KR102335279B1 - Pharmaceutical composition for the prevention or treatment of arthritis comprising the expression or activity inhibitors of ion transporter SLC4A7 as an active ingredient - Google Patents

Abstract

It relates to a pharmaceutical composition for the prevention or treatment of arthritis containing an inhibitor of the expression or activity of the ion transporter SLC4A7 as an active ingredient. Rather, it has an excellent effect in preventing or treating joint deformation and preventing or treating diseases related to bone inflammation by suppressing the expression of bone destructive factors, cell mobility factors, and the like.

Description

Pharmaceutical composition for the prevention or treatment of arthritis comprising the expression or activity inhibitors of ion transporter SLC4A7 as an active ingredient

It relates to a pharmaceutical composition for preventing or treating arthritis, comprising an inhibitor of the expression or activity of the ion transporter SLC4A7 as an active ingredient.

Rheumatoid arthritis is a chronic inflammatory disease that mainly invades the joints, causing pain, swelling, and joint deformation, resulting in functional impairment. It is a disease that is very difficult to control and imposes a great social and economic burden.

Rheumatoid arthritis is the second most common chronic arthritis after osteoarthritis and is the most common cause of inflammatory arthritis. When the synovial membrane is inflamed due to rheumatoid arthritis, various inflammatory cells in the blood gather, and when chronic inflammation occurs along with infiltration of these inflammatory cells, pannus is formed and bone destructive factors are released and Destruction of cartilage and joint deformation are caused by abnormal cell migration and proliferation (Non-Patent Document 1. Arthritis Rheumatol. 2018 Jul; 70(7): 984-999.; Non-Patent Document 2. Immunol Rev. 2010 Jan; 233(1)233-55.;Non-Patent Document 3: Clin Immunol.2005 May;115(2):118-28.). In addition, in rheumatoid arthritis patients, invasion of large joints such as the knee, hip, and shoulder joints is one of the factors with poor prognosis, and permanent joint damage caused by these deformations causes loss of joint function, increasing the burden of social diseases and patients can have a significant impact on the quality of life of

Various drug treatment methods have been provided for the treatment of rheumatoid arthritis, and disease activity can be controlled by such drug treatment. However, intra-articular injection of steroids is often possible in the areas of the joints that are not controlled despite these treatments. These steroid injections are not only localized side effects such as skin discoloration, infection, edema and pain, but also are absorbed systemically, which can cause various systemic side effects, making repeated administration difficult. In addition, although various therapeutic agents for arthritis are being developed, most of these drugs are very expensive, and the reality is that there are many patients who do not respond sufficiently to treatment. Until recently, even for eight FDA-approved rheumatoid arthritis biologics, treatment response rates were still reported to be less than 50%.

Treatments for rheumatoid arthritis are drugs to control the inflammatory response, and include immunosuppressants, specific inflammatory cytokine blockers, steroids, and anti-inflammatory drugs. New drug development has been ongoing for the past 10 years, and from biological DMARDs such as TNF-α inhibitors and IL-6 inhibitors to new low-molecular compounds such as JAK inhibitor Xeljanz, competition in the arthritis treatment market is forming. . However, despite the development and commercialization of these new drugs, most patients need combined use with immunosuppressants, steroids, anti-inflammatory drugs, etc. to control disease activity, which inevitably increases the incidence of side effects.

To date, therapeutics targeting various inflammatory receptors of various immune cells such as T cells and B cells have been developed to control the progression and disease activity rate of rheumatoid arthritis. There are no studies targeting synovial cells, which is one of the pathogenesis. The synovial cell control technology proposed in the present invention proposes a technology different from the case of immune cells (T cells, B cells, etc.) targeted in the prior art and research.

Synovial cells contribute to joint destruction through various mechanisms and mediate inflammatory diseases such as rheumatoid arthritis by exchanging various signals between T cells/B cells (Non-Patent Document 4. Front Immunol. 2019 Jun 19; 10:1395) .; Non-Patent Document 5. Arthritis Res. 2000;2(5):374-8.; Non-Patent Document 6. Int Immunopharmacol. 2019 Aug;73:362-369.). It has been found that excessive proliferation of synovial cells, migration and infiltration into bones are involved in joint deformation and inhibition of joint function (Non-Patent Document 7. Mediat. Inflamm., 2013 (2013), Article 953462, Non-Patent Document 8. J Enzyme Inhib Med Chem. 2019 Dec;34(1):1615-1622.). Recently, attempts have been made to inhibit cell migration by regulating mitogen-activated protein kinase of synovial cells (Non-Patent Document 9. Eur Rev Med Pharmacol Sci. 2019 Apr;23(7);3105-3111), and the synovial membrane in rheumatoid arthritis A pharmacological approach targeting cells is emerging (Non-Patent Document 10. Int Rew Immunol. 2017 Jan 2;36(1):20-30).

