Yukinori Kawahara

Preparation and pharmacological evaluation of captopril sustained-release dosage forms using oily semisolid matrix

Abstract The stability of captopril in the gastrointestinal (GI) tract was estimated by means of in vitro experiments to clarify the reasons why bioavailability of captopril was reduced when administered after meals. It was found that captopril became dramatically unstable in the presence of food, and that the addition of ascorbic acid to the system improved its stability. Oily semisolid matrix (OSSM) and enteric coated granules, ascorbic acid was formulated in both dosage forms, were prepared and administered to beagle dogs to ascertain the stabilization effect in vivo. Both prolonged-action dosage forms showed bioavailability improved, but ascorbic acid worked much more effectively with OSSM. The time courses of captopril plasma concentration, inhibition of angiotensin-converting enzyme (ACE) activity and the inhibition of presser response to angiotension I (AI) were measured following single oral administration of captopril OSSM and conventional tablets (25 mg/T × 2) to non-fasting beagle dogs. The OSSM contained 50 mg of captopril and 250 mg of ascorbic acid. The beagle dogs were pretreated with i.v. administration of AI to obtain a simulated hypertensional state. The OSSM showed longer pharmacological action of captopril than the conventional tablets. The OSSM maintained more than 80% inhibition of ACE activity for over 8.5 h, while conventional tablets could maintain the same effect for only 2.5 h.

Sensitive fluorescence labelling for analysis of carboxylic acids with 4-bromomethyl-6,7-methylenedioxycoumarin

Abstract A fluorescence labelling reagent, 4-bromomethyl-6,7-methylenedioxycoumarin (BrMDC), was synthesized from sesamol and citric acid by Pechman condensation followed by bromination. Nanomole amounts of saturated aliphatic fatty acids were converted into the corresponding fluorogenic esters in the presence of anhydrous potassium carbonate and a crown ether as a catalyst and were separated by reversed-phase high-performance liquid chromatography (HPLC). Quantitative studies revealed that n -caproic acid was esterifted completely at low temperature and with sufficient reproducibility. The detection limit was just below 15 fmol per injection at a signal-to-noise ratio of 3. The fluorescence quenching of the BrMDC derivative was the lowest in conventional mixed solvent systems in comparison with those of two previously reported coumarin compounds. BrMDC was also applied to the simultaneous analysis of some acidic non-steroidal anti-inflammatry agents by reversed-phase HPLC.

Fluorescence derivatization reagent for resolution of carboxylic acid enantiomers by high-performance liquid chromatography

Abstract A novel chiral fluorescence derivatization reagent, (−)-2-[4-(1-aminoethyl)phenyl]-6-methoxybenzoxazole (APMB), was synthesized from 4-(6-methoxy-2-benzoxazolyl)acetophenone in several steps. Enantiomeric carboxylic acids were readily condensed with the chiral reagent in the presence of 2,2′-dipyridyl disulphide and triphenylphosphine. The diastereomeric amides formed were separated on a normal- and a reversed-phase column and were sensitively detected fluorimetrically, at 375 nm with excitation at 320 nm in normal-phase chromatography and at 380 nm with excitation at 320 nm in reversed-phase chromatography. The detection limit of (−)-APMB derivative of 2-phenylpropionic acid was 10 fmol at a signal-to-noise ratio of 3.

Determination of a new angiotensin-converting enzyme inhibitor (CS-622) and its active metabolite in plasma and urine by gas chromatography-mass spectrometry using negative ion chemical ionization

A sensitive and specific gas chromatographic-mass spectrometric method for the simultaneous determination of angiotensin-converting enzyme inhibitor (I, CS-622) and its active desethyl metabolite (II, RS-5139) in plasma and urine was developed. Compound D5-RS-5139 was used as an internal standard and measurements were made by electron-capture negative ion chemical ionization. Extraction from plasma and urine was carried out using Sep-Pak C18 and silica cartridges. The extract of plasma or urine was treated with diazomethane followed by trifluoroacetic anhydride to convert I and II into their methyl ester trifluoroacetyl derivatives. The detection limit of I and II was 0.5 ng/ml in plasma and 5 ng/ml in urine. The proposed method was satisfactory for the determination of I and II in plasma and urine with respect to accuracy and precision. Thus it is suitable for measurement of bioavailability and pharmacokinetics of I and II in body fluids.

