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Background/aims: The aim of this single-center, double-blind study was to investigate the effect of a 4-week once daily administration of 200 mg almorexant on tear film break-up time, spermatogenesis, hormone levels, and pancreatic elastase in stool in healthy male subjects., Methods: Almorexant 200 mg or matching placebo was administered in the evening for 4 weeks once daily to 56 healthy male subjects. Changes in ophthalmological variables, sperm composition, hormone levels, and pancreatic elastase levels in stool were evaluated periodically up to 8 weeks after discontinuation of drug administration. Blood samples for pharmacokinetic measurements were taken after 4 weeks to confirm compliance to study drug intake., Results: The results of this study revealed no treatment effects of almorexant, neither on tear film break-up time nor on other ophthalmological variables investigated during this study. Furthermore, spermatogenesis, hormones of the hypothalamic-pituitary-adrenal and -gonadal axes, and endocrine pancreatic secretion were shown to be not affected by a 4-week once daily administration of almorexant., Conclusion: Almorexant was well tolerated and had no effect on the spectrum of pharmacodynamic variables assessed. Ophthalmology and testicular findings detected in preclinical studies were not observed in this clinical study. Therefore, these preclinical findings appear not to be relevant for humans and do not prevent from conducting larger clinical trials with either healthy subjects or patients., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

An analytical method for the detection of 40 benzodiazepines, (±)-zopiclone, zaleplon and zolpidem in blood and urine by solid-phase extraction liquid chromatography-tandem mass spectrometry was developed and validated. Twenty-nine of 43 analytes were quantified in 0.5 mL whole blood for investigating postmortem, drug-facilitated sexual assault (DFSA) and driving under the influence of drugs cases (DUID). The four different dynamic ranges of the seven-point, linear, 1/x weighted calibration curves with lower limits of quantification of 2, 5, 10 and 20 μg/L across the analytes encompassed the majority of our casework encountered in postmortem, DFSA and DUID samples. Reference materials were available for all analytes except α-hydroxyflualprazolam, a hydroxylated metabolite of flualprazolam. The fragmentation of α-hydroxyflualprazolam was predicted from the fragmentation pattern of α-hydroxyalprazolam, and the appropriate transitions were added to the method to enable monitoring for this analyte. Urine samples were hydrolyzed at 55°C for 30 min with a genetically modified β-glucuronidase enzyme, which resulted in >95% efficiency measured by oxazepam glucuronide. Extensive sample preparation included combining osmotic lysing and protein precipitation with methanol/acetonitrile mixture followed by freezing and centrifugation resulted in exceptionally high signal-to-noise ratios. Bias and between-and within-day imprecision for quality controls (QCs) were all within ±15%, except for clonazolam and etizolam that were within ±20%. All 29 of the 43 analytes tested for QC performance met quantitative reporting criteria within the dynamic ranges of the calibration curves, and 14 analytes, present only in the calibrator solution, were qualitatively reported. Twenty-five analytes met all quantitative reporting criteria including dilution integrity. The ability to analyze quantitative blood and qualitative urine samples in the same batch is one of the most useful elements of this procedure. This sensitive, specific and robust analytical method was routinely employed in the analysis of >300 samples in our laboratory over the last 6 months., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Suvorexant (Belsomra®) is a novel dual orexin receptor antagonist used for the treatment of insomnia. The prevalence of suvorexant in forensic samples is relatively unknown, which demonstrates the need for robust analytical assays for the detection of this sedative hypnotic in forensic toxicology laboratories. In this study, suvorexant was isolated from whole blood using a simple acidic/neutral liquid-liquid extraction followed by analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). Matrix effects were evaluated qualitatively and quantitatively using various extraction solvents, proprietary lipid clean-up devices and source conditions. The method was validated in terms of limit of detection, limit of quantitation, precision, bias, calibration model, carryover, matrix effects and drug interferences. Electrospray is a competitive ionization process whereby compounds in the droplet compete for a limited number of charged sites at the surface. As such, it is capacity-limited, and LC-MS-based techniques must be carefully evaluated to ensure that matrix effects or coeluting drugs do not impact quantitative assay performance. In this report, we describe efforts to ameliorate such effects in the absence of an isotopically labeled internal standard. Matrix effects are highly variable and heavily dependent on the physico-chemical properties of the substance. Although there is no universal solution to their resolution, conditions at the electrospray interface can mitigate these issues. Using this approach, the LC-MS/MS assay was fully validated and limits of detection and quantitation of 0.1 and 0.5 ng/mL suvorexant were achieved in blood., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The orexin system regulates sleep and arousal and is targeted by ACT-541468, a new dual orexin receptor antagonist (DORA). Healthy male subjects received a single oral dose of 5-200 mg to assess safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), mass balance, metabolism, and absolute bioavailability utilizing a 14 C-labeled, orally and intravenously (i.v.) administered microtracer. The drug was safe and well tolerated; the PK profile was characterized by quick absorption and elimination, with median time to reach maximum concentration (t max ) of 0.8-2.8 h and geometric mean terminal half-life (t 1/2 ) of 5.9-8.8 h. Clear dose-related effects on the central nervous system were observed at ≥25 mg, indicating a suitable PK-PD profile for a sleep-promoting drug, allowing for rapid onset and duration of action limited to the intended use. This comprehensive first-in-human study created a wealth of data, while saving resources in drug development., (© 2018 American Society for Clinical Pharmacology and Therapeutics.)

