WO2020136665A1 - Preparation of covalently heparin-polymer conjugate and; use thereof - Google Patents
Preparation of covalently heparin-polymer conjugate and; use thereof Download PDFInfo
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- WO2020136665A1 WO2020136665A1 PCT/IN2019/050331 IN2019050331W WO2020136665A1 WO 2020136665 A1 WO2020136665 A1 WO 2020136665A1 IN 2019050331 W IN2019050331 W IN 2019050331W WO 2020136665 A1 WO2020136665 A1 WO 2020136665A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
- A61L33/0005—Use of materials characterised by their function or physical properties
- A61L33/0011—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
- A61L33/0005—Use of materials characterised by their function or physical properties
- A61L33/0011—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate
- A61L33/0029—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate using an intermediate layer of polymer
Definitions
- This invention i.e. preparation of Heparin - Polymer conjugate by strong chemical covalent bond is conducted in three stages.
- First stage is carried out to determine proper catalyst and/or coupling reagent.
- Second stage is conducted for process optimization of various parameters viz. temperature and time duration as per industrial requirement using three consecutive batches.
- final stage five consecutive batches are prepared and analyzed for activity of Heparin sodium and degradation behaviour of conjugate Heparin - polymer.
- the conjugation reactions are conducted in glass lined reaction with jacket and stirring facilities.
- Heparin sodium (Assay: 214.0 lU/mg; M w : 19.0 kDa), Poly Acrylic acid - PAA (M w : 450 kDa) and Poly L-Lactide - PLLA (M w : 500 kDa) are raw materials for this investigation.
- Poly Acrylic acid (PAA) and Poly L-Lactide (PLLA) are selected as hydrophilic and hydrophobic polymer respectively.
- PLLA is biodegradable polymer, which can be metabolized in human oody via Krebs cycle. It is naturally derived mainly from plant and approved by the Food and Drug Administration (FDA) to be used in surgical sutures, microcapsules, microspheres and implant agent materials.
- FDA Food and Drug Administration
- Acrylic acid and its derivatives are used in disposable diapers, ion exchange resins, and adhesives. It is also popular as thickening, dispersing, suspending, and emulsifying agents in pharmaceuticals, cosmetics and paints. Further, these polymers have terminal hydroxyl and/or amino group in their backbone.
- solvents in this process is based on solubility of polymer, as Heparin sodium is hydrophilic, so, soluble in polar solvents only. If polymer is hydrophilic i.e. soluble in more polar solvent, so, reaction is conducted in polar solvents, such as methanol, acetic acid and water. Selected solvent is methanol in this conjugation reaction. And if polymer is hydrophobic, i.e. soluble in less polar or non-polar, solvent such as chloroform, methylene chloride, benzene, n-hexane, etc. Here, selected solvent is methylene chloride for reaction.
- methylene chloride is also miscible in some polar solvents including methanol, ethanol, ethyl acetate, butanone, etc.
- final product consists of ester or amide group, so, group of coupling reagent and catalyst is selected, which create ester or amide group.
- group of coupling reagent and catalyst is selected, which create ester or amide group.
- two molecules of Heparin sodium are attached with two end group of a polymer molecule. So, selected molar ratio of Heparin sodium, polymer and (catalyst and/or coupling reagent) is 2: 1: 2. All the reactions are conducted at atmospheric pressure with inert nitrogen purging.
- hydrophilicity nature of Heparin-polymer conjugate is depends upon polymer.
- solvent for precipitation and purification step is selected according to hydrophilicity of final product after conjugation.
- Part - 1 and part - II are to be conducted for determine proper catalyst and/or coupling reagent using PAA for esterification and amidification respectively.
- Part - III and part - IV are to be conducted for determine proper catalyst and/or coupling reagent using PLLA for esterification and amidification respectively.
- selected temperature and time duration for conjugation reactions are 50 ⁇ 2 ° C and 2 hours respectively for all the experiments of first stage.
- Heparin sodium and PAA is dissolved in methanol individually. Now, solution of Heparin sodium solution is inserted into reactor. Start agitation and heating upto 50 ⁇ 2 ° C using hot water circulation in jacket.
