“…β-Hydroxy-acetylfentanyl and hydroxyacetyl-fentanyl are novel metabolites of acetylfentanyl. In previous reports, the N-desacyl metabolite (despropionylfentanyl) was found to be formed from both fentanyl and acetylfentanyl 8,13) ; however, this desacyl metabolite was not detected in any cell culture systems in the present study.…”
Section: Identification Of the Metabolites Of Fentanyl Andcontrasting
confidence: 44%
“…Melent'ev et al have reported that acetylfentanyl was metabolized by desphenethylation, deacetylation, and hydroxylation of the phenylethyl moiety followed by further hydroxylation and methylation of one of the hydroxyl groups. 8) However, this group did not confirm the exact chemical structure of the metabolites. In this study, we confirm the exact structure of the metabolites of acetylfentanyl for the first time.…”
Section: Identification Of the Metabolites Of Fentanyl Andmentioning
confidence: 79%
“…7) Acetylfentanyl is metabolized in a similar manner to fentanyl. 8) We evaluated how accurately the in vivo patterns of fentanyl and acetylfentanyl metabolism can be reproduced using h-iPS-HEP.…”
To evaluate the capability of human-induced pluripotent stem cell-derived hepatocytes (h-iPS-HEP) in drug metabolism, the profiles of the metabolites of fentanyl, a powerful synthetic opioid, and acetylfentanyl, an N-acetyl analog of fentanyl, in the cells were determined and analyzed. Commercially available h-iPS-HEP were incubated with fentanyl or acetylfentanyl for 24 or 48 h. After enzymatic hydrolysis, the medium was deproteinized with acetonitrile, then analyzed by LC/MS. Desphenethylated metabolites and some hydroxylated metabolites, including 4′-hydroxy-fentanyl and β-hydroxy-fentanyl, were detected as metabolites of fentanyl and acetylfentanyl in the medium. The main metabolite of fentanyl with h-iPS-HEP was the desphenethylated metabolite, which was in agreement with in vivo results. These results suggest that h-iPS-HEP may be useful as a tool for investigating drug metabolism.Key words induced pluripotent stem cell; hepatocyte; fentanyl; metabolism Human primary hepatocytes (h-PRM-HEP) are widely used for drug metabolism studies because they are highly active in phase I and phase II drug metabolism.1) h-PRM-HEP are regarded as one of the best tools for the investigation of the metabolism of drugs; however, there are problems with their use, including maintaining a stable supply of cells of a consistent quality and occasional significant drops in cell viability resulting from the fragility of the cells.
2)Induced pluripotent stem (iPS) cells, first developed in 2006, can differentiate into many different types of cells, including neurons, cardiomyocytes, and hepatocytes.3) Human iPS cell-derived hepatocytes (h-iPS-HEP) reportedly have catalytic activity from various CYP enzymes, in addition to liver-specific functions, such as albumin secretion and ammonia metabolism.4) Furthermore, h-iPS-HEP are robust and can be supplied stably, making these cells a potentially useful tool for drug metabolism studies.In this study, we investigated the ability of h-iPS-HEP to metabolize drugs using fentanyl and acetylfentanyl (Fig. 1) as model drugs. Fentanyl is a powerful synthetic opioid, which is used as an analgesic in patients with cancer. 5) Acetylfentanyl is an N-acetyl analog of fentanyl, which is used as a substitute for recreational opioid drugs, such as heroin.6) Fentanyl is known to be metabolized by desphenethylation, 4′-hydroxylation, and hydroxylation of the N-propionyl group. 7) Acetylfentanyl is metabolized in a similar manner to fentanyl. 8) We evaluated how accurately the in vivo patterns of fentanyl and acetylfentanyl metabolism can be reproduced using h-iPS-HEP.
