Method for producing low endotoxin chitosan

Abstract

This invention relates to a method for producing low-endotoxin basic chitosan, low-endotoxin basic chitin, low-endotoxin basic chitosan derivatives, or low-endotoxin basic chitin derivatives, and also to a method for producing low-endotoxin neutral chitosan, low-endotoxin neutral chitosan salts, and low-endotoxin neutral chitosan derivatives, as well as products of the above methods. The method includes: contacting chitosan, chitin, chitosan derivatives, or chitin derivatives with an alkaline solution with a concentration less than 0.25 M to form a mixture; allowing the mixture to stand for a period of less than 12 hours; and optionally, drying the mixture. The low-endotoxin basic chitosan can be used to manufacture other useful chitosan products.

Description

[0001] This application is a divisional application of application number 201480036410.X, filed on May 29, 2014, entitled "Method for producing chitosan with low endotoxin". Technical Field

[0002] This invention relates to a method for producing low-endotoxin basic chitosan, and also to a method for producing low-endotoxin neutral chitosan, low-endotoxin neutral chitosan salts and low-endotoxin neutral chitosan derivatives, as well as products related to the above methods. Background Technology

[0003] Chitosan is particularly useful in the preparation of hemostatic materials for controlling bleeding.

[0004] Chitosan is a derivative of solid waste processed from aquatic shellfish and can also be extracted from fungal cultures. Chitosan is a water-insoluble cationic polymer. Before using chitosan in hemostatic materials, it is typically first converted into a water-soluble salt. This allows the chitosan salt to dissolve in the blood, forming a gel that blocks blood flow.

[0005] Because chitosan is readily broken down in the body, chitosan salts are well-suited for the applications described herein. Chitosan is converted to glucosamine by lysozyme and thus naturally excreted from the body. Therefore, there is no need to remove chitosan from the body. Furthermore, chitosan salts exhibit moderate antibacterial properties, thereby reducing the risk of infection when used.

[0006] In order to use chitosan in the preparation of hemostatic materials suitable for controlling bleeding, it is necessary to ensure that chitosan has a sufficiently low concentration of endotoxin.

[0007] Endotoxins are lipopolysaccharides found 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. Endotoxins can act as pyrogens when released into the bloodstream or other tissues where they are not normally detected. Therefore, endotoxins must be removed from pharmaceutically acceptable products.

[0008] The process of treating pyrogens to remove or destroy them (especially endotoxins) is known as "pyrogen removal." Pyrogen removal techniques for materials containing endotoxins include ion exchange chromatography, ultrafiltration, distillation, and various chemical methods designed to destroy endotoxins.

[0009] WO2008063503 relates to a method for removing endotoxins from chitosan, the method comprising the following steps:

[0010] a) Using sterile, pyrogen-free equipment and materials in a sterile environment;

[0011] b) Allows endotoxin-containing chitosan to swell for up to 24 hours;

[0012] c) Dissolve 1 kg / 25 L to 1.5 kg / 25 L of chitosan in a base of 0.01 M to 4.0 M hydroxide;

[0013] d) The chitosan alkaline solution obtained by continuous stirring;

[0014] e) Heat and stir the above solution at 60°C to 100°C for 45 minutes to 4 hours.

[0015] f) Rinse the above solution with up to 10 times its volume of endotoxin-free ultrapure water;

[0016] g) Neutralize the above solution to a pH between 6.8 and 7.5;

[0017] h) Form an ultrapure chitosan slurry with low endotoxin and transfer it to an endotoxin-free closed system;

[0018] i) Remove excess water from the above slurry.

[0019] This is a complex and expensive method, especially requiring sterile equipment and rinsing with 10 times the volume of an endotoxin-free aqueous solution.

[0020] US2006293509 relates to a method for preparing water-soluble chitosan with low endotoxin content, comprising:

[0021] (a) The first time period in which insoluble chitosan is in contact with an alkaline solution for more than 1 hour;

[0022] (b) Advantageously, the non-water-soluble chitosan is rinsed with endotoxin-free water to remove residual alkaline solution;

[0023] (c) In a reaction solution containing a phase transfer agent, the above-mentioned non-water-soluble chitosan is partially acetylated;

[0024] (d) Dissolve partially acetylated water-soluble chitosan in an aqueous solution containing a surfactant and having a pH of about 7.0 to about 7.4;

[0025] (e) Adding a water-miscible solvent to the aqueous solution, and further adjusting the pH of the aqueous solution to at least 8.0 to cause water-soluble chitosan with low endotoxin content to precipitate from the aqueous solution / water-miscible solvent mixture; and

[0026] (f) Optionally, cleaning may be performed with a non-solvent, such as isopropanol.

[0027] However, this method is complex and expensive, and is expected to involve the use of large quantities of endotoxin-free water or other liquids. It also requires the use of a phase transfer agent and takes several hours.

