阅读说明：本技术 生产低内毒素的壳聚糖的方法 (Method for producing low endotoxin chitosan ) 是由 C·哈迪 安德鲁·霍加思 琼·格莱德曼 于 2014-05-29 设计创作，主要内容包括：本发明涉及一种用于生产低内毒素的碱性的壳聚糖、低内毒素的碱性的壳质、低内毒素的碱性的壳聚糖衍生物或低内毒素的碱性的壳质衍生物的方法,并且还涉及一种用于生产低内毒素的中性的壳聚糖、低内毒素的中性的壳聚糖盐和低内毒素的中性的壳聚糖衍生物的方法,以及上述方法的产品。所述方法包括：使壳聚糖、壳质、壳聚糖衍生物或壳质衍生物与碱性溶液接触以形成混合物；使所述混合物放置小于1小时的时段；以及可选地,对所述混合物进行干燥。所述低内毒素的碱性的壳聚糖可用于制造其它有用的壳聚糖类产品。(The present invention relates to a process for producing low endotoxin alkali chitosan, chitin, a derivative or a derivative of chitin, and also to a process for producing low endotoxin neutral chitosan, a chitosan salt and a chitosan derivative, and the products of the above process. The method comprises the following steps: contacting chitosan, chitin, a chitosan derivative or a chitin derivative with an alkaline solution to form a mixture; allowing the mixture to stand for a period of less than 1 hour; and optionally, drying the mixture. The low endotoxin alkali chitosan can be used to make other useful chitosan-based products.)

1. A process for producing low endotoxin alkali chitosan, chitin or a derivative thereof, the process comprising the steps of:

(a) contacting chitosan, chitin, a chitosan derivative or a chitin derivative with an alkaline solution to form a mixture;

(b) allowing the mixture to stand for a period of less than 1 hour; and then

(c) Drying the mixture;

wherein the concentration of the alkaline solution is 0.01M to 0.2M; and

the ratio of the chitosan, chitin, chitosan derivative or chitin derivative to the alkaline solution is from 1:10 to 10: 1.

2. The method of claim 1, wherein the concentration of the alkaline solution is about 0.02M to 0.2M.

3. The method of claim 1, wherein the concentration of the alkaline solution is about 0.1M.

4. The method of any of the preceding claims, wherein the alkaline solution comprises an alkali or alkaline earth metal component selected from one or a combination of a metal hydroxide, a metal carbonate, a metal bisulfite, a metal persilicate, a conjugate base, and ammonium hydroxide.

5. The method of claim 4, wherein the metal is selected from sodium, potassium, calcium, or magnesium.

6. The process according to claim 4, wherein the alkali metal component is selected from sodium hydroxide, potassium hydroxide or sodium carbonate.

7. The method of any one of claims 1 to 3, wherein the alkaline solution is sprayed to the chitosan, chitin, chitosan derivative, or chitin derivative; or mixing the chitosan, chitin, chitosan derivative or chitin derivative with the alkaline solution.

8. The method of any one of claims 1 to 3, wherein the mixture is allowed to stand for a period of less than 3 minutes.

9. The method of claim 8, wherein step (b) comprises drying the mixture immediately after step (a).

10. The method according to any one of claims 1 to 3, wherein the mixture is placed in a clean container and/or under an inert atmosphere.

11. The method of any one of claims 1 to 3, wherein the mixture further comprises a preservative.

12. The method of claim 11, wherein the preservative is selected from silver ions, zinc ions, chlorhexidine, or combinations thereof.

13. A low endotoxin alkali chitosan, chitin, a chitosan derivative or a chitin derivative obtainable by the process of any one of claims 1 to 12.

14. A method of producing a low endotoxin neutral chitosan, chitosan salt or chitosan derivative, the method comprising: a step of contacting the alkaline chitosan prepared by the method of any one of claims 1 to 12 with an acid.

15. The method of claim 14, wherein the step of contacting the alkaline chitosan with an acid is performed prior to the drying step (c) of any one of claims 1 to 12.

16. The method of claim 14 or 15, wherein the acid is sprayed onto the alkali chitosan or the alkali chitosan is mixed with the acid.