3-Bromomethyl-7-methoxy-1,4-benzoxazin-2-one as a highly sensitive fluorescence derivatization reagent for carboxylic acids in high-performance liquid chromatography

Abstract A fluorescence labelling reagent, 3-bromomethyl-7-methoxy-1,4-benzoxazin-2-one (BrMB), was synthesized from 2-amino-5-methoxyphenol and ethyl pyruvate by the Wislecenus reaction followed by bromination. Picomole to nanomole amounts of saturated aliphatic fatty acids were converted into the corresponding fluorogenic esters in the presence of anhydrous potassium carbonate and a crown ether as catalysts. BrMB derivatives of fatty acids were separated on a reversed-phase column and were sensitively detected fluorimetrically at 440 nm with excitation at 345 nm. Quantitative studies revealed that n -caproic acid was esterified completely under mild conditions and with sufficient reproducibility. The detection limit of the BrMB derivative of n -caproic acid was just below 2 fmol.

Design of captopril sustained-release preparation with oily semisolid matrix intended for use in human subjects

Abstract A captopril sustained-release dosage form using oily semisolid matrix (OSSM) has been studied to develop a formulation useful in human treatment. Four OSSMs which had different in vitro dissolution rates were administered to human subjects. The resulting bioavailabilities revealed that the best OSSM suitable for humans had a faster in vitro dissolution rate than that for dogs. Another series of administrations of OSSM also showed that formulated ascorbic acid improved the bioavailability of OSSM, and that the bioavailability was sufficient for a sustained-release dosage form so long as the amount of ascorbic acid in the OSSM was more than 5 times that of the captopril by weight. Pharmacokinetic analysis was performed based on plasma concentrations of captopril in humans ( n = 8) after a single oral administration of conventional tablets or OSSM reformulated for humans. Calculated areas under the curve ( AUC s, 0-∞ h) of plasma captopril concentration were 250.5 for conventional tablets and 283.5 (ng·h/ml) for OSSM. The mean residence times (MRTs) obtained by both formulations were 1.75 h and 3.59 h, respectively. The duration time in which plasma captopril concentration stayed above 50% inhibitory concentration of angiotensin converting enzyme activity was calculated. Total duration time (TDT) per day of conventional tablets (12.5 mg) taken 3 times daily was calculated to be 10.95 h. The TDT of OSSM was 17.00 h when the OSSM (18.75 mg of captopril) was administered twice a day at 12-h intervals. Consequently, OSSM dosed twice a day is expected to show a greater efficiency than conventional tablets taken 3 times daily.

Pharmacokinetics of temocapril hydrochloride, a novel angiotensin converting enzyme inhibitor, in renal insufficiency

SummaryThe pharmacokinetics of temocapril hydrochloride, a novel prodrug-type angiotensin-I converting enzyme (ACE) inhibitor, has been studied in patients with mild (Group II) to severe (Group III) renal insufficiency in comparison with subjects with normal renal function (Group I).The pharmacokinetic parameters of the active diacid metabolite, including Cmax, AUC and half-life (t1/2), showed only slight changes between the three groups: AUC (0–∞) was significantly larger in Group III than Group I, and t1/2 tended to be prolonged in Group III, but the change was not significant.The urinary recovery of the diacid was significantly decreased in Group III. (Group I, 28.1 %, Group II, 21.6 %, Group III, 12.8 %). Compared with other ACE inhibitors, which are mainly excreted through the kidney, the plasma concentration of the active diacid metabolite was hardly influenced by renal function. It was speculated that lowering of the dose of temocapril might be recommended only in patients with severe renal insufficiency.

