Studies Of The Pharmacokinetics Of Neuroinflammatory Inhibitor AD10 And Metabonomic Of Alzheimer’s Disease

Alzheimer’s disease belongs to a neurodegenerative disease. Its aetiology is still unclear. Therefore, no disease-modifying therapeutics is found to either slow or stop the process of the disease till now. One of the direct contributors to synaptic dysfunction and neuronal apoptosis is excessive inflammation in brain. Since target-directed drug discovery approach seems to be ineffective in aspects of AD, neuroinflammation inhibitor based on phenotypic screenings displayes the advantages in the field of drug discovery. AD10 was a neuroinflammation inhibitor, which was discoveried by Guangzhou institutes of biomedicine and health, Chinese Academy of Sciences, based on Minozac(the first neuroinflammation inhibitor in clinical trails). AD10 had improved inhibitory efficacy against interleukin 1 β(IC50 = 0.43 μM). In this study, we further research the saftety and pharmacokinetics of AD10 in rats and dogs in vivo and in vitro. Meanwhile, metabonomics was applied for finding the possible biomarker for Alzheimer’s disease in our study. 1. Building a LC-MS/MS method for the determination of AD10 in plasmaAccording to the food and drug administration guidance on bioanalytical medical method validation in May 2001, a specific, sensitive, rapid and reliable LC-MS/MS method was developed and validated for the determination of AD10 in rat plasma and dog plasma. Plasma samples were processed by liquid-liquid extraction with ether. The results displayed that the linear range was 0.5 – 1500 ng/m L with lower limit of quantification of 0.5 ng/m L. The intraand inter-day precisions were all within 15%, while the accuracy was all between 85% and 115%. The stabilities of AD10 in rat plasma and dog plasma were evaluated by analyzing replicates(n = 3) of plasma samples at the concentrations of 1.0, 20 and 1200 ng/m L, which were exposed to different conditions(time and temperature). The spiked plasma samples were analyzed after storage at room temperature for 6 h, at-20 ?C for one month and after three freeze–thaw cycles from-20 ?C to room temperature. The autosampler stability of AD10 in rat plasma and dog plasma was evaluated by re-injecting the previously injected quality control samples after a period of storage in the autosampler. The matrix effect was evaluated at three concentrations(1.0, 20 and 1200 ng/m L in plasma). Ion suppression or enhancement from the plasma matrix can be negligible under the current conditions. The LC-MS/MS method can be applied for the pharmacokinetics studies of AD10 in rats and dogs. 2. The pharmacokinetics of AD10 in ratsIn this experiment, twenty four rats were used for evaluating the pharmacokinetics of AD10. AD10 was orally administered at the dose of 1 mg/kg, 2 mg/kg and 4 mg/kg, respectively, and intravenous administered at the dose of 2 mg/kg. The pharmacokinetic parameters of AD10 at the oral dose of 1 mg/kg are as follows: AUC(0-∞): 76.5 ± 6.8 μg/L*h; T1/2: 2.2 ± 0.8 h; Cl: 13.2 ± 1.2 L/h/Kg; Cmax: 72.2 ± 5.0 ng/m L. The pharmacokinetic parameters of AD10 at the oral dose of 2 mg/kg are as follows: AUC(0-∞): 152.0 ± 22.8 μg/L*h; T1/2: 2.2 ± 0.2 h; Cl: 13.4 ± 1.9 L/h/Kg; Cmax: 152.7 ± 25.1 ng/m L.The pharmacokinetic parameters of AD10 at the oral dose of 4 mg/kg are as follows: AUC(0-∞): 354.5 ± 20.5 μg/L*h; T1/2: 2.5 ± 0.7 h; Cl: 11.3 ± 0.7 L/h/Kg; Cmax: 336.1 ± 19.4 ng/m L.The pharmacokinetic parameters of AD10 at the intravenous dose of 2 mg/kg are as follows: AUC(0-∞): 1032.3 ± 94.8 μg/L*h; T1/2: 2.3 ± 0.4 h; Cl: 6.4 ± 1.3 L/h/Kg; Cmax: 803.8 ± 109.2 ng/m L. The oral bioavailability of AD10 at the dose of 1 mg/kg, 2 mg/kg and 4 mg/kg is 14.8%, 14.7% and 17.2%, respectively.The linear correlation coefficient between AUC(0-∞) and oral dosage of AD10 was more than 0.99. 