COMT Gene Polymorphism And Levodopa Pharmacokinetics In Chinese Han Population

Catechol-O-methyltransferase(COMT)is an enzyme that plays an important role in deactivation of endogenous or exogenous catechols,such as catecholamines, catechol estrogens,and the metabolism of some drugs including levodopa.The COMT gene in humans is localized to chromosome 22,band q11.2.Human COMT gene contains a common functional polymorphism,with a G→A substitution in nucleotide 1947 of exon 4 altering the amino acid codon at position 108(Val→Met) in COMT protein.Some epidemiological studies have examined associations between the COMT polymorphism and several diseases,including Parkinson's disease, schizophrenia,ovarian cancer,breast cancer and mental depression.However,the results have been quite variable.One reason resulting in the inconsistence might be the different COMT genotype distribution among the research population in these studies.There is limited data available on the COMT gene variants in the Chinese Han populations.Due to the wide variety of nationalities in China and wide regional differences,the study of the polymorphism of nucleotide 1947 of exon 4 (COMTG1947A),especially for the Chinese Fujian Han populations,is still needed. The data of the relationship between COMT polymorphism and COMT activity were mainly from cell model in vitro.Therefore it needs to study the relationship between COMT polymorphism and COMT activity in vivo.Levodopa remains the most effective drug for the treatment of PD.Contin et al showed that there was no effect of COMT genetic polymorphism on the pharmacokinetics of levodopa in PD patients.However,the conclusion was somewhat obscure.In their study,COMT activity had not been measured,thus it was not sure whether COMT activity differentiates among the three genotypes subjects. Furthermore,some pathological and physiological factors,such as age and gender, which might affect the pharmacokinetics of levodopa,had not been excluded in the study.Thus,the effect of COMT polymorphism on the pharmacokinetics of levodopa should be validated further.In the nigrostriatal neurons,where levodopa plays its curative role in PD, levodopa is biotransformed by ALAAD to dopamine.Some studies had been taken on the plasma pharmacokinetics of levodopa,however,the data of that in striatal extracellular fluid was very limited.If pharmacokinetics of levodopa in striatal extracellular fluid was clarified,it would help realize the individualized levodopa therapy.The greatest obstacle in realizing of individualized dose regimen was how to get patient's pharmacokinetics parameters conveniently.Using Bayesian feedback method based on population pharmacokinetic parameters is thought to be the most ideal method to get patient's pharmacokinetics parameters.It is of great importance to take population pharmacokinetics(PPK)research in PD patients to get PPK parameters of levodopa.There is no populalation pharmacokinetic data of LD in Chinese population by now.So it is of great importance to conduct LD population pharmacokinetic study on Chinese population.For these reasons,the techniques such as PCR-RFLP,HPLC-ECD,microdialysis and NONMEM program were used to explore:first,distribution of catechol-O-methyltransferase gene polymorphisms in Han population of China; second,the relationship between COMT gene polymorphism and erythrocytes COMT activity in human;third,the relationship between pharmacokinetics of levodopa and COMT genotypes in Chinese healthy subjects;fourth,the neuropharmacokinetics of levodopa in striatum of rats;fifth,the estimation of clearance of levodopa in PD patients by NONMEM.1.Analysis of COMT gene polymorphisms in Chinese Fujian Han populationCOMT1947 gene in 166 normal Chinese Han people were analysed by PCR and restriction enzyme analysis and compared the genotype and allele frequency of COMT in different populations from different area.The results showed as follows:(1) COMT1947 genotype G/G,G/A andA/A of Han population are 58.4%,36.7%and 4.9%respectively.The allele frequencies of G and A in COMT are 77%and 23% respectively.The distribution of COMT1947 genotype distribution and allele frequency in Han population are almost the same as those in Kenyan,Japanese, Korean and Taiwan Han populations(P＞0.05),but significantly different from that in Southwest Asians and Caucasians,such as U.K.,Finland,Jewish populations (P＜0.01).The frequency of G/G homozygote in COMT1947 is significantly higher than that in Southwest Asians and Caucasians,such as U.K.,Finland,Jewish populations(P＜0.01),while the frequency of A/A homozygote is significantly lower than that in Southwest Asians and Caucasians,such as U.K.,Finland,Israeli Jewish populations(P＜0.01).The results indicate that the distribution of COMT1947 gene polymorphism in Han population is different from that in some other regions,which shows that the difference exists as regard to race and region.2.Relationship between COMT polymorphism and COMT activity in human erythrocytesErythrocytes COMT activity was measured by high performance liquid chromatography with electrochemical detection(HPLC-ECD).The results showed as follows:(1)The HPLC-ECD method,which was accurate and sensitive,was qualified to determine COMT activity in human erythrocytes.