Composition for treating intervertebral disc

Abstract

To provide a composition for replenishing nucleus pulposus.SOLUTION: The present invention provides a composition for replenishing the nucleus pulposus of an intervertebral disc, the composition containing a monovalent metal salt of a low endotoxin alginic acid, being applied to a nucleus pulposus site of a subject, being used to harden a part after application, and having fluidity when applied to the nucleus pulposus site. Accordingly, a composition for replenishing nucleus pulposus capable of promoting regeneration of the nucleus pulposus of an intervertebral disc is provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition for treating intervertebral discs, and in particular to a composition for filling the nucleus pulposus of an intervertebral disc. [Background technology]

[0002] The spine is a rod-shaped skeleton made up of a series of vertebrae that support the trunk and head. The bones are connected by intervertebral discs, which are disc-shaped avascular tissues containing the nucleus pulposus. The annulus fibrosus surrounds the central nerve, and end plates are arranged above and below it. The nucleus pulposus of the intervertebral disc is composed of nucleus pulposus cells and their extracellular matrix, which is a gel-like substance containing a lot of water. It is a highly elastic structure that acts as a cushion to absorb pressure between the vertebrae. The annulus is made up of a layer of fibrocartilage surrounded by a layer of collagen, and is located between the vertebral bodies. It limits rotational movement. The end plates are hyaline cartilage tissue that firmly connects the disc to the vertebral body.

[0003] The nucleus pulposus, located in the center of the intervertebral disc, is unique compared to the annulus fibrosus, endplates, and other cartilage tissues. The main component of the extracellular matrix of the nucleus pulposus is water (70-90%). ; decreases with age), type II collagen (20% of dry weight), proteoglycans ( 50% of the dry weight of collagen compared to other cartilage tissues such as endplates and articular cartilage. It is characterized by a high ratio of proteoglycans to carbohydrates (Non-Patent Document 1). The extracellular matrix of other cartilage tissues, such as articular cartilage, is composed of collagen compared to proteoglycans. The disc's shock absorption function is due to its high water content. This abundant water is mainly bound to the core protein of proteoglycan. It is maintained by the negatively charged glycosaminoglycans that attract water. Furthermore, the structure and size of proteoglycans present in the intervertebral disc are similar to those present in articular cartilage. The difference was particularly marked in the proteoglycans of the nucleus pulposus, unlike the proteoglycans in the has been disclosed (Non-Patent Document 2).

[0004] The nucleus pulposus, annulus fibrosus, and endplates of the intervertebral disc each have different structures and functions. The nucleus pulposus is maintained by a group of cells with different phenotypes. The cells in the annulus fibrosus produce a matrix rich in collagen. The cells in the disc are wrapped in a fiber matrix. It has been reported recently that the phenotype of chondrocytes differs from that of articular chondrocytes. (Non-Patent Document 1).

[0005] Intervertebral discs can become degenerated or damaged due to aging, trauma, disease, etc. cell number, water content, extracellular matrix (type II collagen, aggrecan, etc.) This is a condition in which the functions of the intervertebral disc as a shock absorber are impaired. Disc degeneration and disc damage are specifically caused by disc herniation, disc disease, and spinal Disc damage due to degenerative spondylolisthesis, pyogenic discitis, degenerative spondylosis, spinal stenosis, trauma, etc. For example, in a herniated disc, the annulus fibrosus that covers the nucleus pulposus is deformed or cracked. This causes a herniation and protrusion of the nucleus pulposus from the disc, which then compresses the spinal nerves. This causes pain and paralysis.

[0006] One of the treatments for herniated discs is discectomy (removal), which has a certain degree of effectiveness. However, in the case of discectomy (resection), It is known that degenerative changes in the intervertebral disc may progress if no treatment is given to the surgical site. When a part of the nucleus pulposus is removed by disc nucleotomy, a cavity (referred to herein as a cavity) is formed in the nucleus pulposus site. The nucleus pulposus has almost no self-repair or regeneration ability, so The cavity in the nucleus is easily weakened physically. In addition, fibroblast-like cells accumulate in the cavity, forming the nucleus. The formation of tissue with mechanical properties different from that of the original nucleus pulposus may occur. After discectomy, the recurrence rate of herniation is high. The recurrence rate within 5 years after disc nucleotomy is 4-15%. However, recent long-term data shows that recurrence occurs in the majority of cases after 10 years. If the hernia recurs, another surgery is required, but the spinal nerves are still intact after the first surgery. The spinal nerves are buried in scar tissue, making it difficult to identify their location. Even if the location of the spinal nerve can be confirmed, the scar will be thick and hard, making it difficult to identify the spinal nerve and surrounding tissue. It is extremely difficult to separate the tissue from the graft. Reoperation requires extremely difficult techniques. Therefore, it is necessary to establish a surgical method that does not cause recurrence of herniation or scarring after disc nucleotomy. Standing is required.

[0007] In an attempt to treat disc disease, for example, without removing the nucleus pulposus or annulus fibrosus, A treatment method that introduces polyelectrolyte materials into the disc space A method for the synthesis of polyelectrolytes has been proposed, and alginate is one of many specific examples of such polyelectrolytes. In addition, it is known that glycosaminoglycans and other cartilage-protecting materials are required for the treatment of cartilage damage (Patent Document 1). A method for enhancing the function of the intervertebral disc has been proposed, which involves injecting the chondroprotective material into the disc. One of the many examples is an amphiphilic derivative of sodium alginate ( Patent Document 2) A device for injecting an antacid into an intervertebral disc has also been disclosed (Patent Document 3). In addition to injecting antacids, it is also possible to optionally inject a disc filler. One of the many examples of fillers is calcium or barium cross-linked alginate. However, in these documents, the following are listed as specific examples of the various compounds: These are merely listed as examples, and no specific methods or examples of their use are given. It has not been done.

[0008] Additionally, hydrogels such as alginate are being investigated as materials for filling the nucleus pulposus. When using hydrogels such as phosphates as a nucleus pulposus prosthesis material, their mechanical strength is an issue. The problem is that the shape of the device is maintained for a certain period of time when used in vivo. It was thought that a higher degree was better (Non-Patent Documents 3 to 9).

[0009] Here, alginate is used for the regeneration of cartilage in joints, thoracic wall, intervertebral discs, menisci, etc. It has been proposed (Patent Documents 4 and 5). [Prior art documents] [Patent documents]

[0010] [Patent Document 1] US Patent Application Publication No. 2007 / 0150060 [Patent Document 2] US Patent Application Publication No. 2003 / 0069639 [Patent Document 3] US Patent Application Publication No. 2009 / 0082719

Patent Document 4

Patent Document 5

Non-Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document ⑦

Non-Patent Document 8

Non-Patent Document 9

Summary of the Invention

Problems to be Solved by the Invention

[0012] In the above situation, the object of the present invention is to provide a method for producing a nucleus pulposus capable of promoting the regeneration of the nucleus pulposus of an intervertebral disc. The present invention aims to provide a filling composition that is relatively easy to fill and that does not compress the spinal nerves. The object of the present invention is to provide a composition for nucleus pulposus prosthetic replacement that is less likely to cause any complications. [Means for solving the problem]

[0013] The present inventors have developed a nucleus pulposus made of a biocompatible material as a treatment method for intervertebral disc degeneration and disc damage. In this field of treatment, aluminum has been used as a nucleus pulposus filling material. When using hydrogels such as phosphates, the mechanical properties of the hydrogels must be considered. Strength is an issue, and the shape must be maintained for a certain period of time when used in vivo. However, the present inventors have found that low-endotoxin sodium alginate is the best choice. A composition containing the compound is injected in a sol state into the nucleus pulposus site, and the composition is applied to the surface of the intervertebral disc to prevent leakage. The filling port of the composition is brought into contact with a crosslinking agent to harden a portion of the composition, thereby preventing degeneration of the nucleus pulposus of the intervertebral disc. and increased the proportion of type II collagen-positive cells, which are favorable for nucleus pulposus regeneration, and It was found that this promotes the regeneration of the nucleus pulposus. It also suppresses the degeneration of the entire intervertebral disc tissue, including the annulus fibrosus. I found out how to control it. Based on these findings, the present inventors have conducted further research and have completed the present invention. .

[0014] That is, the present invention is as follows. [1] It is applied to the target nucleus pulposus area and hardens the area after application. A method for treating vertebral vertebrae, comprising the steps of: A composition for filling the nucleus pulposus of an interstitial disc. [1A] Contains low-endotoxin monovalent metal salt of alginic acid, which flows when applied to the nucleus pulposus site. The intervertebral disc is used to harden a portion of the nucleus pulposus after application to the target nucleus pulposus site. A composition for filling the nucleus pulposus. [2] Curing the composition by contacting a crosslinking agent with at least a portion of the surface of the composition. The composition according to [1] or [1A] above, [3] The composition is applied to the nucleus pulposus site through a composition filling port on the surface of the intervertebral disc; A portion of the composition is hardened by contacting a crosslinking agent with the filling port of the composition on the surface of the disc. The composition according to any one of the above [1] to [2], [4] The application of the composition to the nucleus pulposus site is performed by removing at least a portion of the nucleus pulposus. Any one of the above [1] to [3] is carried out by applying the composition to the nucleus pulposus defect. The composition according to item . [5] The viscosity of the fluid composition is 100 mPa·s to 30,000 mPa·s. The composition according to any one of the above [1] to [4], [5A] The apparent viscosity of the fluid composition is measured using a cone-plate viscometer. When measured at 20°C, the viscosity is 100 mPa·s to 30,000 mPa·s.

[0023] - [4] The composition according to any one of the above items. [6] The monovalent metal salt of the low-endotoxin alginic acid is measured by the GPC-MALS method.

[0023] The weight average molecular weight of the polymeric material according to any one of [1] to [5A] above is 80,000 or more. Composition of. [6A] The monovalent metal salt of the low-endotoxin alginic acid is Any of the above [1] to [5A], whose measured weight average molecular weight (absolute molecular weight) is 80,000 or more. The composition according to any one of claims 1 to 4. [7] The composition has a concentration of a monovalent metal salt of low-endotoxin alginic acid of 0.5 w / v The composition according to any one of the above [1] to [6A], wherein the content is 10 w / v % to 5 w / v %. [7A] The composition has a concentration of a monovalent metal salt of low-endotoxin alginic acid of 0.5 w / The composition according to any one of the above [1] to [6A], wherein the content is from 1 wt % to 5 w / w %. [8] The composition is administered in an amount that hardens the composition before application to the nucleus pulposus site of the subject. The composition according to any one of the above [1] to [7A], which does not contain a crosslinking agent. [9] The composition according to any one of [1] to [8] above, wherein the composition does not contain cells. composition.

[10] Any of the above [2] to [9], wherein the crosslinking agent is a divalent or higher metal ion compound. 2. The composition described in item 1. [10A] Divalent or higher metal ion compounds are Ca 2+ , Mg 2+ , Ba 2+ , Sr 2+ mosquito

[10] above, wherein the compound is at least one metal ion compound selected from the group consisting of: composition.

[11] The composition is for treating, preventing, or preventing recurrence of intervertebral disc degeneration and / or disc damage. The composition according to any one of the above [1] to [10A], which is used for suppression.

[12] The disc degeneration and / or disc damage is caused by herniated discs, disc disease, spinal Consists of degenerative spondylolisthesis, pyogenic discitis, degenerative spondylosis, spinal stenosis, and disc damage. The composition according to

[11] above, which is at least one selected from the group consisting of:

[13] The above [1] to

[12] , wherein the composition is in a dry state before application to the nucleus pulposus site. The composition according to any one of the preceding claims. [13A] The composition is in a dry state or in a solution state before application to the nucleus pulposus site. The composition according to any one of [1] to

[12] .

[14] The dry low-endotoxin monovalent metal salt of alginic acid is a freeze-dried product. The composition according to the above

[13] or [13A]. [14A] A method for using a crosslinking agent in which a portion of the composition is hardened in the same manner as when the composition is filled into the nucleus pulposus site. and the ratio used, in vitro, according to Example 4 of the present specification, a 6 mm diameter test The tube was filled with 500 μL of low-endotoxin sodium alginate and cross-linking agent and allowed to stand for 1 hour. After placement, at least 50% of the volume of the composition in the test tube was removed using a syringe with a 21G needle. The composition according to any one of the above [1] to

[14] , which is capable of being inhaled. [14B] The flowable composition is allowed to stand at 20°C for 1 hour, and then The fluidity of the product is such that it can be injected using a syringe needle of size G. Composition of.

[15] The composition according to any one of [1] to [14B] above, and a crosslinking agent A kit for filling a nucleus pulposus of an intervertebral disc, comprising at least:

[16] Methods for treating, preventing, or inhibiting recurrence of intervertebral disc degeneration and / or disc damage It is a law, A composition containing a monovalent metal salt of low-endotoxin alginic acid and having flowability is prepared by the treatment. Apply it to the nucleus pulposus of the intervertebral disc of a subject who needs treatment, prevention, or suppression of recurrence, the method including curing a portion of the applied composition.

