Distinguishing intake of new synthetic cannabinoids ADB-PINACA and 5F-ADB-PINACA with human hepatocyte metabolites and high-resolution mass spectrometry

BACKGROUND: ADB-PINACA and its 5-fluoropentyl analog 5F-ADB-PINACA are among the most potent synthetic cannabinoids tested to date, with several severe intoxication cases. ADB-PINACA and 5F-ADBPINACA have a different legal status, depending on the country. Synthetic cannabinoid metabolites predominate in urine, making detection of specific metabolites the most reliable way for proving intake in clinical and forensic specimens. However, there are currently no data on ADB-PINACA and 5F-PINACA metabolism. The substitution of a single fluorine atom distinguishes the 2 molecules, which may share common major metabolites. For some legal applications, distinguishing between ADB-PINACA and 5F-PINACA intake is critical. For this reason, we determined the human metabolic fate of the 2 analogs. METHODS: ADB-PINACA and 5F-PINACA were incubated for 3 h with pooled cryopreserved human hepatocytes, followed by liquid chromatography- high-resolution mass spectrometry analysis. Data were processed with Compound Discoverer. RESULTS: We identified 19 and 12 major ADB-PINACA and 5F-ADB-PINACA metabolites, respectively. Major metabolic reactions included pentyl hydroxylation, hydroxylation followed by oxidation (ketone formation), and glucuronidation of ADB-PINACA, and oxidative defluorination followed by carboxylation of 5F-ADBPINACA. CONCLUSIONS: Werecommend ADB-PINACA ketopentyl and hydroxypentyl, and ADB-PINACA 5-hydroxypentyl and pentanoic acid, as optimal markers for ADBPINACA and 5F-ADB-PINACA intake, respectively. Since the 2 compounds present positional isomers as the primary metabolites, monitoring unique product ions and optimized chromatographic conditions are required for a clear distinction between ADB-PINACA and 5FADB- PINACA intake

Pharmacodynamic effects, pharmacokinetics, and metabolism of the synthetic cannabinoid AM-2201 in male rats

Novel synthetic cannabinoids are appearing in recreational drug markets worldwide. Pharmacological characterization of these new drugs is needed to inform clinicians, toxicologists, and policy makers who monitor public health. [1-(5-Fluoropentyl)-1H-indol-3-yl](1-naphthyl)methanone (AM-2201) is an abused synthetic cannabinoid that was initially created as a research tool for investigating the endocannabinoid system. Here we measured the pharmacodynamic effects of AM-2201 in rats, and simultaneously determined plasma pharmacokinetics for the parent drug and its metabolites. Male Sprague-Dawley rats were fitted with surgically implanted temperature transponders and indwelling jugular catheters under pentobarbital anesthesia. One week later, rats received subcutaneous injection of AM-2201 (0.1, 0.3, and 1.0 mg/kg) or its vehicle, and serial blood specimens were withdrawn via catheters. Core temperatures and catalepsy were measured just prior to each blood withdrawal, and plasma was assayed for drug and metabolites using liquid chromatography-tandem mass spectrometry. We found that AM-2201 produced dose-related hypothermia and catalepsy that peaked at 2 hours and lasted up to 8 hours. AM-2201 plasma concentrations rose linearly with increasing dose and ranged from 0.14 to 67.9 μg/l. Concentrations of three metabolites, AM-2201 N-(4-hydroxypentyl) (≤0.17 μg/l), naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018) N-(5-hydroxypentyl) (≤1.14 μg/l), and JWH-018 N-pentanoic acid (≤0.88 μg/l) were detectable but much lower. Peak AM-2201, JWH-018 N-(5-hydroxypentyl), and JWH-018 N-pentanoic acid concentrations occurred at 1.3, 2.4, and 6.5 hours, respectively. Concentrations of AM-2201, JWH-018 N-(5-hydroxypentyl), and JWH-018 N-pentanoic acid were negatively correlated with body temperature, but, given the low concentrations of metabolites detected, AM-2201 is likely the major contributor to pharmacodynamic effects under our experimental conditions

In vitro and in vivo human metabolism of a new synthetic cannabinoid NM-2201 (CBL-2201)

