Pharmacokinetics of entacapone, a peripherally acting catechol-O-methyltransferase inhibitor, in man. A study using a stable isotope techique.

OBJECTIVE: This study investigated the pharmacokinetics of the catechol-O-methyltransferase (COMT) inhibitor entacapone by giving simultaneously stable non-radioactive isotope 13C-entacapone intravenously (i.v.) and unlabelled entacapone orally. In comparison with a crossover design, the simultaneous i.v. and oral administration made it possible to minimise intra-individual variation, sample size and the duration of the study and still obtain accurate pharmacokinetic data. METHODS: Eight healthy male volunteers were enrolled in this study. They were given a 20-mg i.v. dose of 13C-entacapone as a 1-mg/ml infusion at a constant rate of 5 mg/min over 4 min and a 100-mg dose of unlabelled entacapone orally immediately after the infusion. Blood samples were drawn at -5 (before onset of infusion), 0 (upon termination of infusion), 2, 5, 10, 20, 30 and 45 min and 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 10 and 12 h after the tablet ingestion. Urine during the 48 h after dosing was collected in fractions. Concentrations of 13C-entacapone and entacapone in plasma samples and urine fractions were determined using gas chromatography-mass spectrometry. RESULTS: The decay of i.v. 13C-entacapone in plasma was tri-exponential and its pharmacokinetics were described using an open three-compartment model. The volume of the central compartment (Vc) and the volume of distribution at steady state (Vss) were 0.08+/-0.03 l/kg and 0.27+/-0.10 l/kg, respectively. Total plasma clearance (Cltot) averaged 11.7+/-1.9 ml/min kg(-1). The half-lives for the distribution phase and for the rapid and terminal elimination phases (t1/2alpha, t1/2beta and t1/2gamma) were 0.05+/-0.01 h, 0.38+/-0.16 h and 2.40+/-1.70 h, respectively. The terminal elimination phase accounted for only 9% of the total area under the plasma concentration-time curve (AUC), which was 409 +/- 98 ng h/ml after the i.v. dose. Oral entacapone was absorbed rapidly with a time to reach the peak concentration (tmax) of 0.9+/-0.4 h, a maximum concentration (Cmax) of 457+/-334 ng/ml and an AUC of 497+/-118 ng h/ml. During the 48 h after dosing, the recovery of free and conjugated unchanged 13C-entacapone in urine was 38.1+/-7.2% of the i.v. dose and the recovery of free and conjugated unchanged entacapone 13.3+/-3.9% of the oral dose. The bioavailability of oral entacapone was 25% based on the AUC values and 35% based on urinary excretion. CONCLUSION: The results of the present study using stable isotope technique indicate that entacapone is rapidly absorbed, distributed to a small volume and rapidly eliminated by mainly non-renal routes. The pharmacokinetic profile of entacapone provides the rationale for a concomitant and frequently repeated simultaneous dosing of entacapone with levodopa and dopa decarboxylase inhibitors in the treatment of Parkinson's disease. This study confirmed the previously published data and fully support the validity of the technique used.

Enzyme-assisted synthesis and structural characterization of nitrocatechol glucuronides.

Enzyme-assisted synthesis and characterization are described for 3-O-beta-D-glucuronides 1b-4b of the aglycons E- and Z-2-cyano-N, N-diethyl-3-(3,4-dihydroxy-5-nitrophenyl)propenamide (entacapone), 1a and 2a, respectively, 3-(3,4-dihydroxy-5-nitrobenzylidene)-2, 4-pentanedione (nitecapone) 3a and 4'-methyl-3, 4-dihydroxy-5-nitrobenzophenone (tolcapone) 4a, and 1-o- and 2-o-glucuronides 5b and 6b of the aglycon 1, 2-dihydroxy-4-nitrobenzene 5a. Liver microsomes from rats pretreated with Aroclor 1254 were used as catalyst in the synthesis. Glucuronidation was regio- and stereoselective in the case of 1a-4a; only one product was observed by HPLC, HPTLC, and NMR. The glucuronidation of 1,2-dihydroxy-4-nitrobenzene 5a resulted in equal amounts of 1-O-beta-D- and 2-O-beta-D-glucuronides. Purification of the crude products by C18 solid-phase extraction and/or flash chromatography gave compounds 1b-6b in 38-98% yields (50-84 mg). The structures of the glucuronides were characterized on the basis of UV and IR spectra and confirmed with FAB-MS and NMR spectroscopy.

