과면역 반응에 의해 유발되는 염증성 질환 치료용 조성물

Detailed description of the invention and specific embodiments

An allogenic (other is) chondrocytes; and TGF-a β expressing chondrocytes; mixing cells containing cell mediated gene therapy number of degenerative arthritis to known. 2 applied to said mixing cells on in both basic research and clinical patient bone arthritis bone arthritis patient's pain, activity and improve cartilage structure has been identified. However, there was not found any transferred their mixing cells.

The present invention victims of the bone arthritis animal model mixed cells after observation of the effect of remodeling pain, anti-inflammatory environment determines cells inducing mixing result, mixing cells treated arthritis in anti-inflammatory cytokines and increases by M2 macrophages is also used for knee joint, to mitigate pain inducing inflammatory joint reproducing inducing environment has been confirmed.

The, mixing cells induce autoimmune billion number properties as signal M2 macrophage-rich environment having been bar, [...] compositions of assumption that effective treatment of inflammatory diseases, rheumatoid arthritis animal model chondrocyte cells expressing TGF-a β establishing chondrocytes in a water vein was administered to the knee joint. As a result, administration at the time of the administration of mixed cells when the middle family goal from the group consisting of reduced edema verify, in addition cytokines that promote a synovial inflammatory reactions induced IL provided 6, IL provided 17A, reduces expression of IL-a 1 beta and TNF a-α has been confirmed. The therapeutic effects of which 26 to 1 mixing cells by administering a single holding is been.

Basing rheumatoid arthritis used as MTX (Methotrexate) number of a substance such as fire extinguishing machine and like oral ulceration and renal cell electrodes simultaneously cause hepatotoxicity risk disclosed. In addition, such as an antibody such as anti-a TNF a-α treatment device to draw earlier patient number are congenital disturb the immune system by expression of tuberculosis (TB) from non-glycosylated tuberculosis from fish oil known. To the W-CDMA, the present invention according to cell therapy for degenerative arthritis clinical trials is like number does not require such a result improve the preservation disclosed. The present invention overcomes the shortcomings of existing rheumatoid arthritis the victims of the mixed cells number identifying new therapeutic number can be possibility that queue.

In one aspect the present invention refers to the, (i) chondrocytes or cartilage cells capable of differentiating into cells; and (ii) encoding TGF-a β gene encoding gene have been introduced chondrocytes or TGF-a β containing said cells capable of differentiating into chondrocytes have been introduced, [...] caused by a composition for treatment of inflammatory diseases are disclosed.

In another aspect the present invention refers to, (i) chondrocytes or cartilage cells capable of differentiating into cells; and (ii) encoding TGF-a β gene encoding gene have been introduced chondrocytes or TGF-a β chondrocytes have been introduced containing cells capable of differentiating into a subject in need thereof the composition caused by the treatment of inflammatory diseases including [...] method are disclosed.

In another aspect the present invention refers to, (i) chondrocytes or cartilage cells capable of differentiating into cells; and (ii) encoding TGF-a β gene encoding gene have been introduced chondrocytes or TGF-a β containing cells capable of differentiating into chondrocytes have been introduced [...] to the therapeutic application of composition caused by an inflammatory disease are disclosed.

In the present invention, the inflammatory disease caused by said [...] rheumatoid arthritis, multiple sclerosis, systemic lupus lupus, myasthenia, colitis, diabetes of type such as Encephalitis number 1 which, among rheumatoid arthritis treatment in particular efficient.

In the present invention, said cartilage progenitor cells or stem cells capable of differentiating into cells be a cartilage cells.

In the present invention, (i) chondrocytes or cartilage cells capable of differentiating into cells; (ii) on TGF-a β gene encoding gene encoding been introduced chondrocytes or TGF-a β cells capable of differentiating into chondrocytes have been introduced; in different mixing ratio can be mixed, preferably 1:1 - 100:1 ratio can have a composition, in more preferably 3:1.

In one aspect of the present invention is a composition for treating an inflammatory disease caused by those who are not [...] transduced chondrocytes (hChonJ cells) expressing TGF-a ß1 chondrocytes (hChonJb #7) and, more specifically human allogenic (other is) chondrocytes (hChonJ) expressing human TGF-a ß1 chondrocytes without using a tool. Various mixing ratio both cells can be mixed. For example, 3:1 ratio of these two cells, i.e. cells can be selected from the group 3 and 1 is in hChonJ hChonJb #7. Said 3:1 ratio mixing cells [Cytotherapy, 2012 Feb; 14 (2): 247 - 256] and process for producing method for the papers American patent number 7,005,127 patent number 7,282,200 call call in U.S. disclosure and over.

