Bioequivalence and pharmacokinetic/pharmacodynamic correlation of clopidogrel in healthy Thai subjects
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Clopidogrel is an antiplatelet drug, selectively binding to the platelet P2Y12 receptor of adenosine diphosphate. Clopidogrel is a prodrug modified through active metabolite in the liver by two steps of CYP enzyme. The active metabolite is responsible for inhibiting platelet aggregation. OBJECTIVE: The study aimed to assess the bioequivalence of clopidogrel 75 mg generic and reference drugs and to investigate the correlation between pharmacokinetics of active metabolites and its antiplatelet activities. MATERIALS AND METHODS: Determination of clopidogrel, carboxylic acid form, and active metabolite were done by liquid chromatography tandem mass spectrometry, and evaluation of platelet function was also investigated by light transmission aggregometer. 20 subjects were randomized and assigned in a crossover design to take a single 75-mg oral dose of clopidogrel generic and reference drugs in two periods with washout. Pharmacokinetic parameters Cmax, AUC0-t, and AUC0-inf of clopidogrel, carboxylic acid form, and active metabolite were analyzed. RESULTS: Bioequivalence could be shown when testing parameters with ANOVA, as 90% confidence intervals were found to be within the acceptance range of 80 - 125%. For the maximum of platelet aggregation after administration of both products, no significant differences were found. Significant correlation of Cmax of clopidogrel active metabolite and maximum platelet aggregation was found after receiving 0 - 6 hours of both formulations. CONCLUSION: The study found bioequivalence of clopidogrel generic and reference drugs. There were also significant correlations between Cmax of clopidogrel active metabolite and maximum platelet aggregation.
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Pharmacokinetics and bioequivalence of a pregabalin 150-mg capsule in healthy Thai subjects.

OBJECTIVE: The purpose of the study was to evaluate the pharmacokinetics and bioequivalence of pregabalin following administration of a 150-mg capsule of test and reference products. METHOD: The study was designed as a randomized, two-treatment, two-period, two-sequence, single-dose crossover with 1-week washout period between period I and period II dosing. 20 healthy male and female Thai subjects were enrolled in the study. Each subject was in fasted state for ~ 10 hours prior to receiving a single oral 150-mg pregabalin capsule. Serial blood samples were collected at pre-dose until 32 hours after drug administration. Plasma samples were extracted by protein precipitation and derivatized with 4-chloro-7-nitrobenzofurazan. Pregabalin plasma concentrations were determined by HPLC method, and pharmacokinetic parameters were calculated. For bioequivalence assessment, the differences of Cmax, AUC0-t, and AUC0-inf means based on ln-transformed data were assessed by the 90% confidence interval (CI). RESULTS: Pharmacokinetic parameters were determined that test and reference products showed 0.96 ± 0.35 and 1.04 ± 0.96 hours for tmax, 4,594.217 ± 834.195 and 4,568.68 ± 573.963 ng/mL for Cmax, 30,048.150 ± 2,998.920 and 29,315.722 ± 2,747.396 ng×h/mL for AUC0-t, 30,594.210 ± 2,872.317 and 29,831.454 ± 2,688.020 ng×h/mL for AUC0-inf, respectively. The 90% CIs of Cmax, AUC0-t, and AUC0-inf for test and reference products were assessed at 95.356 - 104.630%, 99.303 - 105.751%, and 99.373 - 105.788%, respectively. The results were within the acceptance criteria of 80 - 125%. CONCLUSION: Pharmacokinetic parameters of a single oral 150-mg pregabalin capsule in healthy Thai subjects were evaluated and showed rapid absorption. 90% CI for the differences of Cmax, AUC0-t, and AUC0-inf were within the acceptable range of the criteria so that bioequivalence of the test and reference products of pregabalin 150-mg capsule could be concluded.
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Pharmacokinetics and bioequivalence study of irbesartan tablets after a single oral dose of 300 mg in healthy Thai volunteers.

