JP2014156407A - Neuritis or demyelinating disease preventive or therapeutic pharmaceutical composition comprising dock8 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a neuritis or demyelinating disease preventive or therapeutic pharmaceutical composition.SOLUTION: A neuritis or demyelinating disease preventive or therapeutic pharmaceutical composition comprises Dock8 protein or DNA coding for the protein.

Description

  The present invention relates to a pharmaceutical composition for preventing or treating neuroinflammation or demyelinating disease comprising Dock8.

Dock8 (Dedicator of cytokinesis 8) is a member of the Dock family protein and is a guanine nucleotide exchange factor (GEF). Dock8 regulates actin cytoskeleton polymerization through activation of Rho family low molecular weight G proteins. The reorganization of the actin cytoskeleton is related to various cell functions such as cell differentiation / proliferation, migration, and signal transduction (Non-patent Document 1: Kazuhiko Nogata, Takayuki Harada, Biochemistry 84 (5), 368-373, 2012).
In recent years, it has been discovered that a deficiency in the human Dock8 gene causes severe combined immunodeficiency, and several immunological research results of Dock8 have been reported (Non-Patent Document 2: Zhang Q, et al). ., N Engl J Med 361 (21): 2046-2055, 2009; Non-patent document 3: Randall KL, et al., Nat Immunol 10 (12): 1283-1291, 2009; Non-patent document 4: Randall KL, et al., J Exp Med 208 (11): 2305-2320, 2011).
On the other hand, the present inventor has been investigating experimental autoimmune encephalomyelitis (EAE), which is a disease model of multiple sclerosis (MS), for many years. The mechanism of the onset and aggravation of the disease has been explored (Non-Patent Document 5: Guo X, et al., EMBO Molecular Medicine 2 (12): 504-515, 2010). MS is one of the demyelinating diseases of the central nervous system. It is a disease in which visual disorders or movement disorders occur due to lesions in the brain, spinal cord, or optic nerve. Although the number of patients in Japan exceeds 12,000, it is one of the specific diseases designated by the Ministry of Health, Labor and Welfare because the detailed cause has not been elucidated and treatment is difficult. In addition, among optic neuromyelitis (NMO), which was once a type of MS, treatments such as plasma exchange have been carried out for cases positive for aquaporin 4 (AQP4) antibody, but AQP4 antibody The number of positive cases is limited.
Therefore, at present, for neuroinflammation and demyelinating diseases, symptomatic treatment such as administration of steroids and immunosuppressive agents is the center, and it is difficult to say that a fundamental treatment method has been established. As described above, the relationship between the Dock family molecule and immunodeficiency has been reported, but the involvement of the Dock family molecule in neuroinflammation and demyelinating diseases observed in MS has never been investigated.

Kazuhiko Nogata, Takayuki Harada, Biochemistry 84 (5), 368-373, 2012 Zhang Q, et al., N Engl J Med 361 (21): 2046-2055, 2009 Randall KL, et al., Nat Immunol 10 (12): 1283-1291, 2009 Randall KL, et al., J Exp Med 208 (11): 2305-2320, 2011 Guo X, et al., EMBO Molecular Medicine 2 (12): 504-515, 2010

  The present invention has been made in view of such circumstances, and a problem to be solved is to provide a pharmaceutical composition for preventing or treating neuroinflammation or demyelinating disease.

As a result of diligent research to solve the above problems, the inventor of the present invention, in the Dock8 overexpressing mouse uniquely produced by the present inventor, the incidence of EAE decreased, and symptoms of neuroinflammation and demyelination As a result, the present invention has been completed.
That is, the present invention is as follows.

(1) A pharmaceutical composition for preventing or treating neuroinflammation or demyelinating disease, comprising Dock8 protein or DNA encoding the protein.
(2) A pharmaceutical composition for preventing or treating neuroinflammation or demyelinating disease, comprising the following protein (a), (b) or (c):
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 (b) in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6, wherein one or several amino acids are deleted, substituted or substituted Protein (c) comprising an added amino acid sequence and having an action of reducing symptoms of neuroinflammation or demyelinating disease 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 A protein comprising an amino acid sequence having an action of reducing symptoms of neuroinflammation or demyelinating disease (3) comprising a DNA encoding a protein of the following (a), (b) or (c): A pharmaceutical composition for preventing or treating a demyelinating disease.
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 (b) in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6, wherein one or several amino acids are deleted, substituted or substituted Protein (c) comprising an added amino acid sequence and having an action of reducing symptoms of neuroinflammation or demyelinating disease 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 A protein having an amino acid sequence having an action of reducing symptoms of neuroinflammation or demyelinating disease (4) comprising a DNA of the following (a), (b) or (c): A pharmaceutical composition for prevention or treatment.
(A) DNA comprising the base sequence represented by SEQ ID NO: 1, 3 or 5
(B) An action of hybridizing with a sequence consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1, 3 or 5 under stringent conditions and reducing symptoms of neuroinflammation or demyelinating disease DNA encoding a protein having
(C) a protein comprising a base sequence having 60% or more homology to the base sequence represented by SEQ ID NO: 1, 3, or 5 and having an action of reducing symptoms of neuroinflammation or demyelinating disease DNA to do
(5) Any of the above (1) to (4), wherein the neuroinflammation is at least one selected from the group consisting of optic neuritis, neurodegenerative disease, neuralgia, migraine, ischemic stroke and inflammatory bowel disease A pharmaceutical composition according to 1.
(6) The above (1) to (1), wherein the demyelinating disease is at least one selected from the group consisting of multiple sclerosis, acute disseminated encephalomyelitis, progressive multifocal leukoencephalopathy and Guillain-Barre syndrome. The pharmaceutical composition according to any one of 4).
(7) A test substance is brought into contact with a non-human mammal or a biological sample derived from a mammal, and the expression level of Dock8 protein or Dock8 gene in the non-human mammal or the biological sample is measured. A screening method for a prophylactic or therapeutic agent for inflammation or demyelinating disease.
(8) A screening method for a preventive or therapeutic agent for neuroinflammation or demyelinating disease, comprising the following steps.
(A) contacting the test substance with a non-human mammal or a biological sample derived from a mammal;
(B) comparing the expression level of the Dock8 protein or Dock8 gene in the non-human mammal or the biological sample contacted with the test substance with the expression level of the Dock8 protein or Dock8 gene in the control sample; and (c ) Selecting the test substance as a preventive or therapeutic agent for neuroinflammation or demyelinating disease using the comparison result as an index

