| Heparin has its unique advantages as a classical anticoagulant drug, such as strong anticoagulant effect, fast effect and abundant clinical application experience. Despite all these advantages, it has disadvantages as well, such as the need for intravenous administration, short biological half-life, unpredictable pharmacokinetics, individual differences and the risk of bleeding.Compared with heparin, Low molecular weight heparin has more uniform composition, higher anti-FXa/anti FⅡa ratio, quite anti-thrombotic activity of heparin, higher bioavailability, longer half-life in vivo, and smaller risk of bleeding. It can be administered by subcutaneous injection, and the drug pharmacokinetic characteristics can be predicted. Now low molecular weight heparin especially enoxaparin sodium has been widely used in clinical. Enoxaparin sodium is a kind of low molecular weight heparin made from heparin after alkaline degradation of β-elimination. It has the largest clinical indications, and is the best-selling one of all the low molecular weight heparin products. Although low molecular weight heparin including enoxaparin show a significant reduction in the risk of bleeding compared with heparin. But once bleeding occurs, it can not be neutralized by protamine sulfate as effectively as heparin. This study fractionly separated enoxaparin and obtained three components with the method of affinity chromatography. Comparative study of its physical and chemical properties, and biological activity, neutralization degree of protamine sulfate, antithrombotic activity and in vivo pharmacokinetic properties, lays the foundation for more effective and safe foundation for LMWH.In this study, the affinity column was prepared with agarose gel as a carrier and protamine sulfate as a ligand. The affinity chromatography was applied in seperating enoxaparin fractionly and obtaining components with different protamine affinity degree. And the physicochemical properties like IR, NMR, sugar chains (chain mapping), disaccharide composition and molecular weight distribution, were analysis and compared to enoxaparin sodium. The in vitro anticoagulation activity was measured by sheep plasma method and chromogenic substrate method. The neutralized degree of anticoagulation activity by protamine sulfate was determined with chromogenic substrate method. The antithrombus activities of different samples were studied by establishing venous thrombosis model. The pharmacokinetics of samples were determined by chromogenic substrate method after subcutaneous injection to rats. The main research results of this study were as following:1. The affinity column was prepared with CNBr-activted Sepharose4B as a carrier and protamine sulfate as a ligand.2. Using the protamine sulfate affinity column to fractionate enoxaparin sodium, three components were obtained through elution the column with0.05M Tris-HCl, OM NaCl and0.05M Tris-HCl,0.5M NaCl,0.05M Tris-HCl,1.0M NaCl. The low protamine sulfate binding strength(L-EP), middle protamine sulfate binding strength(M-EP) and high protamine sulfate binding strength(H-EP) components were lyophilized after desalting by centrifugal ultrafiltration repeatedly. The yields of L-EP, M-EP and H-EP are56.8%,27.6%and2.6%, separately. Because the yield of H-EP is too low, we did not study this compent.3. The components were determined by UV, IR,’H-NMR and the sugar chain of the components were determined by CTA-SAX HPLC. After enzymolysis completely, the disaccharide composition was determined by SAX-HPLC. GPC-HPLC and multi- angle laser light scatterometer were applied to measure the relative and absolute molecular weight. In addition, SO42-and COO-ratio was measured. The results showed that the binding strength with protamine is related to the molecular weight and charge density of the enoxaparin. The greater the charge density, the molecular weight and the ratio of SO42" and COO-, the combination of the components and the protamine will be stronger.4. The in vitro activity of the components was evaluated. The sheep plasma method was used to determine the anticoagulation activity and the chromogenic substrate method was used to determine the anti-FXa and anti-FⅡa activities. The results showed that the anticoagulant potency of EP, M-EP and L-EP was25.2IU/mg,30.3IU/mg and7.2IU/mg, respectively. The target product M-EP removed the part of low anticoagulant potency. The anti-FXa petency of EP, M-EP and L-EP was112IU/mg,114IU/mg and96IU/mg, respectively. The anti-FIIa potency was32.8IU/mg,38.0IU/mg and28.2IU/mg, respectively. The the target product M-EP has similar antiFXa activity with EP and higher anti FⅡa activity.5. The neutralization degree of the components by protamine sulfate using the chromogenic substrate method. The results showed that the neutralization degree was saturated when adding two times amount of protamine than samples. In this case, the anti FXa contents of EP, M-EP and L-EP were neutralized55.4%,91%and47.9%. The anti FⅡa contents of EP, M-EP and L-EP were neutralized100%,100%and0%. It is concluded that the anticoagulant activity of M-EP was neutralized most and that of L-EP were neutralized least. According to anticoagulant activity, M-EP not only retained the anticoagulant activity of EP, but also improved the neutralization degree by protamine. which was expected to improve drug safety.6. In this study, M-EP and L-EP solution were administered by subcutaneous injection to rats with established venous thrombosis model, as compared to EP. Five indexes were imployed to compare the anticoagulation and antithrombus activities of M-EP and L-EP, they are APTT, PT, venous thrombosis wet weight, Wessler score and coagulation time of caudal vein. The results were as following. The comparison of venous thrombosis wet weight were NS>L-EP>EP>M-EP and significant differences were found in the four groups, which indicated the comparison of the antithrombus efficiency was M-EP>EP>L-EP>NS. The value of other four indexes showed M-EP>EP>L-EP>NS, which indicated the anticoagulation was M-EP>EP>L-EP>NS.7. The pharmacokinetics of samples were determined by chromogenic substrate method after subcutaneous injection to rats. The plasma antiFXa and anti FⅡa activities at various time intervals were determined and the curves of antiFXa and anti FⅡa activity functioned by time were plotted, respectively. As shown from the results, Tmax is2h. In the antiFXa-time curve, Cmax of EP, M-EP and L-EP are1.04IU/ml、1.069IU/ml、0.957IU/ml and AUC are4.589IU*h/mL、6.598IU*h/mL、6.314IU*h/mL, respectively. In the antiFIIa-time curve, Cmax of EP, M-EP and L-EP are0.304IU/ml、0.356IU/ml、0.282IU/ml and AUC are1.987IU*h/mL、2.199IU*h/mL.1.86IU*h/ml, respectively.In conclusion, in this study the component which possess high affinity with protamine sulfate have larger electronic density, higher molecular weight, similar efficiency and more safety as compared to enoxaparin sodium. Furthermore, the protamine sulfate-affinity chromatography established in this study is also suitable for seperating other LMWH products, in other to obtain better novel LMWH products. |
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