• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Procedure for obtaining low molecular weight heparins and low molecular weight heparins obtained by the same. The invention relates to a procedure for obtaining low molecular weight heparins that have great stability. The procedure includes the treatment of depolymerized heparin with H2O2 in a proportion of between 0.04 and 1.0 liters of H2O2 at 33% w/v for each kg of depolymerized heparin. The invention is also directed to a heparin obtained by this process. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2792298A2
    申请号:ES202090024
    申请日:2020-04-27
    公开日:2020-11-10
    发明作者:Rodriguez Guillermo Franco；Aduriz Ibon Gutierro
    申请人:Laboratorios Farmaceuticos Rovi SA；
    IPC主号:
    专利说明:

    [0003] MOLECULAR AND LOW MOLECULAR WEIGHT HEPARINS OBTAINED BY
    [0005] FIELD OF THE INVENTION
    [0006] The present invention relates to a process for the preparation of low molecular weight heparins (LMWH) and to the low molecular weight heparins obtained by that process.
    [0008] BACKGROUND OF THE INVENTION
    [0009] Heparin is a polysaccharide of the family of glycosaminoglycans, formed by uronic acid (L-iduronic acid or D-glucuronic acid) and D-glucosamine, linked alternately. L-iduronic acid can be 2-O-sulphated and D-glucosamine can be N-sulphated and / or 6-O-sulphated, and to a lesser extent N-acetylated or 3-O-sulphated. Heparin is preferably used as a sodium salt, but it can also be used as a salt of other alkali or alkaline earth metals and is mainly used as an antithrombotic and anticoagulant drug.
    [0010] Heparins can be classified according to their molecular weight into unfractionated heparin (UFH), LMWH and very low molecular weight heparin (HMBPM). LMWH and HMBPM come from the depolymerization of the original UFH molecule. Various methods for the preparation of LMWH have been described in the state of the art. One of them corresponds to the alkaline depolymerization by a pelimination mechanism.
    [0011] EP0040144 describes a process for obtaining LMWH by means of a procedure that comprises the steps of transalification of a heparin salt in benzethonium heparinate, esterification of benzethonium heparinate with benzyl chloride, purification and obtaining of the sodium salt of the benzyl ester of heparin , depolymerization with sodium hydroxide with saponification of the ester and purification of the product.
    [0012] EP1070503 describes a process for obtaining LMWH by a process that comprises the steps of transalification of a heparin salt in benzalkonium heparinate, depolymerization in a non-aqueous medium with Triton B and purification of the product.
    [0013] EP2881404 describes a process for obtaining LMWH that comprises a first transalification step, a depolymerization with a phosphazene base or a base derived from guanidine and a last transalification.
    [0014] There is a need for more reproducible and stable processes that allow the obtaining of LMWH, especially LMWH with greater stability.
    [0016] DESCRIPTION OF THE INVENTION
    [0017] The inventors of the present invention have found a method of preparing low molecular weight heparins (LMWH) which exhibit superior stability, while maintaining good anti-FXa and anti-FIIa activity. This method of preparing LMWH comprises treating crude depolymerized heparin with H2O2 in a ratio of 0.04-1.0 liters of 33% w / v H2O2 per kg of depolymerized heparin.
    [0018] Therefore, in a first aspect the invention refers to a process for obtaining low molecular weight heparins with an average molecular weight of between 3 and 3.8 KDa, which comprises the following steps:
    [0019] a) preparing an aqueous solution of sodium heparin;
    [0020] b) adding benzalkonium chloride to the solution of step (a) to obtain benzalkonium heparinate;
    [0021] c) dissolve the benzalkonium heparinate obtained in step (b) in ChCH2, add Triton B and maintain a temperature between 20 and 40 ° C for between 24 and 48 hours; Y
    [0022] d) Carry out at least two treatments with H2O2 of the depolymerized heparin obtained in step c) in a ratio of between 0.04 and 1.0 liters of H2O2 at 33% w / v for each kg of depolymerized heparin, in each treatment .
    [0024] A second aspect of the invention relates to a low molecular weight heparin obtainable by the method of the invention.
    [0026] The LMWH obtained have been found to have a 1,6-anhydrous residue content of between 1 and 15%. Therefore, a third aspect of the invention relates to a low molecular weight heparin with an average molecular weight of between 3 and 3.8 KDa that has a 1,6-anhydrous residue content of between 1 and 15% in the reducing terminal of its oligosaccharide chains.
    [0028] The definitions and embodiments described for one aspect apply equally to all other aspects of the invention.
    [0029] In the present invention, "low molecular weight heparin" or "LMWH" is understood as the definition contained in the document Heparins, Low-Molecular-Mass monograph, 0828, European Pharmacopeia 9th Ed., as the mixture of polysaccharides obtained from heparin and which has an average molecular weight of less than 8,000 Dalton and where at least 60% of its total mass has a molecular weight of less than 8,000 Da. The average molecular weight of the LMWHs of the invention has been determined by the procedure of the European Pharmacopoeia (Ph. Eur. 9th Edition).
    [0030] In the present invention, "1,6-anhydrous residues" are understood to be various chemical groups that are generated in the terminal positions of HPBM during the depolymerization process. Non-limiting examples of these groups are 2-sulfoamino-1,6- anhydro-2-deoxy-pD-glucopyranose (1,6-anhydroglucosamine) and 2-sulfoamino-1,6-anhydro-2-deoxy-pD-mannopyranose (1,6-anhydromanosamine). The amount of these residues in HPBM It is expressed as the percentage of oligosaccharide chains that present these types of residues at their reducing terminal.
