KR102282341B1 - Composition for preventing or treating behcet's diseases containing cd83 inhibitor - Google Patents

Abstract

The present invention provides a composition for preventing or treating Behcet's disease comprising a CD83 inhibitor as an active ingredient. According to the present invention, it was confirmed that the frequency of CD83-expressing cells was significantly high in Behcet's disease mice, and there was a relationship between the increase in CD83-expressing cells and Behcet's disease symptoms. By regulating the expression, it can be effectively used for diagnosis, prevention, and treatment of Behcet's disease. In addition, the therapeutic effect can be further increased by using in combination with existing therapeutic agents for diseases or diseases caused by or associated with CD83 or CD83-expressing cells.

Description

A composition for preventing or treating Behçet's disease comprising a CD83 inhibitor as an active ingredient {COMPOSITION FOR PREVENTING OR TREATING BEHCET'S DISEASES CONTAINING CD83 INHIBITOR}

The present invention relates to a composition for preventing or treating Behcet's disease comprising a CD83 inhibitor as an active ingredient.

Behcet's disease (Behcet's disease) is a rare, intractable inflammatory disease characterized by recurrent aphthous ulcers, uveitis and skin lesions of the oral and/or genital organs. Clinical symptoms are not only skin ulcers, but also joints, central nervous system, stomach, kidneys, genitourinary system, lungs, cardiovascular system, intestinal bleeding, intestinal perforation and other digestive system related symptoms, upper and lower vena cava syndrome, and serious symptoms such as aortic regurgitation. Chronic inflammation is multifaceted. These symptoms are associated with systemic vasculitis and are a central pathophysiological feature of Behcet's disease.

Although the exact cause of Behcet's disease is unknown, it is associated with the autoimmune and autoinflammatory responses triggered by herpes virus infection. In Behcet's disease, cell invasion types include macrophages and dendritic cells, as well as CD4+, CD8+, T cells, and neutrophils. It is also associated with an increase in cytokine production.

Behcet's disease sometimes shows only mild symptoms such as inflammation of the skin mucosa or arthritis, but it can be accompanied by severe sequelae due to severe uveitis or invasion of major organs such as brain, lung, heart, and kidney.

Korean Patent Publication No. 10-2016-0104101 (published on September 2, 2016)

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating Behcet's disease and a drug screening method comprising a CD83 inhibitor as an active ingredient.

In addition, the present invention provides a biomarker composition for diagnosing Behcet's disease, a diagnostic composition, and a diagnostic kit.

The pharmaceutical composition for preventing or treating Behcet's disease according to the present invention may include a CD83 inhibitor as an active ingredient.

A drug screening method for preventing or treating Behcet's disease according to the present invention comprises a first step of treating a test substance in a biological sample isolated from the human body; a second step of measuring the expression or activity level of CD83 or CD83 mRNA in the sample treated with the test substance; and a third step of selecting a test substance in which the expression or activity level of the CD83 or the CD83 mRNA is decreased compared to the control sample.

The biomarker composition for diagnosing Behcet's disease according to the present invention may include CD83 or CD83 mRNA as an active ingredient.

The composition for diagnosing Behcet's disease according to the present invention may include an agent for measuring the expression or activity level of CD83 or CD83 mRNA as an active ingredient.

The kit for diagnosing Behcet's disease according to the present invention may include a composition for diagnosing Behcet's disease.

The pharmaceutical composition according to the present invention can effectively prevent rare and intractable Behcet's disease by including a CD83 inhibitor as an active ingredient, and can treat when it occurs. In addition, by using the biomarker composition, the diagnostic composition, and the diagnostic kit according to the present invention, the likelihood of developing Behcet's disease and the degree of the disease can be significantly predicted or grasped.

In addition, the therapeutic effect can be further increased by using in combination with existing therapeutic agents for diseases or diseases caused by or related to CD83 or CD83-expressing cells.