So far, no research results have been reported on a complex technique for inhibiting migration factors of synovial cells, realizing direct targeting of ion transporters and direct inhibition of mobility, and inhibiting bone destructive factors. Accordingly, the present inventors intend to provide a method for innovatively reducing joint deformation through inhibition of synovial cell mobility and inflammatory response, and suppression of bone destructive factor secretion in order to solve the above problems.

An object of one aspect of the present invention is to provide a composition for preventing or treating arthritis comprising an inhibitor of the expression or activity of the ion transporter SLC4A7 as an active ingredient.

An object of another aspect of the present invention is the ion transporter SLC4A7 protein, or when the expression level of the gene, or the activity of the ion transporter is reduced, the sample is determined as a substance for the prevention or treatment of arthritis, An object of the present invention is to provide a method for screening a substance for preventing or treating arthritis.

In order to achieve the above object, one aspect of the present invention provides a pharmaceutical composition for preventing or treating arthritis containing an inhibitor of the expression or activity of the ion transporter SLC4A7 as an active ingredient.

Another aspect of the present invention comprises the steps of contacting a sample to be analyzed with cells containing the ion transporter SLC4A7, or its gene;

measuring the expression level of the ion transporter SLC4A7 or its gene after the contact, or the activity of the ion transporter SLC4A7; and

As a result of the measurement, when the expression level of the ion transporter SLC4A7 protein, or the gene thereof, or the activity of the ion transporter is reduced, the sample is determined as a substance for preventing or treating arthritis, for the prevention or treatment of arthritis A method for screening a substance is provided.

Inhibitors of the expression or activity of the ion transporter SLC4A7 provided in one aspect of the present invention can inhibit proliferation and migration of synovial cells, as well as osteoclasts, which are important substances for osteoclast differentiation, cell mobility that decomposes joint tissue Since it can suppress the expression of factors and inflammatory factors, it is possible to effectively suppress the fibrosis of synovial cells and the expression of osteoclasts mediated by the inflammatory response of joint deformation according to the progression of rheumatoid arthritis. Through this, by preventing joint deformation caused by the progression of arthritis, normal function of the joint and aesthetic effects can be expected.

1 is a view showing an image confirming the expression change of the ion transporter SLC4A7 in the control (DMEM), synovial fibroblasts of the TNF-α condition through cell migration analysis (migration assay) through a confocal microscope.
FIG. 2 is a diagram showing a graph analyzing the expression level of the ion transporter SLC4A7 in (DMEM), synovial fibroblasts under TNF-α condition.
3 is a view showing the results of confirming the change in the function of the ion channel in the synovial fibroblasts through the pH change in the control (DMEM), TNF-α, and joint fluid conditions of the patient.
FIG. 4 is a graph showing a graph analyzing the results of confirming changes in the function of ion channels in synovial fibroblasts through changes in pH in the control group (DMEM), TNF-α, and joint fluid conditions of patients.
5 is a view showing an image confirming the movement of synovial fibroblasts through cell migration analysis in TNF-α, TNF-α+anti-SLC4A7 conditions through a confocal microscope.
Figure 6 is a graph showing the analysis of the degree of synovial fiber cell migration in the TNF-α, TNF-α + anti-SLC4A7 condition.
7 is a view showing an image confirming the movement of synovial fibroblasts through cell migration analysis in the joint fluid conditions of control (DMEM), TNF-α, DMEM + patients through a confocal microscope.
8 is a graph showing the analysis of the degree of synovial fiber cell migration in the joint fluid condition of a control group (DMEM), TNF-α, and DMEM+ patients.
9 is a view showing an image confirming the movement of synovial fibroblasts through cell migration analysis under the conditions of control (DMEM), TNF-α, DMEM + patient's joint fluid, DMEM + patient's joint fluid + anti-SLC4A7 condition through a confocal microscope.
10 is a graph showing the analysis of the degree of synovial fiber cell migration in the control group (DMEM + patient's joint fluid) and DMEM + patient's joint fluid + anti-SLC4A7 condition.
11 is a control (DMEM), TNF-α, DMEM + joint fluid of patient 2, DMEM + joint fluid of patient 2 + anti-SLC4A7, DMEM + joint fluid of patient 3, DMEM + joint fluid of patient 3 + anti-SLC4A7 through a confocal microscope. It is a diagram showing the image confirming the movement of synovial fiber cells through cell migration analysis.
12 is a control (DMEM), TNF-α, DMEM + joint fluid of patient 2, DMEM + joint fluid of patient 2 + anti-SLC4A7, DMEM + joint fluid of patient 3, DMEM + joint fluid of patient 3 + anti-SLC4A7 synovial fiber cell migration degree It is a diagram showing the analyzed graph.
13 is a graph showing a comparative analysis by extracting only the experimental results under the conditions of MEM + joint fluid of patient 2, DMEM + joint fluid of patient 2 + anti-SLC4A7, DMEM + joint fluid of patient 3, and DMEM + joint fluid of patient 3 + anti-SLC4A7. .
14 is a control (DMEM), TNF-α, TNF-α + anti-SLC4A7, anti-SLC4A7 conditions in the bone destructive factor (RANKL), inflammatory factors (COX-1, COX-2) to confirm the expression level of electrical It is a diagram showing the migrated band.
Figure 15 is a control (DMEM), TNF-α, TNF-α + anti-SLC4A7, anti-SLC4A7 in the conditions of bone destruction factor (RANKL), inflammatory factor (COX-1, COX-2) analysis of the expression level of the graph. It is a drawing showing
16 is a view showing the sequence of sense and antisense nucleotide sequences of siRNA-SLC4A7 used in Example 3 of the present invention.
17 is a view showing the Western blot results confirming whether the SLC4A7 protein is reduced by treating the cells with siRNA-SLC4A7.
18 is a view showing an image confirming HFLS and RA-FLS migration through cell migration analysis in the control (DMEM) and siRNA-SLC4A7 conditions through a confocal microscope.
19 is a diagram illustrating a graph analyzing the degree of migration of human FLS (HFLS) and rheumatoid arthritis (RA-FLS) cells in the control group (DMEM) and siRNA-SLC4A7 conditions.
20 is a diagram showing a schematic diagram of the experimental results provided in one aspect of the present invention.