[17] The disc degeneration and / or disc damage is caused by herniated discs, disc disease, spinal Consists of degenerative spondylolisthesis, pyogenic discitis, degenerative spondylosis, spinal stenosis, and disc damage. The method according to

[16] above, wherein the compound is at least one selected from the group consisting of:

[18] Compositions for the treatment, prevention, or suppression of recurrence of intervertebral disc degeneration and / or disc damage 1. Use of a monovalent metal salt of low-endotoxin alginic acid for producing a composition comprising: The composition is applied to the nucleus pulposus site of a subject and is used to harden the site after application. The above use, which is fluid when applied to the nucleus pulposus site.

[19] The disc degeneration and / or disc damage is caused by a herniated disc, disc disease, or spinal Consists of degenerative spondylolisthesis, pyogenic discitis, degenerative spondylosis, spinal stenosis, and disc damage. The use according to

[18] above, wherein the compound is at least one selected from the group consisting of:

[20] A composition containing a monovalent metal salt of low-endotoxin alginic acid and having flowability. , a subject in need of treatment, prevention, or suppression of recurrence of intervertebral disc degeneration and / or intervertebral disc damage and applying the composition to the nucleus pulposus of the intervertebral disc to harden the applied composition. The present invention relates to a low-endotoxin antibody for use in the treatment, prevention or suppression of recurrence of intervertebral disc injury. Monovalent metal salts of glutamic acid. [Effects of the Invention]

[0015] The present invention provides a composition for nucleus pulposus replacement that can promote regeneration of the nucleus pulposus of an intervertebral disc. The composition of the present invention inhibits degeneration of the entire intervertebral disc tissue, including not only the nucleus pulposus but also the annulus fibrosus. Furthermore, the composition of the present invention can also inhibit the formation of type II collagen in the nucleus pulposus. It has the effect of increasing the proportion of IgG4-positive hyaline chondrocytes.

[0016] In one preferred embodiment of the present invention, the composition of the present invention is used to treat degenerative disc conditions such as herniated discs. For the prevention, treatment, or recurrence prevention of diseases related to the spinal cord, or intervertebral disc damage caused by trauma, etc. It can be used as a nucleus pulposus filling material.

[0017] In addition, the composition of a preferred embodiment of the present invention is injected in a sol state into the nucleus pulposus site using a syringe or the like. It is possible to perform not only direct vision but also percutaneous nucleotomy (approximately 5 mm incision). It is also possible to perform filling under a microscope (approximately 3-4 cm incision) and endoscopy (approximately 1-2 cm incision). This reduces the burden on the patient and the procedure is relatively simple.

[0018] Furthermore, with conventional gel-like nucleus pulposus prosthetic materials, if they were to protrude into the spinal canal, Although there is a risk of compressing and damaging the spinal nerves, the composition of a more preferred embodiment of the present invention Since only the gel is formed, there is little risk of such complications and it is safe.

[0019] The composition of a particularly preferred embodiment of the present invention is effective in preventing the recurrence of herniation after disc nucleotomy (resection). In one preferred embodiment of the present invention, the present invention is directed to the treatment of intervertebral disc degeneration and and / or by applying the composition of the present invention to the nucleus pulposus of an injured intervertebral disc. This reduces the burden on the discs adjacent to the treated disc, preventing degeneration of the adjacent discs. This allows for the reduction and / or mitigation of

[0020] The composition of the present invention satisfies one or more of the above effects. [Brief explanation of the drawings]

[0021] [Figure 1] The viability of cells is shown 6 and 48 hours after serum starvation. Group A: low-endotoxin sodium alginate; and Group B: food-grade sodium alginate. [Figure 2] The apoptotic cell rates are shown 6 and 48 hours after serum starvation in Group A: low-endotoxin sodium alginate; and Group B: food-grade sodium alginate. [Figure 3] 1 is a graph showing the results of evaluation of intervertebral disc tissue according to the Pfirrmann classification 4 weeks after surgery in the normal control group, the aspiration-only group, and the treatment groups (Groups A-1 and A-2). [Figure 4] 1 is a graph showing the results of evaluation of intervertebral disc tissue by MRI index 4 weeks after surgery in the aspiration only group and the treatment groups (Groups A-1 and A-2). [Figure 5] 1 is a graph showing the results of histological evaluation of the severity of intervertebral disc degeneration 4 weeks after surgery in the normal control group, the aspiration only group, and the treatment groups (Groups A-1 and A-2). [Figure 6] (A) Photographs of stained intervertebral disc tissue specimens taken 4 weeks after surgery. Normal control group, aspiration only group, and treatment group (Group A-2). (B) Photographs of stained intervertebral disc tissue specimens taken 12 weeks after surgery. Normal control group, aspiration only group, and treatment group (Group A-2). [Figure 7] This is a graph showing the rate of anti-Type II collagen antibody positive cells relative to the number of cells in intervertebral disc tissue sections 4 and 12 weeks after surgery. The normal control group, the aspiration only group, and the treatment group (Group A-2). [Figure 8] Sheep. Graph showing the results of evaluation by the modified Boos classification 4 weeks after surgery. Normal control group, nucleotomy group, and treatment group. *P<0.05, **P<0.01. [Figure 9] Sheep: Graph showing the results of evaluation by disc height index 4 weeks after surgery. Normal control group, nucleotomy group, and treatment group. *P<0.05. [Figure 10] 1 is a graph showing the rate of anti-Type II collagen antibody-positive cells in cells in sections of sheep intervertebral disc tissue 4 weeks after surgery. Normal control group, nucleotomy group, and treatment group. *P<0.05. [Figure 11] The ratio of sulfated glycosaminoglycans (GAGs) to hydroxyproline (HYPs) in the nucleus pulposus of the intervertebral disc is shown for the nucleectomy group, treatment group, normal control group, and articular cartilage at 4 weeks after surgery. DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention will be described in detail below. 1. Composition of the Present Invention The present invention relates to a composition that is preferably used for filling the nucleus pulposus of an intervertebral disc. The composition of the present invention is applied to the nucleus pulposus site of a subject and is used to harden the area after application. The monovalent metal salt of low-endotoxin alginic acid is used, which has fluidity when applied to the nucleus pulposus site. The composition for nucleus pulposus replacement of an intervertebral disc contains (This may be said.)

[0023] The terms "low endotoxin" and "monovalent metal salt of alginic acid" are as described below.

[0024] An "intervertebral disc" is a cylindrical tissue located between the vertebrae of the spine. It is a disc-shaped avascular tissue, with the nucleus pulposus at the center and the annulus fibrosus surrounding it, and then terminating at the top and bottom. It has a structure with boards arranged in it.

[0025] The "nucleus pulposus" is a gel-like tissue that exists in the center of the intervertebral disc and is composed mainly of nucleus pulposus cells and proteolytic enzymes. It contains mainly water and an extracellular matrix composed of glycans and type II collagen. is thought to have almost no self-repair or regenerative capabilities.

[0026] "Nucleus pulposus replacement" refers to the replacement of the nucleus pulposus with the nucleus pulposus due to aging, trauma, infection, and surgical treatment for these (e.g., vertebral Degenerated, reduced, or removed nucleus pulposus due to disc nucleotomy (resection) etc. In this specification, the term "nucleus pulposus filling" refers to the filling of the missing or removed portion of the nucleus. The term "nucleus pulposus filling composition" used in the present invention is used in the same sense as "nucleus pulposus filling composition." It is synonymous with "thing."

[0027] "Nucleus pulposus site" refers to a site where the nucleus pulposus is present, a site where degeneration or shrinkage of the nucleus pulposus has occurred, Alternatively, it refers to a defect in the nucleus pulposus formed by removing at least a part of the nucleus pulposus, and This also includes the surrounding area of ​​the area where the tumor is located.

[0028] A "subject" is a human or non-human organism, such as birds and non-human mammals (e.g., For example, cows, monkeys, cats, mice, rats, guinea pigs, hamsters, pigs, dogs, and rabbits. , sheep, and horses).

[0029] "Application" means applying the composition of the present invention to the degenerated, reduced, removed or missing part of the nucleus pulposus of the intervertebral disc. This means filling the nucleus pulposus area with an amount sufficient to fill the area.

[0030] "Partially hardening" is as described below.

[0031] The term "containing a monovalent metal salt of low-endotoxin alginic acid" means that the composition of the present invention contains a suitable A sufficient amount of low-endotoxin alginate to regenerate the nucleus pulposus at the applied nucleus pulposus site. This means that the compound contains a monovalent metal salt of the formula:

[0032] "Having liquidity" is as described below.

[0033] "Disc degeneration and / or disc damage" and "treatment, prevention, or suppression of recurrence" are defined below. As stated above.

[0034] The composition of the present invention may be provided in a solution state using a solvent, or in a freeze-dried form (particularly It may be provided in a dry form such as a lyophilized powder or a lyophilized powder. The composition of the present invention is used in a fluid state such as a solution state by using a solvent when applied. The solvent is not particularly limited as long as it is a solvent that can be used in living organisms. For example, water for injection, Purified water, distilled water, ion-exchanged water (or deionized water), Milli-Q water, saline, phosphate Buffered saline (PBS) and the like. Preferably, the solution is used for the treatment of humans and animals. Examples of suitable solutions include water for injection, distilled water, and saline.

[0035] 2. Monovalent metal salts of alginic acid "Monovalent metal salts of alginic acid" are compounds in which the hydrogen atom of the carboxylic acid at the 6th position of alginic acid is replaced by Na + YaK + It is a water-soluble salt made by ion exchange with monovalent metal ions such as Specific examples of monovalent metal salts of ginic acid include sodium alginate and potassium alginate. In particular, commercially available sodium alginate is Preferably, a solution of a monovalent metal salt of alginic acid forms a gel when mixed with a cross-linking agent.

[0036] The "alginic acid" used in the present invention is a biodegradable polymeric polysaccharide, and is a compound of D-mannuron It is a polymer formed by the linear polymerization of two types of uronic acids, L-guluronic acid (M) and L-guluronic acid (G). More specifically, the homopolymer fraction of D-mannuronic acid (MM fraction), L-glucuronic acid The homopolymer fraction of mannuronic acid (GG fraction), and the homopolymer fraction of D-mannuronic acid and L-guluronic acid It is a block copolymer in which the dam-arranged fractions (MG fractions) are randomly linked. The composition ratio of D-mannuronic acid and L-guluronic acid (M / G ratio) is mainly derived from seaweed, etc. The M / G varies depending on the type of organism and is influenced by the habitat and season of the organism. The ratio ranges from a high G type with a ratio of about 0.4 to a high M type with an M / G ratio of about 5.

[0037] Monovalent metal salts of alginic acid are high molecular weight polysaccharides, and it is difficult to accurately determine their molecular weight. However, if the molecular weight is too low, the viscosity will be low and the adhesiveness to the surrounding tissue at the application site will be weak. Moreover, if the molecular weight is too high, it is difficult to manufacture and the solubility is reduced. When it is made into a solution, the viscosity is too high and it becomes difficult to handle. It is difficult to maintain the physical properties during long-term storage. Therefore, the weight average molecular weight is generally 10,000 to 10,000,000, preferably It is in the range of 20,000 to 8,000,000, more preferably 50,000 to 5,000,000. " indicates a numerical range in which the numbers before and after "~" are the minimum and maximum values, respectively. Indicates a range inclusive of the maximum value.

[0038] On the other hand, when measuring the molecular weight of polymeric substances derived from natural products, the values ​​can vary depending on the measurement method. For example, gel permeation chromatography (GPC) or gel filtration chromatography Measurement by size exclusion chromatography (collectively known as size exclusion chromatography) According to the effects shown in the examples of the present invention, the weight average molecular weight is preferably 100,000 or more. , more preferably 500,000 or more, and also preferably 5 million or less, more preferably 3 The preferred range is 100,000 to 5,000,000, and more preferably 50,000,000. The price ranges from 10,000 to 3.5 million yen.

[0039] Also, for example, gel permeation chromatography (GPC) and multi-angle light scattering detector (Multi According to the GPC-MALS method combined with MALS, The weight average molecular weight can be measured by the GPC-MALS method. According to the effects shown in the examples of the present invention, the molecular weight (absolute molecular weight) is preferably 10,000 or more. , more preferably 80,000 or more, and even more preferably 90,000 or more, and preferably 100 10,000 or less, more preferably 800,000 or less, even more preferably 700,000 or less, and particularly preferably The preferred range is 10,000 to 1,000,000, and more preferably 80,000 to 1,000,000. 800,000, even more preferably 90,000 to 700,000, and particularly preferably 90,000 to 500,000. be.

