A rapid, selective and sensitive reverse-phase HPLC methods for the determination of atenolol and chlorthalidone in human serum and whole blood were validated, and applied to the pharmacokinetic study of atenolol and chlorthalidone combination therapy. Atenolol and an internal standard, pindolol, were extracted from human serum by liquid-liquid extraction, and analyzed on a $\mu$-Bondapak C18 $10-{\mu}$ column in a mobile phase of methanol-0.01 M potassium dihydrogenphosphate(30:70, v/v, adjusted to pH 3.5) and fluorescence detection(emission: 300 nm, excitation: 224 nm). Chlorthalidone and an internal standard, probenecid, were extracted form human whole blood by liquid-liquid extraction, and analyzed on a Luna C18 $5-{\mu}$ column in a mobile phase of acetonitrile containing 77% 0.01 M sodium acetate and UV detection at 214 nm. These analysis were performed at three different laboratories using the same quality control(QC) samples. The chromatograms showed good resolution, sensitivity, and no interference by human serum and whole blood, respectively. The methods showed linear responses over a concentration range of 10-1,000 ng/mL for atenolol and 0.05-20 ${\mu}g/mL$ for chlorthalidone, with correlation coefficients of greater than 0.999 at all the three laboratories. Intra- and inter-day assay precision and accuracy fulfilled international requirements. Stability studies(freeze-thaw, short-, long-term, extracted sample and stock solution) showed that atenolol and chlorthalidone were stable. The lower limit of quantitation of atenolol and chlorthalidone were 10 ng/mL and 0.05 ${\mu}g/mL$, respectively, which was sensitive enough for pharmacokinetic studies. These methods were applied to the pharmacokinetic study of atenolol and chlorthalidone in human volunteers following a single oral administration of Hyundai $Tenoretic^{\circledR}$ tablet(atenolol 50 mg and chlorthalidone 12.5 mg) at three different laboratories.
In this study simple and sensitive high performance liquid chromatographic method using a commercially available column, was developed and validated for the determination of zolpidem tartrate in human plasma. The developed method with suitable validation was applied to a bioequivalence study of two different kinds of zolpidem tartrate. Two different formulations containing 10 mg of zolpidem tartate (CAS : 99294-93-6) were compared in 24 healthy male volunteers in order to compare the bioavailability and prove the bioequivalence. The study was performed in an open, single dose randomized, 2-sequence, cross-over design in 24 healthy male volunteers with a one-week washout period. Blood samples for pharmacokinetic profiling were drawn at selected times during 12 h. The mean $AUC_{0-12h}$, $C_{max}$, $T_{max}$ and $T_{1/2}$ were $676.6{\pm}223.4$$ng{\cdot}h{\cdot}mL^{-1}$, $177.4{\pm}34.2$$ng{\cdot}mL^{-1}$, and $0.8{\pm}0.4$ and $3.5{\pm}2.1$, respectively, for the test formulations, and $640.7{\pm}186.6$$ng{\cdot}h{\cdot}mL^{-1}$, $193.0{\pm}64.5$$ng{\cdot}mL^{-1}$, and $0.9{\pm}0.4$ and $2.7{\pm}0.9$, respectively, for the reference formulation. Both primary target parameters $AUC_{0-12h}$ and $C_{max}$ were log-transformed and tested parametrically by analysis of variance (ANOVA). 90% confidence intervals of $AUC_{0-12h}$ and $C_{max}$ were in the range of acceptable limits of bioequivalence (80-125%). Based on these results, the two formulations of zolpidem tartate are considered to be bioequivalent.
Journal of the korean academy of Pediatric Dentistry
/
v.30
no.4
/
pp.626-636
/
2003
Pediococcus pentosaceus K1270 was isolated from naturally fermented kimchi and identified based on the 16S rDNA sequence as well as cultural and biochemical characteristics. This strain strongly inhibited the formation of biofilm by Streptococcus mutans Ingbritt. K1270 also showed antibacterial activity against S. mutans Ingbritt. It was observed that K1270 strain produced hydrogen peroxide on MRS agar supplemented with 3, 3, 5, 5-tetramethylbenzidine (TMB) and peroxidase and the inhibitory effect of K1270 strain on the biofilm formation was reversed by the addition of catalase in part. Culture supernatant of K1270 inhibited the biofilm formation and the multiplication of S. mutans Ingbritt. This inhibitory effect of culture supernatant was decreased slightly by the addition of catalase and abolished by heat or trypsin treatment. Thus, this study suggests that P. pentosaceus K1270 inhibit the biofilm formation through the inhibition of the replication of S. mutans Ingbritt by producing hydrogen peroxide and bacteriocin.
