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

• Abstract
• Introduction
• Materials and Methods
• Results
• Discussion
• References
• Figures
• Tables

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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 (), CYP2C9 (), CYP2D6 (), CYP3A4 (), and baculovirus expressing CYP2C19 (), 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 CL_int with HLM CL_int for several probe substrates.

Both the summed CYP and HLM CL_int determinations were carried out as described under Materials and Methods. The data points represent the mean CL_int determinations, and the error bars reflect the standard deviation from the mean as shown in Table 6. The dotted boxes illustrate HLM CL_ints of 1mg¹ (low clearance) and >65 µl · min¹mg¹ (high clearance). The solid line depicts a linear regression analysis of the data (r² = 0.8, P int = 0.91 × logHLM CL_int + 0.3. The dashed line indicates line of unity.

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

The HLM CL_int determinations were carried out as described under Materials and Methods. The histograms reflect a mean CL_int, 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 CL_int.

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