[0040] Usually, when the molecular weight of a high molecular weight polysaccharide is calculated using the above method, the Measurement errors may occur. For example, if the number is 400,000, the number should be between 320,000 and 480,000, and if the number is 500,000, the number should be between 400,000 and 600,000. If the amount is 10,000 or 1 million, the value can fluctuate within a range of 800,000 to 1.2 million.

[0041] The molecular weight of the monovalent metal salt of alginic acid can be measured by a conventional method. Typical conditions for using gel permeation chromatography to measure molecular weight are as described in the present specification. As described in the Examples, the column is, for example, GMPW-XL x 2 + G2500PW -XL (7.8 mm ID x 300 mm) can be used, and the eluent is, for example, It can be prepared in 200 mM sodium nitrate solution, and pullulan is used as a molecular weight standard. It is possible.

[0042] Typical conditions for using GPC-MALS to measure molecular weight are described in the examples of this specification. As a detector, for example, an RI detector and a multi-atomic optical scattering detector (MALS) are used. You can be there.

[0043] When monovalent metal salts of alginic acid are first extracted from brown algae, they have a large molecular weight and high viscosity. However, the molecular weight becomes smaller and the viscosity becomes lower during the processes of drying and refining with heat. Controlling the manufacturing process temperature and other conditions, selecting the brown algae used as raw materials, and molecular weight fractionation during the manufacturing process By this method, monovalent metal salts of alginic acid with different molecular weights can be produced. Mixing with a different batch of monovalent metal salt of alginic acid with a different molecular weight or viscosity This makes it possible to obtain a monovalent metal salt of alginic acid having the desired molecular weight.

[0044] The monovalent metal salt of alginic acid used in the present invention is preferably a monovalent metal salt of alginic acid. was dissolved in MilliQ water to make a 1 w / w% solution, and the viscosity was measured using a cone-plate viscometer. When viscosity was measured at 0°C, the apparent viscosity was 40 mPa·s to 800 mPa·s. It is preferable that the viscosity is in the range of 50 mPa·s to 600 mPa·s. It is desirable that the apparent viscosity be measured under the conditions described below. In this case, "apparent viscosity" is sometimes simply called "viscosity."

[0045] The alginic acid used in the present invention may be either naturally occurring or synthetic, but the most preferred is naturally occurring alginic acid. Examples of naturally occurring alginic acid include those extracted from brown algae. Brown algae containing alginic acid grow abundantly in coastal areas around the world, but in reality, The seaweed that can be used as a raw material for alginate is limited. Representative examples include Crocystis, European Laminaria and Ascophyllum, and Australian Durvillea. Brown algae that are the source of alginic acid include, for example, Lessonia ia), Macrocystis, Laminaria ria) genus (kelp genus), Ascophyllum genus, Durvillea ( Genus Durvillea, Genus Eisenia, Genus Ecklonia Examples include:

[0046] 3. Low Endotoxin Treatment The monovalent metal salt of alginic acid used in the present invention is a monovalent metal salt of alginic acid with low endotoxin. Low endotoxin means that the endotoxin level is low enough to not cause substantial inflammation or fever. More preferably, it refers to an endotoxin-treated animal. It is preferably a monovalent metal salt of glutamic acid.

[0047] The endotoxin reduction treatment can be carried out by a known method or a method similar thereto. For example, the method of purifying sodium hyaluronate by Kan et al. (see, for example, Japanese Patent Application Laid-Open No. 9-327626) 4001, etc.), the method of purifying β1,3-glucan by Yoshida et al. (see, for example, Patent Publication No. (See, for example, JP-A-8-269102) and biopolymer salts such as alginate and gellan gum. Purification by the method of Williams et al. (see, for example, JP-A-2002-530440) , the method of James et al. (see, e.g., International Publication No. WO 93 / 131) for purifying polysaccharides; No. 36 pamphlet), the method of Lewis et al. (see, for example, U.S. Pat. No. 5,589,591 (see the specification, etc.), the method of purifying alginate by Herman Frank et al. (see, for example, Ap pl Microbiol Biotechnol (1994)40:638-643 The low-enriched ethylene glycol fluoride of the present invention can be achieved by the methods described above. Endotoxin treatment is not limited to these, but also includes washing, filtering (endotoxin removal filter) filtration using electrostatically charged filters, ultrafiltration, columns (endotoxin adsorption affinity Purification using a column such as a purity column, gel filtration column, or ion exchange resin column Adsorption onto aqueous substances, resins or activated carbon, organic solvent treatment (extraction with organic solvents, organic solvent Precipitation and sedimentation by adding an agent), surfactant treatment (for example, JP 2005-036036 (See, for example, the publication No. 2004-2006) or by a suitable combination of these methods. These treatment steps may be appropriately combined with known methods such as centrifugation. It is desirable to select an appropriate amount depending on the type of alginic acid, etc.

[0048] The endotoxin level can be confirmed by known methods, for example, using a Limulus reagent ( LAL (Lysine Alcohol-Lysine Lysine) method, Entospecy (registered trademark) ES-24S set (Seikagaku Corporation) It can be measured by methods such as using the

[0049] The method for treating endotoxins in the monovalent metal salt of alginic acid contained in the composition of the present invention is particularly As a result, the endotoxin content of monovalent metal salts of alginic acid is, but not limited to, When endotoxin was measured using Limulus Agar (LAL) reagent, 500 endotoxins were detected. Preferably, the hydroxyl group is 100 EU / g or less, more preferably 100 EU / g or less. It is particularly preferably 50 EU / g or less, and particularly preferably 30 EU / g or less. The oxidized sodium alginate is available, for example, from Sea Matrix®. (Mochida Pharmaceutical Co., Ltd.), PRONOVA TM UP LVG (FMC BioPolymer r) and other commercially available products.

[0050] 4. Preparation of Solutions of Monovalent Metal Salts of Alginic Acid The compositions of the present invention may be prepared using a solution of a monovalent metal salt of alginic acid. The solution of the monovalent metal salt of an acid can be prepared by a known method or a method similar thereto. That is, the monovalent metal salt of alginic acid used in the present invention is obtained by using the above-mentioned brown algae, It can be produced by known methods such as the acid method and the calcium method. After extracting from these brown algae using an alkaline aqueous solution such as a sodium carbonate solution, Alginic acid can be obtained by adding an acid (e.g., hydrochloric acid, sulfuric acid, etc.), and Salts of alginic acid can be obtained by ion exchange of alginic acid. The solvent for the monovalent metal salt of alginic acid is a solvent that is biocompatible. Examples of suitable water include, but are not limited to, purified water, distilled water, ion-exchanged water, Milli-Q water, and physiological saline. , phosphate buffered saline (PBS), etc. These must be sterilized. It is preferable to use water that has been treated to reduce endotoxins. For example, Milli-Q water is sterilized by filtration. It can be used as such.

[0051] When the composition of the present invention is provided in a dry state such as a freeze-dried product, the above-mentioned solvent may be used. It can be prepared into a fluid solution. Furthermore, all procedures for obtaining the composition of the present invention are carried out at low endotoxin and bacterial levels. For example, the operation should be performed on a clean bench using sterilized equipment. It is preferable to use an endotoxin removal agent, and the equipment used may be treated with a commercially available endotoxin removal agent. stomach.

[0052] 5. Apparent viscosity of the composition of the present invention The composition of some embodiments of the present invention is in the form of a flowable liquid, i.e., a solution. The composition of the present invention is flowable when applied to the nucleus pulposus site. Preferably, the composition of the present invention is prepared by injecting the composition with a 21G syringe needle after leaving the composition at 20°C for 1 hour. The apparent viscosity of the composition of the present invention in this embodiment is such that the effect of the present invention is Although there is no particular limitation as long as the viscosity is obtained, if the viscosity is too low, the adhesiveness to the surrounding tissue of the application site may be poor. Therefore, it is preferable to set the viscosity at 10 mPa·s or more, and more preferably at 100 mPa·s or more. a·s or more, more preferably 200 mPa·s or more, and particularly preferably 500 mPa·s or more If the apparent viscosity is too high, handling may become difficult. It is preferably 50,000 mPa·s or less, more preferably 20,000 mPa·s or less, and even more preferably Preferably, the apparent viscosity is 10,000 mPa·s or less. If the apparent viscosity is 20,000 mPa·s or less, However, the apparent viscosity is 20,000 mPa·s. The above can also be applied using a pressure type or electric type filling device or other means. The preferred range of the viscosity of the light composition is 10 mPa·s to 50,000 mPa·s, more preferably , 100 mPa·s to 30,000 mPa·s, more preferably 200 mPa·s to 20 000 mPa·s, and more preferably 500 mPa·s to 20,000 mPa·s, In particular, the viscosity is preferably 700 mPa·s to 20,000 mPa·s. is 500mPa·s to 10000mPa·s, or 2000mPa·s to 1000 The composition of some embodiments of the present invention may be administered to a subject using a syringe or the like. It is also possible to apply viscosity.

[0053] Measurement of apparent viscosity of compositions containing monovalent metal salts of alginic acid, such as aqueous solutions of alginic acids The viscosity can be measured by a conventional method. For example, the viscosity can be measured by a coaxial double cylindrical rotation viscometer. Viscometer, single cylinder rotational viscometer (Brookfield type viscometer), cone-and-plate rotational viscometer The viscosity can be measured using a viscometer (cone-plate type viscometer) or the like. It is preferable to follow the viscosity measurement method of the Pharmacopoeia (16th edition). In the present invention, viscosity measurement is performed at 20°C. As will be described later, the composition of the present invention is preferably insoluble in a solvent such as cells. In order to accurately measure viscosity, the apparent viscosity of the composition may be determined by measuring the viscosity of the composition containing the substance such as cells. It is preferable to use an apparent viscosity that does not contain

[0054] In the present invention, the apparent viscosity of a composition containing a monovalent metal salt of alginic acid is measured by It is particularly desirable to measure using a cone-plate type viscometer. For example, It is desirable to perform measurements under the following conditions. Prepare the sample solution using MilliQ water. The constant temperature is 20°C. The rotation speed of the cone-plate viscometer is 1 / 2 of the monovalent metal salt of alginic acid. When measuring a 1% solution, use 1 rpm, and when measuring a 2% solution, use 0.5 rpm. The reading time is 2 minutes for the measurement of 1% solution of monovalent metal alginate. The average value from 1 minute to 2 minutes is used. For 2% solution measurements, the measurement is taken for 2.5 minutes, starting from 0.5 The test value is the average value from 2.5 minutes to 3 minutes. The test value is the average value of three measurements.

[0055] The apparent viscosity of the composition of the present invention can be determined by, for example, the concentration of the monovalent metal salt of alginic acid, the molecular weight, or can be adjusted by controlling the M / G ratio, etc.

[0056] The apparent viscosity of a solution of monovalent metal salts of alginic acid is determined by the concentration of the monovalent metal salts of alginic acid in the solution. When the concentration is high, the viscosity is high, and when the concentration is low, the viscosity is low. When the molecular weight is large, the viscosity is high, and when the molecular weight is small, the viscosity is low.

[0057] The apparent viscosity of solutions of monovalent metal salts of alginic acid is affected by the M / G ratio, For example, it is possible to appropriately select alginic acid having a preferable M / G ratio depending on the viscosity of the solution, etc. The M / G ratio of the alginic acid used in the present invention is about 0.1 to 5.0, and preferably It is about 0.1 to 4.0, more preferably about 0.2 to 3.5.

[0058] As mentioned above, the M / G ratio is mainly determined by the type of seaweed used as a raw material. The type of brown algae used affects the viscosity of the solution of the monovalent metal salt of alginic acid. The alginic acid used is preferably selected from the group consisting of Lessonia genus, Macrocystis genus, and Rami genus. Preferably, the brown algae are derived from the brown algae of the genera Naria, Ascophyllum, and Durvillea, and more preferably Lessoni The preferred brown algae are those of the genus Lessonia, and the most preferred are those of Lessonia nigrescens. nigrescens).

[0059] 6. Preparation of the Composition of the Present Invention The composition of the present invention contains a monovalent metal salt of low-endotoxin alginic acid as an active ingredient. The present inventors have investigated the effect of injecting low-endotoxin monovalent metal salt of alginic acid into the marrow of a living body. When filled into the nucleus, the monovalent metal salt of alginic acid itself has a regenerative or therapeutic effect on the nucleus pulposus tissue. It was discovered for the first time that the active ingredient contained low endotoxin alginate. When monovalent metal salts of carboxylic acid are applied to the affected area, they can exert a regenerative or therapeutic effect on nucleus pulposus tissue. It is sufficient that the amount contained is at least 0.1 w / v% or more of the total composition. is preferred, more preferably 0.5 w / v% or more, and even more preferably 1 w / v%. The preferred concentration of the monovalent metal salt of alginic acid in the composition of the present invention is affected by the molecular weight. Although it is difficult to generalize, the preferred range is 0.5 w / v% to 5 w / v%, and more preferably 1 w / v%. 1 w / v% to 5 w / v%, more preferably 1 w / v% to 3 w / v%, and particularly preferably Preferably, the concentration is 1.5 w / v % to 2.5 w / v %. In another embodiment, the composition of the present invention The concentration of the monovalent metal salt of alginic acid in the solution is preferably 0.5 w / w% to 5 w / w%, more preferably Preferably, it is 1 w / w% to 5 w / w%, and more preferably, it is 1 w / w% to 3 w / w%. More preferably, the content may be 1.5 w / w % to 2.5 w / w %.

