JP6253872B2 - Autophagic cell death inhibitor - Google Patents

Description

  The present invention relates to an inhibitor of autophagic cell death containing a low molecular weight hyaluronic acid and the like.

  In many aspects such as differentiation, development, maintenance of an individual, establishment of immune system and nervous system, a cell expresses a gene necessary for cell death by itself and has a system for self-deletion.

  Autophagic cell death, also called type 2 cell death, refers to cell death with autophagy (autophagy). Autophagy is a phenomenon that is essential for the basic metabolism of cells. Cell components that are no longer needed are encapsulated by vesicle-like structures (autophagosomes) together with some cytoplasm, and they receive lysosomal enzymes. It is a mechanism to disassemble.

  Autophagy includes basic autophagy necessary for maintaining intracellular metabolism and homeostasis, and autophagy induced in response to stress. It is known that the loss of basic autophagy will result in death soon after birth. Autophagy is also induced when cells are starved and by pathological conditions such as ischemia and inflammation.

  By the way, cerebral ischemia refers to a state in which sufficient oxygen and nutrients are not supplied to brain tissue as a result of insufficient blood in the brain due to various causes. When cerebral ischemia exceeds the limit, cerebral infarction develops. Cerebral infarction means that nerve cells become necrotic due to lack of oxygen or nutrients due to cerebral ischemia, or become nearly necrotic.

  Cerebral infarction is one of the leading causes of death among Japanese people, often with severe sequelae, and may require long-term care. Cell death is an irreversible phenomenon, and once it occurs, it cannot be recovered.

  Conventionally, as a result of morphological and biochemical studies, it is known that hippocampal pyramidal cell layers after hypoxia-cerebral ischemia are positive for various markers of apoptosis including caspase 3 activation. It was. However, in morphological observation with an electron microscope, cells exhibiting typical apoptosis were hardly observed, while cells exhibiting typical necrosis were hardly observed.

  As a result of producing and analyzing a hypoxia-cerebral ischemic load model using a mouse (Non-patent Document 1) in which Atg7 essential for autophagy is specifically deficient in nerve tissue, the present inventors have analyzed cerebral ischemia. It has been demonstrated that neuronal cell death that occurs occasionally is caused by autophagy that is excessively induced by hypoxic stress (Non-Patent Documents 2 and 3).

  From the above, it was considered that if there is a drug that controls autophagy, the drug may be administered after hypoxia-cerebral ischemic load to reduce the disorder. However, a substance that specifically controls only autophagy has not been found so far.

Komatsu, M. et al., J. Cell Biol., 169: 425-434, 2005 Koike et al., Am. J. Pathol. 172: 454-469, 2008 Uchiyama, Y et al., Autophagy 4: 404-408, 2008

  Then, this invention makes it a subject to provide the pharmaceutical composition which can suppress autophagic cell death.

As a result of repeated studies to solve the above problems, the present inventors have found that low molecular weight hyaluronic acid suppresses autophagic cell death, and have completed the present invention.
That is, the present invention
[1] An autophagic cell death inhibitor comprising a low molecular weight hyaluronic acid or a salt thereof;
[2] The autophagic cell death inhibitor of the above-mentioned [1], wherein the low molecular hyaluronic acid is composed of 2 to 20 sugars.
[3] The autophagic cell death inhibitor according to [1] or [2] above, wherein the low-molecular hyaluronic acid is a hyaluronic acid tetrasaccharide;
[4] The autophagic cell death inhibitor according to any one of [1] to [3], which suppresses autophagic cell death of neurons due to ischemia;
[5] The autophagic cell death inhibitor according to any one of [1] to [4], which is a preventive or therapeutic agent for cerebral ischemic disease; and [6] the cerebral ischemic disease is The autophagic cell death inhibitor according to any one of [1] to [5] above, which is cerebral infarction or hypoxic ischemic encephalopathy,
About.