  According to the present invention, a pharmaceutical composition useful for prevention or treatment of neuroinflammation or demyelinating disease can be provided.

(A) It is a figure which shows the structure of the plasmid used for preparation of the Dock8 overexpression transgenic mouse (Dock8 Tg mouse). (B) It is a figure which shows the result of having quantified the expression level of Dock8 in the T cell (T cell) and dendritic cell (DC) of a wild type mouse (WT) and a Dock8 Tg mouse | mouth. It is a figure which shows the result of having measured the ratio of the subtype of the T cell in a spleen. It is a figure which shows the measurement result of the T cell activity after stimulation by CD3 antibody. It is a figure which shows the measurement result of the antigen presentation ability of a dendritic cell. It is a figure which shows the measurement result of the onset rate of EAE. It is a figure which shows the measurement result of a spinal cord symptom. It is a figure which shows the pathological image of EAE. It is a figure which shows the measurement result of visual dysfunction by EAE.

  Hereinafter, the present invention will be described in detail. The following embodiment is an example for explaining the present invention, and is not intended to limit the present invention to this embodiment alone. The present invention can be implemented in various forms without departing from the gist thereof.

1. Outline The present invention relates to a pharmaceutical composition containing Dock8 protein or DNA encoding Dock8 protein, and relates to a pharmaceutical composition useful for the prevention or treatment of neuroinflammation or demyelinating disease.
Until now, it has been known that the deficiency of the human Dock8 gene causes severe combined immunodeficiency, but the involvement of Dock family molecules in neuroinflammation and demyelinating diseases has not been known.
In contrast, the present inventor produced a mouse that overexpresses the human Dock8 gene and examined the effect of Dock8. As a result, it was unexpectedly found that the incidence of EAE decreased in the mouse and the symptoms of neuroinflammation and demyelination were reduced. Specifically, in about 40% of Dock8 overexpressing mice, the onset of EAE itself was suppressed, and in the remaining 60%, neuroinflammation and mild demyelination were confirmed.
Therefore, Dock8 protein or DNA (gene) encoding Dock8 protein is useful for the prevention or treatment of neuroinflammation or demyelinating diseases. The present invention has been completed based on such findings.

2. Dock8
In the present invention, Dock8 was found to reduce the incidence of EAE and reduce the symptoms of neuroinflammation and demyelination. That is, Dock8 is useful for the prevention or treatment of neuroinflammation or demyelinating diseases. In the present invention, the term “Dock8” means both the Dock8 protein and the DNA encoding the Dock8 protein.

  The Dock8 protein and DNA encoding the protein used in the present invention may be derived from any mammal, and examples of such mammals include mice, rats, rabbits, goats, monkeys. And a human, preferably a mouse, a rat and a human, and more preferably a human.

  In the present invention, amino acid sequences of mouse, rat and human Dock8 proteins are represented by SEQ ID NOs: 2, 4 and 6, respectively. The base sequences of DNAs encoding mouse, rat and human Dock8 proteins are represented by SEQ ID NOs: 1, 3 and 5, respectively. Each amino acid sequence and base sequence is registered with a predetermined accession number (Accession No.) in the GenBank database.

Amino acid sequence of mouse Dock8 protein: XM_488529.2 (SEQ ID NO: 2)
Amino acid sequence of rat Dock8 protein: NM_001037793 (SEQ ID NO: 4)
Amino acid sequence of human Dock8 protein: AB191037 (SEQ ID NO: 6)
Base sequence of DNA encoding mouse Dock8 protein: XM_488529.2 (SEQ ID NO: 1)
Base sequence of DNA encoding rat Dock8 protein: NM_001037793 (SEQ ID NO: 3)
Base sequence of DNA encoding human Dock8 protein: AB191037 (SEQ ID NO: 5)

(1) Dock8 protein As a Dock8 protein used by this invention, the protein containing the amino acid sequence represented by sequence number 2, 4, or 6 is mentioned, for example, Preferably, it is represented by sequence number 2, 4, or 6 It is a protein consisting of an amino acid sequence.

  In addition to the above protein, the Dock8 protein used in the present invention includes an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6. And a protein having an action of reducing the symptoms of neuroinflammation or demyelinating disease.

Examples of the “amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6” include, for example,
(i) 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, more preferably 1 in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6) ) Amino acid sequence from which amino acids are deleted,
(ii) 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, more preferably 1 in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6) Amino acid sequence in which the amino acid of
(iii) 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, more preferably 1) in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 An amino acid sequence added with
(iv) Amino acid sequences mutated by the combination of (i) to (iii) above.