In one aspect of the present invention mixed cells after administration of RAT model is 15 begins to be relaxed when the pain from arthritis after administration, after administration to the pain relief effect is transmitted point 56 (experiment an ending date) is washed out. Such pain relief improvements have been found associated with the arthritis knee joint structure, mixed cells administered peptide effective pain alleviating and both up. I.e., in the present invention method and apparatus for cell mediated gene therapy for HiV therapy pain relief using mixed cells are disclosed.

Another aspect of the present invention mixed cells number 1 and number 2 collagen arthritis RAT model of cartilage tissue administration of collagen to promote the fastness to perform histological analysis result, number 2 collagen [...] vitreous (hyaline) cartilage tissue is developed mixed cells as signal peptides.

Native immune cell (innate immune cell) in macrophage activation phenotype (macrophage) proinflammatory (pro-a inflammatory) properties according to activated "M1 macrophage" activated "M2 macrophage" immune billion number (immunosuppressive) properties to classification with each other. In a key aspect of cytokines is associated with activation of macrophages to IFN-a γ M1, M1 macrophages differentiation induced by pathogen recognition signal is TLR contribute to substrate. M1 TNF α macrophage cells, expression of the resultant high expression of IL provided 12 and IL-a 23 IL provided 10, resulting in tissue damage and cause Th1 Th17 immune response been known for some time. In contrast, increasing the expression level of the expression of the M2 macrophage IL provided 10 IL provided 12 driven by reducing inflammation tissue to promote other.

In one aspect of the present invention expressing TGF β 1 is expressed in human chondrocytes on normal human chondrocytes (human chondrocyte) hChonJ TGF-a β hChonJb #7 in the IL-a 10 M2 macrophages is guided through and inducing the production of a appear, to the generated M2 macrophage IL provided 10 secretion, mixing cells in joint administration of anti-inflammatory environment determined substrate. Mixing cells analyzed result M2 macrophages present animals administered the model a histological can be increased even identify (also 8).

In addition, composition of the present invention immune billion number effect, it is long but oh derived by increased expression of TGF β 1 elimination on number 1 and determined, it is long but oh number 1 of the present invention expression of elimination in animal model of knee joint administration group in one aspect mixing cells present fabrics.

By the reduction of the arginine L - and elimination it is long but oh expressed in the number 1, number effect known as potent immune billion in immune system results and, in addition that it is long but oh induce transcription of number 1 is a method for it is long but oh number TGF-a β elimination is reported etc. (Durante et al. , Circulation, 103 (8), 1121 - 7, 2001). The, macrophage cells in the presence of M2 TGF β 1 may be immune modulating more efficiently determined substrate.

In addition, anti-inflammatory composition of the present invention is effected by administering M2 mixing cells expressing IL provided 10 is concentrated in macrophages activate STAT3 and PI3K, the anti-inflammatory effect by being controlled by a Hmox provided 1 shown substrate.

On the other hand, recent rheumatoid arthritis synovial and present for manufacturing interleukin -1 patient (IL-a 1), interleukin -8 (IL provided 8), tumor necrosis factor (TNF a-α) - α, TGf β (TGF-a β), and a platelet derived growth factor (PDGF) fibroblast growth factor (FGF) such as cytokines and growth factor is detected (Nouri et al. , Clin. Exp. Immunol. 55:275 - 372, 1984; Thornton et al. , Clin. Exp. Immunol. 86:79 - 86, 1991; Saxne, et al. , Arthritis Rheum. 31:1041 - 1045, 1988; Seitz et al. , J. Clin. Invest. 87:463 - 469, 1991; Lafyatis et al. , J. Immunol. 143:1142 - 1148, 1989; Melnyk et al. , Arthrtis Rheum. 33:493 - 500, 1990).

B - cell stimulatory factor 2 or interferon β 2 IL provided 6 is known as cytokines are disclosed. IL provided 6 is involved in lymphocyte activation of B cells as differentiation factor (Hirano T. Et al. , Nature 324, 73 - 76, 1986), various cells from affecting the action of multifunctional cytokines has been found (Akira, S. Et al. , Adv. In Immunology 54, 1 - 78, 1993).

Rheumatoid arthritis synovial or subject's serum no excess soluble IL provided 6 receptor presence on interleukin -6 wirelessly reports in the nanometer range (Houssiau et al. , Arthritis Rheum. 31:784 - 788, 1988; Hirano et al. , Eur. J. Immunol. 18:1797 - 1801, 1988; Yoshioka et al. , Japn. J. Rheumatol. In press). An animal model of rheumatoid arthritis is obtained similar results even since (Takai et at. , Arthritis Rheum. 32:594 - 600, 1989; Leisten et al. , Clin. Immunol. Immunopathol. 56:108 - 115, 1990), IL provided 6 been known factor is related to rheumatoid arthritis.

