WO2007080898A1

WO2007080898A1 - Sustained-release hydrogel preparation

Info

Publication number: WO2007080898A1
Application number: PCT/JP2007/050180
Authority: WO
Inventors: Yasuhiko Tabata; Mahito Hirai
Original assignee: Medgel Corporation; Institute Of Gene And Brain Science
Priority date: 2006-01-10
Filing date: 2007-01-10
Publication date: 2007-07-19
Also published as: JP2007182407A

Abstract

It is intended to provide a sustained-release hydrogel preparation containing a cationic gelatin hydrogel and galectin-1 or a galectin-1 derivative. The sustained-release hydrogel preparation of the invention can control the elution of galectin-1 or the galectin-1 derivative in vivo in a mammal. In the cationized gelatin hydrogel, the substitution ratio of carboxyl groups with amino groups in the gelatin hydrogel may be 10% or more and 60% or less. Further, the water content of the cationized gelatin hydrogel may be 80% or more and 99.8% or less.

Description

Specification

Sustained release Hyde mouth gel formulation

Technical field

[0001] The present invention relates to a sustained-release nose-mouth gel preparation containing galectin 1 or a galectin 1 derivative.

Background art

[0002] In order to keep the drug concentration in a living body constant for a long period of time, a method is known in which the release is controlled by encapsulating the drug in a hydose gel or a microcapsule of a bioabsorbable polymer. . Many types of natural or synthetic polymers such as collagen, gelatin, polylactic acid, polydaricholic acid, and polyγ-glutamic acid have been reported as bioabsorbable polymers used for such purposes. .

[0003] For example, Patent No. 2702729 (Patent Document 1) discloses two types of matrices, a matrix formed by blending a biodegradable polymer with a physiologically active substance, and a matrix composed of a biodegradable polymer alone. Sustained release implants are disclosed that are layered or adjacent.

[0004] JP 2005-511523 (Patent Document 2) includes at least one polycationic polymer complexed with at least one first negative electropharmacologically active agent and is administered to a patient. A sustained release drug delivery composition configured to release the at least one first negatively charged pharmacologically active agent slowly. This document states that the release of a negatively charged (negatively charged) therapeutic agent in its entirety is controlled by the charge interaction between the polycationic polymer and the therapeutic agent. Also described is that this system can be used for the sustained release of negatively charged hydrophilic drugs such as negatively charged oligonucleotides and other active agents having negatively charged peptides and proteins. Have

[0005] In addition, International Publication No. 00-06724 (Patent Document 3) discloses a therapeutic agent for neurological disorders including nerve injury, neurodegeneration, and nerve transplant function decline, comprising galectin 1 or a derivative thereof as an active ingredient. ing. According to this document, this therapeutic agent contains galectin 1 or a derivative thereof in a collagen gel and, if necessary, other neurotrophic factors. It may be added and embedded directly in the neuropathy local area. In this case, necessary components such as a drug and a carrier are enclosed in a tube having a biocompatible material (eg, silicone rubber, collagen, polypropylene, polyester, polyamide, etc.).

However, the prior art described in the above literature has room for improvement in the following points.

[0007] First, with the sustained-release implant of Patent Document 1, when galectin 1 is encapsulated, galectin 1 is eluted immediately in the body, making it difficult to achieve sustained release. It was.

[0008] Second, in the sustained release drug delivery composition of Patent Document 2, the release of the negatively charged therapeutic agent is controlled by the charge interaction between the polycationic polymer and the therapeutic agent. Although the interaction between the negatively charged therapeutic agent and the polycation polymer is not uniquely determined solely by the electrical interaction, various factors such as the size of the molecule and the three-dimensional structure are complicated. It is determined by intertwining. Therefore, even in the sustained release drug delivery composition of Patent Document 2, when galectin 1 is encapsulated, it is difficult to control the elution of galectin 1 in vivo, and it is still difficult to realize sustained release. It was.

[0009] Thirdly, in the therapeutic agent for neuropathy of Patent Document 3, galectin 1 or a derivative thereof is contained in a collagen gel, and if necessary, other neurotrophic factor is added and directly embedded in the neuropathy locally. Since it was in a form and no special treatment was applied to control the elution of galectin 1, it was still difficult to achieve sustained release.

[0010] References cited in the present specification are as follows. All the contents described in these documents are cited herein as part of this specification. Any of these documents is not admitted to be prior art to this specification.

Patent Document 1: Japanese Patent No. 2702729

Patent Document 2: Special Table 2005—511523

Patent Document 3: International Publication No. 00Z06724

Disclosure of the invention

Problems to be solved by the invention

[0011] The present invention has been made in view of the above circumstances, and a galectin in a living body of a mammal.

Controlled release nose mouth gel that can control elution of 1 or galectin 1 derivatives The purpose is to provide a formulation.

Means for solving the problem

[0012] According to the present invention, there is provided a sustained-release hide-mouth gel formulation comprising a cationic gelatinoid mouth gel and galectin 1 or a galectin 1 derivative. According to the present invention, since galectin 1 or galectin 1 derivative interacts with a cationic gelatinoid mouth gel, elution of galectin 1 or galectin 1 derivative can be controlled in the living body of mammals. A sustained-release hide-mouth gel preparation is obtained.

In the present invention, the galectin 1 derivative refers to a compound produced by a change in a small part of the galectin 1 molecule. Galectin 1 derivatives include mutant galectin-1 formed by substituting, deleting, or adding one or more amino acid residues in the amino acid sequence of galectin 1.

The invention's effect

[0014] According to the present invention, since the cationic gelatinoid mouth gel is used, the sustained-release hyde mouth can control the elution of galectin 1 or galectin 1 derivatives in the living body of mammals. A gel formulation is obtained.

Brief Description of Drawings

FIG. 1 is a graph showing the results of an in vitro sustained release test.

FIG. 2 is a graph showing the results of an in vivo short-term sustained release test (first time).

FIG. 3 is a graph showing the results of an in vivo short-term sustained release test (second time).

FIG. 4 is a graph showing the results of an in vivo sustained release test.