Hereinafter, the present invention will be described in detail.

On the other hand, the embodiment of the present invention can be modified in various other forms, the scope of the present invention is not limited to the embodiments described below. In addition, the embodiment of the present invention is provided in order to more completely explain the present invention to those of ordinary skill in the art. Furthermore, in the entire specification, "including" a certain element means that other elements may be further included, rather than excluding other elements, unless otherwise stated.

As described above, although various therapeutic agents for arthritis are being developed, most of the drugs currently on the market are all expensive and there are many problems in patients who do not show sufficient response to treatment. Although efforts are still being made to develop and commercialize new drugs, patients need co-administration of immunosuppressants, steroids, anti-inflammatory drugs, etc. to control their activity, and side effects are inevitable. In contrast, one aspect of the present invention is to provide a technique for controlling synovial cells targeting synovial cells.

Synovial cells are one of the main causes of joint deformity, and proliferation and migration of synovial cells is the main cause of joint deformity. It has been reported that when the joint synovial membrane becomes inflamed due to arthritis, inflammatory cells in the blood gather, and chronic inflammation causes the destruction of cartilage and deformation of the joint due to the formation of pannus and abnormal cell migration. The synovial cells that cause such joint deformation are affected by various inflammatory factors in the joint fluid and secrete inflammatory substances secondarily. In addition, the stimulated synovial cells secrete osteoclasts or move to the site of inflammation. If the secondary inflammatory response and osteoclasts are suppressed without moving the synovial cells to the site of inflammation, joint deformation will not occur. . In contrast, the present inventors tried to find a method to control synovial cells, and as a result, when an inflammatory substance acts on the synovial cells, the ion transporter involved in causing the cell movement works, focusing on the synovial cells. As a result of examining the types of ion transporters that mainly respond to stimulated inflammation, it was confirmed that SLC4A7 was mainly expressed. In addition, when TNF, which is an inflammatory factor, was directly stimulated in cells, it was confirmed that cell mobility was increased, and it was confirmed that SLC4A7, an ion transporter, was placed forward to control cell movement.

In addition, in order to confirm the mechanism for this phenomenon, as a result of treating synovial cells with an antibody complementary to SLC4A7 protein or siRNA complementary to mRNA of SLC4A7 gene, it was confirmed that cell mobility was significantly reduced. In addition, it was confirmed that the mobility of the cells was increased by directly stimulating the joint fluid of the rheumatoid patient to the synovial cells. In addition, treatment with the antibody of the ion transporter SLC4A7 significantly reduced the expression of bone destructive factor (RANKL) and inflammatory factor (COX).

One aspect of the present invention provides a pharmaceutical composition for preventing or treating arthritis, comprising an inhibitor of the expression or activity of the ion transporter SLC4A7 as an active ingredient.