[0060] A monovalent metal salt of alginic acid purified to a desired endotoxin level is used. When the composition is prepared as described above, the endotoxin content of the composition is usually 50% or less. 0 EU / g or less, more preferably 300 EU / g or less, and even more preferably 150 EU / g or less. U / g or less, and particularly preferably 100 EU / g or less.

[0061] The compositions of the present invention are preferably cell-free. In some other embodiments of the present invention, the composition uses cells. Examples of cells include nucleus pulposus cells, stem cells, stromal cells, mesenchymal stem cells, and bone marrow stromal cells. The origin is not particularly limited, but examples include intervertebral disc nucleus pulposus, bone marrow, adipose tissue, and umbilical cord blood. Examples of cells include ES cells and iPS cells. do.

[0062] "Using cells" means using cells from the nucleus pulposus of the intervertebral disc, bone marrow, adipose tissue, umbilical cord blood, etc. as needed. The cells are prepared by collecting and concentrating the target cells and culturing them to increase the amount. Specifically, for example, 1 × 10 4 pcs / ml or more , or 1 × 10 5 / ml or more, preferably 1 x 10 4 pieces / ml~1×10 7 pieces / m The term "cells" refers to the inclusion of 100 cells in the composition of the present invention. Cells may be commercially available. stomach.

[0063] The compositions of the present invention may also contain factors that promote cell growth. Examples of the molecules include BMP, FGF, VEGF, HGF, TGF-β, IGF-1, and P DGF, CDMP (cartilage-derived-morphogenetic protein), CSF, EPO, I These factors include L, PRP (Platelet Rich Plasma), SOX, and IF. It may be produced by recombinant methods or purified from a protein composition. The compositions of some embodiments of the invention do not contain these growth factors. Even in this case, the regeneration of the nucleus pulposus is sufficiently good and is better than when actively promoting cell growth. It is also highly safe.

[0064] The composition of the present invention may also contain a factor that inhibits cell death. Factors that promote the growth of the tumor include caspase and TNFα, and factors that inhibit these include These factors that suppress cell death include antibodies and siRNA. It may be prepared by synthesis or purified from a protein composition. The composition does not contain factors that inhibit these cell deaths. Even in this case, nucleus pulposus regeneration is sufficiently good and compared with the case where cell death is actively suppressed. It is also safer.

[0065] In one embodiment of the present invention, the composition of the present invention comprises a monovalent hydroxyl group of low-endotoxin alginic acid. Other than metal salts, it does not contain any ingredients that exert pharmacological effects on the nucleus pulposus tissue of the intervertebral disc. Even in a composition containing only a monovalent metal salt of hydroxyl alginic acid as an active ingredient, sufficient It may have a regenerative or therapeutic effect on the nucleus pulposus.

[0066] In some embodiments of the present invention, other pharmaceutically active ingredients, conventional stabilizers, Emulsifiers, osmotic pressure adjusters, buffers, isotonicity agents, preservatives, soothing agents, coloring agents, etc., commonly used in pharmaceuticals The composition of the present invention may contain ingredients that can be used in the treatment of various skin conditions.

[0067] 7. Curing of the composition of the present invention The compositions of the present invention are used to harden the area after application to the nucleus pulposus site. "Curing a part" means bringing a crosslinking agent into contact with a part of the fluid composition of the present invention. This means that only a portion of the composition that comes into contact with the crosslinking agent, but not the entire composition, is gelled and hardened. Preferably, a crosslinking agent is brought into contact with at least a portion of the surface of the composition of the present invention having flowability. In some embodiments of the present invention, the composition is "Partially hardening after application to the nucleus pulposus site" means using a crosslinking agent similar to that used for filling the nucleus pulposus site. Using the method and ratio of use described in Example 4 of the present specification, Fill a test tube with 500 μL of low-endotoxin sodium alginate and crosslinker. After standing for 1 hour, at least 50% of the volume of the composition in the test tube is ungelled and the remaining 100% is gelled. The remaining part of the test tube must be filled with at least 50% of the composition volume using a syringe with a 21G needle. This may be indicated by the fact that the composition can be sucked up with a syringe. By showing this property, the composition does not deviate even when compressive force is applied from the cranial and caudal sides of the intervertebral disc after filling. The "at least a portion of the surface of the composition" is, for example, the surface of the nucleus pulposus. The opening on the surface of the disc that leads to the nucleus pulposus is preferably used for applying the composition to the nucleus pulposus area. The opening on the surface of the used intervertebral disc, i.e., the filling port for the composition. By gelling and hardening a portion of the composition, it is possible to effectively prevent the composition from leaking out of the intervertebral disc. The injection port for the composition on the surface of the intervertebral disc can be, for example, a syringe needle or a metal The opening used to fill the composition made by the laser or the scalpel used to remove the herniated disc Preferably, the opening is formed on the surface of the intervertebral disc by the above-mentioned method. is preferably the annulus fibrosus.

[0068] The compositions of the present invention are preferably administered to a subject at the nucleus pulposus site, and the composition is allowed to harden prior to application. Therefore, the composition of the present invention does not contain a large amount of crosslinking agent, and the composition remains hard even after a certain period of time. The predetermined time here is not particularly limited. The curing time is preferably about 30 minutes to 12 hours. For example, the composition was left standing at 20°C for 1 hour, and then a 21G syringe needle was attached to the composition. The composition of some embodiments of the present invention may be a crosslinked composition. Contains no bridging agent.

[0069] The cross-linking agent is a solution of monovalent metal salt of alginic acid, which is used to solidify the surface. There is no particular limitation on the crosslinking agent as long as it can stabilize the polymer. 2 + , Mg 2+ , Ba 2+ , Sr 2+ Divalent or higher metal ion compounds, such as More specifically, crosslinking reagents having two or more valent metal ions are examples of crosslinking reagents. As the ionic compounds, CaCl2, MgCl2, CaSO4, BaCl2, etc. are used, with 2 As a cross-linking reagent having up to four amino groups, a lysyl group (- diaminoalkanes, which may have the formula COCH(NH2)-(CH2)4-NH2 That is, diaminoalkanes and their amino groups are substituted with lysyl groups to form lysylamino groups. Specifically, diaminoethane, diaminopropane, N-(lysine)-2-methylpropane, and derivatives thereof are included. However, they are easily available and have good gel strength, etc. For this reason, a CaCl2 solution is particularly preferred.

[0070] In some embodiments of the present invention, a crosslinking agent is contacted to the surface of the composition of the present invention. The application of the composition of the present invention to the nucleus pulposus site is preferably performed after the composition of the present invention has been applied. There are no particular limitations on the method for contacting a part of the crosslinking agent (e.g., a divalent or higher metal ion). Although not specified, for example, it is possible to inject divalent or higher metal ions using a syringe or sprayer. For example, the crosslinking agent is slowly applied to the surface of the composition. The composition may be continuously applied to the filling port of the composition formed in the intervertebral disc for several to 10-odd seconds. If necessary, a process for removing the cross-linking agent remaining near the filling port may be added. Removal may be, for example, washing the application site with physiological saline or the like.

[0071] The amount of the crosslinking agent used depends on the amount of the composition of the present invention applied, the size of the opening for filling the composition on the surface of the intervertebral disc, and the amount of the crosslinking agent used. It is desirable to appropriately adjust the amount of the nucleus pulposus in the intervertebral disc, taking into consideration the size of the area where the nucleus pulposus is to be applied, etc. To avoid the cross-linking agent having a strong effect on the tissue surrounding the filling hole, do not use an excessive amount of cross-linking agent. The amount of divalent or higher metal ions used should be adjusted so that monovalent metal salts of alginic acid are not present. There are no particular limitations on the amount of the additive, as long as it is capable of solidifying the surface of the composition. For example, when using a 100 mM CaCl2 solution, the filling port on the surface of the intervertebral disc must be 1 mm in diameter. In the case of a CaCl2 solution of about 0.3 ml to 5.0 ml, the amount of CaCl2 solution used should be about 0.3 ml to 5.0 ml. The filling port on the surface of the intervertebral disc is preferably about 0.5 ml to 3.0 ml. When a disc is removed using a scalpel or other device, and the margin is approximately 5mm x 10mm, The amount of 100 mM CaCl2 solution used is preferably about 0.3 ml to 10 ml. The amount of the composition of the present invention at the application site is preferably about 0.5 ml to 6.0 ml. The amount can be increased or decreased as needed while observing the condition of the product.

[0072] If the cross-linking agent contains calcium, the higher the calcium concentration, the faster the gelation will occur. It is also known that calcium can form a firmer gel. Because of its cytotoxicity, if the concentration is too high, it will have a negative effect on the regenerative effect of the composition of the present invention on the nucleus pulposus of the intervertebral disc. Therefore, the surface of the composition containing the monovalent metal salt of alginic acid is hardened. When using a CaCl2 solution, the concentration is preferably 25 mM to 200 mM. More preferably, the concentration is 50 mM to 150 mM.

[0073] In the present invention, it is preferable to add a crosslinking agent to the composition, leave it to stand for a certain period of time, and then It is desirable to remove any crosslinking agent remaining in the applied area by washing, etc. The time is not particularly limited, but is preferably about 1 minute or more, more preferably about 4 minutes or more. Alternatively, it is preferable to leave the composition for about 1 minute to about 10 minutes to gel the surface of the composition. Preferably, the mixture is left standing for about 4 to about 10 minutes, about 4 to about 7 minutes, and more preferably for about 5 minutes. It is preferable to keep the composition and the crosslinking agent in contact for this certain period of time. A crosslinking agent may be added as needed to prevent the liquid surface of the composition from drying out.

[0074] For example, sodium alginate solution was dropped into CaCl2 solution and gelled. However, alginate beads are not suitable for use when pressed against the application site. However, it is necessary to create a device that is appropriate for the size of the application area, and this is difficult to achieve in actual clinical practice. It is technically difficult to use it in this way. In addition, when CaCl2 solution is used as a cross-linking agent, There is also the problem of calcium cytotoxicity, since the calcium ions on the surface of the beads come into contact with the surrounding tissue. In contrast, the composition of the present invention is in the form of a solution, so it can be easily applied to any application site. The composition can be applied to the entire area of ​​application, and the entire area can be covered with the composition, preventing damage to the surrounding tissue. The part of the composition of the present invention that comes into contact with the surrounding tissue keeps the calcium concentration low. The composition of the present invention can be applied to the surrounding tissues without causing any problems with calcium cytotoxicity. The contact area is less affected by the crosslinking agent, so the composition of the present invention can easily penetrate the cells at the application site. Preferably, the composition of the present invention can be applied to the nucleus pulposus site and then contacted with the tissue. After four weeks, it will fuse with the tissue of the body to the point where it is no longer recognizable at the application site. It also has high affinity with living organisms.

[0075] When the composition of the present invention is applied to the nucleus pulposus site, if a part of it is gelled with a crosslinking agent, The composition of the present invention hardens in part at the affected area, and is localized in a state of close contact with the surrounding tissue, and then spreads from the nucleus pulposus to the affected area. In addition, the composition of the present invention adheres closely to the surrounding tissue, The composition of the present invention exerts a more potent effect of regenerating the nucleus pulposus.

[0076] In the examples of the present invention, as a comparative example, the entire filling material filled in the nucleus pulposus site was gelled. When the disc is hardened, compressive force is applied from the craniocaudal side to the disc, and the composition filler inlets on the disc surface On the other hand, when the solution composition of the present invention was injected into the nucleus pulposus area, the hardened gel was observed to escape from the nucleus pulposus area. When this was done, there was no deviation from the filling port on the surface of the intervertebral disc even when compressive force was applied from the craniocaudal side. That is, when the nucleus pulposus is actually filled with the composition of the present invention, the superior effect on the intervertebral disc is It can be said that there is little risk of the filled composition leaking out even when pressure is applied from below.

[0077] In addition, when filling the nucleus pulposus with hardened gel, the hardened gel may protrude into the spinal canal. On the other hand, the solution composition of the present invention does not cause such a problem. The risk of such complications is low.

