JP4463510B2 - Glia scar formation inhibitor - Google Patents

Description

  The present invention relates to an action inhibitor of a substance having an action of promoting morphological change of astrocytes, which contains heparin or a heparin derivative as an active ingredient.

  The present invention also relates to an astrocyte activation inhibitor containing heparin or a heparin derivative as an active ingredient.

  Furthermore, the present invention relates to a glial scar formation inhibitor containing heparin or a heparin derivative as an active ingredient.

  When the central nerve axon of a mature mammal is damaged, the central nerve axon does not regenerate / extend beyond the damaged site. This also contributes to central nervous system diseases, and finding an improvement method has high clinical value. The cause of the central nerve axon not regenerating or elongating is that basic fibroblasts that are self-secreted by glial cells composed of astrocytes, oligodendrocytes, microglia, etc. (a general term for non-neural support cells that make up the central nervous system) Glial cells are activated, proliferated, migrated by the action of cell growth factor (hereinafter referred to as FGF-2), gathered at the damaged site, and the glia cells that became thin due to morphological change adhere to each other by intercellular crosslinking, It is considered to form an aggregate (hereinafter referred to as glial scar).

  In addition, glial scars are not only a physical barrier to nerve axon elongation, but inhibitors such as chondroitin sulfate proteoglycan secreted by glial cells that form glial scars are known to inhibit nerve axon elongation. (Non-Patent Document 1). At present, animal experiments and clinical trials are being carried out from two directions of inhibition of nerve axon elongation and control of glial scar formation (Non-patent Documents 2-4). It has been reported that chondroitinase acts on chondroitin sulfate proteoglycan (Non-Patent Documents 5-6), but other substances and physics secreted by glial cells are reported. The problem of static barriers remained and no effective means were found. The formation of glial scars occurs when growth factors such as FGF-2 bind to the receptor, and signals from outside the cell enter the cell and are transmitted to the nucleus of astrocyte cells. It is thought to be caused by a series of cascades in which regulation causes changes in protein expression. In particular, it has become clear from cultured glial cells (Non-patent Document 7) and injection experiments into damaged brain sites (Non-patent Documents 8-9) that FGF-2 plays an important role in glial cell activation. Yes.

  In order for FGF-2 to act, it is required that FGF-2 binds to a polymer such as heparan sulfate or heparin to form a dimer (Non-patent Document 10), whereas heparin oligomers It has been confirmed in experiments using several cultured cells that it inhibits dimer formation and also inhibits the effects of FGF-2 (Non-patent Documents 11-13). Furthermore, it is known that sulfation of heparin is also important (Non-Patent Document 14).

  In addition, it is known that low molecular weight heparin is used for the prevention and treatment of central nervous system damage and motor neuron disease (Patent Document 1-2), but heparin and heparin derivatives are glial cells due to damage of central nerve axons. The effect of suppressing glial scar formation, which is believed to be caused by the action of FGF-2 that is autocrine from the body, has not been known.

Special Table 2002-500660 Special table 2002-532431 Fawcett, J.W. et al. (1999) Brain Res.Bull., 49, 377-391. Fournier, A.E. et al. (2001) Nature, 409, 341-346. Romero, M.I. et al. (2001) J. Neurosci., 21, 8408-8416. Wickelgren, I. et al. (2002) Science, 297, 178-181. Bradbry, E.J. et al. (2002) Nature, 416, 636-640. Olson, L. et al. (2002) Nature, 416, 589-590. Faber-Elman, A. et al. (1996) J. Clin. Invest., 97, 162-171. Eclancher, F. et al. (1990) Glia, 3,502-509. Eclancher, F. et al. (1996) Brain Res., 737, 201-214. Stauber, D.J. et al. (2000) Proc. Natl. Acad. Sci. USA, 97, 49-54. Habuchi, H. et al. (1992) Biochem. J., 285, 805-813. Maccarana, M. et al. (1993) J. Biol. Chem, 268, 23989-23995. Ornitz, D.M. et al. (1995) Science, 268,432-436. Kreuger, J. et al. (2001) J. Biol. Chem., 276, 30744-30752.

  As described above, various studies have been made on the suppression of glial scar formation, but problems with inhibitors (inhibitors) other than chondroitin sulfate proteoglycans secreted by glial cells and physical barriers remain, and glial scar formation remains. An effective means for suppressing the above has not been found.

