阅读说明：本技术 制备低内毒素壳聚糖的方法 (Method for preparing low endotoxin chitosan ) 是由 J.格拉德曼 C.哈迪 A.霍格思 于 2013-03-25 设计创作，主要内容包括：本发明涉及制备低内毒素碱性壳聚糖的方法,还涉及制备低内毒素中性壳聚糖、壳聚糖盐和壳聚糖衍生物的方法,并涉及这些方法的产物。所述方法包括使壳聚糖与碱性溶液接触,以形成混合物,并让混合物保持至少约12小时。可用低内毒素碱性壳聚糖制备其它有用的基于壳聚糖的产物。(The present invention relates to a process for the preparation of low endotoxin alkali chitosan, to processes for the preparation of low endotoxin neutral chitosan, chitosan salts and chitosan derivatives and to the products of these processes. The method includes contacting chitosan with an alkaline solution to form a mixture and allowing the mixture to stand for at least about 12 hours. Other useful chitosan-based products can be prepared from low endotoxin alkali chitosan.)

1. A method of preparing low endotoxin alkali chitosan, said method comprising the steps of:

(a) contacting chitosan with an alkaline solution to form a mixture; and

(b) the mixture is allowed to stand for at least about 12 hours.

2. The method of claim 1, wherein the method further comprises the step (c) of drying the mixture.

3. The process of claim 1 or 2, wherein the mixture is maintained in step (b) for about 24 hours.

4. The method of any of the preceding claims, wherein the concentration of the alkaline solution is from about 0.01M to about 1M.

5. The method of claim 4, wherein the concentration of the alkaline solution is about 0.02M to 0.2M.

6. The method of claim 5, wherein the concentration of the alkaline solution is about 0.1M.

7. The method of any of the preceding claims, wherein the alkaline solution and chitosan are present in a range of from about 1 part chitosan to about 10 parts alkaline solution to about 10 parts chitosan to about 1 part alkaline solution.

8. The method of any of the preceding claims, wherein the alkaline solution comprises an alkali or alkaline earth metal component selected from the following components, alone or in combination: metal hydroxides, metal carbonates, metal bisulfites, metal persilicates, conjugate bases and ammonium hydroxide.

9. The method of claim 8, wherein the metal is selected from sodium, potassium, calcium, or magnesium.

10. The process of claim 8 or 9, wherein the base component is selected from sodium hydroxide, potassium hydroxide or sodium carbonate.

11. A method according to any one of the preceding claims, wherein the alkaline solution is sprayed onto the chitosan or the chitosan is mixed with the alkaline solution.

12. The process of any of the preceding claims, wherein the mixture is maintained in step (b) for at least 48 hours.

13. The method of claim 12, wherein the mixture is maintained for about 2 to 4 weeks.

14. The method of any one of the preceding claims, wherein the mixture is allowed to remain in a clean container and/or is maintained under an inert atmosphere.

15. The method of any one of the preceding claims, wherein the mixture further comprises a preservative.

16. The method of claim 15, wherein the preservative is selected from the group consisting of silver ions, zinc ions, chlorhexidine, or combinations thereof.

17. The method of any of the preceding claims, wherein the drying step is performed in an oven.

18. A low endotoxin alkali chitosan obtainable by the process of any one of claims 1 to 17.

19. An alkaline chitosan having an endotoxin concentration of less than 100 EU/g.

20. A method of preparing a low endotoxin neutral chitosan, chitosan salt or chitosan derivative comprising the step of contacting an alkaline chitosan prepared by the method of any one of claims 1 to 17 with an acid.

21. The method of claim 20, wherein the step of contacting the alkali chitosan with an acid is performed prior to the drying step (c) of any one of claims 2 to 17.

22. A process according to claim 20 or 21, wherein the acid is sprayed onto the alkali chitosan or the alkali chitosan is mixed with the acid.

23. The process of any one of claims 20 to 22, wherein the acid is selected from the following acids, alone or in combination: organic acids, carboxylic acids, fatty acids, amino acids, lewis acids, monoprotic acids, biprotic acids, polyprotic acids, nucleic acids and inorganic acids.

24. The method of claim 23, wherein the organic acid is selected from the following organic acids, alone or in combination: acetic acid, tartaric acid, citric acid, ascorbic acid, acetylsalicylic acid, gluconic acid and lactic acid.

25. The method of claim 23, wherein the fatty acid is selected from the following fatty acids, alone or in combination: myristoleic acid, palmitoleic acid, cis-6-hexadecenoic acid, oleic acid, elaidic acid, 11-octadecenoic acid, linoleic acid, elaidic acid, alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acidAcid, wood wax acid, wax acid.

26. The method of claim 23, wherein the amino acid is selected from the following amino acids, alone or in combination: histidine, lysine, aspartic acid, glutamic acid, glutamine, glycine, proline and taurine.

27. The method of claim 23, wherein the mineral acid is selected from the following mineral acids, alone or in combination: hydrochloric acid, sulfuric acid and nitric acid.

28. The method of any one of claims 20 to 27, wherein the acid has a concentration of about 1M.

29. A process as claimed in any one of claims 20 to 28 wherein the acid is present as an acid liquor comprising an acid and a non-solvent.

30. The method of claim 29, wherein the non-solvent is selected from the group consisting of ethyl lactate, ethyl acetate, methyl acetate, ethanol, acetone, an 80:20 mixture of ethanolri, or a mixture thereof.

31. The method of claim 29 or 30, wherein the ratio of chitosan to acid liquor is about 5:1 to about 1: 5.

32. The method of any one of claims 20 to 31, wherein the alkali chitosan is mixed with the acid for about 5 minutes.

33. The method of any one of claims 20 to 32, further comprising the step of drying the reaction product.

34. The method of claim 33, wherein the drying step is performed in an oven or by filtering the product through an air dryer.