On the other hand, number number number three cookie troubles [map for developing rheumatoid arthritis clinical biological activity in high - affinity IL provided 17A billion transduction molecule ailim are disclosed. RA general outline demonstrated (PoC) in both basic research, stable doses of MTX combination three cookie troubles [map active RA administering patient was administered. As a result three cookie troubles [map rheumatoid arthritis treatments compared to placebo group has been confirmed that the rapidly improving clinical indications (Hueber et al. (2010) Sci. Transl. Med. 2 (52): 52 - 72). This is particularly likely in patients agent in a rheumatoid arthritis IL provided 17A data indicate are disclosed.

Another aspect of the present invention is effected by administering CFA (complete Freund's adjuvant) rheumatoid arthritis RAT sole administration mixed cells are derived, edema of metatarsal (metatarsal) 28 is verification result, verify (also 10) when the middle family goal from the group consisting of mixed cell administration reduced edema, induced inflammatory response promoting item IFN-a γ Pro non-inflammatory cytokine, IL provided 6, IL provided 17A, IL-a 1 beta and TNF a-α analysis result, MTX can be effectively regulated reducing mixing cell administration group from the group consisting of statistical contrast administration has been confirmed (11 also).

The, composition of the present invention is rheumatoid arthritis synovial joint of a patient in excess secreted inflammatory cytokines IL provided 6, IL provided 17A, IL-a 1 beta and TNF a-α billion number reduced expression of edema flow tides.

Hereinafter, the present invention broadcast receiver through more detailed in the embodiment. In the embodiment for the present invention is only to exemplify these provided, in the embodiment of the present invention interprets these range by one number is not in the person with skill in the art will-case in the art.

In the embodiment 1: arthritis in an animal model for anti-pain relief and Col II mixed cells positive cartilage reproducing induction

Arthritis patient clinical 2 so that an applied to mixture of cells, and articulated to synergistically environment sources capable of liquid therein. In order to identify mechanisms for the treatment of a degenerative arthritis animal model MIA inducing mixing and, von Frey filament test joint histological inspection and by dielectrophoresis.

1 - 1: small MIA induced arthritis animal model number

Small animal model number is 6 week zero male RAT (Spargue-a Dawley, 200 a-225g, country biotech, a compensation) of 0.50 to, animal experiments kolon (a compensation) effective operating life science Committee (IACUC No. KLS IACUC 2013 - 04) with veterinarian in the objects of management are conducting.

To induce arthritis, 60 mg/mL concentration of MIA (monosodium iodoacetate, Sigma, USA) solution using a syringe 50 micro l 31G RAT left knee joint strip was administered.

1 - 2: preparation of cells for cell therapy

Used in the present invention includes quantifying chondrocytes and hChonJ, hChonJb #7 is secreted TGF-a β is that has been transformed to chondrocytes are disclosed. Their number [Cytotherapy, 2012 Feb; 14 (2): 247 - 256] and small ratio method and U.S. patent number 7,282,200 call call in U.S. patent number 7,005,127 disclosure in the nanometer range.

According to a publicly known method of cDNA of TGF-a β hChonJb #7 is injected into cells number can be high pressure liquid coolant. For example, such as a cDNA of metal with hit it is four (methallothionein) promoter (promotor) and TGF-a β [...], such as erythromycin resistance gene Neo vector having publicly known [e.g., Gibco-a BRL yarn pUC19 (containing [...] resistance gene)] included a number of high pressure liquid coolant including vector after TGF-a β cDNA, or liposomes (Lipofectin) method publicly known method such as calcium phosphate in chondrocytes (Calcium Phosphate) method is performed according to number of pectin can be high pressure liquid coolant

1 - 3: process of identifying a peptide effect mixing cells and pain relief

Arthritis induction [...] MIA processing 2 main after mixing cells {(1. 2 × 10⁶ Cells: hChonJ (9 × 10⁵ Cells) + hChonJb #7 (3. 0 × 10⁵ Cells)}, hChonJ (9 × 10⁵ Cells), hChonJb #7 (3. 0 × 10⁵ Cells) or controls {CryoStor provided 10 (CS-a 10)} leftward when the knee joint elevate administration, observation of symptoms, weight main 1 times were measured.