BEST MODE FOR CARRYING OUT THE INVENTION

[0016] In one embodiment of the sustained-release nose-mouth gel formulation of the present invention, the cationic gelatin-hyde mouth gel described above has 10% or more and 60% or less of carboxyl groups in the gelatin-hide mouth gel to amino groups. May be substituted. According to this configuration, the strength of interaction as a total including electrostatic action between galectin 1 or galectin 1 derivative and cationic gelatino or id mouth gel is within a preferable range. Elution rate of galectin 1 or galectin 1 derivative in the mammalian body It will be within the range suitable for the therapeutic purpose.

[0017] In another embodiment of the sustained-release hide-mouth gel preparation of the present invention, the cationic gelatin-hide mouth gel may have a water content of 80% or more and 99.8% or less. According to this configuration, the elution rate of galectin 1 or galectin 1 derivative in the mammal's living body falls within a range suitable for therapeutic purposes such as mammalian nerve regeneration.

[0018] In one embodiment, in the sustained-release nose-mouth gel preparation of the present invention, the aforementioned galectin-11 or galectin-11 derivative may comprise recombinant galectin-11. Here, the recombinant galectin 1 refers to a galectin 1 produced by a genetic recombination technique that does not use natural galectin 1.

[0019] In another embodiment, in the sustained release nose mouth gel preparation of the present invention, the above-described galectin 1 derivative is substituted, deleted, or added to one or more amino acid residues in the amino acid sequence of galectin 1. The mutant galectin 1 may be included.

[0020] In another embodiment, the above-described galectin 1 derivative contains a part of galectin 1 or a part of galectin 1 derivative. But you can. For example, as a part of galectin 1 or a part of galectin 1 derivative, a part of galectin 1 or galectin 1 derivative that is conventionally known to be important for physiological activity can be configured.

[0021] In another aspect, the sustained-release hide-mouth gel formulation of the present invention may be configured to be administered in the vicinity of the spinal cord of a mammal. For example, the sustained-release hide-mouth gel preparation can be configured to have a fluidity that can be filled using an osmotic pump with respect to an injury site of a mammalian spinal cord. Further, by preparing a sustained-release hyde mouth gel preparation using a gelatinoid mouth gel processed into a granular form, it can be configured to be injected onto the damaged site of the spinal cord of a mammal.

Hereinafter, embodiments of the present invention will be described.

[0023] <Gelatin Hyde Mouth Gel>

In this specification, the hyde mouth gel refers to a gel in which an aqueous liquid is held inside a polymer cross-linked structure. Gelatin nose mouth gel is a gel-like substance obtained by hydrating gelatin, and is a kind of bioabsorbable polymer hide mouth gel. More details The raw material for gelatinoid mouth gel is gelatin, which is a protein obtained by heat-denaturing a substance called collagen contained in animal bones and skin and highly purified.

Here, the gelatin derivative has a hydrophobic property such as guazyl group, thiol group, amino group, carboxyl group, sulfate group, phosphate group, alkyl group, acyl group, and benzyl group with respect to gelatin. It includes modified gelatin into which residues and hydrophobic substances with low molecular weight have been introduced.

[0025] Naturally derived gelatin is preferably used as gelatin. Naturally-derived gelatin is a collagen that can be collected from all parts of the body, such as skin, bones, and tendons of various animal species, including cattle, pigs, and fish. It can be obtained by modification by various treatments such as alkaline hydrolysis, acid hydrolysis, and enzymatic decomposition. As the gelatin derivative, modified gelatin of recombinant collagen may be used.

[0026] In a preferred embodiment of the present invention, a positively charged gelatin or id mouth gel is used. The inventor has found that the positively charged gelatin nodule gel force interacts better with galectin-1 or galectin-1 derivatives. The positive charge of the gelatin hydrate gel plays an important role in forming a stable complex with galectin 1 or galectin 1 derivatives. In order to positively charge the gelatin nose mouth gel, for example, it can be cationized by previously introducing an amino group or the like into gelatin. As a result, the binding force between the gelatin hydrate gel and the drug is increased, and a more stable gelatin hydrate gel complex can be formed.

[0027] The step of cationization is not particularly limited as long as it is a method capable of introducing a functional group that becomes cationized under physiological conditions, but a 1, 2 or tertiary amino group is added to the hydroxyl group or carboxyl group of gelatin. Alternatively, a method in which the ammonia group is introduced under mild conditions is preferable. For example, ethylenediamine, N, N-dimethyl-1,3-diaminopropane and other alkyldiamins, trimethylammo-muacetohydrazide, spermine, spermidine or cetylamide hydrochloride, etc. are used as various condensing agents such as 1-ethyl-3. — (3 Dimethylaminopropyl) carbodiimide hydrochloride, cyanuric chloride, N, N, —carbodiimidazole, cyanogen bromide, diepoxy compound, tosyl lipide, jettil triamine— N, N, Ν ', Ν ", N" There is a method of reacting with gelatin using a dianhydride compound such as pentanoic acid dianhydride, trisilk mouthlid or the like. Of these, the method of reacting ethylenediamine or spermidine is preferred because it is simple and versatile.

[0028] At this time, the cation ratio can be used as an index indicating the degree of cation of the cation gelatin. Cationization rate = (number of amino groups per molecule of cation gelatin) Z (number of amino groups per gelatin molecule before cation) Z (number of carboxyl groups per gelatin molecule before cation) * 100 It is expressed in (%).

[0029] The cation ratio is preferably 10% or more and 60%, particularly preferably 30% or more and 50% or less. If the cationic rate is lower than these lower limits, the interaction between the gelatinoid mouth gel and galectin 1 or galectin 1 derivative is weakened. On the other hand, if the cationic rate power S exceeds these upper limits, the cationization of the gelatin nozzle gel may become difficult in the production process.

[0030] In the present invention, in order to obtain a better sustained release control effect of the drug, it is preferable to make the gelatin nozzle gel insoluble or sparingly soluble in water. This makes it possible to freely control the release of the drug according to the biodegradability of the gelatin hydrogel in the living body. In other words, the sustained release rate of the drug can be controlled by decomposing gelatino or id mouth gel in the living body.

[0031] In order to make gelatin hydrate mouth gel water-insoluble or sparingly water-soluble, the molecular chain of gelatin or gelatin derivative is made difficult to dissolve in water by dehydration by chemical method, ultraviolet irradiation or heat. Crosslink. At this time, as the degree of crosslinking increases, the weight ratio (water content) of water in the hydrous hydrate gel decreases.