The proposed method is simple, sensitive and specific Liquid chromatography-tandem mass spectrometry (LCESI-MS/MS) method for the quantification of Entacapone (EA) in human plasma using Entacapone-d10 (EAD10) as an internal standard (IS). Chromatographic separation was performed on Zorbax SB-C18, $2.1{\times}50\;mm$, $5\;{\mu}m$ column, mobile phase composed of 10 mM Ammonium formate (pH 3.0): Acetonitrile (60:40 v/v), with a flow-rate of 0.7 mL/min, followed by Liquid-liquid extraction. EA and EAD10 were detected with proton adducts at m/z $306.1{\rightarrow}233.1$ and $316.3{\rightarrow}233.0$ in multiple reaction monitoring (MRM) positive mode respectively. The method was validated over a linear concentration range of 1.00 - 2000.00 ng/mL with correlation coefficient ($r^2$) $\geq$ 0.9993. Intra and inter-day Precision within 3.60 to 7.30 and 4.20 to 5.50% and Accuracy within 97.30 to 104.20 and 98.30 to 105.80% proved for EA. This method is successfully applied in the bioequivalence study of healthy Indian human volunteers.
The distribution, metabolism and excretion of CKD-602{20(S)-7-[2-(N-Isopropylamino)ethyl]camptothecin HCI), a new camptothecin derivative, were investigated in rats after a sing le administration of CKD-602. 1. The tissue levels of CKD-602 given to mice by the intravenous route at a dose of 20mg/kg were the highest in intestine, followed in descending order by kidney, liver, stomach,lung, heart, spleen and plasma. The concentrations of CKD-602 after 24hrs decreased to less than 2% of the peak level in most tissues except the skin. The urinary and fecal excretion of CKD-602 were 47.6% and 44.4% of the administered dose, respectively, with 0.7% remaining in the rinse. 2. After administration of CKD-602 at 10mg/kg in rats, metabolism of this compound was examined in plasma, urine, and feces. The plasma samples were collected for 24hr, urinary and fecal samples for 72hr. While any peak of CKD-602 in HPLC chromatograms was not detected from plasma and urine it was detected in feces (peaks, 9.8 min). However, additional peak area was about 0.5% of the peak area of parent CKD-602. Therefore, CKD-602 may be eliminated with the parent form and rarely metabolized in the body. 4. After I.v. administration of CKD-602 at 10mg/kg in rats, urinary and fecal excretions were examined for 72hrs post dose period. 87% of total urinary excretion of CKD-602 was excreted within 8hr after administration, 53%, and 32% of total fecal excreted amounts were determined in 0-24 hr and 24-48hr periods, respectively. The total excretion amounts of CKD-602 into urine and feces were 94% of the administered dose.
In order to investigate the effect of the pretreatment with various doses of diltiazem (DTZ) on the pharmacokinetics of indocyanine green (ICG) at steady state, especially the hepatic blood clearance due to the change of hepatic blood flow, the following experiments were carried out with ICG, a hepatic function test marker, not metabolized in liver and only excreted in bile. The intravenous bolus injection ($3,780\mu\textrm{g}$/kg) and the constant-rate infusion ($10,100\mu\textrm{g}$/kg/hr) of ICG into the left femoral vein were made in order to check the steady-state plasma concentration ($C_{ss} of $10\mu\textrm{g}$/ml) of ICG at 20, 25 and 30 min. Following a 90-min washout period, the intravenous bolus injection (108, 430, 860 and $1,720\mu\textrm{g}$/kg) and the constant-rate infusion (108, 433, 866 and $1,730\mu\textrm{g}$/kg/hr) of DTZ into the right femoral vein were made and the achievement of the steady-state plasma levels ($C_{ss} of 50, 200, 400 and 800 ng/ml) of DTZ were conformed at 60, 70 and 80 min. During the steady state of DTZ, the intravenous bolus injection ($3,780\mu\textrm{g}$/kg) and the constant-rate infusion ($10,200\mu\textrm{g}$/kg/hr) of ICG into the left femoral vein were made and also the steady-state plasma concentration of ICG was checked at 20, 25 and 30 min. The plasma concentrations of DTZ and ICG were determined using a high performance liquid chromatographic technique. At the steady state, the hepatic blood clearance of ICG was obtained from the plasma concentration and blood-to-plasma concentration ratio ($R_B$) of ICG. The pretreatment with various doses of DTZ did not influence the plasma concentrations, $R_B$ and plasma free fraction ($f_p$) of ICG. So the hepatic blood clearance of ICG was independent of concentration of DTZ. The hepatic blood clearance of ICG could be affected by both hepatic bood flow and hepatic intrinsic clearance. But there was no change of the hepatic blood clearance of ICG between the control and the DTZ-pretreated rats in this study. So it may be suggested that DTZ does not influence hepatic blood flow.
