Methods for treating immune diseases using calcineurin inhibitors and stem cells

Abstract

The present invention relates to a method for treating immune diseases using a calcineurin inhibitor and stem cells together. The use of a pharmaceutical composition comprising a calcineurin inhibitor and mesenchymal stem cells provided by the present invention can provide a significantly higher level of immunosuppressive activity than that obtained by using a conventional calcineurin inhibitor or mesenchymal stem cells, and therefore can be used to more effectively prevent or treat immune diseases caused by hyperimmunity.

Description

[Technical Field]

[0001] The present invention relates to a method for treating immune disorders using a calcineurin inhibitor in combination with stem cells. [Background technology]

[0002] Immune diseases are caused by damage or malfunction of the in vivo immune system due to various causes, including exogenous, genetic, and endogenous factors, and are characterized by a significant decrease or sudden increase in immunity. In particular, unlike cases of immune decline, where pathological symptoms are relatively predictable, sudden increases in immunity are accompanied by a variety of unpredictable pathological symptoms, and therefore, research into this issue is actively underway.

[0003] Atopic dermatitis (AD), an immune disease caused by a rapid increase in immunity, is a chronic, recurrent skin disease characterized by eczematous skin lesions accompanied by severe itching and epidermal barrier dysfunction. Its onset is known to be due to an excessive type 2 helper T (Th2) cell-mediated inflammatory response, which increases serum total IgE levels and leads to skin barrier dysfunction. Treatments for AD include restoring the epidermal barrier using emollients and anti-inflammatory agents such as corticosteroids or calcineurin inhibitors, with calcineurin inhibitors being the most commonly used treatment. The calcineurin inhibitor binds to FK506-binding protein-12 (FKBP-12) to inhibit the serine / threonine phosphatase calcineurin, subsequently preventing the nuclear potential of nuclear factor of activated T cells (NFAT), thereby suppressing NFAT-mediated transcription of pro-inflammatory cytokines in T cells, thereby improving atopic dermatitis.

[0004] Recently, methods using mesenchymal stem cells (MSCs) have been developed for the treatment of atopic dermatitis. MSCs are capable of self-renewal and differentiation into various lineages for the regeneration of damaged tissues. They are also used as immunomodulators that regulate the proliferation, recruitment, and function of innate and adaptive immune cells. Furthermore, MSCs are expected to have therapeutic effects on atopic dermatitis by inhibiting mast cell degranulation through prostaglandin E2 (PGE2) and TGF-beta (Korean Patent Publication Nos. 10-2009-0111269, 10-2010-0104385, and Korean Patent Registration No. 10-1070730). However, substantial clinical trials have yet to demonstrate specific therapeutic effects on atopic dermatitis. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Korean Patent Publication No. 10-2009-0111269 [Patent Document 2] Korean Patent Publication No. 10-2010-0104385 [Patent Document 3] Korean Patent No. 10-1070730 Summary of the Invention [Problem to be solved by the invention]

[0006] The present inventors have conducted extensive research to develop a method for effectively treating atopic dermatitis using stem cells, and as a result have confirmed that sequential treatment with mesenchymal stem cells and a calcineurin inhibitor increases immunosuppressive activity compared to treatment with either mesenchymal stem cells or a calcineurin inhibitor alone, thereby completing the present invention.

[0007] A primary object of the present invention is to provide a pharmaceutical composition for preventing or treating immune diseases, comprising: a first composition comprising a calcineurin inhibitor or a pharmaceutically acceptable salt thereof; and a second composition comprising stem cells or a culture product thereof.

[0008] Another object of the present invention is to provide a method for treating an immune disease, which comprises sequentially administering a first composition and a second composition contained in the pharmaceutical composition to an individual suffering from the immune disease.

[0009] Another object of the present invention is to provide a use of a calcineurin inhibitor and stem cells for the preparation of a pharmaceutical composition for preventing or treating immune diseases.

[0010] To achieve the above-mentioned object, one embodiment of the present invention provides a pharmaceutical composition for preventing or treating an immune disease, comprising: (a) a first composition comprising a calcineurin inhibitor or a pharmaceutically acceptable salt thereof; and (b) a second composition comprising stem cells or a culture product thereof. [Effects of the Invention]

[0011] The use of a pharmaceutical composition comprising a calcineurin inhibitor and mesenchymal stem cells provided by the present invention can provide a significantly higher level of immunosuppressive activity than that obtained using conventional calcineurin inhibitors or mesenchymal stem cells, and can therefore be used to more effectively prevent or treat immune diseases caused by hyperimmunity. [Brief explanation of the drawings]