    [0031] The terminal 1,6-anhydrous content of LMWH can be obtained by the analytical method described in the document Enoxaparin Sodium monograph, 1097, European Pharmacopeia 9th Ed, described in section Identification B. In this method the molecule is thoroughly depolymerized with a mixture of heparinases I, II and III, separating and quantifying the residues generated by strong anion exchange chromatography (SAX-HPLC). The 1,6-anhydrous content, for example, is determined according to the following formula:
    [0033] % 1,6-anhydrous = Mw • (Ai A2 A3) * 100 / IMwx • Ax
    [0034] where:
    [0035] - Mw, average molecular weight
    [0036] - Mwx, molecular weight of derivative x (see table 1097-1 of the document Enoxaparin Sodium monograph, 1097, European Pharmacopeia 9th Ed)
    [0037] - Ax, area of the peak of derivative x
    [0038] - A1, peak area of the 1,6-anhydrous derivative AIS
    [0039] - A2, peak area of the 1,6-anhydrous derivative AIIS
    [0040] - A3, peak area of the 1,6-anhydrous derivative AIS-IS
    [0041] The proportion of 1,6-anhydroglucosamine residues and 1,6-anhydromanosamine residues in LMWH can be determined by Nuclear Magnetic Resonance (NMR), for example by 1H 13C HSQC. The ratio between both residues can be determined by integrating the signals corresponding to each of these residues in the 1H 13C HSQC spectrum.
    [0042] The anti-FXa and anti-FIIa activity of the LMWHs of the invention has been determined by the chromogenic procedure of the European Pharmacopoeia (Ph. Eur. 9th Edition, Monograph 0828) and has been expressed in international units per mg.
    [0043] The degree of color of LMWH can be determined according to method II of the European Pharmacopoeia chapter 2.2.2.
    [0044] The term "room temperature" refers to a temperature between 20 and 25 ° C.
    [0045] As used herein, the term "about" means ± 10% of the given value.
    [0046] The LMWHs of the invention have an average molecular weight (Mw) of between 3 and 3.8 KDa, preferably between 3 and 3.6 KDa.
    [0047] In one embodiment, the LMWH of the invention has a 1,6-anhydrous residue content of between 1 and 15% at the reducing end of its oligosaccharide chains; preferably between 2 and 13%, more preferably between 4 and 11%.
    [0048] Preferably, the molar ratio of the 1,6-anhydroglucosamine residues in the LMWH of the invention is greater than or equal to that of the 1,6-anhydromanosamine residues. In a particular embodiment, the molar ratio of 1,6-anhydroglucosamine residues: 1,6-anhydromanosamine residues in LMWH is between 1: 1 and 3: 1, preferably between 1: 1 and 2.5: 1 or between 1, 05: 1 and 2.5: 1.
    [0049] In a particular embodiment of the invention, LMWH has an average molecular weight of between 3 and 3.8 KDa, a content of 1,6-anhydrous residues of between 1 and 15% at the reducing terminal of its oligosaccharide chains, and a ratio of 1,6-anhydroglucosamine residues greater than or equal to that of 1,6-anhydromanosamine residues, preferably between 1: 1 and 3: 1.
    [0050] Preferably, the LMWH of the invention has an anti-FXa activity of between 80-120 IU / mg and an anti-FIIa activity of between 5-20 IU / mg. In a particular embodiment, it has an anti-FXa activity of between 95-120 IU / mg and an anti-FIIa activity of between 10-20 IU / mg.
    [0051] In a particular embodiment of the invention, LMWH has an average molecular weight of between 3 and 3.8 KDa, a content of 1,6-anhydrous residues of between 1 and 15% at the reducing terminal of its oligosaccharide chains, and a ratio of 1,6-anhydroglucosamine residues greater than or equal to that of 1,6-anhydromanosamine residues, preferably between 1: 1 and 3: 1, an anti-FXa activity between 80-120 IU / mg and an anti-FIIa activity between 5-20 IU / mg.
    [0052] Preferably, the LMWH of the invention has a color degree greater than or equal to 6 in the range of color reference solutions established in the Pharmacopoeia. European chapter 2.2.2. (method II), for at least 24 months, preferably for at least 36 months, at room temperature and 60% relative humidity. This determination can be made following the method described in the European Pharmacopoeia (chapter 2.2.2; method II), or automatically using a colorimeter.
    [0053] The LMWHs of the invention have been found to exhibit high stability. In particular, they have been observed to be stable for at least 24 months, or even for at least 36 months at room temperature and 60% relative humidity.
    [0054] In one aspect, the invention relates to a process for obtaining low molecular weight heparins with an average molecular weight of between 3 and 3.8 KDa comprising the following steps:
    [0055] a) preparing an aqueous solution of sodium heparin;
    [0056] b) adding benzalkonium chloride to the solution of step (a) to obtain benzalkonium heparinate;
    [0057] c) dissolve the benzalkonium heparinate obtained in step (b) in CH2Ch, add Triton B and maintain a temperature between 20 and 40 ° C for between 24 and 48 hours; Y
    [0058] d) perform at least two treatments with H2O2 of the depolymerized heparin obtained after step c) in a ratio of between 0.04 and 1.0 liters of H2O2 at 33% w / v for each kg of depolymerized heparin, in each treatment .
    [0060] Particular and preferred embodiments for low molecular weight heparin are as previously defined herein.
    [0061] In a particular embodiment, the aqueous sodium heparin solution of step a) is prepared from heparin obtained from the intestinal mucosa of the pig.
    [0062] The addition of Triton B in step c) can be done by one or more sequential additions, for example by 1, 2, 3 or 4 sequential additions of Troton B.