Kim, Boing-Soon;Naidansuren, Purevjargal;Min, Kwan-Sik
Journal of Life Science
/
v.17
no.11
/
pp.1497-1504
/
2007
To investigate the function and secretion of human thrombopoietin (TPO) in mammalian cells, hTPO cDNA was cloned using human liver cDNA, and recombinant hTPO (rec-hTPO) was produced in CHO cell lines. In addition, six N-linked glycosylation sites were substituted for Ala to elucidate the role of each carbohydrate chain. To analyze the biological activity, rec-hTPO protein was injected subcutaneously. Blood was withdrawn for platelet determination. The metabolic clearance rate (MCR) was also analyzed at the 1, 4, 10 and 24 hr after tail vein injection. Wild-type TPO (WT) was efficiently secreted into the medium. However, a hTPO mutant with 116 deleted nucleotides detected by PCR cloning was not secreted. The N-linked glycosylation sites had nearly the same expression quantity as rec-hTPO WT apart from mutants 3 and 4. The glycosylation site of mutant 4 appeared to be an indispensable site for hTPO secretion. Also characterized was the biological activity through an injection with rec-hTPO (10 ng) to ICR mice (7 weeks). The result of the blood analysis showed a considerable increase in the platelet number six days after He injection. To analyze the pharmacokinetics, rec-hTPO was injected into the tail vein (5 ng). The result was 200 pg/ml 1hr after this injection. Following this, it dramatically decreased and virtually disappeared 10 hours after the injection. Thus, rec-hTPO may be a treatment for thrombopenia by the production of the high active rec-hTPO. In addition, hTPO can permit the development of potent new analogues that stimulate the platelet value.
This study was carried out to compare the bioavailability of $Ceclex^{(R)}$ (test drug, cefaclor 250 mg/capsule) with that of $Ceclor^{(R)}$ (reference drug) and to estimate the pharmacokinetic parameters of cefaclor in healthy Korean adult. The bioavailability was examined on 20 healthy volunteers who received a single dose (250 mg) of each drug in the fasting state in a randomized balanced 2-way crossover design. After dosing, blood samples were collected for a period of 6hours. Plasma concentrations of cefaclor were determined using HPLC with UV detection. The pharmacokinetic parameters $(AUC_{0-6hr},\;C_{max},\;T_{max},\;AUC_{int},\;K_e,\;t_{1/2},\;Vd)$ F, and CL/F) were calculated with non-compartmental pharmacokinetic analysis. The ANOVA test was utilized for the statistical analysis of the $T_{max},\;log-transformed\;AUC_{0-6hr}\;log-transformed\;C_{max},\;t_{l/2},\;V_d/F$, and CL/F. The ratios of geometric means of AUC0-6hr and $C_{max}$ between test drug and reference drug were $103.2\%\;(6.74\;{\mu}g{\cdot}hr/ml\;vs\;6.53{\pm}g{\cdot}hr/ml)\;and\;100.4\%\;(4.85\;{\mu}g\ml\;vs\;4.82\;{\mu}g/ml)$, respectively. The $T_{max}$ of test drug and reference drug were $0.9\pm0.38\;hr\;and\;0.83\pm0.34$ hrs, respectively. The $90\%$ confidence intervals of mean difference of logarithmic transformed $AUC_{0-6h},\;and\;C_{max}$ were log $0.98{\sim}log$ 1.08 and log $0.88{\sim}log1.15$, respectively. It shows that the bioavailability of test drug is equivalent with that of reference drug. The estimated half-life of this study was longer $(1.21\pm0.27\;hrs\;vs\;0.5-1\;hr)$, the Vd/F was larger $(68.89\pm25.72L$ vs 24.9L), and the CL/F was higher $(38.62\pm7.09\;L/hr$ vs 24.9 L/hr) than the previously reported values.