[0012] [Figure 1] This is a graph showing the results of comparing the severity of skin lesions in atopic dermatitis animal models treated with pimecrolimus or mesenchymal stem cells. MSC indicates the group administered with stem cells alone, Pime indicates the group administered with pimecrolimus alone, and MSC+Pime indicates the group administered with a combination of stem cells and pimecrolimus. [Figure 2a] 1 is a graph comparing changes in stem cell viability depending on the treatment concentration of pimecrolimus. [Figure 2b] 1 is a graph comparing changes in the proliferation ability of stem cells depending on the treatment concentration of pimecrolimus. [Figure 2c]1 is a graph comparing changes in stem cell cycle profiles depending on the treatment concentration of pimecrolimus. [Figure 2d] 1 is a graph comparing morphological changes in stem cells depending on the treatment concentration of pimecrolimus. [Figure 2e] 1 is a graph comparing changes in the expression levels of stem cell surface markers (CD45, CD34, CD36, CD105, CD29, CD73) by pimecrolimus treatment. [Figure 3] 1 is a graph showing the results of analyzing the effect of pimecrolimus on the immunosuppressive activity of stem cells against ConA-treated PBMC cells. [Figure 4a] 1 is a graph showing the results of comparing the levels of TGF-β expressed from stem cells depending on the treatment concentration of pimecrolimus. [Figure 4b] 1 is a graph showing the results of comparing the levels of PGE2 expressed from stem cells depending on the treatment concentration of pimecrolimus. [Figure 4c] 1 shows a Western blot photograph comparing the levels of COX2 expressed from stem cells depending on the treatment concentration of pimecrolimus, and a graph showing the results of analyzing the density thereof. [Figure 5a] 1 is a graph comparing the immunosuppressive activity obtained by treating stem cells with pimecrolimus under similar conditions. [Figure 5b] 1 is a graph comparing the immunosuppressive activity obtained by first treating with pimecrolimus and then treating with stem cells. [Figure 5c] 1 is a graph comparing the immunosuppressive activity obtained by first treating stem cells and then treating with pimecrolimus. [Figure 5d] 1 is a graph showing the results of analyzing changes in immunosuppressive activity depending on the order of treatment with stem cells and pimecrolimus.

[0013] The term "calcineurin inhibitor" as used herein generally refers to an immunomodulatory substance produced during the fermentation process of microorganisms, and is known to primarily exhibit the effect of suppressing T cell responses. Functionally, the calcineurin inhibitor inhibits the response of calcineurin, a type of phosphorylation enzyme, thereby suppressing the expression of intracellular cytokine genes and, as a result, reducing the secretion of various inflammatory cytokines.

[0014] The calcineurin inhibitor provided by the present invention is not particularly limited thereto, but may be, for example, cyclosporin A, tacrolimus, pimecrolimus, etc., or another example may be pimecrolimus.

[0015] The term "pharmaceutically acceptable salt" as used herein refers to a dosage form that does not impair the biological activity and physical properties of the calcineurin inhibitor. The pharmaceutically acceptable salt includes acid addition salts formed with acids that form non-toxic acid addition salts containing pharmaceutically acceptable anions, such as inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, and hydroiodic acid, organic carboxylic acids such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, and salicylic acid, and sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. For example, pharmaceutically acceptable carboxylate salts include metal salts or alkaline earth metal salts formed with lithium, sodium, potassium, calcium, magnesium, etc., amino acid salts such as lysine, arginine, guanidine, etc., and organic salts such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, diethanolamine, choline, triethylamine, etc. On the other hand, in the case of amlodipine, the besylate salt, which is widely used commercially, is preferred.

[0016] As used herein, the term "stem cells" refers to cells that have excellent proliferation potential and can differentiate into various tissues in the body.

[0017] The stem cells provided in the present invention may be, for example, human adult stem cells, human pluripotent stem cells, induced pluripotent stem cells, animal embryonic stem cells, animal adult stem cells, mesenchymal stem cells, etc., but are not limited thereto. Other examples include mesenchymal stem cells such as adipose-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, myocardial-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, cartilage-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells. In other examples, the various mesenchymal stem cells may be used alone or in various combinations.

[0018] The term "culture product" as used herein refers to the result obtained by culturing the stem cells. The culture product may be, but is not limited to, a whole culture, a culture supernatant obtained from the whole culture, cell bodies obtained from the whole culture, a cell lysate obtained by disrupting the cell bodies, an extract obtained from the cell lysate, a fraction obtained from the extract, etc.

[0019] The term "immune disease" as used herein refers to a disease caused by damage or malfunction of the in vivo immune system due to various causes such as exogenous factors, genetic factors, and endogenous factors.

