| Summary: | 碩士 === 國立臺灣大學 === 毒理學研究所 === 90 === Flunitrazepam (FNZ) is a 7-nitrobenzodiazepam widely used in practice for its hypnotic properties in the treatment of sleep disorders. In 1 to 2 mg dose, it is needed to treat insomnia and as a preoperative sedative. Flunitrazepam has been prescribed by doctors for controlling hyponic, sedative, anxiolytic, muscle-relaxing and anticonvulsant effects of patients. FNZ has also recovered attention as a drug of abuse. For its euphoric effect, FNZ is popular drug abused among young adults. It is often used by rapists, therefore, FNZ has been nicknamed as the “date-rape drugs”. Three main in vivo metabolites of FNZ are 7-aminoflunitrazepam (7-AF), desmethylflunitrazepam (N-DF), and 3-hydroxyflunitrazepam (3-OH). The formation of NDF and 3OH are mediated mainly by CYP 3A4.
7-AF is metabolized through reduction of 7-amino group of FNZ. The role of CYP 450 in the formation of 7-AF is not known. Studies is undertaken using Hep G2 cell line, a liver cell line derived from a human hepatoblastoma, which has been found to express a wide variety of liver-specific metabolic functions, and is free of known hepatotropic viral agents.
The aim of this present study was to investigate the role of CYP 450 in the metabolism of Hep G2 cell lines. The chemical inhibitor (α-thioctic acid), NADPH-cytochrome P450 reductase antibody and pure human reductase microsomes were also used to identify the CYP 450 enzyme responsible for 7-AF formation.
From the present studies, were obtained the following results:
(1) Only 7-AF was formed from FNZ in Hep G2 cell lines. (2) Hep G2 cell was expressed the activity of NADPH-cytochrome P450 reductase. (3) In enzyme kinetics study, Hep G2 cell lines could metabolize FNZ judged from the value of Vmax/Km of 7-AF calculatedly acivity. (4) The specific inhibitor of NADPH-cytochrome P450 reductase, such asα-thioctic acid, inhibited 7-AF formation. (5) The antibody of NADPH-cytochrome P450 reductase also inhibited 7-AF production. (6) 7-AF formed from microsomes. (7) Immunoblotting assay showed that NADPH-cytochrome P450 reductase protein was detected in Hep G2 cell line. Therefore, it is suggested that NADPH-cytochrome P450 reductase has a major role in reductive metabolic pathway from FNZ to 7-AF.
Liver is the primary site of drug metabolism, therefore, it is not surprising that various forms of hepatic disease have been associated with decreased biotransformation of drugs, resulting occasionally in clinically significant toxicity. Hepatitis B is a native desease in Taiwan, more than 15 % population in Taiwan are hepatitis B virus cariers. Several reports suggested that, clinically infection with hepatitis B virus seems to exert a differential effect on metabolism of the drugs. According to this basis, I sought to find out whether hepatitis B viral infection can affect drug metabolism.
Another aim of this present study was to investigate the role of HBV in the metabolism of Hep G2.2.15, a HBV transfected hman hepatoblastoma cell line, which has been shown to be an accurate model of chronic cellular viral infection for in vitro study; and Hep 3B, a well differentiated human hepatoma cell line with HBV DNA integrated. The chemical inhibitor (α-thioctic acid), NADPH-cytochrome P450 reductase antibody were also used to identify the difference between these cell lines.
From the present studies, were obtained the following results:
(1) Only 7-AF was formed from FNZ in these cell lines. (2) Among these cell lines, Hep G2 cell was expressed the highest activity of NADPH-cytochrome P450 reductase. (3) In enzyme kinetics study, judged from the value of Vmax/Km, Hep G2 cell lines could metabolize FNZ to 7-AF better than Hep G2.2.15 and Hep 3B cell lines, which were HBV infected. (4) The specific inhibitorα-thioctic acid, could inhibit 7-AF formation in these cell lines. And the IC50 value of α-thioctic acid is highest in Hep G2 cell line. (5) The activity of NADPH-cytochrome P450 reductase was highest in HeP G2 cell line. (6) Immunoblotting assay showed that, the content of NADPH-cytochrome P450 reductase protein was highest in Hep G2 cell line. Therefore, it is suggested that HBV may affect the metabolism of FNZ to 7-AF, mainly through NADPH-cytochrome P450 reductase.
On the basis of these results, this study indicated that NADPH-cytochrome P450 reductase is involved in the formation of 7-aminoflunitrazepam. And also suggested that the dosage regimen of flunitrazepam for rational prescribing for patients with hepatitis B virus needs to be adjusted. This study also provided an additional example of the effect of hepatitis B virus on the disposition of drug.
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