[0032] Gelatin nodule mouth gels can be insolubilized or hardly soluble by forming chemical crosslinks between molecules of gelatin or gelatin derivatives using various chemical crosslinkers. Examples of the chemical cross-linking agent include water-soluble carbodiimides such as dartal aldehyde such as EDC, such as propylene oxide, diepoxy compounds, hydroxyl groups, carboxyl groups, amino groups, thiol groups, and imidazole groups. An agent can be used. Preference is given to dartalaldehyde. The bioabsorbable polymer may be chemically cross-linked by thermal dehydration treatment, ultraviolet rays, gamma rays, or electron beam irradiation. More In addition, these crosslinking treatments may be used in combination. In addition, the hyde-mouth gel may be prepared by physical crosslinking using salt crosslinking, electrostatic interaction, hydrogen bonding, hydrophobic interaction, and the like.

[0033] In the present invention, when a complex of gelatin hydrate gel and a drug is formed, the drug incorporated in the complex is in accordance with the degradation of the gelatino and id lip gel in vivo. It is gradually released out of the complex. This release rate is determined by the degree of degradation and absorption in the body of the gelatin hydrate gel used, and the strength and stability of the bond between the drug and the gelatinogel in the complex. The degree of degradation and absorption of gelatino and id mouth gels in the living body can be adjusted by adjusting the degree of cross-linking during the preparation of the hide mouth gel.

[0034] When gelatin or a gelatin derivative is used as a raw material for gelatin nose mouth gel, the degree of cross-linking of the hard mouth gel can be evaluated using the water content as an index. The water content is the weight percent of water in the hyde mouth gel with respect to the weight of the swollen hide mouth gel. If the moisture content is high, the degree of cross-linking of the hyde mouth gel is low. Therefore, specifically, the water content showing a preferable sustained release effect is 80 wt% or more and 99.8 wt% or less, particularly preferably 90 wt% or more and 97 wt% or less, and most preferably 92 wt% or more and 97 wt%. It is as follows. If the water content of gelatin or id mouth gel is higher than these upper limits, it becomes difficult to control the release rate of galectin 1, and the desired sustained release effect cannot be obtained.

[0035] Since gelatin or a gelatin derivative is used as the bioabsorbable polymer, the preferred range of the concentration of gelatin or the gelatin derivative and the crosslinking agent in preparing the hydrogen is a concentration of gelatin or gelatin derivative of 1 to 20 wZw%, Agent concentration: 0.01-: LwZ w%. There are no particular limitations on the crosslinking reaction conditions. For example, the reaction can be performed at 0 to 40 ° C, preferably 25 to 30 ° C, for 1 to 48 hours, preferably 12 to 24 hours. In general, as the concentration of gelatin or gelatin derivative, the concentration of crosslinking agent, and the crosslinking time increase, the degree of crosslinking of the hard mouth gel increases and the bioabsorbability decreases.

[0036] The cross-linking reaction of gelatin or gelatin derivatives can also be performed by heat treatment. Examples of crosslinking by heat treatment are as follows. A gelatin film is obtained by casting an aqueous gelatin solution (preferably about 10% by weight) onto a plastic petri dish and air drying. The The film is allowed to stand under reduced pressure, preferably about 110 mmHg, usually 110 to 160 ° C, preferably 120 to 150 ° C, usually 1 to 48 hours, preferably 6 to 24 hours. When the gelatin film is cross-linked by ultraviolet rays, the obtained gelatin film is usually left at room temperature, preferably 0 to 40 ° C. under a sterilizing lamp. In addition, a sponge-like molded body is obtained by freeze-drying a gelatin aqueous solution. This can likewise be crosslinked by heat treatment and UV, gamma and electron beams. Alternatively, the above-mentioned crosslinking methods can be used in combination.

[0037] The shape (three-dimensional structure) of the gelatin hydrated gel for encapsulating galectin 1 or galectin 1 derivatives is not particularly limited, but for example, cylindrical, prismatic, sheet, disc, spherical, paste There is a shape. Columns, prisms, sheets and discs are particularly suitable for use as embedded pieces.

[0038] A cylindrical, prismatic, sheet, or disk-shaped gelatinoid mouth gel is obtained by adding an aqueous solution of a crosslinking agent to an aqueous solution of gelatin or a gelatin derivative, or by adding gelatin or a gelatin derivative to an aqueous solution of a crosslinking agent. It can be prepared by adding it, pouring it into a bowl having a desired shape, and causing a crosslinking reaction. Further, an aqueous crosslinking agent solution may be added to the formed gelatin gel as it is or after drying. To stop the cross-linking reaction, add the ability to contact low molecular weight substances with amino groups such as ethanolamine and glycine, or an aqueous solution with a pH of 2.5 or lower. In order to completely remove the cross-linking agent and low-molecular substances used in the reaction, the obtained gelatinoid mouth gel was washed with distilled water, ethanol, 2-propanol, acetone, etc. and used for preparation of the preparation. The

[0039] The gelatin-hide mouth gel of the present invention can be appropriately used after being cut into an appropriate size and shape, lyophilized and sterilized. For lyophilization, for example, place gelatin-hide mouth gel in distilled water and freeze in liquid nitrogen for 30 minutes or more, or at 80 ° C for 1 hour or more, and then dry in a freeze dryer for 1 to 3 days. This can be done.

[0040] The gelatin-hide mouth gel of the present invention comprises galectin 1 or galectin to be released slowly.

It is possible to form a complex with one derivative by physicochemical interaction. As a result, the gelatin hydrated gel of the present invention is degraded by hydrolysis and oxygen degradation in the living body, or by the action of biologically active substances such as enzymes. It is hydrolyzed.

[0041] <Galectin 1 or Galectin 1 derivative>

The drug used for producing a sustained-release preparation in the present invention is galectin-1 or galectin-1 derivative. Galectin-1 is a cytoplasmic protein belonging to the galectin family. Of these, natural galectin 1 is particularly preferred. The galectin-1 derivative includes mutant galectin-1 obtained by substituting, deleting, or adding one or more amino acid residues in the amino acid sequence of natural galectin-1. Galectin-1 derivatives also include recombinant galectin-1 obtained using a gene recombination technique. When administered to humans, natural human galectin-11 or recombinant human galectin-11 is preferred.

