In vitro metabolic fate of the synthetic cannabinoid receptor agonists (quinolin-8-yl 4-methyl-3-(morpholine-4-sulfonyl)benzoate [QMMSB]) and (quinolin-8-yl 4-methyl-3-((propan-2-yl)sulfamoyl)benzoate [QMiPSB]) including isozyme mapping and carboxylesterases activity testing.

The synthetic cannabinoid receptor agonists (SCRAs) (quinolin-8-yl 4-methyl-3-(morpholine-4-sulfonyl)benzoate [QMMSB]) and (quinolin-8-yl 4-methyl-3-((propan-2-yl)sulfamoyl)benzoate [QMiPSB], also known as SGT-46) are based on the structure of quinolin-8-yl 4-methyl-3-(piperidine-1-sulfonyl)benzoate (QMPSB) that has been identified on seized plant material in 2011. In clinical toxicology, knowledge of the metabolic fate is important for their identification in biosamples. Therefore, the aim of this study was the identification of in vitro Phase I and II metabolites of QMMSB and QMiPSB in pooled human liver S9 fraction (pHLS9) incubations for use as screening targets. In addition, the involvement of human monooxygenases and human carboxylesterases (hCES) was examined. Analyses were performed by liquid chromatography coupled with high-resolution tandem mass spectrometry. Ester hydrolysis was found to be an important step in the Phase I metabolism of both SCRAs, with the carboxylic acid product being found only in negative ionization mode. Monohydroxy and N-dealkyl metabolites of the ester hydrolysis products were detected as well as glucuronides. CYP2C8, CYP2C9, CYP3A4, and CYP3A5 were involved in hydroxylation. Whereas enzymatic ester hydrolysis of QMiPSB was mainly catalyzed by hCES1 isoforms, nonenzymatic ester hydrolysis was also observed. The results suggest that ester hydrolysis products of QMMSB and QMiPSB and their glucuronides are suitable targets for toxicological screenings. The additional use of the negative ionization mode is recommended to increase detectability of analytes. Different cytochrome P450 (CYP) isozymes were involved in the metabolism; thus, the probability of drug-drug interactions due to CYP inhibition can be assessed as low.

In Vitro Metabolic Fate of the Synthetic Cannabinoid Receptor Agonists 2F-QMPSB and SGT-233 Including Isozyme Mapping and Carboxylesterases Activity Testing.

Quinolin-8-yl 3-(4,4-difluoropiperidine-1-sulfonyl)-4-methylbenzoate (2F-QMPSB) and 3-(4,4-difluoropiperidine-1-sulfonyl)-4-methyl-N-(2-phenylpropan-2-yl)benzamide (SGT-233) belong to a new group of synthetic cannabinoid receptor agonists containing a sulfamoyl benzoate or sulfamoyl benzamide core structure. 2F-QMPSB was identified in herbal material seized in Europe in 2018. The aims of this study were the identification of in vitro Phase I and II metabolites of 2F-QMPSB and SGT-233 to find analytical targets for toxicological screenings. Furthermore, the contribution of different monooxygenases and human carboxylesterases to Phase I metabolism was investigated. Liquid chromatography coupled to high-resolution tandem mass spectrometry was used for analysis. Ester hydrolysis was found to be an important step in the metabolism of 2F-QMPSB, which was catalyzed mainly by human carboxylesterases (hCES)1 isoforms. Additionally, nonenzymatic ester hydrolysis was observed in case of 2F-QMPSB. Notably, the carboxylic acid product derived from ester hydrolysis and metabolites thereof were only detectable in negative ionization mode. In case of SGT-233, mono- and dihydroxy metabolites were identified, as well as glucuronides. The cytochrome P450 (CYP) isozymes CYP2C8, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 were found to be involved in the hydroxylation of both compounds. The results of these in vitro experiments suggest that the ester hydrolysis products of 2F-QMPSB and their glucuronides are suitable targets for toxicological screenings. In the case of SGT-233, the mono- and dihydroxy metabolites were identified as suitable screening targets. The involvement of various CYP isoforms in the metabolism of both substances reduces the likelihood of drug-drug interactions due to CYP inhibition.

In Vitro Metabolic Fate of the Synthetic Cannabinoid Receptor Agonists QMPSB and QMPCB (SGT-11) Including Isozyme Mapping and Esterase Activity.

