Pharmacokinetics of Compound D, the Major Bioactive Component of Zingiber cassumunar, in Rats.

Rhizomes of Zingiber cassumunar have been used for many years in traditional Thai medicine as an anti-inflammatory agent. The major bioactive component of this plant is Compound D [E-4-(3', 4'-dimethoxyphenyl)but-3-en-1-ol], which is a strong smooth muscle relaxant, and has antihistamine and anti-inflammatory actions. There is, however, incomplete information available for the pharmacokinetics of Compound D in mammals. In this study, we examined the pharmacokinetic profiles of Compound D in male Wistar rats. A standardized extract of Z. cassumunar containing 4 % w/w Compound D was administered intravenously at 25 mg/kg or by oral gavage at 25, 75, or 250 mg/kg to Wistar rats. Blood, tissues, urine, and feces were collected from 0 to 48 h after dosing and the level of Compound D was determined by liquid chromatography-tandem mass spectrometry. The concentration of Compound D ranged from 10-100 µg/L, reached a maximum approximately 0.15 h after oral dosing. Compound D exhibited an excellent tissue to plasma ratio, ranging from 1- to 1000 in several organs at 1-4 h after oral dosing. Less than 1 % of unchanged Compound D was excreted in the urine and feces. Further studies on tissue uptake and metabolite identification are required to obtain complete pharmacokinetic information and to develop appropriate dosing strategies of Compound D and the standardized extract of Z. cassumunar.

High performance liquid chromatographic determination of 3-methylflavone-8-carboxylic acid, the main active metabolite of flavoxate hydrochloride in human urine.

High performance liquid chromatographic (HPLC) method was presented for the determination of 3-methylflavone-8-carboxylic acid as the main active metabolite of flavoxate hydrochloride (FX) in human urine. The proposed method was based on using CN column with mobile phase consisting of acetonitrile-12 mM ammonium acetate (40:60, v/v) and adjusted to apparent pH 4.0 with flow rate of 1.5 ml min(-1). Quantitation was achieved with UV detection at 220 nm. The proposed method was utilized to the determination of dissolution rate for tablets containing flavoxate hydrochloride. The urinary excretion pattern has been calculated using the proposed method.

Improving the detection limits of antispasmodic drugs electrodes by using modified membrane sensors with inner solid contact.

Three coated wire electrodes (CWEs) for the antispasmodic drugs; dicyclomine (Dc), mebeverine (Mv) and drotaverine (Dv) hydrochlorides were developed. Each electrode based on ion-associate of a heteropoly anion with the drug cation incorporated in membrane sensor modified with graphite and deposited on silver internal solid contact. The influence of addition of graphite to the membranes and the type of the internal solid contact on the potentiometric responses of the electrodes was investigated. The characteristics of the new electrodes were compared to the characteristics of previously reported traditional liquid inner contact electrodes of the same drugs. The lower detection limits of the proposed electrodes were somewhat better than those observed with the corresponding liquid contact ISEs and reached (1.2-2.0)x10(-7)M. The potentiometric selectivity of the CWEs revealed a significant improvement and much faster response times compared to the liquid contact ISEs. The practical utility of each electrode has been demonstrated by using it successfully in potentiometric determination of its respective drug in pharmaceutical preparations both in batch and flow injection conditions. Each electrode was also used as an indicator electrode in the potentiometric titration of the drug against standard silicotungstic acid and in potentiometric determination of the drug concentration in urine samples.

Determination of propiverine and its metabolites in rat samples by liquid chromatography-tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometric (LC-MS-MS) method was developed and validated for the determination of the anticholinergic and antimuscarinc drug propiverine and eight of its metabolites in serum, urine, faeces and different tissue samples of rats. Samples containing propiverine and its metabolites in serum and urine and in the supernatants of faeces and tissue homogenates were extracted and cleaned up using an automated solid phase extraction (SPE) method. An external calibration was used. The analytes were measured employing the multiple reaction monitoring mode (MRM). A sufficient response over the range of 10-1000 ng/ml was demonstrated. The lower limit of quantification of the nine substances was 10 ng/ml. The presented method is suitable for pharmacokinetic or toxicokinetic studies. To look for additional unknown metabolites, the LC-MS-MS system operated in the precursor ion mode using typical product ions of propiverine and of its metabolites. With the help of the chromatographic behaviour and typical fragment ions of the unknown metabolites, it was possible to elucidate their structure. Five until now unknown metabolites were found in the urine and faeces samples. However, without reference substances, a quantification of these analytes was not possible.