[0042] Further, the galectin 1 derivative includes a part of galectin 1 or a part of galectin 1 derivative and having a physiological activity similar to that of galectin 1. Thus, even if a part of the region of galectin 1 or galectin 1 derivative is deleted, if a region known to be important for the physiological activity of galectin 1 remains, galectin- Since it exhibits the same function as 1, it can be used for therapeutic purposes such as nerve regeneration of the spinal cord of mammals.

[0043] Galectins are a family of animal lectins. Galectins are known to have binding specificity for galactose and to characterize the galectin family, and have a predetermined primary amino acid sequence. Galectins are generally soluble and have no metal requirements. Galectins exhibit properties as cytoplasmic proteins, do not have disulfide bonds, additional sugar chains, or signal sequences, and generally have an N-terminal amino acid acetylated. However, the location of galectin expression is not limited to the cytoplasm, but varies with the nucleus, cell surface, and extracellular matrix, and often varies depending on the type of galectin molecule and the tissue and timing of expression.

[0044] There are many unclear points regarding the secretory mechanism of galectin, and a novel secretion model that does not involve a signal sequence is assumed. To date, galectins are known to be widely distributed in vertebrates as well as invertebrates such as nematodes, insects, and sponges, but recently they have galactose binding activity in fungi (mushrooms). The existence of galectins Yes. The life phenomena involved in galectins include development, differentiation, morphogenesis, tumor metastasis, cell death, pricing, etc., but unresolved parts are related to the mechanism of functional expression, particularly in relation to sugar chain recognition. There are many.

[0045] As reported to date, galectins can be classified into three types: proto, chimera, and tandem repeat type, based on the molecular construction mode. On the other hand, it has been proposed that mammalian galectins be called with numbers assigned in order of discovery (order of registration with GenBank). The galectin family has been reported to date with galectin-1-14.

[0046] Galectin 1 is an animal lectin that binds to β-galactosid. Galectin 1 has 6 cysteines in the molecule and exhibits lectin activity only in the reduced state. It has been reported that it is involved in cell adhesion and cell proliferation by binding to glycolipids and glycoprotein sugar chains on the cell surface by lectin activity. Galectin 1 belongs to the above proto type. As functions of galectin 1, for example, apoptotic cell proliferation induced by activated sputum cells, regeneration of mRNA splicing nerve axons (oxidized galectin 1), abnormal neurite outgrowth of olfactory nerve, and the like are known.

[0047] It should be noted that the physiological activity of human togalectin 1 is still unclear and not all of them are divided. However, it has been found that it has a function related to nerve regeneration, which is considered to be applicable to at least regenerative medicine. For example, the nerve regeneration cascade can be moved starting from (acidic 匕 type) galectin 1, and it also acts as a nerve regeneration promoting factor and nerve axon elongation. Therefore, if sustained release in mammals can be achieved, the physiological activity of galectin-1 may be applicable to the treatment of spinal cord injury patients.

[0048] The bioactive action of human galectin 1 is a literature on recombinant human galectin 1, but it is described in the journal "Biochemistry" 72 卷 No. 10 "It is in a reduced state in cells after nerve injury. Galectin 1 is secreted by the regenerative axon Schwann cell, most of which binds to the sugar chains present on the cell surface, and some of the secreted galectin 1 diffuses into the interstitial fluid, but is damaged. The cell has increased permeability through the cell membrane, and galectin 1 diffuses out of the cell.A single molecule of galectin released to the outside of the cell that does not bind to sugar is capable of disulfide binding in an acidic environment. It forms acid 匕 type galectin 1 and exists as a monomer.Oxidized galectin 1 without lectin activity is an isolated nerve cell. Since it does not act directly on cells, it acts as a cytoforce-in-like factor on cell systems that constitute damaged parts other than nerves, such as Schwann cells, fibroblasts, pericytes, and recruitment macrophages, and axon regeneration It is thought that it promotes. There is a detailed description.

[0049] Sustained-release Hyde Mouth Gel Formulation>

The sustained-release hide-mouth gel preparation of the present invention is a sustained-release gelatin-hide mouth gel preparation containing galectin-1 or a galectin-1 derivative. This sustained-release hyde mouth gel preparation can be prepared, for example, by dripping a solution containing galectin 1 or galectin-1 derivative into the freeze-dried gelatino or the id mouth gel described above, or displacing the gelatin hyde mouth gel with galectin 1 or It can be obtained by dipping in a solution containing the galectin 1 derivative and impregnating the galectin 1 or galectin 1 derivative in the hide mouth gel.

[0050] In the sustained release nose mouth gel preparation of the present invention, the galectin for the gelatin nose mouth gel

The molar ratio of 1 or galectin 1 derivative is preferably about 5 times or less. Preferably the further, galectin 1, or galectin 1 induction body with respect to gelatin Nono id port gels are molar ratios of from about 5 to about 1/10 ⁴ times. This impregnation operation is usually completed at 4-37 ° C for 15 minutes-1 hour, preferably at 4-25 ° C for 15-30 minutes, during which time the hydrogen contains galectin 1 or galectin 1 derivatives. The galectin 1 or galectin 1 derivative forms a complex by a physicochemical interaction with the bioabsorbable polymer, and the galectin 1 or galectin 1 derivative is in the hydose mouth gel. Fixed to.

[0051] In addition to the physical interaction such as Coulomb force, hydroxyl bond strength, and hydrophobic interaction, the gelatin hydrated gel thus obtained and galectin 1 or galectin 1 derivative may be coupled with galectin 1 or galectin. It is considered that the functional group of 1 derivative or the coordinate bond between the metal and the functional group on the hyde-mouth gel is involved alone or in combination.

[0052] Other components may be added to the sustained-release nose-mouth gel preparation of the present invention, if desired, depending on the purpose such as the stability of the resulting hide-mouth gel and the sustained release of the drug. Examples of other components include amino sugars or their high molecular weight compounds, chitosan oligomers, and basic amino acids. Examples thereof include basic polymers such as acids or oligomers thereof, high molecular weight polymers, polyallylamine, polyjetylaminoethyl acrylamide, and polyethyleneimine.