    [0063] In a particular embodiment, the Triton B addition in step c) is carried out in a maximum of three sequential additions, i.e. 1, 2 or 3, each with the addition of Triton B in a weight ratio of 0.2: 1 to 0.3: 1 Triton B: benzalkonium heparinate. Preferably, the addition of Triton B in step c) is carried out by three sequential additions of Triton B, and preferably each with the addition of Triton B in a weight ratio of 0.2: 1 to 0.3: Triton B 1: benzalkonium heparinate.
    [0064] In one embodiment of the invention, the addition of Triton B in step c) is carried out by three sequential additions of Triton B, so that after the first addition the reaction is maintained for 6-10 hours until the second addition, after the second addition the reaction is maintained for 12-20 hours until the third addition, and after the third addition the reaction is maintained for 6-10 hours. In a further embodiment, after the first addition the reaction is maintained for 7-9 hours until the second addition, after the second addition the reaction is maintained for 14-18 hours until the third addition, and after the third addition the reaction is maintained. for 7-9 hours. In yet another embodiment, the reaction time after the first, second, and third addition is about 8, about 16, and about 8 hours, respectively.
    [0065] In one embodiment, the temperature in step c) is between 25 and 35 ° C, preferably between 27 and 32 ° C.
    [0066] In a preferred embodiment, the H2O2 treatment in step d) is carried out on an aqueous solution of the depolymerized heparin.
    [0067] Preferably, each H2O2 treatment in step d) is carried out with a ratio of between 0.04 and 0.5 liters of H2O2 at 33% w / v for each kg of depolymerized heparin, preferably between 0.04 and 0.3 liters of H2O2 at 33% w / v for each kg of depolymerized heparin. In a particular embodiment, each treatment with H2O2 in step d) is carried out with a ratio of between 0.04 and 0.2 liters of H2O2 at 33% w / v for each kg of depolymerized heparin. In a preferred embodiment, step d) comprises a first treatment with H2O2 of the depolymerized heparin obtained after step c) in a ratio of between 0.05 and 0.25 liters of H2O2 at 33% w / v per kg of depolymerized heparin, and a second treatment with H2O2 in a ratio of between 0.04 and 0.25 liters of H2O2 at 33% w / v for each kg of depolymerized heparin.
    [0068] In another embodiment, step d) includes three treatments with H2O2.
    [0069] Preferably step d) is carried out at a temperature of between 20 and 50 ° C, preferably between 25 and 45 ° C.
    [0070] Preferably, each H2O2 treatment is carried out for at least 3 hours. For example, for between 3 and 20 hours. In a particular embodiment, the first treatment with H2O2 is carried out for a time between 12 and 20 hours, preferably between 14 and 18 hours. Preferably, the second and subsequent treatments with H2O2 are carried out for a time between 3 and 7 hours, preferably between 4 and 6 hours.
    [0071] In a preferred embodiment, step d) is carried out at a pH between 10.5 and 11.5. Additionally, the process of the invention may include an additional step of precipitation in methanol of the depolymerized heparin after each treatment with H2O2 in step d). In a particular embodiment, two treatments are carried out with H2O2 and a step of heparin precipitation in methanol is carried out. depolymerized between the two H2O2 treatments. In another embodiment, three treatments are carried out with H2O2 and a step of precipitation in methanol of the depolymerized heparin is carried out between the first and second treatment with H2O2 and between the second and third treatment with H2O2.
    [0072] Preferably, after the first treatment with H2O2 in step d), the depolymerized heparin is precipitated from a solution of sodium acetate in methanol.
    [0073] In a preferred embodiment, the LMWH obtained in the process of the invention is purified by precipitation with methanol.
    [0074] The LMWH obtained can be subjected to lyophilization.
    [0076] BRIEF DESCRIPTION OF THE FIGURES
    [0077] Figure 1. 1H NMR spectrum of LMWH obtained by the method of the invention with anomeric zone enlargement.
    [0078] Figure 2. 1H 13C HSQC spectrum of LMWH obtained by the method of the invention with anomeric zone enlargement.
    [0080] EXAMPLES
    [0081] The following specific examples provided below serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and are not to be construed as limitations on the invention claimed herein.
    [0082] Example 1
    [0083] 10 g of sodium heparin are dissolved in purified water and, with stirring, a 50% (w / v) benzalkonium chloride solution is added, forming benzalkonium heparinate. The product formed is washed several times with water to remove excess chlorides and finally the product is dried by lyophilization.
    [0084] The benzalkonium heparinate is dissolved in methylene chloride and the temperature is adjusted to 30 ± 5 ° C. Benzyltrimethylammonium hydroxide (Triton B) 40% is added and it is allowed to react at the temperature indicated above. The addition is repeated two more times.
    [0085] To the aqueous solution of the depolymerized product, H2O2 at 33% w / v is added, particularly at a concentration of 0.1 ± 10% liters of H2O2 per kg of benzalkonium heparinate and it is allowed to react at 30 ± 5 ° C. After the reaction time has elapsed, it is precipitated on a solution of sodium acetate in methanol, the crude low molecular weight heparin being isolated by centrifugation.
    [0086] This crude low molecular weight heparin is dissolved in water and reprecipitated with methanol. The precipitate is dissolved in purified water and treated with H2O233% w / v, specifically, at a concentration of 0.08 ± 10% liters of H2O2 / kg of benzalkonium heparinate at a temperature of 40 ± 2 ° C. After the reaction period, the solution is neutralized and precipitated with methanol.
    [0087] The precipitate is redissolved in purified water and treated again with H2O233% w / v at a temperature of 40 ° C ± 2 ° C. After the reaction period, the solution is neutralized and precipitated with methanol.