In 2014, NM-2201 (CBL-2201), a novel synthetic cannabinoid (SC), was detected by scientists at Russian and US laboratories. It has been already added to the list of scheduled drugs in Japan, Sweden and Germany. Unfortunately, no human metabolism data are currently available, which makes it challenging to confirm its intake, especially given that all SCs investigated thus far have been found to be extensively metabolized. The present study aims to recommend appropriate marker metabolites by investigating NM-2201 metabolism in human hepatocytes, and to confirm the results in authentic human urine specimens. For the metabolic stability assay, 1 µM NM-2201 was incubated in human liver microsomes (HLMs) for up to 1 h; for metabolite profiling, 10 µM of NM-2201 was incubated in human hepatocytes for 3 h. Two authentic urine specimens from NM-2201-positive cases were subjected to β-glucuronidase hydrolysis prior to analysis. The identification of metabolites in hepatocyte samples and urine specimens was achieved with high-resolution mass spectrometry via information-dependent acquisition. NM-2201 was quickly metabolized in HLMs, with an 8.0-min half-life. In human hepatocyte incubation samples, a total of 13 NM-2201 metabolites were identified, generated mainly from ester hydrolysis and further hydroxylation, oxidative defluorination and subsequent glucuronidation. M13 (5-fluoro PB-22 3-carboxyindole) was found to be the major metabolite. In the urine specimens, the parent drug NM-2201 was not detected; M13 was the predominant metabolite after β-glucuronidase hydrolysis. Therefore, based on the results of our study, we recommend M13 as a suitable urinary marker metabolite for confirming NM-2201 and/or 5F-PB-22 intake

Oxytocin by intranasal and intravenous routes reaches the cerebrospinal fluid in rhesus macaques: Determination using a novel oxytocin assay

Oxytocin (OT) is a potential treatment for multiple neuropsychiatric disorders. As OT is a peptide, delivery by the intranasal (IN) route is the preferred method in clinical studies. Although studies have shown increased cerebrospinal fluid (CSF) OT levels following IN administration, this does not unequivocably demonstrate that the peripherally administered OT is entering the CSF. For example, it has been suggested that peripheral delivery of OT could lead to central release of endogenous OT. It is also unknown whether the IN route provides for more efficient entry of the peptide into the CSF compared to the intravenous (IV) route, which requires blood-brain barrier penetration. To address these questions, we developed a sensitive and specific quantitative mass spectrometry assay that distinguishes labeled (d5-deuterated) from endogenous (d0) OT. We administered d5 OT (80 IU) to six nonhuman primates via IN and IV routes as well as IN saline as a control condition. We measured plasma and CSF concentrations of administered and endogenous OT before (t=0) and after (t=10, 20, 30, 45 and 60 min) d5 OT dosing. We demonstrate CSF penetrance of d5, exogenous OT delivered by IN and IV administration. Peripheral administration of d5 OT did not lead to increased d0, endogenous OT in the CSF. This suggests that peripheral administration of OT does not lead to central release of endogenous OT. We also did not find that IN administration offered an advantage compared to IV administration with respect to achieving greater CSF concentrations of OT

Development and Validation of the First Liquid Chromatography-Tandem Mass Spectrometry Assay for Simultaneous Quantification of Multiple Antiretrovirals in Meconium

A novel method for the simultaneous quantification of 16 antiretroviral (ARV) drugs and 4 metabolites in meconium was developed and validated. Quantification of 6 nucleoside/nucleotide reverse transcriptase inhibitors, 2 non-nucleoside reverse transcriptase inhibitors, 7 protease inhibitors, and 1 integrase inhibitor was achieved in 0.25 g of meconium. Specimen preparation included methanol homogenization and solid-phase extraction. Separate positive and negative polarity multiple reaction monitoring mode injections were required to achieve sufficient sensitivity. Linearity ranged from 10 to 75 ng/g up to 2500 ng/g for most analytes and 100–500 ng/g up to 25 000 ng/g for some; all correlation coefficients were ≥0.99. Extraction efficiencies from meconium were 32.8–119.5% with analytical recovery of 80.3–108.3% and total imprecision of 2.2–11.0% for all quantitative analytes. Two analytes with analytical recovery (70.0–138.5%) falling outside the 80–120% criteria range were considered semiquantitative. Matrix effects were −98.3–47.0% and −98.0–67.2% for analytes and internal standards, respectively. Analytes were stable (>75%) at room temperature for 24 h, 4 °C for 3 days, −20 °C for 3 freeze–thaw cycles over 3 days, and on the autosampler. Method applicability was demonstrated by analyzing meconium from HIV-uninfected infants born to HIV-positive mothers on ARV therapy. This method can be used as a tool to investigate the potential effects of in utero ARV exposure on childhood health and neurodevelopmental outcomes