OBJECTIVE: Pharmacokinetics and bioequivalence of 300 mg irbesartan tablets were studied in 26 healthy Thai male volunteers. METHODS: A single oral dose of one 300 mg tablet of the test product and the reference product was given to each volunteer according to a randomized two-way crossover design with 1-week wash out period. Blood samples were collected at predetermined time intervals until 72 hours post dose and irbesartan concentration was quantified with a validated HPLC method. Individual plasma irbesartan concentration-time profile was analyzed for pharmacokinetic parameters. RESULTS: Maximum plasma concentrations (Cmax) of 3,617.19 and 3,295.77 ng/mL for test and reference, respectively, were achieved. Areas under the plasma concentration-time curve; AUC0-t and AUC0-∞ were 15,304.65 and 15,638.90 ng×h/mL for test and 15,389.21 and 15,730.34 ng×h/mL for reference. The median tmax was 1.50 hours and 1.25 hours for test and reference, respectively. Plasma elimination half-lives (t1/2) were 7.35 hours and 8.09 hours for test and reference, respectively. Primary pharmacokinetic parameters Cmax, AUC0-t, and AUC0-∞ were tested parametrically by analysis of variance (ANOVA), and it revealed no statistically significant difference (defined as p < 0.05) between the corresponding Cmax, AUC0-t, and AUC0-∞ with respect to sequence, volunteers, period and formulation. The 90% confidence intervals for the ratio of test and reference product of the parameters Cmax, AUC0-t, and AUC0-∞ were within 80 - 125% (100.13 - 121.40% for Cmax, 90.83 - 106.86% for AUC0-t and 91.11 - 106.55% for AUC0-∞). CONCLUSION: The two products were bioequivalent in terms of both rate and extent of drug absorption into systemic circulation.

The 8-bromobaicalein alleviated chikungunya-induced musculoskeletal inflammation and reduced the viral load in healthy adult mice.

Chikungunya virus is a re-emerging arbovirus that has caused epidemic outbreaks in recent decades. Patients in older age groups with high viral load and severe immunologic response during acute infection are likely to develop chronic arthritis and severe joint pain. Currently, no antiviral drug is available. Previous studies suggested that a flavone derivative, 8-bromobaicalein, was a potential dengue and Zika replication inhibitor in a cell-based system targeting flaviviral polymerase. Here we characterized that 8-bromobaicalein inhibited chikungunya virus replication with EC50 of 0.49 ± 0.11 µM in Vero cells. The molecular target predicted at viral nsP1 methyltransferase using molecular binding and fragment molecular orbital calculation. Additionally, oral administration of 250 mg/kg twice daily treatment alleviated chikungunya-induced musculoskeletal inflammation and reduced viral load in healthy adult mice. Pharmacokinetic analysis indicated that the 250 mg/kg administration maintained the compound level above EC99.9 for 12 h. Therefore, 8-bromobaicalein should be a potential candidate for further development as a pan-arboviral drug.

Pharmacokinetic of gabapentin 600 mg tablet in Thai healthy subjects.

BACKGROUND: Gabapentin is an antiepileptic drug. It is structurally similar to yaminobutyric acid (GABA), which crosses the blood-brain barrier. Gabapentin is absorbed into the blood by the L-amino acid transport system. The oral bioavailability of gabapentin displays dose-dependence. Plasma concentrations ofgabapentin are not directly proportional to dose. Therefore, pharmacokinetic of gabapentin is essential for patients who have to receive gabapentin 600 mg. OBJECTIVE: To investigate the pharmacokinetic of gabapentin 600 mg in Thai healthy subjects. MATERIAL AND METHOD: The present study was performed on 24 healthy Thai male subjects who received a single oral dose of 600 mg gabapentin tablet. Serial blood samples were collected before and to 48 hours after drug administration. Plasma gabapentin concentrations were determined by automated High Performance Liquid Chromatography (HPLC) with UV detector after deproteinized with acetonitrile followed by derivatization with 1-fluoro-2,4-dinitrobenzene. The relevant pharmacokinetic parameters were determined. RESULTS: The mean values of pharmacokinetic parameters (mean +/- SD) were 3.17 +/- 0.80 hour (1.5 to 5.0 hour) for T; 4,853.58 +/- 1,369.67 ng/ml for Cm; 0.11 +/- 0.02 hour for Kel, 6.62 +/- 1.87 hour (4.89 to 11.41 hour) for T1/2; 47,712.88 +/- 12,853.61 ng.hour/ml for AUC0-t, 48,713.20 +/- 12,909.78 ng.hour/ml for AUC0-inf, 5.24 +/- 1.32 L/hour for CI, and 49.28 +/- 15.98 L for Vd. CONCLUSION: The data show the pharmacokinetic parameters of gabapentin 600 mg. These data should be used to support the assignment of therapeutic purposes for patients who have to receive gabapentin 600 mg.

