The Study On The Pharmacokinetic And Bioequivalence Of Methocarbamol In Healthy Volunteers

IntroductionMethocarbamol is a kind of central skeletal muscle relaxant. It is used to treat articular muscle sprain, lumbar muscle strain and ischiodynia, etc. It is also used for the treatment of proliferative rhachitis, rheumatic arthritis and rheumatoid arthritis. The curative effect of methocarbamol is closely related with the dosage used. It can produce the active effect only when it's concentration in blood reaches and maintains a certain level. The curative effect of methocarbamol also depends on the time of administration. Prompt administration has a better effect of treatment.In modern study on the metabolism and pharmacokinetics of drug, the methods commonly used for biological pharmaceutical analysis have many kinds. Properties of high performance liquid chromatography (HPLC) include that the range of application is wide, the efficiency of separation is high and the collection of component is easy. Meanwhile, along with the unceasing renewal and development of detective technology, nowadays HPLC becomes a detective method used most commonly.The purpose of study was to establish a method for the determination of methocarbamol concentration in human plasma with HPLC-UV, which based on the characteristic of conjugated double bonds of molecule having the absorption in ultraviolet spectrum. Methocarbamol capsules made in Tongyuan pharmaceutical factory in Dandong City was taken as tested formulation. The Gaoxiaohongzhuogu capsules (methocarbamol capsules) made in Changzheng Fumin pharmaceutical factory in Shanghai City was taken as reference formulation. The concentrations of each methocarbamol were determined in plasma of 18 healthy volunteers administered with tested and reference formulations. The graph of drug concentration-time curves in the plasma was drawn. According to plasma drug concentration-time data, the areas under the plasma concentration-time curve (AUC) were calculated by the linear trapezoidal method. Plasma elimination half-life (t_1/2 ) was calculated by log-linear regression of the terminal phase of the plasma concentration versus time plot. The major pharmacokinetic parameters and relative bioavailability of tested formulation were obtained. Statistical analyses were performed with collected parameters to evaluate bioequivalence between the two formulations. The study provided scientific experiment evidence for the safe and reasonable usage of methocarbamol capsules.Materials and Methods1,Validation of analysis method(1) Chromatographic conditionChromatographic column: Hypersil ODS2 (150mm×4．6mm, 5μm); mobile phase: 0．05mol/L potassium dihydrogen phosphate buffer solution (pH 4．6) -methanol (69:31, v/v); flow rate: 1．0 ml/min; The UV detection wavelength: 274nm; column temperature: room temperature.(2) Method validationAssay specificity; establishment of standard curve; assay precision; extraction (absolute) recovery; assay accuracy; analyte stability: (short term and room temperature stability, long term and low temperature stability, freezing-thawing stability).2,Trial programEighteen male healthy volunteers were accepted in this study. Their ages and weights were approximated. They had no past medical and allergic history. The physical examination and laboratory tests including blood, function of the liver and kidney, electrocardiogram were made in the first hospital affiliated to the China Medical University and the results examined were normal. They were in a good mental condition and did seldom take medicine in ordinary days. Tobacco and alcohol were forbidden during the trial period. They had stopped using any drugs since 14 days before the trial. 18 male healthy volunteers signed formal consents after they had been informed about objective of the trial, the main effect of pharmacological, possible side effects of the drug, right and interest of the volunteers.Standard two-treatment, two-period and two-sequence 2×2 crossover design was performed. 18 male healthy volunteers were divided into two groups at random. Each group included 9 individuals. Tested and reference formulations were administered to the 18 volunteers according to crossover design. The protocol was as follows: the one group took orally tested formulation, then reference formulation; another group took formulation in reverse. The wash-out period was set to be 7 days. The fasting began at 21pm of last night, and the volunteers received a single oral dose of either tested or reference formulation of 750mg methocarbamol, with 250ml warm boiled water at 7:00am. The uniformed diet was supplied at 4h and 9h after the medication. The blood sample of 5ml was drawn from the median antebrachial vein and collected immediately into the heparinized tubes at 0, 0．33, 0．67, 1．0, 1．33, 1．67, 2．0, 2．5, 3．0, 4．0, 6．0, 9．0, 12．