[0020] In the present invention, the immune disease may be, for example, an immune disease caused by hyperimmunity, or an allergic immune disease or autoimmune disease caused by hyperimmunity, or may be, for example, an allergic immune disease such as hay fever, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, food allergy, or mastocytosis, or may be, for example, an autoimmune disease such as Graves' disease, rheumatoid arthritis, Hashimoto's thyroiditis, systemic lupus erythematosus (lupus), vasculitis, Addison's disease, polymyositis, Sjogren's syndrome, or progressive systemic sclerosis.

[0021] The term "prevention" as used herein means any action of suppressing or delaying the onset of an immune disease by administering the pharmaceutical composition provided by the present invention.

[0022] The term "treatment" as used herein means any action that improves or beneficially alters the symptoms of an individual suspected of or confirmed to have an immune disease by administering the pharmaceutical composition.

[0023] The pharmaceutical composition provided by the present invention comprises a first composition containing a calcineurin inhibitor or a pharmaceutically acceptable salt thereof and a second composition containing stem cells or a culture product thereof, respectively. However, when calcineurin and stem cells are treated simultaneously, the immunomodulatory activity exhibited by these active ingredients is reduced due to mutual interference. Therefore, the pharmaceutical composition provided by the present invention is configured to contain these active ingredients separately in two different compositions, and the terms "first composition" and "second composition" do not imply any order of preparation or administration.

[0024] The calcineurin inhibitor or a pharmaceutically acceptable salt thereof contained in the first composition may be contained in an amount of 0.0001 to 50 wt % based on the weight of the total composition, specifically, 0.01 to 10 wt %, but is not limited thereto.

[0025] In the case of the stem cells contained in the second composition, 1 x 10 5 ~9x10 9 cells / ml, specifically, 5x10 5 ~5x10 9 cells / ml, more specifically 1x10 6 ~1x10 9 It may be contained in amounts of 0.001 to 80, specifically 0.001 to 70, and more specifically 0.001 to 60 wt % in the case of stem cell culture products, but is not limited thereto.

[0026] The first and second compositions may further contain a pharmaceutically acceptable carrier, excipient, or diluent commonly used in the manufacture of pharmaceutical compositions, including non-naturally occurring carriers, such as cryopreservatives, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0027] In addition, the pharmaceutical compositions can be formulated in the form of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, oils, freeze-dried preparations, transdermal absorption agents, gels, lotions, ointments, creams, patches, cataplasms, pastes, sprays, skin emulsions, skin suspensions, transdermal delivery patches, drug-containing bandages or suppositories by conventional methods.

[0028] Specifically, when formulated, the formulation may be prepared using commonly used diluents or excipients such as fillers, weighting agents, binders, wetting agents, disintegrants, and surfactants. Solid formulations for oral administration include, but are not limited to, tablets, pills, powders, granules, and capsules. These solid formulations may be prepared by mixing at least one or more excipients, such as starch, calcium carbonate, sucrose, lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. In addition to oral liquids and liquid paraffin, various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, may also be added to the formulation. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, twin 61, cocoa butter, laurin butter, and glycerogelatin.

[0029] According to one embodiment of the present invention, administration of mesenchymal stem cells to an animal model of atopic dermatitis showed an effect of improving atopic skin lesions. However, it was confirmed that the therapeutic efficacy was lower when pimecrolimus, a calcineurin inhibitor, was administered together with mesenchymal stem cells than when stem cells were administered alone, and the reason for this was analyzed.

[0030] To this end, we have conducted research into the effects of pimecrolimus on stem cells, and as a result have found that pimecrolimus has no effect on various aspects of stem cells, such as viability, proliferation ability, cell morphology, cell cycle, and surface marker expression levels, but it has been confirmed that it has the effect of reducing the immunosuppressive activity of stem cells by suppressing the expression of prostaglandin E2 (PGE2) through the suppression of the expression of COX2 expressed by stem cells.

[0031] As a result of conducting extensive research to develop a method for increasing immunosuppressive activity using both pimecrolimus and mesenchymal stem cells, it was found that the combined treatment of pimecrolimus and stem cells results in a large deviation in immunosuppressive activity. That is, it was confirmed that pimecrolimus can interfere with the immunosuppressive activity of stem cells, and that stem cells also affect the immunosuppressive activity of pimecrolimus. It was also confirmed that sequential treatment of stem cells and pimecrolimus results in a synergistic immunosuppressive activity.