  The autophagic cell death inhibitor of the present invention specifically controls autophagic cell death that has not existed so far, through suppression of autophagic cell death during hypoxia-cerebral ischemia load, Obstacles can be reduced.

  In addition, hyaluronic acid is a substance that is also present in the living body, has no toxicity to cells and individuals, and has a very low possibility of causing side effects, so it is highly useful as a safe pharmaceutical.

FIG. 1 shows the results of hematoxylin-eosin staining of hippocampal pyramidal neurons 24 hours after hypoxia / cerebral ischemic load. Saline is a control group, HA4 is a hyaluronic acid tetrasaccharide administration group, and Large-HA is a large hyaluronic acid administration group. FIG. 2 shows the results of TUNEL staining of hippocampal pyramidal neurons 24 hours after hypoxia / cerebral ischemic load. Saline is a control group, HA4 is a hyaluronic acid tetrasaccharide administration group, and Large-HA is a large hyaluronic acid administration group. FIG. 3 shows the results of TLR2 knockout mice subjected to hypoxia / brain ischemic load and stained in the same manner as in FIG. FIG. 4 shows the result of TUNEL staining of hippocampal pyramidal neurons 24 hours after hypoxia / cerebral ischemic load, as in FIG. Saline is a control group, HA4 is a hyaluronic acid tetrasaccharide administration group, Large-HA is a large hyaluronic acid administration group, TLR-2 is a TLR2 knockout mouse, and TLR-4 is a TLR4 knockout mouse. FIG. 5 shows the results obtained by measuring the expression of NFκB in hippocampal cells after hypoxia / cerebral ischemia load by Western blotting. FIG. 6 shows the results obtained by measuring the expression of IL-1β in hippocampal cells after hypoxia / cerebral ischemia load by a quantitative PCR method. FIG. 7 is a conceptual diagram showing the action of low molecular weight hyaluronic acid in apoptotic cell death. When low molecular weight hyaluronic acid binds to a Toll-like receptor, NFκB is activated and transcription of various proteins is increased. As a result, apoptotic cell death is suppressed.

The present invention provides an autophagic cell death inhibitor.
The autophagic cell death inhibitor of the present invention contains low molecular hyaluronic acid or a salt thereof.

  “Autophagy” is a phenomenon that is essential for the basic metabolism of cells, and wraps obsolete cell components together with some cytoplasm with a vesicle-like isolation membrane (formation of autophagosomes). It is a mechanism that degrades by enzymes. The macromolecular substance taken up into the autophagosome is decomposed into a biologically active monomer under acidic conditions, and many of them are reused in cells.

  According to studies using Atg gene knockout mice identified as autophagy-related genes, the role of autophagy is as follows: (1) supply of amino acids in response to nutrient starvation, (2) non-selective proteolysis (Komatsu et al ., J Cell Biol 169: 425-434, 2005; Nature 441: 880-884), (3) selective degradation of certain proteins via the ubiquitin signal (Komatsu et al., J Cell Biol 169: 425- 434, 2005; Nature 441: 880-884, 2006; Cell 131: 1149-1163, 2007), (4) induction of cell death induced in response to various stresses, etc. .

  In particular, the present inventors have produced and analyzed a hypoxia-cerebral ischemic load model using a mouse (Non-patent Document 1) in which Atg7 essential for autophagy is specifically deficient in nerve tissue. When cerebral ischemia occurs due to oxygen load, autophagy is induced in hippocampal pyramidal neurons, resulting in death due to pycnosis (nuclear enrichment), and this neuronal cell death is also suppressed in caspase 3-deficient mice and CAD-deficient mice. It has been proved that autophagy cell death cannot occur, that is, autophagic cell death occurs through a pathway different from apoptosis (Non-patent Documents 2 and 3).

  As used herein, “autophagic cell death” refers to cell death accompanied by such autophagy (Xu Y., et al., JBC. 2002, 281 (28): 19179-19187).