35. A low endotoxin neutral chitosan, chitosan salt or chitosan derivative obtainable by the method of any one of claims 20 to 34.

36. A neutral chitosan, chitosan salt or chitosan derivative having an endotoxin concentration of less than 100 EU/g.

37. Use of a low endotoxin chitosan salt of claim 35 for the prevention of blood flow.

38. A low endotoxin chitosan salt of claim 35 for use as a haemostat for use in stopping blood flow.

39. A low endotoxin chitosan salt of claim 35 for use in a wound dressing for the superficial non-life threatening or life threatening bleeding.

40. A hemostatic wound dressing comprising the low endotoxin chitosan salt of claim 35.

41. A method of preventing blood flow, the method comprising the steps of: optionally, where possible, cleansing the wound area; applying to the wound area a hemostatic wound dressing comprising a low endotoxin chitosan salt as described herein; and applying a constant pressure to the wound area until a gel mass is formed.

42. A low endotoxin alkali chitosan, neutral chitosan, chitosan salt, chitosan derivative, process or method substantially as described herein in the specification and drawings.

Technical Field

The present invention relates to a process for the preparation of low endotoxin alkali chitosan, to processes for the preparation of low endotoxin neutral chitosan, chitosan salts and chitosan derivatives and to the products of these processes.

Background

Chitosan is particularly useful in the preparation of hemostatic materials for use in controlling bleeding.

Chitosan is a solid waste derivative from the processing of crustaceans and can be extracted from fungal cultures. Chitosan is a water-insoluble cationic polymeric substance. Chitosan is typically first converted to a water-soluble salt before it is used in a hemostatic material. In this way, chitosan salts are soluble in blood to form gels that block blood flow.

Chitosan salts are ideally suited for use in the applications described herein, since chitosan is readily decomposed in the body. Chitosan is converted into glucosamine by lysozyme and is thus naturally excreted from the body. It is not necessary to remove the chitosan from the body. In addition, chitosan salts exhibit mild antibacterial properties, and thus their use reduces the risk of infection.

In order to utilize chitosan in the preparation of hemostatic materials suitable for controlling bleeding, it is necessary to ensure that the chitosan has a sufficiently low endotoxin concentration.

Endotoxin is lipopolysaccharide present on the outer membrane surface of gram-negative bacteria. Endotoxins are highly toxic to mammals, particularly humans, and are notoriously difficult to remove from materials. Endotoxins can become pyrogenic when released into the bloodstream or other tissues where they are not normally found. Thus, endotoxins must be removed from the pharmaceutically acceptable product.

Treatment to remove or destroy pyrogens, particularly endotoxins, is known as the "depyrogen" method. Techniques for depyrogenation of endotoxin-containing materials include ion exchange chromatography, ultrafiltration, distillation, and various chemical methods directed to the destruction of endotoxins.

WO2008063503 relates to a method for removing endotoxin from chitosan, said method comprising the steps of:

a) using sterile, pyrogen-free equipment and materials in a sterile environment;

b) swelling chitosan comprising endotoxin for up to 24 hours;

c) dissolving 1kg/25L to 1.5kg/25L chitosan in 0.01M to 4.0M hydroxide base;

d) continuously stirring the obtained chitosan alkali solution;

e) heating the solution at 60-100 deg.C for 45 min to 4 hr with stirring;

f) washing the solution with up to 10x volume of ultrapure endotoxin-free water;

g) neutralizing the solution to a pH between 6.8 and 7.5;

h) forming ultra-pure low endotoxin chitosan slurry and transferring to an endotoxin-free closed system;

i) excess water is removed from the slurry.

This is a complex and costly process, especially requiring sterile equipment and requiring a 10x volume endotoxin free water rinse solution.

US2006293509 relates to a process for preparing water-soluble chitosan with low endotoxin by:

(a) contacting the water-insoluble chitosan with an alkaline solution for a first period of time greater than 1 hour;

(b) the water-insoluble chitosan is desirably rinsed with endotoxin-free water to remove residual alkaline solution;

(c) partially acetylating a water-insoluble chitosan in a reaction solution containing a phase transfer agent;

(d) dissolving a partially acetylated water soluble chitosan in an aqueous solution comprising a surfactant and having a pH of about 7.0 to about 7.4;

(e) adding a water-miscible solvent to the aqueous solution and further adjusting the pH of the aqueous solution to a pH of at least 8.0 to precipitate water-soluble chitosan having a low endotoxin content from the aqueous solution/water-miscible solvent mixture; and

(f) optionally washed in a non-solvent, e.g., isopropanol.

However, this process is complex and costly, and desirably involves the use of large amounts of endotoxin-free water or other liquid. This process also requires the use of a phase transfer agent and is carried out over several hours.

TW593342 relates to a method for reducing endotoxin in chitosan by:

(a) dissolving chitosan comprising endotoxin in an aqueous solution;

(b) contacting the aqueous solution with a surfactant to form insoluble solids and an aqueous solution having a reduced endotoxin content;

(c) the solids are separated from the aqueous solution using a solid/liquid separation device.

However, this process requires the surfactant to react with the dissolved chitosan to produce insoluble solids. The resulting solid is a mixture of chitosan and a surfactant or a reaction product between chitosan and a surfactant.

Disclosure of Invention

The present invention aims to alleviate the aforementioned difficulties.

According to a first aspect of the present invention there is provided a process for the preparation of low endotoxin alkali chitosan comprising the steps of:

(a) contacting chitosan with an alkaline solution to form a mixture; and

(b) the mixture is allowed to stand for at least about 12 hours.