High-performance liquid chromatographic determination of loxoprofen and its diastereomeric alcohol metabolites in biological fluids by fluorescence labelling with 4-bromomethyl-6,7-methylenedioxycoumarin

A simple and sensitive high-performance liquid chromatographic procedure to determine loxoprofen and its diastereomeric alcohol metabolites in biological specimens is described. The analysis involves liquid-liquid extraction with benzene, pre-column derivatization with a highly fluorogenic reagent, 4-bromomethyl-6,7-methylenedioxycoumarin (BrMDC) and subsequent separation on a reversed-phase column. Loxoprofen, its pharmacologically active metabolite, trans-alcohol, and less active cis-alcohol were completely separated within 20 min with a mobile phase of 55% of aqueous acetonitrile containing acetic acid. Any endogenous substances do not interfere in the analysis of either plasma or urine samples. The quantitation limit was 0.01 micrograms/ml for human plasma and 0.05 micrograms/ml for urine. The method was applied to a pharmacokinetic study in healthy human subjects who had received 60 mg of loxoprofen sodium.

Enantiospecific assay for mammalian carbonyl reductase by liquid chromatography with fluorescence detection

A fluorogenic substrate with an unsymmetrical carbonyl for the sensitive assay of mammalian carbonyl reductase activities, 4-(6-methoxy-2-benzoxazolyl)acetophenone (I), has been prepared. The fluorescence quantum yield of I in acetonitrile is 0.12 at the emission maximum of 448 nm. The corresponding racemic alcohol produced by the chemical reduction of I, (+/-)-sec.-[4-(6-methoxy-2-benzoxazolyl)]phenethyl alcohol (II), exhibits ca. three- to fourteen-fold higher fluorescence at a shorter wavelength emission maximum of 370 nm in conventional solvents. Each enantiomer of II is sufficiently resolved on a chiral cellulose high-performance liquid chromatographic column without derivatization and quantified with high reproducibility. The detection limit for II is 20 fmol per injection at a signal-to-noise ratio of 3. The validity and applicability of I are evaluated with cytosols of mammalian tissues. The optimal pH for metabolic reduction of I in rabbit liver cytosol preparations is 6.2 in the presence of NADPH. The metabolism is proved to be highly stereoselective. The resulting alcohol produced by mammalian tissue preparations, except rabbit kidney, is predominantly of the S-(-)-configuration.

Determination in plasma of angiotensin-converting enzyme inhibitor by inhibitor-binding assay

A method of determining a new angiotensin-converting enzyme inhibitor (CS-622) and its active metabolite (RS-5139) in plasma by inhibitor-binding assay has been developed using high-performance liquid chromatography. The assay is based on the principle that the amount of inhibitor bound to the enzyme is inversely related to the amount of hippuric acid liberated on hydrolysis from the artificial substrate (hippuryl-L-histidyl-L-leucine). Plasma was heated at 60 degrees C for 15 min, to inactivate endogenous enzyme, and preincubated with rabbit-lung angiotensin-converting enzyme at 37 degrees C for 3 min. The artificial substrate (5.75 mg/ml in pH 8.3 phosphate buffer containing sodium chloride) was added to the resulting solution, and the mixture was incubated for 30 min. The reaction was terminated by the addition of 2 M hydrochloric acid. The hippuric acid liberated on hydrolysis was extracted with ethyl acetate and determined by reversed-phase chromatography using methylparaben as an internal standard. The total concentration of the inhibitor and its metabolite were determined by this method after de-esterification by rat-plasma esterase. The standard curve was obtained by the regression analysis of log concentration against logit response. The within-day and day-to-day precision were satisfactory. The proposed method is simple, rapid and sensitive enough to determine angiotensin-converting enzyme inhibitor in plasma.