Whether or not a certain cell death is autophagic, as described above, whether or not cell death occurs in a model animal knocked out of a gene essential for autophagy, or a model animal knocked out of a gene essential for apoptosis The person skilled in the art can determine whether or not the cell death occurs in the above.
It can also be determined by the expression of autophagy marker protein LC3 in dead cells and confirmation of the formation of autophagosomes and autolysosomes by electron microscopy.

  Furthermore, various markers of apoptosis (for example, activation of caspase 3, reduction of cell body and nucleus, condensation of nuclear chromatin, sharp boundary between chromatin and condensed chromatin, formation of apoptotic bodies, etc.) are not observed. In addition, the absence of various markers of necrosis (expansion of organelles such as cell bodies, nuclei, mitochondria, breakdown of cell membrane, outflow of cytoplasmic organelles, etc.) can also be used as a reinforcing material for judgment.

  In this specification, cell death suppression means preventing, delaying, stopping or reducing cell death.

  “Hyaluronic acid” is a kind of glycosaminoglycan (mucopolysaccharide) and has a structure in which a disaccharide unit (GlcAβ1-3GlcNAcβ1-4) of glucuronic acid and N-acetylglucosamine is repeated. Hyaluronic acid has a variety of sizes, from those with a very long chain structure (for example, a molecular weight of 1 million or more) to low molecular weights composed of disaccharides or tetrasaccharides. It is known to show.

  As used herein, the term “low molecular hyaluronic acid” is a term for high molecular hyaluronic acid and is used in its broadest sense. Low molecular hyaluronic acid means, for example, hyaluronic acid composed of 60 sugars or less, 30 sugars or less, 25 sugars or less, or 20 sugars or less. The number of constituent sugars may be an odd number. The sugar located at the non-reducing end may be a saturated sugar or an unsaturated sugar.

  As the low-molecular hyaluronic acid, for example, hyaluronic acid tetrasaccharide (hyaluronic acid composed of tetrasaccharide) can be used. Hyaluronic acid tetrasaccharide has so far been able to protect against traumatic nerve injury, nerve cell activation (Xu H., et al., JBC. 2002, 277 (19): 17308-4), neurite outgrowth, inflammation-related It has been reported that there are physiological activities such as cytokine suppressive action and stress protein expression enhancing action (Japanese Patent Laid-Open No. 2007-291133).

The low molecular weight hyaluronic acid may be obtained by decomposing a natural product or may be artificially synthesized. As a method for decomposing and producing natural products, high molecular hyaluronic acid is isolated and purified from chicken crown, umbilical cord, pig skin, cow skin, fish, microorganisms producing hyaluronic acid according to known methods, and this is enzymatically decomposed. , Chemical decomposition methods, heat treatment methods, and ultrasonic treatment methods. As the enzymatic degradation method, hyaluronidase or chondroitinase can be used, and the chemical degradation method includes, but is not limited to, an alkaline degradation method and a DMSO method. The thus decomposed hyaluronic acid can be further separated into those having a desired molecular weight according to a conventional method using column chromatography or the like.
Artificial synthesis can also be performed by a known method or a method analogous thereto.

  In the present specification, the “hyaluronic acid salt” is not particularly limited, but is preferably a food or a pharmaceutically acceptable salt, and examples thereof include sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt and the like. .

  The autophagic cell death inhibitor of the present invention includes various diseases associated with autophagic cell death (for example, diseases caused by autophagic cell death, and diseases that cause autophagic cell death as a result of the disease) Can be used as a prophylactic or therapeutic agent. Examples of the disease associated with autophagic cell death include ischemic disease and pancreatitis (Hashimoto D. et al., J Cell Biol. 2008; 181 (7): 1065-72).

One embodiment of the autophagic cell death inhibitor of the present invention suppresses autophagic cell death of neurons due to ischemia.
As used herein, “ischemia” refers to local anemia due to a decrease in arterial blood volume.