[0028] TW593342 relates to a method for reducing endotoxins in chitosan, the method comprising:

[0029] (a) Dissolve the endotoxin-containing chitosan in an aqueous solution;

[0030] (b) Contact the above aqueous solution with a surfactant to form an insoluble solid and an aqueous solution with reduced endotoxin content;

[0031] (c) Using solid / liquid separation methods to separate solids and aqueous solutions.

[0032] However, this method requires a surfactant to react with the dissolved chitosan to produce an insoluble solid. The resulting solid will be a mixture of chitosan and the surfactant, or the reaction product between chitosan and the surfactant.

[0033] The present invention aims to solve the above-mentioned problems. Summary of the Invention

[0034] According to a first aspect of the present invention, a method for producing chitosan, chitin, or derivatives thereof with low endotoxin basicity is provided, the method comprising the following steps:

[0035] (a) Contacting chitosan, chitin, chitosan derivatives, or chitin derivatives with an alkaline solution of less than 0.25 M to form a mixture; and

[0036] (b) Allowing the mixture to stand for a period of less than 12 hours.

[0037] The method of the present invention may further include a step (c) of drying the mixture.

[0038] The method of the present invention provides an efficient way to obtain chitosan, chitin, chitosan derivatives, or chitin derivatives with low endotoxin concentrations and alkalinity. 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. Furthermore, it does not require specialized air filtration or sterile conditions. Preferably, the method of the present invention does not include the step of acetylation of chitosan. Furthermore, the method of the present invention does not require the use of endotoxin-free equipment. This reduces the cost of the method compared to methods that require such equipment, and is therefore particularly advantageous.

[0039] The term "chitosan derivative" herein refers to partially deacetylated chitin, which may have different deacetylation percentages as desired. Typically, the partially deacetylated chitin suitable for use in this invention has a degree of deacetylation greater than about 50%, more typically greater than about 75%, and most typically greater than about 85%.

[0040] Furthermore, the term "derivative" in this document encompasses reaction products 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, phosphorylated chitosan, nitrated chitosan, basic chitin, basic chitosan, or metal chelates of chitosan.

[0041] Although the first aspect of the invention provides a method for producing chitosan with low endotoxin, chitin with low endotoxin, or derivatives thereof, for convenience and illustrative purposes, only chitosan is described below.

[0042] Chitosan can be commercially available chitosan, such as food-grade, medical-grade, or pharmaceutical-grade chitosan. Therefore, the method of the present invention can provide low-endotoxin alkaline chitosan from commercially available chitosan. This differs from some methods that remove or reduce endotoxins as part of the chitosan production process. Advantageously, the method of the present invention can be used to provide low-endotoxin alkaline chitosan from prepared chitosan that is unsuitable for medical applications due to its endotoxin concentration.

[0043] As used in this article, alkaline chitosan refers to chitosan compositions having a pH value greater than 7.5.

[0044] The term alkaline solution as used in this article refers to a solution with a pH value greater than 7.5.

[0045] Because the molecular weight of endotoxins can vary significantly, endotoxin concentration is measured as endotoxin units (EUs) per gram of material. The determination of endotoxin concentration is the quantification of endotoxin levels relative to a specific amount of reference endotoxin.

[0046] For example, in this invention, the concentration of endotoxin is determined as endotoxin units (EUs) per gram of chitosan. The term "low endotoxin" as used herein refers to an endotoxin concentration of less than 50 endotoxin units (EUs) per gram of chitosan.

[0047] Therefore, the method of the present invention is suitable for producing basic chitosan with an endotoxin concentration of less than 50 EU / g.

[0048] Preferably, the obtained alkaline chitosan has an endotoxin concentration of less than 30 EU / g, more preferably less than 20 EU / g, more preferably less than 15 EU / g, even more preferably less than 10 EU / g, and most preferably less than 5 EU / g.

[0049] It has been found that low concentrations of alkaline solutions produce products with the desired properties. The concentration of the alkaline solution used in this method can be 0.2 M or lower. Preferably, the concentration of the alkaline solution is about 0.01 M to 0.2 M. More preferably, the concentration of the alkaline solution is about 0.02 M to 0.1 M. The concentration of the alkaline solution can be about 0.04 M to 0.06 M, typically 0.05 M. The concentration of the alkaline solution can be as high as about 0.01 M, 0.05 M, 0.10 M, 0.15 M, 0.20 M, or 0.25 M. Good results have been observed using an alkaline solution with a concentration of 0.1 M.

[0050] In some embodiments, the amount of alkaline solution relative to chitosan can range from about 1 part chitosan to about 10 parts alkaline solution to about 10 parts chitosan to 1 part alkaline solution. Preferably, the amount of alkaline solution relative to chitosan is about 1 part alkaline solution to about 2 parts chitosan, more preferably about 1 part alkaline solution to 1 part chitosan.

