Liquid chromatography-electrospray ionization ion trap mass spectrometry for analysis of in vivo and in vitro metabolites of scopolamine in rats.

In vivo and in vitro metabolism of scopolamine is investigated using a highly specific and sensitive liquid chromatography-mass spectrometry (LC-MSn) method. Feces, urine, and plasma samples are collected individually after ingestion of 55 mg/kg scopolamine by healthy rats. Rat feces and urine samples are cleaned up by a liquid-liquid extraction and a solid-phase extraction procedure (C18 cartridges), respectively. Methanol is added to rat plasma samples to precipitate plasma proteins. Scopolamine is incubated with homogenized liver and intestinal flora of rats in vitro, respectively. The metabolites in the incubating solution are extracted with ethyl acetate. Then these pretreated samples are injected into a reversed-phase C18 column with mobile phase of methanol-ammonium acetate (2 mM, adjusted to pH 3.5 with formic acid) (70:30, v/v) and detected by an on-line MSn system. Identification and structural elucidation of the metabolites are performed by comparing their changes in molecular masses (DeltaM), retention-times and full scan MSn spectra with those of the parent drug. The results reveal that at least 8 metabolites (norscopine, scopine, tropic acid, aponorscopolamine, aposcopolamine, norscopolamine, hydroxyscopolamine, and hydroxyscopolamine N-oxide) and the parent drug exist in feces after administering 55 mg/kg scopolamine to healthy rats. Three new metabolites (tetrahydroxyscopolamine, trihydroxy-methoxyscopolamine, and dihydroxy-dimethoxyscopolamine) are identified in rat urine. Seven metabolites (norscopine, scopine, tropic acid, aponorscopolamine, aposcopolamine, norscopolamine, and hydroxyscopolamine) and the parent drug are detected in rat plasma. Only 1 hydrolyzed metabolite (scopine) is found in the rat intestinal flora incubation mixture, and 2 metabolites (aposcopolamine and norscopolamine) are identified in the homogenized liver incubation mixture.

Structural elucidation of in vivo and in vitro metabolites of anisodine by liquid chromatography-tandem mass spectrometry.

Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESIMSn) was employed to investigate the in vivo and in vitro metabolism of anisodine. Feces, urine and plasma samples were collected after ingestion of 20 mg anisodine to healthy rats. Feces and urine samples were cleaned up by liquid-liquid extraction and solid-phase extraction procedures (C18 cartridges), respectively. Methanol was added to plasma samples to precipitate plasma proteins. Anisodine was incubated with homogenized liver and intestinal flora of rats in vitro, respectively, followed by extraction with ethyl acetate. LC-MSn was used for the separation and identification of the metabolites using C18 column with mobile phase of methanol/0.01% triethylamine solution (2 mM, adjusted to pH 3.5 with formic acid) (60:40, v/v). The results revealed that five metabolites (norscopine, scopine, alpha-hydroxytropic acid, noranisodine and hydroxyanisodine) and the parent drug existed in feces. Three new metabolites (dimethoxyanisodine, tetrahydroxyanisodine and trihydroxy-methoxyanisodine) were identified in urine. Four metabolites (norscopine, scopine, hydroxyanisodine and anisodine N-oxide) and the parent drug were detected in plasma. Two hydrolyzed metabolites (scopine and alpha-hydroxytropic acid) were found in rat intestinal flora incubation mixture, and two metabolites (aponoranisodine and anisodine N-oxide) were identified in homogenized liver incubation mixture.

A dose-ranging study of tiotropium delivered via Respimat Soft Mist Inhaler or HandiHaler in COPD patients.

This was a multicenter, randomized, double-blind within device, parallel-group, dose-ranging study. COPD patients (n = 202; 86% male; mean age: 61 years) were randomized to receive tiotropium 1.25 microg, 2.5 microg, 5 microg, 10 microg, or 20 microg Respimat SMI (a novel, propellant-free device); tiotropium 18 microg HandiHaler; placebo Respimat; or placebo HandiHaler for 3 weeks. The primary endpoint was trough FEV1 on Day 21. Other assessments included FVC, PEFR, rescue medication use, safety, and pharmacokinetics. In general, all active treatments improved the primary and secondary endpoints on Day 21 (steady state) compared with placebo. Tiotropium 5 microg Respimat, 20 microg Respimat, and tiotropium 18 microg HandiHaler were statistically significantly higher than placebo for the primary endpoint (mean change in trough FEV1 was 150 mL (both Respimat doses) versus 20 mL (placebo Respimat); p < 0.05; and 230 mL (HandiHaler) versus -90 mL (placebo HandiHaler); p < or = 0.001). The urinary excretion (up to 2 hours post-dose) of tiotropium 5-10 microg Respimat was comparable with tiotropium 18 microg HandiHaler; the overall incidence of adverse events was comparable across treatment groups. Tiotropium 5 and 10 microg Respimat improve lung function in COPD patients and appear to be comparable with tiotropium 18 microg HandiHaler.

Pharmacokinetics of intravenous, single-dose tiotropium in subjects with different degrees of renal impairment.

