Use of hepatocytes isolated from a liver-humanized mouse for studies on the metabolism of drugs: application to the metabolism of fentanyl and acetylfentanyl

Tatsuyuki Kanamori,Yuko Togawa-Iwata,Hiroyuki Inoue,Kenji Tsujikawa,Kenji Kuwayama,Tadashi Yamamuro,Hiroki Segawa

doi:10.1007/s11419-018-0425-x

Tatsuyuki Kanamori, Yuko Togawa-Iwata + Show 5 more

Open Access

https://doi.org/10.1007/s11419-018-0425-x

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Abstract

PurposeThe usefulness of hepatocytes isolated from a liver-humanized mouse (PXB-cells) as a model in vitro system for the prediction of the in vivo metabolism of new drugs of abuse was evaluated.MethodsFor the drug metabolism study, fentanyl, a powerful synthetic opioid, and acetylfentanyl, an N-acetyl analog of fentanyl, were selected as model drugs. PXB-cells were cultured with the drug for 24–48 h and then the media were collected and analyzed by liquid chromatography/mass spectrometry after deproteinization with acetonitrile.ResultsThe main metabolite formed from fentanyl by PXB-cells was the desphenethylated metabolite (nor-fentanyl), and the other major metabolites formed were 4′-hydroxy-fentanyl, β-hydroxy-fentanyl and (ω-1)-hydroxy-fentanyl. ω-Hydroxy-fentanyl and 4′-hydroxy-3′-methoxy-fentanyl were the minor metabolites. Similar results were obtained for acetylfentanyl. The metabolite profile of fentanyl in PXB-cells was consistent with the in vivo metabolite profile of fentanyl reported previously. Most of the 4′-hydroxy- and 4′-hydroxy-3′-methoxy-metabolites of fentanyl and acetylfentanyl were conjugated in PXB-cells, indicating that PXB-cells had high conjugation enzyme activities. From experiments using human liver microsomes and anti-CYP antibodies, it was revealed that CYP3A4 was involved in the production of nor-fentanyl, β-hydroxy-fentanyl and (ω-1)-hydroxy-fentanyl, while CYP2D6 was partially involved in the production of 4′-hydroxy-fentanyl.ConclusionsOur results indicated that PXB-cells have high activities of phase I and phase II drug-metabolizing-enzymes, can be stably supplied, and are easy to use; thus, PXB-cells are highly useful for the prediction of the in vivo metabolism of drugs of abuse.

Highlights

New drugs of abuse, such as synthetic cannabinoids, synthetic cathinones and fentanyl derivatives, have become widely available around the world
PXB-cells were separated from a liver-humanized mouse and seeded on a cell culture plate (24-wells) by the manufacturer, and the culture plate was supplied for further experiments, thereby eliminating the need for thawing and seeding of the cells. h-PRM-HEP was purchased from Kurabo Industries (Osaka, Japan); β-glucuronidase/aryl sulfatase from Helix pomatia (β-glucuronidase, 32 units/ mL; aryl sulfatase, 102 units/mL) from Merck (Darmstadt, Germany); phosphate-buffered saline (PBS) from Thermo Fisher Scientific (Waltham, MA, USA); anti-cytochrome P450s (CYPs) antibodies, preimmune rabbit IgG and 2-phenyl-2-(1piperidinyl)propane (PPP) from CYP450-GP (Vista, CA, USA); human liver microsomes (HLM) (Ultrapool HLM 150, 6.6 nmol P450/mL) from Corning (Corning, NY, USA)
To clarify which isoform of the CYPs was involved in the production of the metabolites of fentanyl, the relative amounts of four metabolites of fentanyl (nor-fentanyl, 4′-hydroxy-fentanyl, β-hydroxy-fentanyl and (ω-1)-hydroxy-fentanyl) produced by HLM treated with various anti-CYP antibodies were determined (Fig. 5)

Summary

Introduction

New drugs of abuse, such as synthetic cannabinoids, synthetic cathinones and fentanyl derivatives, have become widely available around the world. The countermeasures developed to regulate the abuse of these drugs have been strengthened by the anti-drug authorities in many countries. It is extremely important to understand the metabolic fate of the target drug. The use of h-PRM-HEP is limited by their fragility, poor viability after thawing, and an unstable supply [4]. To overcome these problems, new types of cells and methodologies for cell culture have been investigated.

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