- solution of Heparin sodium and PLLA are prepared using methanol and methylene chloride respectively.
- solution of Heparin sodium solution is inserted into reactor. Start agitation and heating upto 50 ⁇ 2 ° C using hot water circulation in jacket. After achieving desired temperature, selective esterification catalyst and/or coupling reagent are also charged. After 5 - 10 minutes, add polymer solution drop-wise within 20 - 30 minutes with stirring. After addition, maintain the temperature of 50 ⁇ 2 ° C for 2 hours with stirring. Cool the reaction mass upto room temperature. Precipitate out the product using methanol. Filter the final product. It is further purified by dissolving it in methylene chloride.
- a spectrum of Heparin-PAA shows the board stretching of 3600 cm 1 (N-H bond), bending of 1650 cm 1 (amino group) and bending of 1250 cm 1 (sulfonated group). Further, we concluded that better amidification of PLLA and PAA is accomplished using (l-Ethyl-3-(3- dimethyl aminopropyl)-carbodiimide) (EDC) and N-hydroxysuccinimide (NHS) as coupling reagent and catalyst respectively than other investigated amidification composites from the experiments of table 2 and 4.
- EDC l-Ethyl-3-(3- dimethyl aminopropyl)-carbodiimide
- NHS N-hydroxysuccinimide
- Heparin-PLLA shows the two stretching between 3500 - 3300 cm 1 of terminal amino group and disappeared in Heparin-PLLA.
- Heparin-PLLA shows the two bending between 3300 - 3100 cm 1 , which confirmed ester bond in final product.
- a spectrum of Heparin-PAA shows bending of 1650 cm 1 (amino group) and bending of 1250 cm 1 (sulfonated group).
- ACT for Heparin sodium, PAA and PLLA were found to be 220 - 270 (mean: 245) seconds and 120 - 140 (mean: 130) seconds and 120 - 140 (mean: 130) seconds respectively at dosage at 1 lU/mg.
- optimum conditions like temperature and time duration are to be determine for esterification and amidification reaction using [DCC and DMAP] and [EDC and NHS] respectively using three consecutive batches.
- pre-determined conditions i.e. temperature of 40 - 60 °C at interval of 10 °C and time duration of 60 to 300 minutes at interval of 60 minutes for PAA and PLLA.
- Set - I and Set - II are performed using same process as per part - I (for esterification) and part - II (for amidification) respectively for PAA and results are mentioned in table 5 and 6 respectively.
- Platelet Adhesion and Leukocyte Adhesion are performed as per standard method and thereafter, surface morphology is examined by scanning electron microscopy (SEM). Adhesion of platelet and Leukocyte on the surface is manually counted initially and finally; and percentage loss is calculated. Table 9 represents the results of investigated bio-compatibility analysis of Heparin-polymer.
- conjugated - polymers has shown more Heparin activities than hydrophobic polymers due the fact that all the analyses is performed in aqueous medium and Heparin is released more easily from hydrophilic conjugated product. All the analyses concluded that conjugate Heparin- polymer releases Heparin and it is bio-compatible. Further, decomposition studies of Heparin-
- PLLA conjugate is conducted, in which films of PLLA and conjugate PLLA - Heparin are prepared and immerged in PBS solution. After certain time duration, molecular weight by Gel Permeable Chromatography (GPC) is determined. The results indicate that 55 ⁇ 5 and 50 ⁇ 6 % of Heparin -
- PLLA and PLLA respectively are degraded within 15 weeks. It reveals that characteristics of polymer do not changed after conjugation with Heparin.
- the decomposition of Heparin-PAA is not performed as per Heparin-PLLA, because Heparin-PAA is hydrophilic i.e. soluble in water.
- Thermal decomposition studies of Heparin-PAA along-with PAA is carried out using thermo gravimetric analysis (TGA), in which 80 ⁇ 4 % and 86 ⁇ 5 % is accomplished at temperature of 550 °C. From table 9 and decomposition data shown that covalently attached biocompatible Heparin - polymers are prepared without altering the activities of Heparin as well as polymer. Further, conjugate Heparin-polymer is used in various pharmaceutical industries, comprising of medications and medical device products.