[0051] The alkaline solution may include an alkali metal component or an alkaline earth metal component, wherein the alkali metal component or alkaline earth metal component is selected from one or a combination of metal hydroxides, metal carbonates, metal bisulfites, metal persilicates, conjugate bases and ammonium hydroxide.

[0052] Suitable metals include sodium, potassium, calcium, or magnesium.

[0053] Preferably, the alkaline component is sodium hydroxide, potassium hydroxide, or sodium carbonate. Sodium hydroxide is typically used.

[0054] The alkaline solution can be contacted with chitosan by any suitable means known in the art. For example, the alkaline solution can be sprayed onto the chitosan or the chitosan can be mixed with the alkaline solution. Preferably, there is a uniformly distributed alkaline chitosan after contact.

[0055] Preferably, chitosan is mixed with an alkaline solution. At low molecular weights, chitosan can be completely or partially dissolved in the alkaline solution. In step (a), chitosan can be mixed with the alkaline solution for up to about 30 minutes, more preferably about 10 minutes. In some embodiments, chitosan can be mixed with the alkaline solution for more than 30 minutes.

[0056] In some embodiments, chitosan is insoluble in alkaline solutions.

[0057] In some embodiments, chitosan does not swell in alkaline solutions.

[0058] In some embodiments, the alkaline solution wets the chitosan without dissolving or swelling it.

[0059] In some embodiments, the mixture of chitosan and alkaline solution may be intermittently stirred during the duration of step (b).

[0060] The time period during which the mixture of chitosan and alkaline solution is placed allows sufficient endotoxin destruction. The time period during which the mixture of chitosan and alkaline solution is placed is less than 12 hours. It has been found that a shorter time of less than 12 hours during which the mixture of chitosan and alkaline solution with a concentration of less than 0.25 M is placed prior to subsequent processing results in the desired low endotoxin concentration in the resulting alkaline chitosan.

[0061] Advantageously, in the method of the present invention, the mixture can be placed without continuously mixing chitosan and alkaline solution.

[0062] In some embodiments, in step (b), the mixture may be allowed to stand for a period of less than 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour. In step (b), the mixture may be allowed to stand for a period of less than 10 hours, preferably less than 8 hours, more preferably less than 6 hours, even more preferably less than 4 hours, and most preferably less than 2 hours.

[0063] In some embodiments, step (b) may involve allowing the mixture to stand for a period of longer than 1 hour and less than 12 hours, longer than 2 hours and less than 12 hours, longer than 3 hours and less than 12 hours, longer than 4 hours and less than 12 hours, longer than 5 hours and less than 12 hours, longer than 6 hours and less than 12 hours, longer than 7 hours and less than 12 hours, longer than 8 hours and less than 12 hours, longer than 9 hours and less than 12 hours, longer than 10 hours and less than 12 hours, or longer than 11 hours and less than 12 hours. In some embodiments, step (b) may involve allowing the mixture to stand for a period of 1 hour to 11 hours, 1 hour to 10 hours, 1 hour to 9 hours, 1 hour to 8 hours, 1 hour to 7 hours, 1 hour to 6 hours, 1 hour to 5 hours, 1 hour to 4 hours, 1 hour to 3 hours, or 1 hour to 2 hours. Therefore, in some embodiments, the mixture can be left to stand for a period of 2 to 10 hours, 4 to 8 hours, or 5 to 7 hours.

[0064] In some embodiments, in step (b), the mixture may be left to stand for less than 1 hour, including time periods 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. The mixture may also be left to stand for less than 3 minutes, less than 2 minutes, or less than 1 minute.

[0065] In some embodiments, the mixture may be placed only in step (b) for the time period consumed in preparing the mixture for subsequent processing stages (e.g., drying step (c)). Good results were observed when the mixture was dried immediately after contacting the chitosan with the alkaline solution in step (a). In this context, "immediately" means the time period consumed in step (b) for preparing the mixture for drying step (c). Typically, this time period is less than about 30 seconds, preferably less than 20 seconds, and most preferably less than 10 seconds.

[0066] Therefore, according to one aspect of the present invention, a method for producing low-endotoxin basic chitosan, low-endotoxin basic chitin, or derivatives thereof is provided, the method comprising the following steps:

[0067] (a) Contacting chitosan, chitin, chitosan derivatives, or chitin derivatives with an alkaline solution of less than 0.25 M to form a mixture; and

[0068] (b) Dry the mixture immediately.

[0069] In this method, step (b) places the mixture only for the time consumed in preparing it for the next processing stage. For example, the mixture may be placed in step (b) for the time consumed in preparing it for drying. The mixture may then be dried in the drying step (c).

