Jong Won Kwon

Determination of a new phosphodiesterase V inhibitor, DA-8159, in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method using liquid-liquid extraction for sample preparation was developed for the determination of a new phosphodiesterase V inhibitor, DA-8159, in rat plasma and urine using sildenafil citrate as an internal standard. A 100 microl aliquot of 0.1 M Na(2)CO(3) (containing sildenafil citrate, 3 microg/ml as free sildenafil) and a 1 ml aliquot of ether were added to a 100 microl aliquot of biological samples (urine samples were diluted 20 times with distilled water). After vortex centrifugation at 9000 x g for 3 min, the ether layer was collected and dried under nitrogen gas. The residue was reconstituted with a 150 microl aliquot of the mobile phase, centrifuged, and a 100 microl aliquot of the supernatant was injected onto a reversed-phase column. The mobile phases, 20 mM KH(2)PO(4) (pH 4.7):acetonitrile (70:30, v/v for plasma and tissue samples, and 75:25, v/v for urine samples), were run at a flow rate of 1.0 ml/min. The column effluent was monitored by an ultraviolet detector set at 292 nm. The retention times for DA-8159 and the internal standard were approximately 10.7 and 9.1 min, respectively, in plasma and tissue samples and the corresponding values in urine samples were 47 and 33 min. The detection limits for DA-8159 in rat plasma and urine were 20 and 100 ng/ml, respectively. The coefficients of variation of the assay were generally low: below 10% for plasma and 9.9% for urine. No interferences from endogenous substances were found.

The effect of DA-8159 on corpus cavernosal smooth muscle relaxation and penile erection in diabetic rabbits

A previous study showed that DA-8159, a potent type 5 phosphodiesterase inhibitor, enhanced the relaxation of the smooth muscles in the normal rabbit corpus cavernosum. In this study, we investigated the in vitro effects of DA-8159 on cavernosal smooth muscle relaxation and the in vivo erectogenic potential in diabetic rabbits, since erectile dysfunction is a well-known sequela of diabetes mellitus. Diabetes mellitus was induced in male New Zealand White rabbits with alloxan monohydrate. Cavernosal strips from age-matched control and 8-week diabetic animals were mounted in organ baths. The relaxation responses to sodium nitroprusside (10-9∼10-5xa0M), a nitric oxide donor, were assessed in the presence or absence of DA-8159 (10-9∼10-6xa0M). For the penile erection test, DA-8159 was given orally (1~10xa0mg/kg) to diabetic rabbits and the length of the uncovered penile shaft was measured in a time-course manner in the presence or absence of intravenous sodium nitroprusside. The sodium nitroprusside-stimulated relaxations were significantly impaired in the corpus cavernosum from the diabetic group (IC50=1.07×10-6xa0M following 8xa0weeks of diabetes mellitus; compared with 0.48×10-6xa0M for age-matched controls). DA-8159 significantly and dose-dependently enhanced the sodium nitroprusside-stimulated relaxation in the diabetic groups. In addition, DA-8159 induced a dose-dependent penile erection in diabetic rabbits, which was potentiated by intravenous sodium nitroprusside. These results suggest that DA-8159 is an effective treatment for diabetic erectile dysfunction but further evaluation of the efficacy on human needs to be performed.

Effects of protein-calorie malnutrition on the pharmacokinetics of DA-7867, a new oxazolidinone, in rats

The pharmacokinetic parameters of DA‐7867, a new oxazolidinone, were compared after intravenous and oral administration at a dose of 10mg kg−1 to control rats and rats with protein‐calorie malnutrition (rats with PCM). After intravenous administration of 10mg kg−1 DA‐7867 to rats, metabolism of the drug was not considerable and after 14 days approximately 85.0% of the dose was recovered as unchanged drug from urine and faeces. After intravenous administration to rats with PCM, the area under the plasma concentration‐time curve from time zero to time infinity (AUC) was significantly smaller (10800 vs 6990μg min mL−1) compared with control rats. This may have been due to significantly faster total body clearance (CL, 0.930 vs 1.44mL min−1 kg−1). The faster CL in PCM rats could have been due to significantly faster non‐renal clearance (0.842 vs 1.39mL min−1 kg−1 due to significantly greater gastrointestinal (including biliary) excretion; the amount of unchanged DA‐7867 recovered from the entire gastrointestinal tract at 24h was significantly greater (1.19 vs 4.28% of intravenous dose)) because the renal clearance was significantly slower in PCM rats (0.0874 vs 0.0553mL min−1 kg−1). After oral administration to PCM rats, the AUC was significantly smaller compared with control rats (7900 vs 4310μgmin mL−1). This could have been due to a decrease in absorption from the gastrointestinal tract.