(2)Mean erythrocytes COMT activity of G/G,G/A and A/A genotype group were 1.514±0.520 pmol·mg^-1·min^-1 (n=97),1.053±0.453 pmol·mg^-1·min^-1(n=61)and 0.625±0.247 pmol·mg^-1·min^-1(n=8) respectively.(3)Erythrocyte COMT enzyme activity showed significant difference among the three genotype of COMT(P＜0.01).The results indicate that COMT polymorphisms influence COMT activity significantly.COMT activity of G/G genotype is 2-3 folds of that of A/A genotype.3.Relationship between pharmaeokineties of levodopa and COMT genotypes in Chinese healthy subjects Five G/G,five G/A and four A/A genotype male subjects,whose COMT enzyme activity in erythrocytes was determined by a high performance liquid chromatography with electrochemical detection(HPLC-ECD)method,were recruited for the study of levodopa pharmacokinetics from the 166 volunteers.After a single oral dose of Madopar(with 200 mg levodopa and 50 mg benserazide),plasma concentration of levodopa was determined with a HPLC-ECD method.Among the fourteen subjects enrolled in the pharmacokinetics of levodopa,the COMT enzyme activity in erythrocytes in group of G/A genotype(n=5)was significantly lower than that in group of G/G genotype(n=5,P＜0.01),while significantly higher than that in group of A/A genotype(n=4,P＜0.01).The plasma concentration-time profiles of levodopa in the G/G,G/A and A/A groups were all fitted to a two-compartment model.There was no significant difference in pharmacokinetics parameters including t₁/2β,AUC_0-∞, CL/F,C_max,t_maxand V/F(P＞0.05).The results indicated that the pharmacokinetic characteristics of levodopa in healthy Chinese subjects may be not dependent on their COMT genotype status.4.Neuropharmacokinetics of levodopa in striatum of ratsExperiments were performed on 250- to 280g male Sprague-Dawley rats. Dialysate of striatum were collected by microdialysis.The rats were divided by three groups:(â…°)Levodopa/benserazide(48/12 mg·kg^-1,i.g.)administration group,n=7,(â…±) Levodopa(48mg·kg^-1,i.g.)administration group,n=6,(â…²)0.9%sodium chloride solution(i.g.)administration group,n=6.The results showed that irrespctive of LD plus benserazide group or LD group,t_maxof LD in striatal excelluar fluid was slower that that in plasma.C_maxand AUC_0-∞in striatal excelluar fluid were less than those in plasma.C_maxand AUC_0-∞of LD in striatal excelluar fluid of LD plus benserazide group were much higher than those in LD group.Morever,HVA concentration in striatal excelluar fluid of LD plus benserazide group was much higher than that in LD group.These results indicate that the pharmacokinetics of LD in striatal excelluar fluid is not consistent with that in plasma and LD plus benserazide administration can increase significantly the amount that entering central nervous system compared with LD administratration alone.5.Estimation of clearance of levodopa in PD patients by NONMEMForty-one PD patients were recruited in the study.LD concentrations in plasma were determined by HPLC-ECD.Population clearance(CL)was calculated by NONMEM program,with a one compartment model of first order absorption and elimination.The results showed that age,sex,total body weight and the combination with other drugs,including amantadine,benzhexol,piribedil,pergolide and bromocriptine,did not influence CL of levodopa.Daily dose of levodopa and the combination with selegiline were important determinants of CL of levodopa.The final model of clearance was CL/F=(θ₁·D+θ₂)·θ₃,V/F=θ₄·WT,θ₁= 1.81,θ₂=0.012,θ₃= 17.4,θ₄=0.40.Conclusions1.The distribution of COMT1947 polymorphism in Fujian Han population is different from that in some other regions which shows that the difference exists as regard to race and region.2.COMT polymorphisms influence erythrocyte COMT enzyme activity significantly.3.The pharmacokinetic characteristics of levodopa in healthy Chinese subjects may not be dependent on their COMT genotype status.4.The changing rule of LD concentration in striatal excelluar fluid is not consistent with that in plasma.LD plus benserazide administration can increase significantly the amount of LD that entering central nervous system compared with LD administratration alone.5.Age,sex,total body weight and the combination with other drugs,including amantadine,benzhexol,piribedil,pergolide and bromocriptine,did not influence CL of levodopa.Daily dose of levodopa and the combination with selegiline were important determinants of CL of levodopa.The final model of clearance was:CL/F =(θ₁D+θ₂)·θ₃,V/F=θ₄·WT,θ₁=1.81,θ₂=0.012,θ₃=17.4,θ₄=0.40.