[0078] 8. Application of the composition of the present invention The compositions of the present invention can be administered to humans or non-human organisms, such as birds and non-human mammals. (e.g., cows, monkeys, cats, mice, rats, guinea pigs, hamsters, pigs, dogs, To be applied to the nucleus pulposus site of the intervertebral discs of rabbits, sheep, and horses to promote regeneration of the nucleus pulposus It is used for.

[0079] The composition of the present invention is preferably in the form of a flowable liquid, that is, a solution. In the present invention, "having fluidity" means having the property of changing its shape into an amorphous form. It is not necessary for the liquid to have a property of constantly flowing like a solution. For example, the composition can be enclosed in a syringe or the like and injected into the nucleus pulposus of the intervertebral disc. In some embodiments of the present invention, the composition After leaving the specimen at 20°C for 1 hour, the specimen was instilled into the intervertebral disc using a syringe with a 14G to 26G needle. It is desirable that the fluidity be such that it can be injected into the nucleus pulposus site, and more preferably, it is 21G. It is desirable that the composition of the present invention can be injected with a syringe. When the composition is applied, a solvent or the like is used to make the composition fluid as described above. can be done.

[0080] The composition of the present invention in the form of a solution can be dispensed using a syringe, a gel pipette, a dedicated syringe, a dedicated injector, or a dedicated injector. The adhesive can be easily applied to the nucleus pulposus of the intervertebral disc using a filling tool or the like. When the viscosity of the composition of the present invention is high, it becomes difficult to apply it with a syringe. A syringe such as a handheld or electric syringe may be used. It may be applied to the defect of the nucleus pulposus using a rod or the like. When injecting with a syringe, for example, It is preferable to use needles of 14G to 26G or 27G to 14G.

[0081] The method for applying the composition of the present invention to the nucleus pulposus site is not particularly limited, but is preferably a known method. After direct exposure of the affected area by surgical techniques, or under a microscope or endoscope, The composition of the present invention can be applied to the nucleus pulposus site using a syringe, filling device, or the like. In one of the novel embodiments, for example, a needle of a filling instrument is inserted from the surface of the annulus fibrosus toward the nucleus pulposus. The composition of the present invention may be applied to the skin.

[0082] The composition of the present invention is in a liquid form, and therefore can be used to treat any shrinkage of the nucleus pulposus, cavities or defects in the nucleus pulposus, etc. It can be adapted to any shape of nucleus pulposus site, and can fill the entire shrinkage, cavity or defect of the nucleus pulposus. Nucleus pulposus shrinkage, cavities or defects in the nucleus pulposus area are signs of degeneration or damage to the intervertebral disc. It may be caused by a surgical procedure to remove or remove at least a portion of the nucleus pulposus. It may also be caused by suction or by the removal of at least a portion of the nucleus pulposus. It is desirable to apply the composition of the present invention to the defect in the nucleus pulposus formed by the removal.

[0083] The removal of at least a portion of the nucleus pulposus may be performed by, but is not limited to, direct vision, percutaneous surgery, or microscopic surgery. For example, a discectomy performed by an endoscopic or endoscopic method may be performed. A 10-15cm incision is made, and the muscles are separated from the posterior surface of the posterior element of the spine called the vertebral arch, and then the intervertebral arch is removed. The ligament is removed, the nerve and disc herniation are identified, and the herniated disc that is compressing the nerve is removed. The method may be a method in which a laser is irradiated onto the nucleus pulposus to reduce the volume of the nucleus pulposus. It may also be a method of making it so that

[0084] After the composition of the present invention is applied to the nucleus pulposus site, a part of the composition is crosslinked with a crosslinking agent as described above. Can be hardened.

[0085] The amount of the composition of the present invention to be applied may be determined depending on the volume of the application site of the nucleus pulposus to be applied. Although not particularly limited, for example, 0.01 ml to 10 ml, more preferably 0.1 ml The amount of the composition of the present invention is 1 to 5 ml, and more preferably 0.2 to 3 ml. When applying to a defect, it is desirable to inject it so that it fully fills the volume of the defect in the nucleus pulposus area. Desirable.

[0086] The number and frequency of application of the composition of the present invention can be increased or decreased depending on the symptoms and effects. It may be applied only once, or may be applied continuously from once a month to once a year. Alginate is a substance that does not naturally exist in the body of animals, so animals have a specific way of producing alginate. Alginic acid is not degraded by normal hydrolysis in the animal body. It is decomposed gradually in the body, but it is decomposed more slowly in the body than polymers such as hyaluronic acid. Since there are no blood vessels in the nucleus pulposus, if the material is filled into the nucleus pulposus, it is expected that the effect will last for a long period of time. Cut.

[0087] Even if the composition of the present invention is not provided together with cells or growth factors as described above, the present invention When the composition is applied to the nucleus pulposus site, the cells, growth factors, cell death inhibitors, and Other drugs such as those listed above may also be used in combination.

[0088] The composition of the present invention, when applied to the nucleus pulposus area, prevents degenerative changes in the entire intervertebral disc tissue and the nucleus pulposus. Therefore, the composition of the present invention has the effect of suppressing the pulpogenesis of the intervertebral disc and promoting regeneration. It is preferably used as a nucleus filling composition.

[0089] One preferred embodiment of the composition of the present invention is a composition for inhibiting degeneration of an intervertebral disc, more preferably The present invention relates to a composition for inhibiting the degeneration of the nucleus pulposus of an intervertebral disc. The number of cells in the intervertebral disc, water content, and extracellular matrix (type II collagen) change with aging. This refers to a condition in which the amount of enzymes (proteins, aggrecan, etc.) that are reduced causes morphological changes and functional decline. If the disc is moved too far, it will no longer be able to function as a shock absorber. "Suppression of degeneration" means that degenerative changes are suppressed compared to the untreated case, and does not necessarily mean that degeneration is suppressed. It does not mean making someone sexually inactive.

[0090] One embodiment of the composition of the present invention is a composition for regenerating the nucleus pulposus. The purpose is to prevent the accumulation of blast-like cells and regenerate a nucleus pulposus with a high ratio of nucleus pulposus cells. The intention is to regenerate nucleus pulposus tissue rich in type II collagen and proteoglycans. The term "nucleus pulposus regeneration" also includes the inhibition of degeneration of the nucleus pulposus. In one preferred embodiment, the composition of the nucleus pulposus regenerated by applying the composition of the present invention is similar to that of natural normal tissue. It is desirable that the composition be close to that of the normal nucleus pulposus.

[0091] In addition, the composition of a preferred embodiment of the present invention is useful for treating intervertebral disc degeneration and / or disc damage. As used herein, "treatment, prevention, or suppression of recurrence" refers to "Control" means treatment, prevention, suppression of recurrence, reduction, suppression, improvement, elimination, reduction in incidence, delay in onset. This includes delaying disease progression, inhibiting progression, reducing severity, lowering recurrence rate, delaying recurrence, and alleviating clinical symptoms. .

[0092] The preferred embodiments of the composition of the present invention, the method of using the composition, etc. are as described above.

[0093] Disc degeneration and / or disc damage can occur, for example, as a herniated disc, disc disease, or spinal degeneration. selected from the group consisting of spondylolisthesis, pyogenic discitis, degenerative spondylosis, spinal canal stenosis, and intervertebral disc injury. The present invention relates to at least one condition or disease selected from the group consisting of:

[0094] 9.Treatment method The present invention relates to a method for treating intervertebral disc degeneration and / or intervertebral disc damage using the composition of the present invention. Preferably, the treatment method of the present invention is an intervertebral A method for treating, preventing, or inhibiting recurrence of disc degeneration and / or disc damage, comprising: A flowable composition containing a monovalent metal salt of endotoxin alginic acid is used in the treatment, The composition is applied to the nucleus pulposus site of the intervertebral disc of a subject in need of prevention or suppression of recurrence. The method includes curing a portion of the

[0095] The treatment method of the present invention comprises treating at least one part of the nucleus pulposus before applying the composition of the present invention to the nucleus pulposus site. The method may include removing the portion.

[0096] The disc degeneration and / or disc damage may be, for example, a herniated disc, disc disease, or spinal A group consisting of degenerative spondylolisthesis, pyogenic discitis, degenerative spondylosis, spinal canal stenosis, and disc injury The treatment method of some embodiments of the present invention is at least one condition or disease selected from the group consisting of: In the method, the disc degeneration and / or disc damage is a herniated disc, and in particular It is a lumbar disc herniation.

[0097] In some embodiments of the present invention, the composition of the present invention is used to treat intervertebral disc degeneration. In one preferred embodiment of the present invention, the present invention provides a method for suppressing sexual change. The present invention provides a method for regenerating the nucleus pulposus of an intervertebral disc using the composition of the present invention.

[0098] These methods involve the use of a flowable composition containing a monovalent metal salt of endotoxin-containing alginic acid. applying the composition to the nucleus pulposus site of an intervertebral disc of a subject in need of inhibition of intervertebral disc degeneration or nucleus pulposus regeneration; The method includes applying a composition of the present invention to the nucleus pulposus site and allowing the applied portion of the composition to harden. The method may include removing at least a portion of the nucleus pulposus prior to application to the implant.

[0099] Preferred embodiments of the composition of the present invention, specific methods for applying the composition to the nucleus pulposus of an intervertebral disc, and hardness of the composition The method of synthesis and the meaning of the terms are as described above. Other treatment methods and drugs for intervertebral discs may be appropriately combined. The treatment method of the present invention may be carried out in combination.

[0100] In addition, before, at the same time as, or after applying the composition of the present invention to the nucleus pulposus site, Treptomycin, penicillin, tobramycin, amikacin, gentamicin, neomycin Antibiotics such as isin and amphotericin B, aspirin, and nonsteroidal antipyretics (NSAIDs), anti-inflammatory drugs such as acetaminophen, proteolytic enzymes, and adrenal corticosteroids Concomitant medications include drugs such as steroids, HMG-CoA reductase inhibitors such as simvastatin and lovastatin. These drugs may be mixed into the composition of the present invention. may be administered orally or parenterally in combination. Pain medications, neuropathic pain relievers, etc. may be administered orally or parenterally in combination as needed. stomach.

[0101] In some aspects of the present invention, the cells described above are administered to the nucleus pulposus site together with the composition of the present invention. Alternatively, in some aspects of the present invention, the composition of the present invention may be applied to The aforementioned factors that promote cell growth may be applied to the nucleus pulposus site. Therefore, it is also desirable that the composition of the present invention is not used in combination with the above-mentioned cells. However, it is also desirable to use the composition of the present invention in an embodiment in which no factor that promotes cell growth is used in combination. Even without using cells or factors, regeneration of the nucleus pulposus can be promoted.

[0102] The present invention relates to a monovalent metal salt of low-endotoxin alginic acid for producing the composition of the present invention. It also relates to the use of

[0103] The use of the present invention is useful for the treatment, prevention or suppression of recurrence of intervertebral disc degeneration and / or intervertebral disc damage. Use of a monovalent metal salt of low-endotoxin alginic acid for producing a composition for the composition is applied to the nucleus pulposus site of a subject and allowed to harden after application; It has flowability when applied to the nucleus pulposus site.

[0104] The present invention further provides a fluid drug containing a monovalent metal salt of low-endotoxin alginic acid. The composition is administered to a patient in need of treatment, prevention, or suppression of recurrence of intervertebral disc degeneration and / or intervertebral disc damage. and applying the composition to the nucleus pulposus of the subject's intervertebral disc to harden a portion of the applied composition. and / or low-end for use in the treatment, prevention or suppression of recurrence of intervertebral disc injury A monovalent metal salt of the toxin alginic acid is provided.

[0105] 10. Lyophilized preparations and kits The present invention provides a kit for replacing the nucleus pulposus of an intervertebral disc. The kit of the present invention may include the composition of the present invention. The composition of the present invention may be in a solution state or a dry state, but is preferably in a dry state. More preferably, it is a freeze-dried product, and particularly preferably, it is a freeze-dried powder. When the composition is in a dry state, it is desirable to include a solvent for dissolution (eg, water for injection). The kit of the present invention may further comprise a cross-linking agent. The kit of the present invention further includes a crosslinking agent, a syringe, an injection needle, a gel pipette, a dedicated filling device, , instruction manuals, etc.

[0106] A specific example of a suitable kit is: (1) a kit containing low-endotoxin sodium alginate; (1) A vial containing a freeze-dried product (2) An amplifier containing a solvent such as water for injection as a dissolving solution (3) A polymer containing a divalent or higher metal ion compound such as calcium chloride solution as a crosslinking agent. As another example, a kit can be prepared by packing a sample or the like into a single package. The syringe is molded and has two chambers separated by a partition. One chamber contains a monovalent amount of alginate. A metal salt is sealed in one chamber, and a solvent as a dissolving liquid or a solution containing a crosslinking agent is sealed in the other chamber. The partition between the two rooms is designed to be easily opened when needed, and the two are mixed and dissolved when needed. Another example is a kit that contains a pre-filled tablet containing a monovalent metal salt solution of alginic acid. The kit is sealed in a syringe and can be filled as is without any preparation procedures when used. is a kit containing alginate solution and a crosslinking agent in separate syringes, packaged together in a single pack. Alternatively, a vial filled with a monovalent metal salt solution of alginic acid and a vial containing a cross-linking agent are used. The composition of the present invention may be provided as a kit containing an ampoule, etc. The above is as explained above.

