| Pentoxyverine citrate oral disintegrating tablet is a new dosage form developed by Chongqing Guqi Nuo mei Pharmaceutical Co., Ltd. The drug has been approved to conduct bioequivalence test as chemical drug category 5 by SFDA. The approval document number of clinical trial is 2005L00212. Orally disintegrating tablet (ODT) is a novel oral dosage form and becomes R & D hot spots in recent years. This dosage form has many advantages to use, such as convenient oral administration, fast onset of action, high bioavailability and good taste, suitable for young and old, especially to those with mobility problems. Therefore, dosage forms transformation using the efficacy of certain already-listed drugs to orally disintegrating tablets are important to improve the patient's compliance in patients and solve the difficult problems in medication.Objective:To establish a specific, sensitive, and accurate analysis method which is suitable for determination of large-volume biological samples. The method can be used to detect pentoxyverine citrate concentration in plasma. The results of determination can be used to research the bioequivalence of pentoxyverine citrate. Through the studies of pharmacokinetics and relative bioavailability, we can evaluate whether it is bioequivalence between pentoxyverine citrate oral disintegrating tablets developed by Chongqing Guqi Nuomei Pharmaceutical Co., Ltd. and pentoxyverine citrate tablet produced by Tianjin Lisheng Pharmaceutical Co., Ltd.. This study provided the basis for clinical medicine of pentoxyverine citrate oral disintegrating tablets.Methods:The experimental designs are open, fully randomized, double-cycle (Wash period between the two trial was 7 days), double preparations and the same dose of (25 mg) cross-oral administration, self-controlled single-center trial design. Twenty healthy male volunteers were sorted from small to large in turn (1-20). Each subject had a random number generated by computer. The subjects were randomly divided into A or B group by the size of random number. Each group had 10 subjects. In the first test period, one of groups dosed the test formulation, and the other dosed the reference formulation. In the second trial period, it was cross-medication between A and B group using the same dosage. Before and after administration, each subject was collected 4 ml venous blood of elbow at different time points. The blood was collected to heparin tube and the plasma was isolated. Plasma was stored below-20℃until testing.HPLC-MS/ESI+methods was utilized to detect pentoxyverine citrate in plasma of different time. The internal standard was nolatrexed dihydrochloride. Columns: Thermo Hypersil-HyPURITY C18 (150×2.1 mm,5μm); Column temperature: 25℃; Mobile phase:0.1 M ammonium acetate solution (formic acid adjusted pH 4.4):methanol (35:65); Flow rate:0.20 ml/min; MS ionization mode:ESI+; Capillary voltage:3.0 KV; Tapered bore Voltage:30 V; Ion source temperature: 108℃; Off solvent temperature:350℃; Tapered bore anti-blowing flow rate:50 L/h; Off solvent gas flow:350 L/h; Selective ion recording (SIR) m/z 334.1 (Pentoxyverine), m/z 285.0 (Nolatrexed).The blood concentrations of pentoxyverine citrate were calculated using standard curve by the Masslynx 4.0 MS Workstation. The concentrations of accompanied quality control samples were calculated by the same method, in order to assess the reliability of measurement data. The calculation and statistical analysis of pharmacokinetic parameters were completed using DAS2.1.1 software. Prepared plasma concentration-time curve to calculate the main pharmacokinetic parameters (F, Tmax, Cmax, AUC0→15 and AUC0→∞) of the test formulation (T) and the reference formulation (R). After the natural logarithm transformation, the main pharmacokinetic parameters (Cmax, AUC0→15 and AUC0→∞) were used for significance testing by multi-factor analysis of variance (ANOVA) and statistical processing by two one-sided t test and calculating 90% confidence interval of lnAUC and lnCmax. The results were used to evaluate the bioequivalence of two formulations. Equivalent standard are:after natural logarithm transformation, AUC and Cmax were used for significance testing by multi-factor analysis of variance (ANOVA) and statistical processing by two one-sided t test, if the 90% confidence interval of the ratio of lnAUC0→15 and InAUC0→∞for the test formulation and reference formulation falls 80%-125%,90% confidence interval of the ratio of lnCmax falls within 70%-143%, then the test formulation and reference formulation were bioequivaland. Statistical analysis of Tmax was using non-parametric method (Paired Wilcoxon method). It was also used to compare differences between the two preparations.Results:In determining the conditions of the HPLC-MS/ESI+, pentoxyverine citrate plasma drug concentration linear range is 0.78-300 ng·mL-1, quantification limit is 0.78 ng·mL-1. The area ratio (Y) of pentoxyverine and internal standard on the concentration (X), the regression equation is Y= 0.0421X-0.01744, r=0.998. The relative recovery of pentoxyverine is 85%～115%, within days RSD is<15%. The extraction recovery results showed that the average extraction recoveries are 70%～80% when low, medium and high concentrations of pentoxyverine citrate were added into plasma standard blank sample. The average extraction rate of internal standard is approximately 81%. Stability study results showed that pentoxyverine citrate plasma samples were stable with RSD<15% when placed at room temperature for 12h,-20℃repeated freezing and thawing for four times, and-20℃for 23 days.After a single 25 mg oral dose of tested and reference preparations were given to the subjects, the reference and test preparations of Tmax,Cmax,AUC0→15 and AUC0→∞(Mean±Standard Deviation) were as follows:2.525±1.208 h and 1.625±0.754 h; 59.720±33.246 ng/mL and 62.282±33.059 ng/mL; 228.770±129.242 ng-h/mL and 234.442±130.011 ng-h/mL; 244.108±140.729 ng-h/mL and 246.797±136.186 ng-h/mL. The relative bioavailability F of test and reference formulation (evaluation based on AUC0→15) was (112.7±45.7)%.Variance analysis:After the natural logarithmic transformation, the pharmacokinetic parameters (Cmax,AUC0→15 and AUC0→∞) of test and reference formulation were used for variance analysis, the results showed that the order individual, the preparation and during the two weeks, the lnCmax, lnAUC0→15 lnAUCo0→∞, difference of two preparations were not statistically significant (P> 0.05). There was also no residual effect between two cycles.Two one-sided t test:The lnCmax,lnAUC0→15,lnAUC0→∞of two preparations were analyzed by the two one-sided t test. The results showed that the test formulation did not exceed the prescribed reference preparation of high limit and low limit (P<0.05). The ratio of 90% confidence interval of lnAUC0→15,lnAUC0→∞ratio in two preparations were 86.0%～123.3% and 86.5%～121.2% respectively, which falling within the equivalent range of 80%-125%. The ratio of 90% confidence interval of lnCmax was 81.4%-138.4%, also falling within the equivalent range of 80%-125%.Paired Wilcoxon test:Tmax of two formulations were tested using paired Wilcoxon test. The results are statistically significant differences between the two preparations (P<0.05). Tmax of test formulation was significantly faster than the reference.Conclusion:The method of HPLC-MS/ESI+was proven to be specific, sensitive, accurate and suitable for the bioequivalence study of pentoxyverine citrate oral disintegrating tablets.Comparing with pentoxyverine citrate tablets (batch:0711017) produced by Tianjin Lisheng Pharmaceutical Co., Ltd, the pentoxyverine citrate oral disintegrating tablets (batch:071201) developed by Chongqing GuQi Nuomei Pharmaceutical Co., Ltd. were bioequivalent in clinical trial (clinical trial No:2005L00212). |
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