17. The process according to claim 14 or 15, wherein the acid is selected from one or a combination of the following: organic acids, carboxylic acids, fatty acids, amino acids, lewis acids, monoprotic acids, biprotic acids, polyprotic acids, nucleic acids, and inorganic acids.

18. The method of claim 17, wherein the organic acid is selected from one or a combination of the following: acetic acid, tartaric acid, citric acid, ascorbic acid, acetylsalicylic acid, gluconic acid and lactic acid.

19. The method of claim 17, wherein the fatty acid is selected from one or a combination of the following: myristoleic acid, palmitoleic acid, 6-hexadecenoic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, trans-linoleic 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 acid, lignoceric acid, cerotic acid.

20. The method of claim 17, wherein the amino acid is selected from one or a combination of the following: histidine, lysine, aspartic acid, glutamic acid, glutamine, glycine, proline and taurine.

21. The process according to claim 17, wherein the mineral acid is selected from one or a combination of the following: hydrochloric acid, sulfuric acid and nitric acid.

22. The method of claim 14 or 15, wherein the acid has a concentration of about 1M.

23. A process as claimed in claim 14 or 15, wherein the acid is present as an acid liquor comprising the acid and a non-solvent.

24. The method of claim 23, wherein the non-solvent is selected from ethyl lactate, ethyl acetate, methyl acetate, ethanol, acetone, a 80:20 ethanolic mixture, or a mixture thereof.

25. The method of claim 23, wherein the ratio of the chitosan to the acid liquor is about 5:1 to about 1: 5.

26. The method of claim 14 or 15, wherein the alkali chitosan is mixed with the acid for about 5 minutes.

27. The method of claim 14 or 15, further comprising the step of drying the reaction product.

28. A low endotoxin neutral chitosan, chitosan salt or chitosan derivative obtainable by the method of any one of claims 14 to 27.

29. Use of a low endotoxin chitosan salt of claim 28 in the manufacture of a medicament for use in the retardation of blood flow.

30. A low endotoxin chitosan salt of claim 28 for use as a haemostat for stemming blood flow.

31. A low endotoxin chitosan salt of claim 28 for use in a wound dressing for superficial non-life threatening bleeding or life threatening bleeding.

32. A haemostatic wound dressing comprising a low endotoxin chitosan salt of claim 28.

33. Use of a low endotoxin as claimed in claim 28 in the preparation of a haemostatic wound dressing for use in blocking blood flow comprising the steps of: optionally, where possible, cleaning the wound area; applying the hemostatic wound dressing to the wound area; and applying a constant pressure to the wound area until a gel mass is formed.

Technical Field

The present invention relates to a process for producing low endotoxin alkali chitosan, and also relates to a process for producing low endotoxin neutral chitosan, a low endotoxin neutral chitosan salt and a low endotoxin neutral chitosan derivative, and to the products of the above processes.

Background

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

Chitosan is a derivative from solid waste from shellfish processing and can be extracted from fungal cultures. Chitosan is a water insoluble cationic polymeric material. Chitosan is typically first converted to a water-soluble salt before it is used in a hemostatic material. Thus, the chitosan salt can be dissolved in blood to form a gel that blocks blood flow.

Since chitosan is readily decomposed in vivo, chitosan salts are well suited for the applications described herein. Chitosan is converted into glucosamine by lysozyme and is thus naturally excreted from the body. Thereby eliminating the need to remove the chitosan from the body. Furthermore, chitosan salts exhibit moderate antibacterial properties, so that their use reduces the risk of infection.

In order to use chitosan for the preparation of a haemostatic material suitable for controlling bleeding, it must be ensured that the chitosan has a sufficiently low concentration of endotoxin.

Endotoxins are lipopolysaccharides that are present on the surface of the outer membrane of gram-negative bacteria. Endotoxins are highly toxic to mammals, especially humans, and are notoriously difficult to remove from materials. Endotoxin can become a pyrogen when it is released into the blood or other tissues where it is not normally found. Thus, endotoxins must be removed from pharmaceutically acceptable products.

