Pharmacokinetics of metadoxine for injection after repeated doses in healthy volunteers

Alcohol-induced liver disease is one of the main epidemic problems nowadays. Metadoxine is a pyridoxine-pyrrolidone carboxylate with significant scavenging property. Metadoxine is able to accelerate the elimination of alcohol from the blood and tissues, help restore the functional structure of the liver and relieve neuro-psychological disorders associated with alcohol intoxication.1-3 The purpose of the study was to assay the pharmacokinetics of domestic metadoxine after repeated doses. METHODS Subjects Ten male subjects aged 18-40 years, didn't have any clinically significant disease or history of allergy, had not participated in a drug trial within 90 days prior to dosing and whose laboratory values were within the normal range and were eligible for inclusion in the study. Written informed consent was obtained from all subjects prior to enrollment. Drug, reagent and instruments Metadoxine for injection (300 mg/vial, Lot number 030618) and a standard preparation of metadoxine (purity 99.4%) were produced by Zhenyuan Pharmaceutical Company, Zhejiang, China. Acetonitrile (chromatographic pure, Lot number: V06809) was purchased from JT Baker, USA. HPLC (Agilent 1100 series, USA) was used in the assay. Design This was an open-labeled, non-randomized, repeated dose clinical trial. Ten subjects enrolled and received metadoxine 900 mg once daily for 7 days. Pharmacokinetic sampling Blood samples were taken pre-dose on days 1, 4-6 and 7. Blood samples were also taken at 1.5 hours during intravenous drip and at serial points post-dose on day 1 and day 7 ( 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 8, and 12 hours), respectively. Preparation of standards and controls Metadoxine stock solution was made with deionized water and metadoxine standard powder in volumetric flasks and was frozen at —20°C. Drug-free human blood was collected and centrifuged at 3000 r/min for 10 minutes. The pooled blank serum was stored at —20°C until use. Metadoxine stock solution was spiked in blank human serum to make 10 standard solutions (0.09-50 mg/L) and 3 quality controls solutions (0.4, 2, 10 mg/L). Blood sample preprocessing Aliquots of standards, controls and unknown samples (500 μl) were transferred into a test tube. Serum samples were deproteinized by the addition of 200 μl of 10% trichloroacetic acid. After vigorous vortexing and centrifuging at 3000 r/min for 15 minutes, the upper aqueous layer was aspirated for the high-performance liquid chromatography (HPLC) assay. Chromatographic conditions Solid phase: LiChroCART125-4, produced by Merk KgaA Company, Darmstadt, Germany. Specification of chromatographic column: 125×4 mm (5 μm). The mobile phase was composed of 1:9 (v/v) of acetonitrile-phosphate buffer (pH 7, 0.05 mol/L). The flow rate of the mobile phase was 0.8 ml/min. The UV detector was set at 315 nm. The total running time for one sample was 10 minutes. All chromatographic procedures were performed at ambient temperature.4-6 Quantification A standard curve was generated by plotting the peak area of metadoxine against the standard solution concentrations. Weighted (1/concentration2) least square linear regression analyses were applied to obtain the linear regression equation. Concentrations of quality controls and unknown samples were calculated by applying the linear regression equation of the standard curve. Pharmacokinetics and statistical analysis Metadoxine concentrations were analyzed by using the standard one-compartmental model. DAS software was used for pharmacokinetic data processing (t1/2, AUC0-t, CL). The maximum plasma concentration (Cmax) and the time of the maximum concentration (Tmax) were identified from measured samples. Descriptive statistics including mean, standard deviation and relative standard deviation (RSD) were calculated for the plasma concentrations and pharmacokinetic parameters of metadoxine. Student's t test was used to determine significance. RESULTS Validated method of serum analysis Chromatography Under the previous described chromatographic conditions, no endogenous peaks interfered with metadoxine in serum. Linearity and lower limit of quantification (LLQ) The linearity of the assay was assessed with standard curves ranging 0.09—50 mg/L in human serum. The standard curves were generated by plotting the peak area of metadoxine versus the theoretical concentrations. The concentration was calculated by using following equation: Conc. =0.058493×Area+0.007675. The correlation coefficient (r) of the standard curve was 0.99962. The lowest concentration in the standard curves of 0.09 mg/L was chosen as LLQ, which is adequate in analyzing unknown samples. Precision and recovery The inter- and intra-day precisions were estimated by analyzing three quality controls in human serum. Described in Table 1, RSD of metadoxine were all less than 6%, which was within the acceptable range for bioanalytical quantification. The extraction efficiency of metadoxine in human serum is sufficient, larger than 99%.Table 1: Recovery and precision of metadoxine in serum by HPLC (n=5)Subjects The mean age, weight, and height of 10 subjects were (22.10±2.47) years, (68.80±5.87) kg, and (176.40±6.02) cm, respectively. No adverse drug reaction occurred during the study. Plasma concentrations of metadoxine The plasma concentrations of metadoxine are shown in Fig. The Cmax on days 1, 4-6 and 7 were (8.61±1.03), (8.08±1.62), (8.33±2.03), (8.70±1.69) and (7.61±1.54) mg/L, respectively. The Cmin on days 4-6 were (0.12±0.08), (0.18±0.08) and (0.17±0.06) mg/L, respectively.Fig.: Linear plot of the plasma concentration of metadoxine versus time following administration of repeated intravenous doses of 900 mg of metadoxine for 7 days.Pharmacokinetic parameters The main plasma pharmacokinetic parameters of metadoxine are shown in Table 2. The t1/2 on day 1 and day 7 were (0.85±0.09) and (0.77±0.09) hours, respectively. AUC0-12 (mg·L-1) on day 1 and day 7 were (25.56±4.56) and (23.05±4.74) mg·L-1, respectively. And Cmax on day 1 and day 7 were (8.61±1.03) and (7.61±1.54) mg/L, respectively. There was no statistically significant difference between the parameters on day 1 and day 7.Table 2: Main pharmacokinetic parameters of metadoxineDISCUSSION It is well-known that metadoxine can rapidly and effectively improve liver function in alcoholic patients. Our study was to assay the pharmacokinetics of metadoxine after repeated intravenous doses in healthy volunteers. Before this study, we evaluated the pharmacokinetics of metadoxine after single dosing.4-6 The results showed that the systemic exposure increased approximately linearly with dose over the range of 600 to 1200 mg. The differences of t1/2 among 3 dose groups were not significant, which demonstrated that the different doses did not have the effect on the pharmacokinetic profile of metadoxine. In this study, we compared the major pharmacokinetic parameters of metadoxine on day 1 with those on day 7 of treatment. The results showed that the pharmacokinetics, based on Cmax and AUC, after systemic exposure on day 7 were comparable to those observed on day 1. And the parameters t1/2 and CL on day 1 and day 7 were also comparable. According to our results, there was no evidence of the pharmacokinetic profile of metadoxine changes and there was no evidence of accumulation during systemic exposure of metadoxine upon repeat dosing. Finally, based on our previous study and other studies,7,8 900-mg intravenous metadoxine once daily was recommended for the patients with ethanol intoxication.