    [0088] The purified product is dissolved in water and lyophilized, obtaining 6.00 g of low molecular weight heparin and an average molecular weight of 3241 Da, an anti-FXa activity of 118 IU / mg and an anti-FIIa activity of 13. 7 Ul / mg and has a content of 1,6-anhydrous residues between 1 and 15% at the reducing terminal of its oligosaccharide chains, where the proportion of 1,6-anhydroglucosamine residues is greater than or equal to that of residues 1, 6 anhydromanosamine, unlike other low molecular weight heparins.
    [0089] This low molecular weight heparin also has the advantage of being stable at room temperature for 24-36 months, presenting a color grade> 6 measured with a LICO® brand colorimeter that relates color according to the reference color range established in the Pharmacopoeia. (Ph. Eur. (2.2.2, Method II)).
    [0090] The degree of color, a parameter directly related to the stability of the product, made it possible to determine that the final packaged product was stable at room temperature in an interval between 24 and 36 months.
    [0091] Example 2
    [0092] Starting from 10 g of sodium heparin and repeating the steps indicated in Example 1, 6.01 g of low molecular weight heparin are obtained and an average molecular weight of 3259 Da, an anti-FXa activity of 103 IU / mg and a anti-FIIa activity of 15.2 IU / mg. This low molecular weight heparin has the characteristic of having at the reducing end of its oligosaccharide chains a content of 1,6-anhydrous residues between 1 and 15% where the proportion of 1,6-anhydroglucosamine residues is greater than that of 1,6 anhydromanosamine residues, unlike other low molecular weight heparins.
    [0093] This low molecular weight heparin also has the advantage of being stable at room temperature for 24-36 months, presenting a color grade> 6 measured with a LICO® brand colorimeter that relates color according to the reference color range established in the Pharmacopoeia. (Ph. Eur. (2.2.2, Method II)).
    [0094] The degree of color, a parameter directly related to the stability of the product, made it possible to determine that the final packaged product was stable at room temperature in an interval between 24 and 36 months.
    [0095] Example 3
    [0096] 10 g of sodium heparin are dissolved in purified water and, with stirring, a 50% (w / v) benzalkonium chloride solution is added, forming benzalkonium heparinate. The product formed is washed several times with water to remove excess chlorides and finally the product is dried by lyophilization.
    [0097] The benzalkonium heparinate is dissolved in methylene chloride and the temperature is adjusted to 30 ° C ± 5 ° C. Benzyltrimethylammonium hydroxide 40% (Triton B) is added and it is allowed to react at the temperature indicated above. The addition is repeated two more times.
    [0098] To the aqueous solution of the depolymerized product, H2O2 at 33% w / v is added, particularly at a concentration of 0.1 ± 10% liters of H2O2 / kg of benzalkonium heparinate and it is allowed to react at 30 ± 5 ° C. After the reaction time has elapsed, it is precipitated on a solution of sodium acetate in methanol, the crude low molecular weight heparin being isolated by centrifugation.
    [0099] This crude low molecular weight heparin is dissolved in water and reprecipitated with methanol. The precipitate is dissolved in purified water and treated with H2O233% w / v, specifically, at a concentration of 0.08 ± 10% liters of H2O2 / kg of benzalkonium heparinate at a temperature of 40 ± 2 ° C. After the reaction period, the solution is neutralized and precipitated with methanol.
    [0100] The purified product is dissolved in water and lyophilized, obtaining 5.21 g of low molecular weight heparin and an average molecular weight of 3269 Da, an anti-FXa activity of 119 IU / mg and an anti-FIIa activity of 13. 70 IU / mg and presents at the reducing terminal of its oligosaccharide chains a content of 1,6-anhydrous residues between 1 and 15% where the proportion of 1,6-anhydroglucosamine residues is greater or equivalent than that of residues 1, 6 anhydromanosamine, unlike other low molecular weight heparins.
    [0101] This low molecular weight heparin also has the advantage of being stable at room temperature for 24-36 months, presenting a color grade> 6 measured with a LICO® brand colorimeter that relates color according to the reference color range established in the Pharmacopoeia. (Ph. Eur. (2.2.2, Method II)).
    [0102] The degree of color, a parameter directly related to the stability of the product, made it possible to determine that the final packaged product was stable at room temperature in an interval between 24 and 36 months.
    [0103] Example 4
    [0104] Starting from 10 g of sodium heparin and repeating the steps indicated in example 1, 6.25 g of low molecular weight heparin are obtained with an average molecular weight of 3172 Da, an anti-FXa activity of 108 IU / mg and a anti-FIIa activity of 13.1 IU / mg and a profile of 1,6-anhydrous residues in the reducing terminal of its oligosaccharide chains between 1 and 15% where the proportion of 1,6-anhydroglucosamine residues is greater or equivalent than that of the 1,6 anhydromanosamine residues, unlike other low molecular weight heparins, such as enoxaparin.
    [0105] This low molecular weight heparin also has the advantage of being stable at room temperature for 24-36 months, presenting a color grade> 6 measured with a LICO® brand colorimeter that relates color according to the reference color range established in the Pharmacopoeia. (Ph. Eur. (2.2.2, Method II)).
    [0106] The degree of color, a parameter directly related to the stability of the product, made it possible to determine that the final packaged product was stable at room temperature in an interval between 24 and 36 months.