1 shows the change in symptoms before and after CD83 siRNA treatment in a Behcet's disease-like mouse model according to an experimental example of the present invention.
2 is a graph showing the frequency of CD83-expressing (CD83+) cells in the intraperitoneal cavity of a Behcet's disease-like mouse model according to an experimental example of the present invention.
3 shows changes when CD83 siRNA treatment is stopped in a Behcet's disease-like mouse model according to an experimental example of the present invention.
4 is an image observed with a transmission electron microscope after CD83 siRNA treatment in dendritic cells isolated from a Behcet's disease-like mouse model according to an experimental example of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention showed that the frequency of CD83-expressing cells in Behcet's disease mice was significantly higher when comparing the Behcet's disease mice with asymptomatic control mice, and an inhibitor of CD83 reduced the frequency of CD83-expressing cells. By confirming that it can be utilized as a treatment for Behcet's disease, the present invention has been completed.

As used herein, "prevention" refers to any action of suppressing or delaying the onset of Behcet's disease or at least one symptom of Behcet's disease by administration of the pharmaceutical composition according to the present invention. Also includes treatment of subjects in remission of Behcet's disease to prevent or prevent recurrence.

As used herein, "treatment" refers to any action that improves or advantageously changes the symptoms, such as alleviating, reducing or eliminating at least one symptom of Behcet's disease or Behcet's disease by administration of the pharmaceutical composition according to the present invention .

As used herein, "diagnosis" is to determine whether or not the onset of Behcet's disease or the possibility (risk) of the onset, and to determine the subject's susceptibility to at least one symptom of Behcet's disease or Behcet's disease, To determine whether a subject currently has Behcet's disease, to determine the prognosis of a subject with Behcet's disease, or to use therametrics (e.g., an object to provide information about efficacy of treatment) monitoring the status of the Also includes primary diagnosis of clinical condition or diagnosis of recurrent disease.

As used herein, the term "pharmaceutical composition" refers to a composition administered for a specific purpose, which is administered to prevent or treat Behçet's disease for the purpose of the present invention.

As used herein, the term "biomarker" is an indicator that can detect changes in the body, and can measure a normal or pathological state of a living organism, that is, a diagnosis of Behcet's disease, a degree of response to a drug, and the like.

The present invention provides a pharmaceutical composition for preventing or treating Behcet's disease comprising a CD83 inhibitor as an active ingredient.

In the present invention, "CD83 (Cluster of Differentiation 83)" is a human protein encoded by the CD83 gene, and is a single-pass type I membrane glycoprotein belonging to the immunoglobulin superfamily. (glycoprotein). CD83 is a cell surface marker mainly expressed on mature dendritic cells (DCs), and is strongly expressed along with costimulatory molecules such as CD80 and CD86 during the maturation of dendritic cells. In addition, CD83 can also be expressed on activated B and T cells and minimally on immature blood dendritic cells (BDC) and monocyte-derived dendritic cells (MoDC).

In the present invention, "dendritic cells (dendritic cells, DC)" is an immune cell constituting the immune system of a mammal, and acts as a professional antigen presenting cell (professional antigen presenting cell). Their main function is to process antigenic substances, express them on the cell surface and present them to T cells. In this way, they act as messengers between the innate and adaptive immune systems.

Molecules that begin to be expressed upon dendritic cell differentiation and activation assist in linking innate and adaptive immunity. Because dendritic cells exert control over the immune response, activated dendritic cells can be a target for intervention across a number of immunological diseases, including malignant and autoimmune diseases. The CD83 inhibitor according to the present invention can modulate the activation and immune response of these dendritic cells.

In the composition according to the present invention, the CD83 inhibitor may be selected from the group consisting of an antibody that specifically binds to CD83, a peptide, a peptide mimetics, an aptamer, a compound, and a natural product.

In addition, the CD83 inhibitor may be selected from the group consisting of antisense nucleotides complementary to CD83 mRNA, small interfering RNA (siRNA), and short hairpin RNA (shRNA), but is limited thereto. it's not going to be

In the composition according to the present invention, the small interfering RNA (hereinafter, siRNA) may have a nucleotide sequence represented by SEQ ID NO: 1 or 2. The total length of the siRNA may be 10 to 80 bases, preferably 15 to 60 bases, more preferably 20 to 40 bases. The nucleotide sequences of SEQ ID NOs: 1 and 2 are as follows.

