First Characterization of AKB-48 Metabolism, a Novel Synthetic Cannabinoid, Using Human Hepatocytes and High-Resolution Mass Spectrometry

Gandhi, Adarsh; Zhu, Mingshe; Pang, Shaokun; Wohlfarth, Ariane; Scheidweiler, Karl B.; Liu, Huafen; Huestis, Marilyn A.

doi:10.1208/s12248-013-9516-0

Cited by 75 publications

(91 citation statements)

References 23 publications

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“…This might be attributed to electron cloud distribution differences between indole and indazole rings. Likewise, no indazole-hydroxylated metabolites were observed for AKB-48 and AB-PINACA (19,34 ). Both AM-2201 and THJ-2201 underwent oxidative defluorination with subsequent carboxylation and glucuronidation (11,16 ).…”

Section: Thj-018 and Thj-2201 Metabolism Compared With Jwh-018 And Ammentioning

confidence: 94%

High-Resolution Mass Spectrometry for Characterizing the Metabolism of Synthetic Cannabinoid THJ-018 and Its 5-Fluoro Analog THJ-2201 after Incubation in Human Hepatocytes

Diao

Wohlfarth

Pang³

et al. 2016

Clinical Chemistry

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BACKGROUND Despite increasing prevalence of novel psychoactive substances, no human metabolism data are currently available, complicating laboratory documentation of intake in urine samples and assessment of the drugs' pharmacodynamic, pharmacokinetic, and toxicological properties. In 2014, THJ-018 and THJ-2201, synthetic cannabinoid indazole analogs of JWH-018 and AM-2201, were identified, with the National Forensic Laboratory Information System containing 220 THJ-2201 reports. Because of numerous adverse events, the Drug Enforcement Administration listed THJ-2201 as Schedule I in January 2015. METHODS We used high-resolution mass spectrometry (HR-MS) (TripleTOF 5600+) to identify optimal metabolite markers after incubating 10 μmol/L THJ-018 and THJ-2201 in human hepatocytes for 3 h. Data were acquired via full scan and information-dependent acquisition triggered product ion scans with mass defect filter. In silico metabolite predictions were performed with MetaSite and compared with metabolites identified in human hepatocytes. RESULTS Thirteen THJ-018 metabolites were detected, with the major metabolic pathways being hydroxylation on the N-pentyl chain and further oxidation or glucuronidation. For THJ-2201, 27 metabolites were observed, predominantly oxidative defluorination plus subsequent carboxylation or glucuronidation, and glucuronidation of hydroxylated metabolites. Dihydrodiol formation on the naphthalene moiety was observed for both compounds. MetaSite prediction matched well with THJ-018 hepatocyte metabolites but underestimated THJ-2201 oxidative defluorination. CONCLUSIONS With HR-MS for data acquisition and processing, we characterized THJ-018 and THJ-2201 metabolism in human hepatocytes and suggest appropriate markers for laboratories to identify THJ-018 and THJ-2201 intake and link observed adverse events to these new synthetic cannabinoids.

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Section: Thj-018 and Thj-2201 Metabolism Compared With Jwh-018 And Ammentioning

confidence: 94%

High-Resolution Mass Spectrometry for Characterizing the Metabolism of Synthetic Cannabinoid THJ-018 and Its 5-Fluoro Analog THJ-2201 after Incubation in Human Hepatocytes

Diao

Wohlfarth

Pang³

et al. 2016

Clinical Chemistry

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“…Organic solvent percentage (methanol and DMSO, respectively) was <1%. One hundred-microliter samples were collected at 0, 3,8,13,20,30,45, and 60 min and added to 100 μL ice-cold acetonitrile. Samples were centrifuged in a 5804r bench top centrifuge (Eppendorf, Hamburg, Germany) (15,000g, 4°C, 10 min), supernatant was removed and stored at −80°C.…”

Section: Metabolic Stability Assessment With Hlmmentioning

confidence: 99%

“…Epoxide formation with subsequent hydrolysis to dihydrodiol 9.8 8.10E+04 (11) 5.70E+04 (11) 6.10E+04 (10) 8.50E+06 (5) 1.70E+07 (4) 1.70E+06 (9) 5.20E+06 (2) 5.00E+05 (2) 5.30E+05 (2) 2.30E+06 (11) 2.90E+06 (10) (6) 8.50E+06 (7) 2.60E+06 (6) 2.40E+06 (6) 2.60E+05 (6) 2.50E+05 (7) 3.20E+05 (14) (7) 9.20E+06 (6) 2.20E+06 (8) 2.20E+06 (8) A13 Amide hydrolysis+oxidation 13.38 3.20E+05 (7) 2.60E+05 (7) 2.70E+05 (6) 1.50E+07 (2) 2.40E+07 (3) 6.90E+06 (4) 8.60E+06 (8) 1.10E+07 (4) 1.30E+07 (5) 7.30E+06 (3) 8.20E+06 (4) A16 Amide hydrolysis+ketone formation 14.32 3.90E+05 (5) 3.50E+05 (4) 3.60E+05 (5) 4.70E+07 (1) 6.70E+07 (1) 2.40E+07 (1) 3.00E+07…”