As a result of intensive studies to solve the above problems, the present inventors have found that heparin or a heparin derivative is secreted by glial cells, particularly astrocytes, and suppresses the action of a substance having an action for promoting morphological change of astrocytes. And heparin or a heparin derivative was found to be effective as an astrocyte activation inhibitor, and further effective as a glial scar formation inhibitor.
That is, the present invention is as follows.

  This action inhibitor of a substance having an astrocyte shape change promoting action, containing heparin or a heparin derivative as an active ingredient.

An astrocyte activation inhibitor containing heparin or a heparin derivative as an active ingredient.
A glial scar formation inhibitor containing heparin or a heparin derivative as an active ingredient.

  By using the inhibitor of the present invention containing heparin or a heparin derivative as an active ingredient, the same action of a substance having a morphological change action of astrocytes such as FGF-2 secreted from astrocytes is suppressed, or astrocytes By suppressing the activation of glia, the formation of glial scars due to the formation of aggregates by the adhesion of the glial cells that are thinned due to morphological changes to each other by intercellular crosslinking can be effectively suppressed.

Hereinafter, the present invention will be described in detail by the best mode for carrying out the invention.
<Heparin or heparin derivative>
The origin of heparin that can be used in the present invention is not particularly limited, and heparin extracted and purified from organs of mammals such as pigs and cows (intestine, lung, liver, kidney, blood vessels, etc.) can be used.
In addition, the heparin derivative used in the present invention is not particularly limited. For example, heparin oligosaccharide obtained by reducing the molecular weight of heparin, desulfated heparin obtained by desulfating a sulfate group bonded to the hydroxyl group of heparin, and heparin reduced in molecular weight. And desulfated heparin oligosaccharides obtained by desulfating the same.

  As the heparin oligosaccharide, heparin oligosaccharides obtained by any method such as those produced by chemical synthesis, those produced by chemical degradation, those produced using glycolytic enzymes can be used, The size is usually 4 to 12 sugars, preferably 6 to 10 sugars, more preferably 6 to 8 sugars.

  Further, as the desulfated desulfated heparin, specifically, 6-O-desulfated heparin (hereinafter referred to as 6DSH) in which a sulfate group bonded to the 6-position hydroxyl group of the glucosamine residue in heparin is desulfated, 2-O-desulfated heparin (hereinafter referred to as 2DSH) in which the sulfate group bonded to the hydroxyl group at the 2-position of the uronic acid residue in heparin is desulfated, and the sulfate group bonded to the 2-position amino group of the glucosamine residue are Examples include desulfated N-desulfated heparin (hereinafter referred to as NDSH), among which 2DSH and 6DSH are preferable.

  Furthermore, as the desulfated heparin oligosaccharide, specifically, a 6-O-desulfated heparin oligosaccharide (hereinafter, referred to as a sulfated heparin oligosaccharide in which the sulfate group bonded to the 6-position hydroxyl group of the glucosamine residue in the heparin oligosaccharide is desulfated). 6DSH oligo), 2-O-desulfated heparin oligosaccharide (hereinafter referred to as 2DSH oligo) in which the sulfate group bonded to the hydroxyl group at the 2-position of the uronic acid residue in heparin oligosaccharide is desulfated. Of these, 6DSH oligo is particularly preferable.

  As a method for desulfating a sulfate group bonded to the 6-position hydroxyl group of heparin or heparin oligosaccharide, the sulfate group bonded to the 6-position hydroxyl group of the glucosamine residue is selectively or specifically removed. If it is a desulfation method, it will not specifically limit.

Examples of such a method include a method using solvolysis (Japanese Patent Publication No. 9-512822), a method using a silylating agent (WO96 / 01278), and the like.
In addition, as a method for desulfating a sulfate group bonded to the hydroxyl group at the 2-position of the uronic acid residue of heparin or heparin oligosaccharide, the sulfate group bonded to the hydroxyl group at the 2-position of the uronic acid residue is selectively or specific. The method is not particularly limited as long as it can be desulfated.

  For example, as a method for selectively desulfating a sulfate group bonded to the hydroxyl group at the 2-position of a uronic acid residue, the method of Jaseja et al. (Jaseja et al., Can. J. Chem., 67, 1449 (1989)).

  Furthermore, as a method for desulfating the sulfate group bonded to the 2-position amino group of the glucosamine residue of heparin or heparin oligosaccharide, the sulfate group bonded to the 2-position amino group of the glucosamine residue is selectively or specifically removed. There is no particular limitation as long as it is a desulfation method.