Pharmacokinetics of levosimendan in healthy volunteers and patients with congestive heart failure.

Levosimendan is a new inodilatory agent that sensitizes troponin-C in heart muscle cells to calcium, thus improving contractility. The pharmacokinetics of levosimendan were evaluated using a double-isotope technique in eight healthy volunteers and in eight patients with mild congestive heart failure (CHF). A single i.v. dose of 0.50 mg 14C-labeled levosimendan and a single oral dose of 0.50 mg 13C15N-labeled levosimendan were administered concomitantly. The elimination half-lives (mean +/- SD) of levosimendan were 0.96 +/- 0.16 h in healthy volunteers and 1.03 +/- 0.11 h in patients. The respective figures for total drug were 5.73 +/- 1.53 h and 5.23 +/- 0.99 h. Clearances of levosimendan averaged 359 +/- 69 ml/min in healthy volunteers and 296 +/- 61 ml/min in patients and of total drug 104 +/- 15 and 85 +/- 20 ml/min, respectively. Volumes of distribution at steady state were for levosimendan 21.9 +/- 5.9 L in healthy volunteers and 19.5 +/- 4.5 L in patients and for 14C-drug 27.9 +/- 5.3 L and 23.8 +/- 2.8 L, respectively. The bioavailability of oral levosimendan was 85 +/- 6% in healthy volunteers and 84 +/- 4% in patients.

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.

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.

Determination of nitecapone and (13C6)nitecapone in human plasma by gas chromatography/mass spectrometry.

A gas chromatographic/mass spectrometric method is developed and validated for simultaneous determination of nitecapone and its 13C6-labelled analogue in human plasma using (2H6,13C6)nitecapone as internal standard. The method involves extraction of the analytes from plasma to ethyl acetate-hexane mixture (20:80) and conversion to bis(trimethylsilyl) ethers prior to determination by gas chromatography/mass spectrometry using selected ion monitoring. The quantification range is 0.5-2000 ng ml-1. Precision ranges from 11.3% (coefficient of variation) at low levels to 2.4% at high levels. Recovery is about 50% in the whole range. The method is applied to a pharmacokinetic study where nitecapone diluted with 14C-labelled nitecapone is given intravenously concomitantly with an oral dose of (13C6)nitecapone.

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.

Steady state pharmacokinetics of the new antitussive compound vadocaine hydrochloride.

Vadocaine hydrochloride (2',4'-dimethyl-6'-methoxy-3-(2-methyl-piperidyl) propionanilide hydrochloride, OR K-242-HCl) is a novel compound, which chemically resembles amide-type local anaesthetics. Vadocaine has been proved to be an antitussive drug both in animal and in human studies. The steady state pharmacokinetic profile of vadocaine and its main metabolite in healthy volunteers after a dosage of 30 mg t.i.d. for seven days were studied. The safety of vadocaine was evaluated by ECG monitoring and by hematological and biochemical laboratory tests. Blood and urine samples were taken on the 1st, 3rd and 7th days of the study. The peak concentrations of vadocaine were on the 1st day 72.9 +/- 6.5 ng/ml at 1 h, on the 3rd day 86.4 +/- 10.3 ng/ml at 1.5 h, and on the 7th day 86.4 +/- 7.0 ng/ml at 1.5 h. AUC0-infinity values were 327.0 +/- 43.1, 449.6 +/- 81.0 and 430.5 +/- 77.1 (ng/ml)h, respectively. No side-effects or any clinically significant changes in ECG or laboratory tests were detected during the study. According to the serum concentrations and pharmacokinetic parameters the steady state level was achieved already after the third 30 mg dose of vadocaine. No cumulation of intact compound or its metabolite was seen during the study.

Hydrolysis of 2'-esters of erythromycin.