The treatment of pyrogens to remove or destroy pyrogens, particularly endotoxins, is known as a "depyrogenation" process. Techniques for depyrogenation of endotoxin-containing materials include ion exchange chromatography, ultrafiltration, distillation, and various chemical methods aimed at destroying endotoxin.

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

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

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

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

d) continuously stirring the resulting chitosan alkali solution;

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

f) Rinsing the solution with endotoxin-free ultrapure water up to 10 times the volume;

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

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

i) excess water is removed from the slurry.

This is a complicated and expensive process, especially requiring sterile equipment and rinsing with 10 volumes of endotoxin-free aqueous solution.

US2006293509 relates to a method for preparing water-soluble chitosan with low endotoxin comprising:

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

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

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

(d) dissolving a partially acetylated water soluble chitosan in an aqueous solution containing 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 cause precipitation of water soluble chitosan having a low endotoxin content from the aqueous solution/water miscible solvent mixture; and

(f) optionally, washing is performed with a non-solvent (non-solvent), such as isopropanol.

However, this method is complex and expensive, and is expected to involve the use of large amounts of endotoxin-free water or other liquid. This process also requires the use of a phase transfer agent and takes several hours.

TW593342 relates to a method of reducing endotoxin in chitosan, the method comprising:

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

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

(c) solid and aqueous solutions are separated by means of solid/liquid separation.

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

The present invention aims to solve the above-mentioned problems.

Disclosure of Invention

According to a first aspect of the present invention there is provided a process for producing low endotoxin alkali chitosan, chitin or a derivative thereof, the process comprising the steps of:

(a) contacting chitosan, chitin, a chitosan derivative or a chitin derivative with an alkaline solution to form a mixture; and

(b) the mixture was allowed to stand for a period of less than 1 hour.

The method of the present invention may further comprise the step (c) of drying the mixture.

The process of the present invention provides an efficient way to obtain an alkaline chitosan, chitin, chitosan derivative or chitin derivative with a low endotoxin concentration. Advantageously, the method 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. Further, no special air filtration or sterile conditions are required. Preferably, the method of the invention does not comprise a step of acetylating chitosan. Further, the process of the invention does not require the use of endotoxin-free equipment. This is particularly advantageous as it reduces the cost of the process compared to processes requiring such equipment.

The term "chitosan derivative" refers herein to partially deacetylated chitin, which may have different percentages of deacetylation, as desired. Typically, partially deacetylated chitin suitable for use in the present invention has a degree of deacetylation greater than about 50%, more typically greater than about 75%, and most typically greater than about 85%.

Furthermore, the term "derivative" herein encompasses the reaction product of chitosan or chitin with other compounds. Such reaction products include, but are not limited to, carboxymethyl chitosan, hydroxybutyl chitin, N-acyl chitosan, O-acyl chitosan, N-alkyl chitosan, O-alkyl chitosan, N-alkylene chitosan, O-sulfonyl chitosan, sulfated chitosan, phosphated chitosan, nitrated chitosan, basic chitin, basic chitosan, or metal chelates of chitosan, and the like.

Although the first aspect of the present invention provides a process for producing low endotoxin chitosan, chitin or a derivative thereof, chitosan is described below for convenience and illustrative purposes only.

The chitosan may be a commercially available chitosan, such as food grade, medical grade, or pharmaceutical grade chitosan. Thus, the method of the invention enables the provision of low endotoxin alkali chitosan from commercially available chitosan. This is in contrast to some methods of removing or reducing endotoxin as part of the chitosan production process. Advantageously, the method of the present invention can be used to provide low endotoxin alkali chitosan from prepared chitosan, which is not suitable for use in the medical field due to its endotoxin concentration.

The term basic chitosan as used herein refers to a chitosan composition having a pH greater than pH 7.5.

The term alkaline solution as used herein relates to a solution having a pH value of more than pH 7.5.

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

For example, in the present invention, the concentration of endotoxin is determined as endotoxin units per gram of chitosan (EU). The term "low endotoxin" as used herein refers to an endotoxin concentration of less than 50 Endotoxin Units (EU) per gram of chitosan.