[0053] The sustained-release hide-mouth gel formulation of the present invention can be administered to a living body by an arbitrary method. However, galectin 1 or galectin 1 derivative is directionally and continuously released at a specific target site. For this purpose, local administration is particularly preferred. The sustained-release Hyde Mouth Gel preparation can be further released by mixing with a pharmaceutically acceptable carrier (stabilizer, preservative, solubilizer, pH adjuster, thickener, etc.) as necessary. A formulation can be prepared. A known carrier can be used as such a carrier. Furthermore, various additives for adjusting the sustained release effect can be included. In formulating a sustained-release hide-mouth gel preparation, it is further desirable to go through a sterilization process such as sterilization filtration.

[0054] The sustained-release hide-mouth gel formulation of the present invention can be formulated into various shapes depending on the purpose. For example, solid, semi-solid preparations such as granular, circular and prismatic, sheet, disk, stick, and rod shapes can be mentioned. Preferably, it is a solid preparation excellent in sustained release effect at a specific target site and suitable for local administration. Furthermore, it can also be used as a paste-form preparation having fluidity. For example, a sustained-release hide-mouth gel preparation formulated in a sheet form is suitable for local implantation. In addition, any sustained release nose mouth gel preparation can be used in combination with another material depending on the use site. For example, a sustained-release hide-mouth gel preparation may be used by mixing with a paste-like substance for the purpose of fixing to a specific site.

[0055] The dosage of the sustained-release hide-mouth gel formulation of the present invention can be appropriately selected so as to be sufficient to bring about a therapeutic response. Usually a dose is also selected in the range of about 0.01 to about 10,000 g, preferably about 0.1 to about 1,000 g per adult patient, which is placed or infused at the lesion or surrounding area can do. In addition, if the effect is insufficient with a single dose, multiple doses can be administered.

[0056] The sustained-release nodule gel preparation of the present invention has the sustained-release effect and the stable effect of galectin 1 or galectin 1 derivative, so that galectin 1 or galectin 1 derivative can be controlled at a desired site. It can be released for a long time with a high release rate. Therefore, the action of galectin 1 or galectin 1 derivative is within the lesion site. It is effectively demonstrated. The sustained-release Hyde Mouth gel formulation is preferred to be applied around nerves when nerve regeneration of spinal injury is the goal.

[0057] The forces described above regarding the various aspects of the present invention are examples of the present invention, and various configurations other than those described above may be employed.

[0058] The contents of all patents and references explicitly cited herein are hereby incorporated by reference as part of this specification. In addition, the contents described in the specification and drawings of Japanese Patent Application No. 2006-002648, which is the application on which the priority of the present application is based, are cited herein as part of this specification.

Example

[0059] Hereinafter, the present invention will be further described by way of examples. The present invention is not limited to these examples.

[0060] <1. Preparation of experimental sample>

In the following examples, the names of E50, E10, E3, EO.5, etc. were given to the resulting cationized gelatin depending on the amount of ethylenediamine that was calorie to the gelatin during the preparation of the cationic gelatin. The numbers represent the amount (molar ratio) of ethylenediamine added to the carboxyl group in gelatin when preparing cationic gelatin. In addition, the preparation method of the following cationized gelatin shows the preparation method of E50 as an example, and the amount of cationized gelatin represented by other abbreviations is appropriately changed in the amount (molar ratio) to which ethylenediamine is added. Can be produced.

[0061] (i) Cationized gelatin

E50, E10, E3, EO.5, E50, E10, E3, EO.5 and other indicators indicating the degree of catalysis of cationic gelatin actually replace what percentage of carboxyl groups are replaced by amino groups. Can be shown. For example, E50 is about 50%, E10 is about 43%, E3 is about 30%, and E0.5 is about 14%. Cationic gelatins such as E50, E10, E3, and EO.5 were cross-linked with glutaraldehyde to prepare cationic gelatino and id mouth gels of E50, E10, E3, and EO.5.

[0062] The cation ratio was confirmed by quantifying amino groups by the TNBS method after preparation of cationized gelatin. This quantitative method is a general method often used for quantitative determination of amino groups. Ma TNBS means sodium 2,4,6-tri-trobenzenesulfonate dihydrate.

[0063] Gelatin (molecular weight 100000, derived from pig skin, isoelectric point 9.0, provided by Nitta Gelatin) 20. Add 500 ml of 0.1 M phosphate buffer (pH = 5.0) to Og, and at room temperature. 40 after swelling for 1 hour. C, stirred for 1 hour to dissolve. Ethylenediamine (Wako Pure Chemicals, code 053- 0093 6, lot No. CEN-3313) After adding 55.8 g, the pH was adjusted to 5 with concentrated hydrochloric acid (Nacalai Tester, code 18 321- 05, lot No. V5H6401). After preparation to 0, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (Nacalai Testa, code 15 022-02, lot No. M5B7686) 10.7 g was added. The total amount was adjusted to 1 L using 1 M diphosphate buffer (ρΗ = 5.0), and the reaction was performed by stirring at 40 ° C. for 18 hours. The solution was placed in a cellulose tube (Sanko Junyaku Co., Ltd., UC30-32-100, molecular weight cut off 12000-14000) and dialyzed against distilled water for 3 days. The obtained aqueous solution was frozen at −80 ° C. and lyophilized to obtain E50 cationized gelatin.

[0064] To 1.0 g of E50 cationized gelatin, 9.0 ml of distilled water was added and swollen at room temperature for 1 hour. C, stirred for 1 hour to dissolve. 500 1 pieces were cast on a 2 x 2 cm ² polypropylene balance dish (BIOBIK) and allowed to stand at 4 ° C for 12 hours for gelation. 0.01 ml of hydrochloric acid (Nacalai Tester, code 37318-75, lot No. L5F5508) and 42 ml of acetone (Kishida Chemical, code 000-00306) were mixed and divided into 20 ml each in a glass petri dish. 25% glutaraldehyde aqueous solution (Nacalai Testa, code 1 7003-92, lot No. M5B8118) was added to each petri dish, 12.8, 24.8, 62. respectively.

