post-mortem blood was developed and validated, and subsequently used to analyse samples from 33 deaths previously screened as testing positive for flualprazolam in Sweden and Finland. Most of the cases in the study were accidental deaths (61 %) or suicides (18 %). The median (range) flualprazolam concentration was 18.0 (3.0-68) ng/g. The majority of the deceased were male (82 %) and the median age was 30 years. The median age in the Swedish cases was significantly higher (35 years) than in the Finnish cases (23 years) (p

Objectives: This study aimed to compare the risk of fractures, acute myocardial infarction, atrial fibrillation, and ventricular arrhythmia among Danish citizens aged ≥ 65 which were new users of promethazine or domperidone, triazolam, loratadine, and betahistine. Secondly, the study aimed to perform a risk stratification to identify the most relevant predictors for the study outcomes. Methods: The study period was 01/01/2015 to 31/12/2016. The data sources were the Danish registers. Each patient was followed for 90 days. A logistic regression model was used to compute the unadjusted and adjusted odds ratios (OR), and a conditional inference tree was used to identify the most relevant predictors for the study outcomes. Results: Promethazine had a higher risk of hospitalization for atrial fibrillation than loratadine and betahistine (OR 1.58; 95% CI 1.07–2.63 and OR 3.22; 95% CI 1.69–7.14, respectively). For fractures, acute myocardial infarction, and ventricular arrhythmia hospitalizations, no statistically significant differences were found among drugs under investigation. The medical history of cardiac arrhythmia (OR 4.14; 95% CI 2.94–5.78, p < 0.0001) was the most relevant predictor for atrial fibrillation hospitalizations. Conclusion: This study found an increased risk of atrial fibrillation hospitalization among promethazine users, and the risk was higher among patients with prior cardiac arrhythmia.

1-Aminobenzotriazole (ABT) is a mechanism-based inactivator of major cytochrome P450 (CYP) enzymes, which is used in multiple mechanistic studies. The purpose was to evaluate the effect of 2 and 16-h pretreatment regimens of ABT on the exposures of triazolam in rat. Another objective was to evaluate the effect of ABT on gastric emptying of acetaminophen. Plasma area under the curve (AUC) of triazolam was increased by 101-fold and 81-fold for the rats pretreated with ABT at 2 and 16 h, respectively, compared to control rats. Time to reach maximum concentration was 0.3, 4.8 and 3.7 h in control, 2 and 16-h pretreatment animals, respectively. In the case of acetaminophen, where Tmax was not delayed, the mean absorption time (MAT) in control, 2 and 16 h ABT pretreatment groups were 0.3, 4.6 and 2.9 h, respectively, suggesting delayed absorption. This hypothesis was further supported by GastroPlusTM simulation. In summary, extent of triazolam absorption was increased to a similar extent with both 2 and 16 h ABT pretreatment regimens, suggesting that either of the regimen can be used to increase parent exposures in rat. With ABT pretreatment, delayed absorption of triazolam and acetaminophen was observed, as suggested by delay in Tmax and MAT, respectively.

Flualprazolam is a nonregistered drug in the benzodiazepine family and constitutes a new psychoactive substance (NPS). Since 2014, a growing number of designer benzodiazepines have become available over the Internet and on the counterfeit drug market. In June 2019, a cluster of patients intoxicated with flualprazolam was identified by the Oregon Poison Center. As an emerging drug of abuse, the clinical characteristics of flualprazolam have been poorly characterized thus far. Over a one-week period, 6 teenagers presented to local emergency departments after ingesting illegally obtained counterfeit alprazolam, which led to sedation. Other symptoms included slurred speech, confusion, and mild respiratory depression. All 6 patients had resolution of their symptoms within 6 hours of ingestion. Blood and urine samples, as well as a tablet fragment, were obtained from 3 patients. The tablet and biological samples were analyzed by using liquid chromatography–quadrupole time-of-flight mass spectrometry and were found to contain the NPS flualprazolam without other drugs or intoxicants. With this case series, we add to the medical literature a clinical description of an emerging drug of abuse. Flualprazolam appears to share the clinical properties of other benzodiazepines. As flualprazolam and other NPSs become more common, physicians must be aware of their availability and characteristics. Sedation lasting

In this study, an alternative method for synthesizing magnetic cobalt adeninate metal-organic frameworks was developed, and the synthesized materials were examined for their potential application for separating and enriching benzodiazepines from complex samples. Benzodiazepines, widely used as hypnotics, muscle relaxants, sedatives, and anxiolytics, are a class of drugs that require accurate detection and monitoring. Results showed that Fe3 O4 nanoparticles could be well anchored onto the external surface of cobalt adeninate metal-organic frameworks by using amino-silane as a linkage. Their adsorption of benzodiazepines was mainly promoted by intermolecular hydrogen binding, π-π interactions and electrostatic attraction. Their potential application was evaluated by extraction of benzodiazepines in urine and wastewater samples prior to liquid chromatography with mass spectrometry. Under optimum conditions, the calibration curves were linear with a correlation coefficient of ≥0.9928 in the concentration range of 10-5000 ng/L for lorazepam and 5-5000 ng/L for estazolam, chlordiazepoxide, alprazolam, midazolam and triazolam. The limits of detection were in the range of 0.71-2.49 ng/L. The percent of extraction recoveries were 80.2-94.5% for urine and 84.1-94.4% for wastewater, respectively. Results suggested that magnetic cobalt adeninate metal-organic frameworks could potentially be a promising material for enriching benzodiazepines from urine and wastewater with high accuracy and precision.

The potential consequences of drug-drug interaction on the strategies adopted by anti-doping laboratories to report an adverse analytical finding for morphine were investigated. We evaluated in vitro the effects of 14 drugs on the principal metabolic pathways of morphine. The selected drugs are among those most commonly used by the athletes, none of them presently included in the World Anti-Doping Agency (WADA) Prohibited List. The non-prohibited drugs included 4 antifungals (fluconazole, itraconazole, ketoconazole, and miconazole), 6 benzodiazepines (alprazolam, bromazepam, clonazepam, lorazepam, lormetazepam, and triazolam), and 4 non-steroidal anti-inflammatory drugs (diclofenac, ibuprofen, ketoprofen, and nimesulide). The in vitro assays were based on the use of either human liver microsomes or uridine 5'-diphospho-glucuronosyl-transferases. Morphine and its glucuronides were determined by developed liquid chromatography-mass spectrometry procedure after dilution with an aqueous solution containing their deuterated isotopologues as internal standards. Morphine is mainly excreted as phase II metabolites: about 70% of the parent compound is found to be biotransformed by UGT2B7 to morphine-3-glucuronide (6065%) and morphine-6-glucuronide (5-10%). A reduction of the enzymatic activity of the UGT2B7 was recorded in the presence of 9 of the 14 drugs under investigation (ketoconazole, miconazole, itraconazole, diclofenac, ibuprofen, clonazepam, lorazepam, lormetazepam, and triazolam), with a consequent significant reduction of the levels of the glucuronide metabolites. This phenomenon in vivo may affect the rate of the urinary excretion of morphine with the risk of reporting "false negative" results, especially in case of results close to the decision limit value set by WADA.