Thus, the process of the invention is suitable for the production of alkaline chitosan having an endotoxin concentration of less than 50 EU/g.

Preferably, the resulting alkaline chitosan has an endotoxin concentration of less than 30EU/g, more preferably less than 20EU/g, more preferably less than 15EU/g, even more preferably less than 10EU/g and most preferably less than 5 EU/g.

It has been found that in the present process, low concentration alkaline solutions are preferred. The concentration of the alkaline solution used in the present process may be from about 0.01M to about 1M. Preferably, the concentration of the alkaline solution is less than 1M. More preferably, the concentration of the alkaline solution is about 0.02M to 0.25M, and even more preferably, the concentration of the alkaline solution is about 0.04M to 0.06M, typically 0.05M. The concentration of the alkaline solution may be up to 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 were observed with a 0.1M concentration of 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 up to about 10 parts chitosan to 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 1 part chitosan.

The alkaline solution may include an alkali metal component or an alkaline earth metal component selected from one or a combination of a metal hydroxide, a metal carbonate, a metal bisulfite, a metal persilicate, a conjugate base, and ammonium hydroxide.

Suitable metals include sodium, potassium, calcium or magnesium.

Preferably, the alkaline component is sodium hydroxide, potassium hydroxide or sodium carbonate. Usually, sodium hydroxide is used.

The alkaline solution may be contacted with the chitosan by any suitable means 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, there is a uniform distribution of post-contact alkaline chitosan.

Preferably, the chitosan is mixed with an alkaline solution. At low molecular weights, chitosan may be completely or partially dissolved in alkaline solutions. In step (a), chitosan may be mixed with the alkaline solution for up to about 30 minutes, more preferably for about 10 minutes. In some embodiments, the chitosan may be mixed with the alkaline solution for more than 30 minutes.

In some embodiments, the chitosan is insoluble in alkaline solutions.

In some embodiments, the chitosan does not swell in alkaline solutions.

In some embodiments, the alkaline solution wets the chitosan without dissolving or swelling the chitosan.

In some embodiments, the mixture of chitosan and alkaline solution may be intermittently stirred for the duration of step (b).

The mixture of chitosan and alkaline solution is left for a period of time sufficient to destroy endotoxin. The mixture of chitosan and alkaline solution is allowed to stand for a period of less than 1 hour. It has been found that leaving the mixture of chitosan and alkaline solution for a short period of less than 1 hour prior to subsequent treatment will result in the desired low endotoxin concentration in the resulting alkaline chitosan.

When the mixture was allowed to stand for less than 1 hour, a suitably low concentration of endotoxin was observed. From the viewpoint of handling, the shorter the time the mixture of chitosan and alkaline solution is left to stand, the better the effect. Advantageously, in the method of the invention, the mixture can be left without continuous mixing of the chitosan and the alkaline solution.

In some embodiments, the mixture can be allowed to stand for a period of time of less than 60 minutes, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes.

Preferably, the mixture is allowed to stand for a period of less than 3 minutes, more preferably less than 2 minutes, most preferably less than 1 minute.

Preferably, the mixture is left in step (b) only for the period of time consumed to prepare the mixture for the subsequent processing stage (e.g. drying step (c)). It was observed that if the mixture was dried within 1 hour, the endotoxin concentration decreased with time (about 1 to 3 weeks).

Good results were observed when the mixture was dried immediately after contacting the chitosan with the alkaline solution in step (a). In this context, it immediately means that the mixture is only left in step (b) for the period of time during which the mixture prepared for drying step (c) is consumed. Typically, this period is less than about 30 seconds, preferably less than 20 seconds, and most preferably less than 10 seconds.

Thus, according to one aspect of the present invention, there is provided a process for producing low endotoxin alkali chitosan, chitin or a derivative thereof, said process comprising the steps of:

(a) contacting chitosan, chitin, a chitosan derivative or a chitin derivative with an alkaline solution to form a mixture; and

(b) the mixture was immediately dried.

In this process, the mixture is left in step (b) for the period of time it takes to prepare it for the next treatment stage. For example, the mixture may be left in step (b) for the period of time it takes to prepare it for drying. The mixture may then be dried in a drying step (c).

