Inhibitory monoclonal antibodies to human cytochrome P450 1A2: analysis of phenacetin O-deethylation in human liver

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Abstract

Human cytochrome P450 1A2 metabolizes a large number of common drugs and engages in carcinogen metabolism and activation. Baculovirus-expressed 1A2 was used to immunize mice producing hybridomas yielding monoclonal antibodies (MAbs). Three of 2050 clones assayed yielded the MAbs, MAb 26–7-5, MAb 951–-1, MAb 1812–2-4, which were specific for 1A2 as assessed by enzyme-linked immunosorbent assay and immunoblots. The three MAbs inhibited lA2-cata!ysed metabolism of phenacetin, 7-ethoxycoumarin, chlorzoxazone and phenanthrene by more than 85%. The MAbs were highly specific to 1A2 and did not inhibit 11 other human P450s. The phenancetin O-deethylation activity varied from 0.44–2.49 nmol/min/nmol P450 in eight human liver microsomes samples. MAb 26–7-5 inhibited lA2-dependent phenacetin O-deethylation in these samples by 64–84% indicating the amount of 1A2 contribution to this reaction and in addition a role for other P450s in the O-deethylation. Independent analysis of recombinant human P450s showed that 1A1, 1A2, 2A6 and 2C19 exhibited phenacetin Odeethylation activity, with 1A1 and 1A2 being the most active followed by 2C19 and 2A6. Eight other P450s were inactive towards phenacetin O-deethylation. The role of different P450 in eight liver samples was analysed with specific individual inhibitory MAbs. Inhibitory antibodies to 1A2, 2C8/9/18/19, 2A6, 2D6, 2E1, and 1A1 were combinatorially added to the microsomes. The O-deethylation activity was inhibited by antibodies to 1A2 (64–4%), to 2C19 (4.6–0%) and to 2A6 (0–8.8%). The total activity inhibited by antibodies to P450 2E1, 2D6 and 1A1 was less than 4.5%, indicating a minor role for these P450s in phenancetin metabolism in human liver microsomes. Thus, 1A2, 2C19 and 2A6 are the dominant P450s for phenacetin O-deethylation. These studies demonstrate the use of inhibitory MAbs to P450s for a simple and precise assessment of the quantitative role of each P450 in the metabolism of substrates, including drugs, carcinogens, mutagens, environmental Chemicals and endobiotics.

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