A MONOCLONAL-ANTIBODY INHIBITORY TO HUMAN P450 2D6 - A PARADIGM FOR USE IN COMBINATORIAL DETERMINATION OF INDIVIDUAL P450 ROLE IN SPECIFIC DRUG TISSUE METABOLISM

Human cytochrome P450 2D6 metabolizes more than 50 common drugs and is polymorphically expressed, with 5-10% of the population lacking expre ssion caused by mutant genes. This may result in a defective and toxic response in deficient individuals treated with 2D6 drug substrates. B aculovirus-expressed 2D6 was used to immunize mice for hybridoma produ ction and two clones yielded monoclonal antibodies, that were positive against 2D6 by ELISA and inhibited 2D6 catalysed metabolism of bufura lol, dextromethorphan and phenanthrene by more than 90%. The inhibitor y activity was highly specific to 2D6 and the monoclonal antibodies di d not bind to 11 other P450s, nor inhibit seven human P450s tested. An alysis of eight human liver microsome samples showed that their basal bufuralol 1'-hydroxylase activity varied from 6.7-83.5 pmol min(-1) nm ol(-1) P450. The monoclonal antibody 512-1-8 inhibited 2D6-dependent b ufuralol 1'-hydroxylase in these samples by 10-70% indicating a widely variable role for 2D6 in human liver bufuralol 1'-hydroxylase activit y and a role for other P450s in bufuralol metabolism. Independent anal ysis of several recombinant human P450s showed that 2D6, 2C8, 2C9, 2C1 9 and 1A2 exhibited bufuralol 1'-hydroxylase activity with 2D6 and 2C1 9 being the most active. Further analysis of three liver samples was m ade with individual inhibitory monoclonal antibodies. Inhibitory antib odies to 2D6, 2B6, 2E1, 2C8/9/19, 3A4 and 1A2 were added to the micros omes either singly or additively. Inhibitory activity of bufuralol 1'- 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 eac h of these P450s in the bufuralol 1'-hydroxylase of human liver. The m onoclonal antibodies to 2B6, 2E1 and 3A4 were not inhibitory, indicati ng that these enzymes play no role in bufuralol 1'-hydroxylase metabol ism. When the three antibodies to 2D6, 2C8/9/19 and 1A2, respectively, were all added, the total bufuralol 1'-hydroxylase of the liver sampl es was inhibited by more than 90%, indicating that the latter P450s ca talyse all of liver bufuralol 1'-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, carc inogens, mutagens, toxic chemicals and endobiotics.