According to another aspect of the present invention there is provided a process for the preparation of low endotoxin alkali chitosan comprising the steps of:

(a) contacting chitosan with an alkaline solution to form a mixture;

(b) allowing the mixture to stand for at least about 12 hours; and

(c) the mixture is dried.

The process of the present invention provides an efficient way to obtain alkaline chitosan with low endotoxin concentration. The process advantageously does not require a washing step, a rinsing step, the use of surfactants or phase transfer agents, sterile equipment and/or the use of endotoxin-free water. In addition, no special air filtration or sterile conditions are required. The process of the invention preferably does not comprise a step of acetylating chitosan.

The term "alkaline solution" as used herein refers to a solution having a pH greater than pH 7.5.

Since the molecular weight of endotoxins can vary significantly, endotoxin concentration is measured in units of Endotoxin (EU)/gram of material. The determination of endotoxin concentration is a quantification of endotoxin levels relative to a specific amount of a reference endotoxin.

For example, in the present invention, endotoxin concentration is measured in units of Endotoxin (EU)/gram of chitosan. The term "low endotoxin" as used herein means having an endotoxin concentration of less than 100 Endotoxin Units (EU)/gram of chitosan.

Thus, the process of the invention is suitable for the preparation of alkaline chitosan having an endotoxin concentration of less than 100 EU/g.

Preferably the resulting alkali chitosan has an endotoxin concentration of less than 50EU/g, more preferably less than 20EU/g, even more preferably less than 15EU/g, most preferably less than 10 EU/g.

It has been found that low concentration alkaline solutions are preferred in the process of the present invention. The concentration of the alkaline solution used in the process may be from about 0.01M to about 1M. Preferably the concentration of the alkaline solution is less than 1M. Preferably the concentration of the alkaline solution is from about 0.02M to 0.2M, even more preferably the concentration of the alkaline solution is from about 0.04M to 0.06M, typically 0.05M. The concentration of the alkaline solution may be about 0.01M, 0.05M, 0.10M, 0.15M, 0.20M, 0.25M, 0.30M, 0.35M, 0.40M, 0.45M, 0.50M, 0.55M, 0.60M, 0.65M, 0.70M, 0.75M, 0.80M, 0.85M, 0.90M, or 0.95M. Good results have been observed with a concentration of 0.1M alkaline solution.

In some embodiments, the amount of alkaline solution to chitosan may range from about 1 part chitosan to about 10 parts alkaline solution to about 10 parts chitosan to about 1 part alkaline solution. Preferably, the amount of alkaline solution to chitosan is about 1 part alkaline solution to about 2 parts chitosan, more preferably about 1 part alkaline solution to about 1 part chitosan.

The alkaline solution may comprise an alkali or alkaline earth metal (alkaline earth) component selected from the following, alone or in combination: metal hydroxides, metal carbonates, metal bisulfites, metal persilicates (persilicates), conjugate bases and ammonium hydroxides.

Suitable metals include sodium, potassium, calcium or magnesium.

Preferably, the alkali component is sodium hydroxide, potassium hydroxide or sodium carbonate. Sodium hydroxide is generally used.

The alkaline solution may be contacted with chitosan by any suitable method known in the art. For example, the alkaline solution may be sprayed onto the chitosan, or the chitosan may be mixed with the alkaline solution. Preferably, the chitosan is homogeneously distributed in the alkali contact.

Preferably, the chitosan is mixed with an alkaline solution. At low molecular weights, chitosan may be completely or partially soluble in alkaline solutions. The chitosan may be mixed with the alkaline solution for up to about 30 minutes, more preferably about 10 minutes. In some embodiments, the chitosan may be mixed with the alkaline solution for greater than 30 minutes. In some embodiments, the mixture of chitosan and alkaline solution may be stirred intermittently for the duration of step (b).

The mixture of chitosan and alkaline solution is maintained for a period of time in which the endotoxin is destroyed by the alkali. The mixture of chitosan and alkaline solution is maintained for at least about 12 hours. It has been found that the longer the time the mixture of chitosan and alkaline solution is kept, the lower the endotoxin concentration of the resulting alkaline chitosan. A suitably low endotoxin concentration has been observed when the mixture has been maintained for about 12 hours. Another advantage of the method of the present invention is that the mixture can be maintained without the need to constantly mix the chitosan with the alkaline solution.

In some embodiments, the mixture can be maintained for at least about 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 hours.

Preferably, the mixture is maintained for at least 48 hours.

In some embodiments, the mixture can be maintained for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 days or longer.

In some embodiments, the mixture is maintained for about 2 to 4 weeks (or 14 to 30 days) or more. Preferably, the mixture is maintained for between 24 hours and 70 days, more preferably between 7 days and 35 days, and most preferably between 14 days and 21 days.

Good results have been observed by contacting chitosan with 0.1M sodium hydroxide solution and maintaining the mixture for about 12 to 16 days, preferably about 14 days.

The mixture can be maintained at room temperature and pressure. Room temperature and pressure refer to temperatures of about 20-25 c and pressures of about 1 atmosphere (atm). Advantageously, the mixture need not be maintained in a sterile environment.

Preferably the mixture is stored in a clean container. The mixture may be stored under an inert atmosphere.

The mixture may further comprise a preservative. Advantageously, the preservative may eliminate the risk of microbial growth that may occur, for example, when the mixture is maintained for an extended period of time. The preservative may be any preservative that is biocompatible and suitable for use in an alkaline environment. Suitable preservatives include silver ions, zinc ions, chlorhexidine (chlorohexidine), or combinations thereof.