The mixture may be left at room temperature and pressure until the end of step (b). For room temperature and pressure, a temperature of about 20 ℃ to 25 ℃ and a pressure of about 1 atmosphere (atm) are indicated. Advantageously, the mixture does not need to be placed in a sterile environment.

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

The mixture may further comprise a preservative. Advantageously, the preservative may eliminate the risk that microbial growth may occur, for example, when the mixture is left 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 drying step may be carried out by any conventional drying means known in the art. Preferably, the drying step is performed in an oven or by filtration through an air dryer. Again, no specific sterile environment is required for this drying step.

It has been found that once the mixture is dried in the drying step, the endotoxin level of the dried mixture does not increase significantly over time. Indeed, as mentioned above, it has been observed that endotoxin levels can decrease over time. This has the advantage of allowing the mixture to be stored for a certain period of time before further processing.

Thus, a low endotoxin alkali chitosan having an endotoxin concentration of less than 50EU/g is provided. The low endotoxin alkali chitosan may be water insoluble. At low molecular weights, 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, chitin or a derivative thereof obtainable by the process described herein.

According to another aspect of the present invention there is provided an alkaline chitosan, chitin or derivatives thereof comprising an endotoxin concentration of less than 50 EU/g.

Preferably, the alkaline chitosan, alkaline chitin or derivatives thereof have an endotoxin concentration of less than 30EU/g, preferably less than 20EU/g, more preferably less than 15EU/g, even more preferably less than 10EU/g and most preferably less than 5 EU/g.

The low endotoxin alkali chitosan, low endotoxin alkali chitin or derivatives thereof may comprise an alkali solution having a concentration of about 1M or less. Preferably, the concentration is about 0.5M or less, more preferably about 0.25M or less, or even more preferably about 0.2M or less, and most preferably about 0.1M or less.

Low endotoxin alkali chitosan may be used as an intermediate in the preparation of other chitosan products such as derivatives or copolymers, or as an intermediate in the preparation of low molecular weight chitosan or chitosan oligosaccharides. 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 fibres, fabrics, coatings, films, gels, solutions, sheets or foams of chitosan species.

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, for example, carboxymethyl chitosan, hydroxyethyl chitosan, acyl chitosan, alkyl chitosan, sulfonyl chitosan, phosphorylated chitosan, alkylene chitosan, metal chelates, chitosan chloride, chitosan lactate, chitosan acetate, chitosan malate, chitosan gluconate.

Thus, according to a further aspect of the present invention there is provided a process for producing a low endotoxin neutral chitosan, chitosan salt or chitosan derivative, comprising the step of contacting an alkaline chitosan prepared by the above process with an acid.

The method can provide medically useful neutral chitosan, neutral chitosan salts or other chitosan derivatives that are neutral with low concentrations of endotoxin.

The step of contacting the alkali chitosan with an acid may be performed before the drying step (c) in the process for producing alkali chitosan with low endotoxin described above.

Alternatively, the step of contacting the alkali chitosan with an acid may be performed after the drying step (c) in the method for producing alkali chitosan with low endotoxin described above. In such embodiments, the process for producing a low endotoxin neutral chitosan, chitosan salt or chitosan derivative may comprise a further drying step after the step of contacting the alkali chitosan with an acid. This drying step may be carried out by any conventional drying means known in the art. Preferably, the drying step is performed in an oven or by product filtration through an air dryer.

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

Preferably, the alkaline chitosan is mixed with an acid.

Herein, neutral chitosan refers to a chitosan composition having a pH value between about pH 6.5 to about pH7.5, and preferably about pH7.

Thus, to prepare a neutral chitosan, an alkaline chitosan may be mixed with an appropriate volume and/or concentration of acid to form 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 excess of acid in a volume and/or concentration necessary to provide a neutral chitosan.

Suitable acids for use in the present invention may be selected from one or a combination of the following: 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 one or a combination of the following: acetic acid, tartaric acid, citric acid, ascorbic acid, acetylsalicylic acid, gluconic acid and lactic acid.