[0070] The mixture can be left at room temperature and room pressure until the end of step (b). Room temperature and room pressure refer to a temperature of about 20°C to 25°C and a pressure of about 1 atmosphere (atm). Advantageously, the mixture does not need to be left in a sterile environment.

[0071] Preferably, the mixture is stored in a clean container. The mixture may also be stored under an inert atmosphere.

[0072] The mixture may further include a preservative. Advantageously, the preservative can eliminate, for example, the risk of microbial growth that may occur when the mixture is left for an extended period of time. The preservative can be any preservative that is biocompatible and suitable for alkaline environments. Suitable preservatives include silver ions, zinc ions, chlorohexadine, or combinations thereof.

[0073] The method of the present invention may or may not include a drying step. The drying step can be performed by any conventional drying means known in the art. Preferably, the drying step is performed in an oven or by filtering air through an air dryer. Again, a specific sterile environment is not required for this drying step.

[0074] 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. This is advantageous for allowing the mixture to be stored for a certain period of time before further processing.

[0075] Therefore, a low-endotoxin basic chitosan is provided, having an endotoxin concentration of less than 50 EU / g. This low-endotoxin basic chitosan may be water-insoluble. At low molecular weights, low-endotoxin basic chitosans may exhibit some water solubility.

[0076] According to another aspect of the invention, low-endotoxin basic chitosan, low-endotoxin basic chitin, or derivatives thereof are provided that can be obtained by the methods described herein.

[0077] According to another aspect of the invention, basic chitosan, chitin, or derivatives thereof are provided, comprising an endotoxin concentration of less than 50 EU / g.

[0078] Preferably, the alkaline chitosan, alkaline chitin, or their derivatives have an endotoxin concentration of less than 30 EU / g, preferably less than 20 EU / g, more preferably less than 15 EU / g, even more preferably less than 10 EU / g, and most preferably less than 5 EU / g.

[0079] Low-endotoxin basic chitosan, low-endotoxin basic chitin, or their derivatives comprise an alkaline solution with a concentration of less than 0.25 M. Preferably, the concentration is about 0.2 M or less, more preferably about 0.15 M or less, or even more preferably about 0.1 M or less.

[0080] Low-endotoxin basic chitosan can 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 basic chitosan can also be used as a raw material for the preparation of other forms of chitosan or derivatives or copolymers (such as chitosan fibers, fabrics, coatings, films, gels, solutions, sheets or foams).

[0081] Specifically, low-endotoxin basic chitosan can be used to prepare other useful chitosan products with low concentrations of endotoxin, including neutral chitosan and chitosan salts and other chitosan derivatives, such as carboxymethyl chitosan, hydroxyethyl chitosan, acyl chitosan, alkyl chitosan, sulfonyl chitosan, phosphorylated chitosan, alkylene chitosan, metal chelates, chitosan chlorides, chitosan lactates, chitosan acetates, chitosan malates, and chitosan gluconates.

[0082] Therefore, according to another aspect of the present invention, a method for producing low-endotoxin neutral chitosan, low-endotoxin neutral chitosan salt, or low-endotoxin neutral chitosan derivative is provided, the method comprising the step of contacting the alkaline chitosan prepared by the above method with an acid.

[0083] This method can provide medically useful neutral chitosan, neutral chitosan salts, or other neutral chitosan derivatives with low concentrations of endotoxin.

[0084] The step of contacting the basic chitosan with the acid can be performed before the drying step (c) in the method described above for producing basic chitosan with low endotoxin.

[0085] Alternatively, the step of contacting the basic chitosan with acid can be performed after the drying step (c) in the method for producing low-endotoxin basic chitosan described above. In this embodiment, the method for producing low-endotoxin neutral chitosan, low-endotoxin neutral chitosan salts, or low-endotoxin neutral chitosan derivatives may include a further drying step after the step of contacting the basic chitosan with acid. This drying step can be performed by any conventional drying means known in the art. Preferably, the drying step is performed in an oven or by filtering the product through an air dryer.

[0086] The acid can be brought into contact with the basic chitosan by any suitable means known in the art. For example, the acid can be sprayed onto the basic chitosan or the basic chitosan can be mixed with the acid.

[0087] Preferably, the alkaline chitosan is mixed with an acid.

[0088] In this document, neutral chitosan refers to a chitosan composition having a pH value between about 6.5 and about 7.5, and preferably about 7.

[0089] Therefore, to prepare neutral chitosan, alkaline chitosan can be mixed with an acid of appropriate volume and / or concentration to form a neutral solution with a pH between 6.5 and 7.5. The volume and / or concentration of acid required to neutralize this alkaline chitosan will depend on the pH of the alkaline chitosan.

[0090] Alternatively, to prepare chitosan salts or chitosan derivatives, alkaline chitosan can be mixed with an excess of acid in volume and / or concentration required to provide neutral chitosan.