Abstract
Present invention discloses preparation of covalent conjugate Heparin – polymer using carboxyl group of Heparin sodium and terminal hydroxyl and/or group of amino group of polymer. This attachment is conducted for increasing the compatibility of polymer without affecting the properties of polymer and Heparin. For conjugation, the chemical reaction is conducted using catalyst and coupling reagent in presence of solvent. The ACT and TBA method is accomplished to determine total Heparin content of final product. For bio-compatibility, Protein Adsorption, APTT, PT, Platelet Adhesion and Leukocyte Adhesion of conjugate material are investigated. Degradation study of Heparin -PLLA is also carried. Covalently attached Heparin – Polymer is further used in various pharmaceutical industries.
Description
PREPARATION OF COVALENTLY HEPARIN-POLYMER CONJUGATE AND; USE
THEREOF
Description
This invention i.e. preparation of Heparin - Polymer conjugate by strong chemical covalent bond is conducted in three stages. First stage is carried out to determine proper catalyst and/or coupling reagent. Second stage is conducted for process optimization of various parameters viz. temperature and time duration as per industrial requirement using three consecutive batches. In final stage, five consecutive batches are prepared and analyzed for activity of Heparin sodium and degradation behaviour of conjugate Heparin - polymer. For all stages, the conjugation reactions are conducted in glass lined reaction with jacket and stirring facilities. Also, Heparin sodium (Assay: 214.0 lU/mg; Mw: 19.0 kDa), Poly Acrylic acid - PAA (Mw: 450 kDa) and Poly L-Lactide - PLLA (Mw: 500 kDa) are raw materials for this investigation. Poly Acrylic acid (PAA) and Poly L-Lactide (PLLA) are selected as hydrophilic and hydrophobic polymer respectively. PLLA is biodegradable polymer, which can be metabolized in human oody via Krebs cycle. It is naturally derived mainly from plant and approved by the Food and Drug Administration (FDA) to be used in surgical sutures, microcapsules, microspheres and implant agent materials. Poly. Acrylic acid and its derivatives are used in disposable diapers, ion exchange resins, and adhesives. It is also popular as thickening, dispersing, suspending, and emulsifying agents in pharmaceuticals, cosmetics and paints. Further, these polymers have terminal hydroxyl and/or amino group in their backbone.
Selection of solvents in this process is based on solubility of polymer, as Heparin sodium is hydrophilic, so, soluble in polar solvents only. If polymer is hydrophilic i.e. soluble in more polar solvent, so, reaction is conducted in polar solvents, such as methanol, acetic acid and
water. Selected solvent is methanol in this conjugation reaction. And if polymer is hydrophobic, i.e. soluble in less polar or non-polar, solvent such as chloroform, methylene chloride, benzene, n-hexane, etc. Here, selected solvent is methylene chloride for reaction. Because, methylene chloride is also miscible in some polar solvents including methanol, ethanol, ethyl acetate, butanone, etc. Further, final product consists of ester or amide group, so, group of coupling reagent and catalyst is selected, which create ester or amide group. Here, two molecules of Heparin sodium are attached with two end group of a polymer molecule. So, selected molar ratio of Heparin sodium, polymer and (catalyst and/or coupling reagent) is 2: 1: 2. All the reactions are conducted at atmospheric pressure with inert nitrogen purging. Here, hydrophilicity nature of Heparin-polymer conjugate is depends upon polymer. If polymer is hydrophilic like PAA, then final conjugated product is hydrophilic. And if polymer is hydrophobic like PLLA, then final product is hydrophobic. Therefore, solvent for precipitation and purification step is selected according to hydrophilicity of final product after conjugation.