Effects of antimuscarinic drugs on both urinary frequency and cognitive impairment in conscious, nonrestrained rats.

Recent studies indicate a risk of learning and memory impairments when patients with senile dementia are treated with antimuscarinic drugs. In this study, we compared the effectiveness of propiverine hydrochloride (propiverine) and oxybutynin chloride (oxybutynin) on the increased urinary frequency and cognitive impairment induced by nucleus basalis magnocellularis (nBM) lesioning in conscious and nonrestrained rats. For examination of bladder function, nBM-lesioned rats were given total parenteral nutrition regimens for 8 days. Propiverine administered orally at 0.3, 3 and 30 mg/kg on the postoperative day 7 significantly lessened the increase in the frequency of voiding caused by the nBM lesion, whereas oxybutynin administration did not show any improvement at 0.1 or 1 mg/kg but did so at 10 mg/kg. To examine the memory impairment, we trained nBM-lesioned rats in an 8-arm radial maze task for 20 days and then evaluated the effectiveness of oral drug administration on 19th and 20th radial maze performance. The higher rate of errors caused by nBM lesioning was significantly aggravated by oxybutynin at 30 and 100 mg/kg. Propiverine showed slight aggravation of errors, but with no statistical significance at any dose, 30, 100 or 300 mg/kg. These results suggest that propiverine has comparatively less effect on the cognitive impairment than oxybutynin.

[Bioavailability of phloroglucinol in man]. / Biodisponibilité du phloroglucinol chez l'homme.

This work reports a bioavailability study between two oral dosage forms containing 125.2 mg active phloroglucinol. Twelve healthy volunteers subjects received a sublingual administration of both dosage forms, a flash liberation tablet and a freeze-dried reference tablet (lyoc), according to a randomized and cross-over design. An accurate, sensitive and specific high performance liquid chromatographic method was developed for the determination of free phloroglucinol as well as its conjugated metabolites, that allowed as to clarify phloroglucinol pharmacokinetic behaviour in man, specially its important metabolisation, its poor systemic bioavailability after oral administration and its total urinary elimination mainly under metabolized form. Total plasmatic phloroglucinol pharmacokinetic profiles led to pertinent parameters needed for statistical bioequivalence study, i.e. T1/2 alpha, T1/2 beta, AUC, Tmax, Cmax and MRT. The mean comparative values of these parameters showed the equivalent performances of both oral dosage forms studied and the statistical tests performed (ANOVA, Westlake and two one-sided t test) concluded to their bioequivalence.

Determination of quinidine, dihydroquinidine, (3S)-3-hydroxyquinidine and quinidine N-oxide in plasma and urine by high-performance liquid chromatography.

A specific and sensitive method for the quantitation of quinidine, (3S)-3-hydroxyquinidine, quinidine N-oxide, and dihydroquinidine in plasma and urine has been developed. The method is based on a single-step, liquid-liquid extraction procedure, followed by isocratic reversed-phase high-performance liquid chromatography, with fluorescence detection. After extraction from 250 microliters plasma and 100 microliters urine, the limit of determination is 10 nM and 25 nM, respectively. For the use as standards, commercially available quinidine was purified from dihydroquinidine; quinidine N-oxide was synthesized.

The metabolism of mebeverine in man: identification of urinary metabolites by gas chromatography/mass spectrometry.

