Micellar electrokinetic capillary chromatography method for direct determination of glucuronides of entacapone and its (Z)-isomer in human urine.

This paper describes the validation of a micellar electrokinetic capillary chromatography method for the direct determination of the 3-O-glucuronides of entacapone and its (Z)-isomer, the main urinary metabolites of entacapone in humans. Entacapone is a novel drug which, as a potent inhibitor of catechol-O-methyltransferase (COMT), is used as an adjunct in the standard therapy of Parkinson's disease. The 3-O-glucuronide of another COMT inhibitor, nitecapone, was used as internal standard (I.S.). The validation experiments were performed by using spiked urine samples that were extracted with Sep-Pak C18 cartridges before analysis. Determinations were carried out in a buffer of pH 7.0 containing 25 mM of phosphate, 50 mM of borate and 20 mM of sodium dodecyl sulfate, and by applying 15 kV over a 67 cm (60 cm to the detector) x 75 microns fused-silica capillary. UV detection was at 335 nm. The validity of the method was assessed by investigating the identity of the analytes, selectivity, limit of quantitation, linearity, within-day precision, extraction recovery, between-day precision and accuracy, electroosmotic flow stability and analyte stability. The method proved to be reproducible, sufficiently selective and accurate. Extraction recoveries of the analytes were > 94%. The limit of quantitation (LOQ) was 2 micrograms/ml and the assay was linear in the range 2-150 micrograms/ml with correlation coefficients better than 0.999 for both glucuronides. The repeatability of the method, expressed as the ratio of corrected peak area of the analytes to that of I.S., gave RSD values of < 5% even at the LOQ. Between-day precision (RSD) was < 7.5% for both glucuronides at 7.5 micrograms/ml. Determination of the glucuronide concentrations in urine samples of 34 patients treated with entacapone either orally (200 mg) or intravenously (25 mg) showed the method to be suitable for monitoring the concentrations of the glucuronide of entacapone after both oral and intravenous administration and those of the glucuronide of its (Z)-isomer after oral administration. The limited long term stability of the system requires, however, frequent recalibration in applications involving long sample series.

Separation of the glucuronides of entacapone and its (Z)-isomer in urine by micellar electrokinetic capillary chromatography.

A micellar electrokinetic capillary chromatography (MECC) method was developed for the separation of the 3-O-glucuronides of entacapone and its (Z)-isomer, the two main urinary metabolites of entacapone in humans. Entacapone is a novel, potent inhibitor of catechol-O-methyltransferase (COMT) intended for use as an adjunct in the treatment of Parkinson's disease. Urine samples spiked with synthetic 3-O-glucuronides were used to study the effects of running buffer pH, composition and applied voltage on separation of the closely migrating glucuronides. The 3-O-glucuronide of nitecapone, was used as internal standard. The greatest improvement in separation was achieved by increasing the running buffer ionic concentration. Changes in pH had little effect on the separation, whereas increase in sodium dodecyl sulfate (SDS) concentration slightly improved resolution. Baseline separation and good selectivity relative to urine components were achieved by using a phosphate (25 mM)-borate (50 mM)-SDS (20 mM) running buffer, pH 7.0, in a 75 microm x 60/67 cm fused-silica capillary at 15 kV and a 335 nm cut-off filter in the UV detector. The limits of detection (LOD) at a signal-to-noise ratio of 3 were about 0.25 microg/ml (5.2 x 10(-7) M) (injection 0.5 p.s.i./8 s). The linear detection range was 2-100 microg/ml (r2>0.999). Good repeatability of injection and relative migration times were obtained.

Automated analysis of levosimendan in human plasma by on-line dialysis and liquid chromatography.

A fully automated method for quantitation of levosimendan, (R)-(-)-[[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl) phenyl]hydrazono]propanedinitrile, in human plasma is described. The method involves on-line dialysis of the samples, trace enrichment of the dialysates, and reversed-phase high-performance liquid chromatography with UV detection at 380 nm. An internal standard was used to compensate for variations in the dialysis rate caused by temperature fluctuations. The precision and accuracy of the method were good. The between-day variation (RSD) was 2.7% at a plasma concentration of 15 ng/mL and 1.7% at 450 ng/mL. The limit of quantitation was 5 ng/mL with an RSD of 4.0%. The completion time of the assay was 19 min.

COMT inhibition by entacapone does not affect growth hormone or prolactin secretion in healthy volunteers.

