The stimulant designer drug mephedrone is a derivative of cathinone - a monoamine alkaloid found in khat - and its effect resembles that of 3,4-Methylenedioxymethamphetamine (MDMA). Abuse of mephedrone has been documented since 2007; it was originally a 'legal high' drug, but it has now been banned in most Western countries. Using cDNA-expressed CYP enzymes and human liver microsomal preparations, we found that cytochrome P450 2D6 (CYP2D6) was the main responsible enzyme for the in vitro Phase I metabolism of mephedrone, with some minor contribution from other NAPDH-dependent enzymes. Hydroxytolyl-mephedrone and nor-mephedrone were formed in vitro, and the former was purified and identified by nuclear magnetic resonance (NMR). In four forensic traffic cases where mephedrone was detected, we identified hydroxytolyl-mephedrone and nor-mephedrone again; as well as 4-carboxy-dihydro-mephedrone, which has been previously described; and two new metabolites: dihydro-mephedrone and 4-carboxy-mephedrone. Fragmentation patterns for all detected compounds were determined by a UPLC-QTOF/MS(E) system, and a fragmentation pathway via a conjugated indole structure was proposed for most of the metabolites. Blood concentrations in the forensic traffic cases ranged from 1 to 51 µg/kg for mephedrone, and from not detected to 9 µg/kg for hydroxytolyl-mephedrone. In one case, urine concentrations were also determined to be 700 µg/kg for mephedrone and 190 µg/kg for hydroxytolyl-mephedrone. All compounds were detected or quantified with an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) system and an ultra performance liquid chromatography-time of flight/mass spectrometry (UPLC-TOF/MS) system.
New types of synthetic cannabinoid designer drugs are constantly introduced to the illicit drug market to circumvent legislation. Recently, N-(1-Adamantyl)-1-(5-fluoropentyl)-1H-indazole-3-carboxamide (5F-AKB-48), also known as 5F-APINACA, was identified as an adulterant in herbal products. This compound deviates from earlier JHW-type synthetic cannabinoids by having an indazole ring connected to an adamantyl group via a carboxamide linkage. Synthetic cannabinoids are completely metabolized, and identification of the metabolites is thus crucial when using urine as the sample matrix. Using an authentic urine sample and high-resolution accurate-mass Fourier transform Orbitrap mass spectrometry, we identified 16 phase-I metabolites of 5F-AKB-48. The modifications included mono-, di-, and trihydroxylation on the adamantyl ring alone or in combination with hydroxylation on the N-fluoropentylindazole moiety, dealkylation of the N-fluoropentyl side chain, and oxidative loss of fluorine as well as combinations thereof. The results were compared to human liver microsomal (HLM) incubations, which predominantly showed time-dependent formation of mono-, di-, and trihydroxylated metabolites having the hydroxyl groups on the adamantyl ring. The results presented here may be used to select metabolites specific of 5F-AKB-48 for use in clinical and forensic screening.
A broad forensic screening method for 256 analytes in whole blood based on a fully automated SPE robotic extraction and ultra-high-performance liquid chromatography (UHPLC) with TOF-MS with data-independent acquisition has been developed. The limit of identification was evaluated for all 256 compounds and 95 of these compounds were validated with regard to matrix effects, extraction recovery, and process efficiency. The limit of identification ranged from 0.001 to 0.1 mg/kg, and the process efficiency exceeded 50% for 73 of the 95 analytes. As an example of application, 1335 forensic traffic cases were analyzed with the presented screening method. Of these, 992 cases (74%) were positive for one or more traffic-relevant drugs above the Danish legal limits. Commonly abused drugs such as amphetamine, cocaine, and frequent types of benzodiazepines were the major findings. Nineteen less frequently encountered drugs were detected e.g. buprenorphine, butylone, cathine, fentanyl, lysergic acid diethylamide, m-chlorophenylpiperazine, 3,4-methylenedioxypyrovalerone, mephedrone, 4-methylamphetamine, p-fluoroamphetamine, and p-methoxy-N-methylamphetamine. In conclusion, using UHPLC-TOF-MS screening with data-independent acquisition resulted in the detection of common drugs of abuse as well as new designer drugs and more rarely occurring drugs. Thus, TOF-MS screening of blood samples constitutes a practical way for screening traffic cases, with the exception of δ-9-tetrahydrocannabinol, which should be handled in a separate method.
Abuse of the stimulant designer drug methylone (methylenedioxymethcathinone) has been documented in most parts of the world. As with many of the new designer drugs that continuously appear in the illicit drug market, little is known about the pharmacokinetics of methylone. Using in vitro studies, CYP2D6 was determined to be the primary enzyme that metabolizes methylone, with minor contributions from CYP1A2, CYP2B6, and CYP2C19. The major metabolite was identified as dihydroxymethcathinone, and the minor metabolites were N-hydroxy-methylone, nor-methylone, and dihydromethylone. Measuring the formation of the major metabolite, biphasic Michaelis-Menten kinetic parameters were determined: V max,1 = 0.046 6 0.005 (S.E.) nmol/min/mg protein, K m,1 = 19.0 6 4.2 mM, V max,2 = 0.22 6 0.04 nmol/min/mg protein, and K m,2 = 1953 6 761 mM; the low-capacity and high-affinity contribution was assigned to the activity of CYP2D6. Additionally, a time-dependent loss of CYP2D6 activity was observed when the enzyme was preincubated with methylone, reaching a maximum rate of inactivation at high methylone concentrations, indicating that methylone is a mechanism-based inhibitor of CYP2D6. The inactivation parameters were determined to be K I = 15.1 6 3.4 (S.E.) mM and k inact = 0.075 6 0.005 minute 21.
5-Hydroxytryptamine 3 (5-HT 3) receptors are ligand-gated ion channels that mediate neurotransmission by serotonin in the central nervous system. Pharmacological inhibition of 5-HT 3 receptor activity has therapeutic potential in several psychiatric diseases, including depression and anxiety. The recently approved multimodal antidepressant vortioxetine has potent inhibitory activity at 5-HT 3 receptors. Vortioxetine has an inhibitory mechanism that differs from classic 5-HT 3 receptor competitive antagonists despite being believed to bind in the same binding site. Specifically, vortioxetine shows partial agonist activity followed by persistent and insurmountable inhibition. We have investigated the binding mode of vortioxetine at the human 5-HT 3A receptor through computational and in vitro experiments to provide insight into the molecular mechanisms behind the unique pharmacological profile of the drug. We find that vortioxetine binds in a manner different from currently known 5-HT 3A orthosteric ligands. Specifically, while the binding pattern of vortioxetine mimics some aspects of both the setron class of competitive antagonists and 5-hydroxytryptamine (5-HT) with regards to interactions with residues of the aromatic box motif in the orthosteric binding site, vortioxetine also forms interactions with residues not previously described to be important for the binding of either setrons or 5-HT such as Val202 on Loop F. Our results expand the framework for understanding how orthosteric ligands drive 5-HT 3 receptor function, which is of importance for the potential future development of novel classes of 5-HT 3 receptor antagonists.
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