The urinary metabolites of orally administered mebeverine hydrochloride (270 mg) were studied in five healthy volunteers with the aid of gas chromatography/mass spectrometry. Mebeverine, which is an ester of veratric acid and 4-(ethyl-[2-(4-methoxyphenyl)-1-methylethyl]amino)butan-1-ol, was completely hydrolysed to the corresponding acid and alcohol moieties. The acid moiety was subsequently O-demethylated to vanillic acid and isovanillic acid, which in turn were further O-demethylated to protocatechuic acid. The alcohol moiety was O-demethylated to the corresponding phenol 4-(ethyl-[2-(4-hydroxyphenyl)-1-methylethyl]amino)butan-1-ol. In 24 hr, 44% of the dose was accounted for as follows: Veratric acid 32%, vanillic acid 2.7%, isovanillic acid 6.5%, 4-(ethyl-[2-(4-methoxyphenyl)-1-methylethyl]amino)butan-1-ol 0.9% and 4-(ethyl-[2-(4-hydroxyphenyl)-1-methylethyl]amino)butan-1-01 2.1%. Only trace amounts of protocatechuic acid were found in the urine. The results indicated that the metabolites were mostly excreted as conjugates. The total excretion of the acid moiety, unchanged or in the form of metabolites was 97.6%. The corresponding value for the alcohol moiety was 5.5%.

Application of radioreceptor assays for systematic toxicological analysis--1. Procedures for radioreceptor assays for antihistaminics, anticholinergics and benzodiazepines.

Radioreceptor assays can be a useful tool for systematic toxicological analysis in that they can be applied for the detection of an entire pharmacological class of drugs. In the present paper procedures for radioreceptor assays for benzodiazepines, anticholinergics and antihistaminics have been described in detail. The development of the assay for antihistaminics in urine is given in order to illustrate the prerequisites for these types of assays with regard to the incubation conditions. In part 2 the applicability of the three assays for systematic toxicological analysis will be evaluated on the basis of testing a large number of urine samples after administration of a selected number of drugs to healthy volunteers and patients.

Application of radioreceptor assays for systematic toxicological analysis--2. Theoretical considerations and evaluation.

In this paper the applicability of radioreceptor assays for systematic toxicological analysis will be evaluated on a theoretical basis as well as on the basis of the outcomes of the analysis of a large number of urine samples collected after administration of a selected number of drugs to healthy volunteers and patients. Many drugs and other substances of toxicological relevance exert their action through an interaction with one or more receptor (sub)types. Whether the number of persons are using particular drugs intentionally or unintentionally, radioreceptor assays can be a useful tool for systematic toxicological analysis in that they can be applied to the identification of entire pharmacological classes of substances as well as pharmacologically active metabolites. In part 1 of this paper detailed procedures for radioreceptor assays for benzodiazepines, anticholinergics and antihistaminics have been described in detail in order to illustrate not only the potentials but also the limitations of assay conditions. Fifteen drugs were administered to patients and volunteers and urine samples were collected and determined with the three radioreceptor assays. The results of this study underline the theoretical applicability of receptor assays in systematic toxicological analysis though sample pretreatment procedures may contribute to an improvement in sensitivity and applicability to other biofluids.

Determination of the R,R- and S,S-enantiomers of vamicamide in human serum and urine by high-performance liquid chromatography on a Chiral-AGP column.

An enantioselective liquid chromatographic assay for the determinations of the R,R- and S,S-enantiomers of vamicamide, a potent anticholinergic drug, in human serum and urine is described. Racemic vamicamide and internal standard were purified from biological fluids using a two-step extraction procedure involving diethyl ether and 0.1% phosphoric acid. The overall recoveries of racemic vamicamide and internal standard were greater than 80%. The purified samples were measured by high-performance liquid chromatography on a Chiral-AGP column with ultraviolet absorbance detection at 260 nm. The standard curves for the analytes were linear from 10 to 200 ng/ml in serum and from 0.25 to 50 micrograms/ml in urine. The quantification limit of both enantiomers was 10 ng/ml for serum and 250 ng/ml for urine. Both intra-day and inter-day accuracy and precision data showed good reproducibility of the method. The assay has been applied for the analysis of vamicamide enantiomers in serum and urine samples from a healthy volunteer.