The process of the present invention may or may not include a drying step. The drying step may be performed by any conventional drying method known in the art. The drying step is preferably carried out in an oven or by filtration through an air dryer. Also, the drying step does not require special sterile equipment.

It has been found that once the mixture has been dried in the drying step, the endotoxin level of the mixture is no longer significantly increased. This is advantageous for further processing of the mixture.

Accordingly, the present invention provides low endotoxin alkali chitosan having an endotoxin concentration of less than 100 EU/g. The low endotoxin alkali chitosan may be water insoluble. At low molecular weight, low endotoxin alkali chitosan may exhibit some water solubility.

According to another aspect of the present invention there is provided a low endotoxin alkali chitosan obtainable by the process described herein.

According to another aspect of the present invention there is provided an alkaline chitosan having an endotoxin concentration of less than 100 EU/g.

The alkaline chitosan preferably has an endotoxin concentration of less than 50EU/g, more preferably less than 20EU/g, even more preferably less than 15EU/g, most preferably less than 10 EU/g.

Other chitosan products, such as derivatives or copolymers, or low molecular weight chitosans or chitosan oligosaccharides may be prepared using low endotoxin alkali chitosans. Low endotoxin alkali chitosan may also be used as a starting material for the preparation of other forms of chitosan or derivatives or copolymers, such as chitosan-based fibers, fabrics, coatings, films, gels, solutions, sheets or foams.

In particular, low endotoxin alkali chitosan may be used to prepare other useful chitosan products having low endotoxin concentrations, including neutral chitosan and chitosan salts and other chitosan derivatives, such as carboxymethyl chitosan, hydroxyethyl chitosan, acyl chitosan, alkyl chitosan, sulfonyl chitosan, phosphorylated chitosan, alkylidene chitosan, metal chelates, chlorinated chitosan, lactic chitosan, acetic chitosan, malic chitosan, gluconic chitosan.

Thus, according to another aspect of the present invention there is provided a method of preparing a low endotoxin neutral chitosan, chitosan salt or chitosan derivative, the method comprising the step of contacting an alkaline chitosan prepared by the method described herein with an acid.

This method can provide medical neutral chitosan, chitosan salts or other chitosan derivatives with low concentrations of endotoxins.

In the method of the first aspect of the present invention, the step of contacting the alkali chitosan with an acid may be performed before the drying step.

The acid may be contacted with the alkaline chitosan by any suitable method known in the art. For example, the acid may be sprayed onto the alkali chitosan, or the alkali chitosan may be mixed with the acid.

Preferably, the alkali chitosan is mixed with an acid.

Herein, neutral chitosan refers to chitosan having a pH between about 6.5 and about 7.5, preferably about 7.

Thus, to prepare neutral chitosan, alkaline chitosan may be mixed with an acid of suitable volume and concentration to produce a neutral solution having a pH between 6.5 and 7.5. The volume and/or concentration of acid required to neutralize the alkaline chitosan will depend on the pH of the alkaline chitosan.

Alternatively, to prepare a chitosan salt or chitosan derivative, the alkaline chitosan may be mixed with an acid in excess of the volume and concentration required to provide a neutral chitosan.

Suitable acids for use in the present invention may be selected from the following acids, alone or in combination: organic acids, carboxylic acids, fatty acids, amino acids, lewis acids, monoprotic acids, biprotic acids, polyprotic acids, nucleic acids and inorganic acids.

Suitable organic acids may be selected from the following, alone or in combination: acetic acid, tartaric acid, citric acid, ascorbic acid, acetylsalicylic acid, gluconic acid and lactic acid.

Suitable fatty acids may be selected from the following fatty acids, alone or in combination: myristoleic acid, palmitoleic acid (palmitoleic acid), cis-6-hexadecenoic acid (sapienic acid), oleic acid, elaidic acid, 11-octadecenoic acid, linoleic acid, elaidic acid, alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acidAcid, wood wax acid, wax acid.

Suitable amino acids may be selected from the following amino acids, alone or in combination: histidine, lysine, aspartic acid, glutamic acid, glutamine, glycine, proline and taurine.

Suitable mineral acids may be selected from the following mineral acids, alone or in combination: hydrochloric acid, sulfuric acid and nitric acid. The acid selected for neutralization is preferably hydrochloric acid.

The acid may have a concentration of about 0.001M acid to the highest possible acid concentration. For example, a typical maximum concentration for sulfuric acid is about 98% sulfuric acid. The acid may have a concentration of about 0.01M to 5M, 0.01M to 3M, or 0.1M to 2M. Preferably the acid has a concentration of about 1M. The concentration of the acid may be about 0.01M, 0.05M, 0.10M, 0.15M, 0.20M, 0.25M, 0.30M, 0.35M, 0.40M, 0.45M, 0.50M, 0.55M, 0.60M, 0.65M, 0.70M, 0.75M, 0.80M, 0.85M, 0.90M, 0.95M, or 1.0M.

The acid may be present as an acid solution comprising the acid and a non-solvent. The non-solvent may be any solvent in which chitosan is insoluble. Typical non-solvents include ethyl lactate, ethyl acetate, methyl acetate, ethanol, acetone, or mixtures thereof. Preferred non-solvents include ethyl acetate or ethanol. More preferably, the non-solvent comprises 80:20 ethanol/water. It has been advantageously observed that the reaction proceeds at a faster rate using a non-solvent comprising a mixture of 80:20 ethanol and water.

The ratio of chitosan to acid liquor may be from about 5:1 to about 1: 5. The ratio of chitosan to acid liquor is preferably about 2: 1.

