WO2020071520A1 - 間葉系幹細胞の動員活性を有するペプチド - Google Patents
間葉系幹細胞の動員活性を有するペプチドInfo
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- WO2020071520A1 WO2020071520A1 PCT/JP2019/039232 JP2019039232W WO2020071520A1 WO 2020071520 A1 WO2020071520 A1 WO 2020071520A1 JP 2019039232 W JP2019039232 W JP 2019039232W WO 2020071520 A1 WO2020071520 A1 WO 2020071520A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present application relates to a peptide having activity of mobilizing mesenchymal stem cells, a composition for mobilizing mesenchymal stem cells, and a therapeutic agent for diseases based on mobilization of mesenchymal stem cells.
- Mesenchymal stem cells contained in bone marrow fluid and the like have the ability to differentiate into various tissues (pluripotency) such as bone, cartilage, fat, muscle, nerve, and epithelium.
- various tissues such as bone, cartilage, fat, muscle, nerve, and epithelium.
- Attempts to perform regenerative medicine (cell transplantation therapy) using bone marrow-derived mesenchymal stem cells grown by the method described above have been widely performed.
- the collection of bone marrow blood containing mesenchymal stem cells is performed by an open technique in which a thick needle is repeatedly inserted into the iliac bone, so that the burden on the donor is large.
- mesenchymal stem cells gradually lose their proliferative ability and pluripotency as they are continuously subcultured in vitro.
- CPC cell processing center
- the purpose of this application is to develop a new regenerative medicine technology that can overcome the problems of cell transplantation treatment.
- the present inventors have designed an artificially sequenced peptide based on the results of unique research conducted so far, and have found that the peptide exhibits an activity of recruiting mesenchymal stem cells into peripheral blood. In addition, they have found that the artificial sequence peptide has a therapeutic effect on inflammatory bowel disease, atopic dermatitis, and cerebral infarction. Based on these findings, a new regenerative medicine technology that can overcome the problems of cell transplantation therapy is provided.
- a composition for use in recruiting mesenchymal stem cells to peripheral blood comprising a peptide selected from: (A) a peptide comprising the amino acid sequence of SEQ ID NO: 1; (B) a peptide containing an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 1; and (c) about 90% or more of the amino acid sequence of SEQ ID NO: 1
- a composition for use in treating a disease or pathological condition in a subject by recruiting mesenchymal stem cells to peripheral blood comprising a peptide selected from: (A) a peptide comprising the amino acid sequence of SEQ ID NO: 1; (B) a peptide containing an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 1; and (c) about
- composition according to [2], wherein the disease or pathological condition is selected from an inflammatory disease, an autoimmune disease, a tissue damage, a disease involving ischemia or necrosis, and a fibrotic disease.
- the disease or pathological condition is an inflammatory bowel disease.
- the disease or pathological condition is ulcerative colitis.
- the composition according to [2], wherein the disease or pathological condition is atopic dermatitis.
- the composition according to [2], wherein the disease or pathological condition is cerebral infarction.
- a composition for use in treating a disease selected from inflammatory bowel disease, atopic dermatitis and cerebral infarction comprising a peptide selected from: (A) a peptide comprising the amino acid sequence of SEQ ID NO: 1; (B) a peptide containing an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 1; and (c) about 90% or more of the amino acid sequence of SEQ ID NO: 1 A peptide comprising an amino acid sequence having the sequence identity of [10] A peptide selected from: (A) a peptide comprising the amino acid sequence of SEQ ID NO: 1; (B) a peptide containing an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 1; and (c) about 90% or more of the amino acid sequence of SEQ ID NO: 1 A peptide comprising an amino acid sequence having the sequence identity of
- the disease or pathological condition is an inflammatory bowel disease.
- the disease or pathological condition is ulcerative colitis.
- the disease or pathological condition is atopic dermatitis.
- the disease or pathological condition is cerebral infarction.
- a method for treating a disease in a subject selected from inflammatory bowel disease, atopic dermatitis and cerebral infarction comprising administering to the subject an effective amount of a peptide selected from: (A) a peptide comprising the amino acid sequence of SEQ ID NO: 1; (B) a peptide containing an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 1; and (c) about 90% or more of the amino acid sequence of SEQ ID NO: 1
- a peptide comprising an amino acid sequence having the sequence identity of [B1] A peptide selected from the following for use in recruiting mesenchymal stem cells to peripheral blood: (A) a peptide comprising the amino acid sequence of SEQ ID NO: 1; (B) a peptide containing an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 1; and (c) about 90%
- [C7] The use according to [C2], wherein the disease or pathological condition is atopic dermatitis.
- a peptide selected from the following in the manufacture of a medicament for the treatment of a disease selected from inflammatory bowel disease, atopic dermatitis and cerebral infarction (A) a peptide comprising the amino acid sequence of SEQ ID NO: 1; (B) a peptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 1; and (c) about 90% or more of the amino acid sequence of SEQ ID NO: 1
- FIG. 4 is a diagram plotting the number of colonies obtained by culturing peripheral blood 14 hours after administration of physiological saline or peptide.
- saline indicates a control group
- “1r10” indicates a peptide 1r10 administration group.
- the number of colonies was shown as a value per volume of peripheral blood (about 800 ⁇ L) collected from one mouse.
- the long bar indicates the average value and the short bar indicates the standard deviation.
- SEM mean ⁇ standard error
- ** p ⁇ 0.05 (Student's ttest) indicates a peptide 1r10 administration group.
- the horizontal axis indicates the number of days after the start of drinking of the aqueous solution of dextran sulfate sodium (DSS). It is a graph which shows the stool score of a mouse (mean ⁇ standard error (SEM)). In the graph, “saline” indicates a control group, and “1r10” indicates a peptide 1r10 administration group.
- the horizontal axis indicates the number of days after the start of drinking of the aqueous solution of dextran sulfate sodium (DSS). It is a graph which shows the DAI score of a mouse (mean ⁇ standard error (SEM);) * p ⁇ 0.05 (Mann Whitney U test)).
- saline indicates a control group
- “1r10” indicates a peptide 1r10 administration group.
- the horizontal axis indicates the number of days after the start of drinking of the aqueous solution of dextran sulfate sodium (DSS). It is a graph which shows the colon length 10 days after the start of drinking of the aqueous solution of sodium dextran sulfate (DSS) (mean ⁇ standard error (SEM)).
- SEM standard error
- FIG. 1 Shows the margins of the pinna where desquamation is easily observed. It is a graph which shows the transition of the pinna thickness during the peptide administration period (the vertical axis is a relative value when the peptide administration start date is 100%). Each point represents the mean value and the standard error (* p ⁇ 0.05; Repeated one way ANOVA test; post hoc Tukey-Kramer test). 4 is a representative photograph showing an H.E.-stained image of an auricular skin section 4 days after the start of peptide administration. The arrow in the photograph shows an example of the measured dermis thickness. It is a graph (Box-Whisker @ plot) which shows the dermis thickness of each group on the 4th day after peptide administration start.
- the middle Box and Whisker indicate the median and quartile and maximum / minimum, respectively. Diamond in the box indicates the average value. (** p ⁇ 0.01 Mann Whitney U-test; ⁇ ⁇ ⁇ 7 mice in each group x 9 measurements per mouse.) It is a photograph (Laminin (green)-CD45 (red)-DAPI (blue)) which shows the multiple fluorescent immunostaining image of the pinna skin section on day 4 after the start of peptide administration. Laminin was used to label structures in tissues (blood vessels, basement membrane, nerves), and DAPI was used as a counterstain for DNA of each cell.
- the present inventors have so far found substances having an action of activating stem cells in a living body or mobilizing them to damaged tissues through peripheral circulation, and these substances are a new type of substance that can overcome the weakness of cell therapy.
- HMGB1 High mobility group box 1 released from necrotic tissue
- PDGFR ⁇ Platelet-derived growth factor factor that plays a role in inducing tissue regeneration in vivo living cells ⁇ (PDGFR ⁇ ) positive cells
- MSCs mesenchymal stem cells
- the present inventors have also found that a fragment peptide of HMGB1 exhibits a peripheral blood mobilization activity and a tissue regeneration-inducing activity of mesenchymal stem cells.
- the present inventors have designed an artificial sequence peptide having a specific amino acid sequence based on the results of a unique study conducted so far, and have found that the activity of the peptide to mobilize mesenchymal stem cells in peripheral blood is improved. could be confirmed.
- the present inventors have also found that the above-mentioned artificial sequence peptide has a therapeutic effect on inflammatory bowel disease. Specifically, it was confirmed that the artificial sequence peptide improved the DAI (disease activity index) score, which is an index of disease activity evaluation, in a mouse inflammatory bowel disease model.
- DAI disease activity index
- the present inventors have also found that the above-described artificial sequence peptide has a therapeutic effect on atopic dermatitis. Specifically, it was confirmed that the artificial sequence peptide suppressed desquamation and edema, suppressed increase in dermis thickness, and suppressed infiltration of CD45-positive cells in a mouse model of atopic dermatitis.
- the present inventors have also found that the above artificial sequence peptide has a therapeutic effect on cerebral infarction. Specifically, it was confirmed that the artificial sequence peptide reduced the cerebral infarction lesion in a rat cerebral infarction model.