Tiotropium, a new potent anticholinergic bronchodilator, is excreted mainly by the kidney. To investigate the pharmacokinetics of tiotropium in renal impairment, the authors evaluated the pharmacokinetics and safety after administration of a single dose of intravenous tiotropium 4.8 microg, given as an infusion over 15 minutes in subjects with normal renal function and a wide range of renal impairment based on measured creatinine clearance (normal: > 80 mL/min, n = 6; mild impairment: > 50-80 mL/min, n = 5; moderate impairment: 30-50 mL/min, n = 7; severe impairment: < 30 mL/min, n =6). As expected for a drug excreted predominantly in unchanged form by the kidneys, tiotropium plasma concentrations increased as renal impairment worsened, with mean values of 55.5 (16.2 percent geometric coefficient of variation [%gCV]), 77.1 (20.1 %gCV), 101 (29.8 %gCV), and 108 (27.3 %gCV) pgh/mL for AUC(0-4h) in the normal renal function and the mild, moderate, and severe renal impairment groups, respectively. The percentage of tiotropium dose excreted unchanged in the urine decreased from 60.1% of dose (17.7 %gCV) to 59.3% (14.4 %gCV), 39.9% (34.5 %gCV), and 37.4% (10.2 %gCV) in the normal renal function and the mild, moderate, and severe renal impairment groups, respectively. Plasma protein binding of tiotropium did not significantly change in the renal-impaired subjects. Two subjects with normal renal function experienced headache 10 hours after the infusion, which was mild and transient. No adverse events occurred in subjects with renal impairment. There were no clinically relevant changes in blood pressure, pulse rate, 12-lead ECG, physical examination, hematology, or clinical chemistry, compared with baseline values, in any subject after intravenous administration of tiotropium. Tiotropium should only be used in patients with moderate to severe renal insufficiency if the potential benefit outweighs the potential risks.

A sensitive radioreceptor assay for determining scopolamine concentrations in plasma and urine.

A sensitive and reliable procedure for the quantitation of low picogram levels of scopolamine in plasma and urine is described. The method consists of two steps, a preparative extraction step using C18 columns (Sep-Pak), followed by an analytical quantitation step involving a muscarinic radioreceptor assay. The extraction efficiency of the C18 columns was 85-95% for both plasma and urine over a wide concentration range. When [3H]methyl scopolamine is used as a tracer, the assay can detect picogram concentrations (greater than 25 pg) of scopolamine (base) in plasma and urine. The applicability of the procedure for therapeutic drug monitoring of scopolamine was demonstrated by using the method to determine plasma levels in humans after transdermal administration.

Discontinuous oral absorption of cimetropium bromide, a new antispasmodic drug.

The pharmacokinetic profiles of cimetropium bromide, after either intravenous injection of 10 mg or oral ingestion of 200 mg, were determined in eight healthy volunteers. After intravenous administration, the plasma levels and urinary excretion indicated that the drug is distributed and eliminated at a rapid rate (terminal half-life, 50 +/- 8 min) and that urinary excretion is not the exclusive route of elimination (46 +/- 2%) of the administered dose). After oral administration, a low percentage of the drug is absorbed (1-4% of the administered dose), however, the amount is sufficient for therapeutic effect. The absorption is discontinuous, with two distinct phases, and ends abruptly during the second phase.

[Biochemical studies with oxitropium bromide. 1. Pharmacokinetics and metabolism in the rat and dog]. / Biochemische Untersuchungen mit Oxitropiumbromid. 1. Pharmakokinetik und Metabolismus in Ratte und Hund.

The bronchospasmolytic drug (8r)-6 beta, 7 beta-epoxy-8-ethyl-3 alpha-[(-)-tropoyloxy]-1 alpha H, 5 alpha H-tropanium bromide (oxitropium bromide, Ba 253 BR, Ventilat) was tested pharmacokinetically as a 14C labelled substance in rats and dogs. Following oral administration low concentrations of radioactivity persisting over several hours were measured in the blood of dogs and rats. The active ingredient which can be separated from the metabolites by thin layer chromatography and quantified via the radioactivity reaches a maximum in the rat plasma after 1 to 2 h; it is then eliminated from the blood with a half-life of approx. 4 h. Following intravenous administration the radioactivity measured directly (active ingredient + metabolites) is distributed rapidly into the tissue of the rat and the dog. The distribution phase is followed by a relatively fast elimination phase ending in the terminal elimination phase approx. 1 h after administration. Rats and dogs eliminate the radioactivity mainly with the feces after oral administration, whereas following intravenous administration the rat eliminates about half with the feces and half via the kidneys. Biliary excretion of the rat is 12% after oral and 14% after intravenous administration. The rat absorbs 14% and the dog 28% of dose. Five metabolites have been demonstrated in the urine of the rat and the dog. Metabolism takes place exclusively in the tropaic acid part of the molecule and by hydrolysis of the compound.

[Biochemical studies with oxitropium bromide. 2. Pharmacokinetics and metabolism in humans]. / Biochemische Untersuchungen mit Oxitropiumbromid. 2. Pharmakokinetik und Metabolismus im Menschen.

The present paper reports on the human pharmacokinetics of (8r)-6 beta, 7 beta-epoxy-8-ethyl-3 alpha-/(-)-tropoyloxy/-1 alpha H, 5 alpha H-tropanium bromide (oxitropium bromide, Ba 253 BR, Ventilat) after intravenous and oral administration as well as following inhalation. The 14C-labelled substance was used. The concentrations of radioactivity measured in the plasma after i.v. administration show a biphasic course, a rapid alpha phase and a terminal phase (t 1/2 = 1.5 h). Once the alpha phase has passed the radioactivity concentrations measured after i.v. administration of 1 mg are comparable with those after 20 mg administered orally. The concentration course after inhalation corresponds essentially to the course after oral administration of lower doses. The cumulative renal excretion of the radioactivity is 68-78% for i.v. administration, 13% for oral administration, and 10% for inhalation. 7% (i.v.), 77% (p.o.) and 88% (inhalation) is excreted in the faeces. Oxitropium bromide is rapidly hydrolysed after oral administration. As little as 4 h later only 2-3% of intact active ingredient is found, whereas there is 85% of the hydrolysed product in the urine. A similar distribution pattern is observed in urine samples taken later. Some other metabolites are also recorded in minimal quantities. After i.v. administration, too, the hydrolysed product is excreted as the main component.