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Preliminary Study of the in vitro Interaction between Alcohol, High‐Dose Flunitrazepam and its Three Metabolites using Human Liver Microsomes

2005; Wiley; Volume: 96; Issue: 1 Linguagem: Inglês

10.1111/j.1742-7843.2005.pto960113.x

1742-7843

Einosuke Tanaka, Takako Nakamura, Tasaru Terada, Tatsuo Shinozuka, Katsuya Honda,

Alcohol Consumption and Health Effects

Many adverse drug-drug interactions of toxicological and clinical interest appear to be attributable to pharmacokinetic change that can be understood in terms of alterations in hepatic drug metabolic pathways catalyzed by the cytochrome P450 (CYP) system. The two major reasons for drug interactions involving the CYP system are induction and inhibition, with inhibition appearing to be more important as far as documented clinical problems are concerned. Flunitrazepam, 7-nitrobenzodiazepine, is used as intermediate-acting antianxienty drug and hypnosedative, and its effect appears at low concentrations (Druid et al. 2001). Flunitrazepam has been reported to be the most frequently used benzodiazepine and widely involved in polydrug abuse (Navaratnam et al. 1990; Barnas et al. 1992; San et al. 1993; Woods and Winger 1997). In addition, it is often used in combination with alcohol (Simmons & Cupp 1998; Daderman & Lidberg 1999). Its quick onset of sedation, amnesic properties, and additive effects with alcohol have led to its reputation as a party drug, club drug, or date rape drug. Three main in vivo metabolites of flunitrazepam are two oxidized metabolites (desmethylflunitrazepam and 3-hydroxyflunitrazepam) and one reduced metabolite (7-aminoflunitrazepam) (Woods & Winger 1997). Hesse et al. (2001) and Kilicarslan et al. (2001) have reported that the polymorphic enzymes, CYP2C19 and CYP3A4, play an important role in mediating flunitrazepam demethylation, which may alter the efficacy and safety of the drug, while CYP3A4 catalyzes the formation of 3-hydroxyflunitrazepam. Alcohol (ethanol) is one of the most widely used social drugs in the world. Sometimes toxic interactions occur involving a combination of those two drugs (National Research Institute of Police Science 1900–2002). We have already described the toxicological interactions between alcohol and benzodiazepines, and the combined use of benzodiazepines and alcohol in fatal poisoning (Tanaka & Misawa 1998; Tanaka 2002). Fatal poisoning involving co-administration of alcohol and benzodiazepine, especially flunitrazepam, continues to be a serious problem (Tanaka & Misawa 1998; Tanaka 2002). In this study we investigated the in vitro interaction between alcohol and flunitrazepam at therapeutic concentrations using human liver microsomes. Chemicals. NADPH was purchased from Oriental Yeast Co. (Tokyo, Japan). Ethizolam (internal standard, I.S.) and alcohol were purchased from Wako (Osaka, Japan). Ketoconazole and sulfaphenazol were purchased from Sigma Chemical Co. (St. Louis, MO, USA) Flunitrazepam and its two metabolites (desmethylflunitrazepam and 3-hydroxyflunitrazepam) were kindly provided by Hoffmann-La Roche (Basel, Switzerland). 7-Aminoflunitrazepam was purchased from Wako Pure Chemical Ind. (Osaka, Japan). All other chemicals and reagents used were of the highest commercially available quality. Human liver microsomes. Microsomes from three pooled human livers (Catalogue No.: H003, HH13, H032) were obtained from Daiichi Pure Chemical Co. (Tokyo, Japan). Enzyme assay. The incubation mixture contained protein (0.15 mg), 0.1 M potassium phosphate buffer (pH 7.4), 0.1 mM NADPH, flunitrazepam (substrate concentration: 200– 5000 nM, therapeutic level 15–50 nM, toxic level 160 nM) and alcohol (20–80 mM, toxic level 20–40 mM, fatal level 70–80 mM) in a total volume of 0.5 ml. Incubations were initiated following a 3 min. preincubation at 37 ° by the addition of NADPH and generally carried out for 20 min. in a shaking water-bath at 37 °. The reaction was terminated by adding 50 μl 1N NaOH and 1.5 ml tert-butyl methyl ether containing 0.5 μg/ml ethizolam (I.S.). After vortex mixing for 5 min., the tubes were centrifuged at 1,200×g for 5 min. The organic phase was transferred to a clean conical tube and evaporated in a water-bath at about 40 ° under a gentle stream of nitrogen. The residue was dissolved in 200 μl mobile phase and 50 μl injected into the HPLC system. The substrate was delivered in 