- mesenchymal stem cells exert anti-inflammatory, immunomodulatory and anti-fibrotic effects.
- mesenchymal stem cells also have pluripotency capable of differentiating into various tissues, it is well known to those skilled in the art that they exert the action of promoting regeneration of damaged tissues. Therefore, when an artificial sequence peptide having an activity of recruiting mesenchymal stem cells into peripheral blood is administered to a subject, the mesenchymal stem cells are recruited into peripheral blood, and the anti-inflammatory and immunomodulating effects of the mesenchymal stem cells are obtained. It is considered that the anti-fibrotic effect and the tissue regeneration-promoting effect (due to the differentiation of mesenchymal stem cells and / or the inflammation) result in a therapeutic effect on various diseases.
- artificial sequence peptide refers to a peptide having an amino acid sequence that does not exist in nature.
- the “artificial sequence peptide” is also simply referred to as “artificial peptide”.
- the present application provides a composition containing a specific artificial sequence peptide for use in recruiting mesenchymal stem cells to peripheral blood.
- composition of the present application for mobilizing mesenchymal stem cells to peripheral blood can be used as a pharmaceutical composition or a reagent composition.
- pharmaceutical composition is used interchangeably with “medicine,” “drug,” or “pharmaceutical composition”
- reagent composition is used interchangeably with “reagent.”
- compositions of the present application for recruiting mesenchymal stem cells to peripheral blood can be used for treating a disease or pathological condition in a subject, for example, by recruiting mesenchymal stem cells to peripheral blood.
- the mesenchymal stem cells mobilized into peripheral blood using the composition for mobilizing mesenchymal stem cells to peripheral blood of the present application are collected outside the body after concentration, and concentrated to obtain a disease or pathological condition in the subject. It is also possible to use for the treatment of.
- the present application also provides the use of certain artificially sequenced peptides in the manufacture of a medicament or reagent for recovering mesenchymal stem cells outside the body.
- composition of the present application for recruiting mesenchymal stem cells to peripheral blood can be used, for example, for basic research and clinical research.
- Examples of basic research and clinical research include, but are not limited to, studies on mobilization of mesenchymal stem cells in vitro, studies on mobilization of mesenchymal stem cells in experimental animals, and the like.
- the present application also provides for the use of certain artificially sequenced peptides in the manufacture of a medicament or reagent for basic or clinical research.
- composition of the present application for recruiting mesenchymal stem cells to peripheral blood may contain one or more artificial sequence peptides.
- mesenchymal stem cells are collected from bone marrow or other tissues (blood, for example, cord blood, skin, fat, pulp, etc.) and cultured as adherent cells on a culture dish (plastic or glass). -Cells that can proliferate and have the ability to differentiate into mesenchymal tissues such as bone, cartilage, fat, and muscle. In one embodiment, the mesenchymal stem cells also have the ability to differentiate into epithelial tissue and neural tissue. In one embodiment, the mesenchymal stem cell is a cell capable of forming a colony.
- the mesenchymal stem cells in the present application may exist as a heterogeneous cell population including not only stem cells in a narrow sense (cells having self-renewal ability and differentiation ability) but also progenitor cells. Alternatively, it may also include differentiated cells in addition to stem cells and progenitor cells in a narrow sense. In one embodiment, the mesenchymal stem cells may be constituted only by stem cells in a narrow sense.
- a precursor cell is defined as a cell having a unidirectional differentiation ability to a specific tissue cell other than a blood system, and has a differentiation ability to a mesenchymal tissue, an epithelial tissue, a nerve tissue, a parenchymal organ, and a vascular endothelium.
- examples of the mesenchymal stem cells include, but are not limited to, bone marrow mesenchymal stem cells and bone marrow-derived mesenchymal stem cells.
- “Bone marrow mesenchymal stem cells” are cells present in bone marrow, which can be collected from bone marrow, cultured and proliferated as adherent cells on a culture dish (plastic or glass), and used for bone, cartilage, and fat. And cells having the characteristic of having the ability to differentiate into mesenchymal tissues such as muscle.
- bone marrow mesenchymal stem cells also have the ability to differentiate into epithelial tissue and neural tissue.
- the bone marrow mesenchymal stem cells are cells capable of forming colonies.
- the term “bone marrow mesenchymal stem cells” is used interchangeably with “bone marrow mesenchymal stromal cells”, “bone marrow pluripotent stem cells” or “bone marrow pluripotent stromal cells”.
- bone marrow-derived mesenchymal stem cells refers to bone marrow mesenchymal stem cells recruited from the bone marrow to the outside of the bone marrow, such as peripheral blood sampling, mesenchymal tissues such as fat, epithelial tissues such as skin, and brain. Such cells can be obtained by collecting from nerve tissue such as.
- ⁇ bone marrow derived mesenchymal stem cells '' is used interchangeably with ⁇ bone marrow derived mesenchymal stromal cells '', ⁇ bone marrow derived pluripotent stem cells '' or ⁇ bone marrow derived pluripotent stromal cells '' Can be
- bone marrow mesenchymal stem cells and bone marrow-derived mesenchymal stem cells are directly collected after collection, or by administering cells once adhered to a culture dish to a damaged part of a living body, for example, keratinocytes constituting skin, etc. It also has the ability to differentiate into epithelial tissues and nervous tissues that make up the brain.
- Bone marrow mesenchymal stem cells and bone marrow-derived mesenchymal stem cells include osteoblasts (which can be identified by inducing differentiation and calcium deposits) and chondrocytes (alcian blue staining positive, safranin-O staining positive, etc.) ), Adipocytes (specifiable by Sudan III staining, etc.), mesenchymal cells such as fibroblasts, smooth muscle cells, skeletal muscle cells, stromal cells, tendon cells, nerve cells, pigment cells Epidermal cells, hair follicle cells (express the cytokeratin family, hair keratin family, etc.), epithelial cells (e.g., epidermal keratinocytes, intestinal epithelial cells express the cytokeratin family, etc.), endothelial cells, liver, It is preferable to have the ability to differentiate into cells of a solid organ such as kidney and pancreas, but the cells after differentiation are not limited to the above cells.
- PDGFR ⁇ , PDGFR ⁇ , Lin, CD45, CD44, CD90, CD29, Flk-1, negative, CD105, CD73, CD73, CD90, CD71, Stro- All or some of 1 positive, CD106 positive, CD166 positive, CD31 negative, CD271 positive, and CD11b negative can be exemplified, but are not limited thereto.
- rat mesenchymal stem cell marker examples include PDGFR ⁇ -positive, CD44-positive, CD54-positive, CD73-positive, CD90-positive, CD105-positive, CD29-positive, CD271-positive, CD31-negative, and CD45-negative. It is not limited to.
- the mesenchymal stem cells are PDGFR ⁇ -positive mesenchymal stem cells, PDGFR ⁇ -positive bone marrow-derived mesenchymal stem cells, PDGFR ⁇ -positive bone marrow-derived cells, and are obtained by bone marrow collection (bone marrow cell collection) or peripheral blood collection. Cells obtained as adherent cells by culturing mononuclear cell fraction cells in the obtained blood can be exemplified, but not limited thereto. Examples of PDGFR ⁇ -positive mesenchymal stem cells include PDGFR ⁇ and CD44-positive cells, PDGFR ⁇ and CD90-positive cells, PDGFR ⁇ and CD105-positive cells, PDGFR ⁇ and CD29-positive cells, and the like. Can be In one embodiment, the PDGFR ⁇ -positive mesenchymal stem cells may be CD44-negative cells.
- the present application provides a composition comprising a specific artificial sequence peptide for use in treating a disease or pathological condition in a subject by recruiting mesenchymal stem cells to peripheral blood.
- compositions of the present application for use in treating a disease or pathological condition in a subject by recruiting mesenchymal stem cells to peripheral blood can be used as pharmaceutical compositions.
- the subject in the present application is not particularly limited, and includes mammals, birds, fish, and the like.
- mammals include humans and non-human animals, such as humans, mice, rats, monkeys, pigs, dogs, rabbits, hamsters, guinea pigs, horses, sheep, and whales, but are not limited thereto. is not.
- the term "subject” is used interchangeably with "patient”, “individual” or "animal”.
- compositions for use in treating a disease or pathological condition in a subject by recruiting mesenchymal stem cells to peripheral blood of the present application can include one or more artificial sequence peptides.
- the treatment of a disease or pathological condition is selected from, for example, anti-inflammatory treatment, immunomodulation treatment, tissue regeneration induction treatment, and tissue fibrosis suppression treatment, but is not limited thereto.
- the disease or pathological condition is selected from, but not limited to, for example, an inflammatory disease, an autoimmune disease, a tissue damage, a disease involving ischemia or necrosis, and a fibrotic disease.
- examples of the inflammatory disease include, but are not limited to, inflammatory bowel disease and atopic dermatitis.
- Autoimmune diseases include, but are not limited to, for example, inflammatory bowel disease.
- examples of fibrotic diseases include, but are not limited to, pulmonary fibrosis.