3. The pharmacokinetics of AD10 in dogsIn this experiment, twenty four dogs were used for evaluating the pharmacokinetics of AD10. AD10 was orally administered at the dose of 1 mg/kg, 2 mg/kg and 4 mg/kg, respectively, and intravenous administered at the dose of 2 mg/kg. The pharmacokinetic parameters of AD10 at the oral dose of 1 mg/kg are as follows: AUC(0-∞): 125.5 ± 15.9 μg/L*h; T1/2: 3.0 ± 0.6 h; Cl: 8.1 ± 1.0 L/h/Kg; Cmax: 164.7 ± 15.1 ng/m L. The pharmacokinetic parameters of AD10 at the oral dose of 2 mg/kg are as follows: AUC(0-∞): 249.3 ± 28.3 μg/L*h; T1/2: 3.2 ± 0.8 h; Cl: 8.1 ± 0.9 L/h/Kg; Cmax: 314.3 ± 19.0 ng/m L.The pharmacokinetic parameters of AD10 at the oral dose of 4 mg/kg are as follows: AUC(0-∞): 506.2 ± 59.5 μg/L*h; T1/2: 3.4 ± 0.5 h; Cl: 8.0 ± 0.9 L/h/Kg; Cmax: 622.5 ± 74.7 ng/m L.The pharmacokinetic parameters of AD10 at the intravenous dose of 2 mg/kg are as follows: AUC(0-∞): 989.1 ± 65.3 μg/L*h; T1/2: 2.7 ± 0.9 h; Cl: 2.0 ± 0.2 L/h/Kg; Cmax: 757.5 ± 170.1 ng/m L. The oral bioavailability of AD10 at the dose of 1 mg/kg, 2 mg/kg and 4 mg/kg is 25.4%, 25.2% and 31.5%, respectively. The linear correlation coefficient between AUC(0-∞) and oral dosage of AD10 was more than 0.99. 4. The stability of AD10 in liver microsomes systemIn this experiment, the stability of AD10 was evaluated in RLM, DLM, MLM and HLM, respectively. The clearances of AD10 in in RLM, DLM, MLM and HLM are 320.2 ± 13.4 m L·min-1·mg-protein-1, 289.1± 7.3 m L·min-1·mg-protein-1, 272.5± 10.7 m L·min-1·mg-protein-1 and 92.8± 6.7 m L·min-1·mg-protein-1, respectively. The results displayed that AD10 will be eliminated slower in human liver microsomes. These provide some valuable information for the further pharmacology study of AD10. 5. The inhibitory effects of AD10 on the activities of P450Drug interaction plays an important role in new drug development. In this experiment, the inhibitory effects of AD10 on the activities of major human cytochrome P450 are assessed by isoform-specific probe reactions and the determination of IC50 values of the reactions. AD10 has weak inhibition towards CYP3A4/5 and CYP2D6, displaying IC50(15 μM and 26 μM, respectively). However, no inhibitions are found towards CYP1A2,CYP2C8,CYP2C9,CYP2C19 and CYP2B6. The results provide useful information for the further pharmacology of AD10. 6. Discovery of potential biomarkers for AD in rats based on metabonomicsMetabonomics becomes a popular “omics” since the late 1990 s. At present, metabonomics has been successfully applied in biomedicine, disease diagonosis and searching biomarker and so on. In this experiment, metabonomics are used for the discovery of biomarker in AD based on UPLC-TOF-MS.(1) Describe and introduce the objectives of metabonomics and analytical techniques.(2) UPLC-TOF-MS method was used for the determination of samples in AD rats. The data were analysed with pattern recognition methods.(3) By searching in the database, the structure of the molecular was comfirmed with product ion scan and literatures.(4) Eight potential biomarkers in plasma in correlation with AD were found including LPC C16:0, LPC C18:0, LPC C18:1, LPC C18:2, LPC C20:4, phytosphingosin e, dihydrosphingosine and tryptophan. The different mechanisms on the AD correlated with eight biomarkers were illuminated: LPCs, sphingosine and tryptophan were related to the phospholipid metabolism, ceramide metabolism and kynurenine pathway in brain, respectively. The found biomarkers for AD were very important to the study on the pathogenesy, diagnosis and clinical research of AD.