Hydrolysis rates were determined for 2'-acetyl erythromycin, three of its homologues (2'-propionyl, 2'-butyryl and 2'-valeryl) and 2'-ethylsuccinyl erythromycin in buffer solution (pH 7.0, 37 degrees C) and in human plasma (37 degrees C) at concentrations of 5 and 100 mg/l. Ester concentrations were measured by fast atom bombardment mass spectrometry. Hydrolysis in buffer followed pseudo first-order kinetics. The half-lives of the esters ranged from 24.3 to 89.5 min (2'-ethyl-succinyl less than 2'-acetyl less than 2'-propionyl less than 2'-valeryl less than 2'-butyryl). Hydrolysis in plasma followed more complex kinetics. The half-lives were generally longer than in buffer and dependent on the initial ester concentration. Apparent first-order half-lives ranged from 35.5 to 492 min (2'-ethylsuccinyl less than 2'-acetyl less than 2'-propionyl less than 2'-butyryl less than 2'-valeryl). In case of homologous esters, the stabilizing effect of plasma and concentration dependence varied with lipophilicity. In buffer solution the hydrolysis half-life of 2'-acetyl erythromycin was dependent on the concentration of alpha 1-acid glycoprotein. Of the homologous esters, 2'-acetyl erythromycin was hydrolysed most rapidly, but it was hydrolysed more slowly than 2'-ethylsuccinyl erythromycin.

Fate of single oral doses of erythromycin acistrate, erythromycin stearate and pelleted erythromycin base analysed by mass-spectrometry in plasma of healthy human volunteers.

The kinetics of erythromycin acistrate (EA). a new ester prodrug of erythromycin, were studied in three comparative, randomized, cross-over studies in 29 healthy volunteers. A new mass-spectrometric method was used to assay separately erythromycin, 2'-acetyl erythromycin and their anhydro (spiroketal) forms. In Part I, the total antibiotic concentration was higher after EA than after erythromycin stearate (ES; 1.8-fold) and enterocoated pellets of erythromycin base (EB, enterocapsules; 1.4-fold). In plasma, however, only about one third of 2'-acetyl erythromycin was hydrolysed to active erythromycin. Moreover, after unprotected EA tablets, a considerable proportion of erythromycin and 2'-acetyl erythromycin was inactivated by gastric acid as reflected by high concentrations of respective anhydro (spiroketal) forms. In Part II, the unprotected (regular tablets) and acid-protected tablets (dissolution starts at pH 4.5) were compared. The protected tablet, albeit not an enterotablet, was not destroyed by gastric acid. Its absorption was slightly delayed but the bioavailability was good. In this study, the absorption of total antibiotic was 2.8-fold (unprotected tablet) and 3.9-fold (protected tablet) that after enterocapsules. In Part III, the bioavailabilities of 200 and 400 mg tablets (both acid-protected) were equal.

Comparisons of verapamil administration twice and three times daily in hypertension.

The antihypertensive effect of verapamil administered either two or three times daily was compared in a double blind, cross over study in 15 patients with mild to moderate essential hypertension. During the dose titration period with t.i.d. administration, normotension was obtained with daily doses of 240 mg in 7 patients, 360 mg in 7 patients and with 480 mg in one patient. Systolic and diastolic blood pressures (BP) in the supine (13/14 mmHg) and standing (14/14 mmHg) positions were significantly (p less than 0.001) reduced during the dose titration period. There was no statistically significant difference in the BP values between the twice and three times a day regimens. Five to 7-fold individual variations in the serum verapamil concentrations between individuals were observed. No significant correlations were found between serum drug concentrations and the change in mean arterial BP. There was no significant difference in the incidence of side effects between the two treatment regimens. Our results suggest that verapamil administered twice daily is effective in mild to moderate hypertension.

Quantitative determination of erythromycin 2'-ethylsuccinate in human plasma by fast atom bombardment mass spectrometry.

A method for simultaneous quantitative determination of erythromycin 2'-ethylsuccinate and erythromycin in human plasma is described. After extracting the deuterium-labelled internal standards and analytes from alkalinized plasma to diethyl ether the extracts were analysed by fast atom bombardment mass spectrometry. The quantification limit for 2'-ethylsuccinate ester was 50 ng ml-1 and for erythromycin 100 ng ml-1. The calibration curves were linear up to 5 micrograms ml-1 for both. The precision of the method at higher concentration was less than 2% and at the quantification limit approximately 6% for both analytes.