  Examples of such methods include Nagasawa, k. Et al., (1979) J. Biochem., 86, 1323-1329, Nagasawa, k. And Inoue, Y. (1980) Methods Carbohydr. Chem., 8, 287. -289, Ayotte, L. and Perlin, AS (1986) Carbohydr. Res., 145, 267-277.

  Furthermore, heparin or a heparin derivative can be easily concentrated and purified by solvent precipitation using an appropriate solvent, ultrafiltration, column chromatography, dialysis, lyophilization, or a combination thereof.

  The heparin or heparin derivative, which is an active ingredient of the inhibitor of the present invention, can be used in a free form or a salt form thereof. Examples of the alkali salt include sodium salt, calcium salt, and potassium. Examples include alkali metal salts such as salts, alkaline earth metal salts such as magnesium salts and calcium salts, ammonium salts, and tributylamine salts. Alkali metal salts are preferable, and sodium salts are particularly preferable.

<The action inhibitor of a substance having a shape change action of astrocytes>
The action inhibitor (hereinafter referred to as “the present invention inhibitor 1”) of the substance having the shape-changing action of astrocytes of the present invention will be described.

  Here, the “substance having an astrocyte morphological change action” is a substance that is self-secreted by glial cells and acts on the astrocytes to promote the morphological change (transformation) of the cells. Specific examples include growth factors such as FGF-2, but the substance is not limited to this as long as it has a similar action.

  The heparin or heparin derivative, which is an active ingredient of the inhibitor 1 of the present invention, is not limited as long as it can suppress the action of a substance having an astrocyte shape-changing action, but 6-10 sugar heparin oligosaccharides, 2DSH 6DSH, 2DSH oligo and 6DSH oligo are preferable, and 6-8 sugar heparin oligosaccharide and 6DSH oligo are more preferable.

  The effect of the inhibitor 1 of the present invention can be confirmed by adding various target heparins and derivatives thereof to the medium, culturing astrocytes in the presence of FGF-2, and examining the influence on the cell area. it can. As a result of confirmation by this method, for heparin oligosaccharides, for example, 6 to 10 sugars suppressed the decrease of the cell area of astrocytes more than administration of FGF-2 alone, and 6 sugars increased the cell area of astrocytes. Regarding desulfated heparin, 2DSH, 18-saccharide 6DSH oligo and 2DSH oligo suppressed the reduction of the cell area of astrocytes, whereas NDSH did not suppress the decrease of the cell area of astrocytes. In the present invention, the reduction in the cell area of astrocytes means a change in the morphology of astrocytes.

<Astrocyte activation inhibitor>
The astrocyte activation inhibitor of the present invention (hereinafter referred to as “the present invention inhibitor 2”) will be described.
As used herein, astrocyte activation refers to an astrocyte shape-changing agent that is self-secreted by glial cells, typified by growth factors such as FGF-2. It means to wake up.

  The heparin or heparin derivative, which is an active ingredient of the inhibitor 2 of the present invention, is not limited as long as it can suppress astrocyte activation, but it is described in <Inhibitor of action of a substance having an astrocyte shape-changing action>. Preferred are heparin or heparin derivatives.

<Glia scar formation inhibitor>
The glial scar formation inhibitor of the present invention (hereinafter referred to as “the present invention inhibitor 3”) will be described.
Here, glial scar formation refers to an astrocyte shape-changing agent that is secreted by glial cells such as FGF-2, which activates astrocytes to cause morphological changes and reduces the cell area to make them thinner. It means that the sites adhere to each other by intercellular crosslinking to form an aggregate.

  The heparin or heparin derivative that is an active ingredient of the inhibitor 3 of the present invention is not limited as long as it can suppress glial scar formation, but is described in <Suppressor of action of substance having astrocytic shape change action>. Heparin or heparin derivatives are preferred.

<Dosage form, administration route>
In addition, the above-described inhibitor of the present invention is administered to a living body to prevent astrocytes from undergoing morphological changes due to growth factors or the like in the administered central nervous disease, or to prevent astrocytes from being activated. By inhibiting the formation of glial scars, it can be used as a medical drug for treating, preventing, and suppressing the progression of the disease. The dosage form and route of administration can be appropriately selected according to the nature and severity of the target disease. For example, they can be formulated as such or with other pharmacologically acceptable carriers, excipients, disintegrants, binders, lubricants, diluents, etc., for example, powders, granules, fine granules, It can be safely administered orally or parenterally, such as dry syrup, liquid, tablet, capsule, injection, suppository, vaginal, ointment, gel, and spray. In particular, it is preferable to administer parenterally as a suppository, a vaginal agent, an ointment, a gel, or a spray.