We studied the effects of entacapone, a novel inhibitor of the enzyme catechol-O-methyltransferase (COMT), on spontaneous and levodopa (LD) modulated secretion of growth hormone (GH) and prolactin (PRL) in 12 healthy male volunteers. The study had a double-blind, cross-over design with two experimental settings. In the first setting the subjects received a single oral dose of 400 mg of entacapone or matching placebo in a randomized order. In the second setting, a single oral dose of 300 mg of LD and 75 mg of carbidopa was administered concomitantly with either 400 mg of entacapone or matching placebo in a randomized order. Entacapone had no effect on resting levels of GH, but PRL concentrations in plasma were slightly lower after entacapone than after placebo. As expected, LD/carbidopa increased the concentration of GH and decreased that of PRL. The effects of LD were not influenced by concomitant administration of entacapone. Compared with the administration of LD/carbidopa together with placebo, concomitant administration of entacapone increased the AUC of LD by 29% and reduced the AUC of 3-O-methyldopa (a metabolite of LD produced by COMT) by 69%. Entacapone appears not to enhance the effects of LD on hypothalamic-pituitary function, although the LD dose used may have been bigger than optimal for detection of a small modulatory influence.

Enantiomeric bioanalysis of simendan and levosimendan by chiral high-performance liquid chromatography.

rac-Simendan, (+/-)-(R,S)-[[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)-phenyl ] hydrazono]propanedinitrile, and the levorotatory enantiomer levosimendan, are drug candidates intended for the treatment of congestive heart failure. An enantiospecific high-performance liquid chromatographic (HPLC) method suitable for determination of the ratio of the enantiomer concentrations in blood plasma samples was developed. Direct resolution of the enantiomers was achieved by using a chiral beta-cyclodextrin stationary phase in reversed phase mode. With an eluent containing 24-33% of methanol in a 0.5% (v/v) triethylammonium acetate buffer, pH 6.0, and a flow rate of 1 ml/min, a resolution (1.2-1.6) adequate for the determinations was achieved. By using UV detection, the relative concentration of the enantiomers in plasma was assessed down to 10 ng/ml. For the racemate, the results indicated a slightly enantioselective disposition and plasma protein binding in rat, dog, and man. The pure enantiomer, levosimendan, was found not to isomerize in vivo.

Effect of entacapone, a COMT inhibitor, on the pharmacokinetics and metabolism of levodopa after administration of controlled-release levodopa-carbidopa in volunteers.

We studied the effect of entacapone, a catechol-O-methyltransferase (COMT) inhibitor, on the pharmacokinetics and metabolism of levodopa after administration of a controlled-release (CR) levodopa-carbidopa preparation (Sinemet CR) in an open, randomized trial in 12 healthy male volunteers. The inhibition of soluble COMT (S-COMT) in red blood cells (RBCs) was also measured. Single graded doses of entacapone (100-800 mg) were administered concomitant with a single oral dose of CR levodopa, or CR levodopa was given without entacapone (control treatment), at least 1 week apart. Plasma concentrations of levodopa, 3-O-methyldopa (3-OMD), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), carbidopa, and entacapone were determined for pharmacokinetic calculations. Entacapone decreased dose-dependently the activity of S-COMT in RBCs with a maximal inhibition of 66% after the highest dose (800 mg). Entacapone increased the area under the plasma concentration-time curve (AUC) of levodopa; the increase was highest (33%) after the 400-mg dose. Entacapone did not influence time to maximal concentration (Tmax) of levodopa. Entacapone was absorbed faster than levodopa from the CR preparation. The AUCs of 3-OMD and HVA decreased and that of DOPAC increased dose-dependently after entacapone, maximally by 69, 38, and 74%, respectively. Higher doses of entacapone (400 mg and 800 mg) decreased the AUC, but not Tmax of carbidopa. Over the dose range studied, entacapone was well tolerated. Entacapone is an effective COMT inhibitor. It improves the pharmacokinetic profile of levodopa when used in combination with a CR levodopa preparation, as it does with a standard levodopa preparation. The results justify further clinical studies with entacapone in combination with CR preparations of levodopa.

Metabolite profiles of two [14C]-labelled catechol O-methyltransferase inhibitors, nitecapone and entacapone, in rat and mouse urine and rat bile.