In some embodiments, the low endotoxin alkali chitosan may be mixed with the acid for about 5 minutes. The reaction is then allowed to occur as the mixture dries.

The solution obtained from the mixture of alkaline chitosan and acid may contain an acid salt. The basic solution and acid are preferably selected to ensure that the acid salt formed is biocompatible. For example, the alkaline solution may comprise sodium hydroxide and the acid may comprise hydrochloric acid. In this example, the acid salt is the biocompatible salt sodium chloride.

The acid salt is generated as a by-product of the reaction between the basic chitosan and the acid.

The presence of acid salts in the product can affect the effectiveness of the resulting chitosan product. For example, chitosan gels to a lesser extent in saline solution than in water, and to an even lesser extent in double strength saline solution. The double concentration saline solution referred to herein is considered to have a sodium chloride amount of 1.8%. It is therefore desirable to have as low an amount of acid salt as possible in the resulting chitosan product, ideally with little or substantially no difference in the level of acid salt to the effectiveness of the chitosan product.

It has been surprisingly found that the use of an alkaline solution at a concentration of about 0.01M to about 0.1M produces the desired low endotoxin concentration while also producing less acid salt by-product in subsequent processes to give neutral chitosan, chitosan salts or chitosan derivatives. Advantageously, less acid salt by-product means that the resulting chitosan product will have improved gelling in use relative to products comprising higher amounts of acid salt. The process of the present invention can provide a chitosan product having suitably low amounts of acid salts without the need to wash or rinse the chitosan product. This also has the additional advantage that no endotoxin-free water is required in the washing or rinsing step.

It has also been found that the use of the low concentration alkaline solutions described herein results in little viscosity reduction of chitosan when preparing neutral chitosan, chitosan salts or chitosan derivatives. By low concentration of base is meant from about 0.01M to about 1M, preferably less than 1M, more preferably from about 0.02M to about 0.2M. In some embodiments, the base concentration may be 0.05M. In some embodiments, the base concentration may be as described above. Thus, advantageously, the use of low concentration alkaline solutions in the process rarely destroys chitosan. Thus, endotoxin can be removed from chitosan while producing only minimal viscosity changes. It is desirable that the chitosan viscosity be reduced by less than about 25%, preferably less than about 15%, more preferably less than about 10% during the process.

Where the process provides low endotoxin neutral chitosan, the product is suitable as a starting material for the preparation of other chitosan-based products. One particular use is in the preparation of chitosan salts whose absorption properties make them ideal hemostatic formulations for use in controlling bleeding. Preferably the chitosan salt is water soluble.

Thus, in another embodiment of the invention, a low endotoxin chitosan salt may be prepared by contacting a low endotoxin neutral chitosan prepared by the methods described herein with an acid.

The acid can be any acid suitable for providing the desired chitosan salt. For example, if chitosan acetate is desired, acetic acid may be used, and if chitosan succinate is desired, succinic acid, and the like may be used. Low endotoxin chitosan salts may be prepared in the methods of the invention using any of the acids described herein.

The process for preparing a low endotoxin chitosan salt or chitosan derivative may further comprise the step of drying the mixture of low endotoxin neutral chitosan and acid. The drying step may be performed by any conventional drying method known in the art. The drying step is preferably carried out in an oven or by filtering the product through an air dryer.

Thus, the present invention provides a low endotoxin neutral chitosan, chitosan salt or chitosan derivative having an endotoxin concentration of less than 100 EU/g.

The low endotoxin neutral chitosan may be water insoluble.

The low endotoxin chitosan salt may be water soluble.

According to another aspect of the invention there is provided a low endotoxin neutral chitosan, chitosan salt or chitosan derivative obtainable by any of the methods described herein.

According to another aspect of the invention there is provided a neutral chitosan, chitosan salt or chitosan derivative having an endotoxin concentration of less than 100 EU/g.

The neutral chitosan, chitosan salt or chitosan derivative preferably has an endotoxin concentration of less than 50EU/g, more preferably less than 20EU/g, even more preferably less than 15EU/g, most preferably less than 10 EU/g.

The low endotoxin chitosan salts of the present invention are suitable for use as haemostats for arresting blood flow.

Thus, according to another aspect of the present invention there is provided a low endotoxin chitosan salt as described herein for use as a haemostat in stopping blood flow.

The low endotoxin chitosan salt can be added into wound dressing for treating hemorrhage without or without critical life on the surface.

Thus, according to another aspect of the present invention there is provided a low endotoxin chitosan salt as described herein for use in a wound dressing for superficial non-or life threatening bleeding.

The low endotoxin chitosan salt of the invention is suitable for preparing the hemostatic wound dressing for stopping blood flow. According to another aspect of the present invention there is provided a haemostatic wound dressing comprising a low endotoxin chitosan salt as described herein.

According to another aspect of the present invention there is provided a haemostatic material comprising a low endotoxin chitosan salt as described herein.

The hemostatic material and/or chitosan salt may be in any suitable form, such as a particulate, powder, granular, flake, fiber, gel, foam, sheet, film, or liquid form.

According to another aspect of the present invention, there is provided a method of stopping blood flow, the method comprising the steps of: optionally, where possible, cleansing the wound area; applying to the wound area a hemostatic wound dressing comprising a low endotoxin chitosan salt as described herein; and applying a constant pressure to the wound area until a gel mass is formed.

Preferably, the constant pressure is applied to the wound area for about 3 minutes or more.

