WO2006067920A1

WO2006067920A1 - Pharmaceutical for bone marrow stem cell transplantation therapy

Info

Publication number: WO2006067920A1
Application number: PCT/JP2005/020697
Authority: WO
Inventors: Hiroshi Shimizu; Riichiro Abe; Daisuke Inokuma
Original assignee: National University Corporation Hokkaido University
Priority date: 2004-12-21
Filing date: 2005-11-11
Publication date: 2006-06-29
Also published as: JPWO2006067920A1

Abstract

A pharmaceutical comprising as an active ingredient CTACK (T-cell attracting chemokine) that is effective in the regenerative therapy utilizing bone marrow stem cells, especially the regenerative therapy of skin diseases utilizing transplanted bone marrow stem cells. In particular, the pharmaceutical provides a novel therapeutic method for congenital skin diseases whose fundamental treatment has been regarded as being difficult, for example, congenital epidermolysis bullosa.

Description

Specification

Drugs for bone marrow stem cell transplantation

Technical field

[0001] The present invention opens to the use of chemokines in a new treatment method by skin cell regeneration for congenital skin diseases that have been reported as intractable diseases, such as congenital epidermolysis bullosa.

Background art

[0002] Most congenital or inherited diseases are caused by the presence of mutations or deletions in proteins or genes that are required for the maintenance of normal living organisms that impair their original functions. Disease. Therefore, it is generally considered difficult to cure fundamentally by surgical treatment or pharmaceutical treatment by administration of drugs or the like.

[0003] One of such congenital diseases in skin tissue is congenital epidermolysis bullosa (hereinafter referred to as EB). EB is congenitally fragile and is understood as a collective term for congenital diseases that easily cause blistering on the skin, even when weak external force causes dissociation of the epidermal dermis (see, for example, Toshiaki Saida et al. Today's skin disease treatment policy ", 3rd edition, published by medical school, pages 308-309, non-patent literature 1).

[0004] EB is a genetically diverse disease, and blister formation is based on the force generated at any site under electron microscope observation. Epidermis cell blister formation (simple type), blister formation (junction type) And dermal blister formation (nutrient disorder type) directly below (Hiroshi Shimizu, “Classification of epidermolysis bullosa”, previous training course required A course materials, Japan Dermatological Association resident member Issued by the association, 2002 'Non-patent document 2). In the simple type, keratin 5 or keratin 14 gene, in the junction type in laminin 5 gene, in the nutritional disorder type VII collagen gene, mutation or injury is considered to be the cause of the onset, The

[0005] For EB and hereditary diseases, there are currently only therapies that give antibiotics or anti-inflammatory agents as symptomatic external treatments that are not effective radical treatments so far. It is. For this reason, effective treatments include the development of so-called gene therapy that restores the function of genes that have been identified as abnormal (Daisuke Sawamura et al., “ "Possibility of gene therapy against", 2002, West Japan Dermatology, separate volume, Vol. 64, No. 3, pp. 277-280 (Non-patent Document 3).

[0006] Non-Patent Document 1: edited by Toshiaki Saida et al., “Today's skin disease treatment policy”, third edition, published by Medical School, pages 308-309.

Non-patent document 2: Hiroshi Shimizu, “Classification of epidermolysis bullosa”, previous training course required A course material, published by the Japan Dermatological Association Residents Committee, 2002.

Non-Patent Document 3: Daisuke Sawamura et al., “Possibility of gene therapy for epidermolysis bullosa”, 2002 West Japan Dermatology, separate volume, Vol. 64, No. 3, pp. 277-280.

Disclosure of the invention

Problems to be solved by the invention

[0007] However, gene therapy is routinely performed, and although it is a treatment that can be obtained, it is difficult to be more versatile! New development of EB therapy is desired. .

[0008] The present invention particularly relates to a method for effectively treating or ameliorating a hereditary disease without effective pharmaceutical treatment, such as congenital epidermolysis bullosa, by a method other than gene therapy, It provides a useful medicine.

Means for solving the problem

[0009] The present inventors have found that CTACK (T-cell attracting chemo kine), which is a chemokine expressed in the skin, attracts bone marrow stem cells to the skin tissue to which this is administered (chemoatratat), and finally epidermal cells ( bone marrow derived-keratinocytes (BMDK), and it has been found that combining this with bone marrow stem cell transplantation can treat skin diseases such as EB, The present invention has been completed.

That is, the present invention relates to a regenerative therapeutic drug using bone marrow stem cells containing CTACK as an active ingredient, and particularly to a regenerative therapeutic drug using transplanted bone marrow stem cells. In particular, the present invention relates to a pharmaceutical comprising CTACK as an active ingredient that differentiates transplanted bone marrow stem cells into epidermal cells.

[0011] Regenerative treatment using bone marrow stem cells is expected as a treatment for various diseases such as angiogenesis therapy, heart failure, and liver regeneration, and the bone marrow transplant promotion foundation (http: 〃 www.jmdp.or. jp / index.html) establishes so-called bone marrow bank to promote effective use of bone marrow stem cells is doing. Attempts have also been made to treat bone diseases by regenerating the bone marrow stem cell skin for skin diseases. However, CTACK's chemotolatato effect on bone marrow stem cells and the differentiation induction effect on epidermal cells and its bone marrow There is no report on its use for stem cell transplantation therapy.