- Diseases involving tissue damage, ischemia or necrosis include, but are not limited to, for example, inflammatory bowel disease and cerebral infarction.
- Inflammatory bowel diseases include, but are not limited to, ulcerative colitis and Crohn's disease.
- the term “activity of mobilizing mesenchymal stem cells into peripheral blood” is used interchangeably with “activity of increasing the abundance of mesenchymal stem cells in peripheral blood”.
- the activity of recruiting the mesenchymal stem cells of the artificial sequence peptide into the peripheral blood in the present application is as follows: i) collecting the peripheral blood from the individual to which the artificial sequence peptide was administered, and from the individual not receiving the artificial sequence peptide; And cultivation (several days to 10 days), counting the number of colonies formed, and ii) the formed colonies have an ability to adhere to a solid phase and proliferate (self-replicating ability). It can be evaluated by confirming that it has the ability to differentiate into osteoblasts, chondrocytes and adipocytes.
- the red blood cells may be removed from the peripheral blood by a desired method before inoculating the collected peripheral blood into the culture dish.
- the artificial sequence peptide in the present application can be obtained as a recombinant by incorporating DNA encoding the peptide into an appropriate expression system, or can be artificially synthesized. Therefore, the artificial sequence peptide in the present application includes a peptide produced using cells and an artificially synthesized peptide (so-called synthetic peptide).
- DNA encoding the peptide may be incorporated into an appropriate expression system and expressed.
- Hosts applicable to the present application include, but are not limited to, prokaryotic cells and eukaryotic cells.
- Examples of the host applicable to the present invention also include bacteria (eg, Escherichia coli), yeast, animal cells (eg, mammalian cells such as HEK293 cells and CHO cells, insect cells such as silkworm cells), and plant cells. But not limited to these.
- Examples of the host / vector system applicable to the present invention include an expression vector pGEX and E. coli. Since pGEX can express a foreign gene as a fusion protein with glutathione S-transferase (GST) (Gene, 67: 31-40, 1988), pGEX incorporating DNA encoding the artificial sequence peptide of the present invention is heat-treated. It is introduced into an E. coli strain such as BL21 by shock, and after an appropriate culture time, isopropylthio- ⁇ -D-galactoside (IPTG) is added to induce the expression of the GST fusion peptide. Since GST in the present application is adsorbed to glutathione sepharose 4B, the expression product can be easily separated and purified by affinity chromatography.
- GST glutathione S-transferase
- a host / vector system for obtaining a recombinant of the artificial sequence peptide of the present invention the following can be applied in addition to the above.
- an expression vector for a fusion protein using a tag or the like is commercially available.
- the genetically modified product of the present application also includes a recombinant with a tag and a partial peptide thereof.
- the tag added to the artificial sequence peptide of the present application is not particularly limited as long as it does not affect the activity of the artificial sequence peptide of the present application.
- a histidine tag eg, 6 ⁇ His, 10 ⁇ His
- an HA tag e.g., 6 ⁇ His, 10 ⁇ His
- an HA tag e.g., 6 ⁇ His, 10 ⁇ His
- an HA tag e.g, 6 ⁇ His, 10 ⁇ His
- a FLAG tag e.g, 6 ⁇ His, 10 ⁇ His
- GST tag e.g., T7-tag, HSV-tag, E-tag, lck tag, B-tag and the like.
- yeast As for yeast, it is known that yeast belonging to the genus Pichia is effective for expressing proteins having sugar chains.
- an expression system using a baculovirus vector using an insect cell as a host is also useful (Bio / Technology, # 6: 47-55, # 1988).
- transfection of a vector using a promoter such as CMV, RSV, or SV40 is performed using mammalian cells, and all of these host / vector systems express the artificial sequence peptide of the present invention. Can be used as a system.
- plasmid vectors retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors Sendai virus vector, Sendai virus envelope vector
- the gene can also be introduced using a virus vector such as a papilloma virus vector, It is not limited to these.
- the vector may contain a promoter DNA sequence that effectively induces gene expression, a factor that controls gene expression, and a molecule that is necessary for maintaining DNA stability.
- the obtained artificial sequence peptide of the present invention can be isolated from the inside or outside of the host cell (such as a medium) and purified as a substantially pure and uniform peptide.
- the separation and purification of the peptide may be carried out by using the separation and purification methods used in ordinary peptide purification, and are not particularly limited. For example, chromatographic columns, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. If combined, the peptide can be separated and purified.
- chromatography examples include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography and the like (Marshak et al., GieStrategies for Protein Purification and Characterization: A Laboratory). Course Manual. Ed Daniel R. Cold Spring Harbor Laboratory Press, 1996). These chromatographys can be performed using liquid phase chromatography, for example, liquid phase chromatography such as HPLC and FPLC.
- the artificial sequence peptide of the present invention is preferably a substantially purified peptide.
- substantially purified means that the degree of purification of the artificial sequence peptide of the present invention (the ratio of the artificial sequence peptide of the present invention to the entire protein component) is 50% or more, 60% or more, 70% or more, 80%
- the above means 90% or more, 95% or more, 100% or close to 100%.
- the upper limit close to 100% depends on the purification technique and analysis technique of those skilled in the art, and is, for example, 99.999%, 99.99%, 99.9%, 99% and the like.
- the substantially purified artificial sequence peptide is included in the substantially purified artificial sequence peptide even if it is purified by any purification method.
- any purification method for example, the above-described chromatography column, filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc.
- a substantially purified artificial sequence peptide can be exemplified, but is not limited thereto.
- the artificial sequence peptide of the present invention can also be artificially synthesized.
- a peptide can be chemically synthesized by a method such as a peptide liquid phase synthesis method and a peptide solid phase synthesis method.
- the peptide solid phase synthesis method is one of the methods generally used when chemically synthesizing a peptide. Using polystyrene polymer gel beads with a surface modified with amino groups and having a diameter of about 0.1 mm as a solid phase, the amino acid chains are extended one by one by a dehydration reaction. When the target peptide sequence is completed, it is cut out from the solid phase surface to obtain the target substance.
- Pharmaceutically acceptable salts include, but are not limited to, hydrochloride, acetate, trifluoroacetate and the like.
- the artificial sequence peptide of the present application may be in the form of a solvate of the peptide or a solvate of a pharmaceutically acceptable salt of the peptide.
- the solvate refers to a solute molecule in which an arbitrary number of solvent molecules are coordinated, and includes, for example, a hydrate, but is not limited thereto.
- the amino acid length of the artificial sequence peptide in the present application includes, for example, a range of 25 to 35 amino acids, 20 to 40 amino acids, 10 to 50 amino acids, 10 to 70 amino acids, 10 to 100 amino acids, but is not limited thereto. is not.
- examples of the artificial sequence peptide include a peptide selected from the following: (I) an artificial sequence peptide comprising the amino acid sequence of SEQ ID NO: 1; (Ii) an artificial sequence peptide consisting of the amino acid sequence of SEQ ID NO: 1; (Iii) an artificial sequence peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1; (Iv) an artificial sequence peptide comprising an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 1; (V) an artificial sequence peptide consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 1; (Vi) an artificial sequence peptide comprising an amino acid sequence having about 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1; (Vii) an artificial sequence peptide consisting of an amino acid sequence having about 90% or more sequence identity with the amino
- the “several” includes, for example, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 pieces.
- “about 90% or more” means, for example, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more. % Or more, about 98% or more or about 99% or more.
- stringent conditions can refer to, for example, 6 ⁇ SSC, 40% formamide, hybridization at 25 ° C., and 1 ⁇ SSC, washing at 55 ° C. Stringency depends on conditions such as salt concentration, formamide concentration, and temperature, and those skilled in the art can set these conditions to obtain the required stringency.
- a highly homologous base sequence can show, for example, about 60% or more, about 70% or more, or about 80% or more identity.
- “functionally equivalent” means functionally equivalent in terms of the activity of recruiting mesenchymal stem cells into peripheral blood.
- the artificial sequence peptide selected from the above (i) to (ix) is an artificial sequence peptide having an activity of mobilizing mesenchymal stem cells into peripheral blood. Therefore, these artificial sequence peptides have the effect of recruiting mesenchymal stem cells into peripheral blood, and have the therapeutic effect on inflammatory diseases, autoimmune diseases, tissue damage, diseases associated with ischemia or necrosis, and fibrotic diseases. It is thought to have.
- the present application also provides an artificial sequence peptide selected from (i) to (ix) above.
- amino acid sequence described in SEQ ID NO: 1 is the amino acid sequence of the artificial sequence peptide 1r10 of the present application.
- the nucleotide sequence of SEQ ID NO: 2 is an example of the nucleotide sequence of the DNA encoding the artificial sequence peptide 1r10 of the present application.
- Another DNA sequence encoding the artificial sequence peptide 1r10 of the present application is prepared by a method (reverse translation) of converting an amino acid residue of the artificial sequence peptide 1r10 to a corresponding codon using a codon table known to those skilled in the art. Can be. Reverse translation can be performed using various software (including programs, algorithms, and the like) developed for analysis of amino acid and nucleic acid sequences, if desired.