Part - 1 and part - II are to be conducted for determine proper catalyst and/or coupling reagent using PAA for esterification and amidification respectively. Part - III and part - IV are to be conducted for determine proper catalyst and/or coupling reagent using PLLA for esterification and amidification respectively. Initially, selected temperature and time duration for conjugation reactions are 50 ± 2 °C and 2 hours respectively for all the experiments of first stage. For Part - I and Part - II, Heparin sodium and PAA is dissolved in methanol individually. Now, solution of Heparin sodium solution is inserted into reactor. Start agitation and heating upto 50 ± 2 °C using hot water circulation in jacket. After achieving desired temperature, selective esterification catalyst and/or coupling reagent are also charged. After 5 - 10 minutes, add
polymer solution drop-wise within 20 - 30 minutes with stirring. After addition, maintain the temperature of 50 ± 2 °C for 2 hours with stirring. Cool the reaction mass upto room temperature. Precipitate out the product using methylene chloride. Filter the final product and purified it with dissolving it in methanol. Re-precipitate it with methylene chloride. Filter the precipitates and dry it under vacuum at 30 - 35 °C for 12 hours. Further, the product is hydrophilic, so, ACT and TBA method is conducted onto direct product to analyze the total Heparin content. Experiments are conducted as per following table - 1 and 2 using select catalyst and coupling reagent to form ester and amide bond respectively. In all of these analyses (ACT and TBA), PAA and Heparin sodium are taken as reference materials
For Part - III and Part - IV, solution of Heparin sodium and PLLA are prepared using methanol and methylene chloride respectively. Now, solution of Heparin sodium solution is inserted into reactor. Start agitation and heating upto 50 ± 2 °C using hot water circulation in jacket. After achieving desired temperature, selective esterification catalyst and/or coupling reagent are also charged. After 5 - 10 minutes, add polymer solution drop-wise within 20 - 30 minutes with stirring. After addition, maintain the temperature of 50 ± 2 °C for 2 hours with stirring. Cool the reaction mass upto room temperature. Precipitate out the product using methanol. Filter the final product. It is further purified by dissolving it in methylene chloride. Add water in it and stir for 30 minutes, so that unreacted Heparin is come out in water. The reaction mass is undergo layer separation for 30 minutes. Aqueous layer is collected and called as "wash-water". Purified final product is precipitate out using methanol from organic layer. It is dried under vacuum at
30 - 35 °C for 12 hours. Further, the product is hydrophobic, so, total Heparin content is not determined from the product directly. Here, remaining (un-reacted) Heparin is analyzed from
"wash-water" derived from purification step. Wash-water is undergone analysis of ACT and TBA method, which give the value of un-reacted Heparin. Thereafter, ACT, TBC and total Heparin content is calculated from the values of un-reacted Heparin. Experiments are conducted as per following table - 3 and 4 using different catalyst and coupling reagent to form ester and amide bond respectively. In all of these analyses (ACT and TBA), PLLA and Heparin sodium are taken as reference materials. Also, ACT and TBA analyses were conducted at temperature of 37 °C for 5 minutes.
From the experiments of table 1 and 3, we concluded that better esterification for PLLA and PAA respectively is accomplished using Dicyclohexylcarbodiimide (DCC) and N, N' - Dimethylamino pyridine (DMAP) as coupling reagent and catalyst respectively than other investigated esterification compositions. FT-IR spectrum of conjugate Heparin - PAA using DCC and DMAP along-with PAA and Heparin sodium is performed, in which PAA shows the board stretching between 3500 - 3200 cm 1 of terminal hydroxyl group and disappeared in Heparin- PAA. Heparin-PAA shows the bending of 1750 cm , which confirmed ester bond in final product. A spectrum of Heparin-PAA shows the board stretching of 3600 cm 1 (N-H bond), bending of 1650 cm 1 (amino group) and bending of 1250 cm 1 (sulfonated group). Further, we concluded that better amidification of PLLA and PAA is accomplished using (l-Ethyl-3-(3- dimethyl aminopropyl)-carbodiimide) (EDC) and N-hydroxysuccinimide (NHS) as coupling reagent and catalyst respectively than other investigated amidification composites from the experiments of table 2 and 4. Further, FT-IR spectrum of conjugate Heparin - PLLA using EDC and NHS is shown in figure along-with PAA and Heparin sodium is performed, which PLLA
Table 1: Feasibility analysis for catalyst and coupling reagent for ester bond using PAA
Table 3: Feasibility analysis for catalyst and coupling reagent for ester bond using PLLA
shows the two stretching between 3500 - 3300 cm 1 of terminal amino group and disappeared in Heparin-PLLA. Heparin-PLLA shows the two bending between 3300 - 3100 cm 1, which confirmed ester bond in final product. A spectrum of Heparin-PAA shows bending of 1650 cm 1
(amino group) and bending of 1250 cm 1 (sulfonated group). In all above experiments, ACT for Heparin sodium, PAA and PLLA were found to be 220 - 270 (mean: 245) seconds and 120 - 140 (mean: 130) seconds and 120 - 140 (mean: 130) seconds respectively at dosage at 1 lU/mg. Here, standard curves of in-vitro ACT [activated clotting time (seconds) vs. concentration of Heparin (IU/mg)] and TBA [absorbance at 631 nm vs. concentration of Heparin (IU/mg)] were prepared for pristine Heparin and thereafter, Total Heparin content (IU/mg) is calculated from the value of ACT and TBA analysis. In this way, suitable catalyst and coupling reagent is selected by changing them to get strong covalent bond, because, catalyst and coupling reagent may be varies according to terminal group of polymer.