Suvorexant is a dual orexin receptor antagonist, recently approved by USFDA for the treatment of insomnia. It is drug-of-abuse and listed in Schedule IV drug of the Controlled Substances Act. In this study, a simple and highly sensitive UPLC-MS/MS assay was developed and fully validated for the determination of suvorexant in rat plasma. Both suvorexant and internal standard (rivaroxaban; IS) were separated on Aquity BEHTM C18 column after the extraction form plasma using diethyl ether as extracting agent. An isocratic mobile phase, consisting of acetonitrile and 10 mM ammonium acetate in composition ratio of 85:15 was eluted at flow rate of 0.3 mL/min. Both suvorexant and IS were eluted within one min with total run time of 1.5 min only. The ionization was performed on electrospray ionization interface in positive mode by multiple reaction monitoring. Precursor to product ion transition of 451.12 > 104.01 for qualifier and 451.12 > 186.04 for quantifier were used for suvorexant whereas 436.10 > 144.93 for IS, respectively. The calibration curves in plasma were linear in the concentration range of 0.33-200 ng/mL (r2 ≥ 0.995) having limit of detection and limit of quantification of 0.10 and 0.33 ng/mL, respectively. All the validation parameters results were found to be within the acceptable limits according to the "Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines" and can be implemented for forensic analysis., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

A method, using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS), was developed for the determination of suvorexant (MK-4305, Belsomra(®)), a selective dual orexin receptor antagonist for the treatment insomnia, in human plasma over the concentration range of 1-1000ng/mL. Stable isotope labeled (13)C(2)H3-suvorexant was used as an internal standard. The sample preparation procedure utilized liquid-liquid extraction, in the 96-well format, of a 100μL plasma sample with methyl t-butyl ether. The compounds were chromatographed under isocratic conditions on a Waters dC18 (50×2.1mm, 3μm) column with a mobile phase consisting of 30/70 (v/v %) 10mM ammonium formate, pH3/acetonitrile at a flow rate of 0.3mL/min. Multiple reaction monitoring of the precursor-to-product ion pairs for suvorexant (m/z 451→186) and (13)C(2)H3-suvorexant (m/z 455→190) on an Applied Biosystems API 4000 tandem mass spectrometer was used for quantitation. Intraday assay precision, assessed in six different lots of control plasma, was within 10% CV at all concentrations, while assay accuracy ranged from 95.6 to 105.0% of nominal. Quality control (QC) samples in plasma were stored at -20°C. Initial within day analysis of QCs after one freeze-thaw cycle showed accuracy within 9.5% of nominal with precision (CV) of 6.7% or less. The plasma QC samples were demonstrated to be stable for up to 25 months at -20°C. The method described has been used to support clinical studies during Phase I through III of clinical development., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Suvorexant is an orexin receptor antagonist for treating insomnia. The maximum approved dose in the United States and Japan is 20 mg. We evaluated suvorexant effects on respiration during sleep in a randomized, double-blind, 3-period crossover study of healthy adult men (n = 8) and women (n = 4) ≤ 50 years old who received single-dose suvorexant 40 mg, 150 mg, and placebo. Respiration during sleep was measured by oxygen saturation (SpO2 , primary end point) and the Apnea Hypopnea Index (AHI). The study was powered to detect a reduction greater than 5% in SpO2 . There was no effect of suvorexant on mean SpO2 during sleep. The mean (90%CI) treatment differences versus placebo were -0.3 (-1.2-0.6) for 40 mg and 0.0 (-0.9-0.9) for 150 mg. There were no dose-related trends in individual SpO2 values. Mean SpO2 was >96% for all treatments during total sleep time and during both non-REM and REM sleep. There was no effect of either suvorexant dose on AHI. The mean (90%CI) treatment differences versus placebo were 0.8 (-0.7-2.3) for 40 mg and -0.2 (-1.7-1.3) for 150 mg. Suvorexant was generally well tolerated; there were no serious adverse experiences or discontinuations. These data from healthy subjects suggest that suvorexant lacks clinically important respiratory effects during sleep at doses greater than the maximum recommended dose for treating insomnia., (© 2015, The American College of Clinical Pharmacology.)