[0065] Five pieces of hide-mouthed gel per petri dish were immersed in the crosslinking solution and subjected to a crosslinking reaction at 4 ° C for 24 hours. After the reaction, the Hyde mouth gel was immersed in an aqueous solution of 0.1M glycine (Wako Pure Chemicals, code 077-007 35, lot No. SDN—1C62) for 1 hour at room temperature to block unreacted aldehyde groups. The glycine was washed by immersing in distilled water for 1 hour. The obtained nose mouth gel was freeze-dried to obtain cationic gelatin-hide mouth gels with E50 of water content 99.3%, 96.4% and 91.8%, respectively.

[0066] It should be noted that the cationized gelatin of E10, E3, and E0.5 can be appropriately selected by changing the molar ratio of ethylenediamine added in the same manner as the production method of the cationized gelatin of E50. It was produced by adjusting the degree of ON. The water content of E10, E3, and EO.5 cationic gelatin nozzles was crosslinked so that the water content was 80% or more and 99.8% or less.

[0067] (ii) PI = 5

PI = 5 is gelatin with an isoelectric point or PI of 5 derived from bovine bone and treated with alkali. This PI = 5 gelatin was cross-linked with glutaraldehyde to produce a PI = 5 gelatin hydrogen.

[0068] (iii) PI = 9

PI = 9 is gelatin with an isoelectric point or PI of 9 derived from pig skin and acid-treated. This PI = 9 gelatin was cross-linked with glutaraldehyde to produce a PI = 9 gelatin-hide mouth gel.

[0069] (iv) Anionized gelatin (anionization rate 61%)

In contrast to cationized gelatin, key-on gelatin (key-on ratio 61%) is a gelatin with reduced amino groups. The angel gelatin was prepared by introducing succinic acid hydrate into the amino group of Pl = 5 gelatin. The cationization rate is an index indicating the percentage of succinic anhydride introduced in the original P1 = 5 gelatin amino acid group. The amino group of the anion gelatin obtained by the above TNBS method is quantified. It can be obtained by In other words, the ionization rate of 61% means that 61% of the original amino group has been replaced by succinic anhydride. This gel gelatin was cross-linked with dartalaldehyde to produce a gel gelatin gelatin.

[0070] In more detail, ァ gelatin (a ァ rate of 61%) is a DMSO solution containing 12.5% pl = 5 gelatin in 16.2 g of 6.0% succinic anhydride DMSO It was prepared by adding 9.05 g of the solution, stirring at 37 ° C for 1 hour, dialyzing, and freeze-drying. Also, the formula: aionization rate = (number of amino groups per molecule of gelatin) Ζ (ρΙ = 5 number of amino groups per gelatin molecule) * 100 (%) holds.

[0071] (v) SM50

SM50 is a cationized gelatin in which ethylenediamine is replaced with spermine for the preparation method of E50 cationized gelatin. The SM50 cationized gelatin was cross-linked with glutaraldehyde to produce a SM50 cationic gelatin hydrated gel. [0072] (vi) Hydrophobic group-introduced gelatin (CIO)

To prepare a hydrophobic group-introduced gelatin, first, 0.186 g of dodecylamine was added to a solution of 1. Og pl = 5 gelatin / 15 ml 0.1 M MES (pH 5.6). After that, it was adjusted to ρΗ5.6 with concentrated hydrochloric acid. 0.00962g 1-Ethyl 3- (3 dimethylaminopropyl) carbodiimide hydrochloride / 10ml 0.1M MES-nofa (pH 5.6) solution was added to this gelatin solution and reacted at 40 ° C for 18 hours. . The solution was dialyzed and then lyophilized to obtain hydrophobic group-introduced gelatin. This hydrophobic group-introduced gelatin was cross-linked with dartal aldehyde to prepare a hydrophobic group-introduced gelatin hydrated gel.

[0073] (vii) PLA-g gelatin (molecular weight 1000, graft weight 1.24)

In order to prepare PLA-g-gelatin, first, 1. 30 g of DMSO was added to Og pl = 5 gelatin and stirred at 37 ° C. for 2 days. PLA (Molecular weight 1000) 37.5 mg, disuccil carbonate 115 mg, N, N, monodimethylaminopyridine 55. lmg was added to DMSO 30 ml and stirred at 37 ° C for 6 hours to activate the terminal hydroxyl group of PLA. . This PLA DMSO solution was added to the gelatin-DMSO solution and reacted at 37 ° C. for 12 hours. The resulting solution was dialyzed and freeze-dried to obtain PLA-g-gelatin. This PLA-g-gelatin was cross-linked with dartal aldehyde to produce a PLA-g gelatin nose mouth gel.

[0074] (viii) Cholesterol g-gelatin (graft amount 13)

Cholesterol _g— To prepare gelatin, first, DMS015g was added to 1.0 g of gelatin with pl = 5 and stirred for 2 days to dissolve. Thereafter, 05 g of DMS was added to 28.8 mg of discyl carbonate. Furthermore, DMS05g was added to 13.8 mg of N, N, monodimethylaminopyridine. Next, 14.5 mg of cholesterol was dissolved in 10 ml of pyridine. Then, a dimethyl carbonate DMSO solution, an N, N, -dimethylaminopyridine-D MSO solution, and a cholesterol pyridine solution were mixed and stirred at 60 ° C. for 3 hours. This solution was slowly added to gelatin-DMSO solution with stirring and reacted at 37 ° C overnight. The reaction solution was dialyzed, filtered and freeze-dried to obtain cholesterol-g-gelatin. Cholesterol _g -gelatin was cross-linked with glutaraldehyde to prepare cholesterol g gelatin-hide mouth gel.

[0075] (ix) E50 + hydrophobic group-introduced gelatin E50 + hydrophobic group-introduced gelatin was prepared so that the mixing ratio of E50 and hydrophobic group-introduced gelatin was 1: 1. This E50 + hydrophobic group-introduced gelatin was cross-linked with dartalaldehyde to prepare an E50 + hydrophobic group-introduced gelatin nose mouth gel.

