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A fully automated assay to determine the enzymology of drug oxidation by …


Biology Articles » Biochemistry » Enzymology » Automated Definition of the Enzymology of Drug Oxidation by the Major Human Drug Metabolizing Cytochrome P450s » Figures

Figures
- Automated Definition of the Enzymology of Drug Oxidation by the Major Human Drug Metabolizing Cytochrome P450s

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Fig. 1.   CYP1A2, -2C9, -2C19, -2D6, and -3A4-dependent clearance of propranolol.

The determination of the CYP-dependent propranolol metabolism using the automated assay is as described under Materials and Methods. Aliquots were taken at 0, 5, 10, 15, and 20 min, and the amount of propranolol remaining in the incubation media is reflected by the peak area after HPLC-fluorescence detection. The data represent propranolol clearance by E. coli membranes expressing CYP1A2 (black-square), CYP2C9 (), CYP2D6 (black-diamond), CYP3A4 (black-down-triangle), and baculovirus expressing CYP2C19 (black-triangle), as described under Materials and Methods. The solid lines indicate linear regression of the data

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Fig. 2.   Comparison of summed CYP CLint with HLM CLint for several probe substrates.

Both the summed CYP and HLM CLint determinations were carried out as described under Materials and Methods. The data points represent the mean CLint determinations, and the error bars reflect the standard deviation from the mean as shown in Table 6. The dotted boxes illustrate HLM CLints of -1mg-1 (low clearance) and >65 µl · min-1mg-1 (high clearance). The solid line depicts a linear regression analysis of the data (r2 = 0.8, P int = 0.91 × logHLM CLint + 0.3. The dashed line indicates line of unity.

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Fig. 3.   Determination of propranolol CLint at different concentrations of HLM.

The HLM CLint determinations were carried out as described under Materials and Methods. The histograms reflect a mean CLint, and the error bars give the standard deviation from the mean. Experiments were carried out in duplicate a minimum of three times.

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Fig. 4.   Metabolite profile for the CYP-dependent clearance of dextromethorphan.

The identification of CYP-dependent dextromethorphan metabolite formation is as described under Materials and Methods. The data from this laboratory, where dextromethorphan was incubated at 30 µM, are compared with that of Von Moltke et al. (1998) where the data reflect CLint.

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