MATERIALS AND METHODSMaterials Authentic standards of fentanyl, acetylfentanyl, and their metabolites were synthesized in our laboratory. cis-3-Methylfentanyl was synthesized in our laboratory by the method reported previously. 9) β-Glucuronidase/aryl sulfatase (from Helix pomatia; β-glucuronidase, 32 units/mL; aryl sulfatase, 102 units/mL) was purchased from Merck (Darmstadt,
“…β-Hydroxy-acetylfentanyl and hydroxyacetyl-fentanyl are novel metabolites of acetylfentanyl. In previous reports, the N-desacyl metabolite (despropionylfentanyl) was found to be formed from both fentanyl and acetylfentanyl 8,13) ; however, this desacyl metabolite was not detected in any cell culture systems in the present study.…”
Section: Identification Of the Metabolites Of Fentanyl Andcontrasting
confidence: 44%
“…Melent'ev et al have reported that acetylfentanyl was metabolized by desphenethylation, deacetylation, and hydroxylation of the phenylethyl moiety followed by further hydroxylation and methylation of one of the hydroxyl groups. 8) However, this group did not confirm the exact chemical structure of the metabolites. In this study, we confirm the exact structure of the metabolites of acetylfentanyl for the first time.…”
Section: Identification Of the Metabolites Of Fentanyl Andmentioning
confidence: 79%
“…7) Acetylfentanyl is metabolized in a similar manner to fentanyl. 8) We evaluated how accurately the in vivo patterns of fentanyl and acetylfentanyl metabolism can be reproduced using h-iPS-HEP.…”
To evaluate the capability of human-induced pluripotent stem cell-derived hepatocytes (h-iPS-HEP) in drug metabolism, the profiles of the metabolites of fentanyl, a powerful synthetic opioid, and acetylfentanyl, an N-acetyl analog of fentanyl, in the cells were determined and analyzed. Commercially available h-iPS-HEP were incubated with fentanyl or acetylfentanyl for 24 or 48 h. After enzymatic hydrolysis, the medium was deproteinized with acetonitrile, then analyzed by LC/MS. Desphenethylated metabolites and some hydroxylated metabolites, including 4′-hydroxy-fentanyl and β-hydroxy-fentanyl, were detected as metabolites of fentanyl and acetylfentanyl in the medium. The main metabolite of fentanyl with h-iPS-HEP was the desphenethylated metabolite, which was in agreement with in vivo results. These results suggest that h-iPS-HEP may be useful as a tool for investigating drug metabolism.Key words induced pluripotent stem cell; hepatocyte; fentanyl; metabolism Human primary hepatocytes (h-PRM-HEP) are widely used for drug metabolism studies because they are highly active in phase I and phase II drug metabolism.1) h-PRM-HEP are regarded as one of the best tools for the investigation of the metabolism of drugs; however, there are problems with their use, including maintaining a stable supply of cells of a consistent quality and occasional significant drops in cell viability resulting from the fragility of the cells.
2)Induced pluripotent stem (iPS) cells, first developed in 2006, can differentiate into many different types of cells, including neurons, cardiomyocytes, and hepatocytes.3) Human iPS cell-derived hepatocytes (h-iPS-HEP) reportedly have catalytic activity from various CYP enzymes, in addition to liver-specific functions, such as albumin secretion and ammonia metabolism.4) Furthermore, h-iPS-HEP are robust and can be supplied stably, making these cells a potentially useful tool for drug metabolism studies.In this study, we investigated the ability of h-iPS-HEP to metabolize drugs using fentanyl and acetylfentanyl (Fig. 1) as model drugs. Fentanyl is a powerful synthetic opioid, which is used as an analgesic in patients with cancer. 5) Acetylfentanyl is an N-acetyl analog of fentanyl, which is used as a substitute for recreational opioid drugs, such as heroin.6) Fentanyl is known to be metabolized by desphenethylation, 4′-hydroxylation, and hydroxylation of the N-propionyl group. 7) Acetylfentanyl is metabolized in a similar manner to fentanyl. 8) We evaluated how accurately the in vivo patterns of fentanyl and acetylfentanyl metabolism can be reproduced using h-iPS-HEP.
MATERIALS AND METHODSMaterials Authentic standards of fentanyl, acetylfentanyl, and their metabolites were synthesized in our laboratory. cis-3-Methylfentanyl was synthesized in our laboratory by the method reported previously. 9) β-Glucuronidase/aryl sulfatase (from Helix pomatia; β-glucuronidase, 32 units/mL; aryl sulfatase, 102 units/mL) was purchased from Merck (Darmstadt,
“…One possible reason is that β-hydroxy-fentanyl may undergo further metabolism in vivo. On the other hand, it is highly probable that 4′-hydroxy-3′-methoxy-fentanyl may be detected in the biological specimens from fentanyl users because the 4′-hydroxy-3′-methoxy metabolite was reportedly detected for acetylfentanyl, a structurally similar analog of fentanyl [13]. …”
Section: Resultsmentioning
confidence: 99%
“…Overall, higher amounts of metabolites were formed in PXB-cells than in h-PRM-HEP; in particular, hydroxyacetyl-fentanyl was detected only in the medium of PXB-cells. Melent’ev et al [13] analyzed urine samples from acetylfentanyl users and detected various metabolites, such as nor-acetylfentanyl, hydroxylated metabolites, and a hydroxylated and methoxylated metabolite, although the exact structures of these metabolites were not confirmed. In the present study, our results showed that the metabolites of acetylfentanyl reported by Melent’ev et al were formed by PXB-cells and their chemical structures were confirmed.Fig.…”
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.
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