Kinetics of propiverine as assessed by radioreceptor assay in poor and extensive metabolizers of debrisoquine.

A pharmacokinetic study with 30 mg propiverine p.o. was performed in healthy volunteers (10 males, 6 females, age 36-56 years, body weight 55-100 kg, body height 162-184 cm, Broca index 0.96-1.19). 8 of them were poor and 8 extensive metabolizers of the debrisoquine type hydroxylation polymorphism. The total anticholinergic activity of the parent compound and active metabolites was measured with a radioreceptor assay calibrated with the metabolite M2. The affinity of this metabolite to the muscarinic receptors was similar to that of atropine. The urinary excretion of 3 major metabolites was determined with TLC and densitometry. Arterial blood pressure, heart rate, diameter of pupils, accommodation and parotic salivary flow were also measured. The concentrations of anticholinergic equivalents of propiverine were below 1 ng/ml of M2. 1.4-6.0% of the dose were excreted as N-oxidized metabolites into the urine. The poor and extensive metabolizers of debrisoquine did not differ significantly with regard to the concentration time behaviour of the active drug components, pattern of major metabolites, adverse drug reactions or any pharmacodynamic parameters measured.

Determination of 3-methylflavone-8-carboxylic acid, the main metabolite of flavoxate, in human urine by capillary electrophoresis with direct injection.

The effects of tetraalkylammonium salts and sodium dodecyl sulphate on the migration behaviour of human urinary components and other negatively charged or neutral solutes were investigated. The sulphate acted mainly on hydrophobic and positively charged substances, whereas the ammonium salts acted mainly on negatively charged solutes. By choosing the components of the eluent carefully, the free and conjugate forms of 3-methylflavone-8-carboxylic acid (MFA) in human urine, the major metabolites of flavoxate, could be simultaneously determined without pretreatment, using fenprofen as an internal standard. The calibration curve of MFA was linear in the range 1-50 micrograms/ml and the detection limit was 0.2 microgram/ml, which covered the urine levels encountered in pharmacokinetic studies. The intra-day and inter-day precisions of the method, expressed as the relative standard deviation, were less than 2 and 3%, respectively. This method was successfully applied to an excretion study of MFA in eight healthy volunteers, and the results were in agreement with data in the literature obtained by gas chromatography.

Studies on the metabolic clearance of ciclotropium to alpha-phenylciclopentylacetic acid using a new enantiospecific metabolite assay.

Ester hydrolysis represents an important biotransformation pathway for various parasympatholytic agents. Cleavage of the ciclotropium ester bond results in the formation of alpha-phenylciclopentylacetic acid (PCA). The relevance of this metabolic route for ciclotropium bromide (HIT-PCE, CAS 85166-20-7) including its stereochemical aspects was studied in a preliminary pharmacokinetic study. An enantiospecific assay for biological material was developed that is based on chiral derivatization of PCA with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDAC) and the primary amine S-FLOPA, a chiral coupling component for carboxylic acids derived from S-flunoxaprofen, followed by HPLC resolution. R-(--)-Ibuprofen was used as internal standard. From plasma or urine PCA can be extracted into n-hexane/ethanol (9:1) at pH 4 under addition of sodium chloride. Derivatization with EDAC/FLOPA was performed under addition of 1-hydroxybenzotriazole in anhydrous dichloromethane that contained trace amounts of pyridine (ambient temperature; 2 h reaction time). The chromatographic separation was performed on a silica gel stationary phase (Zorbax Sil) using n-hexane-chloroform-ethanol (100:10:1, by vol.) as mobile phase (flow rate, 2 ml/min; fluorescence-detection, 305/355 nm; elution order of the derivatives, (-) before (+)). Limit of quantification was 1.0 ng/ml for plasma and 10 ng/ml for urine. In the pharmacokinetic study in two healthy volunteers who received a single i.v. dose of 10 mg ciclotropium race-mate the PCA concentrations in plasma were below the detection limit, but approx. 1.5% of the administered dose were excreted into urine as the respective glucuronides.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of the quaternary compound ciclotropium in human biological material after hydrolysis and derivatization with the fluorophor flunoxaprofen chloride.