In the present specification, the “cerebral ischemic disease” is not particularly limited as long as it is a disease causing cerebral ischemia, and includes various diseases caused by cerebral ischemia, or those causing cerebral ischemia as a result of the disease. . Cerebral ischemia refers to a state in which brain blood is insufficient and sufficient oxygen and nutrients are not supplied to brain tissue.
Examples of cerebral ischemic diseases include, but are not limited to, cerebral infarction and hypoxic ischemic encephalopathy.

  In the present specification, “prevention or treatment of cerebral ischemic disease” means alleviation of symptoms of cerebral ischemic disease, stop or delay of progression, and the like.

As shown in Fig. 7 (quoted from Bourguinon, LYW, Wong, G., et al, Cytoskeleton published on line DOI 10.1002 / cm. 20554 (2011)), low molecular hyaluronic acid (LMW-HA) containing oligosaccharides When acting on cell surface Toll-like receptors 2 and 4 (hereinafter also referred to as “TLR2” and “TLR4”, respectively) and CD44, the following sequence of events is induced: (1) CD44 -AFAP-110-TLR2 / TLR4-MyD88 complex is formed; (2) AFAP-110 and actin bind to activate cytoskeleton; (3) Simultaneously with (2) MyD88 / NF- κB is activated and translocated into the nucleus; (4) Transcriptional activity of genes such as IL-1β is activated; (5) Increased synthesis of various cytokines and chemokines, for example, increased expression of heat shock proteins Thus, apoptotic cell death is suppressed. AFAP indicates Actin Filament Associated Protein, and MyD indicates Myloidoid Differentiation factor.
However, as shown in Examples described later, in hippocampal cells caused by ischemic injury, low molecular weight hyaluronic acid suppresses the expression of NFκB and also the expression of IL-1β downstream thereof, rather than Toll. It was found to inhibit the function of the receptor. This suggests that cell death in ischemic injury is not apoptotic cell death, and the low molecular weight hyaluronic acid in the present invention specifically suppresses autophagic cell death, not apoptotic cell death. it can.

  The autophagic cell death inhibitor of the present invention can be administered by injection, nasal, oral, transdermal, inhalation, etc., and depending on the administration method, it is a liquid (for example, injection), a dispersion, a suspension. , Tablets, pills, powders, suppositories, capsules, granules, powders, ointments, patches and the like.

  Formulation can be performed according to a known method, and so long as it does not affect the effect of the present invention, a stabilizer, an emulsifier, an osmotic pressure adjuster, a pH adjuster, a buffer, an isotonic agent, a preservative. Ingredients such as carriers and additives usually used in medicine, such as soothing agents, coloring agents, excipients, binders, lubricants, disintegrating agents, and the like can be used.

Examples of carriers and additives include pharmaceutically acceptable organic solvents such as water, saline, phosphate buffer, dextrose, glycerol, ethanol, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, poly Sodium acrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, Examples include, but are not limited to, stearic acid, human serum albumin, mannitol, sorbitol, lactose, surfactants, etc. It is not.
The dose is not particularly limited and can be appropriately determined according to the target disease, the age, weight, etc. of the patient. For example, 0.05 to 50 mg / kg or 0.1 to 3 mg / kg per day It can be administered in one or more divided doses.

[Experimental animals]
All animal experiments in this study are in accordance with the “Act on the Protection and Control of Animals (Animal Protection Law)”, “Guidelines for Proper Implementation of Animal Experiments” and “Guidelines on Animal Experiments at Juntendo University School of Medicine” by the Science Council of Japan. (Animal experiment protocol; approval number 230036 "Molecular cell biological study on the breakdown of autophagy / lysosomal proteolytic system in nervous system"). Wild-type newborn C57BL / 6J mice were purchased from Oriental Bioservice, Japan SLC, C57BL / 6 strain Toll-like receptor (TLR) -2 and -4 knockout mice.