In second stage, optimum conditions like temperature and time duration are to be determine for esterification and amidification reaction using [DCC and DMAP] and [EDC and NHS] respectively using three consecutive batches. Following four set of experiment are conducted at pre-determined conditions i.e. temperature of 40 - 60 °C at interval of 10 °C and time duration of 60 to 300 minutes at interval of 60 minutes for PAA and PLLA. Set - I and Set - II are performed using same process as per part - I (for esterification) and part - II (for amidification) respectively for PAA and results are mentioned in table 5 and 6 respectively. Set - Ill and Set - IV are using same process as per part - III (for esterification) and part - IV (for amidification) respectively for PLLA and results are mentioned in table 7 and 8 respectively.
Table 5: Process optimization for preparation of conjugated PAA - Heparin having ester bond
These values are expressed as mean, n = 3. From table 5 and 7, it is concluded that optimum parameters viz. temperature and time duration are selected for esterification reaction are 50 °C and 180 minutes respectively for esterification for PAA/PLLA. Also, table 6 and 8
revealed that temperature and time duration are selected for amidification reaction hydroxyl group are 40 °C and 120 minutes respectively for PAA/PLLA. In this way, process optimization of covalently conjugate Heparin - polymer is accomplished by altering temperature and time duration to get best fitted parameter for covalent conjugation. Because, process parameters may be varies according to properties of polymer and catalyst and/or coupling agent.
After finalization of coupling reagent and catalyst; and process parameters, five consecutive batches are conducted using these selected catalyst and coupling reagent; and optimized parameters. The compatibility analysis of five batches is carried out using APTT, PT, Protein Adsorption, Platelet Adhesion and Leukocyte Adhesion with PLLA and PAA as control. Films [Surface area: 4 cm2 (2 X 2 cm)] of conjugate Heparin products (Heparin-PLLA and Heparin-PAA) and pristine polymers (PLLA and PAA) are prepared and undergoes various analyses. APTT, PT and Protein Adsorption are conducted as per standard methods. Also, Platelet Adhesion and Leukocyte Adhesion are performed as per standard method and thereafter, surface morphology is examined by scanning electron microscopy (SEM). Adhesion of platelet and Leukocyte on the surface is manually counted initially and finally; and percentage loss is calculated. Table 9 represents the results of investigated bio-compatibility analysis of Heparin-polymer.