[0091] Suitable acids for use in this invention may be selected from one or a combination of the following: organic acids, carboxylic acids, fatty acids, amino acids, Lewis acids, monoprotic acids, diprotic acids, polyprotic acids, nucleic acids, and inorganic acids.

[0092] 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.

[0093] Suitable fatty acids can be selected from one or a combination of the following: myristicinic acid, palmitoleic acid, 6-hexadecenoic acid, oleic acid, trans oleic acid, isoleic 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, tetracosanoic acid, and ceric acid.

[0094] Suitable amino acids can be selected from one or a combination of the following: histidine, lysine, aspartic acid, glutamic acid, glutamine, glycine, proline, and taurine.

[0095] Suitable inorganic acids may be selected from one or a combination of the following: hydrochloric acid, sulfuric acid, and nitric acid. Preferably, hydrochloric acid is selected as the acid for neutralization.

[0096] The acid may have a concentration from about 0.001M to the maximum possible concentration of the acid. For example, for sulfuric acid, the maximum concentration is typically about 98% sulfuric acid. The acid may have a concentration from 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 as high as 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.

[0097] The acid may be present in the form of an acidic solution containing both 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 includes ethyl acetate or ethanol. More preferably, the non-solvent includes an 80:20 ethanol:water ratio. Advantageously, it has been observed that the reaction proceeds at a faster rate when using a non-solvent containing an 80:20 ethanol to water ratio.

[0098] The ratio of chitosan to acid solution can be from about 5:1 to about 1:5. Preferably, the ratio of chitosan to acid solution is about 2:1.

[0099] In some embodiments, the low-endotoxin basic chitosan can be mixed with 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 can then be continued to dry the mixture.

[0100] The product obtained from a mixture of alkaline chitosan and an acid may contain an acid salt. Preferably, the alkaline solution and the acid are selected to ensure that the formed acid salt is biocompatible. For example, the alkaline 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.

[0101] The aforementioned acid salts are formed as byproducts of the reaction between basic chitosan and acid.

[0102] It has been found that the presence of acid salts in the product can affect the usability of the obtained chitosan product. For example, it has been observed that the degree of gelation of chitosan in saline solution is less than that in water, and even less in double-concentration saline solution. Double-concentration saline solution refers to a solution containing 1.8% sodium chloride. Therefore, ideally, the amount of acid salts in the obtained chitosan product should be as low as possible, and ideally, the level of these acid salts should have little or no effect on the effectiveness of the chitosan product.

[0103] Surprisingly, it has been found that using alkaline solutions with low concentrations, such as less than 0.25 M, and preferably from about 0.01 M to about 0.2 M, produces the desired low endotoxin concentration while generating fewer byproducts, acid salts, in subsequent processes for the production of neutral chitosan, chitosan salts, or chitosan derivatives. Advantageously, it has been found that fewer acid salt byproducts compared to products containing higher amounts of acid salts result in improved gelling properties of the chitosan product in use. The method of the present invention can provide chitosan products with appropriately low amounts of acid salts without the need for washing or rinsing the chitosan product. This also provides the advantage of eliminating the need for endotoxin-free water in washing or rinsing steps.

[0104] It has also been found, as mentioned above, that when producing neutral chitosan, chitosan salts, or chitosan derivatives, using a low-concentration alkaline solution results in less reduction in the viscosity of the chitosan.

[0105] A low concentration of alkaline solution refers to less than 0.25 M, preferably about 0.01 M to about 0.2 M. In some embodiments, the alkaline solution concentration can be 0.02 M to 0.1 M, preferably 0.05 M to 0.1 M. Good results were observed using an alkaline solution concentration of about 0.1 M. In some embodiments, the alkaline solution concentration can be as described above. Therefore, advantageously, the use of a low concentration of alkaline solution in this method results in less damage to chitosan. Thus, endotoxins can be removed from chitosan while causing only a small change in viscosity. Ideally, the viscosity of chitosan is reduced by about 25%, preferably by about 15%, and more preferably by about 10% in the above method.

[0106] This method provides a neutral chitosan with low endotoxin content, which serves as an intermediate in the production of other chitosan products. A specific application is the production of chitosan salts, whose absorbability makes them desirable in hemostatic agents used to control bleeding. Preferably, the chitosan salts are water-soluble.

[0107] Therefore, in another embodiment of the present invention, a low-endotoxin chitosan salt can be prepared by contacting a low-endotoxin neutral chitosan produced by the method described above with an acid.

[0108] The acid described above can be any acid suitable for providing the desired chitosan salt. For example, if chitosan acetate is required, acetic acid can be used; if chitosan succinate is required, succinic acid can be used, etc. Any acid required herein can be used to produce chitosan salts with low endotoxin content in this method.

