ABSTRACT
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.
Subject(s)
Aryl Hydrocarbon Hydroxylases/metabolism , Mixed Function Oxygenases/metabolism , Muscarinic Agonists/pharmacokinetics , Pilocarpine/analogs & derivatives , Pilocarpine/pharmacokinetics , Cytochrome P-450 CYP2A6 , Humans , Male , Microsomes, Liver/metabolism , Muscarinic Agonists/urine , Pilocarpine/urineABSTRACT
Concentrations of pilocarpine in plasma, saliva and urine from three healthy male volunteers were measured using a fluorescence derivatisation method, following administration of a single 10 mg oral dose. Pharmacokinetic parameter values were estimated from concentration-time profiles. Linear correlations between plasma and saliva pilocarpine concentrations (r2=0.945, n=10, p<0.001; r2=0.954, n=12, p<0.001) and plasma concentrations and salivation rate (r2=0. 863, n=12, p<0.001; r2=0.862, n=15, p<0.001) were established. Pilocarpine and an unidentified metabolite, respectively 20.3% and 34.7% of the oral dose, were excreted into urine.
Subject(s)
Muscarinic Agonists/pharmacokinetics , Pilocarpine/pharmacokinetics , Saliva/metabolism , Adult , Area Under Curve , Biotransformation , Half-Life , Humans , Male , Middle Aged , Muscarinic Agonists/blood , Muscarinic Agonists/urine , Pilocarpine/blood , Pilocarpine/urine , Pilot Projects , Salivation/drug effectsABSTRACT
A method is described for the determination of pilocarpine and its degradation products isopilocarpine, pilocarpic acid and isopilocarpic acid in human plasma and urine. The method is based on a simple sample preparation step -- ultrafiltration for plasma and dilution for urine samples -- followed by a reversed-phase liquid chromatographic separation of the analytes and detection by means of tandem mass spectrometry. Parameters affecting the performance of these steps are discussed. The high sensitivity and selectivity of the method allow low ng/ml concentrations to be determined for all compounds in plasma and undiluted urine, which enables the investigation of the metabolic fate and elimination of pilocarpine after oral administration to humans.
Subject(s)
Chromatography, High Pressure Liquid/methods , Mass Spectrometry/methods , Pilocarpine/analogs & derivatives , Pilocarpine/analysis , Evaluation Studies as Topic , Humans , Pilocarpine/administration & dosage , Pilocarpine/blood , Pilocarpine/urine , UltrafiltrationABSTRACT
Two inpatients of one hospital ward died. Pilocarpine poisoning was suspected and subsequently confirmed by analysis of urine. The circumstantial evidence strongly suggested that the food given to the patients in the ward had been adulterated. Police inquiries failed to elicit any further information, and open verdicts were returned at the inquest. Precautions taken subsequently to prevent a similar event--sealing food containers and trolleys--entailed a capital cost of 43,000 pounds. In addition, food stores were kept locked and tighter control kept on drugs stored in ward pharmacies.
