Eight inhibitory monoclonal antibodies define the role of individual P-450S in human liver microsomal diazepam, 7-ethoxycoumarin, and imipramine metabolism

Eight inhibitory monoclonal antibodies (MAbs) individually specific to huma n cytochrome P-450 (P-450) 1A1, 1A2, 2A6, 2B6, 2C subfamily (2C8, 2C9, 2C18 and 2C19), 2D6, 2E1, and 3A4/5 were used to define the role of single P-45 0s in the metabolism of diazepam (DZ), 7-ethoxycoumarin (7-EC), and imipram ine (IMI) in human liver microsomes (HLM). The MAbs were added combinatoria lly to six HLM samples. With DZ as a substrate, more than 80% of temazepam (TMZ) formation was inhibited in all six samples by the addition of MAb to 3A4/5, indicating an 80% contribution of 3A4/5 to TMZ formation. Nordiazepa m formation was inhibited with MAbs to 2B6 (6-23%), 2C subfamily (12-61%) a nd 3A4/5 (14-45%). The MAbs to 1A1, 1A2, 2A6, 2D6, and 2E1 did not inhibit TMZ or nordiazepam formation; this indicates their noninvolvement in DZ met abolism. The MAb-defined P-450 contribution to 7-EC O-deethylation in six H LM samples was 17 to 60% for 2E1, 15 to 46% for 2A6, and 5 to 22% for 1A2, reflecting the role and variation of each P-450 in this activity. MAbs to 1 A1,the 2C subfamily, 2D6, and 3A4/5 did not affect 7-EC metabolism in the H LM samples. IMI is metabolized mainly to 2-hydroxyimipramine by expressed 2 C19 and 2D6, and desipramine (DIM) by expressed 1A2, 2C18, 2C19 and 2D6. Ex pressed 1A1, 2C9, and 3A4 showed low activities for the formation of DIM. O f six HLM samples, five showed IMI hydroxylation activity (0.35-2.6 nmol/mi n/nmol P-450) while one (HL43) lacked hydroxylation activity. All six HLM s amples showed N-deethylation activity (0.74-1.4 nmol/min/nmol P-450). The M Ab-determined contribution of 2D6 and 2C19 to 2-hydroxyimipramine formation ranged from 47 to 90% and from 0 to 49%, respectively, while HL43 did not show P-hydroxylation. The role of P-450s involved in DIM formation varied f or 2C19 (13-50%), 1A2 (23-41%), and 3A4 (8-26%). These studies demonstrate a system for identifying the quantitative metabolic role of single P-450s a nd their interindividual variability in a tissue containing multiple P-450s . The system using inhibitory MAbs is simple, precise, and applicable to an y P-450-mediated catalytic activity including that for drugs, carcinogens, mutagens, toxic chemicals and endobiotics.