  In the present invention, “the action of reducing the symptoms of neuroinflammation or demyelinating disease” refers to the action of reducing the symptoms of neuroinflammation or demyelinating diseases such as optic neuritis and multiple sclerosis. “Symptoms” of neuroinflammation or demyelinating diseases include, for example, demyelination, immune responses in nerves (eg inflammation), degeneration of nerve axons, paralysis of part or whole body of body, numbness, sensory disturbance, visual function Examples include, but are not limited to, disability, double vision, movement paralysis, gait disturbance, and urination / defecation disorder. In addition, “having an action of reducing the symptoms of neuroinflammation or demyelinating disease” means an action of reducing the symptoms of neuroinflammation or demyelinating diseases of a protein comprising the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, preferably 90% or more It means to show.

  The effect of reducing the symptoms of neuroinflammation or demyelinating disease can be confirmed using, for example, the method described in Example 3 of the present specification. For example, an experimental autoimmune encephalomyelitis (Experimental) which is a disease model of multiple sclerosis in a mouse in which a mutant of a protein containing the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 is overexpressed. By developing autoimmune encephalomyelitis (EAE) and confirming the degree of EAE symptoms, it is possible to confirm whether the protein has an action of reducing the symptoms of neuroinflammation or demyelinating diseases.

  In addition to the amino acid sequence represented by SEQ ID NO: 2, 4 or 6, the Dock8 protein used in the present invention has 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2, 4 or 6. And a peptide having an amino acid sequence having an action to reduce symptoms of neuroinflammation or demyelinating disease. Such peptides include about 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% with respect to the amino acid sequence shown in SEQ ID NO: 2, 4 or 6. Or having an amino acid sequence having a homology of 99% or more and having an action of reducing symptoms of neuroinflammation or demyelinating disease (substantially the amino acid sequence represented by SEQ ID NOs: 2, 4, and 6) Equivalent amino acid sequences). For homology, homology search such as FASTA, BLAST, PSI-BLAST, etc. can be used on a homology search site using the Internet, for example, Japan DNA Data Bank (DDBJ). In addition, a search using BLAST can be performed in the National Center for Biotechnology Information (NCBI).

In order to introduce a mutation into DNA encoding the protein in order to prepare a protein having the above mutation, a mutation introduction kit using a site-directed mutagenesis method such as Kunkel method or Gapped duplex method, for example, QuikChange ^TM Site-Directed Mutagenesis Kit (Stratagene), GeneTailor ^TM Site-Directed Mutagenesis System (Invitrogen), TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc .: Takara Bio) Etc. can be used. Further, methods such as site-specific mutagenesis described in “Molecular Cloning, A Laboratory Manual (4th edition)” (Cold Spring Harbor Laboratory Press (2012)) and the like can be used.

The Dock8 protein of the present invention can be prepared using a known method. Specifically, it can be produced by the following method.
(i) Preparation of expression vector
The expression vector for producing Dock8 protein is not particularly limited as long as it is retained in the host cell to be expressed, and examples thereof include plasmid vectors, virus vectors, bacteriophages and the like. Examples of plasmid vectors include, but are not limited to, pME18S, pcDNA3, pBR322, pUC18, pUC19, pUC118, pUC119, pBluescript, and the like. Examples of viral vectors include adenovirus vectors, AIDS virus vectors, lentivirus vectors, and vectors derived therefrom.
As a technique for incorporating the DNA encoding the Dock8 protein of the present invention into a vector, a technique of ligating with a ligase after digestion with an appropriate restriction enzyme or the like is employed (see Molecular cloning etc. above).
For the DNA encoding the Dock8 protein of the present invention, a primer for amplifying the DNA region is designed, the region is amplified using a PCR method or the like, and then only a desired portion is linked to the vector using a restriction enzyme or ligase. Can be made.

(ii) Transformation The host used in the present invention may be any host that expresses the DNA encoding the Dock8 protein after the DNA encoding the Dock8 protein has been introduced. Examples of such a host include, but are not limited to, mammalian cells, bacteria such as bifidobacteria, lactic acid bacteria, and E. coli, insect cells, yeasts, and molds.
Introduction of the recombinant DNA into the host can be performed by a known method. Examples of the method for introducing the vector into a host include a calcium phosphate method, a DEAE-dextran method, an electroporation method, and a cationic lipid method.
Confirmation of the introduction of DNA can be performed using a selectable marker gene (for example, ampicillin resistance gene, neomycin resistance gene, tetracycline resistance gene, kanamycin resistance gene, etc.).

(iii) Production of Dock8 Protein The Dock8 protein of the present invention can be obtained by culturing the transformant containing DNA encoding the Dock8 protein or a mutant thereof, and collecting the protein from the culture.
“Culture” means any of culture supernatant, cultured cells, cultured cells, or disrupted cells or cells. The transformant is cultured according to a usual method used for host culture.
When the Dock8 protein is accumulated in the cells or cells after the culture, the Dock8 protein is collected by crushing the cells or cells by performing a homogenizer treatment or the like. When Dock8 protein is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, the Dock8 protein is collected from the culture solution by an ammonium sulfate precipitation operation or the like, and further isolated and purified using various chromatography or the like as necessary.

In the present invention, it is possible to produce Dock8 protein by employing a cell-free protein synthesis system without using any living cells.
The cell-free protein synthesis system is a system that synthesizes a protein in an artificial container such as a test tube using a cell extract. For example, it reads mRNA information and synthesizes a protein on a ribosome. The cell-free protein synthesis system used in the present invention includes a cell-free transcription system that synthesizes RNA using DNA as a template.
In the present invention, cell-free protein synthesis can also be performed using a commercially available kit. Examples of such kits include reagent kits PROTEIOS ^™ (Toyobo), TNT ^™ System (Promega), synthesizer PG-Mate ^™ (Toyobo), RTS (Roche Diagnostics) and the like.
The obtained Dock8 protein can be purified by appropriately selecting chromatography. Confirmation that the Dock8 protein has been isolated and purified can be performed by SDS-PAGE or the like.