[0032] Specifically, it was confirmed that immunosuppressive activity can be significantly increased by first treating with pimecrolimus and then treating with stem cells after a certain period of time, or by first treating with stem cells and then treating with pimecrolimus after a certain period of time, and that the most effective immunosuppressive activity can be obtained by first treating with pimecrolimus and then treating with stem cells after a certain period of time.

[0033] Another aspect of the present invention provides a method for preventing or treating an immune disease, comprising administering the pharmaceutical composition to an individual who has or may have an immune disease.

[0034] At this time, the definitions of the calcineurin inhibitor, stem cells, immune disease, prevention and treatment are as explained above.

[0035] As used herein, the term "administration" refers to the act of introducing a given substance into an individual in an appropriate manner.

[0036] The term "individual" as used herein refers to all animals, including humans, such as rats, domestic animals, and the like, that have or may develop the immune disease. Specific examples include mammals, including humans.

[0037] The method for preventing or treating an immune disease provided by the present invention may specifically include the step of administering pharmaceutically effective amounts of the first composition and the second composition to an individual suffering from an immune disease.

[0038] The method of administering the first composition and the second composition provided by the present invention is not particularly limited thereto, but may be, for example, a method in which the first composition is administered first once or multiple times, and then the second composition is administered once or multiple times; or, as another example, a method in which the second composition is administered, and then the first composition or the second composition is administered separately once or multiple times.

[0039] As another example, the second composition may be administered once or multiple times, followed by administering the first composition once or multiple times. As another example, the first composition may be administered, followed by administering the first composition or the second composition individually once or multiple times.

[0040] In addition, the administration of the first composition and the second composition can be carried out once or repeatedly. If the administration is repeated, the number of times the first composition is administered and the number of times the second composition is administered can be the same or different.

[0041] As an example, the compositions may be administered in the order of the first composition, the second composition, and the first composition, or in the order of the second composition, the first composition, and the second composition. As another example, the compositions may be administered in the order of the first composition, the second composition, the first composition, and the second composition, or in the order of the second composition, the first composition, the second composition, and the first composition. As another example, the compositions may be administered in the order of the first composition, the second composition, the first composition, the second composition, and the first composition, or in the order of the second composition, the first composition, the second composition, the first composition, and the second composition. As another example, the compositions may be administered in the order of the first composition, the second composition, the first composition, the second composition, the first composition, and the second composition, or in the order of the second composition, the first composition, the second composition, the first composition, the second composition, and the first composition. As another example, the first composition can be administered one or more times first, followed by one or more administrations of the second composition, and then one or more administrations of the first composition, or vice versa.

[0042] As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment and which does not cause side effects. The effective dose level can be easily determined by one skilled in the art depending on factors including the patient's sex, age, weight, health condition, type and severity of the disease, drug activity, sensitivity to the drug, administration method, administration time, administration route, and excretion rate, treatment period, concomitant or concurrently used drugs, and other factors well known in the medical field.

[0043] Specifically, the composition of the present invention can be administered at a daily dose of 0.0001 to 100 mg / kg body weight, more specifically 0.001 to 100 mg / kg body weight, based on the solid content. The recommended dose can be administered once a day or in divided doses.

[0044] In the method for preventing or treating an immune disease of the present invention, the route and mode of administration of the composition are not particularly limited and may be any route and mode of administration as long as the composition containing the composition can reach the desired site. Specifically, the composition may be administered via various routes, such as oral or parenteral, and non-limiting examples of the administration route include subcutaneous, oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, or inhalation.

[0045] Another aspect of the present invention provides a use of a calcineurin inhibitor and stem cells for the preparation of a pharmaceutical composition for the prevention or treatment of immune diseases. DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention will be described in more detail with reference to the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0047] Example 1: Effects of pimecrolimus and mesenchymal stem cells on atopic dermatitis animal models First, an animal model of atopic dermatitis was created.

[0048] Specifically, we created an animal model of atopic dermatitis by removing the hair from under the ear to above the tail of NC / Nga mice (male, 7 weeks old). We then applied 100 mg / head of Df (Dermatophagoides farinae; Biostir-AD, Biostir, Kobe, Japan) with a cotton swab to the shaved back and behind the ears twice a week for 3 weeks (6 times in total) to induce skin lesions.

[0049] Next, pimecrolimus, a known calcineurin inhibitor, and human UCB-MSCs were administered individually or in combination to the animal model, and changes in skin lesions were analyzed for 7 days.

[0050] Specifically, Df was applied as the final treatment, and after 3 days, pimecrolimus or stem cells were applied to the Df-treated skin lesions. Pimecrolimus was applied at a dose of 0.2 g / head once a day for 7 days, and stem cells were applied at a dose of 2 × 10 6 Stem cells were subcutaneously injected at a certain number of cells. Mice without atopic dermatitis were used as the negative control group (NC), and mice without pimecrolimus or stem cell treatment after atopic dermatitis induction were used as the positive control group (PC).