Drawings

Embodiments of the present invention will now be further described in the following non-limiting examples, with reference to the accompanying drawings, in which:

FIG. 1 is a graph showing the effect of different concentrations of acid salt by-product on the viscosity of chitosan products in different media;

FIG. 2 is a graph showing the effect of treatment with acid on chitosan polymer viscosity;

FIG. 3 is a graph showing saline penetration in two different hemostatic materials of the invention;

FIG. 4 is a graph showing the time to blood coagulation for two different hemostatic materials of the present invention;

fig. 5 is a graph showing the percent hemostasis for the cut-over-the-abdomen test of two different hemostatic materials of the present invention.

Detailed Description

Endotoxin assay

1. USP (United states Pharmacopeia) extraction solution (4.6ml 1M HCl and 45.4ml endotoxin free water) was prepared as described for the USP test for chitosan endotoxin;

2. the chitosan product was extracted by adding 0.1g of test chitosan product to 9.9ml of USP extraction solution and holding at 37 ℃ for 48 hours;

after 3.48 hours, 100 μ l of the extract was diluted in 0.9ml of endotoxin-free water; and is

4. More than 100 μ l of material was mixed in 100 μ l Endotoxin Specific (ES) buffer provided by Charles River.

Endosafe-PTS (Endosafe-PTS) made of FDA (food and drug administration) approved disposable columns^TMThe resulting extract was tested using a handheld spectrophotometer. The extraction procedure used 2000x dilution and a minimum assay limit of 10 EU/g.

Examples

Example 1:

50g of chitosan (Primex Icelland) were mixed with 50g of 1M NaOH for 30 minutes. The resulting wet alkaline chitosan crumb was held at room temperature for 48 hours. And then dried in an oven on a tray at 40 ℃.

Initial endotoxin of raw material chitosan: 383EU/g

Drying the treated alkaline chitosan: 10.6EU/g

Control (0.1g endotoxin-free water used during extraction instead of chitosan): <10EU/g

Example 2:

50g of chitosan (Primex Icelland) were mixed with 50g of 0.5M NaOH for 10 minutes. The resulting wet alkaline chitosan crumb was held at room temperature for 72 hours. And then dried in an oven on a tray at 40 ℃.

Initial endotoxin of raw material chitosan: 383EU/g

Drying the treated alkaline chitosan: 38.3EU/g

Example 3:

50g of chitosan (Primex Icelland) were mixed with 50g of 0.2M NaOH for 10 minutes. The resulting wet alkaline chitosan crumb was kept at room temperature for 7 days. And then dried in an oven on a tray at 40 ℃.

Initial endotoxin of raw material chitosan: 383EU/g

Drying the treated alkaline chitosan: 27.9EU/g

Example 4:

50g of chitosan (Primex Icelland) were mixed with 50g of 0.1M NaOH for 10 minutes. The resulting wet alkaline chitosan crumb was kept at room temperature for 14 days. And then dried in an oven on a tray at 40 ℃.

Initial endotoxin of raw material chitosan: 383EU/g

Drying the treated alkaline chitosan: 12.7EU/g

Example 5:

50g of chitosan (Primex Icelland) were mixed with 100g of 0.2M NaOH for 10 minutes. The resulting wet alkaline chitosan crumb was kept at room temperature for 2 days. And then dried in an oven on a tray at 40 ℃.

Initial endotoxin of raw material chitosan: 383EU/g

Drying the treated alkaline chitosan: 35.7EU/g

Example 6:

50g of chitosan (Primex Icelland) were mixed with 100g of 0.1M NaOH for 10 minutes. The resulting wet alkaline chitosan crumb was kept at room temperature for 2 days. And then dried in an oven on a tray at 40 ℃.

Initial endotoxin of raw material chitosan: 383EU/g

Drying the treated alkaline chitosan: 49.3EU/g

Example 7:

50g of chitosan powder (Cognis, Germany) was mixed with 50g of 0.1M NaOH for 10 minutes. The resulting wet alkaline chitosan crumb was kept at room temperature for 7 days. And then dried in an oven on a tray at 40 ℃.

Initial endotoxin of raw material chitosan: 45.3EU/g

Drying the treated alkaline chitosan: <10EU/g

Control (0.1g endotoxin-free water used during extraction instead of chitosan): <10EU/g

Example 8:

50g of chitosan powder (Cognis, Germany) were mixed with 50g of 0.05M NaOH for 10 minutes. The resulting wet alkaline chitosan crumb was kept at room temperature for 7 days. And then dried in an oven on a tray at 40 ℃.

Initial endotoxin of raw material chitosan: 45.3EU/g

Drying the treated alkaline chitosan: <10EU/g

Control (0.1g endotoxin-free water used during extraction instead of chitosan): <10EU/g

Example 9:

50g of chitosan powder (Cognis, Germany) was mixed with 50g of 0.025M NaOH for 10 minutes. The resulting wet alkaline chitosan crumb was kept at room temperature for 7 days. And then dried in an oven on a tray at 40 ℃.

Initial endotoxin of raw material chitosan: 45.3EU/g

Drying the treated alkaline chitosan: <13.5EU/g

Control (0.1g endotoxin-free water used during extraction instead of chitosan): <10EU/g

This process can also be scaled up and used to prepare larger batches. Examples 10 and 11 were prepared in a class 100,000 clean room (US FED STD 209E clean room standard) commonly used in medical device manufacturing.

Example 10:

3.5kg of chitosan powder (Primex Icelland) was mixed with 3.5kg of 0.1M NaOH for 30 minutes. The resulting wet alkaline chitosan crumb was kept at room temperature for 14 days. Then dried by filtration through an air dryer at 40 ℃.