[0076] (X) Galactose-introduced gelatin

To prepare galactose-introduced gelatin, pl = 9 gelatin lOOmg is dissolved in 0.1M carbonic acid buffer solution 10ml (pH = 9.5) and galactopyranosyl mono-ferrous isothiocyanate 8. 60mg Was stirred at room temperature for 20 hours. Then, the unreacted galactoviranosyl roof isothiocyanate was removed using a PD-10 column, and the amount of unreacted galactoviranosyl roof isothiocyanate was measured by measuring the absorbance at 282 nm. The graft amount was calculated. The graft amount was the amount of galactose introduced per molecule of gelatin and was 25.6 mol Z mol gelatin. This galactose-introduced gelatin was crosslinked with glutaraldehyde to prepare a galactose-introduced gelatin nose mouth gel.

[0077] (xi) Mannose-introduced gelatin

To prepare mannose-introduced gelatin, dissolve 1 = 9 gelatin 100111 ₈ in 10 ml of 0.1 M carbonate buffer (pH = 9.5), add 8.83 mg of mannopyranosyl monophenol-isoisothiocyanate, and The mixture was stirred for 20 hours. Then, using a PD-10 column, unreacted mannobilanosyl roof eluisothiocyanate was removed, and the amount of unreacted mannopyranosyl phenol diisothiocyanate was determined by measuring the absorbance at 282 nm. The graft amount was calculated. The graft amount was the amount of mannose introduced per molecule of gelatin and was 29.2 mol Z mol gelatin. This mannose-introduced gelatin was cross-linked with glutaraldehyde to prepare a mannose-introduced gelatin nose mouth gel.

[0078] 2. Recombinant human galectin 1 in vitro sustained release test>

100 μg Zml recombinant human galectin 1 (TECHNE Corporation, code 21152X) / PBS 200 μ Na I (Perkinelmer life and analytical sciences, code NEZ033 1122904) / 0. LMNaOH aqueous solution 5 1 and 0.2 mg / ml chloramine T , Code 08005- 52, lot No. M2T9497) Z ○ 5M potassium phosphate buffer (pH = 7.5, 0.5M NaCD lOO 1), shake for 2 minutes, 4mgZml sodium disulfite (Nacalai Tester, code 316—09, lot No. M7B95) Aqueous solution 1 00 μl was added and shaken for another 2 minutes.

[0079] The reaction solution was passed through a PD-10 column (amersham biosciences ゝ code 17—0851-01), and a fraction of every 500 liters was obtained using an auto well Gamma system (Aloka, ARC-300). Measured and separated into labeled recombinant human galectin-11 and unreacted Na ¹²⁵ I.

[0080] 20 μ1 of the recombinant human galectin 1 aqueous solution radiolabeled using the chloramine T method as described above, PI = 5, PI = 9, cationized gelatin, E50, SM50, hydrophobic group-introduced gelatin, Gelatin Hyde Mouth Gel 2 mg of E 50 + hydrophobic group-introduced gelatin, PLA-g-gelatin, cholesterol-g-gelatin was dropped and impregnated at 4 ° C overnight. Place this in a sample tube and add 1 ml of PBS (Nissui Pharmaceutical, code 05913) 37. C, penetrated at 100 rpm.

[0081] After 0.5, 1, 2, 4, 8, 12, and 24 hours, the entire PBS was extracted to obtain a Sampnore solution. 1 ml of PBS was newly added, followed by shaking at 37 ° C. The radioactivity of the sample solution was measured and the remaining amount was measured. Figure 1 shows the measurement results obtained.

[0082] As shown in Fig. 1, in the in vitro sustained-release test, various types of hide-mouth gels as described above were used to confirm by experiments the best gel-and-mouth gel for sustained release of recombinant human galectin-1. It was. As a result, the release amount of recombinant human galectin 1 was abruptly increased in gelatin nodule gels with PI = 5, cholesterol-g-gelatin, and gelatinized gelatin (aionization rate 61%). Sufficient sustained release of recombinant human galectin-1 was not achieved.

[0083] However, PI = 9, E50 + hydrophobic group-introduced gelatin, PLA-g—gelatin, hydrophobic group-introduced gelatin, SM50 cationized gelatin, and E50 cationized gelatin The increase was suppressed, and it was confirmed that the sustained release of recombinant human galectin 1 was improved. That is, by using cationic gelatino, id mouth gel, high isoelectric point _P H, gelatin hyde mouth gel or hydrophobic gelatin hyde mouth gel, the sustained release of recombinant human galectin-11 is improved in vitro. Confirmed to do. Of these, it was concluded that E50 and SM50 Hyde Mouth Gels were the most excellent in sustained release, so the following additional experiments were conducted using E50 as the center.

[0084] <3. Recombinant human galectin-1 in vivo short-term sustained release test (first time)> Recombinant human galectin 1 201 1 radiolabeled using the chloramine T method as described above, pl = 9, E50, SM50, hydrophobic group-introduced gelatin, hydrophobic group-introduced gelatin + E 50, PLA —G—Various gelatin hydrated gels of gelatin were dropped into 2 mg and impregnated with 4 ° C overnight. The hide mouth gel was implanted subcutaneously in the back of the mouse and removed one day later, and the remaining amount of the radioactive capsule was measured. Figure 2 shows the measurement results obtained.

As a result, in vivo, in the case of E50, SM50, hydrophobic group-introduced gelatin + E50 using a cationic gelatin-hydrated mouth gel, recombinant human galectin 1 compared to other experimental samples. As a result, it was confirmed that the sustained release property of the thread-replaceable human galectin 1 with a large residual amount of the protein was improved. That is, it was confirmed that the sustained release of recombinant human galectin 1 was improved even in vivo by using cationic gelatino or id mouth gel. On the other hand, in the experimental samples of PI = 9, hydrophobic group-introduced gelatin and PLA-g gelatin, which showed an effect of improving sustained release at the in vitro mouth, sufficient recombinant humans with a low residual amount of thread and human galectin 1 were obtained. Galectin 1 was effective in improving the sustained release of galectin.

[0086] <4. Recombinant human galectin— 1 in vivo short-term sustained release test (second time)>

Recombinant human galectin 1 20 1 radiolabeled using the chloramine T method as described above shows pl = 9, galactose-introduced gelatin, mannose-introduced gelatin, E50, SM 50, hydrophobic group-introduced, hydrophobic group It was added dropwise to 2 mg of various gelatin-hide gels of + E50 and impregnated with 4 ° C overnight. The Hyde mouth gel was implanted subcutaneously in the back of the mouse and removed one day later, and the residual amount of radioactivity was measured. The obtained measurement results are shown in Fig. 3.