These values are expressed as mean, n = 5. The results of table indicates that APTT and PT of all conjugate products are higher compared their respective control viz. PLLA and PAA. The percentage losses of Platelet and Leukocyte values for conjugated Heparin-polymer are shown decreasing, while comparing their control polymers. In these analyses, hydrophilic
Table 9: Bio-compatibility analysis of covalent Heparin-polymer
conjugated - polymers has shown more Heparin activities than hydrophobic polymers due the fact that all the analyses is performed in aqueous medium and Heparin is released more easily from hydrophilic conjugated product. All the analyses concluded that conjugate Heparin- polymer releases Heparin and it is bio-compatible. Further, decomposition studies of Heparin-
PLLA conjugate is conducted, in which films of PLLA and conjugate PLLA - Heparin are prepared and immerged in PBS solution. After certain time duration, molecular weight by Gel Permeable
Chromatography (GPC) is determined. The results indicate that 55 ± 5 and 50 ± 6 % of Heparin -
PLLA and PLLA respectively are degraded within 15 weeks. It reveals that characteristics of polymer do not changed after conjugation with Heparin. The decomposition of Heparin-PAA is not performed as per Heparin-PLLA, because Heparin-PAA is hydrophilic i.e. soluble in water. Thermal decomposition studies of Heparin-PAA along-with PAA is carried out using thermo gravimetric analysis (TGA), in which 80 ± 4 % and 86 ± 5 % is accomplished at temperature of 550 °C. From table 9 and decomposition data shown that covalently attached biocompatible Heparin - polymers are prepared without altering the activities of Heparin as well as polymer. Further, conjugate Heparin-polymer is used in various pharmaceutical industries, comprising of medications and medical device products.
Claims
1. Present invention is carried out for enhancement the compatibility of polymers by covalently attaching Heparin sodium using coupling reagent and catalyst without compromising the characteristics of polymer and Heparin sodium. The covalently conjugate Heparin - polymer is analyzed for bio-compatibility and used in pharmaceutical industries.
2. According to claim 1, polymers are selected from the group comprising of hydrophilic and hydrophobic polymers
3. According to claim 1, polymers are selected from the group comprising terminal group of both hydroxyl groups or both amino groups or one hydroxyl end group and one amino end group. But end groups of polymer are not limited to them.
4. According to claim 2, hydrophilic polymers are selected from the group comprising of poly acrylic acid, poly ethylene glycol, poly ethylene oxide, polyvinyl pyrrolidone, polymethacrylate, polyvinyl alcohol, polyacrylamide, poly(N-isopropylacrylamide), poly(2-oxazoline), polyethylenimine, polyoxazoline, polyphosphazene, poly (N-isopropylacrylamide), polymaleic anhydride, polyja - substituted acrylic acid], poly(methacrylic acid), polymethacrylate, poly amino acids, polyether polyvinyl alcohol, polyvinylhalide, polyurea and polyvinyl acetate; and their copolymer, but not limiting only to them.
5. According to claim 2, hydrophobic polymers are selected from the group comprising of poly Lactide, poly glycolide, poly caprolactam, polychloroprene, chitosan, polyurea, poly vinyl chloride, polyisobutylene, polydienes, polyacetylene, polyamide, polyester, poly anhydride, polycarbonate, polyurethane, polyacrylonitrile, polysulfide, polyheterocyclic, polysiloxane, polyphosphate, polysilsesquioxane and polystyrene; and their copolymer, but not limiting only to them.
6. According to claim 1, wherein carboxyl acid group of Heparin is reacted with terminal hydroxyl and/or amino group of polymer to yield conjugated product having ester or amide group respectively using selective coupling reagent and catalyst. But the groups of polymer are not limited to them.
7. According to claim 1, procedure for conjugation is selected according to solubility of polymer. If polymer is hydrophilic i.e. soluble in more polar solvents, than, Heparin sodium and polymer is dissolved in polar solvent individually. Now, solution of Heparin sodium solution is inserted in apparatus having heating and stirring facility. Start agitation and heating upto 20 - 90 °C. After achieving desired temperature, selective catalyst and/or coupling reagent are also charged. Add polymer solution drop-wise within 5 - 60 minutes with stirring. After addition, maintain the temperature of 20 - 90 °C for 1 - 8 hours with stirring. Cool the reaction mass upto room temperature. Precipitate out the product using less polar or non-polar solvent. Filter the final product and purified it with dissolving it in polar solvent. Re-precipitate it with less polar or non polar solvent. Filter the precipitates and dry it under vacuum at 20 - 40 °C for 2 - 12 hours. Further, ACT and TBA method is conducted on final pure product to analyze the total Heparin content. But the process of Heparin conjugation with hydrophilic polymer is not limited to this.