    [0107] Example 5
    [0108] Starting from 10 g of sodium heparin and repeating the steps indicated in Example 1, 6.34 g of low molecular weight heparin are obtained with an average molecular weight of 3347 Da, an anti-FXa activity of 110 IU / mg and a anti-FIIa activity of 14.7 IU / mg and a profile of 1,6-anhydrous residues at the reducing terminal of its oligosaccharide chains between 1 and 15% where the proportion of 1,6-anhydroglucosamine residues is greater or equivalent than that of the 1,6 anhydromanosamine residues, unlike other low molecular weight heparins, such as enoxaparin.
    [0109] This low molecular weight heparin also has the advantage of being stable at room temperature for 24-36 months, presenting a color grade> 6 measured with a LICO® brand colorimeter that relates color according to the reference color range established in the Pharmacopoeia. (Ph. Eur. (2.2.2, Method II)). The degree of color, a parameter directly related to the stability of the product, made it possible to determine that the final packaged product was stable at room temperature in an interval between 24 and 36 months.
    [0110] Example 6
    [0111] Starting from 10 g of sodium heparin and repeating the steps indicated in Example 1, 7.11 g of low molecular weight heparin are obtained with an average molecular weight of 3400 Da, an anti-FXa activity of 119 IU / mg and a anti-FIIa activity of 15.0 IU / mg and a profile of 1,6-anhydrous residues in the reducing terminal of its oligosaccharide chains between 1 and 15% where the proportion of 1,6 residues anhydroglucosamine is higher than that of the 1,6 anhydromanosamine residues, unlike other low molecular weight heparins, such as enoxaparin.
    [0112] This low molecular weight heparin also has the advantage of being stable at room temperature for 24-36 months, presenting a color grade> 6 measured with a LICO® brand colorimeter that relates color according to the reference color range established in the Pharmacopoeia. (Ph. Eur. (2.2.2, Method II)). The degree of color, a parameter directly related to the stability of the product, made it possible to determine that the final packaged product was stable at room temperature in an interval between 24 and 36 months.
    [0113] Example 7
    [0114] Starting from 10 g of sodium heparin and repeating the steps indicated in Example 1, 6.92 g of low molecular weight heparin are obtained and an average molecular weight of 3328 Da, an anti-FXa activity of 117 IU / mg and a anti-FIIa activity of 14.9 IU / mg and and a 1,6-anhydrous residue profile in the reducing terminal of its oligosaccharide chains between 1 and 15% where the proportion of 1,6-anhydroglucosamine residues is greater or equivalent than that of the 1,6 anhydromanosamine residues, unlike other low molecular weight heparins, such as enoxaparin.
    [0115] This low molecular weight heparin also has the advantage of being stable at room temperature for 24-36 months, presenting a color grade> 6 measured with a LICO® brand colorimeter that relates color according to the reference color range established in the Pharmacopoeia. (Ph. Eur. (2.2.2, Method II)). The degree of color, a parameter directly related to the stability of the product, made it possible to determine that the final packaged product was stable in an interval between 24 and 36 months.
    [0116] Example 8
    [0117] Starting from 10 g of sodium heparin and repeating the steps indicated in Example 3, 7.04 g of low molecular weight heparin are obtained with an average molecular weight of 3331 Da, an anti-FXa activity of 113 IU / mg and a anti-FIIa activity of 15.3 IU / mg and a 1,6-anhydrous residue profile at the reducing terminal of its oligosaccharide chains between 1 and 15% where the proportion of 1,6-anhydroglucosamine residues is greater or equivalent than that of the 1,6 anhydromanosamine residues, unlike other low molecular weight heparins, such as enoxaparin.
    [0118] This low molecular weight heparin also has the advantage of being stable at room temperature for 24-36 months, presenting a color grade> 6 measured with a LICO® brand colorimeter that relates color according to the reference color range established in the Pharmacopoeia. (Ph. Eur. (2.2.2, Method II)). The degree of color, A parameter directly related to the stability of the product, it allowed to determine that the final packaged product was stable at room temperature in an interval between 24 and 36 months.
    [0119] Example 9
    [0120] Starting from 10 g of sodium heparin and repeating the steps indicated in Example 3, 7.09 g of low molecular weight heparin are obtained with an average molecular weight of 3366 Da, an anti-FXa activity of 115 IU / mg and a anti-FIIa activity of 16.0 IU / mg and a 1,6-anhydrous residue profile at the reducing terminal of its oligosaccharide chains between 1 and 15% where the proportion of 1,6-anhydroglucosamine residues is greater or equivalent than that of the 1,6 anhydromanosamine residues, unlike other low molecular weight heparins, such as enoxaparin.
    [0121] This low molecular weight heparin also has the advantage of being stable at room temperature for 24-36 months, presenting a color grade> 6 measured with a LICO® brand colorimeter that relates color according to the reference color range established in the Pharmacopoeia. (Ph. Eur. (2.2.2, Method II)). The degree of color, a parameter directly related to the stability of the product, made it possible to determine that the final packaged product was stable at room temperature in an interval between 24 and 36 months.
    [0122] Comparative Example 10 (without treatment with H 2 OR 2 )
    [0123] 10 g of sodium heparin are dissolved in purified water and, with stirring, a 50% (w / v) benzalkonium chloride solution is added, forming benzalkonium heparinate. The product formed is washed several times with water to remove excess chlorides and finally the product is dried by lyophilization.
    [0124] The benzalkonium heparinate is dissolved in methylene chloride and the temperature is adjusted to 30 ± 5 ° C. Benzyltrimethylammonium hydroxide 40% (Triton B) is added and it is allowed to react at the temperature indicated above. The addition is repeated two more times.
    [0125] The solution of the depolymerized product is precipitated on a solution of sodium acetate in methanol, the crude low molecular weight heparin being isolated by centrifugation.
