COMPETITIVE-INHIBITION OF COUMARIN 7-HYDROXYLATION BY PILOCARPINE ANDITS INTERACTION WITH MOUSE CYP 2A5 AND HUMAN CYP 2A6

1 We have shown earlier that pilocarpine strongly inhibits mouse and h uman liver coumarin 7-hydroxylase activity of CYP 2A and pentoxyresoru fin O-deethylase activity of CYP 2B in vitro. Since pilocarpine, like coumarin, contains a lactone structure we have studied in more detail its inhibitory potency on mouse and human liver coumarin 7-hydroxylati on. 2 Pilocarpine was a competitive inhibitor of coumarin 7-hydroxlase in vitro both in mouse and human liver microsomes although it was not a substrate for CYP 2A5. K-i values were similar, 0.52 +/- 0.22 mu M in mice and 1.21 +/- 0.51 mu M in human liver microsomes. 3 Pilocarpin e induced a type II difference spectrum in mouse, human and recombinan t CYP 2A5 yeast cell microsomes, with K-a values of 3.7 +/- 1.6, 1.6 /- 1.1 and 1.5 +/- 0.1 mu M, respectively. 4 Increase in pH of the inc ubation medium from pH 6 to 7.5 increased the potency of inhibition of coumarin 7-hydroxylation by pilocarpine. 5 Superimposition of pilocar pine and coumarin in such a way that their carbonyls, ring oxygens and the H-7' of coumarin and N-3 of pilocarpine overlap yielded a common molecular volume of 82%. 6 The results indicate that pilocarpine is a competitive inhibitor and has a high affinity for mouse CYP 2A5 and hu man CYP 2A6. In addition the immunotype nitrogen of pilocarpine is coo rdinated towards the haem iron in these P450s.