A simple LC-MS/MS method for pharmacokinetic study of carvedilol and 4/-hydroxyphenyl carvedilol at a low dose.

Background and purpose: The study was aimed at validating a simple, rapid, and low-cost LC-MS/MS method for carvedilol and 4/-hydroxyphenyl carvedilol assay in human plasma. The validated method was applied to investigate the pharmacokinetics after a low dose of 6.25 mg. carvedilol. Experimental approach: In this study, the plasma was extracted by liquid-liquid extraction and evaporated the organic layer to dryness, then both analytes in the residue were reconstituted and detected by LC- MS/MS. The method was validated following the guideline on bioanalytical method validation. Thirty-one healthy volunteers participated in the pharmacokinetic study. After 10 h of fasting, each volunteer received one tablet of 6.25 mg carvedilol orally. Blood samples were collected at 16 prescheduled time points. The plasma samples were analyzed for pharmacokinetics. Findings/Results: The method was linear over a range of 0.050-50.049 ng/mL for carvedilol and 0.050- 10.017 ng/mL for 4/-hydroxyphenyl carvedilol. Crucial validated results reached the requirements of selectivity, accuracy, precision, and stability. Pharmacokinetics of carvedilol and 4/-hydroxyphenyl carvedilol were evaluated which showed Cmax at 21.26 ± 9.23 and 2.42 ± 2.07 ng/mL; AUC0-t 66.95 ± 29.45 and 5.93 ± 3.51 ng.h/mL; AUC0-inf 68.54 ± 30.11 and 6.78 ± 3.49 ng.h/mL; and T1/2 6.30 ± 1.95 and 6.31 ± 6.45 h, respectively. Conclusion and implications: The validated method was able to detect and quantify both analytes in plasma samples and can be applied to the pharmacokinetic study of carvedilol and 4/-hydroxyphenyl carvedilol after receiving carvedilol at 6.25 mg orally.

Development and validation of an ultra-performance liquid chromatography mass spectrometry/mass spectrometry method for simultaneous quantification of total and free mycophenolic acid and its metabolites in human plasma.

A reliable method has been validated using ultra-performance liquid chromatography mass spectrometry (MS)/MS for simultaneous evaluation of human plasma concentration of mycophenolic acid (MPA) and its major metabolites both total and free form. All analytes were extracted from plasma by simple protein precipitation procedure with methanol. Samples for determination of their free form concentration require a preanalytic spin through an ultrafiltration system. The chromatographic separation was completed using C18column at 0.3 ml/min with a gradient condition. Method validation was performed as the United State Food and Drug Administration guidelines for bio-analytical methods concerning precision, accuracy, linearity, selectivity, recovery, and matrix effect. Linearity was obtained over concentration of 0.05-4, 0.5-60, and 0.025-3 µg/ml for total MPA, mycophenolic acid glucuronide (MPAG) and mycophenolic acid acyl-glucuronide (AcMPAG), respectively. The linearity of the method for free form of analytes was confirmed in the range of 10-500, 125-10,000, and 0.5-300 ng/ml for MPA, MPAG, and AcMPAG, respectively. The intra- and interday accuracy ranged from 85.73%-102.01% for total form, and 87.23%-111.89% for free form, and the precisions of all analytes were lower than 15%. The mean recoveries of the analytes ranged from 85.54% to 94.76% and the matrix factor ranged from 0.88-1.06. The developed method is rapid, sensitive and convenient for pharmacokinetic study or therapeutic drug monitoring in patients after oral administration of enteric-coated mycophenolate sodium or mycophenolate mofetil.