[0051] The changes in the skin lesions were analyzed by measuring and comparing the severity of the skin lesions. Four symptoms, erythema, scar / dryness, edema, and erosion, were scored on a scale of 0 to 3 (0: none, 1: mild, 2: moderate, 3: severe) (Figure 1).

[0052] Figure 1 is a graph showing the results of comparing the severity of skin lesions in animal models of atopic dermatitis treated with pimecrolimus or mesenchymal stem cells. MSC indicates the group administered with stem cells alone, Pime indicates the group administered with pimecrolimus alone, and MSC+Pime indicates the group administered with a combination of stem cells and pimecrolimus.

[0053] As shown in Figure 1, administration of stem cells alone was effective in alleviating atopic dermatitis lesions, but administration of stem cells in combination with pimecrolimus reduced the effect of alleviating atopic dermatitis lesions.

[0054] From the above results, it was confirmed that pimecrolimus appears to inhibit the therapeutic effect of stem cells on atopic dermatitis. However, this result was interpreted as being contrary to the expectation of a synergistic effect between stem cells and calcineurin inhibitors, which are known to be therapeutic agents for atopic dermatitis.

[0055] Example 2: Analysis of the effects of pimecrolimus on stem cells themselves Assuming that the results obtained in Example 1 were due to the effect of pimecrolimus on stem cells, the effect of pimecrolimus on stem cells itself was analyzed.

[0056] First, stem cells (hUCB-MSCs) were cultured and treated with various concentrations of pimecrolimus for 3 days, and the changes in stem cell viability were compared using the MTT assay (Figure 2a).

[0057] FIG. 2a is a graph comparing the change in stem cell viability depending on the treatment concentration of pimecrolimus.

[0058] As can be seen in Figure 2a, we confirmed that pimecrolimus in the range of 0.1 to 1 μg / ml did not alter the viability of stem cells.

[0059] Next, we cultured stem cells (hUCB-MSCs) and treated them with various concentrations of pimecrolimus for 3 days, and then compared the changes in the proliferation ability of the stem cells (Figure 2b).

[0060] FIG. 2b is a graph comparing the change in proliferation ability of stem cells depending on the treatment concentration of pimecrolimus.

[0061] As can be seen in Figure 2b, it was confirmed that pimecrolimus in the range of 0.01 to 100 ng / ml did not alter the proliferation ability of stem cells.

[0062] Next, we cultured stem cells (hUCB-MSCs) and treated them with various concentrations of pimecrolimus for 3 days, and then compared the changes in the cell cycle profile of the stem cells using cell cycle analysis (Figure 2c).

[0063] FIG. 2c is a graph comparing changes in stem cell cycle profiles depending on the treatment concentration of pimecrolimus.

[0064] As can be seen in Figure 2c, we confirmed that pimecrolimus in the range of 0.01–100 ng / ml did not alter the cell cycle profile of stem cells.

[0065] Next, stem cells (hUCB-MSCs) were cultured and treated with various concentrations of pimecrolimus for 3 days, and the morphological changes of the stem cells were compared (FIG. 2d).

[0066] FIG. 2d is a graph comparing the morphological changes of stem cells depending on the treatment concentration of pimecrolimus.

[0067] As can be seen in Figure 2d, it was confirmed that pimecrolimus in the range of 0.01 to 100 ng / ml did not change the morphology of stem cells.

[0068] Finally, we treated stem cells (hUCB-MSCs) with 100 ng / ml pimecrolimus for 3 days while culturing them, and then compared the expression levels of stem cell surface markers (CD45, CD34, CD36, CD105, CD29, and CD73) using flow cytometry (Fig. 2e).

[0069] FIG. 2e is a graph comparing changes in the expression levels of stem cell surface markers (CD45, CD34, CD36, CD105, CD29, CD73) following pimecrolimus treatment.

[0070] As shown in Figure 2e, it was confirmed that the expression levels of stem cell surface markers were not altered by treatment with 100 ng / ml pimecrolimus.

[0071] Taking the above results together, it was found that pimecrolimus had no effect on the stem cells themselves.

[0072] Example 3: Analysis of the effect of pimecrolimus on the immunoregulatory capacity of stem cells From the results of Example 2, it was confirmed that pimecrolimus had no effect on stem cells themselves, so whether pimecrolimus interferes with the immune regulatory ability of stem cells was analyzed.