8. According to claim 2, if polymer is hydrophobic i.e. soluble in less polar or non-polar, than solution of Heparin sodium and PLLA are prepared using polar and less polar or non-polar solvent respectively. For this reaction, less polar solvent is selected that also miscible in polar solvent such
as methylene chloride. Now, solution of Heparin sodium solution is inserted in apparatus having heating stirring facility. Start agitation and heating upto 20 - 90 °C. After achieving desired temperature, selective catalyst and/or coupling reagent are also charged. Add polymer solution drop-wise within 5 - 60 minutes with stirring. After addition, maintain the temperature of 20 - 90 °C for 1 - 8 hours with stirring. Cool the reaction mass upto room temperature. Precipitate out the product using polar solvent. Filter the final product. It is further purified by dissolving it in less polar or non-polar solvent. Add water in it and stir for 10 - 180 minutes, so that unreacted Heparin is come out in water. The reaction mass is undergo layer separation for 10 - 180 minutes. Aqueous layer is collected and called as "wash-water". Purified final product is precipitate out using methanol from organic layer. It is dried under vacuum at 20 - 40 °C for 2 - 24 hours. Further, ACT and TBA method is conducted on wash-water to analyze the total Heparin content. But the process of Heparin conjugation with hydrophobic polymer is not limited to this.
9. According to claim 1, biocompatibility analyses are selected from the group comprising of APTT, PT, Protein Adsorption, Platelet Adhesion and Leukocyte Adhesion, but not limiting only to them.
10. According claim 1, pharmaceutical industry are selected from the group comprising of medications and medical devices in their products and intermediates. Medication products and intermediates are selected from the group comprising of anesthetics, analgesics, antipyretics, anti-inflammatory, antiallergics, antidotes, anticonvulsants, antiepileptics, anti-infective, antimigraine, antineoplastic, androgens, immunologicals, muscle relaxants, cholinesterase inhibitors, ophthalmological, oxytocics and antioxytocics, psychotherapeutic, peritoneal dialysis, antitussives, antiasthmatic, blood products and plasma substitutes, but not limiting only to them. Medical device products and intermediates are selected from the group comprising of vascular grafts, stents, heart valves, catheters, in vitro diagnostic devices perfusion set, intro ocular lenses, i.v. cannulae, bone cements, blood grouping sear, skin ligatures, sutures, staplers, intrauterin devices, tubal rings, surgical dressing, umbilical tapes, blood component bags, scalp vein set, orthopaedic implants, internal prosthetic replacement, metered dose inhaler, spinal needles, endotracheal tubes, introducer sheath, annuloplasty ring, cardiac patch, cochlear implant, extension tube, close wound drainage set, needle, heart lung pack, cuff, tracheotomy tube, hemodialysis tubing set, bload tubing set, arterial venous tubing set, Artificial Hips, heart Pacemakers, Breast Implants, Spine Screws, Rods and Artificial Discs, Intra uterine Devices, knee, ear tube, , but not limiting only to them.
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CN111893642A (en) * | 2020-08-10 | 2020-11-06 | 四川大学 | Halamine polymer antibacterial and antiviral nanofiber membrane and preparation method thereof |
CN113372494A (en) * | 2021-06-01 | 2021-09-10 | 河南驼人医疗器械研究院有限公司 | Anticoagulation polymer coating material and preparation method thereof |
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US7396541B2 (en) * | 2004-06-18 | 2008-07-08 | Advanced Cardiovascular Systems, Inc. | Heparin prodrugs and drug delivery stents formed therefrom |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111893642A (en) * | 2020-08-10 | 2020-11-06 | 四川大学 | Halamine polymer antibacterial and antiviral nanofiber membrane and preparation method thereof |
CN111893642B (en) * | 2020-08-10 | 2022-05-17 | 四川大学 | Halamine polymer antibacterial and antiviral nanofiber membrane and preparation method thereof |
CN113372494A (en) * | 2021-06-01 | 2021-09-10 | 河南驼人医疗器械研究院有限公司 | Anticoagulation polymer coating material and preparation method thereof |
CN113372494B (en) * | 2021-06-01 | 2023-07-14 | 河南驼人医疗器械研究院有限公司 | Anticoagulant polymer coating material and preparation method thereof |
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