Pharmacokinetics And Tissue Distribution Of Melatonin In Rats

Objective:A high performance liquid chromatograph (HPLC) method for analyzing the concentration of melatonin(MT) in biological sample was established. And the aim of this study was to compare the pharmacokinetics (PK) parameters and tissue distribution of MT in liver, spleen, thymus, joint afert intragastric(ig) administration of MT in healthy rats and in rats with adjuvant arthritis(AA).Methods:The HPLC assay was established by Hypersil C18 column(4.6mm×150mm, 5μm)with 20mmol·L^-1 balanced solution (sodium acetate trihydrate-glacial acetic acid) (PH=3.4)-methanol (65:35) as mobile phase at a flow rate of 1.0mL·min^-1.The detection wavelength was Ex=285nm, Em=345nm.20μL of sample was injected into the ODS2 column where the temperature was 35℃.The biological sample was extracted with acetoacetate for HPLC analysis. The biological sample sample were collected at different time after intragastric administration of MT in healthy rats and in rats with AA.The plasma concentration-time curve and the pharmacokinetic parameters were calculated with DAS ver 2.0 practical pharmacokinetics program.Results:1.The linearity range for MT in plasma in rats was obtained from 0.1ng·mL^-1 to 200.00ng·mL^-1 and the linear regression equation was y=406.22x+9.16(r = 0.9995, 0.1^-10ng·mL^-1)and y=419.70x^-166.57(r=0.9999, 10-200ng·mL^-1). The linearity range for MT in tissue was obtained from 0.5ng·mL^-1 to 50ng·mL^-1 and the linear regression equation of liver, spleen, thymus, joint each was y=418.59x^-101.20(r=0.9995), y=410.56x-47.21(r=0.9998) , y=357.59x +198.47(r=0.9995) , y=333.07x +239.01(r=0.9995).The limits of detection for MT was 0.1ng·mL^-1. The recoveries was more than 90%(n=5),The relative standard deviations of the intra-day and inter-day were less than 10%(n=5). Biological sample was stable in ordinary temperature or in freezing during 7 or 30 days.2. The mean plasma concentrations of MT after ig MT with single dose in rats(10,100,1000μg·kg^-1) could be fitted to a two-compartment model with a weigh of 1.the main PK parameters of MT in rats were as follows: Ka为0.130±0.046,0.160±0.095,0.165±0.055 min^-1; V1为9.758±3.238,7.041±3.447,11.113±5.066 L·kg^-1; V为17.108±4.763,10.204±1.028,14.428±2.850 L·kg^-1; T1/2α为68.574±51.920,54.914±38.310,71.839±47.907min; T1/2β为153.085±42.788,107.834±11.683,135.230±15.546min; CL为0.080±0.023,0.066±0.007,0.074±0.011 L·min^-1; Tmax为16.000±2.236,16.000±2.236,16.000±2.236min; Cmax为0.737±0.111,10.212±1.333,71.870±3.194 mg·L^-1; AUC(0-tn)为106.435±32.719,1247.744±68.519,11061.007±1023.445 mg·min·L^-1; AUC(0-∞)为118.274±32.161,1312.534±85.220,12179.390±1128.002 mg·min·L^-1; MRT(0-tn)为117.718±7.409,109.276±3.184,120.385±3.043min; MRT(0-∞)为162.271±17.812,127.759±7.718,156.642±5.437min.3. The mean plasma concentrations of MT after ig MT with multidoses in healthy rats(10,100,1000μg·kg^-1) could be fitted to a two-compartment model with a weigh of1.the main PK parameters of MT in healthy rats were as follows: Ka为0.078±0.008,0.090±0.024,0.216±0.033 min^-1; V1为2.702±0.677,2.694±1.242,10.230±1.309 L·kg^-1; V为8.185±0.660,12.128±4.506,14.682±2.117 L·kg^-1; T1/2α为15.459±2.455,18.416±5.693,21.062±9.007min; T1/2β为137.000±19.282,140.566±33.092,55.173±8.632min; CL为0.042±0.003,0.060±0.021,0.185±0.012 L·min^-1; Tmax为17.000±2.739,17.000±2.739,16.000±2.236min; Cmax为1.442±0.064,15.460±0.782,80.611±3.968 mg·L^-1; AUC(0-tn)为168.503±10.993,1201.139±147.323,6371.039±346.453 mg·min·L^-1; AUC(0-∞)为192.657±16.249,1341.117±145.527,7472.260±611.520 mg·min·L^-1; MRT(0-tn)为112.602±6.635,94.050±4.614,107.754±6.213min; MRT(0-∞)为163.222±15.872,138.664±11.553,171.921±23.839min.The mean plasma concentrations of MT after ig MT with multidoses in rats with AA(10,100,1000μg·kg^-1)could be fitted to a two-compartment model with a weigh of1.the main PK parameters of PF in rats with AA were as follows: Ka为0.165±0.069,0.160±0.023,0.247±0.034 min^-1; V1为1.936±0.814,1.997±0.330,5.995±0.784L·kg^-1; V为5.630±0.537,4.495±1.167,38.147±11.275 L·kg^-1; T1/2α为11.315±5.811,20.422±8.623,14.075±2.410min; T1/2β为240.600±30.883,125.465±33.174,156.216±57.370min; CL为0.016±0.003,0.025±0.001,0.176±0.036 L·min^-1; Tmax为11.000±2.236,11.000±2.236,10.000±0.000min; Cmax为2.641±0.318,31.848±1.388,112.130±5.596 mg·L^-1; AUC(0-tn)为380.531±38.149,3362.177±116.266,5155.851±304.508 mg·min·L^-1; AUC(0-∞)为546.324±76.177,3798.224±162.236,5364.670±330.444 mg·min·L^-1; MRT(0-tn)为137.945±3.615,103.090±3.095,75.357±3.194min; MRT(0-∞)为286.141±28.786,149.874±12.439,90.585±5.977min Conclusion:1. The method was sensitive, simple, rapid and accurate. The study provides a method for determination of MT in the biological samples, and to obtain the pharmacokinetic parameters.2. The mean plasma concentrations of MT after ig MT with single dose in rats could be fitted to a two-compartment model with a weigh of 1.the main PK parameters of MT in rats showed its quick and widespread distribution, slow elimination, low clearance , long mean residence time and high bioavailability.3. Comparing with ig MT with multidoses in rats with AA and in healthy rats, concentration,ka,AUC(0-tn),AUC(0-∞),MRT(0-tn),MRT(0-∞) significantly further increased, CL significantly further decreased in low and midest dose groups, and AUC(0-tn),AUC(0-∞),MRT(0-tn),MRT(0-∞) significantly further decreased in high dose groups.4. Comparing with ig MT with midest dose in rats with AA and in healthy rats after ig MT, concentration of MT in liver, spleen, thymus and joint significantly further increased in 5min point,concentration of MT in liver, thymus and joint significantly further increased in 15min point,and concentration of MT in spleen and joint significantly further increased in 90min point.