Metabolism of Carfentanil, an Ultra-Potent Opioid, in Human Liver Microsomes and Human Hepatocytes by High-Resolution Mass Spectrometry

Abstract

Carfentanil is an ultra-potent synthetic opioid. No human carfentanil metabolism data are available. Reportedly, Russian police forces used carfentanil and remifentanil to resolve a hostage situation in Moscow in 2002. This alleged use prompted interest in the pharmacology and toxicology of carfentanil in humans. Our study was conducted to identify human carfentanil metabolites and to assess carfentanil's metabolic clearance, which could contribute to its acute toxicity in humans. We used Simulations Plus's ADMET Predictor™ and Molecular Discovery's MetaSite™ to predict possible metabolite formation. Both programs gave similar results that were generally good but did not capture all metabolites seen in vitro. We incubated carfentanil with human hepatocytes for up to 1 h and analyzed samples on a Sciex 3200 QTRAP mass spectrometer to measure parent compound depletion and extrapolated that to represent intrinsic clearance. Pooled primary human hepatocytes were then incubated with carfentanil up to 6 h and analyzed for metabolite identification on a Sciex 5600+ TripleTOF (QTOF) high-resolution mass spectrometer. MS and MS/MS analyses elucidated the structures of the most abundant metabolites. Twelve metabolites were identified in total. N-Dealkylation and monohydroxylation of the piperidine ring were the dominant metabolic pathways. Two N-oxide metabolites and one glucuronide metabolite were observed. Surprisingly, ester hydrolysis was not a major metabolic pathway for carfentanil. While the human liver microsomal system demonstrated rapid clearance by CYP enzymes, the hepatocyte incubations showed much slower clearance, possibly providing some insight into the long duration of carfentanil's effects.

Fentanyl, fentanyl analogs and novel synthetic opioids: A comprehensive review

“…Like fentanyl, analogs such as alpha-methylfentanyl, alfentanil, butyrfentanyl, carfentanil, and sufentanil are primarily metabolized via the CYP 3A4 hepatic pathway, generating N-dealkylated metabolites that are primarily inactive (Guitton et al, 1997;Sato et al, 2010, Feasel et al, 2016). An important point to note is that sufentanil and alfentanil are metabolized into the same Ndealkylated product, making forensic distinction impossible when only this metabolite is identified (Guitton et al, 1997).…”

“…Carfentanil has 12 metabolites and while phase I biotransformations dominate metabolite formation, the Ndealkylated norcarfentanil is not the only abundant metabolite. The other is formed after monohydroxylation at the piperidine ring (Feasel et al, 2016). An in vivo evaluation of butyrfentanyl in a post-mortem case revealed hydroxylation of the butanamide side chain followed by subsequent oxidation to the carboxylic acid, representing the major metabolic step (Steuer et al, 2016).…”

“…This is attributed to an easily accessible ester group allowing for rapid hydrolysis by circulating blood esterases. The N-dealkylated metabolite is minor, but shares the same structure as carfentanil's nor-metabolite, making laboratory distinction difficult (Feasel et al, 2016).…”

“…In those studies, potency was also described in terms of mean effective dose and lethal dose. Measured values from in vivo studies are commonly compared to those from in vitro guinea pig ileum assays which, historically, have been the goldstandard for μ-opioid receptor pharmacology (Hayes et al, 1985;Feasel 2016). …”

“…In recent years, LC-HRMS has been used for the detection of synthetic opioids such as butyrfentanyl, 4-fluorobutyrfentanyl, acetylfentanyl, 4-methoxybutyrfentanyl, furanylfentanyl, acrylfentanyl, 4Cl-iBF, 4F-iBF, THF-F, cyclopentylfentanyl, AH-7921, MT-45, and U-47700 in individual cases, case series, outbreaks, and epidemiological surveillance efforts (Backberg et al, 2015;Wohlfarth et al, 2016;Helander et al, 2016;Schneir et al, 2017;Armenian et al, 2017;Breindahl et al, 2017;Jones et al, 2017;Steuer et al, 2016;Vo et al, 2017;Fleming et al, 2017;Guerrieri et al, 2017;Helander et al, 2017;Goggin et al, 2017;Watanabe et al, 2017;Fels et al, 2017). It has also been used for the elucidation of the metabolic pathways of emerging synthetic opioids, such as, AH-7921, butyrfentanyl, carfentanil, U-47700, furanylfentanyl, acetylfentanyl, acrylfentanyl, and 4F-iBF Jones et al, 2017;Steuer et al, 2016;Feasel et al, 2016;Fleming et al, 2017;Goggin et al, 2017;Watanabe et al, 2017). LC-HRMS has clearly emerged as the predominant method for the detection of novel synthetic opioids, however, this technology is not readily available in most routine clinical and forensic laboratories.…”