    [0126] This crude low molecular weight heparin is dissolved in water and reprecipitated with methanol. The precipitate is dissolved in purified water at a temperature of 40 ± 2 ° C at pH 11. After 5 hours, the solution is neutralized and precipitated with methanol.
    [0127] The purified product is dissolved in water and lyophilized, obtaining 5.28 g of low molecular weight heparin and an average molecular weight of 3280 Da, an anti-FXa activity of 112 IU / mg and an anti-FIIa activity of 13.70 IU / mg and has a 1,6-anhydrous residue content of 18.5% at the reducing terminal of its oligosaccharide chains. In addition, the ratio of 1,6-anhydroglucosamine residues to that of 1,6-anhydromanosamine residues is analyzed, unlike the previous examples, 1,6-anhydromanosamine residues are higher than 1,6-anhydroglucosamine.
    [0128] The degree of stability correlated with the color of the sample is also analyzed, observing that from the tenth month at room temperature it presents a color degree of 4 measured with a LICO® brand colorimeter that relates the color according to the chromatic range of reference established in the Pharmacopoeia (Ph. Eur. (2.2.2, Method II)).
    [0129] The degree of color, a parameter directly related to the stability of the product, made it possible to determine that the final packaged product was not stable for more than 9 months at room temperature, which is why the H2O2 additions seem to be responsible for this behavior.
    [0130] Comparative example 11 (depolymerization at 60 ° C)
    [0131] 10 g of sodium heparin are dissolved in purified water and, with stirring, a 50% (w / v) benzalkonium chloride solution is added, forming benzalkonium heparinate. The product formed is washed several times with water to remove excess chlorides and finally the product is dried by lyophilization.
    [0132] The benzalkonium heparinate is dissolved in methylene chloride and the temperature is adjusted to 60 ± 5 ° C. Benzyltrimethylammonium hydroxide 40% (Triton B) is added and it is allowed to react at the temperature indicated above. The addition is repeated two more times.
    [0133] To the aqueous solution of the depolymerized product, hydrogen peroxide at 33% w / v is added, particularly at a concentration of 0.1 ± 10% liters of H2O2 / kg of benzalkonium heparinate and it is allowed to react at 30 ± 5 ° C . After the reaction time has elapsed, it is precipitated on a solution of sodium acetate in methanol, the crude low molecular weight heparin being isolated by centrifugation.
    [0134] This crude low molecular weight heparin is dissolved in water and reprecipitated with methanol. The precipitate is dissolved in purified water and treated with 33% w / v hydrogen peroxide, specifically, at a concentration of 0.08 ± 10% liters of H2O2 per kg of benzalkonium heparinate at a temperature of 40 ± 2 ° C. After the reaction period, the solution is neutralized and precipitated with methanol.
    [0135] The precipitate is redissolved in purified water and treated again with hydrogen peroxide 33% w / v at a temperature of 40 ± 2 ° C. After the reaction period, the solution is neutralized and precipitated with methanol.
    [0136] The purified product is dissolved in water and lyophilized, obtaining 5.10 g of low molecular weight heparin and an average molecular weight of 2341 Da, an anti-FXa activity of 109 IU / mg and an anti-FIIa activity of 2, 8 IU / mg and presents at the reducing terminal of its oligosaccharide chains a content of 1,6-anhydrous residues of 21% where the proportion of 1,6-anhydroglucosamine residues is much lower than that of 1,6-anhydromanosamine residues.
    [0137] The degree of stability correlated with the color of the sample is also analyzed, observing that from the tenth month at room temperature it presents a color degree of 4 measured with a LICO® brand colorimeter that relates the color according to the chromatic range of reference established in the Pharmacopoeia (Ph. Eur. (2.2.2, Method II)).
    [0138] The degree of color, a parameter directly related to the stability of the product, made it possible to determine that the final packaged product was not stable for more than 10 11 months at room temperature, requiring a cold chain for the stability of the product to exceed 12 months.
    [0139] Example 12
    [0140] The products obtained in examples 1 to 11 are analyzed to determine percentage content of 1,6-anhydrous residues in a concrete way, according to the method described in the document Enoxaparin Sodium monograph, 1097, European Pharmacopeia 9th Ed, described in the Identification section B, obtaining the following results.
    [0143] Example 13
    [0145] The product obtained in Example 1 is analyzed by nuclear magnetic resonance, specifically by the 1H-NMR and 1H-13C HSQC experiments.
    [0146] 1H-NMR spectroscopy has been the most widely used technique for the study of these compounds, given that it is an abundant nucleus with a high gyromagnetic ratio. The region between 1.8-2.1 ppm comprises the signals corresponding to the N-acetyl groups or the methyl groups of the reducing terminals that can be included synthetically. The region between 2.8-4.6 ppm, comprises most of the signals of the saccharide ring and presents a high degree of overlap between them, which makes it difficult to extract structural information directly from this zone. Between 4.6 - 6.0 ppm are the signals corresponding to the anomeric protons. Since it is an area much less populated with signals, it is possible to extract a great deal of information from it. Furthermore, in the case of LMWHs obtained by the p-elimination mechanism, they also contain the signals corresponding to the H4 of the non-reducing terminals of the molecule.
    [0147] Specifically, the signals corresponding to 1,6-anhydrous residues appear at 5.57 and 5.62 ppm, which correspond to the anomeric proton of the 1,6-anhydromanosamine and 1,6-anhydroglucosamine structures, respectively, and such as describes the literature.