Suitable fatty acids may be selected from one or a combination of the following: myristoleic acid, palmitoleic acid, 6-hexadecenoic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, trans-linoleic 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 acid, lignoceric acid, cerotic acid.

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

Suitable inorganic acids may be selected from one or a combination of the following: hydrochloric acid, sulfuric acid and nitric acid. Preferably, the acid selected for neutralization is hydrochloric acid.

The acid may have a concentration of about 0.001M acid up to the maximum possible concentration of acid. For example, for sulfuric acid, a maximum concentration of about 98% sulfuric acid is typical. 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 acid may be present in a concentration of up to 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 an acid and a non-solvent. The non-solvent may be any solvent in which chitosan is insoluble. Common non-solvents include ethyl lactate, ethyl acetate, methyl acetate, ethanol, acetone, or mixtures thereof. Preferably, the non-solvent comprises ethyl acetate or ethanol. More preferably, the non-solvent comprises 80:20 ethanol to water. Advantageously, it has been observed that the reaction proceeds at a faster rate with a non-solvent containing ethanol at a ratio of 80:20 to water.

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

In some embodiments, the low endotoxin alkali chitosan may be mixed with the acid for up to about 30 minutes or less, more preferably about 10 minutes or less, and most preferably about 5 minutes or less. The reaction may then be continued to dry the mixture.

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

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

It has been found that the presence of acid salts in the product can affect the usefulness of the resulting chitosan product. For example, it has been observed that chitosan forms gels to a lesser extent in saline solutions, and to an even lesser extent in double concentrated saline solutions, as compared to chitosan in water. Reference herein to double concentrated brine is to an amount having 1.8% sodium chloride. Thus, the amount of acid salt in the resulting chitosan product is desirably made as low as possible, and desirably, the level of such acid salt should have little or no effect on the effectiveness of the chitosan product.

Surprisingly, it has been found that the use of an alkaline solution having a low concentration, such as less than 0.25M, preferably from 0.01M to 0.2M, and more preferably from about 0.01M to about 0.1M, results in the desired low endotoxin concentration while producing fewer by-products, acid salts, in the subsequent process for producing neutral chitosan, chitosan salts or chitosan derivatives. Advantageously, it has been found that less acid salt by-product results in a chitosan product having improved gelling properties in use relative to products containing higher amounts of acid salt. The method of the present invention is capable of providing a chitosan product with a suitably low amount of acid salt without the need to wash or rinse the chitosan product. This also allows to increase the advantage that no endotoxin-free water need be used in the washing or rinsing step.

It has also been found that, as described above, when producing neutral chitosan, chitosan salts or chitosan derivatives, using a low concentration of alkaline solution results in less reduction in the viscosity of chitosan.

By low concentration alkaline solution is meant from about 0.01M to about 1M, preferably less than 1M, more preferably from about 0.02M to about 0.25M. In some embodiments, the alkaline solution concentration may be 0.02M to 0.1M, preferably 0.05M to 0.1M. Good results were observed with an alkaline solution concentration of about 0.1M. In some embodiments, the alkaline solution concentration may be as described above. Thus, advantageously, the use of low concentration alkaline solutions in the present process results in less damage to the chitosan. Thus, endotoxin can be removed from chitosan while only causing a slight change in viscosity. It is desirable for the viscosity of chitosan to be reduced by about 25%, preferably by about 15%, and more preferably by about 10% in the above process.

The process provides low endotoxin neutral chitosan, which is provided as an intermediate in the production of other chitosan-based products. One particular application is in the production of chitosan salts whose absorptive properties make them desirable in hemostatic formulations for 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 produced by the method described herein above with an acid.

The acid may be any acid suitable for providing the desired chitosan salt. For example, if chitosan acetate is desired, acetic acid may be used; if chitosan succinate is desired, succinic acid or the like may be used. Any acid required herein may be used in the present method to produce a low endotoxin chitosan salt.

The process for producing a low endotoxin chitosan salt or chitosan derivative may further comprise the step of drying the mixture of low endotoxin neutral chitosan and an acid. This drying step may be carried out by any conventional drying means known in the art. Preferably, the drying step is performed in an oven or by product filtration through an air dryer.