The metabolites of two inhibitors of catechol O-methyltransferase, nitecapone [3-(3,4-dihydroxy-5-nitrobenzylidene)2,4-pentanedione] and entacapone [(E)-2-cyano-N,N-diethyl-3-(3,4-dihydroxy-5-nitrophenyl)propenamide++ +], excreted in urine and bile by rats and in urine by mice, were compared and quantified by using HPLC with radiochemical detection after administration of [14C]-labelled compounds. With the exception of 3-O-methylated nitecapone, no major metabolites were found in rat bile that were not found in rat urine. For both compounds the major biotransformations were the same in the mouse and the rat. However, a bisulfite adduct of nitecapone was found in rat urine only, and reduction of the C = C and C = O groups of the nitecapone side chain was more extensive in the mouse. After entacapone administration, the products of amide N-dealkylation were more abundant in rat urine than in mouse urine. Most of the dose was excreted in urine and bile as O-conjugates. Most abundant were the O-glucuronides, while smaller amounts of O-sulfates and O-methylated metabolites were found in both species. One non-glucuronide glycoside of entacapone was found in urine of both rats and mice.

Inhibition of soluble catechol-O-methyltransferase and single-dose pharmacokinetics after oral and intravenous administration of entacapone.

The inhibition of soluble catechol-O-methyl-transferase (S-COMT) in red blood cells (RBCs) by entacapone, and the pharmacokinetics of entacapone after single oral (5-800 mg) and i.v. (25 mg) doses have been examined in an open study in 12 healthy young male volunteers. Oral entacapone dose-dependently decreased the activity of S-COMT in RBCs with a maximum inhibition of 82% after the highest dose (800 mg). The inhibition of S-COMT in RBCs was reversible and the activity recovered within 4-8 h. Entacapone showed linear pharmacokinetics over the dose range studied: Cmax and AUC were correlated with the dose of the drug. Oral absorption of entacapone was fast, with a tmax ranging from 0.4 to 0.9 h, depending on the dose. Systemic availability of entacapone varied between 30 and 46%. Entacapone was rapidly eliminated by metabolism with a half-life of 0.27-0.30 h after oral doses of 5 to 50 mg. After doses from 100 to 800 mg the disposition was best described by two phases with a t1/2 alpha of 0.27-0.37 h and t1/2 beta of 1.59-3.44 h. Over the dose range studied, the single oral and i.v. doses of entacapone were well tolerated. No haematological, biochemical or haemodynamic adverse effects were seen. The results show that entacapone is an orally effective and reversible COMT inhibitor in man and has simple, linear pharmacokinetics.

Identification of major urinary metabolites of the catechol-O-methyltransferase inhibitor entacapone in the dog.

Metabolites of entacapone, (E)-2-cyano-N,N-diethyl-3-(3,4-dihydroxy-5-nitrophenyl) propenamide, a potent inhibitor of catechol-O-methyltransferase, were isolated from dog urine. After hydrolysis of glucuronides and sulfates, 5 metabolites were identified in addition to unchanged entacapone by HPLC with diode-array UV detection, electron ionization mass spectrometry and IR spectroscopy. The (Z)-isomer of entacapone was the most abundant phase I metabolite while less abundant metabolites were formed through cleavage or reduction of the side chain carbon-carbon double bond, hydrolysis of the amide bond or through hydration of the nitrile group. The most abundant urinary metabolites were glucuronides. The glucuronidation site of these ortho-nitrocatechols was shown to be the hydroxyl meta to the nitro group.

The effect of catechol-O-methyl transferase inhibition by entacapone on the pharmacokinetics and metabolism of levodopa in healthy volunteers.

We studied the effect of inhibiting the enzyme catechol-O-methyltransferase (COMT) by a novel COMT inhibitor, entacapone, on the pharmacokinetics and metabolism of levodopa in 12 healthy male volunteers. Single increasing oral doses of entacapone (50-400 mg) were administered concomitantly with a single oral dose of levodopa/carbidopa (100/25 mg). The subjects were treated with carbidopa (100 mg t.i.d.) for 1 day prior to the administration of study drugs. Plasma concentrations of levodopa; its metabolites 3-O-methyldopa (3-OMD), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA); as well as carbidopa and entacapone were determined for pharmacokinetic calculations. Entacapone dose-dependently increased the area under the plasma concentration-time curve (AUC) of levodopa; the increase was 65% after the 400 mg dose of entacapone. Neither Cmax nor Tmax of levodopa was statistically significantly influenced by entacapone. Entacapone dose-dependently decreased the AUC of 3-OMD, maximally by 58%. The AUC of DOPAC was statistically significantly increased but no change in the AUC of HVA was observed after entacapone. No drug-related adverse events or hemodynamic effects were observed. The in vivo biochemical effects of entacapone indicate that it is an orally active COMT inhibitor and that it may improve the therapeutic efficacy of levodopa in Parkinson's disease.

