Pharmacokinetic Study Of Lamotrigine In The Treatment Of Epilepsy Based On Metabolic Enzymes,Transporters And Receptors Genetic Polymorphisms

Part 1: Determination of lamotrigine in human serum by LC-MS/MSObjective: To develop a LC-MS/MS method for determination of lamotrigine in human serum.Methods: Agilent Eclipse plus C₁₈?4.6 mm×100 mm,3.5 ?m?was used to separate the analyses,and the mobile phase was methanol-water?85:15?system containing with 5 mmol·L^-1 ammonium format at a flow rate of 0.5 mL·min^-1.The lamotrigine-¹³C₃ were employed as the internal standard,the human serum sample was precipitated by methanol.Detecting the serum drug concentration of lamotrigine?m/z 256.00?144.90?and ISTD?m/z 258.00?144.90?was performed with multiple reactions monitoring?MRM?using positive electrospray ionization?ESI?by triple quadrupole mass spectrometer,and applied in clinical samples.Results: The linear calibration curve of lamotrigine had greatly linearity?R² =0.9996,n =8?within the range of 0.2²5 ?g·m L^-1.The accuracy of LLOQ after continuous sampling was 95.90¹01.30%.The high,medium and low concentration RSD of inter-?n =6?and intra-?n =3?day precisions were less than 3.01% and accuracy were within 100.45¹05.90%.The extraction recoveries of lamotrigine were 98.40%,100.18%,and 101.10%,respectively.The high and low concentration internal standard normalization matrix factors were 106.56% and 99.44%.Conclusion: This method is simple,sensitive and efficiency to detect lamotrigine in human serum,and the internal impurity doesn't interference the simultaneously determination of lamotrigine and ISTD.Part 2: Analysis of the lamotrigine-rifampin interaction in epileptic patients with bacterial infectionObjective: To explore the impacts of lamotrigine?LTG?– rifampin?RFP?interaction on LTG serum concentrations and symptoms of epilepsy,while epileptic patients with bacterial infections received antibacterial therapy.Methods: The clinical data of sixteen epileptic patients with variety of microbial infection treated with LGT and RFP were analyzed retrospectively.The standardized concentration of LTG(CD_RLTG)was monitored by LC-MS/MS and analyzed before,during and after the combined use of LTG and RFP,as well as the differences in seizure frequency and epileptic symptoms were observed.Results: The CD_RLTG of patients after combine with RFP three days were significantly persistent decline.Compared with before add-on therapy?n =16?,the reduce ratio during treatment six days?n =6?was 44.23 ± 15.78% and fourteen days?n =9?was 66.19 ± 15.73%.The differences of the two days were significant?P <0.05?.The CD_RLTG values could be declined greatly from 2.65 ?g·ml^-1·kg·mg^-1 to 0.38 ?g·ml^-1·kg·mg^-1.Most patients would be suffered seizure during add-on therapy 3⁵ days later,but symptoms or frequency seems not to be exacerbated;Symptoms in patients were more mitigated than previous period of treatment.Conclusion: RFP significantly repeatedly reduces the concentration of LTG in epileptic patients significantly,while the reduction meaningfully enhance as the co-administration time lengthen.Anti-microbial treatment may have auxiliary effective for epileptic patients with microbial infection.Part 3: Distribution frequency of lamotrigine metabolic enzymes,drug targets and transporters gene polymorphismObjective: To investigate the distribution frequency of metabolic enzymes,drug targets and transporters gene polymorphism,which were associated with lamotrigine in Chinese epileptics.Methods: A total of 99 participants were enrolled between July 2014 and January 2017.Genomic DNA was extracted from whole blood by phenol-chloroform extraction.The genetic polymorphisms of the genes were detected by Singer or SnapShots methods.The representative of population groups was analyzed by Hardy-Weinberg equilibrium.Results: The A allele of UGT1A4-70C>A?rs6755571?wasn't discovered,The G allele distribution of UGT1A4-142T>G was 21.73%,which was higher than most of Europe and parts of Asia people crowd.MDR1-G2677 was non-synonymous mutation,GG genotype was 29.29% and the patients amount of AA or TT was 16.The genotype frequency of SCN1A-3115 G>A GG,GA or AA were 46.46%,44.44%,9.09%,respectively.The genotype frequency of UGT1A4,UGT2B7,MDR1,ABCG2,ABCC,SLC22A1 and SCN1 A were accord with Hardy-Weinberg equilibrium law.Conclusion: The UGT1A4-70C>T?P24T?CC genotype frequency of Chinese epileptics was 100%.There may be a racial and regional differences between Asiatic and European in UGT1A4-142T>G.The frequency of each genotype has a population representative.Part 4: Population pharmacokinetic?PPK?model of lamotrigine in Chinese epilepticsObjective: To establish the population pharmacokinetic?PPK?model of lamotrigine in Chinese epileptics,and evaluate the effect of genetic polymorphism on lamotrigine PK parameters.Methods: The concentration,demographic,drug interaction and genetic polymorphism data of 89 patients were collected to build the PPK model of lamotrigine.The PPK model was established with NONMEM according to a one-compartment model,and estimate the whole body clearance and distribution capacity.The significant variables were screened by using forward inclusive and backward removed methods.Normalized Prediction Distribution Errors?NPDE?was applied to estimate the precision of the final model parameter values.Results: The final regression model for LTG was as follows: CL_?1?= ?₁ ×?1-?₄ × VPA?×?1 + ?₅ × RFP?× e^ETA1;The predicted value of CL in basic model was 1.1L/h,?_VPA was-0.385,?_RFP was 0.647;V?1?= ?₂ × e_ETA2,V in basic model was 12.7 L,the population typical values of Ka was 1.97 L/h.The AA genotype of SLC22A1-1222A>G could increase lamotrigine clearance,and the GG genotype of ABCG2-34G>A reduces lamotrigine distribution volume by approximately 42.0%.While the MDR1-2677 TT genotype and-C3435 T genotype TT exist at the same time,the V value was increased by approximately 136.0%.The individual variation of CL and V were 0.152 and 0.20 respectively.Conclusion: Valproic acid could reduce lamotrigine clearance rate of about 38.5%;and rifampicin could increase lamotrigine clearance rate of about 64.7%.SLC22A1-1222A>G,ABCG2-34G>A and MDR1-2677 G and-C3435 T could affect lamotrigine clearance and distribution volume in vivo.Such as age,weight,gender or other genetic polymorphisms had no significant effect on lamotrigine clearance.