[0107] This kit can be used, for example, in the treatment method of the present invention.

[0108] All literature and publications mentioned in this specification are hereby incorporated by reference for any purpose. The entire contents of which are incorporated herein by reference. The scope of claims in the foundational Japanese patent application, Patent Application No. 2016-058396 (filed March 23, 2016) This application includes the disclosure of the specification and drawings. [Example]

[0109] The present invention will be described in more detail with reference to the following examples, but it should be understood that the present invention is limited to these examples. It shouldn't be.

[0110] Example 1: Effect of low-endotoxin sodium alginate on human intervertebral disc cells 1-(1) Isolation and culture of non-degenerated human intervertebral disc cells Nucleus pulposus tissue was extracted from human non-degenerated intervertebral disc tissue and purified with 0.25% collagenase (Wako). The marrow was treated with DMEM (Dulbecco's modified Eagle's medium) at 37°C for 4 hours. The nucleus pulposus cells were isolated. The obtained nucleus pulposus cells were treated with 1% penicillin / streptomycin, 1.25 μg / ml Fungizone (Invitrogen), 10% FBS (fetal bovine serum albumin), The cells were cultured in DMEM containing 100 μm under conditions of 37°C, 5% CO2, and 20% O2. Cells from passage 2 were used in the experiments.

[0111] 1-(2) Preparation and culture of alginate beads (A) low-endotoxin sodium alginate and (B) food Commercial grade sodium alginate (Wako Pure Chemical Industries, Ltd., 199 -09961) and two types of sodium alginate (Mochida Pharmaceutical Co., Ltd.) , human nucleus pulposus cells were cultured and compared.

[0112] Using Milli-Q water, sodium alginate solutions with a concentration of 2 w / v % were prepared. 4.0 × 10 human nucleus pulposus cells obtained in (1) above 6 The cells were then treated with these alginate carbonates. The cells were then suspended in 1.0 ml of sodium solution. The nucleus pulposus cells-containing alginate was extracted by dropping it into a 0.2 mM calcium chloride aqueous solution and leaving it for 10 minutes. Beads containing a sodium phosphate solution were prepared.

[0113] The beads were washed twice with 0.9% saline and then diluted with 1% penicillin / streptomycin. Place the cells in DMEM culture medium containing 1.25 μg / ml fungizone and 10% FBS. The cells were then cultured in a 3D environment at 37°C, 5% CO2, and 20% O2. The cells were collected after 8 hours, 7 days, 14 days, and 28 days and used for the following evaluation.

[0114] [Table 1]

[0115] 1-(3) Evaluation of cell viability The beads were collected 48 hours, 7 days, 14 days, and 28 days after the start of culture and incubated at 4°C for 55 min. The beads were dissolved by immersing them in an aqueous solution of 100 mM sodium citrate for 20 minutes, and then centrifuged. The cells were harvested by detachment. The obtained cells were stained with PI (Protein Iodide) and observed under a confocal laser microscope ( Calcein AM-positive cells were observed as live cells, and PI-positive cells were observed as live cells. Dead cells were analyzed using ImageJ (National Institutes of Health, Bethesda, MD, USA). The viable cell rate was calculated using n = 5 for each time point at which cells were collected.

[0116] In the examples of the present specification, the statistical analysis results are expressed as mean ± standard deviation (mean ± SD). Student's t-test was used for comparison between two groups, and Steel-Dwass test was used for comparison between multiple groups. Statistical significance was considered to be <0.05.

[0117] As a result, Group A (low endotoxin sodium alginate) and Group B (food grade Sodium alginate) was added at 48 hours, 7 days, 14 days, and 28 days after the start of culture. At both points, the viability of the cells was approximately 90%, and no difference in viability was observed between the two groups. It was.

[0118] 1-(4) Evaluation of apoptotic cells As in 1-(3), the cells were cultured for 48 hours, 7 days, 14 days, and 28 days after the start of culture. The beads were collected, washed twice with PBS, and then the cells were collected and 3.6 × 10 5 individual cells Annexin V-fluorescein isothiocyanate(FITC) Apoptosis Detection Kit II(BD Bios Sciences, San Jose, CA, USA) and then labeled using a flow cytometer (FACS Cant; Apoptotic cells were counted using a FITC / PI assay (BD biosciences, CA, USA). Apoptotic cells and FITC+ / PI+ were considered as late apoptotic cells. The FITC- / PI- cells were defined as live cells, and the ratio of live cells to total cells was calculated. The ratio of apoptotic cells to total cells was also evaluated. The number of participants was set to n=5.

[0119] As a result, Group A (low endotoxin sodium alginate) and Group B (food grade Sodium alginate) was added at 48 hours, 7 days, 14 days, and 28 days after the start of culture. At each time point, the viable cell rate was approximately 90%, and the apoptotic cell rate was approximately 10%. No difference was observed between the two groups.

[0120] 1-(5) Evaluation of cells under serum starvation induction The human intervertebral disc is an avascular area and is in a low-nutrient environment, so we simulated the environment inside the intervertebral disc. Cell culture experiments were performed under serum starvation and evaluated.

[0121] The beads were collected on day 7 of the 3D culture in 1-(2) and washed twice with PBS. Serum-free DMEM, 1% penicillin / streptomycin, 1.25 μg / ml The beads were added to Fungizone medium and incubated at 37°C, 5% CO2, and 20% O2. After the start of serum starvation, the cells were incubated for 6 hours and 48 hours, respectively. The cells were collected and evaluated using a confocal laser microscope and a flow cytometer. Evaluation was carried out using a meter.

[0122] As a result, when evaluated using a confocal laser microscope under serum starvation conditions, Group A (low endogenous Toxin B (sodium alginate) and group B (food grade sodium alginate) There was no significant difference in the viable cell rate between the two groups after 6 hours and 48 hours. After 48 hours, Group A (low endotoxin sodium alginate) outperformed Group B (food grade There was a tendency for the cell viability to be higher compared to sodium alginate.

[0123] In addition, evaluation using a flow cytometer showed no difference between the two groups in the samples taken 6 hours later. After 48 hours, Group A (low endotoxin sodium alginate) showed a significant improvement over B. The viability of cells was significantly higher than that of the food-grade sodium alginate group, and the apoptosis rate was significantly higher. The rate of ptosis cells was significantly lower (Figures 1 and 2).

[0124] The induction of serum starvation in this study simulates the avascular, low-nutrient environment of the human intervertebral disc nucleus pulposus. This is the intended test. Cultivation of nucleus pulposus cells using low-endotoxin sodium alginate , compared with food-grade sodium alginate, under serum starvation induction These results suggest that the cells are highly resistant to apoptosis. Sodium alginate has a higher solubility in the nucleus pulposus of the intervertebral disc compared to food-grade sodium alginate. It has been suggested that when filled into the nucleus pulposus, it does not induce apoptosis of nucleus pulposus cells and maintains cell viability. was done.

[0125] Example 2: Low-endotoxin sodium alginate solution in a rabbit intervertebral disc nucleus pulposus defect model Application of Two low-endotoxin sodium alginate solutions were used in rabbit intervertebral disc nucleus pulposus defect models. Each was filled with a different solution and the effects were evaluated.

[0126] 2-(1) Creation of a rabbit intervertebral disc nucleus pulposus defect model Japanese white rabbits weighing 3.2-3.5 kg were anesthetized with pentobarbital intravenously. Local anesthesia with 1% xylocaine was administered, and the nucleus pulposus tissue of the intervertebral disc was aspirated using an 18G needle. A disc nucleus pulposus defect model was created. Aspiration was performed on the L2 / 3 and L4 / 5 intervertebral disc nucleus pulposus. The L3 / 4 disc was left as a normal disc (normal control). group).

[0127] 2-(2) Filling of low-endotoxin sodium alginate solution The following two types of low-endotoxin sodium alginate were used. The endotoxin content of each low-endotoxin sodium alginate was less than 50g / EU. The apparent viscosity and weight average molecular weight are shown in Table 2. Apparent viscosity of sodium alginate Measurements were performed using a rotational viscometer (cone-plate type) in accordance with the viscosity measurement method of the Japanese Pharmacopoeia (16th edition). The specific measurement conditions are as follows: Preparation of sample solution The preparation was carried out using MilliQ water. The measuring instrument was a cone-plate type rotational viscometer (viscosity viscoelasticity The measurement device used was a Rheostress RS600 (Thermo Haake GmbH) sensor: 35 / 1. The number of rotations is 1 rpm when measuring 1 w / w% sodium alginate solution, and 2 w / w% sodium alginate solution. When measuring thorium solution, the rpm was set to 0.5. When measuring 1 w / w% solution, the reading time was Measure for 2 minutes and take the average value from 1 minute to 2 minutes. When measuring a 2 w / w% solution, take the average value from 2.5 minutes. The measurement was made for 0.5 minutes to 2.5 minutes, and the average value was taken as the average of three measurements. The measurement temperature was 20°C.

[0128] The weight average molecular weight of each sodium alginate was determined by gel permeation chromatography (G Measurements were performed using two methods: GPC and GPC-MALS. The measurement conditions were as follows: do.

[0129] [Pretreatment method] After adding the eluent to the sample and dissolving it, the sample was filtered through a 0.45 μm membrane filter and used as the measurement solution. The solution was made into a liquid. (1) Gel permeation chromatography (GPC) measurement [Measurement conditions (relative molecular weight distribution measurement)] Column: TSKgel GMPW-XL x 2 + G2500PW-XL (7.8 mm I .D.×300mm×3 pieces) Eluent: 200mM sodium nitrate aqueous solution Flow rate: 1.0mL / min Concentration: 0.05% Detector: RI detector Column temperature: 40℃ Injection volume: 200μL Molecular weight standards: standard pullulan, glucose

[0130] (2)GPC-MALS measurement [Refractive index increment (dn / dc) measurement (measurement conditions)] Suggestive refractometer: Optilab T-rEX Measurement wavelength: 658nm Measurement temperature: 40℃ Solvent: 200 mM sodium nitrate aqueous solution Sample concentration: 0.5 to 2.5 mg / mL (5 concentrations)

[0131] [Measurement conditions (absolute molecular weight distribution measurement)] Column: TSKgel GMPW-XL x 2 + G2500PW-XL (7.8 mm I .D.×300mm×3 pieces) Eluent: 200mM sodium nitrate aqueous solution Flow rate: 1.0mL / min Concentration: 0.05% Detector: RI detector, light scattering detector (MALS) Column temperature: 40℃ Injection volume: 200μL

[0132] [Table 2]

[0133] Dissolve each low-endotoxin sodium alginate in Milli-Q water to prepare a 2 w / v% solution. did. The needle was inserted from the side of the intervertebral disc toward the nucleus pulposus, and low-endotoxin sodium alginate was injected. After the injection needle was removed, 20 μl of 102 mM sodium solution was injected into the nucleus pulposus defect. A calcium chloride solution was poured over the samples for a few seconds (referred to as Group A-1 and Group A-2, respectively). (These are referred to as the treatment groups.)

[0134] The group that underwent only nucleus pulposus aspiration was designated the aspiration alone group (aspiration alone group).

[0135] Four weeks after surgery, the animals were euthanized by overdosing with pentobarbital, and the lumbar vertebrae were removed and the intervertebral disc tissue was circulated. Group A-2 was also evaluated 12 weeks after surgery. Eight cases were evaluated in each group.

[0136] 2-(3) Evaluation of intervertebral disc tissue using MRI and Pfirrmann classification T2-weighted sagittal images of the intervertebral disc were taken using a 7.0-Tesla MR scanner (Unity Inova, Varian). T2-weighted MRI images of the intervertebral disc show changes due to disc degeneration. To assess the severity of disc degeneration, scores were calculated using the Pfirrmann classification. The grade is an index that evaluates the degeneration of the intervertebral disc on MRI in five stages (Grade 1: normal to Grade 2: normal). Grade 5: Severe degeneration). The evaluation criteria for disc degeneration are shown in Table 3 (Spine (Phila Pa. 1976). 2001;26(17) 1873-8).

[0137] [Table 3]

[0138] As a result, the Pfirrmann classification, which scores the severity of disc degeneration, showed that In the study, the aspiration only group and the treatment group (Group A-1 and Group A-2) were compared with the normal control group. Compared with group A-1, the scores were significantly higher, indicating disc degeneration. The scores were significantly lower in the A-2 group compared with the A-1 group, and degeneration was suppressed. In the suction-only group, no significant difference was observed in the scores, but the A-2 group showed a higher score than the suction-only group. There was a tendency for the core to have low values ​​(Figure 3).

