In silico, in vitro, and in vivo human metabolism of acetazolamide, a carbonic anhydrase inhibitor and common "diuretic and masking agent" in doping.

Acetazolamide (ACZ) is a carbonic anhydrase inhibitor prescribed for the treatment of various pathologies. It is also used in doping and is prohibited in and out of sportive competitions. ACZ was reported not to undergo metabolization. However, the detection of ACZ metabolites may be critical for documenting ACZ use. We aimed to further investigate ACZ metabolic fate in humans. ACZ putative metabolites were generated in silico to assist in metabolite identification. ACZ was incubated with primary human hepatocytes to identify in vitro metabolites (10 µmol/l ACZ and 106 cells/ml), and urine and plasma samples from patients receiving a single 5.0 mg/kg BW PO ACZ dose were analyzed to confirm the results in vivo. Analyses were performed with reversed-phase liquid chromatography and hydrophilic interaction chromatography coupled with high-resolution tandem mass spectrometry (RPLC-HRMS/MS and HILIC-HRMS/MS, respectively). Data were screened with a software-assisted targeted/untargeted workflow. ACZ was quantified in urine samples with creatinine normalization. We identified two metabolites in hepatocyte incubations and three additional metabolites in urine and plasma. Major transformations included cysteine conjugation, glucuronidation, and N-acetylation. All metabolites were detected in plasma, 1.5 h after intake. Major metabolites were detected in urine from 0.25 to 24 h (last collection) after intake. As opposed to the literature, ACZ does undergo metabolization in humans. We propose ACZ, ACZ-Cys, and N-acetyl-ACZ in urine, and ACZ and N-acetyl-ACZ in plasma as specific biomarkers of ACZ intake in doping.

ß'-Phenylfentanyl Metabolism in Primary Human Hepatocyte Incubations: Identification of Potential Biomarkers of Exposure in Clinical and Forensic Toxicology.

From 2014 onwards, illicit fentanyl and analogues have caused numerous intoxications and fatalities worldwide, impacting the demographics of opioid-related overdoses. The identification of cases involving fentanyl analogues is crucial in clinical and forensic settings to treat patients, elucidate intoxications, address drug use disorders and tackle drug trends. However, in analytical toxicology, the concentration of fentanyl analogues in biological matrices is low, making their detection challenging. Therefore, the identification of specific metabolite biomarkers is often required to document consumption. ß'-Phenylfentanyl (N-phenyl-N-[1-(2-phenylethyl)-4-piperidinyl]-benzenepropanamide) is a fentanyl analogue that was first detected in Sweden in 2017 and has recently reemerged onto the American illicit drug market. There is little data available on ß'-phenylfentanyl effects and toxicokinetics and its metabolism is yet to be studied. We aimed to investigate ß'-phenylfentanyl human metabolism to identify potential biomarkers of use. To assist in ß'-phenylfentanyl metabolite identification, a list of putative reactions was generated using in silico predictions with GLORYx freeware. ß'-phenylfentanyl was incubated with cryopreserved 10-donor-pooled human hepatocytes, analyses were performed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS-MS) and data were processed using a partially automated targeted/untargeted approach with Compound Discoverer. We identified 26 metabolites produced by N-dealkylation, oxidation, hydroxylation, O-glucuronidation, O-methylation and combinations thereof. We suggest ß'-phenylnorfentanyl (N-phenyl-N-4-piperidinyl-benzenepropanamide) and further metabolites 1-oxo-N-phenyl-N-4-piperidinyl-benzenepropanamide and 1-hydroxy-N-phenyl-N-4-piperidinyl-benzenepropanamide as major biomarkers of ß'-phenylfentanyl use. In silico predictions were mostly wrong, and ß'-phenylfentanyl metabolic fate substantially differed from that of a closely related analogue incubated in the same conditions, highlighting the value of the experimental assessment of new psychoactive substance human metabolism. In vivo data are necessary to confirm the present results. However, the present results may be necessary to help analytical toxicologists identify ß'-phenylfentanyl-positive cases to provide authentic samples.

Melatonin Treatment in Patients with Burning Mouth Syndrome: A Triple-Blind, Placebo-Controlled, Crossover Randomized Clinical Trial.

AIMS: To evaluate the efficacy of melatonin compared to placebo in reducing pain associated with burning mouth syndrome (BMS), as well as side effects of treatment and effects on sleep quality, anxiety, and serum and salivary melatonin levels. METHODS: In this triple-blind, randomized clinical trial, 20 BMS patients (mean age ± standard deviation: 64.4 ± 11.5 years; range: 35 to 82 years) were enrolled to receive melatonin (12 mg/day) or placebo for 8 weeks in a crossover design. After treatment, changes in pain from baseline were ascertained by patient self-assessment with a verbal category scale and a visual analog scale. Secondary outcomes included evaluation of changes in sleep quality and anxiety. Data were subjected to analysis of variance (ANOVA), Fisher exact test, paired t test, Wilcoxon signed rank test, or chi-square test, as appropriate. RESULTS: Melatonin was not superior to placebo in reducing pain. Melatonin significantly improved anxiety scores, though without strong clinical relevance. Independent of treatment, sleep quality did not significantly change during the trial, although melatonin slightly increased the number of hours slept. After active treatment, the mean ± standard error serum melatonin level peaked at 1,520 ± 646 pg/mL. A generally safe pharmacologic profile of melatonin was observed, and the placebo and melatonin treatments resulted in similar adverse effects. CONCLUSION: Within the limitations of this study, melatonin did not exhibit higher efficacy than placebo in relieving pain in BMS patients.