0.1% (final concentration, v/v) methanol. Determination of flunitrazepam and its metabolites. The HPLC equipment consisted of a pump (Model CCPS, Tosho, Tokyo, Japan) and a variable-wavelength UV detector (Model UV-8020, Tosho, Tokyo, Japan). Separation was achieved using a C18 reversed-phase column (150 mm×4.6 mm I.D., particle size 5 μm, Inersil ODS-3 (GL Science, Tokyo, Japan). The mobile phase was 10 mM KH2PO4-methanol-acetonitrile (pH6.8) (50:35:15, v/v/v) and the flow rate was 0.75 ml/min. The absorbance of the eluate was monitored at 220 nm. All instruments were operated at ambient laboratory temperature (23 °). The retention time of 7-aminoflunitrazepam, desmethylflunitrazepam, 3-hydroxyflunitrazepam, flunitrazepam and diazepam (I.S.) in a spiked sample of human liver microsomes was 5.0, 11.5, 12.4, 16.9 and 25.0 min., respectively. However, 7-aminoflunitrazepam from the reduced metabolite was not detected after enzyme assay. The lower level of quantification of desmethylflunitrazepam and 3-hydroxyflunitrazepam was <50 nM and <30 nM, respectively. The within-day and between-day precision and accuracy were all <10%. Statistical analysis was performed by Student's t-test for unpaired samples. Differences were considered significant when P<0.05. Results are expressed as the mean±S.E. The production of desmethylflunitrazepam and 3-hydroxyflunitrazepam was significantly inhibited by 18–42% and 10–38%, respectively, by alcohol (20–80 mM) in a concentration-depend manner (table 1). Sometimes the serious toxic interactions or side effects occur after the administration of combinations of some drugs. Flunitrazepam (RohypnolTM) is one of the widely used “Club Drugs” (Smith et al. 2002). The sedative and toxic effects of flunitrazepam are aggravated by concurrent use of alcohol. Recently, Assaf & Abdel-Rahman (1999) reported serious hepatotoxicity of produced by a combination of flunitrazepam and alcohol using hepatocytes isolated from male Sprague-Dawley rats. The purpose of this study was investigate the interaction involving flunitrazepam and alcohol using human liver microsome over the therapeutic range in vitro. In man, flunitrazepam is oxidized to two major metabolites (desmethylflunitrazepam and 3-hydroxyflunitrazepam), catalyzed by CYP2C19, and CYP3A4, and reduced to 7-aminoflunitrazepam (Woods & Winger 1997; Hesse et al. 2001; Kilicarslan et al. 2001). In this study, the production of desmethylflunitrazepam was significantly inhibited by alcohol (about 20–40%), and weak inhibition of 3-hydroxyflunitrazepam (about 10–40%) was observed. The reasons for this discrepancy may be due to a difference in affinity for the drug metabolizing enzymes involved in the production of desmethylflunitrazepam and 3-hydroxyflunitrazepam, although this was not investigated in the present study. The liver is the major site of alcohol metabolism, and hepatic alcohol dehydrogenase is the enzyme primarily responsible for this process. Long-term intake of large amounts of alcohol induces pathways of metabolism that are independent of alcohol dehydrogenase (Lieber 1994; Asai & Imaoka 1996; Kitson 1996; Song 1996). Other enzymes, in particular the microsomal alcohol-oxidizing system, involving CYP2E1, are also involved at higher dose levels of alcohol, and these metabolize up to 10% of the ingested alcohol (Lieber 1994; Asai & Imaoka 1996; Kitson 1996; Song 1996). Acute alcohol ingestion causes competitive inhibition of the enzymes involved in alcohol catabolism, including CYP1A2, CYP2E1 or CYP3A activity, while chronic exposure to ethanol may produce induction (Lieber 1994; Asai & Imaoka 1996; Kitson 1996; Song 1996). An increasing number of reports indicate that flunitrazepam is frequently abused, often in combination with alcohol (Hermansson 1998; Simmons & Cupp 1998). Yamamoto et al. (2000) described a case report involving fatal acute alcohol and flunitrazepam intoxication in an aldehyde dehydrogenase heterozygote. In this case the blood alcohol concentration in a sample taken from the femoral vein was 2.00 mg/ml but the flunitrazepam concentration was not determined. In conclusion, these results using a human liver microsomal preparation show that the formation of both metabolites of flunitrazepam is only weakly inhibited by ethanol. In future, it is necessary to use different human microsomes to establish the existence of CYP genetic polymorphisms and in vivo clinical studies.