Initial endotoxin of raw material chitosan: 288EU/g

Drying the treated alkaline chitosan: 10.2EU/g

Control (0.1g endotoxin-free water used during extraction instead of chitosan): <10EU/g

Example 11:

3.5kg of chitosan (Primex Icelland) powder was mixed with 3.5kg of 1M NaOH for 30 minutes. The resulting wet alkaline chitosan crumb was held at room temperature for 24 hours. Then dried by filtration through an air dryer at 40 ℃.

Initial endotoxin of raw material chitosan: 288EU/g

Drying the treated alkaline chitosan: 15.3EU/g

Control (0.1g endotoxin-free water used during extraction instead of chitosan): <10EU/g

This process can be used on chitosan in different physical forms, such as chitosan fibers or chitosan fabrics.

Example 12:

10g of chitosan fibers (1.8dtex X28 mm) were mixed with 10g of 0.1M NaOH for 10 minutes. The resulting wet alkali chitosan fibers were kept at room temperature for 2 days. And then dried in a laboratory oven on a tray at 40 ℃.

Initial endotoxin of raw chitosan fiber: 88EU/g

Drying the treated alkaline chitosan fibers: <10EU/g

Control (0.1g endotoxin-free water used during extraction instead of chitosan): <10EU/g

Example 13:

5g of chitosan nonwoven (60gsm) was mixed with 5g of 0.1M NaOH for 10 minutes. The resulting wet alkaline chitosan fabric was kept at room temperature for 2 days. And then dried in a laboratory oven on a tray at 40 ℃.

Initial endotoxin of raw material chitosan fabric: 401EU/g

Drying the treated alkaline chitosan fabric: <78EU/g

Control (0.1g endotoxin-free water used during extraction instead of chitosan): <10EU/g

The following examples 14 and 15 utilize a different base than sodium hydroxide.

Example 14:

50g of chitosan (Primex Icelland) were mixed with 50g of 0.2M KOH (potassium hydroxide) for 30 minutes. The resulting wet alkaline chitosan crumb was kept at room temperature for 7 days. And then dried in a laboratory oven on a tray at 40 ℃.

Initial endotoxin of raw material chitosan: 383EU/g

Drying the treated alkaline chitosan: 14.4EU/g

Control (0.1g endotoxin-free water used during extraction instead of chitosan): <10EU/g

Example 15:

50g of chitosan (Primex Icelland) were mixed with 50g of 0.5M sodium carbonate for 30 minutes. The resulting wet alkaline chitosan crumb was kept at room temperature for 7 days. And then dried in a laboratory oven on a tray at 40 ℃.

Initial endotoxin of raw material chitosan: 383EU/g

Drying the treated alkaline chitosan: 25.8EU/g

Control (0.1g endotoxin-free water used during extraction instead of chitosan): <10 EU/g.

Examples 1-15 all relate to the preparation of low endotoxin alkali chitosan. This low endotoxin alkali chitosan can then be used as a starting material for the preparation of other chitosan-based products. For example, alkaline chitosan may be neutralized to pH 7 by adding a low level of a suitable acid that reacts with a base to produce a biocompatible salt to produce neutral chitosan. For example, if sodium hydroxide is used in an alkaline solution, it can be neutralized by the addition of hydrochloric acid. The product will contain low amounts of residual sodium chloride.

Example 16:

20g of wet alkaline chitosan crumb from example 4 was weighed into a beaker. This contained 10g of chitosan and 10g of 0.1M NaOH. To neutralize the NaOH, 1g of 1M HCl was required. This was mixed with 9g of ethanol in a separate beaker. The acid liquor was then mixed into the wet alkaline chitosan crumb and stirred for 5 minutes. The resulting mixture was then dried in a laboratory oven at 40 ℃. It contains 0.29% sodium chloride.

Example 17:

20g of wet alkaline chitosan crumb from example 4 was weighed into a beaker. This contained 10g of chitosan and 10g of 0.1M NaOH. To neutralize NaOH, 1g of 1M acetic acid was required. This was mixed with 9g of ethanol in a separate beaker. The acid liquor was then mixed into the wet alkaline chitosan crumb and stirred for 5 minutes. The resulting mixture was then dried in a laboratory oven at 40 ℃. It contained 0.5% sodium acetate.

Low endotoxin water soluble chitosan salts or other chitosan derivatives may also be prepared using the low endotoxin alkali chitosan produced in examples 1-15. Advantageously, this can be achieved without the need for a sterile environment, without the use of extensive, costly endotoxin-free water, and without the need for rinsing or washing. For example, low endotoxin alkali chitosan may be reacted with a greater level of a suitable acid. A small portion of the acid will react with the base to produce a biocompatible salt.

Example 18:

3.1kg of dried low endotoxin alkali chitosan from example 10 was weighed into a stainless steel mixer in a class 100,000 clean room. 3.6kg of lactic acid was premixed with 0.9kg of endotoxin-free water. This was sprayed onto the chitosan while the mixer was running. The resulting material was dried by filtration through an air dryer at 40 ℃.

The material was found to be completely water soluble.

Initial endotoxin of raw material chitosan: 288EU/g

Drying the treated alkaline chitosan: 10.2EU/g (example 10)

Drying the water-soluble chitosan: 13.8EU/g

Control (0.1g endotoxin-free water used during extraction instead of chitosan): <10EU/g

Example 19:

3.1kg of dried low endotoxin alkali chitosan from example 11 was weighed into a stainless steel mixer in a class 100,000 clean room. 3.9kg of lactic acid was premixed with 0.9kg of endotoxin-free water. This was sprayed onto the chitosan while the mixer was running. The resulting material was dried by filtration through an air dryer at 40 ℃.

The material was found to be completely water soluble.