0 h after the medication. After standing for 5min, the samples were centrifuged at 3000rpm for 10min to get plasma. The plasma samples were stored at-20℃until analysis.3,Determination of plasma drug concentrationPlasma samples of the volunteers were taken out from a refrigerator. To melting 200μl aliquot of plasma, 25μl ciprofloxacin inter-standard solution (20μg/ml) and 120μl protein precipitant (10% trichloroacetic acid solution) were added. The mixed samples were vortex-mixed for 1min. After standing for 30min, the samples were centrifuged at 10,000rpm for 10min and the supernatant was filtered by 2．2μm filter membrane. The 50μl of filter liquor was injected into HPLC system and the concentration of methocarbamol was determined. 4,Data processingPlasma drug concentration-time data were input into the computer. Statistical analysis was performed by means of 3P97 software (Practical Pharmacokinetic Program, 1997) established by Chinese Pharmacological Society. The pharmacokinetic parameters and bioavailability were estimated and bioequivalence was evaluated.Results1,Determination methodSelective detection wavelength was 274nm. Peak shapes of ciprofloxacin as internal standard and methocarbamol were clear. There was no impurity peak interfering with determination. Baseline was smooth. Retention time of ciprofloxacin and methocarbamol was 5．5min and 9．4min, respectively. Methocarbamol and ciprofloxacin can be isolated well. Standard curve of methocarbamol was obtained from the concentration range of 0．2μg/ml to 20μg/ml in human plasma. Linear correlation of standard curve was excellent. The results showed that precision of method was good and plasma extraction recovery (absolute recovery) reached above 80%. The range of relative recovery for methocarbamol was from 102．68% to 116．27%, which was accorded with the demand of the determination of biological samples. The analytes were found to be stable in human plasma for 24h at room temperature and for 30 days under-20℃freezer conditions. The analytes in the plasma were also shown to be stable after three cycles of freeze (-20℃)-thaw (room temperature).2,Experimental objectAverage age of 18 volunteers was 22．4±0．8 years old, body weight was 75．2±3．5kg and body height was 178．1±3．4cm. Body mass index (BMI) was 23．7±0．6kg/m²ï¼ŽThe results of blood and urine routine examination and electrocardiogram showed to be in normal range. There were no abnormalities of blood pressure and blood biochemistry index. 3,Plasma concentration of methocarbamol in the volunteers administered tested and reference formulation4,Plasma concentration-time curve5,The estimation of pharmacokinetic parameters and relativebioavailabilityThe statistical analysis was performed by 3P97 (practical pharmacokinetic program, 1997) and pharmacokinetic parameters were obtained. The relative bioavailability was calculated by the values of AUC_0→t from tested and reference formulation. After the volunteers received a single oral dose of 750mg tested and reference methocarbamol, T_max of in plasma were 0．80±0．23h and 0．85±0．23h respectively, C_max were 14．34±2．99μg/ml and 13．26±2．84μg/ml, t_1/2 were 1．84±0．62h and 1．96±0．68h, AUC_0→t were 33．97±8．36μg·h/ml and 32．66±10．19μg·h/ml, AUC_{0→∞} were 35．13±8．72μg·h/ml and 33．83±10．00μg·h/ml. The relative bioavailability of methocarbamol capsule was calculated from AUC_0→t and the F_0-t was 106．0±12．2%.6,The evaluation of bioequivalenceAnalysis of variance (ANOVA) of the main pharmacokinetic parameters was performed after log-transformation. The results showed that the values of AUC_{0→∞},AUC_0→t,C_max and T_max between the two methocarbamol formulations were no significant different (P>0．05) . Furthermore, two one-sided test and (1-2α) % confidence intervals also showed that AUC_0→t and C_max between two formulations were no significant different (P>0．05) . In addition, the 90% confidence intervals (CI) for AUC_0→t was 100．4～110．4%, and for C_max was 100．9～116．2%. The results of statistical analysis showed that the two formulations had the same biological effects.Conclusion(1) The method for the determination of methocarbamol concentration in plasma with HPLC-UV was established in the study. The method was specific, sensitive and accurate for determination of methocarbamol concentration in plasma. The performance of the method was simple, the cost was low, the precision was good and the recovery was high. It can be extensively applied to the study on analysis of chemical drug and bioequivalence.(2) Plasma concentration of methocarbamol in 18 healthy volunteers administered tested and reference formulation was determined. According to the analysis of pharmacokinetic parameters, it was found that tested and reference formulations of methocarbamol were bioequivalent. The results indicated that the metabolism of methocarbamol capsules made in Dandong Tongyuan in the body was very similar with the methocarbamol drug sold in the market, made in ShanghaiChangzheng Fumin pharmaceutical factories. The tested formulation--methocarbamol capsule can be used to the clinic safely and effectively.