[0139] In the evaluation of the A-2 group 12 weeks after surgery, the score of the A-2 group was significantly higher than that of the aspiration only group. The values ​​were significantly lower.

[0140] 2-(4) Evaluation of intervertebral disc tissue using MRI index Analyze 10.0 software (AnalyzeDirect, Overland Park, KS, USA) was used to analyze the sagittal section. The MRI index (product of the mean signal intensity of the nucleus pulposus and the area of ​​the nucleus pulposus) was measured and quantitatively evaluated. The MRI index of the intervertebral disc in each group is calculated based on the MRI index of the normal control group, which is set at 100. The results were evaluated as a percentage of the total number of patients with pulmonary embolism (Spine (Phila Pa 1976). 2005 Jan 1;30(1):15-24).

[0141] As a result, 4 weeks after surgery, MRI indices of the treatment groups (A-1 and A-2 groups) were x was significantly higher than in the aspiration-only group, indicating that degenerative changes were suppressed (Fig. 4). In the evaluation of the A-2 group 12 weeks after surgery, the MRI index of the A-2 group was This was significantly higher than in the aspiration only group.

[0142] 2-(5) Histological evaluation After MRI, tissue specimens of the intervertebral discs were prepared. The samples were fixed in 10% formaldehyde. The specimens were then decalcified with 10% EDTA (pH 7.5) and embedded in paraffin. Paraffin sections were deparaffinized with xylene, treated with alcohol, washed with water, and then subjected to HE The annulus fibrosus was stained with Nishim staining and Safranin-O staining. The classification of the intervertebral disc tissue was performed using the classification of Spine (Phila Pa 1976). 1998;23(14):1531-8. The overall degree of degeneration was scored. Nishimura et al.'s classification is as follows: .

[0143] Grade 1: Mildly serpentine with rupture Grade 2: Moderately serpentine with rupture Grade 3: Severely serpentine with mildly reversed ) Grade 4: Severely reversed contour Grade 5: Indistinct

[0144] The severity of disc degeneration was evaluated histologically, and at 4 weeks after surgery, the aspiration-only group showed a significantly higher severity than the control group. , and the treatment groups (A-1 and A-2 groups) showed significantly higher Nish scores compared to the normal control group. The scores according to the classification by Imura et al. were significantly higher, suggesting degeneration. The treatment groups (A-1 and A-2) had significantly lower scores than the aspiration-only group. Degeneration was suppressed (Fig. 5).

[0145] In the evaluation of the A-2 group 12 weeks after surgery, the A-2 group showed significantly higher blood pressure than the aspiration only group. The scores were significantly lower in the normal control group, the suction only group, and the A-2 group at 4 weeks after surgery. Photographs of tissue specimens at 12 weeks are shown (Figure 6).

[0146] 2-(6) Immunohistological evaluation The tissue specimens prepared in 2-(5) were incubated with anti-Type I collagen antibody and anti-Type II collagen antibody. Immunohistological staining was performed using anti-IgE antibody in the nucleus pulposus of the intervertebral discs. The number of positive cells in the field was counted.

[0147] As a result, the rate of anti-Type I collagen antibody positive cells relative to the number of cells in the section was 4 weeks after surgery. At each time point, the normal control group, the aspiration only group, and the treatment groups (Groups A-1 and A-2) No differences were observed between groups.

[0148] On the other hand, the rate of anti-type II collagen antibody positive cells was 4 weeks after surgery. Compared with the suction-only group, both the aspiration-only group and the treatment group (A-1 and A-2 groups) had significantly lower The values ​​showed a decrease in extracellular matrix production, which is seen in normal disc tissue. In groups A-1 and A-2, the number of anti-type II collagen antibody positive cells was higher than that in the aspiration only group. The rate was significantly higher.

[0149] In the evaluation of group A-2 12 weeks after surgery, anti-type II collagen antibody-positive cells were observed in group A-2. The cell rate was significantly higher than that of the aspiration only group, and no difference was observed between the group and the normal control group. The normal control group, the suction only group, and the A-2 group at 4 and 12 weeks after surgery The graph of the percentage of cells positive for anti-type II collagen antibody against the number of cells in the intervertebral disc nucleus pulposus tissue section is shown below. Figure 7 shows that Type II collagen antibody-positive cells indicate the presence of hyaline cartilage-like cells. Endotoxin-containing sodium alginate solution was used to investigate the effects of endotoxin on the nucleus pulposus of rabbit intervertebral discs. At 12 weeks after surgery, the percentage of hyaline cartilage-like cells was comparable to that of the normal control group. was shown to be recovering.

[0150] From the above, it was found that the low-endotoxin sodium alginate solution can be used to fill the nucleus pulposus defect. It has been revealed that this suppresses degenerative changes in the entire intervertebral disc tissue and the nucleus pulposus, and promotes regeneration. In addition, the low-endotoxin sodium alginate A-1 and A-2 showed similar vertebral activity. The effect of inhibiting disc degeneration and regenerating the disc was observed.

[0151] From the viewpoint of handling, the 2% solution of A-2 has a higher viscosity than the 2% solution of A-1. This has the advantage that it is difficult for fluid to flow back from the filling area when filling the nucleus pulposus, and it is easy to distinguish from body fluids. The 2% solution of A-2 has an appropriate viscosity for filling and is easy to handle. I found out that...

[0152] Example 3: Examination of injection methods for low-endotoxin sodium alginate solution 3-(1) Investigation of the injection method of low-endotoxin sodium alginate solution The following two injection methods (i) and (ii) were used to administer low-endotoxin sodium alginate solution. The solution was injected into the lumbar vertebrae of a sheep cadaver and evaluated.

[0153] (i) As described in Example 2, the low-endotoxin alginate A-2 described in Example 2 was used. After injecting a 2% sodium phosphate solution into the area where the nucleus pulposus was partially removed, A method of contacting sodium alginate with 100 mM calcium chloride solution (The area that comes into contact with the sodium solution hardens.) (ii) A-2: 2% low-endotoxin sodium alginate solution and 100 mM potassium chloride A technique in which calcium solution is injected into the area where the nucleus pulposus is partially removed at the same time in a 1:1 ratio. (The entire sodium alginate hardens.) (ii) Low-endotoxin sodium alginate solution and calcium chloride solution Each was placed in a separate syringe and injected simultaneously into the defect in the nucleus pulposus of the intervertebral disc using a 22G needle. This was carried out by

[0154] As a result, when the method (i) was performed on the intervertebral discs of sheep cadavers, the injection technique was difficult. On the other hand, method (ii) uses a low-endotoxin sodium alginate solution and It was thought that the reproducibility of achieving a uniform mixing ratio with calcium chloride solution was low. In addition, if part of the space gels during filling, the remaining space can be filled with gel without any gaps. Furthermore, it was unclear whether the technique (ii) could be used to fill the nucleus pulposus of the intervertebral disc with hardened gel. After filling, when compressive force is applied to the disc from the cranial and caudal sides, the hardened gel is released from the holes on the side of the disc where the gel was injected. In the case of method (i), the gel did not escape outside the intervertebral disc. It was.

[0155] Compared with method (ii), method (i) can reduce the calcium concentration in the intervertebral disc and The advantage of method (ii) is that the hardened gel penetrates the spinal column. If the needle were to protrude into the tube, there is a risk of serious nerve damage. The risk of this is low.

[0156] From the above, method (i), i.e., applying low-endotoxin sodium alginate solution to the vertebrae, The technique of injecting calcium chloride solution into the nucleus pulposus of the disc and then applying it to the surface of the disc is This study suggests that the use of sodium alginate toxin is a suitable method for nucleus pulposus implantation.

[0157] 3-(2) Mechanical testing using sheep cadavers 3-(1) The sheep cadaver intervertebral disc (low end of A-2) prepared by the method (i) After injecting a 2% solution of sodium alginate into the area of ​​the partially removed nucleus pulposus of the intervertebral disc, (100 mM calcium chloride solution was contacted near the hole of the injection needle) Approximately 1 hour after the procedure, an Instron 5943 (Instron) was used to inject the -3 Repeated compression and extension forces of 1000 to 300N were applied to the specimen. The sodium alginate solution was then injected into the hole, and the hole was observed for any deviation. The sodium glutamate solution was stained with 0.05% toluidine blue for improved visibility. We used the following.

[0158] As a result, no leakage of the sodium alginate solution from the injection port to the outside of the intervertebral disc was observed. Therefore, after injecting the sodium alginate solution into the nucleus pulposus of the intervertebral disc, The method of hardening the area with a cross-linking agent is to change the position after filling with sodium alginate solution. It has been suggested that this filling method can withstand the compressive and tensile forces exerted on the intervertebral discs by walking, etc. Ta.

[0159] Example 4: Examination of the properties of low-endotoxin sodium alginate solution after injection After injecting a low-endotoxin sodium alginate solution into the nucleus pulposus of the intervertebral disc, The calcium chloride solution is brought into contact with the sodium phosphate solution inlet and the contact area is hardened. When the low-endotoxin sodium alginate solution was injected, how did it act in the nucleus pulposus of the intervertebral disc? To predict what properties the compound might have, the following in vitro studies were carried out.

[0160] 4-(1) Test method Using the following three methods, X, Y, and Z, alginate was added to a micro test tube (diameter 6 mm, height 25 mm). Add sodium solution to each test tube and let it stand on its side for 1 hour, 24 hours, and 48 hours. After one week and one week later, the properties of the test substance in the test tube were evaluated. In the extraction model, the annulus fibrosus was excised to a size of 5mm x 3mm and the nucleus pulposus was removed. The size of the 6mm diameter tube is relatively close to that of the ivo test tube and can be manipulated in a test tube. A micro test tube was selected. The low-endotoxin sodium alginate of Example 2, A-2, was used. The test was carried out at room temperature (20°C) after dissolving the drug in saline to prepare a 2 w / v% solution.

[0161] [Table 4]

[0162] 4-(2) Results The 2 w / v% sodium alginate solution used in the test had a higher viscosity than saline. When the vial is tilted, the liquid level moves slowly. I was able to smoke it with it on.

[0163] The characteristics of group X were almost the same from 1 hour to 1 week after administration. The surface area of ​​the sodium glutamate solution was gel-like for about 2-3 mm, but the rest of the solution was solid. No crust was observed and the sol was in a sol state. Most of the sol-like part was It was possible to aspirate the solution with a syringe, but compared with the 2 w / v% sodium alginate solution, The viscosity was high and it took a long time to suction.

[0164] The characteristics of group Y were almost the same from 1 hour to 1 week after administration. The area had turned into a jelly-like gel, and a small amount of water-like liquid was observed in some areas. It was speculated that this was a "syringe" phenomenon, where the liquid separates and seeps out due to shrinkage. It was not possible to aspirate the fluid using a syringe with a needle attached.

[0165] The characteristics of group Z were almost the same from 1 hour to 1 week after the procedure. A cloudy white gelatinous mass was formed in some parts, while the rest was a watery liquid. The gelled portion was aspirated using a syringe with a 21G needle. It was impossible to do so.

[0166] As described above, the method of group X is a method that imitates the injection method into the nucleus pulposus of the intervertebral disc performed in the example of the present invention. When sodium alginate solution was injected into the nucleus pulposus of the intervertebral disc, sodium alginate The gel solution was predicted to exist in a sol state. On the other hand, both groups Y and Z existed in a gel state. As confirmed in Example 3 of the present invention, after filling the nucleus pulposus with gel, When compressive force is applied from the side, the hardened gel may come off from the side of the intervertebral disc into which the gel was injected. There were concerns about the possibility of this happening.

[0167] Example 5: Low-endotoxin sodium alginate solution in sheep intervertebral disc nucleus pulposus defect model Application of A low-endotoxin sodium alginate solution was administered to a sheep intervertebral disc nucleus pulposus defect model. The effect was evaluated using seven sheep (male, Suffolk breed) weighing 40 kg to 60 kg. The following evaluations were performed using the L1 / 2, L2 / 3, L3 / 4, and L4 / 5 intervertebral discs.

[0168] 5-(1) Creation of a sheep intervertebral disc nucleus pulposus defect model The sheep were anesthetized, and the intervertebral disc was exposed using an electric scalpel. mm x 3 mm, and then forceps were inserted through the hole to remove 0.10 g of nucleus pulposus. A nucleus pulposus defect model was prepared. The size of the annulus fibrosus resection and the amount of nucleus pulposus removed were determined in advance. The size of the annulus fibrosus resection was determined by a sheep disc degeneration test using nucleotomy. The results were analyzed using 5mm x 3mm and 10mm x 3mm, and the extent of disc degeneration was evaluated according to the amount of nucleus removed. The 5mm x 3mm diameter was selected because progression was observed. The amount of nucleus removed was 0.02g, 0.05g, and 0. The amount of nucleus pulposus extracted from sheep, 0.1g, is equivalent to 1g in humans. This is 1.2g, which is closest to the amount of nucleus pulposus extracted in human clinical practice. Selected.