Initial endotoxin of raw material chitosan: 288EU/g

Drying the treated alkaline chitosan: 15.3EU/g (example 4)

Drying the water-soluble chitosan: 14.6EU/g

Control (0.1g endotoxin-free water used during extraction instead of chitosan): <10EU/g

In another embodiment, low endotoxin chitosan derivatives, such as carboxymethyl chitosan, may also be prepared using low endotoxin alkali chitosan as a starting material.

Example 20:

20g of wet alkaline chitosan crumb from example 1 was weighed into a beaker. This contained 10g of chitosan and 10g of 1M NaOH. In a separate beaker, a mixture of 5g of sodium chloroacetate and 5g of water and 10g of ethanol was prepared. The mixed liquor was then stirred into the wet alkaline chitosan crumb. The temperature was then raised to 60 ℃ and held for 4 hours. The resulting mixture was washed 3 times with 10g of ethanol to remove any residual sodium chloroacetate before drying in the laboratory oven at 40 ℃.

Effect of acid salts on viscosity:

reacting a low endotoxin alkali chitosan with an acid to produce a neutral pH chitosan or chitosan salt, producing an acid salt by-product. The presence of such by-products can affect the properties of the chitosan product. For example, the level of by-products can affect the viscosity of the chitosan product in saline.

Referring to FIG. 1, there is shown the results of sodium lactate added to brine in varying concentrations and the effect on the viscosity of 2g of current commercially available chitosan product CELOX samples in 20g of solution in different media after 3 minutes.

The base medium was saline from body fluids to which various levels of sodium lactate were added. Sodium lactate represents a by-product of the reaction between sodium hydroxide and lactic acid.

The results are shown in table 1 and fig. 1.

TABLE 1

As is clear from FIG. 1, the viscosity of CELOX in the medium decreased with increasing salt levels added. Thus, advantageously results in the chitosan product of the present invention having only low levels of residual salt by-products.

Effect of low concentration alkaline solution on viscosity:

the low endotoxin alkali chitosan prepared in example 10 was tested to demonstrate the effect of acid treatment on the viscosity of the chitosan polymer, which is considered a measure of molecular weight. The test was carried out according to the following method steps:

a) weigh out 5g of the low endotoxin alkali chitosan particles prepared in example 10;

b) weighing 4.95g of acetic acid into a 600ml beaker;

c) 490.05g of deionized water was added to a beaker to make up 495g of a 1% acetic acid solution;

d) place the beaker on a stirrer plate and start stirring (increasing as the solution viscosity increases);

e) adding chitosan particles to an acetic acid solution;

f) the solution is often looked at until all particles have dissolved and the level of agitation is increased as the solution viscosity increases, if necessary;

g) the solution was maintained for a total of 24 hours as measured when the acetic acid solution was introduced from the chitosan particles;

h) coupling the spindle 64 to a Brookfield viscometer

i) Set the rotor to 10 rpm;

j) inserting the rotor into the solution to the rotor mark, starting the viscometer and stabilizing it;

k) the viscosities (cPs) were recorded at selected time intervals.

The results of the above viscosity test are shown in table 2 below and fig. 2. For each batch, three readings were averaged over each time interval.

TABLE 2

It can be inferred from the viscosity measurements shown in FIG. 2 that the low endotoxin alkali chitosan polymers prepared with 0.1M sodium hydroxide solution were stable in molecular weight for several weeks.

Effect of reducing concentration of alkaline solution

The effect of using a lower concentration alkaline solution in the method of the invention was tested in three tests, focusing on (1) the percent permeability of the saline into the test sample; (2) the time at which the blood coagulates; and (3) percent hemostasis for the in-vivo model of the upper abdominal incision.

Referring to fig. 3, the results of the permeability test are shown.

The general test method is as follows: 5ml of distilled water was added to the test tube. Add 1 drop of red food dye to water. A3 g sample of hemostatic powder was poured gently on the water to form a layer. After 1 minute, the distance that the water moves into the hemostatic powder was measured and recorded as percent penetration.

As can be seen in fig. 3, the hemostatic powder prepared with 1.0M sodium hydroxide was less stable over time than the hemostatic powder prepared with 0.1M sodium hydroxide, and the increased penetration of saline solution into the particles indicated that there was more opportunity for blood to pass through the particles without forming a gel plug.

Referring to fig. 4, the results of the blood clotting test are shown.

The general test method is as follows: 0.75g of the hemostatic powder of the sample was added to the tube, and 5ml of heparinized rabbit blood was added thereto. The tube was then inverted and the time taken for the blood to fully coagulate into a gel mass was recorded.

As can be seen in FIG. 4, the hemostatic powder prepared with 1.0M sodium hydroxide takes longer to clot blood than the hemostatic powder prepared with 0.1M sodium hydroxide. The time was about 3 times longer, which relates to lower hemostatic properties (data have shown that a period of more than 180 seconds does not result in 100% hemostasis in the low pressure, intermediate volume in vivo hemorrhage model).

Referring to fig. 5, the results of the hemostasis test are shown.

The general test method is as follows: the upper abdominal artery of the pig model (non-heparinized) was cut 3-5 cm. A particulate hemostatic material was applied and pressurized for 1 minute. If re-bleeding occurs, additional pressurization is applied for 1 minute.

As can be seen in fig. 5, the hemostatic particles prepared with 1.0M sodium hydroxide gave 60% of the tested hemostasis compared to 100% of the hemostatic particles prepared with 0.1M sodium hydroxide.

The test results show that the lower the concentration of alkaline solution used in preparing the hemostatic material of the invention, the better the permeability, blood clotting and hemostasis of the material perform.

It will of course be appreciated that the invention is not intended to be limited to the embodiments described above, which are described as examples only.