An inhibitory monoclonal antibody to human cytochrome P450 2A6 defines itsrole in the metabolism of coumarin, 7-ethoxycoumarin and 4-nitroanisole inhuman liver

Cytochrome P450 (CYP) 2A6 is an important enzyme catalysing the metabolism of many drugs, procarcinogens and promutagens. Its role in human liver meta bolism of coumarin, 4-nitroanisole, 4nidrophenol and 7-ethoxycoumarin was a nalysed with an inhibitory monoclonal antibody (MAb) to CYP2A6. MAbs were d erived from a panel of 16 hybridomas which yielded positive enzyme-linked i mmunosorbent assay (ELISA) results or Immunoblots against CYP2A6. The hybri domas were selected from more than 500 clones generated by the fusion of my eloma cells with spleen cells of mice immunized with purified baculovirus-e xpressed human CYP2A6. The MAbs obtained from four of the 16 hybridomas exh ibited strong inhibitory activity to CYP2A6-catalysed phenanthrene metaboli sm. MAb 151-45-4 was positive and highly specific to CYP2A6 as determined b y ELISA and immunoblot, and showed no cross-reactivity with recombinant hum an CYP 1A1, 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5, as tested with ELISA and immunoblot analyses. MAb 151-45-4 specifically inhibited CYP2A6- catalysed metabolism of phenanthrene, 4-nitroanisole, 4-nitrophenol, coumar in and 7-ethoxycoumarin each by 94-99% and did not inhibit their metabolism catalysed: by 10 other human CYPs. The potent inhibitory effect of MAb 151 -45-4 was used to define the contribution of human CYP2A6 to the metabolism of coumarin, 4-nitroanisole and 7-ethoxycoumarin in seven human liver micr osome samples. Coumarin metabolism in all of the seven samples was inhibite d by greater than 94% by MAb 151-45-4 which indicates that essentially all microsome mediated coumarin metabolism in human liver is catalysed only by CYP2A6. Inhibition of 4-nitroanisole and 7-ethoxycoumarin metabolism by ant i 2A6 MAb ranged from 22-65% and 8-24%, respectively. The degree of inhibit ion defines the contribution of CYP2A6 activity to the 4-nitroanisole and 7 -ethoxycoumarin metabolism in human liver and the range reflects the variab ility among samples. The inhibitory antibody to CYP2E1 was used to determin e its role in 4-nitroanisole and 7-ethoxycoumarin metabolism in seven human liver samples. The addition of both MAbs to CYP2A6 and 2E1 to the microsom e samples defined combinatorially the relative role of CYP2A6 and 2E1 in th e metabolism of 4-nitroanisole and 7-ethoxycoumarin. Pharmacogenetics 9:229 -237 (C) 1999 Lippincott Williams & Wilkins.