Received for publication August 1, 2007.
Revised December 18, 2007.
Accepted for publication December 18, 2007.

High Affinity Binding of ³[H]-Cimetidine to a Heme-Containing Protein in Rat Brain

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 presently 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 (CYP), the effects of non-selective and isoform-selective CYP inhibitors were studied. The heme inhibitor KCN and the non-selective CYP 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 CYPs 1A2, 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 CYPs, 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 CYPs, eight recombinant human CYPs and three recombinant rat CYPs did not exhibit 3HCIM binding. Inhibition by KCN and metyrapone suggests that 3HCIM binds to a heme-containing brain protein (possibly a CYP). However, results with selective CYP inhibitors, recombinant CYPs, and a CYP antibody did not identify a 3HCIM-binding CYP isoform. Finally, CC12 is a new, potent inhibitor of CYP2B6 and CYP2C19 which may be a valuable tool for CYP research.

Key words: CYP inhibition, CYP2C, cytochrome P450, cytochrome P450 catalyzed oxidations, cytochrome P450 isoforms, human CYP enzymes, imidazole drugs