[0012] The present invention uses CTACK in order to improve the efficiency in skin regenerative medicine as a treatment for skin diseases.

[0013] CTACK in the present invention is a protein having a total of 112 amino acid residues described in International Patent Publication WO98Z23750, or a force in which some of the amino acid residues are different from those described in the same publication. It means a protein that retains chemotratat activity against stem cells and BM DK-inducing activity.

[0014] To date, CTACK functions have been known to include the selective attraction of CLA + memory T cells, skin dendritic cells (Langelnos cells), and their effects on their precursors. It has been reported that a certain healing effect is expected for skin disorders involving the immune system, such as psoriasis, skin cancer, inflammation, allergy, dermatitis, wound healing, infection, etc. Publication WO98Z23750, Janine Morales, et al., Proc. Natl. Ac ad. Sci. No. 157-165, 2002).

[0015] According to these reports, CTACK binds to CCR10, which is a chemokine receptor, and chemostratates cells such as CLA + cells, T cells, rod cells or rod precursors, and these are dermis of the skin. It has been reported that by moving into the layer and into the Z or epidermal layer, it shows therapeutic effects on skin diseases associated with immune system cells. However, it has been reported that CTA CK has the function of chemostratating bone marrow stem cells different from immune system cells and further inducing differentiation into epidermal cells! Wow! /.

[0016] The present invention uses CTACK's differentiation-inducing function for bone marrow stem cells, so that the use of CTACK can be applied to the treatment of skin diseases related to immune cells that have been proposed in the past, and to the application frame. It expands to the treatment of new skin diseases located outside.

[0017] The present invention can induce differentiation of bone marrow stem cells into epidermal cells in the field of skin damage, and is a congenital skin disease that has been considered to be particularly difficult to cure, such as congenital skin diseases. It is possible to provide a new treatment method and a pharmaceutical for the treatment of sexual epidermolysis bullosa.

Brief Description of Drawings

[0018] FIG. 1 shows chemotract activity of CTACK against bone marrow stem cells by in vitro test.

[FIG. 2] FIG. 2 shows the activity of inducing differentiation of CTACK into bone marrow stem cells into epidermal cells in an in vivo test.

[FIG. 3] FIG. 3 shows the differentiation-inducing activity of CTACK to epidermal cells against bone marrow stem cells in mice pretreated with G-CSF.

FIG. 4 shows C TACK differentiation-inducing activity into epidermal cells against bone marrow stem cells in mice with an increased number of CD34-positive bone marrow stem cells in peripheral blood.

BEST MODE FOR CARRYING OUT THE INVENTION

[0019] CTACK used in the present invention can be prepared recombinantly in accordance with the description of International Patent Publication No. WO98Z23750 or the literature of Morales et al.

[0020] For example, using a probe having the whole or part of the base sequence of the gene encoding CTACK described in International Patent Publication No. WO98Z23750, cloning may be performed according to the description in the publication. Also, DNA encoding the full length of CTACK can be synthesized using a DNA synthesizer or the like, and can be prepared recombinantly using this. CTACK, which is commercially available from R & D System of Minneapolis, USA or other institutions, may also be used.

[0021] Further, even if the amino acid sequence or DNA sequence specifically described in the international publication or the like is used, for example, a bone marrow stem cell comprising an amino acid sequence in which one or more amino acids are substituted, deleted, and Z or added. Polypeptides having chemoretorato activity against BMDK and activity to induce BMDK can also be used in the present invention.

[0022] For example, due to the physical properties such as the charge, size, and hydrophobicity of amino acid residues, the mutation is highly conserved, such as glycine (Gly) and proline (Pro), Gly and alanine ( Ala) or valine (Val), leucine (Leu) and isoleucine (lie), glutamic acid (Glu) and glutamine (Gin), aspartic acid (Asp) and asparagine (Asn), cysteine (Cys) and threonine (Thr), Examples include substitution between Thr and serine (Ser) or Ala, lysine (Lys) and arginine (Arg).

[0023] Similarly, a gene encoding a powerful CTACK or a variant thereof can also be used for recombinant production of CTACK and the like.

[0024] In addition, a vector holding a DNA encoding CTACK, a host to be transformed with the vector, and the like may be used by selecting a combination described in the preceding literature or other general-purpose combinations. In addition to plasmids, various viruses such as baculovirus, retrovirus, vaccinia virus and the like can also be used.

[0025] Expression may be performed under the control of a promoter sequence specific to the CTACK gene, or another appropriate expression promoter may be used by connecting or replacing it with a promoter sequence specific to the CTACK gene. The promoter used in this case may be appropriately selected according to the host and the purpose of expression. For example, when the host is E. coli, the 1S host such as T7 promoter, lac promoter, trp promoter, λ PL promoter is yeast. In some cases, PH05 promoter, GAP promoter, ADH promoter and the like can be exemplified by SV40-derived promoter, retrovirus promoter and the like when the host is an animal cell, but of course, it is not limited thereto.

