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The in vitro biotransformation of 7-alkoxycoumarin by rat liver microsomes was studied to develop a simple and accurate assay procedure for 7-alkoxycoumarin

To investigate whether the free-drug theory is accurate in that only unbound drug is available for drug metabolism or enzyme inhibition. The effect of addition of rat liver cytosol to an in vitro system using human liver microsomes was examined by measuring the catalytic activities of CYP2C9 (tolbutamide and diclofenac) and CYP3A4 (terfenadine). And, the results were compared with those obtained when human serum albumin (HSA) was added to microsomes as far as unbound drug concentrations were concerned. After addition of rat liver cytosol, the unbound Km value (Km,u) for terfenadine metabolism by CYP3A4, and the unbound Ki value of miconazole (Ki,u) for CYP2C9 were smaller than for the controls. Addition of HSA resulted in smaller Km,u values for diclofenac and terfenadine metabolism by CYP2C9 and CYP3A4, respectively, and the Ki,u value for ketoconazole inhibition of CYP3A4 was also reduced. These results suggest protein-facilitated effects on drug metabolism and enzyme inhibition for both CYP2C9 and CYP3A4. However, no protein-facilitated drug metabolism was observed for tolbutamide in the presence of HSA or cytosol, or for diclofenac in the presence of cytosol. Protein-facilitated enzyme inhibition did not occur with miconazole in the presence of HSA or with ketoconazole in the presence of rat liver cytosol. Protein-facilitated metabolism and enzyme inhibition were observed for CYP2C9 and CYP3A4 in five cases but there was no obvious pattern of enzyme, substrate, or binding protein specificity. Further investigations are necessary to clarify the relevance of these results to in vivo observations.

Abstract-Enzyme distribution in rat liver lobes was investigated using liver homogenate as the enzyme source. When the content or the activity was expressed on the basis of unit (gram) wet weight of liver tissue, the protein concentration was almost the same throughout the liver, but several enzymes were distributed heterogeneously within the liver. Cytochrome P-450 monooxygenase activity was higher in the median and right lobes of livers, while the left lobe contained a higher concentration of mitochondrial enzymes. Phenobarbital induced cytochrome P-450 monooxygenase, and the activity was still higher in the median and right lobes even after the pretreatment of rats with phenobarbital. On the other hand, administration of 8-naphthoflavone resulted in a uniform increase of cytochrome P-450 (P-448) content throughout the liver to almost the same concentration. Increase of cytochrome P-448-dependent O-dealkylation activity of 7 alkoxycoumarin by the administration of 8-naphthoflavone was much greater in the left lobe compared to that in the median and the right lobes. Heterogeneous distribution of the enzymes in various liver lobes was also determined employing liver samples obtained from various physiological states of rats (fasting, glucose refeeding after fasting and hyperthyroidism), although the cytochrome P-450 content and drug-metabolizing activity were altered markedly depending upon the physiological states of the rats.

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