Hepatic cytochrome P450 2E1 activity in nonalcoholic fatty liver disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a worldwide phenomenon spanning all the continents. The pathogenesis of NAFLD has not been completely elucidated. One hypothesis is that hepatic cytochrome P450 2E1 (CYP2E1) plays an important role in increasing the lipid peroxidation and oxidative stress in NAFLD. OBJECTIVE: The aim of the present study was to examine hepatic CYP2E1 activity in patients with NAFLD. MATERIAL AND METHOD: Healthy subjects were included. After an overnight fasting, the subjects were orally administered 400 mg chlorzoxazone (CHZ) and serial blood samples were collected at 0 (predose), 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6 and 8 hours after dosing. For patients with NAFLD, plasma samples were collected at 0 (predose), 1.5, 2, 2.5 and 3 hours after dosing. Plasma CHZ and 6-hydroxychlorzoxazone (6-OH-CHZ) was assayed by reversed-phase high-performance liquid chromatography (HPLC) with UV detector. Hepatic CYP2E1 activity was calculated by using concentration ratio of 6-OH-CHZ / CHZ. RESULTS: High concentration levels of CHZ and 6-OH-CHZ in healthy subjects were found between 1.5 to 3 hours after the dose. At 1.5 to 3 hours, the concentration ratio of 6-OH-CHZ / CHZ of patients with NAFLD seemed to be more than of healthy subjects. The time point which showed most different was 2.5 hours. (0.40 +/- 0.27 vs. 0.25 +/- 0.12 microg/ml, respectively, p = 0.10). CONCLUSION: Although significant difference of the concentration ratio of 6-OH-CHZ / CHZ between the two groups was not exhibited, the data demonstrated the possibility of the increasing hepatic CYP2E1 activity in NAFLD. The concentration ratio of 6-OH-CHZ / CHZ at the point 2.5 hours may be the best index for measuring hepatic CYP2E1 activity in NAFLD.

A study on the pharmacokinetics of chlorzoxazone in healthy Thai volunteers.

BACKGROUND: Chlorzoxazone (CHZ), a centrally acting skeletal muscle relaxant, is metabolized to 6-hydroxychlorzoxazone (6-OH-CHZ) by CYP2E1. CHZ can be used as an in vivo probe of CYP2E1 activity in patients with liver diseases. Pharmacokinetics of CHZ in Thai subjects should be studied for application to Thai patients. OBJECTIVE: The purpose of the present study was to determine clinical pharmacokinetics of CHZ and 6-OH-CHZ. MATERIAL AND METHOD: Ten healthy Thai volunteers were included. After an overnight fasting, the volunteers were orally administered 400 mg CHZ and serial blood samples were collected at 0 (predose), 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, and 8 hours after dosing. Plasma CHZ and 6-OH-CHZ were assayed by reversed-phase high-performance liquid chromatography (HPLC) with UV detector. The pharmacokinetic parameters including maximum concentration (Cmax), time to reach maximum concentration (Tmax), area under the concentration-time curve (AUC0-8 and AUC0-infinity), elimination half-life (t1/2), elimination rate constant (K(el)), oral clearance (Cl), and volume of distribution (Vd) were determined. RESULTS: CHZ was absorbed into systemic circulation with time to reach maximum concentration (Tmax) of 2.00 +/- 0.82 hrs and maximum concentration (Cmax) of 7.15 +/- 2.09 microg/ml. It was metabolized to 6-OH-CHZ with Tmax of 3.05 +/- 1.17 hrs and Cmax of 1.77 +/- 0.50 microg/ml. The extent of CHZ absorption (area under the concentration-time curve, AUC) was 25.47 +/- 7.11 and 27.52 +/- 8.05 microg x hr/ml for AUC0-8 and AUC0-infinity respectively. The AUC0-8 and AUC0-infinity of 6-OH-CHZ were 7.32 +/- 2.21 and 8.50 +/- 2.78 microg x hr/ml, respectively. The elimination rate constant (K(el)) was 0.48 +/- 0.10 and 0.40 +/- 0.13 hr-1 for CHZ and 6-OH-CHZ, respectively The elimination half-life (t1/2) was 1.49 +/- 0.32 and 1.95 +/- 0.73 hours for CHZ and 6-OH-CHZ, respectively. Oral clearance (Cl) and volume of distribution (Vd) of CHZ was found to be 15.77 +/- 4.81 (L/hr) and 33.13 +/- 9.75 L, respectively. CONCLUSION: An oral dose of 400 mg CHZ was used to probe for the pharmacokinetic characteristics of this drug in Thai volunteers. Those parameters reflected absorption, distribution, and elimination of CHZ in healthy Thai volunteers.