[0026] CTACK is administered to patients who have undergone bone marrow stem cell transplantation in the form of a solution in which CTACK is dissolved in an appropriate buffer, for example, physiological saline, an emulsion in which an appropriate emulsifier is added, and the like. Either a solution or a sheet form such as a nonwoven fabric holding emulsion can be used. These can be used by directly applying to or pasting the affected area.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

Example 1

[0028] The chemotolatato activity of CTACK on bone marrow stem cells was confirmed by an in vitro test.

[0029] Bone marrow cells were isolated from the bone marrow of mice (C57BL6) according to the method of Wiley et al. (Current Protocols of Immunology, ISS N 0-471-52276-7). Then expressed on the cell surface Antibody staining using an antibody specific for CD34 antigen (PharMingen) was performed, and CD34 positive bone marrow stem cells were separated using FACSVantage (BD Bioscience).

[0030] 1 to 10 ⁵ CD34 positive bone marrow stem cells suspended in DMEM medium in the upper insert of a 3 μm pore insert (Kurashiki Boseki Co., Ltd.) dissolved in the same medium in the lower plate 0 to 500 ngZml CTACK (Manufactured by R & D System) or stromal cell derived factor-1 (stroma cell derived factor-1, SDF-1) was added and incubated at 37 ° C. After 4 hours, the number of CD34-positive bone marrow stem cells that had migrated to the lower plate was counted with a hemocytometer.

[0031] As a result, CTACK chemostratated CD34-positive bone marrow stem cells in a concentration-dependent manner at concentrations of lOngZml or higher (Fig. 1).

Example 2

[0032] The induced differentiation activity of bone marrow stem cells at the site of CTACK skin injury was confirmed by in vivo tests.

[0033] From a transgenic mouse (GFP mouse, distributed by Jackson Laboratories, USA) prepared using a recombinant gene obtained by placing the GFP protein gene under the control of the β-actin promoter, the same procedure as in Example 1 was performed. Bone marrow stem cells were isolated.

[0034] After these cells were injected into the bone marrow of a wild-type mouse strain (C5 7BI / 6) whose bone marrow cells had been destroyed by 850crad radiation treatment, the cells were raised under standard conditions for 4 weeks. Only GFP chimeric mice that emit GFP fluorescence were created.

[0035] The chimeric mice were divided into 4 groups, and immediately after the creation of a skin defect wound with a diameter of 6 mm on the back of each chimeric mouse, 1 group was CTACK (R & D System, IngZ phosphate buffered saline 30 1) SDF—l (lngZ phosphate buffered saline 30 1) in group 2, secondary lymphoid tissue chemokine (IngZ phosphate buffered saline 30 / zl) in group 3, In group 4, only the same buffer was administered locally around the wound, and the animals were raised under standard conditions until the wound was completely healed (about 28 days).

[0036] After breeding, the wound is excised, embedded in OCTcompound, and a 5 μl thick tissue piece is prepared. Antibodies and fluorescent dyes against keratin 14, CTACK, and other chemokines (FITC, RI TC) Each antibody reaction was diluted 100-fold with a secondary antibody bound to Fluorescent tissue staining was performed at room temperature for 1 hour.

[0037] The stained tissue pieces were observed using a confocal laser microscope (Laser Scanning Confocal Imaging System MRC 1024, manufactured by Bio-Rad), and GFP positive and keratin 14 positive cells were detected. The percentage of total epidermal cells was measured as the epidermal cells.

[0038] As a result, the proportion of bone marrow stem cell-derived epidermal cells significantly increased in the group receiving CTACK (Fig. 2).

Example 3

[0039] Prior to CTACK administration, Example 1 except that 150 g / kg / day of G-CSF was administered for 3 consecutive days to increase the number of CD34-positive bone marrow stem cells in peripheral blood (approximately 100 times). In the same manner as above, the ratio of epidermal cells differentiated from bone marrow stem cells in the total epidermal cells was measured (Fig. 3).

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[0040] In addition, CD34 positive cells were separated from bone marrow cells of GFP mice using MACS (magnetic activated cell sorting, Miltenyl Biotec), and these cells (5 x 10 ⁵ cells) were separated from normal mice. Tail force was also injected intravenously to increase the number of CD34-positive bone marrow stem cells in peripheral blood, increasing the number of epidermal cells differentiated from bone marrow stem cells in the total epidermal cells. Measured (Figure 4).

As a result, it was possible to confirm a larger number of bone marrow stem cell-derived epidermal cells than in Example 1.

Claims

The scope of the claims

[1] A regenerative treatment drug using bone marrow stem cells containing CTACK as an active ingredient.

[2] The medicament according to claim 1, which is a regenerative treatment using transplanted bone marrow stem cells.

[3] The medicament according to claim 1 or 2, wherein the regenerative treatment is a skin regenerative treatment.

[4] The medicament according to claim 3, which is a treatment for regenerating skin by differentiating transplanted bone marrow stem cells into epidermal cells.