High-performance liquid chromatographic analysis of DA-7867, a new oxazolidinone, in human plasma and urine and in rat tissue homogenates.

An HPLC method was developed for the determination of a new oxazolidinone, DA-7867 (I), in human plasma and urine and in rat tissue homogenates. To 100 microl of biological sample, 300 microl acetonitrile and 50 microl methanol containing 10 microg/ml DA-7858 (the internal standard) were added. After vortex-mixing and centrifugation, the supernatant was evaporated under a gentle stream of nitrogen. The residue was reconstituted in 100 microl of the mobile phase and a 50-microl aliquot was injected directly onto the reversed-phase (C(18)) column. The mobile phase, 20 mM KH2PO4:acetonitrile (75:25, v/v) was run at a flow rate of 1.5 ml/min and the column effluent was monitored by a UV detector set at 300 nm. The retention times of I and DA-7858 were approximately 6.5 and 8.7 min, respectively. The detection limits of I in human plasma and urine and in rat tissue homogenates were 20, 20, and 50 ng/ml, respectively.

Effects of cysteine on the pharmacokinetics and pharmacodynamics of intravenous and oral azosemide in rats with protein-calorie malnutrition

The effects of cysteine on the pharmacokinetics and pharmacodynamics of azosemide were investigated after intravenous (10 mg/kg) and oral (20 mg/kg) administration to male Sprague-Dawley rats fed on 23% protein diet (control rats), and 5% protein diet with (rats with PCMC) or without (rats with PCM) oral cysteine (250 mg/kg, twice daily for the fourth week) for 4 weeks. After intravenous administration to rats with PCMC, some pharmacokinetic parameters restored fully or more than the level of control rats; the time-averaged nonrenal clearance (2.70 versus 2.32 ml/min/kg) and apparent volume of distribution at steady state (160 versus 189 ml/kg) were comparable to those in control rats, however, the terminal half-life (34.7 versus 57.2 min) and mean residence time (73.3 versus 99.3 min) were significantly shorter, area under the plasma concentration-time curve from time zero to time infinity (AUC, 1930 versus 2680 microg min/ml) was significantly smaller, and time-averaged renal (2.24 versus 1.21 ml/min/kg) and total body (CL, 4.98 versus 3.65 ml/min/kg) clearances were significantly faster than those in control rats. This could be mainly due to significantly faster renal clearance and at least partly due to increased cytochrome P450 1A2 activity by cysteine supplementation. After intravenous administration to rats with PCMC, the total amount of 8-hr urinary excretion of unchanged azosemide was significantly greater (457 versus 305 microg/g body weight), however, the 8-hr urine output (15.3 versus 31.1 ml/g kidney) was not significantly different between control rats and rats with PCMC. This could be due to the fact that urine output seemed to reach an upper plateau from 10 mg/kg dose of azosemide in rats.

Effects of cysteine on the pharmacokinetics of intravenous 2-(allylthio)pyrazine, a new chemoprotective agent, in rats with protein-calorie malnutrition.

The effects of cysteine on the pharmacokinetics of 2-(allylthio)pyrazine (2-AP) were investigated after intravenous administration of the drug (50 mg/kg) to control (Sprague-Dawley) rats (4-week fed on 23% casein diet), and rats with protein-calorie malnutrition (PCM, 4-week fed on 5% casein diet) and PCMC (PCM with 250 mg/kg of oral cysteine, twice daily starting from the fourth week). In rats with PCM, the area under the plasma concentration-time curve from time zero to time infinity (AUC) of 2-AP was significantly smaller than that in control rats. However, in rats with PCMC, the AUC of 2-AP was significantly greater than that in control rats and rats with PCM. This could be due to significantly greater formation of M4 in rats with PCM and significantly smaller formation of M4 in rats with PCMC than that in control rats. In rats with PCMC, some pharmacokinetic parameters of 2-AP restored fully or more than the levels of control rats. For example, in rats with PCMC, the apparent volume of distribution at steady state of 2-AP (7290, 16,600, and 7050 ml/kg for control rats, and rats with PCM and PCMC, respectively), the percentage of dose excreted in 24-h urine as unchanged 2-AP (0.242, 0.727, and 0.130%), and the amount excreted in 24-h urine as M4 (100, 228, and 51%) were comparable to those in control rats. However, the AUC (739, 434, and 1240 microg/min/ml) and total body clearance (67.7, 115, and 40.2 ml/min/kg) of 2-AP were significantly greater and slower, respectively, than those in control rats. This could be at least partly due to increase in S-methyltransferase activity (to form M4) in rats with PCM and greater restoration of its activity (decrease in its activity) in rats with PCMC.