[0109] The method for producing low-endotoxin chitosan salts or chitosan derivatives may further include a step of drying a mixture of low-endotoxin neutral chitosan and an acid. This drying step can be performed by any conventional drying means known in the art. Preferably, the drying step is performed in an oven or by filtering the product through an air dryer.

[0110] Thus, neutral chitosan, chitosan salts, or chitosan derivatives with low endotoxin concentrations of less than 50 EU / g are provided.

[0111] Neutral chitosan with low endotoxin content may be non-water-soluble.

[0112] Low endotoxin chitosan salts can be water-soluble.

[0113] According to another aspect of the invention, a low-endotoxin neutral chitosan, chitosan salt, or chitosan derivative is provided that can be obtained by any of the methods described herein.

[0114] According to another aspect of the invention, neutral chitosan, chitosan salts or chitosan derivatives with an endotoxin concentration of less than 50 EU / g are provided.

[0115] Neutral chitosan, chitosan salts, or chitosan derivatives may have an endotoxin concentration of less than 30 EU / g, preferably less than 20 EU / g, more preferably less than 15 EU / g, even more preferably less than 10 EU / g, and most preferably less than 5 EU / g.

[0116] The low endotoxin chitosan salt of the present invention is suitable as a hemostatic agent that blocks blood flow.

[0117] Therefore, according to another aspect of the invention, a low-endotoxin chitosan as described above is provided for use as a hemostatic agent for blocking blood flow. The low-endotoxin chitosan salt can be used as a hemostatic agent for internal or external bleeding. For chitosan salts used in surgeries involving internal bleeding, an endotoxin concentration of less than 5 EU / g is required.

[0118] The low-endotoxin chitosan salt of the present invention can be incorporated into wound dressings for superficial non-life-threatening or life-threatening bleeding.

[0119] Therefore, according to another aspect of the present invention, a low-endotoxin chitosan salt as described above is provided for wound dressings for superficial non-life-threatening or life-threatening bleeding.

[0120] The low-endotoxin chitosan salt of the present invention is suitable for preparing hemostatic wound dressings for blocking blood flow. According to another aspect of the invention, a hemostatic wound dressing containing a low-endotoxin chitosan salt as described herein is provided.

[0121] According to another aspect of the invention, a hemostatic material containing a chitosan salt with low endotoxin content as described herein is provided.

[0122] The hemostatic material and / or chitosan salt may be in any suitable form, such as granules, powders, particles, flakes, fibers, gels, foams, sheets, films or liquids.

[0123] According to another aspect of the present invention, a method for blocking blood flow is provided, comprising the steps of: optionally, cleaning a wound area where possible; applying a hemostatic wound dressing comprising a low-endotoxin chitosan salt to the wound area; and applying constant pressure to the wound area until a gel mass is formed.

[0124] It is preferable to apply normal pressure to the wound area for 3 minutes or longer.

[0125] 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 effect. Attached Figure Description

[0126] Embodiments of the present invention will now be further described with reference to the accompanying drawings and through the following non-limiting examples, wherein:

[0127] Figure 1 This is a diagram illustrating the effect of different concentrations of acid salt byproducts on the viscosity of chitosan products in different media. Detailed Implementation

[0128] Endotoxin test

[0129] 1. Prepare the USP extract (4.6 mL of 1M HCl and 45.4 mL of endotoxin-free water) according to the detailed description of the chitosan endotoxin test in the USP (United States Pharmacopeia).

[0130] 2. Add 0.1 g of the test chitosan product to 9.9 mL of USP extraction solution and incubate at 37°C for 48 hours;

[0131] 3. After 48 hours, dilute 100 μl of the extract in 0.9 ml of endotoxin-free water; and

[0132] 4. Mix 100 μl of the above solution with 100 μl of endotoxin-specific (ES) buffer provided by Charles River.

[0133] use -PTS TM The obtained extract was tested using a handheld spectrophotometer and an FDA-approved single-use kit. The extraction process used a 2000-fold dilution, and the limit of detection was 10 EU / g.

[0134] Example

[0135] Example 1:

[0136] Mix 50g of chitosan with 50g of 0.1M NaOH for 10 minutes. Then immediately dry the resulting wet, alkaline chitosan flakes in a fluidized bed dryer at 40°C.

[0137] Initial endotoxin level of unprocessed chitosan: 64.8 EU / g

[0138] Dry, processed, alkaline chitosan: 16.3 EU / g

[0139] Example 2:

[0140] Mix 50g of chitosan with 50g of 0.05M NaOH for 10 minutes. Then immediately dry the resulting wet, alkaline chitosan flakes in a fluidized bed dryer at 40°C.

[0141] Initial endotoxin level of unprocessed chitosan: 64.8 EU / g

[0142] Dry, processed alkaline chitosan: 20.0 EU / g

[0143] Example 3:

[0144] Mix 50g of chitosan with 50g of 0.01M NaOH for 10 minutes. Then immediately dry the resulting wet, alkaline chitosan flakes in a fluidized bed dryer at 40°C.

