<i>In Vitro</i> Phase I Metabolism of the Recently Emerged Synthetic MDMB-4en-PINACA and Its Detection in Human Urine Samples

Öztürk, Yeter Erol; Yeter, Oya

doi:10.1093/jat/bkaa017

Cited by 27 publications

(18 citation statements)

References 5 publications

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“…Ester hydrolysis plus dehydrogenation metabolite (B20) was the second most abundant major metabolite in the HLM model, similar to the in-vivo findings where it is the second most prevalent metabolite detected in 18/20 urine samples (Table 2). Ester hydrolysis with dehydrogenation formed invivo in this study was also reported among other indazole carboxamide type SCBs with tert-leucine methyl ester moieties such as 5F-MDMB-PINACA [15] and MDMB-4en-PINACA [39,40]. This biotransformation was reported by both Krotulski et al [5].…”

Section: Discussionsupporting

confidence: 84%

Monitoring metabolism of synthetic cannabinoid 4F-MDMB-BINACA via high-resolution mass spectrometry assessed in cultured hepatoma cell line, fungus, liver microsomes and confirmed using urine samples

et al. 2020

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Purpose A tert-leucinate derivative synthetic cannabinoid, methyl (2S)-2-([1-(4-fluorobutyl)-1H-indazole-3-carbonyl]amino)-3,3-dimethylbutanoate (4F-MDMB-BINACA, 4F-MDMB-BUTINACA or 4F-ADB) is known to adversely impact health. This study aimed to evaluate the suitability of three different modes of monitoring metabolism: HepG2 liver cells, fungus Cunninghamella elegans (C. elegans) and pooled human liver microsomes (HLM) for comparison with human in-vivo metabolism in identifying suitable urinary marker(s) for 4F-MDMB-BINACA intake. Methods Tentative structure elucidation of in-vitro metabolites was performed on HepG2, C. elegans and HLM using liquid chromatography–tandem mass spectrometry and high-resolution mass spectrometry analysis. In-vivo metabolites obtained from twenty authentic human urine samples were analysed using liquid chromatography–Orbitrap mass spectrometry. Results Incubation with HepG2, C. elegans and HLM yielded nine, twenty-three and seventeen metabolites of 4F-MDMB-BINACA, respectively, formed via ester hydrolysis, hydroxylation, carboxylation, dehydrogenation, oxidative defluorination, carbonylation or reaction combinations. Phase II metabolites of glucosidation and sulfation were also exclusively identified using C. elegans model. Eight in-vivo metabolites tentatively identified were mainly products of ester hydrolysis with or without additional dehydrogenation, N-dealkylation, monohydroxylation and oxidative defluorination with further oxidation to butanoic acid. Metabolites with intact terminal methyl ester moiety, i.e., oxidative defluorination with further oxidation to butanoic acid, were also tentatively identified. Conclusions The in-vitro models presented proved useful in the exhaustive metabolism studies. Despite limitations, HepG2 identified the major 4F-MDMB-BINACA ester hydrolysis metabolite, and C. elegans demonstrated the capacity to produce a wide variety of metabolites. Both C. elegans and HLM produced all the in-vivo metabolites. Ester hydrolysis and ester hydrolysis plus dehydrogenation 4F-MDMB-BINACA metabolites were recommended as urinary markers for 4F-MDMB-BINACA intake.

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Section: Discussionsupporting

confidence: 84%

Monitoring metabolism of synthetic cannabinoid 4F-MDMB-BINACA via high-resolution mass spectrometry assessed in cultured hepatoma cell line, fungus, liver microsomes and confirmed using urine samples

et al. 2020

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“…Based on this finding, its metabolite, MDMB‐4en‐PINACA 3,3‐dimethylbutanoic acid (Figure 2), was prioritized for addition to the library database for identification in cases where urine samples were submitted. Metabolic profiles for methyl ester synthetic cannabinoids, including MDMB‐4en‐PINACA, have been well documented, and this species was reported as a primary metabolite and reasonably biomarker for monitoring use of MDMB‐4en‐PINACA 16,17 …”

Section: Resultsmentioning

confidence: 99%

“…Norman et al reported the detection of MDMB‐4en‐PINACA on drug‐infused papers from Scottish prisons in June 2019 15 . Ozturk et al reported the detection of MDMB‐4en‐PINACA in an herbal product from Turkey in April 2019 16 . In this study, toxicology testing of urine samples collected from suspected drug‐using individuals was monitored for the presence of MDMB‐4en‐PINACA metabolites; researchers identified 56 positive specimens (2.6% positivity).…”

Section: Introductionmentioning

confidence: 90%

“…The metabolism of MDMB‐4en‐PINACA has been studied by two research groups, each concluding that the ester hydrolysis product (MDMB‐4en‐PINACA 3,3‐dimethylbutanoic acid) is a primary metabolite and an appropriate marker for monitoring use (Figure 2). 16,17 The presence of MDMB‐4en‐PINACA in medicolegal death investigations and impaired driving investigations has not been reported.…”

Section: Introductionmentioning

confidence: 99%

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The next generation of synthetic cannabinoids: Detection, activity, and potential toxicity of pent‐4en and but‐3en analogues including MDMB‐4en‐PINACA