[Hypoxia / cerebral ischemic load]
Hypoxia / cerebral ischemic stress was observed in 7-day-old neonatal mice in the Rice-Vannucci model (Rice JE III, Vannucci RC, Brierley JB: The influence of immaturity on hypoxic-ischemic brain damage in the rat. Ann Neurol 1981, 9: 131-141) was modified. After ischemia by ligating the left common carotid artery of the mouse under anesthesia with 2% isoflurane, recovery was awaited at 37 ° C. for 1 hour. Thereafter, a low oxygen load was applied by placing the container in a container adjusted to an oxygen concentration of 8% and a room temperature of 37 ° C. for 25 to 30 minutes. After hypoxic / cerebral ischemia, newborn mice are immediately returned to their womb and are subjected to molecular, biochemical, or histological analysis after 1, 3, 6, 12, 24 hours, or 7 days. It was done. This method is known to induce cell death of pyramidal neurons in the ischemic hippocampus (Sheldon RA, Sedik C, Ferriero DM: Strain-related brain injury in neonatal mice subjected to hypoxia-ischemia. Brain Res 1998, 810: 114-122; Ness JM, Harvey CA, Strasser A, Bouillet P, Klocke BJ, Roth KA: Selective involvement of BH3-only Bcl-2 family members Bim and Bad in neonatal hypoxia-ischemia. 2006, 1099: 150-159).

[Hyaluronic acid administration]
Hyaluronic acid tetrasaccharide was administered from the abdominal cavity at a concentration of 10 mg / kg (body weight) at two points 12 hours before and immediately before hypoxia / cerebral ischemia. Large hyaluronic acid (molecular weight 800 kDa) was administered at a molar concentration equivalent to hyaluronic acid tetrasaccharide. In the control group, physiological saline was administered.

[Histochemical and morphological analysis]
Hypoxic / cerebral ischemic mice were exsanguinated under anesthesia, perfusion-fixed with 0.1 mol / L phosphate buffer (pH 7.2) containing 4% sucrose, the brain was removed, and the fixative was used overnight. Immersion fixed. For the paraffin section, a 4 μm sample was prepared using a microtome (RM2245; Leica) after dehydration. Cryosections were immersed in 0.1 mol / L phosphate buffer (pH 7.2) containing 15% and 30% sucrose, embedded in OCT compound (Miles, Elkhart, IN), and cryostat (CM3050; Leica). 10 μm sections were prepared.
The results are shown in FIG. The hippocampus is the most vulnerable area to ischemic invasion. Chromatin aggregation and cytoplasm loss were observed in the large hyaluronic acid administration group and the control group, whereas the hyaluronic acid tetrasaccharide administration group showed a marked improvement in prognosis.

In order to examine the cytoprotective effect of hyaluronic acid tetrasaccharide in more detail, TUNEL staining was subsequently performed as an indicator of cell death. TUNEL staining was performed according to a previous paper (Gavrieli Y, Sherman Y, Ben-Sasson SA: Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992, 119: 493-501). Briefly, the sections are 100 U / ml terminal deoxynucleotidyl transferase (TdT; Promega) and 10 nmol / L biotinylated 16-2'-dUTP (Roche Diagnostics) in TdT buffer (100 mmol / L sodium cacodylate, pH 7.0, 1 (mmol / L cobalt chloride, 50 μg / ml gelatin) at 37 ° C. for 1 hour. For detection, streptavidin-conjugated Alexa Fluor 488 (Invitrogen, San Diego, Calif.) Was reacted at room temperature for 1 hour.
The results are shown in FIG. The number of TUNEL-positive pyramidal neurons in the hippocampal CA area 24 hours after hypoxia / cerebral ischemic load was larger in the hyaluronic acid tetrasaccharide group (204 ± 22 per section) than in the large hyaluronic acid group (517 per section). (± 112) and the control group (438 ± 152 per section) were found to be reduced by half.