Thus, a low endotoxin neutral chitosan, chitosan salt or chitosan derivative having an endotoxin concentration of less than 50EU/g is provided.

The low endotoxin neutral chitosan may be water insoluble.

The low endotoxin chitosan salt may be water soluble.

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

According to a further aspect of the present invention there is provided a neutral chitosan, chitosan salt or chitosan derivative having an endotoxin concentration of less than 50 EU/g.

The neutral chitosan, chitosan salt or chitosan derivative may have an endotoxin concentration of less than 30EU/g, preferably less than 20EU/g, more preferably less than 15EU/g, even more preferably less than 10EU/g and most preferably less than 5 EU/g.

The low endotoxin chitosan salt of the present invention is suitable for use as a hemostatic agent for arresting blood flow.

Thus, according to a further aspect of the present invention there is provided a low endotoxin chitosan as described above for use as a haemostat for use in the retardation of blood flow. Low endotoxin chitosan salts can be used as hemostatic agents for internal or external bleeding. For chitosan salts used in surgery for internal bleeding, endotoxin concentrations of less than 5EU/g are required.

The low endotoxin chitosan salts of the present invention may be incorporated into wound dressings for superficial non-life threatening bleeding or life threatening bleeding.

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

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

According to a further 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 is in any suitable form, such as granular, powdered, granular, flaky, fibrous, gel, foam, sheet, film, or liquid form.

According to yet another aspect of the present invention, there is provided a method of blocking blood flow, comprising the steps of: optionally, where possible, cleaning the wound area; applying a haemostatic wound dressing comprising a low endotoxin chitosan salt to the wound area; and applying a constant pressure to the wound area until a gel mass is formed.

Preferably, atmospheric pressure is applied to the wound area for a period of 3 minutes or more.

Advantageously, the lower the concentration of the alkaline solution used in preparing the hemostatic material of the present invention, the better the permeability of the material, and the better the blood clotting and hemostatic effects.

Drawings

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

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

Detailed Description

Endotoxin test

1. Preparing USP extract (4.6mL of 1M HCl and 45.4mL of endotoxin free water) according to the USP (United states Pharmacopeia) specifications for the chitosan endotoxin test;

2. 0.1g of the test chitosan product was added to 9.9mL of USP extract and left at 37 ℃ for 48 hours;

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

4. mu.l of the above solution was mixed in 100. mu.l of endotoxin-specific (ES) buffer supplied by Charles River.

By usingThe resulting extracts were tested using a hand-held spectrophotometer and an FDA approved disposable kit was utilized. The extraction procedure utilized 2000-fold dilutions and the minimum test detection limit was 10 EU/g.

Examples

Example 1:

50g of chitosan was mixed with 50g of 1M NaOH for 10 minutes. The resulting wet, alkaline chitosan crumb was then immediately dried on a 40 ℃ fluid bed dryer.

Initial endotoxin of raw chitosan: 64.8EU/g

Dried treated alkaline chitosan: <5EU/g

Example 2:

50g of chitosan was mixed with 50g of 0.1M NaOH for 10 minutes. The resulting wet, alkaline chitosan crumb was then immediately dried on a 40 ℃ fluid bed dryer.

Initial endotoxin of raw chitosan: 64.8EU/g

Dried treated alkaline chitosan: 16.3EU/g

Example 3:

50g of chitosan was mixed with 50g of 0.05M NaOH for 10 minutes. The resulting wet, alkaline chitosan crumb was then immediately dried on a 40 ℃ fluid bed dryer.

Initial endotoxin of raw chitosan: 64.8EU/g

Dried treated alkaline chitosan: 20.0EU/g

Example 4:

50g of chitosan was mixed with 50g of 0.01M NaOH for 10 minutes. The resulting wet, alkaline chitosan crumb was then immediately dried on a 40 ℃ fluid bed dryer.

Initial endotoxin of raw chitosan: 64.8EU/g

Dried treated alkaline chitosan: <30EU/g

The method can be scaled up and performed in larger batches.

The method is applicable to chitosan in different physical forms, such as chitosan fibers or chitosan fabrics.