The ion transporter SLC4A7 expression inhibitor may be an antisense nucleotide complementary to the mRNA of the SLC4A7 gene, short hairpin RNA (shRNA), small interfering RNA (siRNA) or ribozyme. have. The sense sequence of the siRNA is shown in SEQ ID NO: 1, and the antisense sequence of the siRNA is shown in SEQ ID NO: 2.

The antisense nucleotide binds (hybridizes) to the complementary base sequence of DNA, immature-mRNA, or mature mRNA as defined in Watson-Click base pairing to disrupt the flow of genetic information from DNA to protein. The nature of antisense nucleotides specific to a target sequence makes them exceptionally versatile. Since antisense nucleotides are long chains of monomeric units, they can be readily synthesized for the target RNA sequence. Many recent studies have demonstrated the usefulness of antisense nucleotides as biochemical means for studying target proteins (Rothenberg et al., J. Natl. Cancer Inst., 81:1539-1544, 1999). Since there have been many recent advances in oligonucleotide chemistry and nucleotide synthesis exhibiting improved cell adsorption, target binding affinity and nuclease resistance, the use of antisense nucleotides can be considered as a new type of inhibitor.

The peptide mimetics inhibits the activity of the ion transporter SLC4A7 protein, which inhibits the binding domain of the ion transporter SLC4A7 protein. Peptide mimetics may be peptides or non-peptides and are amino acids joined by non-peptide bonds, such as psi bonds (Benkirane, N., et al. J. Biol. Chem., 271:33218-33224, 1996). can be configured. In addition, "conformationally constrained" peptides, cyclic mimetics, cyclic mimetics comprising at least one exocyclic domain, a binding moiety (binding amino acid) and an active site It may be metric. Peptide mimetics is structured similarly to the secondary structural properties of the ion transporter SLC4A7 protein and is either an antibody (Park, BW et al. Nat Biotechnol 18, 194-198, 2000) or a soluble receptor (Takasaki, W. et al. Nat Biotechnol 15, 1266-1270, 1997) can mimic the inhibitory properties of large molecules, and it can be a novel small molecule that can act with an effect equivalent to that of a natural antagonist (Wrighton, NC et al. Nat Biotechnol 15, 1261-1265, 1997).

The aptamer is a single-stranded DNA or RNA molecule, and has high affinity and high affinity for specific chemical or biological molecules by an evolutionary method using an oligonucleotide library called SELEX (systematic evolution of ligands by exponential enrichment). It can be obtained by isolating an oligomer that has selectivity and binds (C. Tuerand L. Gold, Science 249, 505 - 510, 2005; AD Ellington and JW Szostak, Nature 346, 818 - 822, 1990; M. Famulok, et al. al., Acc. Chem. Res. 33, 591 - 599, 2000; DS Wilson and Szostak, Annu. Rev. Biochem. 68, 611 - 647, 1999). Aptamers can specifically bind to a target and modulate the activity of the target, eg, block the ability of the target to function through binding.

The antibody can specifically and directly bind to the ion transporter SLC4A7 to effectively inhibit the activity of the ion transporter SLC4A7. The antibody specifically binding to the ion transporter SLC4A7 is preferably a polyclonal antibody or a monoclonal antibody. The antibody specifically binding to the ion transporter SLC4A7 may be prepared by a method known to those skilled in the art, or a commercially known ion transporter SLC4A7 antibody may be purchased and used. The antibody can be prepared by injecting the immune-causing ion transporter SLC4A7 protein into an exogenous host according to a conventional method known to those skilled in the art. External hosts include mammals such as mice, rats, sheep, and rabbits. The immunogen is injected by an intramuscular, intraperitoneal or subcutaneous injection method, and can generally be administered together with an adjuvant to increase antigenicity. Antibodies can be isolated by routinely collecting blood from an external host and collecting sera that show an established titer and specificity for an antigen.

The inhibitor of the activity of the ion transporter SLC4A7 may be a peptide, peptide mimetics, matrix analogue, aptamer or antibody that complementarily binds to the SLC4A7 protein.

The pharmaceutical composition may inhibit proliferation of synovial cells, or movement of synovial cells to bone.

The pharmaceutical composition may be one that inhibits the expression of RANKL (Receptor Activator of Nuclear factor Kappa-B ligand), or COX (cyclooxygenase, cyclooxygenase), and the COX (cyclooxygenase, cyclooxygenase) is COX- 1 or COX-2.

The type of arthritis is not limited, but may be, for example, psoriatic arthritis, degenerative arthritis, osteoarthritis, or rheumatoid arthritis.