As a result, in vivo, in the case of E50, SM50, hydrophobic group-introduced gelatin + E50 using a cationic gelatin-hide mouth gel, recombinant human galectin 1 compared to other experimental samples. As a result, it was confirmed that the sustained release property of the thread-replaceable human galectin 1 with a large residual amount of the protein was improved. That is, it was confirmed in the same way in a second experiment that the sustained release of the thread-replaced human galectin-1 was improved even in vivo by using cationic gelatino and id mouth gel.

[0088] On the other hand, in the experimental sample of PI = 9, hydrophobic group-introduced gelatin, which showed an effect of improving sustained release in vitro, the remaining recombinant human galectin 1 remained in the same manner in the second experiment. A small amount of recombinant human galectin 1

. In addition, galactose-introduced gelatin and mannose-introduced gelatin have some effects of improving the sustained release of recombinant human galectin 1. Compared with E50, SM50, and hydrophobic group-introduced gelatin + E50, The effect of improving the sustained release of galectin 1 was not sufficient.

[0089] <5. Recombinant human galectin 1 in vivo sustained release test>

Recombinant human galectin 1 20 1 radiolabeled using the chloramine T method as described above, various cationic gelatin nose mouth gels (water content 91.8) with different water contents as shown in FIG. , 96.4, 99.3 wt%) 2 mg, and impregnated with 4 ° C overnight.

[0090] The radioactivity of this recombinant human galectin 1-containing hyde mouth gel was measured using an autowell Gamma system, and implanted subcutaneously in the back of a mouse (ddY mouse, female, 5 weeks old). The samples were taken out on the 3rd, 7th and 14th days, and the residual amount of radioactivity of the hyde mouth gel was measured. Figure 4 shows the measurement results obtained. Here, we show the results for the degradation test and the control test. The degradation of the cationic gelatin gelatin and id-mouthed gel radiolabeled according to the control test ί, Bolton-hunter (Perkinelmer life and analytical sciences, code NEZ033 1122904) was carried out in the same manner.

[0091] As a result, in the in vivo degradation test, when the water content of the cationic E50 gelatin hydrogen was 91.8 wt%, 96.4 wt%, 99.3 wt%, the water content was 91.8 wt%. In the case of 96.4 wt%, it was confirmed that the sustained release property of recombinant human galectin 1 with a large amount of remaining recombinant human galectin 1 was improved. Among these, when the water content was 96.4%, the sustained release property of the recombinant human galectin-11 was particularly improved. In other words, it was confirmed that the sustained release of recombinant human galectin 1 was improved in an in vivo degradation test by using a cationic E50 gelatin-hide mouth gel with a water content of 97 wt% or less. In addition, it was confirmed that the sustained release of recombinant human galectin 1 was further improved in an in vivo degradation test by using a cationic E50 gelatin hyde mouth gel having a water content of 92 wt% or more and 97 wt% or less.

[0092] <6. Bioactivity of Galectin 1>

Using the same mice as in the in vivo sustained-release test system described above, the physiology of galectin 1 The activity was confirmed. After exposing the sciatic nerve of an adult mouse under anesthesia and destroying it by freezing damage, cut the length of the destroyed site to 7 mm and send oxidized galectin 1 to the destroyed site using an osmotic pump. Thus, the effect on axonal regeneration was examined. At this time, the high bone nerve constituting the sciatic nerve of the mature rat was cut under anesthesia. The other end of a silicon tube (filled with collagen gel supplemented with acid-bacteria galectin-11) with one end closed with glass beads was sutured to the central cut end, and the effect was examined.

[0093] As a result, when acid-kappa galectin 1 was actually applied to an in vivo sciatic nerve injury model, promotion of Schwann cell proliferation / migration and enhancement of axon regeneration were observed. When the antibody was acted on, the phenomena related to nerve regeneration were all reduced. From these results, it is suggested that acid-gale galectin 1 can be applied to regenerative medicine for sciatic nerve injury by sustained release in vivo.

[0094] The present invention has been described based on the embodiments. It is to be understood by those skilled in the art that this embodiment is merely an example, and various modifications are possible, and such modifications are within the scope of the present invention.

For example, in the above examples, recombinant human galectin 1 was used as galectin 1, but natural human galectin 1 may be used, or a polypeptide composed of a part thereof may be used. This is also because the physiological activity of human galectin 1 is exhibited in this case, and the effect of nerve regeneration is obtained in the same manner as in the above examples.

Industrial applicability

[0096] The sustained-release hide-mouth gel preparation according to the present invention is useful for the treatment of neuropathy.

Claims

The scope of the claims

[1] Cationic gelatin-hide mouth gel;

Galectin 1 or galectin 1 derivative,

A sustained-release hide-mouth gel formulation.

[2] In the sustained release hyde mouth gel preparation according to claim 1,

The cationic gelatin-hide mouth gel is

A sustained-release hide-mouth gel formulation in which 10% or more and 60% or less of the carboxyl groups of the gelatinoid mouth gel are substituted with amino groups.

[3] The sustained-release hide-mouth gel preparation according to claim 1 or 2,

The cationic gelatin-hide mouth gel is

A sustained-release hide-mouth gel preparation having a water content of 80% or more and 99.8% or less.

[4] The sustained-release hide-mouth gel preparation according to any one of claims 1 to 3, and

The sustained release hyde mouth gel preparation, wherein the galectin 1 or galectin 1 derivative comprises a thread-replaceable galectin 1.

[5] The sustained-release hide-mouth gel preparation according to any one of claims 1 to 4, and

The galectin 1 derivative is

A sustained-release hide-mouth gel preparation comprising mutant galectin 1 in which one or more amino acid residues in the amino acid sequence of galectin 1 are substituted, deleted, or added.

[6] In the sustained-release hide-mouth gel preparation according to claim 5,

The galectin 1 derivative is

A sustained-release hide-mouth gel preparation comprising a part of the galectin 1 or a part of the galectin 1 derivative.

[7] The sustained-release hide-mouth gel preparation according to any one of claims 1 to 6, and

It is configured to be administered in the vicinity of the spinal cord of mammals!