- an effective amount of the artificial sequence peptide of the present application or a pharmaceutical composition containing the same (hereinafter, referred to as a pharmaceutical composition, etc.) is administered to a subject for treatment of the diseases and conditions described herein.
- an effective amount refers to an amount sufficient to treat the disease or pathological condition described herein. Treatment herein includes, but is not limited to, relief, delay, arrest, improvement, remission, cure, cure, and the like.
- the administration site of the pharmaceutical composition or the like of the present application is not limited, and may be a site where a symptom of a disease or a pathological condition appears, or the vicinity thereof, a site different therefrom (other site), a site where a symptom of the disease or a pathological condition appears. Distant from the site, distant from the site where the symptoms of the disease or pathological condition appear, or distant and ectopic to the site where the symptoms of the disease or pathological condition appear. Even if administered, the pharmaceutical composition and the like of the present application can exert their effects.
- the pharmaceutical composition or the like of the present application may be a tissue different from a tissue in which symptoms of a disease or a pathological condition appear, a tissue apart from a tissue in which symptoms of a disease or a pathological condition appears, or a tissue in which symptoms of a disease or a pathological condition appear.
- the effect can be exerted when administered to any tissue, such as tissue that is distal, or that is distant and ectopic to the tissue that exhibits symptoms of the disease or pathological condition.
- the administration method of the pharmaceutical composition and the like of the present application includes oral administration and parenteral administration.
- parenteral administration method include intravascular administration (intra-arterial administration, intravenous administration, etc.), intramuscular administration, subcutaneous administration, Examples include, but are not limited to, intradermal, intraperitoneal, nasal, pulmonary, and transdermal administrations.
- the pharmaceutical composition or the like of the present application can be administered systemically or locally (for example, subcutaneously, intradermally, skin surface, eyeball or eyelid conjunctiva) by injection administration, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection and the like. , Nasal mucosa, buccal and gastrointestinal mucosa, vaginal / intrauterine mucosa, or site of injury).
- a cell that secretes the artificial sequence peptide a gene therapy vector into which DNA encoding the artificial sequence peptide has been inserted, and a pharmaceutical composition containing these can also be used. .
- the administration method can be appropriately selected depending on the age and symptoms of the patient.
- the dose can be selected within the range of 0.0000001 mg to 1000 mg per 1 kg of body weight per administration.
- the dose can be selected in the range of 0.00001 to 100000 mg / body per patient.
- administration may be performed such that the amount of the artificial sequence peptide is within the above range. it can.
- the pharmaceutical composition in the present application is not limited to these dosages.
- the pharmaceutical composition of the present application can be formulated according to a conventional method (for example, Remington's Pharmaceutical Science, Latest Edition, Mark Publishing Company, Easton, USA), which contains both pharmaceutically acceptable carriers and additives.
- a conventional method for example, Remington's Pharmaceutical Science, Latest Edition, Mark Publishing Company, Easton, USA
- pharmaceutically acceptable carriers and additives There may be.
- surfactants, excipients, coloring agents, flavors, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, fluidity enhancers, flavoring agents And the like, but not limited thereto, and other commonly used carriers can be used as appropriate.
- peptide C57BL / 6J mice (8 weeks old, male, weighing 25 g) were prepared and divided into a group to which the artificial sequence peptide 1r10 was administered and a control group.
- the peptide was administered by injecting a solution of peptide 1r10 adjusted to a concentration of 1 ⁇ g / ⁇ L using physiological saline as a solvent into the tail vein at an amount of 100 ⁇ L / animal (4 mg / kg as a peptide dose).
- physiological saline was injected into the tail vein in an amount of 100 ⁇ L / animal.
- the medium was replaced with a fresh one twice a week.
- the cells on the plate were stained using Differential Quik Stain Kit (Sysmex Corporation, Cat No. 16920), and the number of colonies containing 50 or more cells was counted.
- HMGB1 peptide 1-44 a peptide consisting of amino acid residues 1-44 of human HMGB1 protein (hereinafter referred to as HMGB1 peptide 1-44) having the activity of mobilizing mesenchymal stem cells into peripheral blood
- peripheral blood is administered on a solid phase. All colonies obtained as a result of culturing also have solid phase adhesion and self-replication ability, have been confirmed to be PDGFR ⁇ -positive, and have clustered transcriptome analysis data to perform gene-ontology analysis. From the results, it has been confirmed that the mesenchymal stem cells have a characteristic gene expression profile.
- colonies obtained as a result of culturing peripheral blood on solid phase are mesenchymal stem cells, and the increase in the number of colonies detected in solid phase culture of peripheral blood indicates an increase in the number of mesenchymal stem cells in peripheral blood it is conceivable that.
- mesenchymal stem cells hardly exist in peripheral blood, so it is considered that the increased amount of mesenchymal stem cells was mobilized into peripheral blood from tissues other than peripheral blood (eg, bone marrow).
- the number of colonies detected in solid-phase culture of peripheral blood after administration of a test substance can be used as an indicator of the activity of the test substance to mobilize mesenchymal stem cells into peripheral blood.
- mice to which peptide 1r10 was administered the number of colonies obtained on the plate by culturing peripheral blood-derived cells was larger than that in mice to which physiological saline was administered (FIG. 1).
- IBD inflammatory bowel disease
- the peptide was administered at a concentration of 10 mL / kg of a peptide 1r10 solution adjusted to a concentration of 0.5 mg / mL using physiological saline as a solvent.
- the dose of the peptide was 5 mg / kg).
- physiological saline was infused into the tail vein at a dose of 10 mL / kg on days 1, 4, 7, 8, and 9 after the start of drinking of the DSS aqueous solution.
- the length of the large intestine (from the colon to the rectum) collected 10 days after the start of DSS drinking was measured using a digital caliper.
- the stool score (total of the “stool condition” score and “bleeding” score scored as described in Table 1 above) and DAI score 8 to 10 days after the start of drinking of the DSS aqueous solution are shown in FIGS. 3 and 4, respectively. (See “saline” for the control group and “1r10” for the peptide-administered group). Both the stool score and the DAI score showed lower values in the peptide administration group than in the control group.
- the colon length of the peptide-administered group was longer than that of the control group (FIG. 5; see “saline” for the control group and “1r10” for the peptide-administered group).
- IBD inflammation of the mucous membrane of the intestinal tract occurs, erosions and ulcers are formed, and symptoms such as narrowing and shortening of the intestinal tract may occur.
- weight loss occurs as another symptom of IBD, and the cause is considered to be inferior nutritional status due to inflammation or tissue damage (such as ulcer) occurring in the intestinal mucosa.
- intravenous injection of mesenchymal stem cells in an animal IBD model improves various symptoms including weight loss, intestinal epithelial damage, and infiltration of inflammatory cells. The improvement of such symptoms is attributable to suppression of inflammation of the intestinal mucosa by the anti-inflammatory action of mesenchymal stem cells and, as a result, promotion of regeneration of mucosal tissues.
- Drugs MC903 (generic name: Calcipotriol) was used to induce atopic dermatitis (Atopic dermatitis; hereinafter also referred to as “AD”) in mice.
- the peptide 1r10 (TFA salt) described in Example 1 was used as a test substance.
- Such MC903-induced AD model elicits a series of type 2 immune response cascades by artificially inducing TSLP secretion from epithelial cells actually seen in AD patients, causing a short-term (1- (2 weeks) (Li M et al., Proc Natl Acad Sci US A. 2006; 103: 11736-41.).
- the peptide 1r10 solution was injected into the tail vein at a volume of 5 mL / kg (1.5 mg / kg as peptide dose).
- the control group was adjusted to 0.3 mg / mL using physiological saline twice a day, once on the grouping day (Day 15) and once during the following four days (Day 16 to Day 19).
- the solution of bovine serum albumin (BSA) was injected into the tail vein in a volume of 5 mL / kg (1.5 mg / kg as a dose of BSA).
- mice were placed under the influence of isoflurane anesthesia, euthanized by cervical dislocation, and the inflammatory site on the pinna was quickly removed while preventing protein denaturation of the sample on ice.
- the skin in the surrounding area was cut off and collected.
- the collected pinna skin was immersed in 10% Formalin, and fixed overnight at 4 ° C. while shaking on ice using a shaker. After the fixed skin is thoroughly washed with phosphate buffered saline (PBS) and trimmed, an auto-embedder (Excelsior ES; Thermo Scientific ) To make a paraffin block.
- PBS phosphate buffered saline
- the slices were sliced (5 ⁇ m thick) with a rotating microtome (HM 325; Thermo Sceintific), fixed on a slide glass, and subjected to various stainings.
- HM 325 rotating microtome
- hematoxylin and eosin (HE) staining was performed on paraffin slices (5 ⁇ m thick) of the skin tissue prepared as described above, and the dermis thickness was measured. For details, three sections were prepared for each individual, and for each section, the center of inflammation (center), head side (Head), and tail side (Tail) were photographed with a microscope (Keyence BZ-X710). (That is, a total of 9 photographs per individual). Next, a vertical line from the epidermis-Dermis junction to the subcutaneous fat-cartilage junction is drawn from the photographed image, and only the longest vertical line in the visual field is selected to measure the dermal thickness. Used as value.