The pharmaceutical composition for the prevention or treatment of arthritis containing the inhibitor of the expression or activity of the ion transporter SLC4A7 of the present invention as an active ingredient preferably contains 0.0001 to 50% by weight of the active ingredient based on the total weight of the composition, but limited thereto doesn't happen

The pharmaceutical composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration. The pharmaceutically acceptable carrier may be used in a mixture of saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and one or more of these components, and, if necessary, an antioxidant , buffers, bacteriostatic agents, and other conventional additives may be added. In addition, by additionally adding diluents, dispersants, surfactants, binders and lubricants, it can be formulated into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets, and can act specifically on target organs. A target organ-specific antibody or other ligand may be used in combination with the carrier. Furthermore, it can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA). have.

Preferably, the nucleic acid or vector is used in injectable form. Accordingly, it may be admixed with any pharmaceutically acceptable vehicle for injectable compositions, particularly for direct injection into the area to be treated. The pharmaceutical compositions of the present invention may include, inter alia, lyophilized compositions which allow the composition of an injectable solution upon addition of an isotonic sterile solution or dry particularly sterile water or suitable physiological saline. Direct injection of nucleic acids into a patient's tumor is advantageous as it allows the therapeutic efficiency to be focused on the infected tissue. The dosage of the nucleic acid used can be controlled by various parameters, in particular the gene, the vector, the mode of administration used, the disease in question or, alternatively, the length of treatment required. In addition, the range varies depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and the severity of the disease. The daily dose is about 0.0001 to 100 mg/kg, preferably 0.001 to 10 mg/kg, and it is preferable to administer it once or several times a day in divided doses.

Another aspect of the present invention comprises the steps of contacting a sample to be analyzed with cells containing the ion transporter SLC4A7, or its gene;

measuring the expression level of the ion transporter SLC4A7 or its gene after the contact, or the activity of the ion transporter SLC4A7; and

As a result of the measurement, when the expression level of the ion transporter SLC4A7 protein, or the gene thereof, or the activity of the ion transporter is reduced, the sample is determined as a substance for preventing or treating arthritis, for the prevention or treatment of arthritis A method for screening a substance is provided.

At this time, the expression level is immunoprecipitation (immunoprecipitation), radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemistry, RT-PCR, Western blot (Western Blotting) and flow cytometry (FACS) group consisting of It can also be measured with any one selected from

Hereinafter, Examples and Experimental Examples of the present invention will be specifically illustrated and described below. However, the Examples and Experimental Examples to be described below are only illustrative of a part of the present invention, and are not limited thereto.

<Example 1> Confirmation of the effect of inhibiting the migration of synovial fibroblasts by the ion transporter SLC4A7 antibody

1-1. Confirmation of SLC4A7 expression in synovial fibroblasts (cell migration assay, migration assay)

Fibroblast-like synoviocytes (FLS) are activated by inflammatory factors because they are cells with the characteristic of migrating to inflamed areas. Expression of the ion transporter SLC4A7 was confirmed in the migrated synovial fibroblasts through a cell migration assay capable of confirming the movement of synovial cells.

Cell migration analysis is performed by preparing DMEM (Dulbecco's modified eagle medium) medium, which is a control condition, on the lower plate using a transwell, and treating the inflammatory factor TNF-α with 10 ng/ml as an experimental group. It was tested under conditions similar to inflammation. The synovial fibroblasts of rheumatoid arthritis were treated with a number of 5x10 ^{4 in} the gastric chamber, and then cultured at 37° C. for 6 hours, and then the synovial fibroblasts migrated in the transwell direction in the gastric chamber were fluorescently stained. Fluorescence staining was performed after fixing the synovial fibroblasts with methanol, then treated with the SLC4A7 antibody, and after 1 hour of treatment with a secondary antibody, rhodamine, the change in expression was confirmed at a wavelength of 510 nm through a confocal microscope. . The results are shown in FIGS. 1 and 2 .

As can be seen in FIGS. 1 and 2 , as the synovial fibroblasts migrated to the transwell treated with the inflammatory factor, the expression of the ion transporter SLC4A7 increased approximately twofold. Through this, it was confirmed that the expression of the ion transporter SLC4A7 was increased by inflammation in synovial fibroblasts.

1-2. Confirmation of ion channel (NBC) activity in synovial fibroblasts under inflammatory conditions

In order to confirm the change in the function of the ion channel in the synovial fibroblast cells in the inflammatory factor TNF-α and the joint fluid condition of the patient, the pH change in the cell (FIG. 3) and the activity of the ion channel NBC (Sodium/Bicarbonate Cotransporter) (FIG. 4) were investigated. measured.