The process may also utilize a different base, such as potassium hydroxide, instead of sodium hydroxide.

Examples 1 to 4 relate to the production of low endotoxin alkali chitosan. The low endotoxin alkali chitosan can then be used as a feedstock for the production of other chitosan-based products. For example, basic chitosan can be neutralized to pH7 to form neutral chitosan by adding low levels of a suitable acid that will react with a base to form a biocompatible salt. For example, if sodium hydroxide is used in the alkaline solution, it can be neutralized by adding hydrochloric acid. The product will contain a small amount of residual sodium chloride.

The low endotoxin alkali chitosan formed in examples 1 to 4 can also be used to produce low endotoxin water soluble chitosan salts or other chitosan derivatives. Advantageously, this can be achieved without the need for a sterile environment, without the need for large amounts of expensive endotoxin-free water, and without the need for rinsing or cleaning. For example, low endotoxin alkali chitosan can be reacted with higher levels of a suitable acid. A small portion of the acid will react with the base to produce a biocompatible salt.

In another embodiment, low endotoxin alkali chitosan may also be used as a derivative for producing low endotoxin chitosan, such as carboxymethyl chitosan.

Effect of acid salts on viscosity

Low endotoxin alkali chitosan is reacted with an acid to produce a chitosan or chitosan salt of neutral pH, producing an acid salt by-product. The presence of this by-product 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, the results of adding sodium lactate to various concentrations of brine are shown and result in various concentrations of brine after 3 minutes versus the current commercial chitosan product2g of sample in a solution of 20g of different media.

The base medium may be saline from body fluids to which different levels of sodium lactate are 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, as the level of added salt increases, the medium becomes more neutralThe viscosity of (3) is decreased. Thus, it is beneficial that only low levels of residual salt byproducts are present in the chitosan product of the present invention.

Effect of Low concentration alkaline solution on viscosity

The low endotoxin alkali chitosan of the present invention may be tested to confirm the effect of treatment with acid on the viscosity of the chitosan polymer, which is considered a measure of molecular weight. The test comprises the following method steps:

a) weighing 5g of low endotoxin alkali chitosan particles;

b) 4.95g of acetic acid were weighed into a 600mL beaker;

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

d) place the beaker on a stirring table and start stirring (increasing with increasing viscosity of the solution);

e) adding chitosan particles into an acetic acid solution;

f) checking the solution regularly until all particles are dissolved and if necessary increasing the level of agitation as the viscosity of the solution increases;

g) the solution was left to stand for a total of 24 hours from the introduction of the chitosan particles into the acetic acid solution;

h) attach spindle number 64 to brookfield viscometer;

i) the rotor was set at 10 rpm;

j) insert the rotor in the solution up to the mark on the rotor and turn on the viscometer and stabilize it;

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

Reducing the effect of the concentration of alkaline solutions

The effect of using a lower concentration of alkaline solution in the method of the invention can be tested in three experiments, focusing on: (1) percent penetration of saline into the test sample; (2) the period of blood coagulation; and (3) percent hemostasis in the upper abdominal segment in the in vivo model.

The general test method for (1) percent penetration into saline is as follows: 5mL of distilled water was added to the test tube. A drop of red food dye was added to the water. A 3g sample of hemostatic powder was tapped on top of the water, forming a layer. After 1 minute, the distance traveled to the hemostatic powder by the water was measured and recorded as percent penetration.

The general test method for (2) the period of blood clotting is as follows: a 0.75g sample of hemostatic powder was added to the test tube, to which was added 5mL of heparinized rabbit blood. Subsequently, the test tube was inverted and the time spent with blood fully coagulated into a coagulum was recorded.

The general test method for (3) percent hemostasis in the upper abdominal section in the in vivo model is as follows: a 3cm to 5cm fracture was made in the upper abdominal artery of a pig model (not heparinized). Hemostatic material in granular form was applied and compression was applied for 1 minute. If re-bleeding occurs, an additional 1 minute of compression is performed.

It will of course be appreciated that the invention is not limited to the foregoing embodiments which have been described above by way of example only.