Abstract

[³H]Cimetidine (3HCIM) specifically binds to an unidentified site in the rat brain. Because recently described ligands for this site have pharmacological activity, 3HCIM binding was characterized. 3HCIM binding was saturable, heat-labile, and distinct from the histamine H₂ receptor. To test the hypothesis that 3HCIM binds to a cytochrome P450 (P450), the effects of nonselective and isoform-selective P450 inhibitors were studied. The heme inhibitor KCN and the nonselective P450 inhibitor metyrapone both produced complete, concentration-dependent inhibition of 3HCIM binding (K_i = 1.3 mM and 11.9 μM, respectively). Binding was largely unaffected by inhibitors of CYP1A2, 2B6, 2C8, 2C9, 2D6, 2E1, and 19A1 but was eliminated by inhibitors of CYP2C19 (tranylcypromine) and CYP3A4 (ketoconazole). Synthesis and testing of CC11 [4(5)-(benzylthiomethyl)-1H-imidazole] and CC12 [4(5)-((4-iodobenzyl)-thiomethyl)-1H-imidazole] confirmed both drugs to be high-affinity inhibitors of 3HCIM binding. On recombinant human P450s, CC12 was a potent inhibitor of CYP2B6 (IC₅₀ = 11.7 nM), CYP2C19 (51.4 nM), and CYP19A1 (140.7 nM) and had a range of activities (100–494 nM) on nine other isoforms. Although the 3HCIM binding site pharmacologically resembles some P450s, eight recombinant human P450s and three recombinant rat P450s did not exhibit 3HCIM binding. Inhibition by KCN and metyrapone suggests that 3HCIM binds to a heme-containing brain protein (possibly a P450). However, results with selective P450 inhibitors, recombinant P450 isoforms, and a P450 antibody did not identify a 3HCIM-binding P450 isoform. Finally, CC12 is a new, potent inhibitor of CYP2B6 and CYP2C19 that may be a valuable tool for P450 research.