Identification of major metabolites of the catechol-O-methyltransferase inhibitor nitecapone in the rat and dog.

Metabolites of nitecapone [3-(3,4-dihydroxy-5-nitrobenzylidene)-2,4-pentanedione], a potent catechol-O-methyltransferase inhibitor with gastroprotective and antiulcerogenic effects, were isolated by extraction and HPLC from dog and rat urine after enzymatic hydrolysis and as glucuronic acid and sulfate conjugates. Eight and 10 nonconjugated metabolites and unchanged nitecapone were found in hydrolyzed dog and rat urine, respectively, and identified by HPLC with diode-array UV detection, electron ionization mass spectrometry, and IR spectroscopy. In both species the main phase I metabolic pathways were: 1) reduction of the side chain carbon-carbon double bond and carbonyl groups and 2) cleavage of the side-chain double bond, giving an aromatic aldehyde that was partly oxidized to the corresponding carboxylic acid. These phase I metabolites and unchanged nitecapone were excreted in urine mainly as their glucuronides and sulfates in both species. Additionally, the 3-O-methylated metabolite, not found in urine, was identified in rat plasma.

Identification of major metabolites of the catechol-O-methyltransferase inhibitor entacapone in rats and humans.

Metabolites of entacapone [(E)-2-cyano-N,N-diethyl-3-(3,4-dihydroxy-5-nitrophenyl)propenamide++ +], a potent inhibitor of catechol-O-methyltransferase, were isolated from human and rat urine. After hydrolysis of glycosides and sulfates, four human and eight rat metabolites were identified, in addition to unchanged entacapone by HPLC with diode-array UV detection, electron ionization mass spectrometry, and IR spectroscopy. In man 10% of an oral dose was excreted in urine during 8 hr. The glucuronides of entacapone and its (Z)-isomer represented about 70 and 25% of the urinary metabolites, respectively. The (Z)-isomer of entacapone and two less abundant urinary metabolites, formed through cleavage or reduction of the side chain carbon-carbon double bond, were also formed in an erythrocyte incubation. The (Z)-isomer was the only phase I metabolite found in addition to entacapone in human plasma. The nitro group of entacapone seems to hinder methylation of the catechol hydroxyls in man, because no methylation products were detected. Twenty-four hr after iv administration of 14C-labeled entacapone to rats, over 50% was excreted in the feces and approximately 35% extensively metabolized in the urine. Entacapone and its phase I metabolites were excreted mainly as glucuronides and sulfates in rat urine. The most abundant urinary metabolite was the glucuronide of entacapone. Unchanged, N-dealkylated, and O-methylated entacapone, the (Z)-isomer of entacapone, and 3,4-dihydroxy-5-nitrobenzaldehyde were found in both plasma and urine from rats. Two minor urinary metabolites were formed through reduction of the side chain carbon-carbon double bond and through acetylation of the amino group resulting from nitro reduction.

Liquid chromatographic determination of a new catechol-O-methyltransferase inhibitor, entacapone, and its Z-isomer in human plasma and urine.

Assay procedures for analysis of entacapone, (E)-2-cyano-N,N-diethyl-3-(3,4-dihydroxy-5-nitrophenyl)-propenamide++ +, and its Z-isomer in human plasma and urine are described. The methods were based on reversed-phase liquid chromatography with amperometric detection. Entacapone and its Z-isomer were extracted with n-hexane-ethyl acetate mixtures after acidification with hydrochloric acid. From urine extracts the analytes were back-extracted into phosphate buffer (pH 7.2). During sample treatment 1-2% of entacapone was changed to the Z-isomer. With recoveries exceeding 75% the relative standard deviations for within-day precision were less than 11% for plasma and less than 6% for urine at the quantitation limit (10 ng ml-1) and less than 6% for both methods at higher concentrations (20-2000 ng ml-1). The assays were specific with respect to all known metabolites and selective, sensitive and precise enough for determination of entacapone and its Z-isomer in plasma and urine down to 10 ng ml-1. The methods are thus suitable for the kind of pharmacokinetic studies exemplified in this paper.

Determination of a catechol-O-methyltransferase inhibitor, nitecapone, in human plasma and urine by liquid chromatography.

