Influences Of Vitamin D3on Metformin Pharmacokinetics In Mice

A HPLC method of quantitation of metformin in mouse serum was validated in this article. By this method, influences of vitamin D3(VD3) on metformin pharmacokinetics in mice were studied.Objective:To validate a reported HPLC method of metformin quantitation; to confirm whether VD3has influences on metformin pharmacokinetics in mice.Method:1. Validation of the HPLC methodAcetonitrile is a commonly used reagent to remove proteins from biological samples. One double serum samples were dealed with acetonitrile which is twice the volume of one sample while another double samples with acetonitrile which is three times the volume of one sample. Residual protein content of the samples was quantitated by Coomassie brilliant blue method to verify the effect of acetonitrile on removing protiens. Concentration gradient metformin in saline and serum solutions were prepared and determined by HPLC method under the selected chromategraphic conditions. The standard curves of metformin were obtained and the range of liner and quantitation were confirmed. Prepared metformin serum samples with content of100.500and1000ug/mL, and control serum samples were measured by HPLC to calculate the intra-day precision, inter-day precision and recovery rate. The HPLC spectrograms of metformin samples and serum samples were compared with each other. Merformin serum samples frozen and stored for3days and samples dealed with acetonitrile and frozen and stored for7days were measured by HPLC to study the stability of samples after frozen.2. Influences of VD3on metformin pharmacokinetics in miceThe serum concentrations of metformin in mice after ig administration at dose of80，240and720mg/kg were determined at different time points, and then the pharmacokinetic parameters were analyzed by DAS software. After pre-treatment with VD3ig at dose of200.600and1800IU/kg for14days, the mouse serum concentrations of metformin，which was ig administrated at dose of80,240and720mg/kg at the fifteenth day, were determined at different time points, and then the pharmacokinetic parameters were analyzed by DAS software. The serum concentrations of metformin in mice after iv administration at dose of80mg/kg were measured. After the pre-treatment of VD3ig at dose of200IU/kg for14days, and iv administration at dose of80mg/kg at the fifteenth day, the serum concentrations of metformin in mice were measured. Then the pharmacokinetic parameters were analyzed by DAS software.Results:1. Using acetonitrile twice the volume of serum samples, the average of residual protein content of the double samples was0.01mg/mL; while using acetonitrile three times the volume of serum samples, the average value was-0.5μg/mL。The standard curves of metformin in saline and serum solutions were obtained and the liner range of metformin was0.25～1000μg/mL, R2>0.99. The inter-day precisions of low, medium and high-concentration samples were2.23%,1.33%and0.73%, the intra-day precisions were3.79%,1.97%and1.80%,and the recovery rates were13.58%,105.05%and99.59%; respectively. After frozen for3days or dealed with acetonitrile and frozen for7days, the changes of sample recovery rate were no more than15.43%.2. The C-t curves and main pharmacokinetic parameters in each group were obtained by pharmacokinetic analysis.Without pre-treatment, the main pharmacokinetic parameters of metformin after ig administration at dose of80,240and720mg/kg were:t1/2β(min)289.476,238.169，276.627; Vd(L/kg)24.950,28.132,31.731; CL(L/min/kg)0.060,0.082,0.080; Cmax(mg/L)4.35,15.28,27.19; AUC0-∞(mg/L*min)1339.087,2931.336,9055.721. Without pre-treatment. the main pharmacokinetic parameters of metformin after iv administration at dose of80mg/kg were: tl/2β(min)153.349; Vd(L/kg)17.282; CL(L/min/kg)0.028; AUC0-∞(mg/L*min)2890.478. The F of metformin after ig administration at dose of80mg/kg was46.33%.After pre-treatment with VD3at the low dose, the main pharmacokinetic parameters of metformin after ig administration at dose of80,240and720mg/kg were:tl/2β(min)202.455,211.363,390.808; Vd(L/kg)18.226,27.573,51.711; CL(L/min/kg)0.062,0.090,0.092; Cmax(mg/L)6.34,15.64,36.40; AUCO-oo(mg/L*min)1282.057,2654.163,7850.301. After pre-treatment with VD3at the low dose, the main pharmacokinetic parameters of metformin after iv administration at dose of80mg/kg were:tl/2P(min)108.752; Vd(L/kg)16.011; CL(L/min/kg)0.035; AUC0-∞(mg/L*min)2288.313. The F of metformin after ig administration at dose of80mg/kg was56.03%.After pre-treatment with VD3at the medium dose, the main pharmacokinetic parameters of metformin after ig administration at dose of80,240and720mg/kg were:tl/2(3(min)134.042,347.953,336.691; Vd(L/kg)10.227,30.363,49.526; CL(L/min/kg)0.053,0.060,0.102; Cmax(mg/L)8.27,17.96,24.91; AUC0-∞(mg/L*min)1512.791,3967.899,7061.600.After pre-treatment with VD3at the high dose, the main pharmacokinetic parameters of metformin after ig administration at dose of80,240and720mg/kg were:tl/2β(min)380.393,290.440,265.282; Vd(L/kg)23.983,19.917,26.509; CL(L/min/kg)0.044,0.048,0.069; Cmax(mg/L)5.53,19.19,35.62; AUC0-∞(mg/L*min)1830.628,5049.019,10394.75; respectively.Conclusions:1. The measuring method used in this research is suitable for metformin pharmacokinetic studies, and it is reliable and accurate.2. The pharmacokinetic characteristics of metformin match two-compartment model. The elimination of metformin matches first-order kinetic. These characteristics are not changed by pre-treatment with VD3. This preliminary study shows that VD3almost does not affect the motablism and elimination of metformin, but may affect the absorbtion and distribution of high dose of metformin.