Quinolin-8-yl 4-methyl-3-(piperidine-1-sulfonyl)benzoate (QMPSB) and quinolin-8-yl 4-methyl-3-(piperidine-1-carbonyl)benzoate (QMPCB, SGT-11) are synthetic cannabinoid receptor agonists (SCRAs). Knowing their metabolic fate is crucial for the identification of toxicological screening targets and to predict possible drug interactions. The presented study aimed to identify the in vitro phase I/II metabolites of QMPSB and QMPCB and to study the contribution of different monooxygenases and human carboxylesterases by using pooled human liver S9 fraction (pHLS9), recombinant human monooxygenases, three recombinant human carboxylesterases, and pooled human liver microsomes. Analyses were carried out by liquid chromatography high-resolution tandem mass spectrometry. QMPSB and QMPCB showed ester hydrolysis, and hydroxy and carboxylic acid products were detected in both cases. Mono/dihydroxy metabolites were formed, as were corresponding glucuronides and sulfates. Most of the metabolites could be detected in positive ionization mode with the exception of some QMPSB metabolites, which could only be found in negative mode. Monooxygenase activity screening revealed that CYP2B6/CYP2C8/CYP2C9/CYP2C19/CYP3A4/CYP3A5 were involved in hydroxylations. Esterase screening showed the involvement of all investigated isoforms. Additionally, extensive non-enzymatic ester hydrolysis was observed. Considering the results of the in vitro experiments, inclusion of the ester hydrolysis products and their glucuronides and monohydroxy metabolites into toxicological screening procedures is recommended.

Flubromazolam-Derived Designer Benzodiazepines: Toxicokinetics and Analytical Toxicology of Clobromazolam and Bromazolam.

Flubromazolam is widely known as highly potent designer benzodiazepine (DBZD). Recently, the two flubromazolam-derived new psychoactive substances (NPS) clobromazolam and bromazolam appeared on the drugs of abuse market. Since no information concerning their toxicokinetics in humans is available, the aims of the current study were to elucidate their metabolic profile and to identify the isozymes involved in their phase I and phase II metabolism. In vitro incubations with pooled human liver S9 fraction were performed and analyzed by liquid chromatography coupled to orbitrap-based high-resolution tandem mass spectrometry (LC-HRMS-MS). Biosamples after the ingestion of bromazolam allowed the identification of metabolites in human plasma and urine as well as the determination of bromazolam plasma concentrations by LC-HRMS-MS using the standard addition method. In total, eight clobromazolam metabolites were identified in vitro as well as eight bromazolam metabolites in vitro and in vivo. Predominant metabolic steps were hydroxylation, glucuronidation and combinations thereof. Alpha-hydroxy bromazolam glucuronide and bromazolam N-glucuronide are recommended as screening targets in urine. Bromazolam and its alpha-hydroxy metabolite are recommended if conjugate cleavage is part of the sample preparation procedure. The bromazolam plasma concentrations were determined to be 6 and 29 µg/L, respectively. Several cytochrome P450 (CYP) and uridine 5'-diphospho-glucuronosyltransferase (UGT) isozymes were shown to catalyze their metabolic transformations. CYP3A4 was involved in the formation of all phase I metabolites of both NPS, while UGT1A4 and UGT2B10 catalyzed their N-glucuronidation. Several UGT isoforms catalyzed the glucuronidation of the hydroxy metabolites. In conclusion, the determined bromazolam plasma concentrations in the low micrograms per liter range underlined the need for sensitive analytical methods and the importance of suitable urine screening procedures including DBZD metabolites as targets. Such an analytical strategy should be also applicable for clobromazolam.

Toxicokinetic Studies and Analytical Toxicology of the New Synthetic Opioids Cyclopentanoyl-Fentanyl and Tetrahydrofuranoyl-Fentanyl.

The growing number of new synthetic opioids (NSO) on the new psychoactive substances (NPS) market bears new challenges in toxicology. As their toxicodynamics and particularly their toxicokinetics are usually unknown, impact on human health is not yet fully understood. Detection of the 2 NSO cyclopentanoyl-fentanyl (CP-F) and tetrahydrofuranoyl-fentanyl (THF-F) was first reported in 2016. Both were involved in several fatal intoxication cases, but no detailed information about their toxicological characteristics is available so far. The main purpose of this study was therefore to investigate the in vitro toxicokinetics and in vivo analytical toxicology of CP-F and THF-F by means of liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). These studies included metabolic stability, phase I and II metabolism, isozyme mapping, plasma protein binding and detectability in LC-HRMS/MS standard urine screening approaches (SUSA) using rat urine samples. In total, 12 phase I metabolites of CP-F and 13 of THF-F were identified, among them 9 metabolites described for the first time. Overall, N-dealkylations, hydroxylations and dihydroxylations were the main metabolic reactions. The cytochrome P450 (CYP) isozymes mainly involved were CYP2D6 and CYP3A4, leading to elevated drug levels and intoxications in CYP2D6 poor metabolizers. CP-F showed a high plasma protein binding of 99%, which may increase the risk of toxicity by simultaneous intake of other highly bound drugs. Detectability studies showed that neither the parent compounds nor their metabolites were detectable in rat urine using LC-HRMS/MS SUSA. However, a more sophisticated analytical strategy was successfully applied and should be used for analytical confirmation of an intake of CP-F and/or THF-F.