| Background: The drug –drug metabolic interaction of anti-tuberculous drugs with other medication is becoming a very common problem in clinical practice, which can leads to poor prognosis and serious adverse reaction. Cytochrome P450 enzyme system is key enzymes dealing with drug metabolism, among which isoenzyme CYP1A2 and 3A4 take part in 80% drug's metabolism. As we know, isoniazid is a inhibitor of some Cytochrome P450 isoenzymes, but rifampicin is a inductor of some Cytochrome P450 isoenzymes, yet what is the combination of the two drugs? Furthermore, most of these informations were from animal experiments. As there are species differences between animal and human , are the changes in human beings same as in animal? It is difficult to study the influence of the combination of isoniazid and rifampicin to the activity of Cytochrome P450 of human being , because of the reasons of medical ethics. Recent studies showed, human primary hepatocytes are the most similar system to liver in vivo in studying drug metabolism and hepatotoxicity. So we took the primary human hepatocytes as trial system to study the influence of isoniazid and rifampicin to the activities of CYP1A2 and 3A4, and the influence of these two drugs to the expression of mRNA of these two CYP450 isoenzymes.The informations of hepatotoxicity of anti-tuberculous drugs are merely from animal experiments or clinical epidemic investigation. Because of species difference and confounding factors, their reality has been questioned. In this studying , we also applled human primary hepatocytes to observe the hepatotoxicity of isoniazid and rifampicin .Objective :(1)To study the influence of either single or combinative using isoniazid and rifampicin to the activity of CYP1A2 and 3A4. (2) To observe the influence of either single or combinative using isoniazid and rifampicin to the expression of CYP1A2mRNA and 3A4mRNA , to explore the mechanism of influence of these two drugs to the activity of CYP1A2 and 3A4. (3) To study the hepatotoxicity of either single or combinative using isoniazid and rifampicin.Method: Part one: Hepatocytes were isolated from human liver, cultivated for 3 days.Then added isoniazid(25uM , 50uM ), rifampicin(12.5uM, 25uM )and both of them (forCYP1A2:rifampicin12.5uM & isoniazid50uM, rifampicin25uM & isoniazid50uM; for CYP3A4:rifampicin12.5uM & isoniazid25uM, rifampicin25uM & isoniazid25uM, rifampicin25uM&50uM)respectively. After cultured for 2 days added substrate (forCYP1A2: phenacetin ; for CYP3A4: testosteone), 1 hour later measured metabolite of them(forCYP1A2: acetaminophen ; for CYP3A4: 6?-testosteone) with HPLC to assess the activity of CYP1A2 and 3A4.Part two: Hepatocytes were isolated from human liver, cultivated for 3 days. Then added isoniazid, rifampicin and both of them respectively, at the concentration of which as those in Part one.With RT-PCR technique detected the expression of CYP1A2mRNA and 3A4mRNA , to explore the mechanism of these drug's influence to activities of CYP1A2 and 3A4.Part three:Hepatocytes were isolated from human liver, added isoniazid (5uM,10uM, 25uM, 50uM), rifampicin(5uM,10uM, 25uM, 50uM)and both of them (R5uM & I5uM,R10uM & I10uM&,R25 uM & I25uM, R50uM & I50uM) in cell petri dish respectively, Then cultivated them for 24 hours, and detected the activity of ALT and AST in culture medium.Results:Part one:The activity of CYP1A2 in isoniazid groups with the concentration 25uM and 50uM was lower than that in control group(P<0.01), but not less than 50% of that in control group. The activity of CYP1A2 in rifampicin group with the concentration 12.5uM was higher than that in control group (P<0.05), but was not 2 folds or more of that in control group.There was no statistical difference in activities between rifampicin with 25uM and control groups(P>0.05); The activity of CYP1A2 of combinative the groups with isoniazid and rifampicin was lower than that of the control group(P<0.01), but was not less than 50% of that of control group.The activit... |
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