Identification of Metabolite Biomarkers of the Designer Hallucinogen 25I-NBOMe in Mouse Hepatic Microsomal Preparations and Human Urine Samples Associated with Clinical Intoxication

Abstract

'NBOMe' (dimethoxyphenyl-N-[(2-methoxyphenyl)methyl]ethanamine) derivatives are a new class of designer hallucinogenic drugs widely available on the Internet. Currently, 2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25I-NBOMe) is the most popular abused derivative in the USA. There are little published data on the absorption, metabolism and elimination of 25I-NBOMe, or any of the other NBOMe derivatives. Therefore, there are no definitive metabolite biomarkers. We present the identification of fifteen 25I-NBOMe metabolites in phase I and II mouse hepatic microsomal preparations, and analysis of two human urine samples from 25I-NBOMe-intoxicated patients to test the utility of these metabolites as biomarkers of 25I-NBOMe use. The synthesis of two major urinary metabolites, 2-iodo-4-methoxy-5-[2-[(2-methoxyphenyl) methylamino]ethyl]phenol (2-O-desmethyl-5-I-NBOMe, M5) and 5-iodo-4-methoxy-2-[2-[(2-methoxyphenyl)methylamino]ethyl]phenol (5-O-desmethyl-2-I-NBOMe), is also presented. Seven phase II glucuronidated metabolites of the O-desmethyl or the hydroxylated phase I metabolites were identified. One human urine sample contained 25I-NBOMe as well as all 15 metabolites identified in mouse hepatic microsomal preparations. Another human urine sample contained no parent 25I-NBOMe, but was found to contain three O-desmethyl metabolites. We recommend β-glucuronidase enzymatic hydrolysis of urine prior to 25I-NBOMe screening and the use of M5 as the primary biomarker in drug testing.

Characterization of the hepatic cytochrome P450 enzymes involved in the metabolism of 25I‐NBOMe and 25I‐NBOH

“…The metabolism of 25I‐NBOMe has recently been considered in several in vivo studies . Caspar et al .…”

“…Poklis et al identified one main monohydroxylated metabolite of 25I‐NBOMe in the two cases included in their study, but lacking primary reference standards, it was not possible to determine the exact location of the hydroxylation. They were able to determine that the hydroxylation was located at the methoxybenzyl moiety based on the fragmentation pattern . Our results support their identification, and using a reference standard exact location of the main monohydroxylated metabolite was determined.…”

“…However, we also showed that 25I‐NBOH is liable to direct glucuronidation, which decreases the influence of CYP genetic polymorphisms or DDI. Also, all oxidative metabolites of 25I‐NBOMe identified here have been reported as conjugated metabolites present in urine and so are subject to a phase II metabolism step …”

“…Based on in vitro clearance data obtained using human liver microsomes (HLM), 10 investigated NBOMe derivatives had higher intrinsic clearance (CL int ) compared to 11 phenethylamines . Based on case reports, the major urine metabolites of 25I‐NBOMe appeared to be the glucuronic acid conjugates of the three possible O‐ demethylated metabolites (2‐ O ‐desmethyl‐25I‐NBOMe, 5‐ O ‐desmethyl‐25I‐NBOMe, and 25I‐NBOH) together with 2C‐I and an undetermined monohydroxylated metabolite of 25I‐NBOMe . A cytochrome P450 enzyme (CYP) screen demonstrated involvement of CYP1A2, 2C9, 2C19, and CYP3A4 in the oxidative metabolism of 25I‐NBOMe, but the relative importance of each enzyme was not further investigated …”

Recommendations for specimen collection for NBOMe analysis in clinical toxicology

“…However, it should be noted that in one case we did not detect 25I-NBOMe in urine from an intoxicated patient collected 13 hours post-admission. Four O-desmethyl metabolites were the only 25I-NBOMe analogs detected (5). With the exception of 2CT-NBOMe, 2-[2,5 dimethoxy-4-(methylthio) phenyl]-N-(2-methoxybenzyl)ethanamine, which is unstable in urine at room temperature, we have found NBOMe derivatives are stable in urine at room temperature for 3 days and frozen for at least one month (6).…”