    [0148] Another two-dimensional experiment of particular importance for the structural characterization of this type of compound is the 1H-13C HSQC (Heteronuclear Single-Quantum Correlation), which correlates chemical shifts of proton with chemical shifts of carbon 13, and allows to assign the primary structures of oligosaccharides derived from GAGs and the monosaccharide composition.
    [0149] The increase in spectral dispersion achieved with this two-dimensional technique allows the quantification of the integrals of the signals that are superimposed on the corresponding one-dimensional spectra.
    [0150] The signals corresponding to the 1,6-anhydro residues appear at 5.57-103.9 and 5.61-104.3 ppm, which correspond to the anomeric proton of the 1,6-anhydromanosamine and 1,6-anhydroglucosamine structures, respectively, and as described in the literature and thus observed in Figures 1 and 2 for the sample of the product obtained in Example 1.
    [0151] Example 14
    [0152] The products obtained in Examples 1 to 10 were analyzed to determine the residue content of 2-sulfo-amino-1,6-anhydro-2-deoxy-pD-glucopyranose (1,6-anhydroglucosamine or 1,6-an .A) and 2-sulfo-amino-1,6-anhydro-2-deoxy-pD-mannopyranose (1,6-anhydromannosamine or 1,6-an.M), according to the procedure known in the state of the art for the 1H-13C HSQC experiments, obtaining the following results.
    [0153]
    权利要求:
    Claims (20)
    [1]
    1. Procedure for obtaining low molecular weight heparins with an average molecular weight between 3 and 3.8 KDa that comprises the following steps:
    a) preparing an aqueous solution of sodium heparin;
    b) adding benzalkonium chloride to the solution of step (a) to obtain benzalkonium heparinate;
    c) dissolve the benzalkonium heparinate obtained in step (b) in ChCH 2 , add Triton B and maintain a temperature between 20 and 40 ° C for between 24 and 48 hours; Y
    d) carry out at least two treatments with H 2 O 2 of the depolymerized heparin obtained after step c) in a ratio of between 0.04 and 1.0 liters of H 2 O 2 at 33% w / v for each kg of depolymerized heparin, in each treatment.
    [2]
    2. Process according to claim 1, wherein the Triton B addition of step (c) is made in a maximum of three sequential additions, each one with the addition of Triton B in a 0.2 weight ratio: 1 to 0.3: 1 of Triton B: benzalkonium heparinate.
    [3]
    3. Process according to any one of claims 1 or 2, wherein the temperature of step (c) is between 25 and 35 ° C, preferably between 27 and 32 ° C.
    [4]
    4. Process according to any one of claims 1 to 3, wherein step (c) comprises three sequential additions of Triton B, so that after the first addition the reaction is maintained for 6-10 hours until the second addition, after the second addition the reaction is maintained for 12-20 hours until the third addition, and after the third addition the reaction is maintained for 6-10 hours.
    [5]
    5. Process according to any one of claims 1 to 4, wherein each treatment with H 2 O 2 in step d) is carried out with a ratio of between 0.04 and 0.5 liters of H 2 O 2 at 33% p / v per kg of depolymerized heparin, preferably between 0.04 and 0.3 liters of H 2 O 2 at 33% w / v for each kg of depolymerized heparin.
    [6]
    6. Process according to any one of claims 1 to 5, wherein step (d) is carried out at a temperature between 20 and 50 ° C, preferably between 25 and 45 ° C.
    [7]
    7. Process according to any one of claims 1 to 6, wherein step (d) is carried out at a pH between 10.5 and 11.5 and preferably for at least 3 hours each treatment with H 2 O 2 .
    [8]
    8. Process according to any one of claims 1 to 7, wherein step d) comprises a first treatment with H 2 O 2 of the depolymerized heparin obtained after step c) in a ratio of between 0.05 and 0.25 liters of H 2 O 2 at 33% w / v for each kg of depolymerized heparin, and a second treatment with H 2 O 2 in a ratio of between 0.04 and 0.25 liters of H 2 O 2 at 33% w / v for each kg of depolymerized heparin.
    [9]
    9. Process according to any one of claims 1 to 8, wherein the depolymerized heparin is precipitated in methanol between each treatment step with H 2 O 2 .
    [10]
    10. Process according to any one of claims 1 to 9, wherein the obtained low molecular weight heparin is purified by precipitation with methanol.
    [11]
    11. Process according to any one of claims 1 to 10, wherein the obtained low molecular weight heparin is subjected to lyophilization.
    [12]
    12. Low molecular weight heparin with an average molecular weight between 3 and 3.8 KDa, obtained by the process according to any one of claims 1 to 11.
    [13]
    Low molecular weight heparin according to claim 12, characterized in that it has at the reducing end of its oligosaccharide chains a content of 1,6-anhydrous residues of between 1 and 15%, preferably between 4 and 11%.
    [14]
    Low molecular weight heparin according to any one of claims 12 or 13, wherein the molar ratio of the 1,6-anhydroglucosamine residues is greater than or equal to that of the 1,6-anhydromanosamine residues.
    [15]
    Low molecular weight heparin according to any one of claims 12 to 14, characterized in that it has an anti-FXa activity of between 80-120 IU / mg and an anti-FIIa activity of between 5-20 IU / mg.
    [16]
    Low molecular weight heparin according to any one of claims 12 to 15, wherein said heparin has a color degree greater than or equal to 6 in the range of color reference solutions established in European Pharmacopoeia 2.2.2. Method II, for at least 24 months, preferably for at least 36 months, at room temperature.
    [17]
    17. Low molecular weight heparin with an average molecular weight between 3 and 3.8 KDa, characterized in that it has a content of 1,6-anhydrous residues of between 1 and 15% at the reducing terminal of its oligosaccharide chains, preferably between 4 and 11%.