(2) DNA encoding Dock8 protein
In the present invention, the DNA encoding the Dock8 protein includes, in addition to the DNA encoding the Dock8 protein or a variant thereof, for example, a DNA containing the base sequence represented by SEQ ID NO: 1, 3, or 5; Is DNA which consists of a base sequence represented by sequence number 1, 3, or 5.

  The DNA encoding the Dock8 protein used in the present invention includes, in addition to the above DNA, a sequence consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1, 3 or 5 under stringent conditions. And a DNA encoding a protein that has a function of reducing the symptoms of neuroinflammation or demyelinating disease.

  In the present invention, “DNA that hybridizes under stringent conditions” is, for example, a colony hybridization method using, as a probe, all or part of a polynucleotide comprising a base sequence complementary to the base sequence of SEQ ID NO: 1. It can be obtained by using a plaque hybridization method or a Southern hybridization method. As a hybridization method, for example, a method described in “Molecular Cloning, A Laboratory Manual (4th edition)” (Cold Spring Harbor Laboratory Press (2012)) can be used.

  In the present invention, the “stringent conditions” may be any of low stringent conditions, medium stringent conditions, and high stringent conditions. “Low stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 32 ° C. The “medium stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, and 42 ° C. “High stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 50 ° C. Under these conditions, it can be expected that DNA having higher homology can be efficiently obtained as the temperature is increased. However, factors affecting the stringency of hybridization include multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration, and those skilled in the art can select these factors as appropriate. By doing so, it is possible to achieve the same stringency.

  The DNA encoding the Dock8 protein used in the present invention contains a nucleotide sequence having 60% or more homology with the nucleotide sequence represented by SEQ ID NO: 1, 3, or 5, and is associated with neuroinflammation or depletion. Also included is a DNA encoding a protein having an action of reducing the symptoms of myelopathy. As such DNA, when calculated using homology search software such as FASTA, BLAST and the like using the default parameters, the nucleotide sequence represented by SEQ ID NO: 1, 3 or 5 is 60% or more and 70%. As mentioned above, DNA having homology of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more can be mentioned.

  The DNA encoding the Dock8 protein or a variant thereof is, for example, based on the nucleotide sequence described in the NCBI GenBank database etc., “Molecular Cloning, A Laboratory Manual (4th edition)” (Cold Spring Harbor Laboratory Press (2012) ) And the like can be obtained using a known genetic engineering technique.

3. Pharmaceutical Composition The Dock8 protein of the present invention has a preventive or therapeutic effect on neuroinflammation or demyelinating diseases. Therefore, the pharmaceutical composition containing Dock8 protein or DNA encoding Dock8 protein can be used as a pharmaceutical composition for preventing or treating neuroinflammation or demyelinating disease. Here, the Dock8 protein of the present invention includes those expressed by overexpression of the protein.
In the present invention, “neuroinflammation” means a pathological condition in which the intrinsic cells of the nervous system play an important role. Examples of such diseases include optic neuritis, neurodegenerative diseases, neuralgia, migraine, Examples include ischemic stroke and inflammatory bowel disease.

In the present invention, the term “demyelinating disease” means that the nerve myelin sheath is damaged, and examples of such diseases include multiple sclerosis, acute disseminated encephalomyelitis, progressive Examples include multifocal leukoencephalopathy and Guillain-Barre syndrome, and multiple sclerosis is preferred.
In the present invention, “prevention” means that the pharmaceutical composition of the present invention is brought into contact (for example, administration) with the subject before the onset of the disease, thereby suppressing the onset rate of the disease compared to the case where the subject is not in contact (for example, administration). ) And alleviating symptoms after the onset of the disease, and does not necessarily mean completely suppressing the onset.

In the present invention, “treatment” refers to reducing the symptom of the disease by bringing the pharmaceutical composition of the present invention into contact with the subject after the onset of the disease (for example, administration), as compared with the case of not making contact. It does not necessarily mean that the symptoms of the disease are completely suppressed. The onset of a disease means that symptoms of the disease appear in the body.
“Symptoms” of neuritis or demyelinating diseases include, for example, demyelination, immune responses in nerves (eg inflammation), degeneration of nerve axons, paralysis of part or whole body of body, numbness, sensory disturbance, visual function Examples include, but are not limited to, disability, double vision, movement paralysis, gait disturbance, and urination / defecation disorder.
The “nerve” in the present invention includes both the central nerve (brain and spinal cord) and the peripheral nerve.

  The pharmaceutical composition of the present invention can contain the Dock8 protein of the present invention, DNA encoding the Dock8 protein, a vector expressing the Dock8 protein, and a pharmaceutically acceptable carrier. “Pharmaceutically acceptable carrier” refers to any carrier (liposome, lipid vesicle, micelle, etc.), diluent, excipient, wetting agent suitable for the pharmaceutical composition for prevention or treatment of neuroinflammation or demyelinating disease , Buffers, suspending agents, lubricants, adjuvants, emulsifiers, disintegrants, absorbents, preservatives, surfactants, colorants, flavoring agents, or sweeteners.

  The pharmaceutical composition of the present invention comprises an injection, a lyophilized product, a tablet, a hard capsule, a soft capsule, a granule, a powder, a pill, a syrup, a suppository, a buccal, an ointment, a cream, and an eye drop. Etc. can be used. Liquid preparations such as injections may be in the form of powders for preparation (for example, freeze-dried powders) that are dissolved in physiological saline before use.