Using BCECF-AM, which can measure changes in intracellular pH, EIPA (5-(N-Ethyl-N-isopropyl)Amiloride), which inhibits NHE (Sodium/Hydrogen Exchanger), _{is mixed with HCO 3} ^- solution to ion channel It was allowed to measure only the activity of NBC. As can be seen from FIG. 3 , when the HCO ₃ ^{- solution without Na +} is given, the pH of the cells is decreased. If an HCO ₃ ^- solution ^{containing Na + is given} to the outside after a few minutes, ^{Na +} and HCO ₃ ^- enter the cell together. At this time, the pH in the cell is increased by _{HCO 3} ^{- (basic component) entering the cell.} It was measured how much the function of NBCe1 changed through the change (gradient) with time of this increased pH. As a result, it was found that the activity of the ion channel NBC was significantly increased in the TNF-α condition and the patient's joint fluid condition (SF, patinet) than in the DMEM condition as a control. Specifically, relative to the control condition, the amount of change in pH per second increased by about 1.75 times in the TNF-α condition, and the amount of change in pH per second increased by about 2.5 times in the joint fluid condition of the patient. Through this, it was confirmed that the activity of ion channels in synovial fibroblasts was increased under inflammatory conditions.

1-3. Confirmation of the effect of inhibiting the migration of synovial fibroblasts by the SLC4A7 antibody in the inflammatory condition caused by the TNF-α factor

In Example 1-1, it was confirmed that the migration of synovial fibroblasts increased and the expression of the ion transporter SLC4A7 increased under the inflammatory factor TNF-α condition. In Example 1-3, the migration effect of synovial fibroblasts by the SLC4A7 antibody under the conditions of the inflammatory factor TNF-α was confirmed through cell migration analysis.

The lower plate of the transwell was treated with 10 ng/ml of TNF-α, a condition similar to inflammation of rheumatoid arthritis, and the upper chamber was treated with 5x10 ⁴ synovial fibroblasts, and as an experimental group, ionized water at a concentration of 1 mg/ml It was co-treated with the SLC4A7 antibody and incubated for 6 hours. After that, only the filter of the Transwell was obtained and the synovial fibroblasts present in the filter were fixed with methanol using methanol, treated with a dye capable of staining the nucleus of the cell, and incubated for 30 minutes. After washing the filter with a physiological buffer solution (PBS), the nucleus of the cells was observed at a wavelength of 405 nm through a confocal microscope. The results are shown in FIGS. 5 and 6 .

As can be seen in FIGS. 5 and 6 , when synovial fibroblasts migrating by stimulation of the inflammatory factor TNF-α were treated with the SLC4A7 antibody (anti-SLC5A7), the movement was inhibited at a level of about 60%. . Through this, it was confirmed that the migration of synovial fibroblasts due to inflammation can be effectively inhibited using the SLC4A7 antibody.

1-4. Confirmation of the effect of inhibiting the migration of synovial fibroblast cells by the SLC4A7 antibody in the joint fluid condition of arthritis patients

In Example 1-4, it was confirmed that the same effect as stimulation of TNF-α was shown using the joint fluid of an actual rheumatoid arthritis patient, and the movement of synovial fibroblasts by the joint fluid of a rheumatoid arthritis patient could be inhibited using the ion transporter SLC4A7 antibody. check that it can be done.

First, in order to check whether the joint fluid of an actual rheumatoid arthritis patient has the same effect as the stimulation of TNF-α, that is, increases the migration of synovial fibroblasts, prepare DMEM medium as a control in the lower plate of the transwell, and in the lower plate as an experimental group. A medium inducing cell migration was prepared by mixing the TNF-α 10 ng/ml-treated medium and the joint fluid of a rheumatoid arthritis patient 1:1 with the DMEM medium. Thereafter, cell nuclei were observed using cell migration analysis in the same manner as in Examples 1-3. The results are shown in FIGS. 7 and 8 .

As can be seen in FIGS. 7 and 8 , it can be seen that the experimental group treated with the joint fluid of rheumatoid arthritis patients causes migration of synovial fibroblasts similar to the experimental group treated with TNF-α.

Next, in order to check whether the migration of synovial fibroblasts by the joint fluid of rheumatoid arthritis patients can be inhibited using the ion transporter SLC4A7 antibody, 5x10 ⁴ synovial fibroblasts are treated in the upper chamber, and the lower plate of the transwell DMEM, TNF-α 10 ng/ml, and joint fluid of a 67-year-old rheumatoid arthritis patient were treated respectively, and the experimental group was treated with the ion transporter SLC4A7 antibody 1 mg/ml in the upper chamber of the medium treated with the patient's joint fluid on the lower plate. did. Thereafter, cell nuclei were observed using cell migration analysis in the same manner as in Examples 1-3. The results are shown in FIGS. 9 and 10 .