Kim, Chong-Kook;Jeong, Eun-Ju;Lee, Eun-Jin;Shin, Hee-Jong;Lee, Won-Keun
Journal of Pharmaceutical Investigation
/
v.23
no.1
/
pp.41-49
/
1993
The bioequivalence of two omeprazole enteric-coated products was evaluated in 16 normal male volunteers (age 26-32 yr, body weight 57-75 kg) following single oral administration. Test product was enteric-coated KD-182 tablet (Chong Kun Dang Corp., Korea) and reference product was $Rosec^{\circledR}$ capsule containing enteric-coated pellets of omeprazole (Yuhan Corp., Korea). Both products contain 20 mg of omeprazole. One tablet or capsule of the test or the reference product was administered to the volunteers, respectively, by randomized two period cross-over study ($2\;{\times}\;2$ Latin square method). Average drug concetrations at each sampling time and pharmacokinetic parameters calculated were not significantly different between two products(p>0.05); the area under the concentrationtime curve to last sampling time (8 hr) $(AUC_{0-8hr})$$(1946.5{\pm}675.3\;vs\;2018.3{\pm}761.6\;ng{\cdot}hr/ml)$, AUC from time zero to infinite $(AUC_{o-\infty})$$(2288.6{\pm}1212.8\;vs\;2264.9{\pm}1001.3\;ng{\cdot}hr/ml)$, maximum plasma concentration $(C_{max})$$(772.5{\pm}283.3\;vs\;925.8{\pm}187.7\;ng/ml)$, time to maximum plasma concentration $(T_{max})$$(2.38{\pm}1.06\;vs\;2.34{\pm}1.09\;hr)$, apparent elimination rate constant $(k_{\ell})$$(0.5339{\pm}0.2687\;vs\;0.5769 {\pm}0.2184\;hr^{-I})$, apparent absorption rate constant $(k_a)$$(1.1536{\pm}0.5278\;vs\;0.9739{\pm}0.9507 hr^{-1})$ and mean residence time (MRT) $(3.13{\pm}0.73\;vs \;3.41{\pm}1.04\;hr)$. The differences of mean $(AUC_{0-8hr})$, $C_{max}$, $T_{max}$ and MRT between the two products (3.69, 19.83, 1.32 and 8.99%, respectively) were less than 20%. The power $(1-{\beta})$ and treatment difference $(\triangle)$ for $AUC_{o-8hr}$$C_{max}$ and MRT were more than 0.8 and less than 0.2, respectively. Although the power for $T_{max}$ was under 0.8, $T_{max}$ of the two products was not significantly different each other(p>0.05). These results suggest that the bioavailability of KD-182 tablet is not significantly different from that of $Rosec^{\circledR}$ capsule. Therefore, two products are bioequivalent based on the current results.
To develop a sustained-release preparation containing ketorolac tromethamine, two sustained-release pellet formulations were evaluated with a pharmacokinetic study as compared with a conventional commercial tablets (10 mg $Tarasyn^{TM}$, Roche Korea Ltd.). Two sustained-release formulations were as follows; formulation A was composed of an inner layer containing 75% of drug coated with $Eudragit^{TM}$ RS 100 membrane and an outer layer containing 25% of drug mixed with $Eudragit^{TM}$ NE30D, and formulation B was composed of only an inner layer containing 100% of drug coated with $Eudragit^{TM}$ RS 100 membrane. The dissolution test was performed for two formulations. In case of conventional tablets, 2.5 mg of drug per a dose was administered orally into male Albino rabbit (2.0-2.3 kg of body weight) 3 times at intervals of 4 hours. In case of two sustained formulations, 7.5 mg of drug was administered once orally. Blood samples were withdrawn periodically after the administration, and the blood concentration was determined by HPLC. The conventional tablets showed very high peak-trough fluctuation between administered doses, but two sustained formulations showed less fluctuation. Formulation A with the loading dose showed the time to reach minimum effective concentration (MEC) i.e. the onset time was less than 20 min, while Formulation B had more than 1 hr of the onset time. Formulation A had the more constant plasma level than formulation B. However, formulation B had a time lag, so the plasma level was less than MEC for an initial period of 1 hr. In formulation A, the plasma level was maintained within the therapeutic window $(0.3-5\;{\mu}g/ml)$ for a long period. Formulation A was thought to be an ideal sustained-release formulation for ketorolac tromethamine oral delivery system.
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