Salvinorin A is the main active psychoactive ingredient in Salvia divinorum, a Mexican plant that has been widely available as a hallucinogen in recent years. The aims of this study were to investigate the stability of salvinorin A in rat plasma, esterases responsible for its degradation, and estimation of the degradation products. The apparent first-order rate constants of salvinorin A at 37 degrees C, 25 degrees C, and 4 degrees C were 3.8 x 10(-1), 1.1 x 10(-1), and < 6.0 x 10(-3) h(-1), respectively. Salvinorin A degradation was markedly inhibited by the addition of sodium fluoride, an esterase inhibitor. Moreover, phenylmethylsulfonyl fluoride (serine esterase inhibitor) and bis-p-nitrophenylphosphate (carboxylesterase inhibitor) also inhibited salvinorin A degradation. In contrast, little or no suppression of the degradation was seen with 5,5'-dithiobis-2-nitrobenzoic acid (arylesterase inhibitor),ethopropazine (butyrylcholinesterase inhibitor), and BW284c51 (acetylcholineseterase inhibitor). These findings indicated that carboxylesterase was mainly involved in the salvinorin A hydrolysis in rat plasma.4. The degradation products of salvinorin A estimated by liquid chromatography-mass spectrometry included the deacetylated form (salvinorin B) and the lactone-ring-open forms of salvinorin A and salvinorin B. This lactone-ring-opening reactions were involved in calcium-dependent lactonase.

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,

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Segmental hair analysis is used to estimate the time of drug intake at monthly precision in drug-related crimes. Previously, we advanced this analytical method to specify the day of drug intake by cutting a strand of hair into 0.4-mm segments, which correspond to daily hair growth. Herein, we investigated the distributions of 7 compounds in a strand of hair using micro-segmental analysis. Several strands of hair were collected 33.1-229.4 days after subjects were administered 4 pharmaceutical products that contained 10 drugs in single doses within 32 hours. The administered drugs and resulting metabolites were extracted from 0.4-mm hair segments and quantified using liquid chromatography-tandem mass spectrometry. Acidic and neutral compounds were detected at low amounts in any of the hair segments analyzed. Epinastine, fexofenadine, dihydrocodeine, chlorpheniramine, and the chlorpheniramine metabolite, desmethylchlorpheniramine each was localized to 2 regions within a strand of hair. By contrast, methylephedrine and its metabolite, ephedrine, each was localized to only a region. Among 20 individual strands of hair associated with different subjects and head regions, few differences in the shapes of drug concentration-hair segment curves for each compound were detected. Our data indicated that 2 mechanisms for drug uptake into hair can operate depending on drug properties and that co-administered drugs can be localized to different regions in a strand of hair. Micro-segmental analysis may aid in the identification of the day of drug intake and help to elucidate the mechanisms of drug uptake into hair.

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