Von Frey filament test result of reducing inflammatory observed, also as shown in the variation 2, numerical pain after administration reduced from 15 cells after a transparent conductive layer, such effect is terminated blade (56 after administration of the lowest and) been duration to the lesion. Expressing TGF-a β hChonJb #7 (3. 0 × 10⁵ Cells) when administered group, pain relief is observed from 15 but, its effect is a full-length after main 6 as signal peptides. As compared to, normal human chondrocytes is hChonJ (9 × 10⁵ Cells) group consisting in administering pain relieving effective appears not.

In order to identify whether a peptide is associated with pain relieving effective of mixing, two left knee joint and an aqueous 5 group of experiments, Masson's trichrome staining and H&E through histological analysis (also 3 and 4 also) conducting dyeing.

Controls (CS-a 10) administration group was hChonJ and peptide is uncertain. In contrast, other mixed cell administration group showed more interbody inhibin hChonJb #7 administration group.

In addition, immunohistochemistry dyeing result, also 5 as shown in the variation, in type II collagen content higher than the mixing cell administration group administration has been confirmed that when the cartilage hChonJb #7, these results from mixing cell administration group near interbody vitreous cartilage that his car.

For joint state improving method for assessing, papers [Kobayasi, K. , J. Such as, J. Vet. Med. Sci. , 65:1195 - 1199, 2003] is a dyeing method H&E pathological results have pathological score evaluating joint tissue tissue, to abstract the result was below table 2. Most are improved interbody below experiment results cells processing group.

In the embodiment 2: mixing cells influence on analysis processing anti-inflammatory cytokine secretion

Mixing in an animal model of processing (synovial fluid) cytokines expression profile in an influence on synovial joint steel has been confirmed.

Macrophage derived in order to identify whether the site of administration to cells, in the presence of CD68 positive cells present therefrom.

The present micro m thickness tissue slide number after a small gun every grudge paraffin pak paragraph 4, CD68 antibody (Novus, Co, USA) was embodiment dyeing using immunohistochemistry.

As a result, as shown in the variation of Figure 7 A, CD68 positive cells large mixing cell administration group been increased.

The IL-a 10 pain relieving effective exhibits the known. Based on arthritis model produced by administering the reporting of on mixing cells, expression of IL provided 10 has been confirmed. Expression of cytokines in 96 well plate assay induced arthritis RAT synovial IL-a 10 (# RECYTMAG-a 65K, Milipore, USA) using magnetic bead panel were measured.

As a result, as shown in the group from the group consisting of mixed cell administration of Figure 6 B hChonJ administration has been confirmed that the expression of IL provided 10 increases. Histological analysis results from the group consisting of mixed cells IL provided 10 positive cells also increased administration as signal peptides. These results indicate that the administration of anti-inflammatory cytokines induce arthritis knee joint mixed in cells that exhibits a IL-a 10.

In the embodiment 3: M2 macrophage derived rich environment for identifying type of mixing

M1 and M2 which is essentially two forms of type is type-macrophage colony stimulating factor, macrophage colony stimulating factor responsible to M1 type acute inflammation, high levels of inflammatory cytokines such as IL provided 12 and TNF a-α and secreted, M2 macrophage wound healing and anti-inflammatory control functions, used for plays an important role.

In the embodiment 2, since high levels of from the group consisting of mixed cell administration IL provided 10 expression is verified, mixing cells induced arthritis in animal cells are induced to function key whether secretion of knee, M1 macrophages present in animal model of CD86 expression of M2 macrophage marker number 1 is a marker expression has been confirmed that it is long but oh moieties.

To this end, it is long but oh CD86 antibody (AbD serotec, NC, USA) using a gun every grudge pak paragraph number 1 antibodies (BD Bioscience, USA) present elimination and mouse immune tissue chemical dyeing his paraffin.

As a result, as shown in the fig. 8, a signal detected from the from the group consisting of mixed with cells while expression of CD86 contrast administration, it is long but oh mixed cell administration appeared to possess a significantly higher from the group consisting of moieties to number 1. This mixing cells to induce macrophage M2 at or.

In the embodiment 4: mixing cells induced by M2 macrophage rich environment for identifying: RNA

In the embodiment 3 M2 macrophages in order to identify gene expression in induction in identifying, isolating total RNA from animal model of present, M1 and M2 macrophages expression of gene has been confirmed.

Cells using RNeasy Lipid Tissue Mini kit (QIAGEN, USA) to separate when the RNA is present in RAT processing, (Invitrogen, USA) RNA isolated from cDNA using SuperScriptTM III First provided Strand Synthesis System and copiers.