  The pharmaceutical compositions of the invention can be administered locally or systemically by any means known to those skilled in the art. As the administration route of the pharmaceutical composition of the present invention, both oral administration and parenteral administration are possible. In the case of parenteral administration, intra-tissue administration (subcutaneous administration, intraperitoneal administration, intramuscular administration, intravenous administration) Etc.), intradermal administration, topical administration (such as transdermal administration) or rectal administration. The pharmaceutical composition of the present invention can be administered in a dosage form suitable for these administration routes.

  The dosage of the pharmaceutical composition of the present invention varies depending on factors such as the age, weight, health status, sex, symptom, administration route, number of administrations, dosage form, etc. of the subject, and the specific administration procedure is determined by those skilled in the art. Can be set. For example, for an adult, when the pharmaceutical composition of the present invention is administered as a tablet, 0.1 μg to 10 g, preferably 1 μg to 1 g, more preferably 10 μg to 100 mg should be administered 1 to 5 times a day. Can do.

  The administration timing can be determined appropriately according to the symptoms, and multiple doses can be administered simultaneously or separately over time. In addition, the pharmaceutical composition of the present invention may be administered to a subject before the onset of the disease or may be administered after the onset of the disease.

  The pharmaceutical composition of the present invention can be administered to a mammal as a subject. Examples of mammals include mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, goats, pigs, sheep, cows, horses, monkeys, humans, and the like.

  Furthermore, when the pharmaceutical composition of the present invention is used as a gene therapy agent, in addition to the method of directly administering the pharmaceutical composition of the present invention by injection, there is a method of administering a vector incorporating a DNA encoding Dock8 protein. Can be mentioned. That is, the “DNA encoding the Dock8 protein” in the present invention includes an expression vector containing DNA encoding the Dock8 protein. Examples of the vector include a plasmid vector, an adenovirus vector, an adeno-associated virus vector, a herpes virus vector, a vaccinia virus vector, a retrovirus vector, a lentivirus vector, and the like, and can be efficiently administered by using a virus vector. .

More specifically, for example, a Dock8 expression vector prepared by inserting a DNA encoding a Dock8 protein into a pCAGGS vector (Niwa H. et al., Gene 108: 193-200, 1991) This is preferable because it can be overexpressed systemically. The pCAGGS vector is a vector having a so-called CAG promoter (a structure in which a Cytomegalovirus enhancer and a chicken β-actin promoter are connected) and a polyA signal site of a rabbit β-globin gene.
In the present invention, an expression vector containing a DNA encoding Dock8 protein or a pharmaceutical composition containing the same is administered to a subject, and the inflammation or demyelinating disease is prevented or treated by overexpressing the Dock8 protein in the subject. be able to. That is, the pharmaceutical composition of the present invention can be used in a method for preventing or treating neuroinflammation or demyelinating disease.

  It is also possible to introduce the pharmaceutical composition of the present invention into a phospholipid vesicle such as a liposome and administer the vesicle. The endoplasmic reticulum holding Dock8 protein or DNA encoding Dock8 protein is introduced into a predetermined cell by the lipofection method. Then, the obtained cells are systemically administered, for example, intravenously or intraarterially. It can also be administered locally to the brain or the like. For example, when the above-mentioned pharmaceutical composition is administered to an adult, 0.1 μg / kg to 1000 mg / kg per day, preferably 1 μg / kg to 100 mg / kg per day can be administered. A person skilled in the art can also use a commercially available kit suitable for gene transfer.

  Furthermore, the vector expressing the Dock8 protein of the present invention can be transformed into a host such as bifidobacteria, lactic acid bacteria, yeast, filamentous fungi, etc., and the transformant can be used for the prevention or treatment of neuroinflammation or demyelinating diseases. These transformants can be used as they are, or can be appropriately formulated into the above-mentioned dosage forms and used as the pharmaceutical composition of the present invention.

4). Screening method The present invention was completed based on the knowledge that neuroinflammation or demyelinating disease can be prevented or treated by overexpression of Dock8, and the expression level of Dock8 protein or Dock8 gene was used as an index. Alternatively, a substance that increases the expression level of the Dock8 gene can be screened as a preventive or therapeutic agent for neuroinflammation or demyelinating disease.

  That is, the present invention is characterized in that a test substance is brought into contact with a non-human mammal or a biological sample derived from a mammal, and the expression level of Dock8 protein or Dock8 gene in the non-human mammal or the biological sample is measured. A screening method for a preventive or therapeutic agent for neuroinflammation or demyelinating disease is provided.

In another aspect, the present invention provides a screening method for a prophylactic or therapeutic agent for neuroinflammation or demyelinating disease, comprising the following steps.
(A) contacting the test substance with a non-human mammal or a biological sample derived from a mammal;
(B) comparing the expression level of the Dock8 protein or Dock8 gene in the non-human mammal or the biological sample contacted with the test substance with the expression level of the Dock8 protein or Dock8 gene in the control sample; and (c ) Selecting the test substance as a preventive or therapeutic agent for neuroinflammation or demyelinating disease using the comparison result as an index

  In the present invention, the “test substance” refers to a substance that may have some influence on the expression of Dock8 protein or Dock8 gene. For example, nucleic acids (DNA, RNA, cDNA, etc.) and vectors and antibodies containing them , Peptides, proteins, synthetic compounds and the like.

In the present invention, the term “non-human mammal” means a mammal other than human, for example, mouse, rat, hamster, guinea pig, rabbit, cat, dog, goat, pig, sheep, cow, horse, monkey, etc. Is mentioned. Among non-human mammals, rodents, particularly mice or rats, which have relatively short ontogeny and biological cycle and are easy to reproduce, are preferred.
The non-human mammal may be a normal animal or an animal that has developed neuroinflammation or a demyelinating disease.