- paraffin slices (5 ⁇ m thick) prepared as described above were subjected to fluorescent immunostaining using anti-Laminin antibody and anti-CD45 antibody (Laminin For detection, Anti-Laminin (Sigma Aldrich; catalog number L9393) as a primary antibody, Donkey anti-Rabbit IgG (H + L), Alexa Fluor 488 (Thermo Fisher Scientific; catalog number A-21206) as a secondary antibody
- Aminin For detection, Anti-Laminin (Sigma Aldrich; catalog number L9393) as a primary antibody
- Donkey anti-Rabbit IgG (H + L) Alexa Fluor 488 (Thermo Fisher Scientific; catalog number A-21206)
- Goat Anti-mouse CD45 Goat Anti-mouse CD45 (R & D Systems; catalog number AF114) was used as the primary antibody
- Cy3 AffiniPure Bovine Anti-Goat IgG (H + L) Jackson ImmunoReseach, Inc.
- Pinna thickness (edema) The transition of pinna thickness (edema) during the peptide administration period is shown in FIG. 7 (see “Control” for the control group and “1r10” for the peptide administration group). In the peptide administration group, a statistically significant decrease in pinna thickness was observed as compared to the control group.
- Dermal thickness As a result of HE staining of skin tissue sections, the control group showed an increase in dermis thickness (cell infiltration and vascular permeability associated with the immune response caused by AD) that were not seen in non-AD-induced mice. (See FIG. 8; “Control” for the control group and “1r10” for the peptide-administered group). On the other hand, in the peptide-administered group, a statistically significant decrease in dermal thickness was observed as compared to the control group (FIG. 9; see “Control” for the control group and “1r10” for the peptide-administered group). Also, when peptide 1r10 was administered at a dose of 0.5 mg / kg, a statistically significant decrease in dermal thickness was observed as compared to the control group (data not shown).
- the right common carotid artery, right external carotid artery and right internal carotid artery were exposed, and the right common carotid artery and right external carotid artery were ligated with suture.
- No. 4 nylon thread (obturator) was inserted through the bifurcation of the right external carotid artery and the right internal carotid artery to occlude the right middle cerebral artery (MCA) .
- MCA middle cerebral artery
- a peptide 1r10 solution adjusted to a concentration of 1 mg / mL in physiological saline was used to administer the peptide at a volume of 2 mL / kg. This was performed by injecting into the tail vein at a dose of 2 mg / kg).
- the control group received saline infusion in the tail vein at a volume of 2 mL / kg at 105 minutes and 24 hours after right MCA occlusion.
- Brain slices are 4 mm in front of Bregma, 2 mm in front of Bregma, and on Bregma, referring to Paxinos and Watson brain diagrams (Paxinos G. and Watson C., The rat brain in stereotaxic coordinates, second edition.Academic Press Inc .; 1986). , 2 mm posterior to Bregma, 4 mm posterior to Bregma, and 6 mm posterior to Bregma.
- the brain sections were immersed and stained in a 1 w / v% solution of 2,3,5-Triphenyltetrazolium chloride (TTC) at room temperature and photographed. The obtained photographs were image-analyzed, the cerebral infarct area and the cross-sectional area of the brain were determined by actual measurement, and the cerebral infarct volume and total brain volume from 4 mm before Bregma to 6 mm after Bregma were calculated by the following formulas. .
- V Cerebral infarct volume or total brain volume (mm 3 ) a: Area of cerebral infarct lesion or cross-sectional area of brain on 4 mm section of Bregma (mm 2 ) b: Area of cerebral infarct lesion or cross-sectional area of brain on 2 mm cross section in front of Bregma (mm 2 ) c: Area of cerebral infarct lesion on Bregma cross section or cross-sectional area of brain (mm 2 ) d: Area of cerebral infarct lesion or cross-sectional area of brain on 2 mm cross section behind Bregma (mm 2 ) e: Area of cerebral infarct lesion or cross-sectional area of brain on 4 mm cross section behind Bregma (mm 2 ) f: Area of cerebral infarct lesion or cross-sectional area of brain on 6 mm section after Bregma (mm 2 )
- the cerebral infarct volume ratio of the control group and the peptide administration group was 34.1 ⁇ 1.1% and 27.2 ⁇ 1.2% (mean ⁇ standard error), respectively (FIG. 12; “Control” for the control group, See “1r10” for the administration group).
- the cerebral infarct volume ratio in the peptide-administered group was lower than that in the control group, and a statistically significant difference was recognized (Mann-Whitney U test, p ⁇ 0.01).