Through an ABI7900 after conducting real-a time PCR. 1 μl cDNA PCR reaction mixture is, 0. 2 μm each primer, total 50 μl to 10 μl SYBR Premix Ex Taq (TAKARA, Japan) set to use a transparent conductive layer, the reaction conditions in 10 seconds 95 °C, 60 °C 30 S. in conducting cycle total 40. Gene expression levels can be identified using gene expression vector β - 2 - Δ Δ Ct method when the compared expression dose, 3 shown to a table using qRT-a PCR primer.

As shown by the macrophage gene expression profiles have shown to 9 also concentrated qRT-a PCR result. A large number of CD68 positive cells present kind administration by mixing cells, expression of CD68 cells appeared high mixing group administration. A big difference from the group consisting of TNF a-α is mixed with contrast administration cells cannot. Anti-inflammatory cytokines are expressed remarkably from the group consisting of a mixed cell administration IL provided 10.

In order to identify whether increase protein expression of mRNA expression IL provided 10 leading into, conducting multiple cytokines present [e tax it will reach. As a result, as shown in the variation also 10, vibrates the protein levels of the group consisting of mixed cell administration IL provided 10 as signal peptides. The additional macrophage marker (interleukin 10 receptor alpha subunit) (heme oxygenase 1) mRNA levels of IL provided 10RA Hmox1 significantly increased and in addition has been confirmed.

The classifies said results, when administering cells mixed in arthritis model, the extra M2 derived macrophages can be know.

In the embodiment 5: mixing in cells confirming efficacy in an animal model of rheumatoid arthritis

5 - 1: number small animal model of rheumatoid arthritis

7 week zero number is small animal model of rheumatoid arthritis induced female RAT (160 provided 180g Lewis, central effective, Japan) of 0.50 to, life science kolon animal experiments (a compensation) effective operating Committee (IACUC No. KLS IACUC 2015 - 07) with veterinarian in the objects of management are conducting.

Induce rheumatoid arthritis symptoms, concentration of 5 mg/mL CFA (Complete Freud's Adjuvant, number irradiation: Chondrex, Inc.) solution using a syringe 50 micro l 31G RAT sole under pin was administered. 3 main behind metatarsal edema induced by discrete cosine measure the magnitude rheumatoid arthritis using calipers most edema when test material was administered elevate serious derived Lewis-a rat left knee joint.

5 - 2: animal model test substance administration

Rheumatoid arthritis induced by each population by mary [...] 6, CFA processing 3 main after mixing cells {(1. 2 × 10⁶ Cells: hChonJ (9 × 10⁵ Cells) + hChonJb #7 (3. 0 × 10⁵ Cells)}, hChonJ (9 × 10⁵ Cells), hChonJb #7 (3. 0 × 10⁵ Cells), voice controls (CS-a 10) leftward elevate knee joint was administered. Positive controls in the case of MTX (Methotrexate) 0. A 1 to 4 mg/kg orally 1 weeks by times, total 3 times was administered. 3 metatarsal diameter and body weight were measured all at once.

Table 4 have shown each experiment group administration conditions.

5 - 3: mixing in cells in an animal model of rheumatoid arthritis

Said arthritis model test material administration, using caliper metatarsal (metatarsal) 10 shown to also measure the magnitude results of edema. Controls (CS-a 10), hchonJ, hChonJb #7, Invossa, MTX (Methotrexate) 19 and 26 and the edema size decrease amount measured after administration of controlled pneumatic, mixed cell administration group, the group consisting of statistical significance have shown a decrease amount regulated contrast administration of MTX.

5 - 4: mixing cells by administering cytokine secretion profile analysis

Arthritis model sample administration, the third 28 and effective synovia an aqueous cytokines in RAT left knee joint steel when the analysis, also shown to result 11. In RAT insulin syringe for saline solution after administration to 50 μl syringe 31G synovial extraction is then joint steel directly in recovering his manner an aqueous state 204. The cytokines IFN-a γ, IL provided 6, IL provided 17, when the analyzing IL-a 1 beta and TNF a-α, Rat Cytokine/Chemokine multiplex analysis (RECYTMAG-a 65K, Millipore) using conducting. Pro non-inflammatory cytokine induced inflammatory response promoting item IFN-a γ, IL provided 6, IL provided 17, IL-a 1 beta and TNF a-α analysis results, MTX can be effectively regulated from the group consisting of mixed cell administration group (Methotrexate) reducing statistical contrast administration has been confirmed.

The present invention above described particular content portion that are directionally-shaped conductor, the party industry in this particularly preferred embodiment only the procedure to only aspect which person with skill in the art, the range of the present invention it is apparent that by a number that are not one will. The, substantial range of the present invention defined by appended claim and their equivalent that will.