Study On The Preparation Procedure And Quality Standard Of A New Ultra Low Molecular Weight Heparin

Anticoagulant drugs mainly include heparins and coumarins on the current market. Heparin has been widely used for the prevention and treatment of venous thromboembolism (VTE) since 1934, and its clinical value is still not replaced by any existing drugs. Heparins include unfractionated heparin (UFH, average molecular weight (MWavg)～15000 Da), low molecular weight heparin (LMWH, MWavg 4000-6000 Da) and ultra low molecular weight heparin (ULMWH, MWavg<4000 Da).When UFH is used to prevent and treat VTE, the adverse events like bleeding and heparin induced thrombosis (HIT) occurs for its high molecular weight. LMWH has the same antithrombotic activity to UFH. When LMWH used in clinic, the incidence of adverse event is lower, the bioavailability of subcutaneous injection is higher, the individual differences is smaller than UFH and it does not need laboratory monitoring. Because of these, LMWH has gradually replaced UFH in the prevention and treatment of VTE. ULMWH has the lowest molecular weight, the same or better antithrombotic activity to LMWH and the lowest incidence of bleeding adverse event, so the research of ULMWH is increasing.Currently, ULMWH is mainly prepared by chemical or enzymatic degradation of UFH and chemical synthesis. When using β-elimination to prepare LMWH and ULMWH, the sulfate groups are less destroyed, the anticoagulant activity can be better retained and the reagents are not residued in final product, which make it a good preparation method. In this study, P-elimination was used to prepare a ULMWH with a molecular weight range of 3000 Da to 4200 Da. In order to obtain a stable preparation procedure, we designed the orthogonal experiment to optimize the degradation reaction conditions. According to the obtained preparation procedure, four batches of samples were prepared, and their physicochemical properties were analyzed. According to the results, we established the quality standard in line with Chinese pharmacopoeia requirements. Finally, the structure of samples was analyzed by disaccharide analysis,2D 1H-13C HSQC and reversed-phase ion-pair chromatography mass spectrometry.The results and conclusions obtained in this study are as follows.1 Study on the preparation procedure of ULMWHIt has been reported that ULMWH can be obtained from degradation of UFH in the organic phase. In this study, the degradation reaction is completed in dichloromethane solution of heparin benzethonium chloride salts. The preparation procedure includes three steps, and the degradation reaction is the key step. There are many factors effecting degradation reaction including the ratio of heparin benzethonium chloride salts and benzyl trimethyl ammonium hydroxide, the reaction temperature and the times and the time interval of adding benzyl trimethyl ammonium hydroxide. In order to obtain a stable preparation procedure, we designed four factors and three levels orthogonal experiment. Then the degradation reaction condition was determined as: the ratio of heparin benzethonium chloride salts and dichloromethane is 1:5, the ratio of heparin benzethonium chloride salts and benzyl trimethyl ammonium hydroxide is 3.2:1, the reaction temperature is 35℃, the interval time of adding benzyl trimethyl ammonium hydroxide is 24 h and the times of adding benzyl trimethyl ammonium hydroxide is 5.2 Optimization of the preparation procedure for ULMWHThe boiling point of the solvent used in the degradation reaction is closed to the reaction temperature, so we want to replace it with the high boiling solvent. The sample was obtained by the degradation reaction in which DMF was used as the solvent. The molecular weight of the sample is 13500 Da. Then, using DMSO as solvent, the molecular weight of the sample is 13607 Da. There are many shortcomings in using benzyl trimethyl ammonium hydroxide, such as low boiling point, volatility and toxicity, so we consider switching to its structural analogs, tetramethyl ammonium hydroxide. According to the original conditions, the experiment was completed with tetramethyl ammonium hydroxide. The molecular weight of the sample is 5872 Da. The results of molecular weight showed that the qualified samples can not be acquired by using different solvent or alkali. Although we did not find the appropriate solvent and alkali, it is benefit to improve the security if finding the appropriate solvents with high boiling and low toxicity.3 Formulating the quality standardAccording to the preparation procedure, four batches of samples are prepared. The weights of each batch of samples are 21.0 g,24.9 g,30.46 g and 19.0 g, separately. The physicochemical properties of each batch of samples were analyzed. The molecular weight of each batch of samples was 3378 Da,3638 Da,3820 Da and 3986 Da, separately. The other physicochemical properties showed that there were no significant differences between each batch of samples. Through preparing four batches of samples, we discussed the impact of the sample process on the physicochemical properties. The final yield can be improved by the preparation method of sample 131014B3. Finally, according to the physicochemical properties of each batch of samples and referencing the enoxaparin sodium quality standard in USP, we formulated the quality standard which met the pharmacopoeia requirements.It provided a standard to control the quality.4 The structural analysis of ULMWHThe structural analysis including disaccharide composition analysis,2D 1H-13C HSQC and reversed-phase ion-pair chromatography-mass spectrometry was carried with the four batches of samples. According to the analysis results, there were no significant differences in structure of each sample. The main disaccharides of all the samples are IS (α-â–³UA-2S-[1â†'4]-GlcNS-6S, average 75.545%), IIS (α-â–³UA-[1â†'4]-GlcNS-6S, average 8.563%) and HIS (a-AUA-2S-[1â†'4]-GlcNS, average 7.158%), and the samples have a high degree of sulfation. There was a 4,5-unsaturated uronic acid in the non-reducing end, and the samples have the similar structure to heparin in the reducing end. There is an unknown dehydration structure in the saccharide chains of the samples.In this study, we obtained a feasible preparation procedure of ULMWH. And we formulated the quality standard which met the pharmacopoeia requirements. At last, the structural analysis was carried with each batch of samples. All of these provide a favorable foundation for controlling the quality of ULMWH.