As can be seen in FIGS. 9 and 10 , it was confirmed that the migration of synovial fibroblasts was significantly reduced by about 50% in the group treated with the ion transporter SLC4A7 antibody compared to the control group treated with the joint fluid of the patient.

Next, the movement of synovial fibroblasts was confirmed using cell migration analysis in the same manner as above using the joint fluids of patients with different rheumatoid arthritis 2 and 3, respectively. The results are shown in FIGS. 11 and 12, and in FIG. 13, among the experimental results, the group treated with the joint fluid of arthritis patients (control) and the group treated with the SLC4A7 antibody (anti-SLCA7) to inhibit the movement of synovial fibroblasts Only extracted and shown.

11 to 13 , the ion transporter SLC4A7 antibody can inhibit the migration of synovial fibroblasts, similar to the results of the joint fluid test of arthritis patients. Specifically, as shown in FIG. 13 , in patient 2 (patient2), the migration of synovial fibroblasts was reduced by about 50% by the ion transporter SLC4A7 antibody, and in patient 3 (patient3), the migration was significantly reduced to about 60%. decreased. Through this, it was confirmed that the ion transporter SLC4A7 antibody effectively inhibited the movement of synovial cells by the joint fluid of arthritis patients.

<Example 2> Confirmation of the inhibitory effect on the expression of bone destructive factor (RANKL, Receptor Activator of Nuclear factor Kappa-B ligand) and COX (Cyclooxygenase) by the ion transporter SLC4A7 antibody

Electrophoresis was used to determine whether the antibody of the ion transporter SLC4A7 had the effect of suppressing the expression of RANKL, a substance important for inducing osteoclast differentiation, and the expression of COX, which causes inflammation by synthesizing prostaglandin. and experimented.

After treating normal synovial cells, Human FLS (HFLS), in 6 wells, 10 ng/ml of TNF-α and SLC4A7 antibody were treated together for 48 hours. Thereafter, RNA was isolated from the synovial fibroblasts ^{using the Hybrid-Ribo EX} extraction system, and the concentration of the isolated RNA was measured using a spectrophotometer, ND-1000. Then, using the RT-PCR kit, cDNA was synthesized at 42°C for 1 hour and 94°C for 5 minutes, and mixed with cDNA synthesized with GAPDH, RANKL, COX-1, COX-2 primers at 95°C for 5 minutes, 95°C. 30 sec, 55 ℃ 1 min, and 72 ℃ 1 min were synthesized under the conditions of 45 cycles. The PCR product thus made was loaded on a 1% agarose gel and electrophoresed at 100 mV for 24 minutes, and then the electrophoresed band was confirmed using ^{GelDoc XR.} The results are shown in FIGS. 14 and 15 .

As can be seen in FIGS. 14 and 15 , the expression of RANKL was increased by the inflammatory factor TNF-α, which was again inhibited by the antibody of the ion transporter SLC4A7. it can be seen that Inflammatory factors, COX-1 and COX-2, were also increased by TNF-α, and the expression was more than doubled in COX-2 compared to COX-1, and both COX-1 and COX-2 were ion transporters. It was significantly reduced by the SLC4A7 antibody. Through this, it can be seen that the ion transporter SLC4A7 antibody is effective in preventing or treating arthritis because it can excellently inhibit not only the movement of synovial fibroblasts but also the expression of bone destruction factor (RANKL) and inflammatory factor (COX).

<Example 3> Confirmation of the effect of inhibiting the migration of synovial fibroblasts by siRNA-SLC4A7

The base sequence of siRNA-SLC4A7 used in the present invention (sense of siRNA-SLC4A7: SEQ ID NO: 1; antisense of siRNA-SLC4A7: SEQ ID NO: 2) is shown in FIG. 16 . 17 shows the Western blot results confirming whether the ion transporter SLC4A7 protein is reduced after siRNA-SLC4A7 is treated in the cells. After treatment with siRNA-SLC4A7, it was confirmed that the change in SLC4A7 protein was significantly reduced, and this was used for cell migration experiments.