The quantitative determination of the quaternary spasmolytic compound ciclotropium and its metabolite N-isopropyltropinium is described for human plasma and urine. The analytical procedure consists of ion-pair extraction from biological material, alkaline hydrolysis, subsequent derivatization with the fluorophor flunoxaprofen chloride and separation by high-performance liquid chromatography on a reversed-phase column with fluorimetric monitoring. The detection limits of 0.5 ng/ml in plasma and 10 ng/ml in urine at signal-to-noise ratios higher than 3 permit the determination of pharmacokinetic parameters after therapeutic doses.

Simultaneous determination of isbufylline and its major metabolites in rabbit blood and urine by reversed-phase high-performance liquid chromatography.

A sensitive high-performance liquid chromatographic assay for isbufylline and its major metabolites in rabbit blood and urine is described. After extraction, samples were eluted by a linear reversed-phase gradient. Specimens obtained after intravenous administration of isbufylline to rabbits were analysed to identify and subsequently quantify the potential metabolites. Using the ultraviolet absorption trace on the recorder as a reference, elution fractions were collected and analysed by mass spectrometry with the direct inlet system and gas chromatography-mass spectrometry after derivatization. Seven metabolites were identified and another five quantified. The method is specific, accurate, reproducible and recommended for pharmacokinetic studies.

An apparent fatal overdose of terodiline.

The results of a forensic toxicological investigation on a young man with an unknown cause of death are reported here. Analysis revealed the presence of a possibly fatal level of terodiline in blood and urine. No other drugs were detected. Terodiline was detected by thin-layer and gas-liquid chromatography and identified by gas chromatography/mass spectrometry. Quantification was carried out by a mass fragmentographic procedure using the m/z 100 from terodiline for selective ion monitoring (SIM). The blood and urine concentrations were found to be greater than 10 mg/L, whereas therapeutic concentrations in serum are usually not more than 1 mg/L. Support and confirmation of the laboratory results was provided at the subsequent inquest. It was revealed that the deceased had died from the inhalation of vomit due to an oral overdose of terodiline. To the best of our knowledge this is the first reported death due to fatal poisoning with terodiline in the United Kingdom.

Studies on the metabolic pattern of propiverine in urine after single administration.

In 11 healthy volunteers the metabolic pattern of propiverine [1; alpha, alpha-diphenyl-n-propoxy-1, 2-acetic acid-4-(1-methyl-piperidinyl)ester] was studied in urine after a single i.v. (5 mg) or oral dose (15 mg). To each dose 30.4 microCi (1.11 MBq) 14C-1 were added. The various urine fractions (0-1, 1-4, 4-8, 8-24 h) were extracted by chloroform and ethyl acetate at different pH-values and TLC was performed. The metabolites were identified by comparison of the RF-values of the radiochromatograms with those of the reference compounds after TLC using various solvent mixtures. Evidence for identity of the metabolites was additionally obtained by ester hydrolysis, ether cleavage or reduction with subsequent TLC after elution of the spots from the plate. The formed products were rechromatographed. 1 undergoes an extensive biotransformation: about 70% of the radioactive substances in urine consisted of 19 different metabolites, while 1 amounted to only 3%. Additionally, 3 acidic metabolites of unknown structure were isolated. Due to the metabolic pattern the following reactions of degradation were found: oxidation of the tertiary nitrogen in the piperidinyl moiety yielding N-oxides (40 to 50% of radioactivity), oxidation of one of the three carbon atoms of the propyl side chain, oxidation of the N-methyl group resulting in N-demethylated products, and ester cleavage. Propiverine N-oxide (20 to 25%) was determined as a major metabolite, whereas demethylated products occurred in minute amounts (1%). There was no evidence for oxidation of both phenyl moieties.

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.