[0073] Example 3-1: MLR analysis PBMC cells treated with concanavalin A (ConA) were cultured and then treated with pimecrolimus and mesenchymal stem cells (hUCB-MSCs), either individually or in combination, and the immunosuppressive activity measured in each experimental group was compared (Figure 3). The negative control group (NC) consisted of PBMC cells that were not treated with ConA, not co-cultured with stem cells, and not treated with pimecrolimus, while the positive control group (PC) consisted of PBMC cells that were treated with ConA, not co-cultured with stem cells, and not treated with pimecrolimus.

[0074] FIG. 3 is a graph showing the results of analyzing the effect of pimecrolimus on the immunosuppressive activity of stem cells against ConA-treated PBMC cells.

[0075] As shown in Figure 3, immune activity increased in the positive control group treated with ConA, but it was confirmed that immune activity was suppressed by treatment with stem cells or pimecrolimus. However, it was confirmed that the immunosuppressive activity observed when stem cells were treated alone was reduced by treatment with pimecrolimus.

[0076] Example 3-2: Analysis of expression levels of immune activation regulators Since the results of Example 3-1 confirmed that the immunosuppressive activity of stem cells was inhibited by pimecrolimus, we investigated whether the expression levels of TGF-β or prostaglandin E2 (PGE2), which are immunomodulatory factors expressed by stem cells, were affected by pimecrolimus.

[0077] Specifically, hUCB-MSCs were plated in a 6-well cell culture plate at 1.5 × 10 cells per well. 5After 24 hours of incubation, the cells were treated with various concentrations of pimecrolimus and further cultured for 72 hours. After incubation, the cells were centrifuged to obtain the supernatant, which was then applied to the ELISA kits Human TGF-β1 Quantikine ELISA (R&D Systems, Minneapolis, MN, USA) and Human PGE2 Quantikine ELISA (R&D Systems) to analyze the expression levels of TGF-β and prostaglandin E2 (PGE2) (Figures 4a and 4b).

[0078] FIG. 4a is a graph showing the results of comparing the levels of TGF-β expressed from stem cells according to the treatment concentration of pimecrolimus.

[0079] As shown in FIG. 4a, the level of TGF-β expressed from stem cells treated with pimecrolimus in the range of 0.01 to 100 ng / ml did not show any significant change.

[0080] FIG. 4b is a graph showing the results of comparing the levels of PGE2 expressed from stem cells depending on the treatment concentration of pimecrolimus.

[0081] As shown in FIG. 4b, the level of PGE2 expressed from stem cells treated with pimecrolimus in the range of 0.01 to 100 ng / ml was confirmed to decrease rapidly in proportion to the treatment concentration of pimecrolimus.

[0082] Example 3-3: Analysis of COX2 expression levels The results of Example 3-2 confirmed that treatment of stem cells with pimecrolimus inhibits the expression of prostaglandin E2 (PGE2), a regulator of immune activity. To verify this, the expression level of COX2 was analyzed. Since PGE2 is known to be synthesized from arachidonic acid released from membrane phospholipids by cyclooxygenase (COX), we aimed to determine whether the previously confirmed inhibition of PGE2 expression was due to the inhibition of COX expression.

[0083] Pimecrolimus-treated stem cells were obtained using the method described in Example 3-2, centrifuged to collect the precipitated cells, and then disrupted to obtain cell lysates. Western blot analysis of the cell lysates using COX2 antibody was performed (Figure 4c). GAPDH was used as an internal control.

[0084] FIG. 4c is a photograph of a Western blot showing the results of comparing the levels of COX2 expressed from stem cells depending on the treatment concentration of pimecrolimus, and a graph showing the results of analyzing the density thereof.

[0085] As shown in FIG. 4c, the level of PGE2 expressed from stem cells treated with pimecrolimus in the range of 0.01 to 100 ng / ml was confirmed to decrease rapidly in proportion to the treatment concentration of pimecrolimus.

[0086] Taking the results of Examples 3-1 to 3-3 together, it was found that pimecrolimus acts on stem cells to suppress the expression of COX2, thereby suppressing the expression level of PGE2 in stem cells, and that this suppression of the expression level of PGE2 reduces the immunosuppressive activity of stem cells.

[0087] Example 4: Correlation between stem cells and pimecrolimus on immunosuppressive activity Since it was confirmed through Example 3 that pimecrolimus has the effect of reducing the immunosuppressive activity of stem cells, various conditions were set and the results were analyzed by MLR analysis to analyze such effect in more detail.

[0088] Specifically, concanavalin A (ConA)-treated PBMC cells were cultured for 7 days and treated with pimecrolimus and mesenchymal stem cells (hUCB-MSCs), either individually or in combination, and the immunosuppressive activity measured in each experimental group was compared. At that time, 10 experimental groups were established based on the combination of pimecrolimus and mesenchymal stem cells (Table 1).