- the effect of reducing the cerebral infarction lesion was obtained by administering the artificial sequence peptide of the present invention to a cerebral infarction model rat.
- This is a result of the recruitment of mesenchymal stem cells into peripheral blood by the action of the artificial sequence peptide of the present application, and the cells exerting an anti-inflammatory effect, a trophic effect (secretion of trophic factors), a tissue regeneration effect, and the like. it is conceivable that.
- the artificial sequence peptide of the present application can be used as a therapeutic agent for inflammatory diseases, autoimmune diseases, fibrotic diseases, and diseases involving tissue damage / ischemia / necrosis.
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Abstract
Description
〔1〕
以下から選択されるペプチドを含有する、末梢血への間葉系幹細胞の動員に用いるための組成物:
(a)配列番号:1のアミノ酸配列を含むペプチド;
(b)配列番号:1のアミノ酸配列において1もしくは数個のアミノ酸が置換、欠失、挿入若しくは付加されたアミノ酸配列を含むペプチド;および
(c)配列番号:1のアミノ酸配列と約90%以上の配列同一性を有するアミノ酸配列を含むペプチド。
〔2〕
以下から選択されるペプチドを含有する、末梢血への間葉系幹細胞の動員による、対象における疾患または病的状態の治療に用いるための組成物:
(a)配列番号:1のアミノ酸配列を含むペプチド;
(b)配列番号:1のアミノ酸配列において1もしくは数個のアミノ酸が置換、欠失、挿入若しくは付加されたアミノ酸配列を含むペプチド;および
(c)配列番号:1のアミノ酸配列と約90%以上の配列同一性を有するアミノ酸配列を含むペプチド。
〔3〕
疾患または病的状態の治療が、炎症抑制治療、免疫調節治療、組織の再生誘導治療、および組織の線維化抑制治療から選択される、〔2〕に記載の組成物。
〔4〕
疾患または病的状態が、炎症性疾患、自己免疫疾患、組織の損傷、虚血もしくは壊死を伴う疾患、および線維性疾患から選択される、〔2〕に記載の組成物。
〔5〕
疾患または病的状態が、炎症性腸疾患である、〔2〕に記載の組成物。
〔6〕
疾患または病的状態が、潰瘍性大腸炎である、〔2〕に記載の組成物。
〔7〕
疾患または病的状態が、アトピー性皮膚炎である、〔2〕に記載の組成物。
〔8〕
疾患または病的状態が、脳梗塞である、〔2〕に記載の組成物。
〔9〕
以下から選択されるペプチドを含有する、炎症性腸疾患、アトピー性皮膚炎および脳梗塞から選択される疾患の治療に用いるための組成物:
(a)配列番号:1のアミノ酸配列を含むペプチド;
(b)配列番号:1のアミノ酸配列において1もしくは数個のアミノ酸が置換、欠失、挿入若しくは付加されたアミノ酸配列を含むペプチド;および
(c)配列番号:1のアミノ酸配列と約90%以上の配列同一性を有するアミノ酸配列を含むペプチド。
〔10〕
以下から選択されるペプチド:
(a)配列番号:1のアミノ酸配列を含むペプチド;
(b)配列番号:1のアミノ酸配列において1もしくは数個のアミノ酸が置換、欠失、挿入若しくは付加されたアミノ酸配列を含むペプチド;および
(c)配列番号:1のアミノ酸配列と約90%以上の配列同一性を有するアミノ酸配列を含むペプチド。
〔A1〕
以下から選択されるペプチドの有効量を対象に投与する工程を含む、末梢血へ間葉系幹細胞を動員する方法:
(a)配列番号:1のアミノ酸配列を含むペプチド;
(b)配列番号:1のアミノ酸配列において1もしくは数個のアミノ酸が置換、欠失、挿入若しくは付加されたアミノ酸配列を含むペプチド;および
(c)配列番号:1のアミノ酸配列と約90%以上の配列同一性を有するアミノ酸配列を含むペプチド。
〔A2〕
以下から選択されるペプチドの有効量を対象に投与する工程を含む、末梢血への間葉系幹細胞の動員により、対象における疾患または病的状態を治療する方法:
(a)配列番号:1のアミノ酸配列を含むペプチド;
(b)配列番号:1のアミノ酸配列において1もしくは数個のアミノ酸が置換、欠失、挿入若しくは付加されたアミノ酸配列を含むペプチド;および
(c)配列番号:1のアミノ酸配列と約90%以上の配列同一性を有するアミノ酸配列を含むペプチド。
〔A3〕
疾患または病的状態の治療が、炎症抑制治療、免疫調節治療、組織の再生誘導治療、および組織の線維化抑制治療から選択される、〔A2〕に記載の方法。
〔A4〕
疾患または病的状態が、炎症性疾患、自己免疫疾患、組織の損傷、虚血もしくは壊死を伴う疾患、および線維性疾患から選択される、〔A2〕に記載の方法。
〔A5〕
疾患または病的状態が、炎症性腸疾患である、〔A2〕に記載の方法。
〔A6〕
疾患または病的状態が、潰瘍性大腸炎である、〔A2〕に記載の方法。
〔A7〕
疾患または病的状態が、アトピー性皮膚炎である、〔A2〕に記載の方法。
〔A8〕
疾患または病的状態が、脳梗塞である、〔A2〕に記載の方法。
〔A9〕
以下から選択されるペプチドの有効量を対象に投与する工程を含む、炎症性腸疾患、アトピー性皮膚炎および脳梗塞から選択される対象における疾患を治療する方法:
(a)配列番号:1のアミノ酸配列を含むペプチド;
(b)配列番号:1のアミノ酸配列において1もしくは数個のアミノ酸が置換、欠失、挿入若しくは付加されたアミノ酸配列を含むペプチド;および
(c)配列番号:1のアミノ酸配列と約90%以上の配列同一性を有するアミノ酸配列を含むペプチド。
〔B1〕
末梢血への間葉系幹細胞の動員に用いるための、以下から選択されるペプチド:
(a)配列番号:1のアミノ酸配列を含むペプチド;
(b)配列番号:1のアミノ酸配列において1もしくは数個のアミノ酸が置換、欠失、挿入若しくは付加されたアミノ酸配列を含むペプチド;および
(c)配列番号:1のアミノ酸配列と約90%以上の配列同一性を有するアミノ酸配列を含むペプチド。
〔B2〕
末梢血への間葉系幹細胞の動員による、対象における疾患または病的状態の治療に用いるための、以下から選択されるペプチド:
(a)配列番号:1のアミノ酸配列を含むペプチド;
(b)配列番号:1のアミノ酸配列において1もしくは数個のアミノ酸が置換、欠失、挿入若しくは付加されたアミノ酸配列を含むペプチド;および
(c)配列番号:1のアミノ酸配列と約90%以上の配列同一性を有するアミノ酸配列を含むペプチド。
〔B3〕
疾患または病的状態の治療が、炎症抑制治療、免疫調節治療、組織の再生誘導治療、および組織の線維化抑制治療から選択される、〔B2〕に記載のペプチド。
〔B4〕
疾患または病的状態が、炎症性疾患、自己免疫疾患、組織の損傷、虚血もしくは壊死を伴う疾患、および線維性疾患から選択される、〔B2〕に記載のペプチド。
〔B5〕
疾患または病的状態が、炎症性腸疾患である、〔B2〕に記載のペプチド。
〔B6〕
疾患または病的状態が、潰瘍性大腸炎である、〔B2〕に記載のペプチド。
〔B7〕
疾患または病的状態が、アトピー性皮膚炎である、〔B2〕に記載のペプチド。
〔B8〕
疾患または病的状態が、脳梗塞である、〔B2〕に記載のペプチド。
〔B9〕
炎症性腸疾患、アトピー性皮膚炎および脳梗塞から選択される疾患の治療に用いるための、以下から選択されるペプチド:
(a)配列番号:1のアミノ酸配列を含むペプチド;
(b)配列番号:1のアミノ酸配列において1もしくは数個のアミノ酸が置換、欠失、挿入若しくは付加されたアミノ酸配列を含むペプチド;および
(c)配列番号:1のアミノ酸配列と約90%以上の配列同一性を有するアミノ酸配列を含むペプチド。
〔C1〕
末梢血への間葉系幹細胞の動員のための医薬または試薬の製造における、以下から選択されるペプチドの使用:
(a)配列番号:1のアミノ酸配列を含むペプチド;
(b)配列番号:1のアミノ酸配列において1もしくは数個のアミノ酸が置換、欠失、挿入若しくは付加されたアミノ酸配列を含むペプチド;および
(c)配列番号:1のアミノ酸配列と約90%以上の配列同一性を有するアミノ酸配列を含むペプチド。
〔C2〕
末梢血への間葉系幹細胞の動員による、対象における疾患または病的状態の治療のための医薬の製造における、以下から選択されるペプチドの使用:
(a)配列番号:1のアミノ酸配列を含むペプチド;
(b)配列番号:1のアミノ酸配列において1もしくは数個のアミノ酸が置換、欠失、挿入若しくは付加されたアミノ酸配列を含むペプチド;および
(c)配列番号:1のアミノ酸配列と約90%以上の配列同一性を有するアミノ酸配列を含むペプチド。
〔C3〕
疾患または病的状態の治療が、炎症抑制治療、免疫調節治療、組織の再生誘導治療、および組織の線維化抑制治療から選択される、〔C2〕に記載の使用。
〔C4〕
疾患または病的状態が、炎症性疾患、自己免疫疾患、組織の損傷、虚血もしくは壊死を伴う疾患、および線維性疾患から選択される、〔C2〕に記載の使用。
〔C5〕
疾患または病的状態が、炎症性腸疾患である、〔C2〕に記載の使用。
〔C6〕
疾患または病的状態が、潰瘍性大腸炎である、〔C2〕に記載の使用。
〔C7〕
疾患または病的状態が、アトピー性皮膚炎である、〔C2〕に記載の使用。
〔C8〕
疾患または病的状態が、脳梗塞である、〔C2〕に記載の使用。
〔C9〕
炎症性腸疾患、アトピー性皮膚炎および脳梗塞から選択される疾患の治療のための医薬の製造における、以下から選択されるペプチドの使用:
(a)配列番号:1のアミノ酸配列を含むペプチド;
(b)配列番号:1のアミノ酸配列において1もしくは数個のアミノ酸が置換、欠失、挿入若しくは付加されたアミノ酸配列を含むペプチド;および
(c)配列番号:1のアミノ酸配列と約90%以上の配列同一性を有するアミノ酸配列を含むペプチド。
(i)配列番号:1のアミノ酸配列を含む、人工配列ペプチド;
(ii)配列番号:1のアミノ酸配列からなる、人工配列ペプチド;
