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.

Pharmacokinetics and metabolism of radiolabelled SNI-2011, a novel muscarinic receptor agonist, in healthy volunteers. Comprehensive understanding of absorption, metabolism and excretion using radiolabelled SNI-2011.

The pharmacokinetics and metabolism of SNI-2011 ((+/-)-cis-2-methylspiro[1,3-oxathiolane-5,3'-quinuclidine]monohydrochloride hemihydrate, cevimeline, CAS 153504-70-2), a novel muscarinic acetylcholine receptor agonist developed for the treatment of Sjögen's syndrome, were investigated in six healthy volunteers after a single oral administration of 14C-SNI-2011. After administration, plasma concentrations of the radioactivity and SNI-2011 reached to Cmax at approximately 2 h, and then decreased with t 1/2 of 9 and 4 h, respectively. Cmax and AUC0-infinity of the radioactivity in plasma were 2.2 and 5.0 times higher than those of SNI-2011, respectively. The main excretion route of the radioactivity was urine, and 97.3% of the dose excreted in urine within 168 h, indicating that 14C-SNI-2011 was completely absorbed. The mean recoveries of the metabolites in urine at 24 h after administration were 16.0% for SNI-2011, 35.8% for SNI-2011 trans-sulfoxide (SNI-t-SO), 8.7% for SNI-2011 cis-sulfoxide, 4.1% for SNI-2011 N-oxide, furthermore, two unknown metabolites, UK-1 and UK-2, were detected 14.6% and 7.7%, respectively. LC/MS analysis and hydrolysis studies revealed that UK-1 and UK-2 were glucuronic acid conjugates of SNI-2011 and SNI-t-SO, respectively.

Pharmacokinetics of the M1-agonist talsaclidine in mouse, rat, rabbit and monkey, and extrapolation to man.

1. Talsaclidine is an M1-agonist under development for the treatment of Alzheimer's disease. The aim of the study was to investigate the absorption, distribution, metabolism and excretion (ADME) of single intravenous and oral doses of [14C]-talsaclidine in mouse, rat, rabbit and monkey. Previous data in humans showed that the drug was mainly excreted into the urine as the unchanged parent drug. The hypothesis was tested if animal data of drugs, which are mainly excreted renally, could be extrapolated to human. 2. The apparent volume of distribution at steady-state (V(ss)) was comparable in all animal species (2-5 l x kg(-1)) indicating an extensive distribution of the drug into tissues. The plasma protein binding was low and comparable in all species including man (< or = 7%). Elimination in terms of clearance was rapid-to-moderate depending on the species. The total plasma clearance (Cl) decreased in the order: mouse (128 ml x min(-1) x kg(-1))> rat (73.9) > monkey (10.6). Urinary excretion is the dominant route of excretion (> or = 86%). 3. A good correlation was achieved with human and animal data in allometric scaling of CI and V(ss). This confirms the hypothesis that renal filtration is scalable over the species and, given a comparable protein binding, animal data is predictive for man.

Metabolites of [(14)C]-5-(2-ethyl-2H-tetrazol-5-yl)-1-methyl-1,2,3, 6-tetrahydropyridine in mice, rats, dogs, and humans.

The M1 muscarine agonist, 5-(2-ethyl-2H-tetrazol-5-yl)-1-methyl-1,2, 3,6-tetrahydropyridine (Lu 25-109), is extensively metabolized in mice, rats, dogs, and humans. The metabolite profile after an oral dose of [(14)C]Lu 25-109 was determined in plasma and in urine. Lu 25-109 was metabolized by N-demethylation (Lu 25-077), N-oxidation (Lu 32-181), and N-deethylation (Lu 31-126). In addition, combined N-demethylation and N-deethylation (Lu 31-190), and formation of a pyridine derivative took place (Lu 31-102). Lu 25-109 was also oxidized to pyridinium (Lu 29-297), 3-hydroxy-pyridinium (Lu 35-080), N-deethyl-2-pyridone (Lu 35-026), and a glucuronide of a 4, 6-dihydroxy-pyridinium ("m/z 398") compounds. A glucuronide of a dihydroxylated dihydro-pyridine compound ("m/z 400") was isolated from human urine, but not fully identified. In vitro studies were undertaken to elucidate the order of formation of the metabolites. In human plasma, the concentrations of Lu 25-109 and the pharmacologically active N-demethyl metabolite (Lu 25-077) were small compared with the N-oxide (Lu 32-181) and the N-deethyl-2-pyridone (Lu 35-026) at the first sample time (0.75 h). The N-deethyl metabolite (Lu 31-126) was the major component in human plasma between 3 and 10 h postdose. The major human metabolites in urine (Lu 32-181, Lu 35-026, and Lu 31-126) and the minor metabolites (Lu 25-077, Lu 35-080, Lu 31-190, and Lu 29-297) were all present in urine from rats, dogs, and mice, whereas m/z 398 was present in only mice and humans, and Lu 31-102 in only rats. The minor human metabolite m/z 400 was not detected in mice, rats, or dogs.

A pilot study of the disposition of pilocarpine in plasma, saliva and urine after a single oral dose.

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.

Agents for the treatment of overactive detrusor. IX. Synthesis and pharmacological properties of metabolites of N-tert-Butyl-4,4-diphenyl-2-cyclopentenylamine (FK584) in human urine.

We synthesized the racemates of the five presumed metabolites (1b-f) of (S)-(--)-N-tert-butyl-4,4-diphenyl-2-cyclopentenylamine hydrochloride (FK584, S(--)-1a), a novel agent for the treatment of overactive detrusor syndrome, in order to confirm the structures of the metabolites and also to evaluate their inhibitory activity against detrusor contraction. (+/-)-N-tert-Butyl-4-(4-hydroxyphenyl)- and 4-(4-hydroxyphenyl)- and 4-phenyl-2-cyclopentenylamines (1b--e) were synthesized via 5-(4-methoxyphenyl)- and 5-(4-benzyloxy-3-methoxyphenyl)-5-phenyl-2-cyclopenten-1-one (9g, h), respectively. Compounds 1b-f prepared in this study were identical with the metabolites in human urine in gas chromatography-mass spectrometry and analytical HPLC. The inhibitory activity of compounds 1b-f against detrusor contraction in vitro induced by electrical field stimulation in guinea-pigs was less potent than that of FK584.