Preparation And In Vitro/in Vivo Evaluation Of A Rationally Designed New Low Molecular Weight Heparin

Anticoagulant drugs, currently on the market, are mainly composed of heparin, coumarins, and others. Heparin-class anticoagulants have been used on clinical for nearly80years. They have been widely used in anticoagulation and prevention of thrombosis. Heparin anticoagulants include unfractionated heparin, low molecular weight heparin, ultra low molecular weight heparin, chemically synthesized heparin analogues.Rational design of heparin is also in progress. Heparin’s molecular weight, polydispersity, polysaccharide chain length, resulting in a lot of shortcomings, which are as follows:bleeding side effects, pharmacokinetic characteristics difficult to predict, individual differences. The existing low molecular weight heparin can not be effectively neutralized. Control of the molecular chain length of heparin components, polydispercity, may develop a more superior performance new heparin anticoagulant.The anti-FⅡa activity of heparin requires at least18monosaccharide, then the molecular weight is at least5400Da.To get a relatively small P.D. of the RD-LMWH, it was designed to collect the fragments between5400Da and9OOODa. After the alkaline beta-elimination degradation, we got the product named β-LMWH。We took theβ-LMWH as material, combined with the Bio Gel P-10gel permeation chromatography method,the fragments between5400Da-9OOODa, denoted with RD-LMWH, fragments with molecular weight>9000denoted as LMWH-A, ragments with molecular weight<5400Da were recorded as of LMWH-B. HP and enoxaparin were taken as control. We did a lot of research, including structure certification and physicochemical property analysis, in vitro activity evaluation, in vivo evaluation of antithrombotic activity, determination of pharmacokinetic characteristics.The results and conclusions of our research are as follows.1. Preparation and purification of RD-LMWHUFH has been taken as material.Through the control of β-elimination conditions combinded with gel filtration chromatography, we get the rational designed low molecular weight heparin (RD-LMWH).The total yield is12.3%. The molecular weight nature of RD-LMWH, enoxaparin, and unfractionated heparin are as follows: RD-LMWH:Mp of6570Da,Mw of6550Da, P.D. of1.052,5400Da-9OOODa (%) of76.5%.HP:Mp of15000Da, Mw of15000Da, P.D. of1.5. enoxaparin:Mp of2896Da,Mw of4148Da,P.D. of1.279.It can be seen from the previous results that the RD-LMWH are appropriate in the molecular weight and distribution with the expectations of the experimental design.2. Structure certification and physicochemical property analysis of RD-LMWH(1)We use the disaccharide composition analysis, ultraviolet (UV) spectroscopy, infrared (IR) spectroscopy,1H-NMR, MALDI-TOF method to determine the molecular structure of the product.(2) The multi-angle laser light scattering results are basically the same determination with high performance liquid determination of results. RD-LMWH:Mp of6989Da(0.3%), Mw of6751Da(0.4%). HP:Mp of16079Da(0.2%), Mw of16650(0.3%). enoxaparin:Mp of3735Da(0.2%), Mw of4244Da(0.5%)。 (3)Part of the physical and chemical properties of the samples was determined. The sulfate content determination results:RD-LMWH of27.8%, heparin of28.4%, enoxaparin of27.3%, LMWH-A of27.9%, LMWH-B of26.8%, β-LMWH of27.5%. Sulfate/carboxyl ratio:RD-LMWH of1.97:1, HP of2.05:1, enoxaparin of1.92:1, LMWH-A1.95:1, LMWH-B of1.88:1,β-LMWH of1.94:1. The specific optical rotation results:RD-LMWH of+50.29°,HP of+50.98°,enoxaparin of+43.63°,LMWH-A of+52.58°,LMWH-A of+52.58°,RD-LMWH of+51.96,which meet the quality criteria of heparin anticoagulants.3. In vitro activity evaluation(1)Anticoagulant potency of samples with sheep plasma method are as follows: RD-LMWH of173.2±0.4IU/mg,heparin of204IU/mg, enoxaparin of33.9±3IU/mg, LMWH-A of180.1±0.7IU/mg, LMWH-B of23.4±0.12IU/mg,β-LMWH of138.6±0.4IU/mg.