(iii)配列番号:1のアミノ酸配列の一部からなる、人工配列ペプチド;
(iv)配列番号:1のアミノ酸配列において1若しくは数個のアミノ酸が置換、欠失、挿入若しくは付加されたアミノ酸配列を含む、人工配列ペプチド;
(v)配列番号:1のアミノ酸配列において1若しくは数個のアミノ酸が置換、欠失、挿入若しくは付加されたアミノ酸配列からなる、人工配列ペプチド;
(vi)配列番号:1のアミノ酸配列と約90%以上の配列同一性を有するアミノ酸配列を含む、人工配列ペプチド;
(vii)配列番号:1のアミノ酸配列と約90%以上の配列同一性を有するアミノ酸配列からなる、人工配列ペプチド;
(viii)配列番号:2の塩基配列からなるDNAによりコードされる、人工配列ペプチド;および
(ix)配列番号:2の塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされる、人工配列ペプチド。
人工配列ペプチドによる間葉系幹細胞の動員
i)ペプチドの製造
配列番号:1のアミノ酸配列からなる人工配列ペプチドを化学的に合成した(得られたペプチドはトリフルオロ酢酸(TFA)塩の形態である)。以下、当該ペプチドを「人工配列ペプチド1r10」または「ペプチド1r10」と称する。
C57BL/6Jマウス(8週齢、雄、体重25g)を用意し、人工配列ペプチド1r10を投与する群と対照群に分けた。ペプチドの投与は、生理食塩水を溶媒として1μg/μLの濃度に調整したペプチド1r10の溶液を100μL/匹の量(ペプチドの投与量としては4mg/kg)で尾静脈に注入することにより行った。対照群には、生理食塩水を100μL/匹の量で尾静脈に注入した。
生理食塩水または人工配列ペプチド1r10の投与から14時間後に、全身麻酔下で心臓から末梢血を約800-1000μL採取した(ヘパリンを含有する1 mLシリンジを使用)。赤血球を除去するため、採取した血液と等量のHetasep(STEMCELL Technologies社、Cat No. ST-07906)を加え、100Gで2分間遠心し、室温で15分間インキュベートした後、上清を回収した。当該上清を、末梢血中の有核細胞を含むサンプルとして次の実験に供した。
上記の手順によって得た上清(末梢血由来細胞含有サンプル)を、コラーゲンIでコートされた6ウェルプレート(Corning社、Cat No. 356400)に播種し、MesenCult Expansion Kit(STEMCELL Technologies社、Cat No. ST-05513)を利用して当該キットのマニュアル通りに調製したExpansion Mediumに1% L-glutamine(ナカライテスク社)、10μM ROCK inhibitor(Y27632、Tocris Bioscience社)および 1% ペニシリン/ストレプトマイシン(ナカライテスク社)を含有させた培地(数値はいずれも終濃度)を用いて、37℃、5%CO2、5%O2の条件下で10日間培養した。培養期間中は1週間に2回、培地を新鮮なものに交換した。培養10日目に、Differential Quik Stain Kit(シスメックス株式会社、Cat No. 16920)を用いてプレート上の細胞を染色し、50個以上の細胞を含むコロニーの数をカウントした。
ペプチド1r10を投与したマウスでは、生理食塩水を投与したマウスと比較して、末梢血由来細胞の培養によってプレート上に得られるコロニーの数が多かった(図1)。
炎症性腸疾患に対する人工配列ペプチドの有効性
i)薬剤
デキストラン硫酸ナトリウム(DSS)(分子量36,000~50,000、MP Biomedicals社製、製品番号160110)を精製水に溶解し、2.5%(w/v)のDSS水溶液を調製した。また、実施例1に記載のペプチド1r10(TFA塩)を被験物質として用いた。
BALB/cマウス(9週齢、オス)に2.5% DSS水溶液を7日間自由飲水させ、大腸炎を誘発した。その後、DSSから水道水の飲水に変更し、3日後(DSS水溶液の飲水開始10日後)に大腸採取を行った。
上記の通り作成したIBDモデルマウスをペプチド1r10投与群(n=8)および対照群(n=8)に分けた。ペプチドの投与は、DSS水溶液の飲水開始後1、4、7、8および9日目に、生理食塩水を溶媒として0.5mg/mLの濃度に調整したペプチド1r10の溶液を10 mL/kgの量(ペプチドの投与量としては5mg/kg)で尾静脈に注入することにより行った。対照群には、DSS水溶液の飲水開始後1、4、7、8および9日目に、生理食塩水を10 mL/kgの量で尾静脈に注入した。
DSS水溶液の飲水開始から10日間、土曜・日曜・祝日を除いて毎日、マウスの体重測定と症状(便の状態、および出血)の観察を行い、DAI(disease activity index)スコアを算出して症状の程度を評価した。DAIスコアは、体重減少率、便の状態、および便出血を下記表の通りにスコア付けし、これら3項目のスコアを合計することにより算出した。スコア付けの基準は、Liらの方法(PLoS One. 2015 Dec 7; 10(12):e0144101)を一部参考にした。
試験期間中におけるマウスの体重変化を図2に示す(対照群について「saline」、ペプチド投与群について「1r10」参照)。対照群およびペプチド投与群ともにDSS飲水開始後経時的に体重が減少したが、その減少程度は対照群よりペプチド投与群の方が緩やかであり、DSS水溶液の飲水開始10日後にはペプチド投与群の体重は回復傾向を示した。
アトピー性皮膚炎に対する人工配列ペプチドの有効性
i)薬剤
マウスにアトピー性皮膚炎(Atopic Dermatitis; 以下、「AD」とも称する)を誘導するため、MC903(一般名:Calcipotriol)を用いた。また、実施例1に記載のペプチド1r10(TFA塩)を被験物質として用いた。
C57BL/6マウス(C57BL/6JJcl、7-8週齢、雄、微生物グレードSPF)を日本クレア株式会社より入手し、5日以上動物飼育室で馴化させた後、以下の実験に供した。当該マウスの耳介部皮膚に、エタノールで調整したMC903溶液を、2.25nmol MC903/耳/1回/1日の量で週に5回、2週間にわたって塗布(合計10回塗布)し、アトピー性皮膚炎(AD)を誘導した。かかるMC903誘導性のADモデルは、AD患者において実際に見られる上皮細胞からのTSLP分泌を人工的に誘導することにより一連の2型免疫反応カスケードを惹起させ、AD様病態を短期間(1-2週間)で再現させることができるモデルである(Li M et al., Proc Natl Acad Sci U S A. 2006 ;103 :11736-41.)。また、予備試験として正常マウスとMC903によるAD誘導マウス(誘導開始14日後)の耳介部の外観および組織を観察した結果、AD誘導マウスでは炎症による紅斑、辺縁領域における落屑、角質の苔癬化、表皮のAcanthosis(有棘細胞増殖による表皮肥厚)、局所的なバリアの破壊、真皮への免疫細胞の浸潤、真皮厚の増大、および表皮の一部におけるSpongiosis(海綿状変化)が生じることも確認できている。
MC903の塗布開始(Day 1とする)から2週後(Day 15)に、落屑(Scaling)、紅斑(Erythema)、浮腫(Edema)の主症状から判断し、明らかにADが誘導された個体のみを選抜後、個体間の炎症の重症度の差異を考慮して、耳介厚に基づきグループ間で平均化処置を行い、対照群およびペプチド投与群(各群n=7)に分けた。ペプチドの投与は、群分け日(Day 15)に1回と、その後の4日(Day 16からDay 19)の間に1回の合計2回、生理食塩水を用いて0.3mg/mLに調整したペプチド1r10の溶液を5mL/kgの容量(ペプチドの投与量として1.5mg/kg)で尾静脈に注入することにより行った。対照群には、群分け日(Day 15)に1回と、その後の4日(Day 16からDay 19)の間に1回の合計2回、生理食塩水を用いて0.3mg/mLに調整したウシ血清アルブミン(BSA)の溶液を5mL/kgの容量(BSAの投与量として1.5mg/kg)で尾静脈に注入した。
浮腫および落屑の評価
ペプチド投与期間中(Day 15からDay 19)は、浮腫(Edema)の判定のために耳介厚(Ear Swellings)の測定を毎日実施し、ADの主徴である落屑(Scaling)については耳介部から13-14cmの高さの固定台からデジタルカメラ(E-M1 II; Olympus社)を用いて写真撮影(週3回)により判定を行った。
ペプチドの投与開始後4日目(Day 19)にマウスをイソフルラン麻酔影響下に置き、頸椎脱臼を行って安楽死させ、氷上でサンプルのタンパク変性を防ぎつつ、すばやく耳介の炎症部位およびその周辺領域の皮膚を切り取り回収した。採取した耳介部皮膚は、ピンセットで余分な毛や皮膚を除去した後、10% Formalinに浸漬し、shakerを用いて氷上で振盪させながら、4℃で一晩固定させた。固定後の皮膚をリン酸緩衝食塩水(PBS)でよく洗浄し、トリミングした後、皮膚固有の背腹軸/左右軸の組織の極性に留意しながら自動包埋器(Excelsior ES;Thermo Scientific社)によりパラフィンブロックにした。その後、回転式ミクロトーム(HM 325;Thermo Sceintific社)で薄切(5μm厚)してから、スライドグラス上に固定させ、各種染色に供した。病理学的解析として、組織化学染色および蛍光免疫染色法によりそれぞれ皮膚厚および免疫細胞の浸潤について評価を行った。
炎症部位の治癒状態を定量評価するため、上記の通り作成した皮膚組織のパラフィン薄切(5μm厚)に対してヘマトキシリン・エオジン(H.E.)染色を行い、真皮厚を測定した。その詳細は、1個体につき3枚の切片を用意し、各切片について炎症中心部位(center)、頭側(Head)および尾側(Tail)を1箇所ずつ顕微鏡(Keyence BZ-X710)で撮影した(即ち、1個体につき計9箇所撮影)。次に撮影した画像について表皮真皮接合部(Epidermis-Dermis junction)から皮下脂肪-軟骨接合部(Subcutaneous Fat-Cartilage junction)までの垂線を引いて視野中の最長の垂線のみを選抜し真皮厚の測定値として用いた。
