¹Gifu Pharmaceutical University, Laboratory of Pharmaceutics, Department of Biomedical Pharmaceutics, Gifu, Japan, ²Gifu Pharmaceutical University, Laboratory of Functional Food Development for Health Promotion, Gifu, Japan, ³Gifu Pharmaceutical University, Grobal Regulatory Science, Gifu, Japan, ⁴Gifu University of Medical Science, Laboratory of Pharmacognosy, Gifu, Japan

Background: Structurally modified fentanyls, so-called fentanyl analogs (FAs), represent a global public health threat associated with opioid misuse and overdose. Butyrylfentanyl (BF) and valerylfentanyl (VF), which have an extended propionyl chain of fentanyl, are listed as Schedule I substances by the U.S. Drug Enforcement Administration (DEA) and the United Nations Office on Drugs and Crime (UNODC). In general, the extension of the propionyl chain may enhance brain penetration through increased lipophilicity and/or may decrease the metabolic stability by facilitating enzyme-mediated attack. Alternatively, para-methoxylation of the aniline ring of fentanyl produces 4-methoxyfentanyl (4-MF), which is also classified as a Schedule I substance. This modification may also increase lipophilicity and decrease metabolic stability; however, so far, there is no information available for the metabolic profile of 4-MF. Moreover, no data are also available for FAs bearing dual structural modifications—both an extended propionyl chain and a methoxylated aniline moiety.

Aims & Objectives: We synthesized two FAs, 4-methoxybutyrylfentanyl (4MBF) and 4-methoxyvalerylfentanyl (4MVF) in order to try to investigate the metabolic profiles of 4MBF and 4MVF. The results would be compared to previous reports related to BF and VF to predict their harmfulness.

Method: In vitro metabolism of 4MBF and 4MVF was investigated using human liver microsomes (HLMs) up to 4 h. Metabolites were analyzed and structurally characterized by LCMS-IT-TOF and LC-MS/MS with using the in silico metabolic predictions using GLORYx.

Results: Marked differences in metabolic stability were observed between the two analogs. The in vitro metabolic half-life (T1/2) of 4MBF and 4MVF were 115 and 31 min, respectively. Both 4MBF and 4MVF shared common metabolic pathways, including N-dealkylation (M1), monohydroxylation (M2), and O-demethylation (M3). M1 were major metabolites of both compounds. However, the production of 4-MVF M3 was relatively higher than that of 4-MBF M3.

Discussion & Conclusions: The detection pattern of metabolites is consistent with previous studies using BF and VF^1,2). T1/2 of VF is relatively shorter than T1/2 of BF, suggesting that the extension of the propionyl chain in VF may decrease the metabolic stability by facilitating enzyme-mediated attack. However, previous in vivo pharmacokinetic study in rats (0.3 mg/kg, sc) demonstrated that in vivo T1/2 of BF was mostly similar to that of VF³⁾. These discrepancies might be caused by the introduction of methoxy moiety and/or species differences. Further study is needed to investigate the possible impact of the introduction of methoxy moiety although it has been reported that the pharmacological effect of 4-MF is relatively weaker than that of fentanyl⁴⁾. Comparative studies using BF and VF also would be necessary since no information of T1/2 of BF and VF using HLMs is available. Taken together, these unique results would give us the useful information about the harmfulness of FAs.
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