In order to confirm the inhibitory effect of siRNA-SLC4A7 on the migration of synovial fibroblasts, Human FLS (HFLS) and Rheumatoid arthritis (RA-FLS) cells were pre-plated in 6 wells, and then siRNA-SLC4A7 was transfected for 48 hours. . After 48 hours, each cell was removed using TE, and cell migration analysis was prepared using a transwell. The lower medium of the transwell was treated with DMEM medium, and the control HFLS and RA-FLS cells and the siRNA-treated cells ^{were treated in a number of 5x10 4} and cultured for 6 hours in the upper chamber. Thereafter, the synovial fibroblasts present in the filter were fixed with methanol using methanol, treated with a dye capable of staining the nucleus of the cell, and incubated for 30 minutes. After washing the filter with PBS, cell nuclei were observed at a wavelength of 405 nm through a confocal microscope. The results are shown in FIGS. 18 and 19 .

As can be seen in FIGS. 18 and 19 , DAPI-stained nuclei were not observed in both HFLS and RA-FLS in the siRNA-SLC4A7-treated place compared to the control (NS), which indicates that synovial cells are trans It shows that there is no migration to the filter in the well. Therefore, it can be seen that the ion transporter SLC4A7 is involved in the migration of synovial cells because siRNA-SLC4A7 treatment significantly reduces cell migration of synovial cells. In addition, from the above experimental results, it was confirmed that the siRNA of SLC4A7 can be used to inhibit the migration of synovial cells very well.

Synovial fibroblasts present in the synovial membrane are affected by the TNF-α condition in rheumatoid arthritis. At this time, as SLC4A7, one of the ion channels present in the synovial fibroblast membrane, is recruited into the cell membrane, the mobility of the synovial fibroblasts is activated, and this Movement to other inflammatory sites is increased. In the present invention, using the SLC4A7 antibody and siRNA-SLC4A7 that regulates the activity of the NBC channel, which is an ion channel, through the above experiment, the migration of synovial fibroblasts can be very excellently inhibited, so the ion transporter SLC4A7 or its gene It was confirmed that joint deformation and joint function inhibition can be prevented or treated using an antibody or siRNA (FIG. 20).

As mentioned above, although the present invention has been described in detail through preferred preparation examples, examples and experimental examples, the scope of the present invention is not limited to the characteristic examples, and should be interpreted by the appended claims. In addition, those skilled in the art should understand that many modifications and variations are possible without departing from the scope of the present invention.

<110> Gachon University of Industry-Academic cooperation Foundation <120> Pharmaceutical composition for the prevention or treatment of arthritis comprising an inhibitor of ion transporter SLC4A7 as an active ingredient <130> 2019P-09-044 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 29 <212> RNA <213> Artificial Sequence <220> <223> siRNA-SLC4A7 sense <400> 1 aaaggtggga tccttctata cgcatagaa 29 <210> 2 <211> 33 <212> RNA <213> Artificial Sequence <220> <223> siRNA-SLC4A7 antisense <400> 2 aaaattctat gcgtatagaa ggatcccacc ttt 33

Claims (9)

Antisense nucleotides complementary to the mRNA of the ion transporter SLC4A7 gene, short hairpin RNA (shRNA) or small interfering RNA (siRNA), or an antibody complementary to SLC4A7 protein as an active ingredient A pharmaceutical composition for the prevention or treatment of arthritis, characterized in that it contains.
delete delete According to claim 1,
The pharmaceutical composition is a pharmaceutical composition for preventing or treating arthritis, characterized in that it inhibits proliferation of synovial cells, or movement of synovial cells to bone.
According to claim 1,
The pharmaceutical composition is RANKL (Receptor Activator of Nuclear factor Kappa-B ligand), or COX (cyclooxygenase, cyclooxygenase), characterized in that inhibiting the expression, the prevention or treatment of arthritis pharmaceutical composition.
6. The method of claim 5,
The COX (cyclooxygenase, cyclooxygenase) is a pharmaceutical composition for the prevention or treatment of arthritis, characterized in that COX-1 or COX-2.
According to claim 1,
The arthritis is a pharmaceutical composition for preventing or treating arthritis, characterized in that the disease is selected from the group consisting of psoriatic arthritis, degenerative arthritis, osteoarthritis, and rheumatoid arthritis.
contacting a sample to be analyzed with cells containing the ion transporter SLC4A7 or its gene;
measuring the expression level of the ion transporter SLC4A7 or its gene after the contact, or the activity of the ion transporter SLC4A7; and
As a result of the measurement, when the expression level of the ion transporter SLC4A7 protein, or its gene, or the activity of the ion transporter is reduced, the sample is characterized as comprising the step of determining as a substance for preventing or treating arthritis Methods of screening substances for prophylaxis or treatment.
9. The method of claim 8,
The expression level is selected from the group consisting of immunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemistry, RT-PCR, Western blotting and flow cytometry (FACS). A screening method, characterized in that it is measured by any one.

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