[0089] [Table 1]

[0090] The experimental results of each experimental group were classified and analyzed according to the purpose of analysis (FIGS. 5a and 5b).

[0091] First, we analyzed the results of comparing stem cells and pimecrolimus under similar treatment conditions (Fig. 5a).

[0092] FIG. 5a is a graph comparing the immunosuppressive activity obtained by treating stem cells with pimecrolimus under similar conditions.

[0093] As can be seen in FIG. 5a, when stem cells and pimecrolimus were treated simultaneously, no immunosuppressive activity was observed regardless of the treatment time (experimental groups 1 and 6).

[0094] Furthermore, when stem cells and pimecrolimus were treated separately, although there were some differences depending on the treatment time, the immunosuppressive activity of pimecrolimus was generally superior to that of stem cells (experimental groups 2 and 3; experimental groups 5 and 9).

[0095] Next, we analyzed the results of treating cells with pimecrolimus first and then with stem cells (Fig. 5b). FIG. 5b is a graph comparing the immunosuppressive activity obtained by pimecrolimus first treatment and stem cell second treatment.

[0096] As shown in FIG. 5b, pimecrolimus treatment followed by sequential treatment with stem cells showed the highest immunosuppressive activity among the 10 experimental groups (experimental group 4).

[0097] These results show a comparative advantage when compared with the results of treating pimecrolimus and stem cells individually under similar conditions (experimental groups 2 and 5), and it was analyzed that pimecrolimus and stem cells have a synergistic effect.

[0098] We also analyzed the results of treating stem cells first and then pimecrolimus (Fig. 5c). FIG. 5c is a graph comparing the immunosuppressive activity obtained by first treating with stem cells and then treating with pimecrolimus.

[0099] As shown in Figure 5c, treatment with stem cells followed by pimecrolimus showed comparatively superior immunosuppressive activity compared to treatment with stem cells alone (experimental groups 3 and 7).

[0100] Furthermore, the sequential treatment of stem cells and pimecrolimus, rather than the simultaneous treatment, showed comparatively superior immunosuppressive activity compared to the treatment of stem cells alone (experimental groups 8 and 10).

[0101] Finally, stem cells and pimecrolimus were treated together, and the changes depending on the treatment order were analyzed (FIG. 5d).

[0102] FIG. 5d is a graph showing the results of analyzing changes in immunosuppressive activity depending on the treatment order when stem cells and pimecrolimus were treated together.

[0103] As shown in Figure 5d, simultaneous treatment with stem cells and pimecrolimus did not show any immunosuppressive activity (experimental group 1), whereas sequential treatment with stem cells and pimecrolimus in various combinations of treatment order showed excellent immunosuppressive activity (experimental groups 4 and 7).

[0104] In particular, when pimecrolimus was administered first and then the stem cells were administered later (experimental group 4), it showed relatively superior immunosuppressive activity compared to when stem cells were administered first and then the pimecrolimus was administered later (experimental group 7).

[0105] Taking the above analysis results together, it was confirmed that pimecrolimus and stem cells, which each exhibit immunosuppressive activity, can result in significant deviations in immunosuppressive activity depending on the treatment method used in combination. That is, the results of Example 3 confirmed that pimecrolimus unilaterally interferes with the immunosuppressive activity of stem cells, but in fact, it was confirmed that stem cells can also affect the immunosuppressive activity of pimecrolimus.

[0106] As a result, it was found that the use of stem cells and pimecrolimus together can produce a synergistic effect, but for this to occur, a sequential treatment method, rather than simultaneous treatment, is required. In particular, when using this sequential treatment method, it was further confirmed that the most effective immunosuppressive activity was achieved when pimecrolimus was treated first, followed by the treatment of stem cells.

[0107] From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present invention should be interpreted as including within the meaning and scope of the claims below, and all modifications and variations derived from the equivalent concepts thereof, rather than the above detailed description.

Claims

1. (a) a first composition comprising a calcineurin inhibitor or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutical composition for preventing or treating an immune disease, comprising a second composition containing mesenchymal stem cells, the calcineurin inhibitor is cyclosporin A, tacrolimus, or pimecrolimus; The immune disease is an immune disease caused by hyperimmunity, the first composition is administered one or more times, followed by one or more administrations of the second composition, or the second composition is administered one or more times, followed by one or more administrations of the first composition; the first composition and the second composition are not administered simultaneously; The immunological disease caused by hyperimmunity is an allergic immune disease or an autoimmune disease.