  In the present invention, the “mammal-derived biological sample” means a mammal-derived tissue, cell, a crushed material or extract thereof, and a body fluid itself or a sample containing these, and is not particularly limited. Here, “mammal” includes humans in addition to the non-human mammals. Examples of tissues derived from mammals include brain, spinal cord, heart, lung, kidney, gallbladder, liver, pancreas, spleen, intestine, testis (testis), ovary, uterus, placenta, muscle, blood vessel, bone marrow, thyroid, thymus , Mammary gland, lymph node, blood and the like.

  The “mammal-derived biological sample” can be subjected to any treatment, such as dilution, separation, nucleic acid extraction, etc., on the mammal-derived tissue, cells, crushed material or extract thereof, and body fluid. Samples obtained by performing are also included.

  In the present invention, mammal-derived cells include cells contained in the tissue, organ or body fluid, cultured cells obtained by isolation and culture from the tissue, organ or body fluid, cell lines derived from mammals (cell line). ) And induced pluripotent stem cells (iPS cells). The cells include somatic cells such as nerve cells and immune cells, as well as stem cells. The cultured cells include primary cultured cells and subcultured cells.

  In the present invention, “contact” means that a non-human mammal or a biological sample derived from a mammal and a test substance are present in the same reaction system. For example, the test substance is applied to a non-human mammal. Administering a test substance to a biological sample derived from a mammal, culturing the cell in the presence of the test substance when the biological sample derived from a mammal is a cell, and For example, injecting a test substance.

  In the present invention, the “control sample” refers to a non-human mammal or a biological sample derived from a mammal that has not been contacted with a test substance. The mammal may be normal or may have developed neuroinflammation or demyelinating disease, and those skilled in the art can appropriately select the mammal according to its purpose.

  The expression level of Dock8 protein can be measured by, for example, immunoassay. Examples of immunoassay include radioimmunoassay (RIA), immunofluorescence assay (FIA), immunoluminescence assay, enzyme immunoassay (eg, Enzyme Immunoassay (EIA), Enzyme-linked Immunosorbent assay (ELISA)), Or Western blot method etc. are mentioned. Alternatively, the expression level of Dock8 protein may be measured by immunohistochemistry. Specific examples of immunohistochemistry include enzyme-labeled antibody method or fluorescent antibody method.

  The expression level of Dock8 gene is measured using a known method, for example, Northern hybridization using a probe and / or primer designed based on the base sequence of DNA encoding Dock8 protein, PCR method, RT-PCR method. Can be done. In the present invention, the “Dock8 gene” includes DNAs, mRNAs, cDNAs, and nucleic acids containing these that encode Dock8 proteins.

In the present invention, a test substance is contacted with a non-human mammal or a biological sample derived from a mammal, the expression level of Dock8 protein or Dock8 gene in the non-human mammal or the biological sample is measured, and the measurement result and nerve It can be associated with the prophylactic or therapeutic effect of inflammation or demyelinating disease. Then, a critical value (cut-off value) in the expression level of the Dock8 protein or the Dock8 gene is determined, and a test substance exhibiting an expression level equal to or higher than the cut-off value is used as a prophylactic or therapeutic agent for neuroinflammation or demyelinating disease. It can also be selected as a medicine.
Here, the expression level of the Dock8 protein or Dock8 gene for determining the cut-off value can be determined as follows, for example.
First, a substance that increases the expression level of the Dock8 protein or the Dock8 gene and has been confirmed to have a preventive or therapeutic effect on neuroinflammation or demyelinating disease (for example, a Dock8 protein expression vector) is treated with a subject (for example, a non-human). And the expression level of Dock8 protein or Dock8 gene in the sample derived from the subject is measured. At this time, the number of subjects is 2 or more, for example, 10 or more, 50 or more, 100 or more, or 500 or more. It is also preferable to measure the expression level of the Dock8 protein or Dock8 gene in a sample derived from a subject who has not been administered a substance that increases the expression level of the Dock8 protein or Dock8 gene (control sample). The number of examples of the control sample is, for example, 2 or more, 10 or more, 50 or more, 100 or more, or 500 or more. Then, the cut-off value of the expression level of the Dock8 protein or Dock8 gene is statistically calculated from the whole including both the biological sample group and the control sample group derived from the subject administered the substance that increases the expression level of the Dock8 protein or Dock8 gene. Obtain by processing. Examples of the statistical processing include analysis using a ROC (receiver operating characteristic) curve.

In the present invention, the expression level of Dock8 protein or Dock8 gene in a non-human mammal or a mammal-derived biological sample contacted with a test substance is compared with the expression level of Dock8 protein or Dock8 gene in a control sample. Then, using the comparison result as an index, the test substance can be selected as a preventive or therapeutic agent for neuroinflammation or demyelinating disease.
Specifically, the expression level (A) of a Dock8 protein or Dock8 gene in a non-human mammal or a mammal-derived biological sample contacted with a test substance, and the expression level (B) of a Dock8 protein or Dock8 gene in a control sample When the expression level (A) is higher than the expression level (B), for example, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60 % Or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, or about 100% or more, the test substance is a prophylactic or therapeutic agent for neuroinflammation or demyelinating disease Can be selected.
In addition, the expression level of the Dock8 protein or Dock8 gene in the non-human mammal contacted with the test substance or the biological sample derived from the mammal is statistically compared with the expression level of the Dock8 protein or Dock8 gene in the control sample. When it is significantly high, the test substance can also be selected as a prophylactic or therapeutic agent for neuroinflammation or demyelinating disease. Statistical analysis can be performed using a known method such as a T test.
In the above case, the non-human mammal contacted with the test substance or the biological sample derived from the mammal and the control sample are prepared in 2 or more cases, for example, 3 or more, 5 or more, 10 or more, respectively. Comparing the average value of the expression level of Dock8 protein or Dock8 gene in a non-human mammal contacted with a substance or a biological sample derived from a mammal with the average value of the expression level of Dock8 protein or Dock8 gene in a control sample preferable.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples.

