Studies On Bioavailabilities Of Compound Levodopa Preperations And Genetic Polymorphism

In current clinical, Levodopa(L-DP) is the most effective drug in the treatment of Parkinson’s disease(PD), the immediate precursor of dopamine(DA). It can be easily passed through the blood-brain barrier, and play a key role in the brain by DA which is decarboxylation from L-DP by dopa decarboxylase(DDC). As the DDC not only in the brain, they are also widespread in other organs and vascular wall cells. If we use L-DP alone, only about 1% of the drug can enter the centrum, so the clinical use of L-DP which is always conjunction with peripheral dopa decarboxylase inhibitor (DDCI), such as carbidopa (C-DP) or benserazide (BSZ), which are made of compound preparation, in order to enhance L-DP into the centrum. Among them, C-DP and L-DP controlled released tablets (Sinemet(?)) is the L-DP compound preparation which is more commonly used for the current domestic.After oral administration, L-DP is rapidly absorbed and metabolized quickly in the body. It is mainly metabolised by two ways. One is decarboxylation into DA by the DDC, the other is metabolised as 3-O-methyldopa (3-OMD) by the catechol-O-methyltransferase (COMT). In the circumstances of combination of peripheral DDCI such as C-DP, the COMT metabolism is the main metabolic pathway. Acording to clinical findings, there are large individual differences in plasma concentration after treating with L-DP preparations.Catechol-O-methyltransferase(COMT), is the primary metabolic enzyme of compounds with structure of the catecholamine which is biologically active or toxic in vivo, such as DA and L-DP. COMT is an important metabolic enzyme in the metabolic pathways of L-DP. Differences in COMT activity in vivo have an important effect on metabolism of its substrates. L-DP is a substrate of COMT, therefore. COMT genetic polymorphism may be an important reason for individual differences in the pharmacokinetic parameters of L-DP and its metabolite 3-OMD.The studies investigated the pharmacokinetic process of L-DP and its metabolite 3-OMD after multiple oral three compound levodopa preperations in Chinese healthy male volunteers, and look forvard to choice the best administration strategies of regimen in levodopa therapy in PD patients, then we investigated the relations between COMT genetic polymorphism and pharmacokinetics of L-DP and its metabolite 3-OMD for the potential of trying appropriate single regimen in clinical for PD patients.In the first chapter of this thesis, the high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established and validated for the simultaneous determination of L-DP,3-OMD,BSZ or C-DP in human plasma. The protein precipitation with 2% formic acid in methanol solution was used in pretreatment of human plasma. YMC PACK MB-ODS (150 mm×2.1 mm,3μm) was used as the analytical coloum and maintaind at 50℃. The mobile phase consisted of a mixture of methanol:0.5% formic acid(30:70, v/v)pumped at a flow rate of 0.15 mL/min. The electrospray ion source, positive ion detection, multiple reaction monitoring (MRM) were used in mass spectrum conditions. Flow rates of atomization gas, curtain gas and collision gas were 10 L/min,10 L/min and 4 L/min, respectively. The spray voltage was 5000 V and ion source temperature at 500℃. Benserazide hydrochloride (BSZ) or carbidopa (C-DP) was used as internal standard (IS),respectively. The calibration curves of L-DP,3-OMD and BSZ were linear within the range of 50～1000 ng/ml, and C-DP were linear within the range of 25～1000 ng/ml (r> 0.99)。Lower limit of quantification (LLOQ) for L-DP,3-OMD and BSZ were 50 ng/mL, C-DP was 25 ng/mL. The precision of within-day and between-day was less than 11%. The average recovery was between 95.7%-105.2%. Samples were stable for at least three freeze-thaw cycles and stable for at least 8 h at room temperature and stable for at least 24 h after being extracted. The method established in the paper was found to be accurate and reproducible for quantifying the concentrations of L-DP,3-OMD and BSZ or C-DP, and have successfully be applied in the following clinical pharmacokinetics studies.In the second chapter of this thesis, the multiple-dose pharmacokinetic profiles of L-DP after multiple oral three compound L-DP preperations with or without entacarpone tablets designed as a random,open and three-cross trial in 18 Chinese healthy male volunteers were investigated. The PK parameters were showed in the following table. The relative bioavailability of test 1 and test 2 tablets to reference tablets were (128.77±47.69)% and (80.43±20.11)%. The main PK parameters were tested by 95% confidence interval and two one-side test. The stastical analysis results showed, there were significant differences on the main pharmacokinetic parameters (AUC, Tmax) of L-DP and 3-OMD between the three preparations(P<0.05). Hence, the results of stastical analysis demonstrated that the two test L-DP preparations regimens were nonbioequivalent to the reference regimen.On the basis of AUC, test 1 regimen of sinemet controlled released tablets plus entacarpone tablet showed more higher AUC than that of the test 2 regimen and reference regimen, the DF% is more lower than the test 1 regimen. So, the regimen of test 1 maybe be considered as the best regimen choice in the treatment of PD patients. The CSSmax is almost constant between the three L-DP regimen, these data showed all of the DDC inhibitors (BSZ or C-DP) and the COMT inhibitor(entacarpone used in the trial) have no influence on the dissolution and releasing of levodopa from the test tablets. The volunteers have good tolerance for the three regimens of compound levodopa preperations, no AEs and no laboratory clinical meaningful findings in the trials. The third chapter investigated that the relationship between the main pharmacokinetic parameters of L-DP,3-OMD and the single nucleotide polymorphism (SNP) of COMT G1947A, C1883G and G1966T of COMT. The DNA direct sequencing of SNP of COMT G1947A, C1883G and G1966T were used in 19 Chinese healthy male volunteers. The genotype frequency and allelic frequency of COMT G1947A. C1883G and G1966T were investigated in the the above mentioned volunteers. The effects of COMT G1947A. C1883G genetic polymorphism on the pharmacokinetic parameters(AUC0-∞、Cssmax、Tmax) of L-DP and 3-OMD and the ratios of pharmacokinetic parameters (AUC0-∞Ratio、Cssmax Ratio) of L-DP/3-OMD were investigated. Results showed that COMT G1947A and C1883G were in Hardy-weinberg distribution equilibrium, sample were representative of a group. But SNP detection of COMT G1966T was all genotype of T/T.The study of relevance between COMT genetic polymorphism and pharmacokinetics of L-DP and its metabolite 3-OMD showed that, for COMT C1883G, the datas of AUC0-∞(L-DP,3-OMD) and AUC0-∞Ratio (L-DP/3-OMD) showed a remarkable difference in heterozygous (C/G) group compared with wild-type (C/C) group and the homozygous mutant (G/G) group. Therefore, in terms of clinical medicine, patients with heterozygous (C/G) may need to reduce the dosage of L-DP preparations, whereas patients with homozygous mutant (G/G) may need to increase the dosage of L-DP preparations.The findings in this thesis contribute to declaration of the best therapeutical regimen(Sinemet CR 0.5 tablet+entacapone 1 tablet) of the three L-DP preperations regimens for PD patients, individualized administration of L-DP preperations, and provision of a reference for the rational use of L-DP preparations in patients.