  Further, the amount and dosage of heparin or heparin derivative, which is the active ingredient of the inhibitor of the present invention, in the preparation depends on the administration method, dosage form, purpose of use, specific patient symptoms, patient weight, etc. Although it is a matter which should be determined individually, it is not specifically limited, About 0.1 mg / Kg-about 300 mg / Kg per day as a clinical dosage can be illustrated. Moreover, the administration interval of the above preparation can be about once a day, and can be administered once to several times a day or more.

In addition, the inhibitor of the present invention can be used to suppress morphological changes of astrocytes when culturing astrocytes in vitro.
That is, the inhibitor of the present invention can be used not only as a drug administered to a living body but also for various uses such as regenerative medicine and neuroscience research.

All heparin or heparin derivatives used in the following examples are sodium salts.
<Test Example 1>
Preparation of DMEM high glucose medium
The DMEM high glucose medium was prepared as follows.

DMEM medium (Nissui, product number: 302059157) 1 g, Penicillin-streptomycin-glutamine (Gibco, product number: 10378-016) 1 ml, NaHCO ₃ 120 mg, Horse serum (HS) (Gibco, (Product No. 16050-130) 5 ml, fetal bovine serum 5 ml, and Glucose 350 mg were added with 100 ml of sterilized distilled water, stirred with a stirrer, and filtered using a Millipore filter.

<Test Example 2>
Brains were removed from rats a day after birth of cell cultures of astrocytes , and the cerebellum, midbrain and meninges were removed in cold sterilized PBS to obtain cerebral cortex. Ten cerebral cortices are sliced vertically and horizontally with a scalpel on a 60 mm dish, and 10 ml phosphate buffered saline (PBS) is added twice to the cerebral cortex sections into 50 ml falcon tubes. It was collected. The PBS supernatant was removed by centrifugation at 500 rpm for 1 minute. After 5-6 ml of 12 unit / ml Papain solution was added to the cell sedimentation, the mixture was incubated at 37 ° C. for 10 min for enzyme digestion. After the papain digestion process was repeated again, the papain solution was removed. Subsequently, the DMEM high glucose medium was added, the cells were naturally precipitated, and the supernatant was removed three times to purify the cells. Furthermore, the cell suspension was filtered through a funnel with lens paper, and 5-6 ml of fresh DMEM high glucose medium was added to wash out cells remaining on the lens paper. The filtrate thus obtained was centrifuged at 500 rpm for 80 to 90 minutes, and the supernatant was removed. This process was repeated three times. Thereafter, 3 ml of DMEM high glucose medium was added, and the cells in the medium were made uniform by gently pipetting. As a premise for setting the cell concentration, the cells were counted using a hemocytometer after staining with trypan blue. Based on this measured value, the cell concentration was set to 1 × 10 ⁵ cells / ml ² and cultured at 37 ° C. and 5% CO ₂ for 3 days. At this time, when confluence was reached, the cells were passaged, and the cells that had been passaged twice were used for the assay.

<Test Example 3>
The heparin oligosaccharides (4, 6, 8, 10 sugars) used in the examples described later were prepared by the following method.
To 100 mg of heparin, add and stir 10 mL of nitrous acid solution (pH 1.5) prepared by a known method (Guo, Y. and Conrad, HE (1989) Anal. Biochem., 176, 96-104) did. The reaction mixture was warmed at room temperature for 3 minutes to partially proceed with the nitrite decomposition reaction. The reaction was stopped by adding 1 N Na ₂ CO ₃ and the reaction mixture was concentrated. This reaction mixture containing heparin oligosaccharide was applied to a Cellulofine GCL-25 column (φ3.3 x 30 cm; sold by Seikagaku Corporation) and desalted, and then the BioGel P-6 column ( Size fractionation was applied to φ2.0 x 100 cm). Thus, 10-20 mg of heparin oligosaccharide (4, 6, 8, 10 sugar) was prepared.

<Test Example 4>
Desulfated heparin (2DSH, 6DSH and NDSH) and desulfated heparin oligosaccharide (18 sugar 2DSH oligo and 6DSH oligo) used in Examples described later were prepared by the following known methods.