[0145] Initial endotoxin level of unprocessed chitosan: 64.8 EU / g

[0146] Dry, processed alkaline chitosan: <30 EU / g

[0147] Example 4:

[0148] Mix 50g of chitosan with 50g of 0.1M NaOH for 8 hours. Then immediately dry the resulting wet, alkaline chitosan flakes in a fluidized bed dryer at 40°C.

[0149] Initial endotoxin level of unprocessed chitosan: 64.8 EU / g

[0150] Dry, processed alkaline chitosan: 23.4 EU / g

[0151] This method can be scaled up proportionally and performed in large batches.

[0152] This method is applicable to chitosan in different physical forms, such as chitosan fibers or chitosan fabrics.

[0153] This method can also utilize different bases, such as potassium hydroxide, instead of sodium hydroxide.

[0154] Examples 1 through 4 relate to the production of low-endotoxin basic chitosan. This low-endotoxin basic chitosan can then be used as a raw material for the production of other chitosan products. For example, the basic chitosan can be neutralized to pH 7 to form neutral chitosan by adding a low level of a suitable acid (which will react with the 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.

[0155] The low-endotoxin basic 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 a sterile environment, without the use of large quantities of expensive endotoxin-free water, and without rinsing or washing. For example, the low-endotoxin basic chitosan can be reacted with a suitable acid at a higher level. A small portion of the acid will react with the base to produce a biocompatible salt.

[0156] In another embodiment, low-endotoxin basic chitosan can also be used to produce low-endotoxin chitosan derivatives, such as carbonylmethyl chitosan.

[0157] Effect of acid salts on viscosity

[0158] Reacting low-endotoxin basic chitosan with acid to produce neutral pH chitosan or chitosan salts generates acid salt byproducts. The presence of these byproducts can affect the properties of the chitosan product. For example, the level of these byproducts can affect the viscosity of the chitosan product in brine.

[0159] Reference Figure 1 The study showed the results of adding sodium lactate to saline solutions of different concentrations, and obtained the effects of different saline solutions on currently commercially available chitosan products after 3 minutes. The effect of a 2g sample in 20g solutions of different media.

[0160] The base medium can be saline solution derived from bodily fluids, to which different levels of sodium lactate are added. Sodium lactate represents a byproduct of the reaction between sodium hydroxide and lactic acid.

[0161] The results are shown in Table 1 and Figure 1 middle.

[0162] Table 1

[0163]

[0164] from Figure 1 It is clearly known that as the level of added salt increases, the medium... The viscosity decreases. Therefore, it is advantageous that only low levels of residual salt byproducts are present in the chitosan products of the present invention.

[0165] Effect of low concentration alkaline solution on viscosity

[0166] The low-endotoxin alkaline chitosan of the present invention can be tested to confirm the effect of acid treatment on the viscosity of the chitosan polymer, which is considered a measure of molecular weight. The above test includes the following method steps:

[0167] a) Weigh 5g of low-endotoxin alkaline chitosan granules.

[0168] b) Weigh 4.95 g of acetic acid into a 600 mL beaker;

[0169] c) Add 490.05 g of deionized water to a beaker to obtain 495 g of 1% acetic acid solution;

[0170] d) Place the beaker on the stirring table and start stirring (increase the stirring as the viscosity of the solution increases);

[0171] e) Add the chitosan particles to the acetic acid solution;

[0172] f) Regularly check the above solution until all particles dissolve, and if necessary, increase the stirring level as the solution viscosity increases;

[0173] g) Allow the solution to stand for a total of 24 hours from the time the chitosan particles were introduced into the acetic acid solution;

[0174] h) Attach rotor No. 64 to the Brinell viscometer;

[0175] i) Set the rotor to 10 rpm;

[0176] j) Immerse the rotor in the solution until the mark on the rotor is reached, and turn on the viscometer and allow it to stabilize;

[0177] k) Record viscosity (cPs) at selected time intervals.

[0178] The effect of reducing the concentration of alkaline solution

[0179] The effects of using a lower concentration of alkaline solution in the method of the present invention can be tested in three experiments, focusing on: (1) the percentage of saline solution permeating into the test sample; (2) the time of blood clotting; and (3) the percentage of hemostasis in the upper abdominal segment of the in vivo model.

[0180] The general test method for (1) the percentage of penetration into saline solution is as follows: Add 5 mL of distilled water to the test tube. Add one drop of red food dye to the water. Gently tap 3 g of hemostatic powder sample onto the top surface of the water to form a layer. After 1 minute, measure the distance traveled through the water to the hemostatic powder and record it as the percentage of penetration.