    [18]
    Low molecular weight heparin according to claim 17, wherein the molar ratio of the 1,6-anhydroglucosamine residues is greater than or equal to that of the I, 6 anhydromanosamine residues.
    [19]
    Low molecular weight heparin according to any one of claims 17 to 18, characterized in that it has an anti-FXa activity of between 80-120 IU / mg and an anti-FIIa activity of between 5-20 IU / mg.
    [20]
    Low molecular weight heparin according to any one of claims 17 to 19, wherein said heparin has a color grade greater than or equal to 6 in the range of color reference solutions established in European Pharmacopoeia 2.2.2. Method II, for at least 24 months, preferably for at least 36 months, at room temperature.
    类似技术:
    公开号 | 公开日 | 专利标题
    Chaidedgumjorn et al.2002|Effect of |-and |-linkages of fully sulfated polysaccharides on their anticoagulant activity
    Zhao et al.2013|Structure and anticoagulant activity of fucosylated glycosaminoglycan degraded by deaminative cleavage
    Marais et al.2001|A fucoidan fraction from Ascophyllum nodosum
    ES2817779T3|2021-04-08|Evaluation of heparin preparations
    Mestechkina et al.2010|Sulfated polysaccharides and their anticoagulant activity: A review
    Shively et al.1970|Stoichiometry of the nitrous acid deaminative cleavage of model amino sugar glycosides and glycosaminoglycuronans
    Zou et al.2009|Preparation of sulfated-chitins under homogeneous conditions
    JPH0629282B2|1994-04-20|Depolymerization and supersulfated heparin, method for producing the same and pharmaceutical composition
    CA2285383C|2006-09-26|Glycosaminoglycans having high antithrombotic activity
    US6162797A|2000-12-19|Derivatives of K5 polysaccharide having high anticoagulant activity
    RU2346005C2|2009-02-10|Mixtures of oligosaccharides, which are derivatives of heparin, method of obtaining them and pharmaceutical compositions containing them
    Fu et al.2014|Structure and activity of a new low-molecular-weight heparin produced by enzymatic ultrafiltration
    Guan et al.2016|Comparison of low-molecular-weight heparins prepared from bovine lung heparin and porcine intestine heparin
    Mansour et al.2019|Primary structure and anticoagulant activity of fucoidan from the sea cucumber Holothuria polii
    SI2794665T1|2018-03-30|Non anti-coagulative glycosaminoglycans comprising repeating disaccharide unit and their medical use
    EP2985298B1|2019-09-25|Low-molecular-weight fucosylated glycosaminoglycan derivative containing terminal 2,5-anhydrated talose or derivative thereof
    SI2794667T1|2018-06-29|Low anticoagulant heparins
    Razi et al.1995|Structural and functional properties of heparin analogues obtained by chemical sulphation of Escherichia coli K5 capsular polysaccharide
    Islam et al.2002|Further evidence that periodate cleavage of heparin occurs primarily through the antithrombin binding site
    WO2016159296A1|2016-10-06|Method for sulfating glycosaminoglycan
    AU2014377099B2|2019-05-30|Fuc3S4S substituted oligoglycosaminoglycan and preparation method thereof
    ES2792298A2|2020-11-10|PROCEDURE FOR OBTAINING LOW MOLECULAR WEIGHT HEPARINS AND LOW MOLECULAR WEIGHT HEPARINS OBTAINED BY THE SAME |
    DK3063177T3|2018-10-08|DERIVATIVES OF N-DESULPHATED GLYCOSAMINOGLYCANES AND USE AS MEDICINES
    ES2669727T3|2018-05-29|Procedure for obtaining low and very low molecular weight heparins
    EP2980103A1|2016-02-03|Low molecular weight glycosaminoglycan derivative, pharmaceutical composition thereof, preparation method therefor and use thereof
    同族专利:
    公开号 | 公开日
    ES2792298R2|2021-11-16|
    WO2021219902A1|2021-11-04|
    WO2021219907A1|2021-11-04|
    CN114072430A|2022-02-18|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    FR2482611B1|1980-05-14|1986-03-07|Pharmindustrie|NOVEL SULFATED POLYSACCHARIDES, METHODS FOR THEIR PREPARATION AND THEIR USE AS MEDICAMENTS|
    ES2003197A6|1987-01-05|1988-10-16|Rovi Lab Farmaceut Sa|Process for the depolymerization of heparin for obtaining heparin with a low molecular weight and having an antithrombotic activity.|
    ES2161615B1|1999-07-23|2003-03-16|Rovi Lab Farmaceut Sa|COMPOSITIONS OF HEPARINS OF VERY LOW MOLECULAR WEIGHT.|
    CN102399379B|2010-09-09|2016-08-31|上海喜恩医药科技发展有限公司|Polysaccharide mixture of a kind of heparin derivative and preparation method thereof and pharmaceutical composition|
    ES2445494B1|2012-08-02|2015-03-06|Rovi Lab Farmaceut Sa|Procedure for obtaining low and very low molecular weight heparins|
    法律状态:
    2020-11-10| BA2A| Patent application published|Ref document number: 2792298 Country of ref document: ES Kind code of ref document: A2 Effective date: 20201110 |
    2021-11-16| EC2A| Search report published|Ref document number: 2792298 Country of ref document: ES Kind code of ref document: R2 Effective date: 20211108 |
    优先权:
    申请号 | 申请日 | 专利标题
    PCT/ES2020/070271|WO2021219902A1|2020-04-27|2020-04-27|Method for obtaining low-molecular-weight heparins and low-molecular-weight heparins obtained by same|
    [返回顶部]