Involvement of CYP2A6 in the formation of a novel metabolite, 3-hydroxypilocarpine, from pilocarpine in human liver microsomes.

Pilocarpine is a cholinergic agonist that is metabolized to pilocarpic acid by serum esterase. In this study, we discovered a novel metabolite in human urine after the oral administration of pilocarpine hydrochloride, and we investigated the metabolic enzyme responsible for the metabolite formation. The structure of the metabolite was identified as 3-hydroxypilocarpine by liquid chromatography-tandem mass spectrometry and NMR analyses and by comparing to the authentic metabolite. To clarify the human cytochrome P450 (P450) responsible for the metabolite formation, in vitro experiments using P450 isoform-selective inhibitors, cDNA-expressed human P450s (Supersomes; CYP1A2, -2A6, -2B6, -2C9, -2C19, -2D6, -2E1, and -3A4), and liver microsomes from different donors were conducted. The formation of 3-hydroxypilocarpine in human liver microsomes was strongly inhibited (>90%) by 200 microM coumarin. Other selective inhibitors of CYP1A2 (furafylline and alpha-naphthoflavone), CYP2C9 (sulfaphenazole), CYP2C19 [(S)-mephenytoin], CYP2E1 (4-methylpyrazole), CYP2D6 (quinidine), and CYP3A4 (troleandomycin) had a weak inhibitory effect (<20%) on the formation. The highest formation activity was expressed by recombinant CYP2A6. The K(m) value for recombinant CYP2A6 was 3.1 microM, and this value is comparable with that of human liver microsomes (1.5 microM). The pilocarpine 3-hydroxylation activity was correlated with coumarin 7-hydroxylation activity in 16 human liver microsomes (r = 0.98). These data indicated that CYP2A6 is the main enzyme responsible for the 3-hydroxylation of pilocarpine. In conclusion, we identified a novel metabolite of pilocarpine, 3-hydroxypilocarpine, and we clarified the involvement of CYP2A6 in the formation of this molecule in human liver microsomes.

Involvement of adenosine in depression of synaptic transmission during hypercapnia in isolated spinal cord of neonatal rats.

Adenosine is one of the most important neuromodulators in the CNS, both under physiological and pathological conditions. In the isolated spinal cord of the neonatal rat in vitro, acute hypercapnic acidosis (20% CO2, pH 6.7) reversibly depressed electrically evoked spinal reflex potentials. This depression was partially reversed by 8-cyclopentlyl-1,3-dimethylxanthine (CPT), a selective A1 adenosine receptor antagonist. Isohydric hypercapnia (20% CO2, pH 7.3), but not isocapnic acidosis (5% CO2, pH 6.7), depressed the reflex potentials, which were also reversed by CPT. An ecto-5'-nucleotidase inhibitor did not affect the hypercapnic acidosis-evoked depression. An inhibitor of adenosine kinase, but not deaminase, mimicked the inhibitory effect of hypercapnic acidosis on the spinal reflex potentials. Accumulation of extracellular adenosine and inhibition of adenosine kinase activity were caused by hypercapnic acidosis and isohydric hypercapnia, but not isohydric acidosis. These results indicate that the activation of adenosine A1 receptors is involved in the hypercapnia-evoked depression of reflex potentials in the isolated spinal cord of the neonatal rat. The inhibition of adenosine kinase activity is suggested to cause the accumulation of extracellular adenosine during hypercapnia.