炎症部位の免疫細胞数を定量評価するため、上記の通り作成したパラフィン薄切(5μm厚)に対して抗Laminin抗体および抗CD45抗体を用いて蛍光免疫染色を行った(Lamininの検出には、1次抗体としてAnti-Laminin(Sigma Aldrich社; カタログ番号 L9393)、2次抗体としてDonkey anti-Rabbit IgG (H+L) , Alexa Fluor 488(Thermo Fisher Scientific社; カタログ番号A-21206)を使用した。CD45の検出には、1次抗体としてGoat Anti-mouse CD45 (R&D Systems社; カタログ番号 AF114)、2次抗体として Cy3 AffiniPure Bovine Anti-Goat IgG (H+L) (Jackson ImmunoReseach社; カタログ番号 805-165-180)を使用した)。定量の詳細としては、1個体につき3枚の切片を用意し、各切片における炎症中心部位(center)、頭側(Head)および尾側(Tail)について1箇所ずつ、顕微鏡(Keyence BZ-X710)で高倍率視野(HPF:400x)を撮影した(即ち、1個体につき計9箇所撮影)。次に撮影した画像について視野中のCD45陽性細胞(Cy3の蛍光で検出)をカウントし、測定値として用いた。
上記解析によって得られた測定値または補正後の算出値は、統計解析ソフト(statcel-3(OMS出版)およびExcel統計(BellCurve))を用いて各統計値を計算後、解析/判定を行った。具体的には、事前検定として(1)正規性の検定(歪度/尖度係数でのダゴスティーノ検定; P>0.05を正規分布とみなす。) (2)棄却検定(Grubbs検定;P<0.05の場合、外れ値(outlier)とみなす)を行い、(1)で得られた結果が正規分布を示す場合には群間でt-testを行った。正規分布を示さない場合には群間の順位によるノンパラメトリック検定としてMann whitney U-testを行い、p<0.05の場合に有意差ありと判定した。また時系列における反復測定を主とする体重変化および耳介厚変化については、Repeated one way ANOVA testを実施し、さらにpost hoc testとしてTukey-Kramer testを行った。外れ値の処理に関しては、(a)Grubbs検定でP<0.05となった群内の個体数が当該群の全個体数(n数)に対して10%以下の場合は当該外れ値を欠損値として取り扱い、(b)P<0.05となった個体数がn数に対して10%を超える場合は外れ値を有さない他の個体での平均値を補完的に代入(imputation)する方法を採用した(所謂Mean substitution)。
落屑
ADモデル誘導後の対照群マウスの耳介部では、典型的なAD像である湿疹と乾燥に起因する剥離表皮による落屑が辺縁部に高頻度に生じた(図6、左写真の矢印部分)。一方、ペプチド投与群では、耳介部における落屑の軽減が認められた(図6、右写真の矢印部分)。
ペプチド投与期間中の耳介厚(浮腫)の推移を図7に示す(対照群について「Control」、ペプチド投与群について「1r10」参照)。ペプチド投与群では、対照群と比較して統計的に有意な耳介厚の減少が認められた。
皮膚組織切片に対してH.E.染色を行った結果、対照群においては、AD非誘導マウスでは見られない真皮厚の増大(ADで引き起こされる免疫反応に伴う細胞浸潤と血管透過性の亢進に起因するものと考えられる)が観察された(図8;対照群について「Control」、ペプチド投与群について「1r10」参照)。一方、ペプチド投与群では、対照群と比較して統計的に有意な真皮厚の減少が認められた(図9;対照群について「Control」、ペプチド投与群について「1r10」参照)。また、0.5mg/kgの用量でペプチド1r10を投与した場合にも、対照群と比較して統計的に有意な真皮厚の減少が見られた(データ示さず)。
皮膚組織切片に対して蛍光免疫染色を行った結果、対照群では、ADで引き起こされる免疫反応に伴うCD45陽性免疫細胞の組織内浸潤が血管周囲(Lamininで標識される)を始めとする皮膚全体に見られた(図10の矢印部分;対照群について「Control」、ペプチド投与群について「1r10」参照)。一方、ペプチド投与群では、対照群と比較して統計的に有意なCD45陽性細胞数の減少が認められた(図11;対照群について「Control」、ペプチド投与群について「1r10」参照)。
脳梗塞に対する人工配列ペプチドの有効性
i)薬剤
実施例1に記載のペプチド1r10(TFA塩)を被験物質として用いた。
Crl:CD(SD)ラット(7週齢、雄)を日本チャールス・リバーより入手し、6日間の馴化の後、対照群(n=8)とペプチド投与群(n=8)に分けた。群分けの翌日に、ラットを2%イソフルラン吸入麻酔下(麻酔背景は笑気:酸素 = 7:3)にて仰臥位に固定した。手術中の体温調節を行うためにディジタル温度計に接続した体温計用プローブを直腸内に挿入し、手術前後の直腸温の変化をモニターした。その結果、いずれのラットも直腸温の低下は認められなかったため、加温は実施しなかった。右総頸動脈、右外頸動脈および右内頸動脈を露出し、右総頸動脈および右外頸動脈を縫合糸で結紮した。予めシリコンでコーティングし、19 mmの長さに切断した4号のナイロン糸(栓子)を右外頸動脈と右内頸動脈の分岐部より挿入し、右中大脳動脈(MCA)を閉塞した。右MCA閉塞後、頸部皮膚の縫合を行い、麻酔から解放した。右MCA閉塞後30分に閉塞側の対側前肢の屈曲を確認した。2%イソフルランの吸入麻酔下にて、再び右総頸動脈、右外頸動脈および右内頸動脈を露出し、右MCA閉塞後90分に栓子を抜き、右MCAの血流を再開させた。右MCAの血流再開後に右内頸動脈の結紮および頸部皮膚の縫合を行い、麻酔から解放した。なお、右MCA閉塞手術前にベンジルペニシリンカリウムを20000 units/kgの用量で筋肉内投与した。
ペプチドの投与は、右MCA閉塞後105分および24時間の時点において、生理食塩水を溶媒として1mg/mLの濃度に調整したペプチド1r10の溶液を2mL/kgの容量(ペプチドの投与量として 2mg/kg)で尾静脈に注入することにより行った。対照群には、右MCA閉塞後105分および24時間の時点において、生理食塩水を2mL/kgの容量で尾静脈に注入した。
脳梗塞巣体積率の算出
右MCA閉塞後48時間でペントバルビタールナトリウム(50 mg/kg、腹腔内投与)による麻酔下で動物を断頭し、全脳を摘出して厚さ2 mmの脳切片を作製した。脳切片はPaxinos and Watson脳図譜(Paxinos G. and Watson C., The rat brain in stereotaxic coordinates, second edition. Academic Press Inc.; 1986)を参考に、Bregma前方4 mm、Bregma前方2 mm、Bregma上、Bregma後方2 mm、Bregma後方4 mmおよびBregma後方6 mmの冠状面が得られる位置とした。脳切片は、1 w/v% の2,3,5-Triphenyltetrazolium chloride(TTC)溶液に室温で浸漬・染色して写真撮影を行った。得られた写真を画像解析し、脳梗塞巣面積および脳の断面積を実測値で求め、下記計算式によりBregma前方4 mm~Bregma後方6 mmの脳梗塞巣体積および脳の総体積を算出した。また、脳梗塞巣体積および脳の総体積より脳梗塞巣体積率を算出した(脳梗塞巣体積率(%)=脳梗塞巣体積/脳の総体積×100)。
V:脳梗塞巣体積または脳の総体積(mm3)
a:Bregma前方4 mm断面上の脳梗塞巣面積または脳の断面積(mm2)
b:Bregma前方2 mm断面上の脳梗塞巣面積または脳の断面積(mm2)
c:Bregma断面上の脳梗塞巣面積または脳の断面積(mm2)
d:Bregma後方2 mm断面上の脳梗塞巣面積または脳の断面積(mm2)
e:Bregma後方4 mm断面上の脳梗塞巣面積または脳の断面積(mm2)
f:Bregma後方6 mm断面上の脳梗塞巣面積または脳の断面積(mm2)
対照群およびペプチド投与群の脳梗塞巣体積率は、それぞれ34.1±1.1%および27.2±1.2%(平均値±標準誤差)であった(図12;対照群について「Control」、ペプチド投与群について「1r10」参照)。対照群と比較してペプチド投与群の脳梗塞巣体積率は低い値を示し、統計学的に有意差が認められた(Mann-WhitneyのU検定、p<0.01)。
Claims (10)
- 以下から選択されるペプチドを含有する、末梢血への間葉系幹細胞の動員に用いるための組成物:
(a)配列番号:1のアミノ酸配列を含むペプチド;
(b)配列番号:1のアミノ酸配列において1もしくは数個のアミノ酸が置換、欠失、挿入若しくは付加されたアミノ酸配列を含むペプチド;および
(c)配列番号:1のアミノ酸配列と約90%以上の配列同一性を有するアミノ酸配列を含むペプチド。 - 以下から選択されるペプチドを含有する、末梢血への間葉系幹細胞の動員による、対象における疾患または病的状態の治療に用いるための組成物:
(a)配列番号:1のアミノ酸配列を含むペプチド;
(b)配列番号:1のアミノ酸配列において1もしくは数個のアミノ酸が置換、欠失、挿入若しくは付加されたアミノ酸配列を含むペプチド;および
(c)配列番号:1のアミノ酸配列と約90%以上の配列同一性を有するアミノ酸配列を含むペプチド。 - 疾患または病的状態の治療が、炎症抑制治療、免疫調節治療、組織の再生誘導治療、および組織の線維化抑制治療から選択される、請求項2に記載の組成物。
- 疾患または病的状態が、炎症性疾患、自己免疫疾患、組織の損傷、虚血もしくは壊死を伴う疾患、および線維性疾患から選択される、請求項2に記載の組成物。
- 疾患または病的状態が、炎症性腸疾患である、請求項2に記載の組成物。
- 疾患または病的状態が、潰瘍性大腸炎である、請求項2に記載の組成物。
- 疾患または病的状態が、アトピー性皮膚炎である、請求項2に記載の組成物。
- 疾患または病的状態が、脳梗塞である、請求項2に記載の組成物。
- 以下から選択されるペプチドを含有する、炎症性腸疾患、アトピー性皮膚炎および脳梗塞から選択される疾患の治療に用いるための組成物:
(a)配列番号:1のアミノ酸配列を含むペプチド;
(b)配列番号:1のアミノ酸配列において1もしくは数個のアミノ酸が置換、欠失、挿入若しくは付加されたアミノ酸配列を含むペプチド;および
(c)配列番号:1のアミノ酸配列と約90%以上の配列同一性を有するアミノ酸配列を含むペプチド。 - 以下から選択されるペプチド:
(a)配列番号:1のアミノ酸配列を含むペプチド;
(b)配列番号:1のアミノ酸配列において1もしくは数個のアミノ酸が置換、欠失、挿入若しくは付加されたアミノ酸配列を含むペプチド;および
(c)配列番号:1のアミノ酸配列と約90%以上の配列同一性を有するアミノ酸配列を含むペプチド。
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