Preparation of Dock8 overexpressing transgenic mouse (Dock8 Tg mouse) Construction of human Dock8 expression vector
A human Dock8 expression vector is inserted into the pCAGGS vector (Niwa H. et al., Gene 108: 193-200, 1991) using a DNA encoding the human Dock8 protein (SEQ ID NO: 5) using a conventional genetic engineering technique. Was constructed (FIG. 1A).

2. Gene transfer
Ten-week-old female C57 / BL / 6J mice (Claire Japan) were mated and fertilized eggs were collected from the mice. To the collected fertilized egg, the above 1. The human Dock8 expression vector prepared in 1 was injected by the microinjection method. The fertilized egg injected with the vector was transplanted into the oviduct of a pseudopregnant mouse (C57 / BL / 6J mouse (Claire Japan)). Pseudopregnant mice were produced by mating with infertile male mice and confirming plug positivity.
After the fertilized egg was transplanted into the fallopian tube, it was born by spontaneous delivery to obtain a Dock8 Tg mouse.

3. Confirmation of protein expression After the presence or absence of transgene expression in the born mouse was confirmed by PCR, protein expression was confirmed by Western blot.
As a result, in Dock8 Tg mice, overexpression of the human Dock8 gene could be confirmed in the spleen and thymus important for immune response (FIG. 1B).

Functional analysis of Dock8 Tg mouse In the following examples, the mouse prepared in Example 1 above is used as a Dock8 Tg mouse, and a C57 / BL / 6J mouse into which Dock8 is not introduced is a wild type (WT) mouse. used.

1. Measurement of the proportion of T cell subtypes in the spleen of Dock8 Tg mice In order for a normal immune response to occur, it is important that T cells differentiate normally into each subtype.
First, spleens were taken out from wild type (WT) mice and Dock8 Tg mice, and spleen cells (splenocytes) were purified. Using purified spleen cells, the proportion of T cell subtypes in the spleen was measured by flow cytometry (FACS).
As a result, no abnormality was observed in the number of T cells of each subtype in Dock8 Tg mice (FIG. 2). That is, no abnormal development of T cells in the spleen was observed in Dock8 Tg mice. Splenocytes: spleen cells, CD3 + T cells: total T cells, CD3 + CD4 + T cells: helper T cells, CD3 + CD8 + T cells: killer T cells.

2. Measurement of T cell activity in Dock8 Tg mice Spleen cells purified in the same manner as described above were stimulated by administration of an anti-CD3 antibody, and then cultured for 3 days. Using flow cytometry, cells expressing CD25 or CD69 on the cell surface and the number of cells not expressing were counted. The ratio of the number of cells expressing CD25 or CD69 to CD4 positive cells was compared between WT mice and Dock8 Tg mice.
As a result, in the spleen cells derived from Dock8 Tg mice, the number of positive cells of CD25 or CD69, which is an activation index, decreased as compared with WT mice.
From this result, it was shown that the spleen cells derived from Dock8 Tg mice had a decreased T cell activity compared to WT mice (FIG. 3).

3. Measurement of antigen-presenting ability in dendritic cells of Dock8 Tg mice Lymphocytes derived from avian ovalbumin (OVA) -specific T cell receptor transgenic mice (OT-II mice) were purified. OT-II mice (Barnden MJ et al., Immunol Cell Biol, 76, 34-40, 1998) were used for analysis of antigen presentation ability.
Dendritic cells were purified from bone marrow of WT mice and Dock8 Tg mice, respectively, and OVA was incorporated after culture. The previously purified lymphocytes and dendritic cells incorporating OVA were mixed and co-cultured for 3 days. To measure the activity state of lymphocytes, the rate of cell division and proliferation was measured by flow cytometry.
As a result, there was no difference in antigen-presenting ability of dendritic cells between WT mice and Dock8 mice (FIG. 4). In FIG. 4, CFSE means a fluorescent substance called carboxyfluorescein diacetatesuccinimidyl ester.

1. Examination of preventive and therapeutic effects of Dock8 against neuroinflammation or demyelinating diseases Measuring the incidence of experimental autoimmune encephalomyelitis (EAE) Experimental autoimmune encephalomyelitis (EAE) is a disease model for multiple sclerosis. Myelin oligodendrocyte glycoprotein (MOG) was used to develop EAE. 6-8 week old male mice are immunized by subcutaneous administration of complete Freund's adjuvant containing 100 μg of MOG _35-55 peptide, and pertussis toxin is administered intraperitoneally on days 0 and 2 of immunization. Mice were made. The change in the proportion of individuals who developed hindlimb paralysis was shown in the graph. Furthermore, the severity of the disease state was classified into a score of 1-5. Score 1: Paralyzed at the tip of the tail, Score 2: Paralyzed throughout the tail, Score 3: Abnormal erectile reflex, Score 4: Partial hind limb paralysis, Score 5: Complete hind limb paralysis.
As a result, the EAE incidence in WT mice was 100%, whereas the EAE incidence in Dock8 Tg mice was suppressed to about 60% (FIG. 5). That is, it was shown that about 40% of Dock8 Tg mice suppress the onset of EAE itself.
From these results, it was shown that Dock8 has a preventive effect on neuroinflammation and demyelinating diseases.