[0169] 5-(2) Filling of low-endotoxin sodium alginate solution A-2 of Example 2, low-endotoxin sodium alginate, was dissolved in Milli-Q water at 2 w / v%. The solution was prepared, and 0.10 ml of the solution was injected into the ovine intervertebral disc nucleus pulposus defect prepared in 5-(1) using a syringe. The surface of the injected sodium alginate solution was coated with 102 mM calcium chloride. After leaving it for about 5 minutes, the area where the calcium chloride solution was applied was covered with saline. The lesion was washed with saline and sutured. This was the treatment group (n=11). The group in which only nucleotomy was performed and sutured was designated the nucleotomy group (n = 10). The plates served as normal control group (n=7). Four weeks after surgery, the animals were euthanized by an overdose of pentobarbital, and the lumbar spine was resected and the intervertebral disc tissue was removed. was recovered.

[0170] 5-(3) Histological evaluation According to the description of 2-(5) above, HE staining and Safranin O staining were performed to identify the tissue markings of the intervertebral disc. A revised classification based on the Boos classification (Eur Spine J. 2014 Jan; 23(1):1 The degree of degeneration of the intervertebral disc was evaluated using the MRI scan (9-26. Spine 2002 Vol.27, No.23 p.2631-2644). The classification is shown in Table 5. The maximum score for the intervertebral disc items is 20 points, and the maximum score for the vertebral endplate items is 16 points. The total was scored out of a maximum of 36 points.

[0171] [Table 5]

[0172] The severity of disc degeneration was evaluated histologically, and the results showed that the nucleotomy group had a significantly higher severity of disc degeneration than the non-nucleotomy group at 4 weeks after surgery. , and the treatment group had significantly higher scores according to the classification in Table 5 compared to the normal control group. However, the treatment group showed significantly higher values ​​than the nucleotomy group, suggesting degeneration. The core was significantly lower, and degeneration was suppressed (Figure 8).

[0173] In addition, the disc height index (DHI) was used to evaluate the disc height. The disc height index was calculated by dividing the disc height (average of the anterior, middle, and posterior disc heights) by the mean disc height. The mean value was divided by the anterior-posterior diameter of the disc (Eur Spine J. 2014, 23(1):19-26.). .

[0174] As a result, the disc height index in the nucleotomy group was significantly higher than that in the normal control group. On the other hand, the disc height index in the treatment group was significantly lower than that in the normal control group. This suggests that low-endotoxin sodium alginate It was found that filling the nucleus pulposus with the solution prevented the loss of disc height caused by nucleotomy. (Figure 9).

[0175] 5-(4) Immunohistological evaluation The tissue specimens prepared in 5-(3) above were treated with anti-Type I collagen antibody and anti-Type II collagen antibody. Immunohistological staining was performed using collagen antibody in randomly selected nucleus pulposus of the intervertebral disc. The number of positive cells was counted in five fields.

[0176] As a result, at 4 weeks after surgery, the number of cells in the sections was significantly increased by anti-Type I collagen antibody. The percentage of positive cells was less than 10% in the normal control group, whereas it was 10% in the nucleotomy group and The treatment group showed significantly higher values. However, the treatment group showed significantly higher values ​​than the nucleotomy group. The proportion of cells positive for anti-Type I collagen antibody was significantly lower.

[0177] In addition, 4 weeks after surgery, the number of cells in the sections was positive for anti-Type II collagen antibodies. The percentage of cells was approximately 60% in both the normal control group and the treatment group, with no significant difference. On the other hand, the nucleotomy group showed approximately 40%, which was significantly higher than the normal control group. There were significant differences compared with the control group and the treatment group (Fig. 10). In the present study, the percentage of anti-Type II collagen antibody positive cells was decreased compared to normal, but the low end By filling the patient with sodium alginate solution containing toxin, the normal control group was achieved 4 weeks after surgery. It was shown that the recovery was comparable to that of the control group.

[0178] From the above results, application of low-endotoxin sodium alginate solution to the nucleus pulposus is effective for the intervertebral It was shown that the degeneration of the nucleus pulposus was inhibited and regeneration was promoted. Considering all of these factors, the composition of the present invention is particularly suitable for filling the nucleus pulposus after discectomy. In addition, the treatment group showed a decrease in the disc height index due to nucleotomy. As a result, it is possible to prevent and treat the degeneration of the discs adjacent to the treated disc. It was suggested that this may have the potential to cause and / or reduce the risk of pulmonary embolism.

[0179] Example 6: Examination of the composition of ovine intervertebral disc nucleus pulposus The main components of the extracellular matrix of the nucleus pulposus of the intervertebral disc are water, type II collagen, and protein. It is a glycosaminoglycan that has a stronger affinity to collagen than other cartilage tissues such as intervertebral disc endplates and articular cartilage. It is said that the ratio of proteoglycans to collagen is high. The ratio of glycans was calculated by dividing the ratio of sulfated glycosaminoglycans by the ratio of hydroxyproline (HYP). There is a paper that looked at the ratio of sulfated glycosaminoglycans (GAG) (European Cells and Materials Vol.8. 2004 p.58-64). A low-endotoxin sodium alginate solution was administered to a sheep intervertebral disc nucleus pulposus defect model. After filling, biochemical analysis of the nucleus pulposus tissue was performed and evaluated 4 weeks after surgery according to the above literature. .

[0180] 6-(1) Method The L1 / 2 and L2 intervertebral discs of two male Suffolk sheep weighing 35-60 kg were The sheep intervertebral discs were used for the study according to Example 5. A nucleus pulposus defect model was prepared and filled with 2 w / v% low-endotoxin sodium alginate solution. These were treated (n=4). In accordance with Example 5, only the intervertebral disc nucleus pulposus was removed and sutured. The group was designated as the nucleotomy group (n = 4). The normal control group (n=4) was used. Four weeks after surgery, the nucleus pulposus of the intervertebral disc and the intact femur were Cartilage tissue (articular cartilage) was collected from both sides of the bone, and after pretreatment of the sample, sulfated glycosaminoglycans were analyzed. Glycans (GAGs) and hydroxyprolines (HYPs) were measured.

[0181] The sample was pretreated by freeze-drying the sample, measuring the dry weight, and then adding 1 mL of protease inhibitor to the dried sample. Enzyme solution (pronase (Calbiochem) at a concentration of 1 mg / mL) in 20 mM HEPES buffer ( The mixture was digested at 60°C for 3 hours with stirring every hour. After centrifugation, the resulting supernatant was used as the stock solution for the measurement sample, which was then stored in a refrigerator until the time of measurement.

[0182] Sulfated glycosaminoglycans (GAGs) were measured using the Wieslab® sGAG quantitation system. The amount of GAG was measured using the e kit (Euro Diagnostica, product number: GAG201RUO) according to the instruction manual. The measurement sample stock solution was diluted 100 times with purified water, and the resulting diluted solution was used as the measurement sample. The rank sample was a 100-fold dilution of pronase solution. The measurement wavelength was 620 nm, and the ch The amount of GAG in the sample was calculated using a calibration curve prepared using cysteine ​​6-sulfate (CS-6) as the standard. did.

[0183] Hydroxyproline (HYP) was measured as follows: The stock solution for measurement was purified. The diluted solution was used as the measurement sample. 50 μL of the measurement sample was placed in a vial for hydrolysis, and the same amount of concentrated hydrochloric acid was added. After sealing, the container was hydrolyzed at 120°C for 16 hours. Three hydrolysis samples were prepared per sample. 20 μL of the hydrolyzed sample and 100 μL of the HYP standard solution were placed in a well plate. The sample was dried under reduced pressure at 0°C for 15 hours. 100 μL of purified water was added to the dried sample. According to the method of Woessner (Woessner JF Jr, Arch Biochem Biophys, 93, (1961) p.440-447) Color was developed and the absorbance at 557 nm was measured. The dilution ratio of the measurement sample during hydrolysis and color development was 1. 0 times, the total dilution ratio is 1000 times. The amount of HYP in the sample was calculated.

[0184] 6-(2) Results The amount of GAG and HYP per dry weight of the specimen (μg / mg dry weight) was obtained, and the ratio of GAG to HYP was calculated. The ratio of AG (GAG / HYP) was calculated. The mean and standard deviation were calculated for each group (n = 4) (Table 6 ) The scatter plots for each group are shown in Figure 11.

[0185] [Table 6]

[0186] As a result, the average GAG / HYP value of the normal control group (intervertebral disc nucleus pulposus) was 17.8, and At 4 weeks after surgery, the GAG ​​values ​​of the nucleotomy group and the treatment group were The mean values ​​of / HYP were decreased compared with the corresponding values ​​in the normal control group. Compared with the enucleation group, the mean GAG / HYP values ​​tended to be slightly higher. The nucleus is richer in sulfated glycosaminoglycans (GAGs) / hydroxyproline than articular cartilage. The (HYP) value is high, meaning that the ratio of components that make up the tissue is different, and the nucleus pulposus of the intervertebral disc and the articular cartilage are It was suggested that the tissue characteristics are different between the two. It was thought that there was a possibility of restoring the composition.

Claims

1. It is applied to the nucleus pulposus area of ​​the subject and hardens the area after application. A method for treating intervertebral discs, comprising the step of forming a monovalent metal salt of low-endotoxin alginic acid, which is fluid when used. A composition for filling a nucleus pulposus.

2. Curing the composition includes contacting at least a portion of the surface of the composition with a crosslinking agent. The composition of claim 1 .

3. The composition is applied to the nucleus pulposus site through a composition filling port on the surface of the intervertebral disc. curing a portion of the composition by contacting a crosslinking agent with the filling port of the composition on the disc surface; The composition according to claim 1 or 2.

4. The method and use of the crosslinking agent in which the hardening of a portion of the composition is similar to filling the nucleus pulposus site Using the ratio, a low-enriched 6 mm diameter test tube was prepared in vitro according to Example 4 of the present specification. After filling the tube with 500 μL of sodium alginate containing doxorubicin and a cross-linking agent and leaving it to stand for 1 hour, the test tube was At least 50% of the composition volume in the test tube can be aspirated using a syringe with a 21G needle. The composition according to any one of claims 1 to 3, wherein

5. The composition is applied to the nucleus pulposus site by removing at least a portion of the nucleus pulposus. The method according to any one of claims 1 to 4, wherein the composition is applied to a nucleus-defective part. Finished product.

6. The apparent viscosity of the fluid composition was measured at 20°C using a cone-plate viscometer. The viscosity of any one of claims 1 to 5 is 100 mPa·s to 30,000 mPa·s when measured under the conditions. The composition according to any one of claims 1 to 4.

7. The monovalent metal salt of the low-endotoxin alginic acid was measured by GPC-MALS method. The weight average molecular weight (absolute molecular weight) of the polymer according to any one of claims 1 to 6 is 80,000 or more. Composition of.

8. The composition has a concentration of a monovalent metal salt of low-endotoxin alginic acid of 0.5 w / w% to 5 The composition according to any one of claims 1 to 7, wherein the composition is in a w / w% range.

9. The composition may be provided with an amount of crosslinking agent that hardens the composition prior to application to the nucleus pulposus site of the subject. The composition according to any one of claims 1 to 8, which does not contain an agent.

10. The fluid composition was allowed to stand at 20° C. for 1 hour, and then injected with a 21G syringe. The composition according to any one of claims 1 to 9, which has flowability that allows it to be poured into a container.

11. The composition of any one of claims 1 to 10, wherein the composition does not contain cells.

12. The crosslinking agent according to any one of claims 2 to 11, wherein the crosslinking agent is a divalent or higher metal ion compound. composition.

13. The composition is used for the treatment, prevention or suppression of recurrence of intervertebral disc degeneration and / or intervertebral disc damage. The composition according to any one of claims 1 to 12, which is used for

14. The disc degeneration and / or disc damage may be a herniated disc, disc disease, degenerative spinal syndrome, or the like. vertebral disc injury, spondylosis deformans, spinal canal stenosis, and intervertebral disc injury. The composition according to claim 13, wherein the composition is at least one selected from the group consisting of:

15. Claims 1 to 14, wherein the composition is in a dry state or in a solution state before application to the nucleus pulposus site. The composition according to any one of the preceding claims.

16. The dry monovalent metal salt of low-endotoxin alginic acid is a freeze-dried product. Item 16. The composition according to item 15.

17. 17. A composition comprising at least the composition according to claim 1 and a crosslinking agent. A kit for replacing the nucleus pulposus of an intervertebral disc.

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