Next, FIG. 3 shows the results of observing specimens prepared by the same method as in FIG. 1 for TLR2 knockout mice. In TLR2 knockout mice, as in the case of hyaluronic acid tetrasaccharide administration, the number of hippocampal cell deaths due to ischemic injury decreased.
In addition, TUNEL staining was performed on TLR2 and TLR4 knockout mice in the same manner as in FIG. 2, and the experiment was repeated in the hyaluronic acid tetrasaccharide administration group (HA4), the large hyaluronic acid administration group (HA-L), and the target group (Cont). The result of having performed is shown in FIG. In TLR2 and TLR4 knockout mice, it was confirmed that the number of TUNEL-positive pyramidal neurons was remarkably reduced as in the case of hyaluronic acid tetrasaccharide administration.
From the above, it was suggested that hyaluronic acid tetrasaccharide exhibits the same action as inhibiting the function of Toll-like receptor.

[Western blotting]
Next, the expression of NF-κB in the mouse brain after hypoxia / cerebral ischemia was examined by Western blotting.
Protein from each tissue is RIPA buffer solution (50 mM Tris-HCl (pH7.6) with 150 mM NaCl, 1% NP-40, containing protease inhibitor (Nacalai) and phosphatase inhibitor (Nakarai). 0.5% sodium deoxycholate and 0.1% SDS]. Further, in order to remove the residue, it was centrifuged at 4 ° C and 15,000 rpm for 10 minutes. After assaying the protein concentration by BCA protein assay system (Pierce, Rockford, IL), an equal amount of protein was subjected to Western blotting. The protein suspension was denatured in loading buffer, and then subjected to SDS-PAGE and transferred to Immobilon P-membrane (Millipore). Anti-NF-κB p65 antibody (1: 1000 dilution; Cell signaling technology) and anti-phospho NF-κB p65 (Ser536) antibody (1: 1000; Cell signaling technology) were used as primary antibodies.
The results are shown in FIG. In hippocampal cells undergoing ischemic injury, NFκB, known as a gene expression regulator, is temporarily activated, but it was found that this temporary activation is suppressed by administration of HA4.

[Quantitative PCR]
Next, the expression level of IL-1β in the hippocampus was measured by quantitative PCR.
In performing quantitative PCR, the ischemic hippocampus was dissolved in Isogen (Nippon gene), total RNA was extracted, and cDNA was prepared using Superscript 3 (Invitrogen). The expression level of IL-1β was quantified using a Thunderbird sybr qPCR mix (Toyobo) by Thermal cycler dice (Takara). The primer combinations are as follows.
The results are shown in FIG. The expression level of IL-1β located downstream of NFκB activation was suppressed by the administration of hyaluronic acid tetrasaccharide.
The results in FIGS. 5 and 6 show that cell death due to ischemic injury is different from apoptotic cell death which suppresses hyaluronan tetrasaccharide as a result of activating NFκB through binding to Toll-like receptors. It is.

  The results of the above examples strongly suggest that the neuronal protective effect of hyaluronic acid tetrasaccharide at the time of neonatal hypoxia / cerebral ischemia load in mice.

SEQ ID NO: 1 is the DNA sequence of the forward primer used for quantitative PCR in which the expression level of IL-1β was measured.
SEQ ID NO: 2 is the DNA sequence of the reverse primer used for quantitative PCR in which the expression level of IL-1β was measured.
SEQ ID NO: 3 is the DNA sequence of the forward primer used for quantitative PCR in which the expression level of β-actin was measured.
SEQ ID NO: 4 is the DNA sequence of the reverse primer used for quantitative PCR in which the expression level of β-actin was measured.

Claims (2)

An autophagic cell death inhibitor in the hippocampus comprising a low molecular weight hyaluronic acid tetrasaccharide or a salt thereof. The autophagic cell death inhibitor of Claim 1 which suppresses the autophagic cell death of the nerve cell in the hippocampus by ischemia.