2. Examination of spinal cord symptoms by EAE The degree of spinal cord symptom in individuals who developed EAE in Japan was compared between Dock8 Tg mice and WT mice.
As a result, the spinal cord symptom in the individual who developed EAE in the Dock8 Tg mouse was milder than that in the WT mouse. In addition, in the Dock8 Tg mouse, the symptom gradually decreased (becomes milder) after about 2 weeks after immunization (FIG. 6).
From these results, it was shown that Dock8 has a therapeutic effect on neuroinflammation and demyelinating diseases.

3. Pathological analysis
The spinal cord and optic nerve were collected from WT mice and Dock8 Tg mice that developed EAE, respectively, and pathological analysis was performed. Specifically, pathological findings of demyelination and axonal degeneration in both tissues were observed by Luxol Fast Blue staining of the spinal cord and electron microscope analysis of the optic nerve.
We also observed tissue infiltration of microglia and macrophage cells, which play an important role in chronic inflammation, by tissue immunostaining of the spinal cord using anti-iba1 antibody.
The result is shown in FIG. In FIG. 7, the upper part shows the result in the WT mouse, and the lower part shows the result in the Dock8 Tg mouse. The left column shows the results of Luxol Fast Blue staining of the spinal cord of mice that developed EAE, and the red dotted line shows the demyelinating region. The middle row shows the result of immunostaining with anti-iba1 antibody of the mouse spinal cord that developed EAE, and the arrowhead shows microglia / macrophages infiltrating the lesion. The right column shows an electron micrograph of the optic nerve, and the arrows indicate degenerated axons.
As shown in FIG. 7, in the pathological analysis of the spinal cord, demyelination and reduction of neuroinflammation were observed in Dock8 Tg mice compared to WT mice (left column and middle column in FIG. 7). Moreover, in the electron microscope analysis of the optic nerve, degeneration of the optic nerve axon was remarkably suppressed in the Dock8 Tg mouse compared to the WT mouse (right column in FIG. 7).
From these results, it was shown that Dock8 has a therapeutic effect on neuroinflammation and demyelinating disease in pathological analysis.

4). Visual function analysis
EAE was developed in Dock8 mice and WT mice, respectively, and the presence or absence of visual dysfunction was confirmed. Specifically, multi-regional retinal potentials were analyzed using a device similar to the apparatus (VERIS; Electro-Diagnostic Imaging, Redwood City, CA, USA) used for human visual function tests.
After general anesthesia of the mouse, it was fixed to the measurement table with a contact lens with an electrode attached to the measurement eye. By showing the stimulus image displayed on the monitor for 7 minutes, the electrophysiological reaction in the retina was captured.
As a result, in WT mice, visual function decreased due to the onset of EAE, whereas Dock 8 Tg
In mice, visual function was shown to be normal (FIG. 8).

  From the above results, it was shown that Dock8 has a preventive or therapeutic effect on neuroinflammation and demyelinating diseases.

  According to the present invention, a pharmaceutical composition useful for prevention or treatment of neuroinflammation or demyelinating disease can be provided.

Claims (8)

  A pharmaceutical composition for preventing or treating neuroinflammation or demyelinating disease, comprising Dock8 protein or DNA encoding the protein. A pharmaceutical composition for preventing or treating neuroinflammation or demyelinating disease, comprising the following protein (a), (b) or (c):
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 (b) in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6, wherein one or several amino acids are deleted, substituted or substituted Protein (c) comprising an added amino acid sequence and having an action of reducing symptoms of neuroinflammation or demyelinating disease 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 A protein having an amino acid sequence having an action to reduce symptoms of neuroinflammation or demyelinating disease A pharmaceutical composition for preventing or treating neuroinflammation or demyelinating disease, comprising DNA encoding the following protein (a), (b) or (c).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 (b) in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6, wherein one or several amino acids are deleted, substituted or substituted Protein (c) comprising an added amino acid sequence and having an action of reducing symptoms of neuroinflammation or demyelinating disease 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 A protein having an amino acid sequence having an action to reduce symptoms of neuroinflammation or demyelinating disease A pharmaceutical composition for preventing or treating neuroinflammation or demyelinating disease, comprising the following DNA (a), (b) or (c):
(A) DNA comprising the base sequence represented by SEQ ID NO: 1, 3 or 5
(B) An action of hybridizing with a sequence consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1, 3 or 5 under stringent conditions and reducing symptoms of neuroinflammation or demyelinating disease DNA encoding a protein having
(C) a protein comprising a base sequence having 60% or more homology to the base sequence represented by SEQ ID NO: 1, 3, or 5 and having an action of reducing symptoms of neuroinflammation or demyelinating disease DNA to do   The pharmaceutical according to any one of claims 1 to 4, wherein the neuroinflammation is at least one selected from the group consisting of optic neuritis, neurodegenerative disease, neuralgia, migraine, ischemic stroke and inflammatory bowel disease. Composition.   The demyelinating disease is at least one selected from the group consisting of multiple sclerosis, acute disseminated encephalomyelitis, progressive multifocal leukoencephalopathy, and Guillain-Barré syndrome. The pharmaceutical composition according to item.   A test substance is brought into contact with a non-human mammal or a biological sample derived from a mammal, and the expression level of Dock8 protein or Dock8 gene in the non-human mammal or the biological sample is measured. A screening method for a prophylactic or therapeutic agent for myelopathy. A screening method for a preventive or therapeutic agent for neuroinflammation or demyelinating disease, comprising the following steps.
(A) contacting the test substance with a non-human mammal or a biological sample derived from a mammal;
(B) comparing the expression level of the Dock8 protein or Dock8 gene in the non-human mammal or the biological sample contacted with the test substance with the expression level of the Dock8 protein or Dock8 gene in the control sample; and (c ) Selecting the test substance as a preventive or therapeutic agent for neuroinflammation or demyelinating disease using the comparison result as an index