Assessment of bioavailability of experimental single-unit sustained release tablets of verapamil hydrochloride using the stable isotope technique.

A stable isotope technique has been used to assess the bioavailability of sustained release verapamil products. The test formulations were tablets with a core containing 90 mg of verapamil hydrochloride coated with ethylcellulose film, the permeability of which was controlled using different amounts of hydroxypropyl methylcellulose. A product containing ethylcellulose 75% hydroxypropyl methylcellulose 25% w/w gave a single-unit sustained release tablet of verapamil hydrochloride that allowed a dose interval of 24 h. There was no loss in bioavailability, even though verapamil had extensive first-pass metabolism.

Impairment of captopril bioavailability by concomitant food and antacid intake.

Single oral doses of captopril (50 mg) were given in a randomized cross-over study to 10 healthy volunteers after fasting, after a standardized breakfast (440 kcal; 1804 kJ) or with 50 ml of an antacid suspension. Blood and urine samples were analyzed by gas chromatography. The peak captopril concentrations attained were 701 +/- 81 ng/ml (mean +/- s.e.m.) after fasting, 351 +/- 56 ng/ml with an antacid (p less than 0.05) and 140 +/- 14 ng/ml after a meal (p less than 0.01). The peak concentrations were reached in 0.5, 0.9 and 1.5 h (p less than 0.01), and the areas under the blood concentration-time curves were 782 +/- 86, 456 +/- 60 (p less than 0.05) and 344 +/- 47 ng X h/ml (p less than 0.01), respectively. The relative bioavailability of captopril was 0.66 and 0.48 with antacid and after food, respectively. The deleterious effect of food - but not that of antacid - was reflected as a delayed hypotensive activity of captopril.

Impairing effect of food on ketoconazole absorption.

Single oral doses of ketoconazole (200 mg) or miconazole (250 mg) were given in a randomized cross-over study to 10 healthy volunteers. Ketoconazole with administered (i) after fasting (both brand 1 [Orion Pharmaceutical Co.] and brand 2 [Janssen Pharmaceutica] were tested), (ii) after a standardized meal (660 kilocalories; 2,772 kJ) (brand 1), and (iii) with 300 ml of orange juice (pH 3.8) (brand 1). Miconazole was administered after fasting. Venous blood samples for high-performance liquid chromatography determinations of ketoconazole and gas chromatographic analyses of miconazole were drawn periodically up to 24 h. The concentrations of ketoconazole in sera attained with the two brands were not statistically different. The peak concentrations of ketoconazole attained with brand 1 were 4.1 +/- 0.3 micrograms/ml (mean +/- standard error of the mean) after fasting, 2.3 +/- 0.3 micrograms/ml after the standardized meal (P less than 0.01), and 3.6 +/- 0.2 micrograms/ml with orange juice. The peak concentrations were reached in 1.4, 2.3 (P less than 0.05), and 1.8 h, respectively, whereas the areas under the serum concentration-time curves were 14.4 +/- 2.21, 8.6 +/- 1.33 (P less than 0.05), and 13.4 +/- 1.30 micrograms.h/ml, respectively. The half-lives (1.7 to 2 h) did not vary significantly among the different regimens. Compared with ketoconazole, oral absorption of miconazole was poor (peak concentration, 0.47 +/- 0.7 micrograms/ml; time to reach the peak concentration, 2.6 h; area under the serum concentration-time curve, 1.10 +/- 0.20 micrograms.h/ml).

Quantitative determination of nitroglycerin in human plasma by capillary gas chromatography negative ion chemical ionization mass spectrometry.

A quantitative method for determination of nitroglycerin in human plasma was developed. Nitroglycerin and the internal standard (butane-1,2,4-triyl trinitrate) were extracted from plasma with pentane. The extracts were analysed by gas chromatography mass spectrometry using fused silica capillary columns and electron capture negative ion chemical ionization. The quantitation limit of the method was about 50 pg ml-1. Linear calibration curves were obtained in the range of 50-1600 pg ml-1. Precision at the level of 100 pg ml-1 was 4%.