Methods based on reversed-phase liquid chromatography with amperometric detection have been developed for determination of nitecapone, 3-(3,4-dihydroxy-5-nitrobenzylidene)-2,4-pentanedione, a COMT inhibitor, in human plasma and urine. Nitecapone was extracted with ethyl acetate-hexane mixtures from plasma after acidification with hydrochloric acid and from urine as the tetrabutylammonium ion-pair of its diphenylborate derivative. The recoveries of both methods exceeded 70% and the relative standard deviations for within-day precision were less than 4% and 8% at 50 ng ml-1 and at the quantitation limits, respectively. The methods are selective, sensitive and precise enough for determination of 4-5 ng ml-1 of nitecapone in plasma and urine and are thus suitable for the kind of pharmacokinetic studies exemplified in this paper.

Simultaneous determination of levodopa, its main metabolites and carbidopa in plasma by liquid chromatography.

An ion-pair reversed-phase liquid chromatographic method for the simultaneous determination of levodopa, 3-O-methyldopa, 3,4-dihydroxyphenylacetic acid, homovanillic acid and carbidopa in plasma designed for clinical trials performed to study the effect of peripheral catechol-O-methyltransferase inhibitors on the metabolism of levodopa is described. The high sample throughput of over 50 samples per day of the method makes it ideal for the assay of the large number of samples encountered in clinical trials. After protein precipitation with perchloric acid the analytes are completely separated within 15 min and determined down to a plasma concentration of 20 ng ml-1 using amperometric detection at 800 mV relative to an Ag/AgCl reference electrode. For all analytes the within-day precision defined as a relative standard deviation (n = 8) is lower than 7 and 3% at plasma concentrations of 20 and 40 ng ml-1, respectively. As the method is specific and highly reproducible, the most important factor affecting accuracy is the stability of the analytes during storage and analysis.

Identification of major metabolites of the catechol-O-methyltransferase-inhibitor nitecapone in human urine.

Metabolites of nitecapone [3-(3,4-dihydroxy-5-nitrobenzylidene)-2,4-pentanedione], a potent new catechol-O-methytransferase-inhibitor, were isolated from human urine both after hydrolysis with beta-glucuronidase and as intact conjugates. Seven phase-I metabolites and corresponding glucuronides were identified using electron ionization and fast atom bombardment mass spectrometry, IR spectroscopy, and proton NMR spectrometry. The most abundant metabolite in urine was the glucuronide of unchanged nitecapone, representing 60-65% of the metabolites found. The main phase-I metabolic reaction was reduction of the side chain double bond and carbonyl groups. One of the major metabolites was formed by cleavage of the side chain by retro aldol condensation. All phase-I metabolites were present mainly as their glucuronic acid conjugates. The 3-nitrocatechol-structure of nitecapone seems to hinder nitro-reduction, catechol-O-methylation, and sulfation reactions.

Effect of a novel catechol-O-methyltransferase inhibitor, nitecapone, on the metabolism of L-dopa in healthy volunteers.

A new catechol-O-methyltransferase (COMT) inhibitor, nitecapone, was given in increasing doses of 0-100 mg concomitantly with L-Dopa/carbidopa (100/25 mg or 100/100 mg) to healthy male volunteers. Plasma concentrations of L-Dopa, 3-O-methyldopa (3-OMD), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA), as well as the excretion of catecholamine metabolites in urine were followed to evaluate the changes in the metabolism of L-Dopa after nitecapone. Plasma concentrations of nitecapone and the soluble COMT activity in erythrocytes were also measured. The area under the plasma concentration-time curves (AUC) values for plasma nitecapone, L-Dopa and its metabolites were calculated. Nitecapone dose-dependently inhibited the soluble COMT activity in erythrocytes at 30 min after drug intake. Nitecapone slightly but significantly increased the relative bioavailability of L-Dopa. The AUC values of plasma 3-OMD decreased dose-dependently after nitecapone, and those of HVA decreased less, whereas the AUC values of DOPAC increased significantly. The elevation of the dose of carbidopa from 25 to 100 mg increased the AUC value of L-Dopa, but the effect of nitecapone was not clearly modified. Nitecapone decreased the excretion of the methylated dopamine metabolites 3-methoxytyramine (3-MT) and HVA at an L-Dopa/carbidopa dose of 100/25 mg. At a dose of 100/100 mg, the excretion of metanephrine, in addition to 3-MT and HVA, was also significantly decreased by nitecapone. The biochemical changes in L-Dopa metabolism and erythrocyte COMT activity indicate that nitecapone is an active COMT inhibitor in humans, when given orally in single doses. The changes in L-Dopa metabolism by COMT inhibitor warrant further clinical studies in Parkinson's disease.