Effects of cysteine on the pharmacokinetics of intravenous phenytoin in rats with protein–calorie malnutrition

The effects of cysteine on the pharmacokinetics of phenytoin and one of its metabolites, 5-(p-hydroxyphenyl)-5-phenylhydantoin (pHPPH) were investigated after intravenous administration of phenytoin, 25 mg/kg, to control rats (4-week fed on 23% casein diet) and rats with PCM (protein-calorie malnutrition, 4-week fed on 5% casein diet) and PCMC (PCM with oral cysteine supplementation, 250 mg/kg, twice daily starting from the fourth week). In rats with PCM and PCMC, the phenytoin hydroxylation (to form pHPPH) activities were significantly smaller (164, 103 and 95.3 pmol/min per mg protein for the control rats, and rats with PCM and PCMC, respectively) than that in control rats. In rats with PCMC, the intrinsic clearance of phenytoin, CL(int) was significantly slower than those in control rats and rats with PCM (0.175, 0.131 and 0.044 ml/min). The above data suggested that the formation of pHPPH could be reduced in rats with PCM and PCMC. This was supported by significantly smaller 24-h urinary excretion of pHPPH (54.7, 35.6 and 32.5% of intravenous dose of phenytoin) in rats with PCM and PCMC than that in control rats. In rats with PCM, the maximum velocity (0.344, 0.203 and 0.196 microg/min), apparent volume of distribution in central compartment (44.4, 65.4 and 72.2 ml/kg) of phenytoin, and total area under the plasma concentration-time curve from time zero to time infinity (609, 714 and 1210 microg min/ml), renal clearance (20.5, 13.4 and 4.67 ml/min per kg) and 24-h urinary excretion (54.7, 35.6 and 32.5% of intravenous dose of phenytoin) of pHPPH were not returned to control levels by cysteine supplementation (rats with PCMC). This could be mainly due to the fact that the phenytoin hydroxylation activity in rats with PCMC was not returned to control level.

Penile erectile responses to electric stimulation are enhanced by a new phosphodiesterase type-5 inhibitor

Abstractu2002 Aim:u2002 This study was conducted to investigate the effect of DA‐8159, a new phosphodiesterase type‐5 (PDE5) inhibitor, on electrostimulation‐induced penile erection in rats.

Determination of a new carbapenem derivative, DA-1131, in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the determination of a new carbapenem, DA-1131 (I), in human plasma and urine and in rat blood and tissue homogenates. The method involved deproteinization of the biological samples with 1 volume each of 0.04 M Ba(OH)2 and ZnSO4 aqueous solution. A 50-microliters aliquot of the supernatant was injected onto a C18 reversed-phase column. The mobile phase employed was 0.015 M KH2PO4-acetonitrile (9:1, v/v) with a pH of 5.0. The flow-rate was 0.8 ml/min. The column effluent was monitored by a ultraviolet detector at 300 nm. The retention time of I was 8.0 min. The detection limits of I in human plasma and urine were 0.1 and 0.5 micrograms/ml, respectively. The coefficients of variation of the assay were generally low (below 8.39%) for human plasma and urine, and rat blood and tissue homogenates. No interferences from endogenous substances were observed.

Effects of water deprivation for 72 hours on the pharmacokinetics of a new carbapenem, DA-1131, in rats.

Hormonal, physiological, and biochemical changes occurring in dehydrated patients could alter the pharmacokinetics of the drugs; therefore, the pharmacokinetics of DA-1131, a new carbapenem antibiotic, were investigated after 1-min intravenous administration of the drug at 50 mg/kg to control and 72-hr water-deprived rats. The impaired kidney and liver functions were observed in water-deprived rats on the basis of tissue microscopic examination. After intravenous infusion of the drug to water-deprived rats, the plasma concentrations of DA-1131 were higher and this resulted in a significantly greater total area under the plasma concentration-time curve from time zero to time infinity than those in control rats (4520 versus 3760 microg min/ml). This could be due to significantly slower total body clearance (CL) of DA-1131 in water-deprived rats (9.81 versus 14.1 ml/min/kg). The significantly slower CL of DA-1131 in water-deprived rats was due to significant decrease in both renal clearance (2.87 versus 5.13 ml/min/kg because of a significant decrease in 8-hr urinary excretion of unchanged DA-1131 [28.4 versus 39.9% of the intravenous dose] due to impaired kidney function) and nonrenal clearance (6.82 versus 8.66 ml/min/kg because of a significant decrease in the metabolism of DA-1131 in the kidney, as proved by the significant decrease in total renal DHP-I enzyme activity [1900 versus 2130 mU/each kidney]) in water-deprived rats. Water-deprivation did not alter the affinity of rat tissues to DA-1131.