(2)Anti-FXa potency of samples was determined by chromogenic substrate method. RD-LMWH of166.5IU/mg, HP of193.2IU/mg, enoxaparin of118.7IU/mg. Anti-FⅡa potency of samples was determined by chromogenic substrate method. RD-LMWH of159.6IU/mg, heparin of190.3IU/mg, enoxaparin of31,9IU/mg.(3)The degree of anti-FXa potency neutralized with PS was tested. When PS was added1:1to samples, the anti-FXa potency of RD-LMWH was neutralized more than68%, while heparin of96%, enoxaparin of only23%.Adding an excess (3times) of samples, the anti-FXa potency of RD-LMWH was neutralized more than89%, while heparin of98%, enoxaparin of only30%.(4)The degree of anti-FⅡa potency neutralized with PS. When PS was added1:1to samples, the anti-FXa potency of RD-LMWH was neutralized more than87%,while HP of92%, enoxaparin of only87%.Adding an excess (3times) of samples, the anti-FXa potency of RD-LMWH was neutralized more than97.2%, while HP of97.5%, enoxaparin of only97.1%.(5)PS can be effective in the anti-FIIa titer, but not an effective anti-FXa potency. The degree of anti-FXa and anti-FⅡa potency neutralized with PS:heparin> RD-LMWH> enoxaparin.4. In vivo antithrombotic activity evaluationIn rat carotid artery thrombosis model, the thrombus wet weight, APTT and PT analysis shows that, the RD-LMWH at the dose of3mg/kg and5mg/kg are superior to enoxaparin of3mg/kg.In rat inferior vena cava thrombosis model, the thrombus wet weight, wessler score, APTT and PT analysis show that, the RD-LMWH at the dose of3mg/kg and5mg/kg are superior to enoxaparin of3mg/kg.5. Pharmacokinetic assaysDetermination of pharmacokinetic in New Zealand rabbits was through subcutaneous administration of enoxaparin and RD-LMWH, heparin is administered intravenously, all with3mg/kg. DAS2.0pharmacokinetic software has been used to calculate. The results are as follows.HP:t1/2of0.606±0.187h, AUC(0-t) of6.343±1.467(IU/L*h),Tmax of0.167h, Cmax of5.29±0.638(IU/mL);RD-LMWH t1/2of3.1±0.788h,AUC(0-t)(IU/L*h) of4.1±0.474, Tmax of3.667±0.816h, Cmax of0.49±0.047(IU/mL);enoxaparin t1/2of3.971±1.029h,AUC(0-t) of3.675±0.37(IU/L*h),Tmax of2.667±1.033h,Cmax of0.397±0.029(IU/mL).The APTT and PT of the rabbit blood were also determined.The conclusions are as follows.Tmax:RD-LMWH>enoxaparin>HP.It is because of the large molecular weight of RD-LMWH and the way of injection.t1/2:RD-LMWH>enoxaparin>HP,which was same to the references。From AUC(0-t) and Cmax, it can be seen that RD-LMWH can meet the requirements of rational design.6. Topics summary and innovation(1) Optimization of a β elimination method and gel chromatography to prepare the rationally designed low molecular weight heparin. (2) Analysis of the structure and physicochemical properties of heparin, RD-LMWH enoxaparin. The property of heparin and enoxaparin was also evaluated as comparation.(3)The anti-FXa activity and anti-FⅡa potency of heparin, RD-LMWH, enoxaparin were determined. And it was the first time to measure the degree of RD-LMWH neutralized with protamine sulfate. The property of heparin and enoxaparin was also evaluated as comparation.(4) The RD-LMWH pharmacokinetic evaluation was done for the first time. The property of heparin and enoxaparin was also evaluated as comparation. RD-LMWH After the S.C. administration, the t1/2and AUC (0-t) nature of LMWH was close to enoxaparin, and it can achieve the therapeutic effect of plasma concentration(5)It was the first time to evaluate the effect of RD-LMWH of anti-arterial thrombosis and venous thrombosis. The property of heparin and enoxaparin was also evaluated as comparation. The results showed that RD-LMWH can play an effective anti-artery thrombosis and anti-venous thrombosis activity.