Pharmacokinetics Of Melatonin Controlled-Release Tablet In Healthy Chinese Volunteers

The thesis is composed of two parts:Part I:Development and validation of the HPLC-MS/MS method for the determination of melatonin in human plasma.Part II:The Phase I clinical pharmacokinetic study of melatonin controlled-release tablet in human.The novelty of this thesis:1.It is the first time to report the main pharmacokinetic parameters of melatonin after the single-doses of melatonin controlled-release tablet of 2,4,8 mg and the multiple-dose of melatonin controlled-release tablet of 2 mg in healthy Chinese volunteers.2.It is the first time to report the influence factors including sexual distinction and high fat and high calorie diet on pharmacokinetic parameters of melatonin.Objective:An assay on Chinese healthy subjects was undertaken to investigate pharmacokinetics of melatonin controlled-release tablet.We discussed the effects of pharmacokinetics while concerning diet and gender.The aim was to provide the basis for clinical rational use of drug.Sample preparation methods Melatonin plasma samples were prepared by spiking 50 ?L of the I.S.solution(Melatonin-d7)into 200 ?L of plasma sample.Then plasma samples were extracted with 1 mL ethyl acetate for 3 min,followed by centrifugation at 15000 rpm at 4? for 10 min.The extracting solution was evaporated to dryness at 40?.Then,the dried extract was dissolved in 300 ?L of mobile phase,followed by centrifugation at 15000 rpm at 4? for 10 min.The reconstituted solution was transferred to an auto sampler vial and an aliquot of 40 ?L was injected into the chromatographic system.LC-MS/MS conditions A Xterra(?)RP18 column,3.5 ?m,100 mm x4.6 mm i.d.was used for the separation.For the separation of plasma samples,the mobile phase was water solution containing 1%acetic acid-methanol,at a flow rate of 700 ?L/min.The total run time was within 7 min.The column temperature was maintained at 40 ?.The MS/MS was carried out in the positive ionization mode with melatonin and melatonin-D7(IS)being identified by the multiple reactions monitoring(MRM)at m/z 233.1?174.1 and m/z 240.2?178.0,respectively.Results of method validation Endogenous substances in human plasma didn't interfere the determination.The calibration curves were linear over the range of 0.02000?30.00 ng/mL.The LLOQ was 0.02000 ng/mL.The intra-and inter-assay precisions were all below 15%.The extraction recoveries were 65.0%?59.0%?63.6%.The matrix effect of the method was were 95.5%?97.7%?98.9%.The stock solution was stable for 56 h at ambient temperature and for at least 43 days at 2-8 ?.The plasma samples were stable for 6 h at ambient temperature,during the three freeze-thaw cycles and for at least 31 days at-30?.The post-preparative solution was stable for 8 h at ambient temperature and for 27 h in the autosampler at 4?.Pharmacokinetic parameters of single dose administration After single dose oral administration,the main pharmacokinetic parameters found for melatonin at doses of 2,4,8 mg were as follows:AUC0-10h were(4.693�2.206)ng/mL h,(5.202�4.833)ng/mL h,(11.09�10.00)ng/mL h,AUC0-?were(4.981�2.454)ng/mL h,(5.413�5.003)ng/mL�h,(12.05�11.16)ng/mL.h,Cmax were(1.396�0.685)ng/mL,(1.598�1.600)ng/mL,(3.480�3.137)ng/mL,t1/2 were(2.53�1.10)h,(1.95 �0.51)h,(3.11�1.90)h,Tmax were(1.28�0.85)h,(0.98 � 0.61)h,(0.96 �0.63)h,Vd were(2367�2922)L,(3343�2281)L,(4472�3439)L,CL/F were(645.2�728.0)L/h,(1261 �884)L/h,(1130�726)L/h,respectively.Pharmacokinetic parameters of multiple dose administration After multiple dose oral administration,the main pharmacokinetic parameter of steady state Cav,DF,Ro,Rs,AUC0-10h(AUCss),AUC0-?,Cmax,t1/2,Tmax,Vd,CL/F were(0.259�0.118)ng/mL,(6.749�1.202),(1.510 �0.793),(1.414�0.689),(6.210�2.836)ng/mL�h,(6.538�2.975)ng/mL.h,(1.853�1.008)ng/mL,(2.39 �0.54)h,(0.85�0.60)h,(1279�564)L,(407.4�265.0)L/h,respectively.Pharmacokinetic parameters of single dose administration after high fat and calories diet 10 healthy volunteers received single oral dose of melatonin controlled-release tablet 2 mg in combination with high fat and calories diet,the main pharmacokinetic parameter AUC0-10h,AUC0-?,Cmax,t1/2,TmaX,Vd,CL/F were(4.090�1.58)ng/mL�h,(4.321 � 1.636)ng/mL�h,(1.127�0.381)ng/mL,(2.39�0.54)h,(2.28�1.52)h,(2002�1427)L,(585.0 �396.2)L/h,respectively.Influencing factors of dose,times,diet and sexual on pharmacokinetics of melatonin:? The lnAUC0-10h?lnAUC0-??lnCmax were dose-dependent.The dose-related curve is typically described by equation y = ax + b,and the equation was efficiency because of the p value>0.05.The linearity was assessed by linear regression R.R were less than 0.4,which demonstrated that lnAUC0-10h?lnAUC0-??lnCmax had a bad linear dose-dependent relationship.There were no statistical difference among the dose range for MRT0-t,MRT0-?,t1/2,Vd,CL/F,Tmax(P>0.05).? The AUC0-10h and AUC0-? values were higher in multiple-dosing regimen than the corresponding values obtained after single-dose(day 1)administration with significant difference(P<0.05),while the In Cmax,t1/2,Vd,CL/F and Tmax showed no significant difference between the first and the last dose(P>0.05).The slight accumulation was found following repeat dosing of melatonin(?=24h)with R0 and Rs out of 0.8?1.2.? 10 healthy volunteers received single oral dose of melatonin controlled-release tablet 2 mg in combination with high fat and calories diet,the main pharmacokinetic parameter AUC0-10h,AUC0-?,Cmax were less the corresponding values obtained on the fasting state,and the radio were out of the scope of 80%?125%,Tmax was delayed with significant difference(P<0.05).? Pharmacokinetics parameters of males and females were statistical analyzed.No significant differences(P>0.05)were noted in different group except MRT and CL/F.Conclusion:Over the dose of 2 mg to 8 mg,melatonin is characterized by linear pharmacokinetics.Slight accumulation was found following repeat dosing of melatonin controlled-release tablet 2 mg(t = 24 h),which means degree of absorption is increased by multiple dose administration,with no influence on rate of absorption and metabolism.It is suggested that high fat and calories diet has effect on pharmacokinetics of melatonin controlled-release tablet with absorption degree decreased and absorption rate delayed.Pharmacokinetics parameters of males and females are different while no significant differences were observed except MRT and CL/F.