| Many kinds of drugs are metabolized by the liver. In case of hepatic failure , drugs and toxic substances will accumulate in the body. From the pharma-cokinetic aspects, the half - life of diazepam ( DZP) may be 5 times longer in patients with cirrhosis than in the controls. Moreover, DZP and other benzodiaz-epines have been advocated as sedatives for agitated patients with hepatic en-cephalopathy, despite the risks of induction of hepatic coma by their use.Bioartificial liver ( BAL) support system is currently expected to be a novel therapeutic device for the end stage of hepatic failure. Researchers are using various devices as their BAL. The authors group has been utilizing the 3 - dimensional bioreactor, Kigunasu? as a BAL because of its advantages of high density of cells, reliability during a long - term operation, capability of efficiently performing the exchanges of nutrient, waste products and gases between cells; and medium. This device has also been used by Dr Enosawa. He applied a re combined HepG2 overexpressing glutamine synthetase (HepG2 -GS) , which is involved in the accessory pathway of ammonia removal, in Kigunasu to examine; on ischemic liver failure pigs.However, HepG2 - GS does not include all the functions of hepatocytes. Many kinds of substances and drugs such as testosterone, lidocaine and erythro-mycin are not metabolized by GS, but by cytochrome P450 3A4 that also has a high diazepam metabolism capacity.ObjectsThe author started the current study to provide a clue to the treatment of hepatic failure using an artificial liver device. Such a treatment may also be usefulin a clinical setting of hepatic failure patients overdosed with sedatives such as DZP. Therefore, HepG2 - GS -3A, which overexpresses CYP 3A4 and GS, was applied to Kigunasu as our artificial liver to metabolize diazepam and ammonia. Finally, this BAL was attempted to treat acute hepatic failure dogs overdosed with DZP, to decrease the concentrations of DZP and ammonia in the dogs 'plasma. Thus, the aims of the present study are summarized as follows:1. to estabolish and investigate the in vitro metabolism capacity of diazepam and ammonia by HepG2 - GS - 3 A4,2. to measure diazepam and ammonia metabolism capacity by Kigunasu inoculated with HepG2 - GS -3A4, HepG2 and without cell,3. to treat a canine model combining acute hepatic failure with diazepam overdosage using our BAL.Mefhods and resultsFirst, after the establishment of HepG2 - GS -3A4, its vitro metabolism capacity of diazepam and ammonia were studied in dishes. DZP and its metabolites were checked by HPLC. We found that 0.1 to 30 g/ml DZP, were metabolized by HepG2 - GS -3A4 time and concentration - dependently, and the metabolites of DZP were produced by HepG2 - GS -3A4 time and concentration -dependently, too, within 4 hours. Reduction rate of 0. 1 - 30jxg/ml DZP by HepG2 - GS - 3A4 was 0. 12 + 0. 006 - 3. 213 + 0. 210u,g/ml/h/mg Protein. Km for DZP N - demethylation and 3 - hydroxylation by HepG2 - GS - 3A4 were 204. 34, and 61.58 M. Vm for DZP N - demethylation and 3 - hydroxila-tion by HepG2 -GS -3A4 were 106.90, and 85.13pmol/min/mg Protein.After the incubation of lmM and 0. lmM ammonia with HepG2 - GS -3A4 for 6 hours, ammonia concentration decreased by 34. 3% and 43. 1% , respectively. lmM and 0.1mM ammonia reduction rate by HepG2 - GS - 3A4 were 18.6,234.0 ug/h/mg protein, respectively.Then, 4. 0 x 108hepatocytes were inoculated in a circulatory flow bioreac-tor, Kigunasu, as our BAL, and cultured for 7 days, until the cells were nearly confluent. Thereafter DZP and ammonia in vitro metabolism capacities of ourBAL were checked. DZP and NH4C1 were added into the medium to make the ultimate concentration at 5 ug/ml (DZP) and 2. 5mM (ammonia). Samples were collected at scheduled point times for 24 hours. DZP and its metabolites were measured by HPLC. Ammonia concentration was checked with Berthelot Reaction.From the Kigunasu in vitro study, it was found that the DZP metabolism capacity of Kigunasu with... |
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