Research On Pharmacokinetics/ Pharmacodynamics(PK/PD)and Mechanism Of Action Of D-polymannuronicate

Objective:1. To establish the methodology for the determination of D-polymannuronicate plasma concentration, investigate pharmacokinetics and pharmacodynamics of D-polymannuronicate in rats with intravenous injection and intragastric administration, and provide information for dosage regimen design and the treatment of ischemic cerebrovascular disease. 2. To establish middle cerebral artery occlusion(MCAO)reperfusionin model of rats, observe protection of D-polymannuronicate and determine the contents of MDA,SOD,GSH-PX,TXB2 and 6-Keto- PGF1αin brain tissue of rats that subjected to cerebral ischemia reperfusion , investigate the possible mechanism pharmacological action.3. To observe the effect of anti-thrombus of different molecular weight D-polymannuronicate in rats. Methods:1. Applied aPTT bioanalysis to determine D-polymannuronicate plasma concentration and calculated the parameters of pharmacokinetics by DAS2.1.1. Half life and elimination rate constant were calculated by non-compartment model. The differences of parameters of pharmacokinetics between single and successive administration and between intravenous injection and intragastric administration were analyzed by t-test. 2. To establish the MCAO reperfusion model of rats with nylon line. After successive intravenous injections for 7 days, cerebral infarction area was determined by TTC staining method, cerebral edema was determined by dry-wet weight method, and brain tissue was observed by HE coloretur. At ischemia reperfusion(IR)24h, IR72h, IR144h, brain tissue was made tohomogenate and determined the contents of MDA,SOD,GSH-PX,TXB2,6-Keto- PGF1αrespectively. 3.The normal rats were injected by different molecular weight D-polymannuronicate .To determine the indexs of plasma after 1h. Results:1. Two groups of rats were administered by intravenous injection 9mg/kg and intragastric administration 60mg/kg. The plasma concentration-time curve fitted two compartment model. Terminal elimination rate constant Zata was 0.89 h-1～1.06 h-1 and elimination half life t1/2z was 0.69h～0.78 h,bioavailability was 3%～5%, apparent volume of distribution (Vd )was far less than 1 L?kg-1。After intravenous injection 9mg/kg and intragastric administration 60mg/kg for 7 days, parameters of pharmacokinetics on 7th day were similar with single dose, especially Zata and t1/2z .It indicated that multiple dosing didn't affect elimination and metabolism of this drug and accumulation after multiple dosing was not found. After D-polymannuronicate intravenous injection in rats, anticoagulation increased with plasma concentration. After 6 hours aPTT rebound to ordinary fluctuating level, and anticoagulant effect of DPS disappeared, after 7-day administration maximum of aPTT by intravenous injection was 2 times as single intravenous injection. Nevertheless anticoagulant effect didn't increase apparently. 2. The scores of neurologic impairment gradually decreased, Afte the rats were injected by D-polymannuronicate, Nimodipine, and low molecular weight heparin, Compared with Vehicle group at IR144h(p<0.05), the drugs could reduced cerbral infarct size evidently(p<0.05). After HE coloretur,neurocyte damage and interstitial edema aggravated gradually in Vehicle group, but the death of neurocyte decreased obviously in medication administration groups. Compared with sham operated group, content of MDA increased apparently in Vehicle group(p<0.01). The content of SOD and GSH-PX decreased apparently(p<0.05). The content of MDA decreased obviously to the level of pre-reperfusion at I/R 144h(p>0.05) in the D-polymannuronicate and Nimodipine groups ,compared with Vehicle group(p>0.05).Compared with sham operated group, the content of TXB2 increased apparently at different times(p<0.05),the content of 6-Keto-PGF1 decreased sharply(p<0.01),and the ratio of TXB2/6-Keto- PGF1αincreased evidently(p<0.01)in Vehicle group. D-polymannuronicate group ,the content of TXB2 ischemia-side decreased to compared with sham operated group, Vehicle group and Nimodipine group(p<0.05), the content of 6-Keto-PGF1αincreased evidently at IR144h compared with sham operated group(p>0.05), the ratio of TXB2/6-Keto- PGF1 decreased apparently along with time compared with sham operated group. compared with Vehicle and Nimodipine groups(p<0.05),the tatio of TXB2/6-Keto- PGF1αappeared to degraded tendency , compared with sham operated group(p<0.05), compared with Vehicle and Nimodipine groups at IR144h(p<0.05).3.The APTT extended notably in DPS I group ,PT and FIB had significant differences to compare with ehicle group and LMWH groups, Activity of anti-F IIa of DPS-I was higher than that of LMWH and DPS II and DPS III.The ratio of F Xa/ F IIa of DPS I group was lower than other groups.Conclusion:1. The bioanalysis for the determination of D-polymannuronicate plasma concentration established in this study can satisfy pharmacokinetics of chemicals. The well linearity in D-polymannuronicate plasma concentrations with anticoagulant effect was found. We found that t1/2z of DPS was about 1 hour, and the effect of anticoagulated blood lasted for about 6 hours. Hysteresis effect between maximal effect and plasma drug concentration didn't appear. Single and multiple dosing didn't affect main pharmacokinetics. Drug accumulation didn't appear on the continuous 7 days. The pharmacology, pharmacodynamics and pharmacokinetics of injections were similar with low molecular heparin. The effects of intragastric administration were similar with aspirin. 2. The scores of neurologic impairment had been improved evidently, cerebral infarct size had been reduced and the cerebral ischemia had been protected in MCAO reperfusion rats after successive D-polymannuronicate for 7 days. D-polymannuronicate can eliminate many free radicals in ischemia brain tissue, and mitigate brain injured induced by free radical reaction. In addition, it also can cause the decreasing of the content of TXB2 in ischemia brain tissue. As to 6-Keto- PGF1α, the content decreased firstly, and then rebounded to the level of pre-reperfusion. Moreover, D-polymannuronicate can decrease obviously the ratio of TXB2/6-Keto- PGF1αand inhibit the forming of thrombus. 3. Both activity of anti-F IIa and extension of APTT and effect of anticoagulated blood increase as molecular weight increase in different molecular weight D-polymannuronicate.when molecular weight of D-polymannuronicate diminish, the ratio of anti-F Xa/ F IIa increase,so the effect of anti-thrombus increase and the hemorrhagic tendency weak.