[0181] The general test method for (2) the time of blood clotting is as follows: Add 0.75 g of hemostatic powder sample to a test tube, and add 5 mL of heparinized rabbit blood. Then, invert the test tube and record the time it takes for the blood to completely clot into a clot.

[0182] The general test method for the percentage of hemostasis in the upper abdominal segment of the in vivo model (3) is as follows: A 3-5 cm incision is made in the upper abdominal artery of a porcine model (unheparinized). Hemostatic material in granular form is applied and pressure is applied for 1 minute. If rebleeding occurs, pressure is applied for another minute.

[0183] Of course, it should be understood that the present invention is not limited to the embodiments described above, which are only used as examples.

Claims

1. A method for producing low-endotoxin basic chitosan, low-endotoxin basic chitin, or derivatives thereof, said method comprising the following steps: (a) Contacting chitosan, chitin, chitosan derivatives or chitin derivatives with an alkaline solution of 0.01 M to 0.2 M to form a mixture, wherein the amount of alkaline solution relative to the amount of chitosan, chitin, chitosan derivatives or chitin derivatives is 1 part alkaline solution to 1 part chitosan, chitin, chitin derivatives or chitin derivatives. (b) Allowing the mixture to stand at room temperature for a period of less than 11 hours; and then... (c) Dry the mixture.

2. The method according to claim 1, wherein, The concentration of the alkaline solution is 0.1M.

3. The method according to any one of claims 1 to 2, wherein, The alkaline solution comprises an alkali metal component or an alkaline earth metal component, wherein the alkali metal component or alkaline earth metal component is selected from one or a combination of metal hydroxides, metal carbonates, metal bisulfites and metal persilicates.

4. The method according to any one of claims 1 to 2, wherein, The alkaline solution includes a conjugate base or ammonium hydroxide.

5. The method according to claim 3, wherein, The metal is selected from sodium, potassium, calcium, or magnesium.

6. The method according to claim 3, wherein, The alkali metal component is selected from sodium hydroxide, potassium hydroxide, or sodium carbonate.

7. The method according to any one of claims 1 to 2, wherein, The alkaline solution is sprayed onto the chitosan, chitin, chitosan derivative, or chitin derivative; or the chitosan, chitin, chitosan derivative, or chitin derivative is mixed with the alkaline solution.

8. The method according to any one of claims 1 to 2, wherein, The mixture is left to stand for a period of less than 10 hours.

9. The method according to claim 8, wherein, The mixture is left to stand for a period of less than 8 hours.

10. The method according to any one of claims 1 to 2, wherein, Place the mixture in a clean container and / or under an inert atmosphere.

11. The method according to any one of claims 1 to 2, wherein, The mixture further includes a preservative.

12. The method according to claim 11, wherein, The preservative is selected from silver ions, zinc ions, dichlorophenoxyguanidine, or combinations thereof.

13. A low-endotoxin basic chitosan, a low-endotoxin basic chitin, a low-endotoxin basic chitosan derivative or a low-endotoxin basic chitin derivative that can be obtained by the method of any one of claims 1 to 12.

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

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

16. The method according to claim 14 or 15, wherein, The acid is sprayed onto the alkaline chitosan, or the alkaline chitosan is mixed with the acid.

17. The method according to claim 14 or 15, wherein, The acid is selected from one or a combination of the following: organic acids and inorganic acids.

18. The method according to claim 14 or 15, wherein, The acid is selected from Lewis acids.

19. The method of claim 17, wherein, The organic acid is selected from one or a combination of the following: fatty acids, amino acids, and nucleic acids.

20. The method of claim 17, wherein, The organic acid is a carboxylic acid.

21. The method according to claim 14 or 15, wherein, The acid is selected from one or a combination of the following: monoprotic acid, diprotic acid, and polyprotic acid.

22. The method according to 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.

23. The method according to claim 19, wherein, The fatty acids are selected from one or a combination of the following: myristicinic acid, palmitoleic acid, 6-hexadecenoic acid, oleic acid, trans oleic acid, isoleic acid, linoleic acid, trans linoleic acid, α-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, tetracosanoic acid, and ceric acid.

24. The method according to claim 19, 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.

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

26. The method according to claim 14 or 15, wherein, The acid has a concentration of 1 M.

27. The method according to claim 14 or 15, wherein, The acid exists in the form of an acidic liquid containing the acid and a non-solvent.

28. The method according to claim 27, wherein, The non-solvent is selected from ethyl lactate, ethyl acetate, methyl acetate, ethanol, acetone, a mixture of ethanol and water in a ratio of 80:20, or a mixture thereof.

29. The method according to claim 27, wherein, The ratio of chitosan to acid solution is 5:1 to 1:

5.

30. The method according to claim 14 or 15, wherein, The alkaline chitosan is mixed with the acid for 5 minutes.

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

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