  2DSH was prepared according to the method shown in Piani, S. et al. (1993) J. Carbohydr. Chem., 12, 507-521. 6DSH was prepared according to the method shown in Kariya, Y. et al. (2000) J. Biol. Chem., 275, 25949-25958. NDSH was prepared according to the method shown in Ayotte, L. and Perlin, A. S. (1986) Carbohydr. Res., 145, 267-297.

  Desulfated heparin oligosaccharide was prepared basically according to Test Example 3 described above. That is, nitrous acid decomposition reaction was performed under the same conditions except that 100 mg of each desulfated heparin was used as a starting material and the reaction time was 1 minute. The reaction product desalted with a Cellulofine GCL-25 column (φ3.3 × 30 cm) was applied to a Cellulofine GCL-90 column (φ3.0 × 96 cm) for size fractionation. Thus, about 20 mg of 18-saccharide 2DSH oligo and 6DSH oligo were obtained.

<Example 1>
Examination of FGF-2 concentration causing morphological change in astrocytes
The concentration at which astrocytes undergo morphological changes with FGF-2 alone was examined.
On the first day of the assay, a cover glass (circular) was placed in a 24-well microtiter plate and coated with polyethyleneimine. Next, the cells detached with trypsin were seeded on a cover glass at a concentration of 1 × 10 ₄ cells / cm 2. An appropriate amount of DMEM high glucose medium was added and cultured at 37 ° C. and 5% CO ₂ . On the second day of the assay, the medium was changed from DMEM high glucose medium to serum-free DMEM medium, and FGF-2 and specimen heparin were added at various concentrations. On the third day of the assay, the cells were fixed with 4% paraformaldehyde, and the cytoplasm was stained with a 0.2% eosin solution. The dedicated software (Winroof) was used to calculate the area observed from the cell screen. As a result, the change in the area of the astrocytes was measured using the one-dimensional area under the microscope observation of the astrocytes as an index. As a result, when the FGF-2 concentration was 1 ng / ml, there was a marked decrease in the cell area, and even if the FGF-2 concentration was increased further, there was no significant change. Therefore, the FGF-2 concentration used in the following assay was fixed at 1 ng / ml.

<Example 2>
Examination of Heparin Concentration Affecting on Astrocyte Morphology According to the method of Example 1, the concentration of heparin (SPL, purified product derived from porcine small intestine) that promotes astrocyte morphological change by FGF-2 was examined. As a result, the cell area decreased most at 1 μg / ml of heparin compared to 1 ng / ml of FGF-2. At heparin concentrations higher than this, the cells became too thin and detached when the medium was removed, making measurement impossible. Therefore, the heparin concentration used in the following assay was fixed at 1 μg / ml. That is, heparin 1 μg / ml was used for 1 ng / ml of FGF-2.

<Example 3>
Effect of Heparin Oligosaccharide on Astrocyte Shape Change by FGF-2 Four kinds of heparin oligosaccharides ranging from tetrasaccharide to 10 sugars were examined according to the method of Example 1 to examine the influence of FGF-2 on astrocyte shape change. The inhibition rate of the cell area reduction of 4, 6, 8, 10 heparin oligosaccharide astrocytes relative to the cell area of astrocytes when FGF-2 alone was administered was calculated. Table 1 shows.

6-10 sugars suppressed cell area reduction more than FGF-2 alone administration, especially 6 sugars increased astrocyte cell area. This is considered to be because heparin oligosaccharide suppressed the effect of FGF-2 on astrocyte shape change.

<Example 4>
Effect of Desulfated Heparin on FGF-2-induced Astrocyte Morphological Change According to the method of Example 1, the effect of 2DSH, 6DSH, and NDSH on astrocyte morphological change was examined for various desulfated heparins. The inhibition rate of cell area reduction of 2DSH, 6DSH, NDSH, 18 sugar 2DSH oligo and 6DSH oligo astrocytes relative to the cell area of astrocytes when FGF-2 alone was administered was calculated. It shows in Table 2.

2DSH, 18-saccharide 6DSH oligo and 18-saccharide 2DSH oligo suppressed the reduction of astrocyte cell area, whereas NDSH failed to suppress the reduction of astrocyte cell area.

Claims (2)

A glial scar formation inhibitor containing 2-O-desulfated heparin as an active ingredient. A glial scar formation inhibitor containing 18-saccharide 6-O-desulfated heparin as an active ingredient.