Krotulski

Cannaert

Stove

et al. 2020

Drug Testing and Analysis

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A new class of synthetic cannabinoids has emerged as new psychoactive substances (NPS). Similar in structure to JWH‐022, these substances contain alkene modifications to the tail region of the synthetic cannabinoid core structure, and nomenclature denotes these new analogues as pent‐4en or but‐3en species. Internationally, two analogues from this new series recently emerged: MDMB‐4en‐PINACA and MMB‐4en‐PICA. Previously, data regarding activity and potential toxicity were not available. In vitro assessment of cannabinoid receptor 1 (CB1) activation via the β‐arrestin 2 recruitment was studied for three (3) pent‐4en analogues, one (1) but‐3en analogue, and one (1) principal metabolite. MDMB‐4en‐PINACA (2.47 nM, 239%), MDMB‐4en‐PICA (11.5 nM, 302%), and MDMB‐3en‐BINACA (14.3 nM, 286%) were highly potent and efficacious (comparison: JWH‐018, 25.3 nM, 100%), while the potencies of MMB‐4en‐PICA and MDMB‐4en‐PINACA 3,3‐dimethylbutanoic acid were markedly lower. Modifications to core and tail structural features (i.e., indole vs. indazole) led to relatively small differences in potency, while changes among the head region led to larger differences. Sample‐mining and data‐mining conducted on toxicology samples led to the identification of MDMB‐4en‐PINACA in 25 forensic toxicology cases, including postmortem and impaired driving investigations, with case details and limited histories described herein. Moderate geographical distribution of MDMB‐4en‐PINACA was noted in the United States with emergence in the Northeast, Midwest, South, and West regions. Results from toxicology testing paired with case history show the potential for MDMB‐4en‐PINACA to cause or contribute to impairment or death. Forensic scientists, public health and public safety officials, law enforcement, clinicians, medical examiners, and coroners should consider involvement of emergent synthetic cannabinoids in their work and that new analogues containing an alkene tail can retain similar or increased potency and toxicity.

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“…To date, MMB022 has been identified in seizures in Sweden (July 2018) and in Hungary (February 2019), as well as test purchases in Slovenia (July 2018) and in Germany (January 2019), but not yet reported in biological samples. In comparison, MDMB-4en-PINACA, a synthetic cannabinoid analog with an alkene, has been identified in authentic biological samples, 7,8 and has become increasingly prevalent in infused papers at Scottish prisons since June 2019. 9 Due to the structural similarity, MMB022 might become one of the next prevalent cannabinoids.…”

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confidence: 99%

Metabolism of MMB022 and identification of dihydrodiol formation in vitro using synthesized standards

Watanabe

Dahlén

et al. 2020

Drug Testing and Analysis

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MMB022 (methyl 3-methyl-2-[1-(pent-4-en-1-yl)-1H-indole-3-carboxamido]butanoate) is a new synthetic cannabinoid with an alkene at the pentenyl side chain, a rare functional group for synthetic cannabinoids. Metabolite identification is an important step for the detection of synthetic cannabinoids in humans, since they are generally extensively metabolized. The aims of the study were to tentatively identify in vitro phase I metabolites, to confirm major metabolites using synthesized metabolites, to examine metabolic pathways thoroughly, to study metabolic stability and to suggest metabolites appropriate for urine screening. MMB022 and its synthesized metabolites were incubated with human liver microsomes (HLM) and the supernatants were analyzed by liquid chromatography-quadrupole time-of-flight mass spectrometry. Sixteen metabolites were identified, which were generated via dehydrogenation, dihydrodiol formation, ester hydrolysis, hydroxylation, and combinations thereof. A major biotransformation of the alkene at the pentenyl side chain was confirmed to be dihydrodiol formation. The major metabolites were ester hydrolysis (M15) and dihydrodiol (M8) metabolites, whereas the metabolite derived from the combination of ester hydrolysis and dihydrodiol (M5) was the fourth most abundant metabolite. The metabolic pathways were investigated using synthesized metabolites and revealed that M5 is an end product of the pathways, indicating that it might become a more abundant metabolite in vivo depending on the rate of metabolism in humans. The major pathway of MMB022 to M5 was determined to be via M8 formation. Intrinsic clearance of MMB022 was determined to be 296 mL/min/kg and t 1/2 was 2.1 min, indicating a low metabolic stability. M15, M8, and potentially M5 are suggested as suitable urinary targets.

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In Vitro Phase I Metabolism of the Recently Emerged Synthetic MDMB-4en-PINACA and Its Detection in Human Urine Samples

Cited by 27 publications

References 5 publications

Monitoring metabolism of synthetic cannabinoid 4F-MDMB-BINACA via high-resolution mass spectrometry assessed in cultured hepatoma cell line, fungus, liver microsomes and confirmed using urine samples

Monitoring metabolism of synthetic cannabinoid 4F-MDMB-BINACA via high-resolution mass spectrometry assessed in cultured hepatoma cell line, fungus, liver microsomes and confirmed using urine samples

The next generation of synthetic cannabinoids: Detection, activity, and potential toxicity of pent‐4en and but‐3en analogues including MDMB‐4en‐PINACA

Metabolism of MMB022 and identification of dihydrodiol formation in vitro using synthesized standards

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