Section: A3mentioning

confidence: 99%

“…Further, it is necessary to identify the most prevalent metabolites for synthesis of reference standards for analytical method development. Since the first appearance of synthetic cannabinoids, many metabolism studies with human liver microsomes (HLM), human hepatocytes, and/or authentic specimens elucidated their metabolic pathways (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22).…”

Section: Introductionmentioning

confidence: 99%

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Pentylindole/Pentylindazole Synthetic Cannabinoids and Their 5-Fluoro Analogs Produce Different Primary Metabolites: Metabolite Profiling for AB-PINACA and 5F-AB-PINACA

Wohlfarth

Castaneto

Zhu

et al. 2015

AAPS J

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144

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Abstract. Whereas non-fluoropentylindole/indazole synthetic cannabinoids appear to be metabolized preferably at the pentyl chain though without clear preference for one specific position, their 5-fluoro analogs' major metabolites usually are 5-hydroxypentyl and pentanoic acid metabolites. We determined metabolic stability and metabolites of N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide (AB-PINACA) and 5-fluoro-AB-PINACA (5F-AB-PINACA), two new synthetic cannabinoids, and investigated if results were similar. In silico prediction was performed with MetaSite (Molecular Discovery). For metabolic stability, 1 μmol/L of each compound was incubated with human liver microsomes for up to 1 h, and for metabolite profiling, 10 μmol/L was incubated with pooled human hepatocytes for up to 3 h. Also, authentic urine specimens from AB-PINACA cases were hydrolyzed and extracted. All samples were analyzed by liquid chromatography high-resolution mass spectrometry on a TripleTOF 5600+ (AB SCIEX) with gradient elution (0.1% formic acid in water and acetonitrile). High-resolution full-scan mass spectrometry (MS) and information-dependent acquisition MS/MS data were analyzed with MetabolitePilot (AB SCIEX) using different data processing algorithms. Both drugs had intermediate clearance. We identified 23 AB-PINACA metabolites, generated by carboxamide hydrolysis, hydroxylation, ketone formation, carboxylation, epoxide formation with subsequent hydrolysis, or reaction combinations. We identified 18 5F-AB-PINACA metabolites, generated by the same biotransformations and oxidative defluorination producing 5-hydroxypentyl and pentanoic acid metabolites shared with AB-PINACA. Authentic urine specimens documented presence of these metabolites. AB-PINACA and 5F-AB-PINACA produced suggested metabolite patterns. AB-PINACA was predominantly hydrolyzed to AB-PINACA carboxylic acid, carbonyl-AB-PINACA, and hydroxypentyl AB-PINACA, likely in 4-position. The most intense 5F-AB-PINACA metabolites were AB-PINACA pentanoic acid and 5-hydroxypentyl-AB-PINACA.

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“…In the USA, the Drug Enforcement Administration (DEA) temporarily listed AKB-48 as a Schedule I drug as of May 2013 (17), and the compound is also prohibited in Denmark (22). During the first half of 2013, AKB-48 and its fluorinated derivative, 5F-AKB-48 (Fig.…”

Section: Introductionmentioning

confidence: 99%

CYP3A4 Mediates Oxidative Metabolism of the Synthetic Cannabinoid AKB-48

Holm

Nielsen

Línnet

2015

AAPS J

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Abstract. Synthetic cannabinoid designer drugs have emerged as drugs of abuse during the last decade, and acute intoxication cases are documented in the scientific literature. Synthetic cannabinoids are extensively metabolized, but our knowledge of the involved enzymes is limited. Here, we investigated the metabolism of N-(1-adamantyl)-1-pentyl-1H-indazole-3-carboxamide (AKB-48), a compound identified in herbal blends from 2012 and onwards. We screened for metabolite formation using a panel of nine recombinant cytochrome P450 (CYP) enzymes (CYP1A2, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4) and compared the formed metabolites to human liver microsomal (HLM) incubations with specific inhibitors against CYP2D6, 2C19, and 3A4, respectively. The data reported here demonstrate CYP3A4 to be the major CYP enzyme responsible for the oxidative metabolism of AKB-48, preferentially performing the oxidation on the adamantyl moiety. Genetic polymorphisms are likely not important with regard to toxicity given the major involvement of CYP3A4. Adverse drug-drug interactions (DDIs) could potentially occur in cases with co-intake of strong CYP3A4 inhibitors, e.g., HIV antivirals and azole antifungal agents.

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First Characterization of AKB-48 Metabolism, a Novel Synthetic Cannabinoid, Using Human Hepatocytes and High-Resolution Mass Spectrometry

Cited by 75 publications

References 23 publications

High-Resolution Mass Spectrometry for Characterizing the Metabolism of Synthetic Cannabinoid THJ-018 and Its 5-Fluoro Analog THJ-2201 after Incubation in Human Hepatocytes

High-Resolution Mass Spectrometry for Characterizing the Metabolism of Synthetic Cannabinoid THJ-018 and Its 5-Fluoro Analog THJ-2201 after Incubation in Human Hepatocytes

Pentylindole/Pentylindazole Synthetic Cannabinoids and Their 5-Fluoro Analogs Produce Different Primary Metabolites: Metabolite Profiling for AB-PINACA and 5F-AB-PINACA

CYP3A4 Mediates Oxidative Metabolism of the Synthetic Cannabinoid AKB-48

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