2. The pharmaceutical composition of claim 1, wherein the calcineurin inhibitor is pimecrolimus.

3. 2. The pharmaceutical composition of claim 1, wherein administering the first composition one or more times followed by administering the second composition one or more times, or administering the second composition one or more times followed by administering the first composition one or more times, significantly increases the immunosuppressive activity compared to administering the first composition and the second composition simultaneously.

4. 2. The pharmaceutical composition according to claim 1, wherein the mesenchymal stem cells are selected from the group consisting of adipose-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, myocardium-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, cartilage-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and combinations thereof.

5. (a) a first composition comprising a calcineurin inhibitor or a pharmaceutically acceptable salt thereof; and (b) a composition for immunosuppression, comprising a second composition containing mesenchymal stem cells, the calcineurin inhibitor is cyclosporin A, tacrolimus, or pimecrolimus; The immunosuppressive composition is characterized in that the first composition is administered one or more times, followed by one or more further administrations of the second composition, or the second composition is administered one or more times, followed by one or more further administrations of the first composition, the first composition and the second composition are not administered simultaneously; and The composition, wherein the immunosuppression is immunosuppression in an allergic immune disease or an autoimmune disease.

6. The pharmaceutical composition according to claim 1, wherein the allergic immune disease is atopic dermatitis.

7. 2. The pharmaceutical composition of claim 1, wherein the allergic immune disease is selected from the group consisting of hay fever, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, food allergy, mastocytosis, and combinations thereof.

8. 7. The pharmaceutical composition of claim 6, wherein the autoimmune disease is selected from the group consisting of Graves' disease, rheumatoid arthritis, Hashimoto's thyroiditis, systemic lupus erythematosus (lupus), vasculitis, Addison's disease, polymyositis, Sjogren's syndrome, progressive systemic sclerosis, and combinations thereof.

9. 2. The pharmaceutical composition of claim 1, wherein the first composition is administered one or more times prior to administration of the second composition one or more times.

10. The pharmaceutical composition according to claim 9, wherein the allergic immune disease is atopic dermatitis.

11. A method for sequentially administering pharmaceutical compositions for preventing or treating an immune disease, comprising: (a) a first composition comprising a calcineurin inhibitor or a pharmaceutically acceptable salt thereof; and (b) a second composition containing mesenchymal stem cells; to a hyperimmune non-human subject, the calcineurin inhibitor is cyclosporin A, tacrolimus, or pimecrolimus; The immune disease is an immune disease caused by hyperimmunity, the method comprising administering one or more doses of the first composition followed by one or more further doses of the second composition, or administering one or more doses of the second composition followed by one or more further doses of the first composition; the first composition and the second composition are not administered simultaneously; The method, wherein the immune disease caused by hyperimmunity is an allergic immune disease or an autoimmune disease.

12. The pharmaceutical composition of claim 1 , further comprising a pharmaceutically acceptable carrier, excipient, or diluent.

13. 1. Use of a pharmaceutical composition in the manufacture of a medicament for the prevention or treatment of an immune disease, said pharmaceutical composition comprising: (a) a first composition comprising a calcineurin inhibitor or a pharmaceutically acceptable salt thereof; and (b) a second composition comprising mesenchymal stem cells; the calcineurin inhibitor is cyclosporin A, tacrolimus, or pimecrolimus; The immune disease is an immune disease caused by hyperimmunity, the first composition is administered one or more times, followed by one or more administrations of the second composition, or the second composition is administered one or more times, followed by one or more administrations of the first composition; the first composition and the second composition are not administered simultaneously; The immune disease caused by hyperimmunity is an allergic immune disease or an autoimmune disease.

14. The use according to claim 13, wherein the immune disease caused by hyperimmunity is atopic dermatitis.

15. 14. The use of claim 13, wherein the first composition is administered one or more times, followed by one or more administrations of the second composition.

16. 1. A method of immunosuppression, comprising: (a) a first composition comprising a calcineurin inhibitor or a pharmaceutically acceptable salt thereof; and (b) a second composition containing mesenchymal stem cells; to a hyperimmune non-human subject, the calcineurin inhibitor is cyclosporin A, tacrolimus, or pimecrolimus; the method comprising administering one or more doses of the first composition followed by one or more further doses of the second composition, or administering one or more doses of the second composition followed by one or more further doses of the first composition; the first composition and the second composition are not administered simultaneously; The method, wherein the immune disease caused by hyperimmunity is an allergic immune disease or an autoimmune disease.

17. The pharmaceutical composition according to claim 1, wherein the second composition is administered two or more days after the administration of the first composition.

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