A monoclonal antibody inhibitory to human P450 2D6: a paradigm for use in combinatorial determination of individual P450 role in specific drug tissue metabolism


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Abstract

Human cytochrome F450 2D6 metabolizes more than 50 common drugs and is polymorphically expressed, with 5-10% of the population lacking expression caused by mutant genes. This may result in a defective and toxic response in deficient individuals treated with 2D6 drug substrates. Baculovirus-expressed 2D6 was used to immunize mice for hybridoma production and two clones yielded monoclonal antibodies, that were positive against 2D6 by ELISA and inhibited 2D6 catalysed metabolism of bufuralol, dextromethorphan and phenanthrene by more than 90%. The inhibitory activity was highly specific to 2D6 and the monoclonal antibodies did not bind to 11 other P450s, nor inhibit seven human P450s tested. Analysis of eight human liver microsome samples showed that their basal bufuralol I'-hydroxylase activity varied from 6.7-83.5 pmol min-1 nmol-1 P450. The monoclonal antibody 512-1-8 inhibited 2D6-dependent bufuralol I'-hydroxylase in these samples by 10-70% indicating a widely variable role for 2D6 in human liver bufuralol I'-hydroxylase activity and a role for other P450s in bufuralol metabolism. Independent analysis of several recombinant human P450s showed that 2D6, 2C8, 2C9, 2C19 and 1A2 exhibited bufuralol I'-hydroxylase activity with 2D6 and 2C19 being the most active. Further analysis of three liver samples was made with individual inhibitory monoclonal antibodies. Inhibitory antibodies to 2D6, 2B6, 2111, 2C8/9/19, 3A4 and 1A2 were added to the microsomes either singly or additively. Inhibitory activity of bufuralol I'-hydroxylase was observed with antibodies to 2D6 (14-76%), 2C8/9/19 (24-69%) and 1A2 (2-25%) indicating a variable and different role for each of these P450s in the bufuralol I'-hydroxylase of human liver. The monoclonal antibodies to 2B6, 2E1 and 3A4 were not inhibitory, indicating that these enzymes play no role in bufuralol I'-hydroxylase metabolism. When the three antibodies to 2D6, 2C8/9/19 and 1A2, respectively, were all added, the total bufuralol I'-hydroxylase of the liver samples was inhibited by more than 90%, indicating that the latter P450s catalyse all of liver bufuralol I'-hydroxylase metabolism. These studies demonstrate that inhibitory monoclonal antibodies offer a simple and precise method for assessing the quantitative role of each P450 in the metabolism of a P450 substrate in a tissue, which include drugs, carcinogens, mutagens, toxic chemicals and endobiotics.

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