SEQ ID NO: 1: 5'-GUGCUUUUCAGUCAUCUACAAGCTA -3'

SEQ ID NO:2: 5'-UAGCUUGUAGAUGACUGAAAAGCACUC-3'

In the present invention, "siRNA (small interfering RNA)" is a small RNA fragment with a size of 21-25 nt, which forms a complex with RISC (RNA-induced silencing complex) in a cell and specifically binds to mRNA having a complementary sequence. It is one of the substances inducing RNA interfering (RNAi) that inhibits protein expression after cleaving the target mRNA. As an inhibitor of CD83 according to the present invention, the siRNA can inhibit the CD83 expression in dendritic cells through RNA interference. When CD83 expression is inhibited, activation of T cells by dendritic cells can be inhibited.

In the composition according to the present invention, the siRNA may include a nucleotide sequence of a complementary sequence (antisense) of SEQ ID NO: 1 or 2 (sense, sence). In addition, the siRNA may be a single RNA strand of a stem-loop structure in which SEQ ID NO: 1 or 2 and the complementary sequence of SEQ ID NO: 1 or 2 are linked by a loop.

The siRNA may be formed by chemical synthesis, amplification of a required nucleotide sequence by polymerase chain reaction (PCR), or purifying an existing siRNA by recombinant synthesis.

In the composition according to the present invention, the siRNA may include one or more chemical modifications in the sugar portion, nucleobase portion or internucleotide structure of the RNA. The chemical modification is for improving in vivo stability, conferring resistance to nucleases, and reducing non-specific immune responses. Through the chemical modification, the siRNA does not affect RNAi ability, but increases resistance to nucleases, increases intracellular uptake, improves cell targeting, increases stability, decreases interferon activity, immune response, and sense ( off-target properties such as sense) effect can be improved over unmodified siRNA. Any chemical modification capable of improving the stability and biocompatibility of the siRNA according to the present invention in vivo may be included in the scope of the present invention. The chemical transformation may be made in only one form of transformation, or various chemical transformations may be made together.

As used herein, "antibody" is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site. The antibody in the present invention refers to an antibody that specifically binds to CD83, and the antibody can be prepared according to a conventional method in the art.

In the present invention, "peptide" has the advantage of high binding strength to the target material, and does not undergo denaturation even during heat/chemical treatment. In addition, since the molecular size is small, it can be attached to other proteins and used as a fusion protein. Specifically, it can be used as a diagnostic kit and drug delivery material because it can be used by attaching it to a polymer protein chain.

In the present invention, "aptamer" is a special type of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure and can bind to a target molecule with high affinity and specificity. It refers to a kind of polynucleotide composed of As described above, the aptamer can specifically bind to an antigenic substance in the same way as an antibody, but has higher stability than a protein, has a simple structure, and is composed of a polynucleotide that is easy to synthesize, so it can be used as an alternative to an antibody. can

In the pharmaceutical composition according to the present invention, the pharmaceutical composition may target any disease or disease caused by or associated with CD83 or a cell expressing CD83. For example, it may include Behcet's disease. Behcet's disease (BD) is a rare and incurable chronic inflammatory disease that can invade multiple organs, such as the skin, blood vessels, gastrointestinal tract, central nervous system, heart and lungs, in addition to oral ulcers, genital ulcers, and eye inflammation. The target to be treated is not limited to Behcet's disease. Behcet's disease is a disease in which CD83 expression is high, and inflammatory diseases related to or caused by Behçet's disease and other diseases in which CD83 expression is high may also be included.

The pharmaceutical composition according to the present invention may be prepared by further including an appropriate pharmaceutically acceptable carrier, excipient or diluent according to the formulation. The "pharmaceutically acceptable" refers to exhibiting properties that are not toxic to cells or humans exposed to the pharmaceutical composition.

Carriers, excipients or diluents usable in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.

The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. there is.

When the pharmaceutical composition according to the present invention is formulated in the above form, it may be prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient, for example, starch, calcium carbonate, sucrose or lactose. (lactose), gelatin, etc. can be mixed to prepare it.

In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

In the pharmaceutical composition according to the present invention, the pharmaceutical composition may be administered in a pharmaceutically effective amount. The "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not to cause side effects, and the effective dose level is determined by the patient's health condition, Type, severity, drug activity, sensitivity to drug, administration method, administration time, administration route and excretion rate, treatment period, factors including drugs used in combination or concurrently, and other factors well known in the medical field. .

The amount of the active ingredient used in the pharmaceutical composition according to the present invention may vary depending on the patient's age, sex, weight, and disease, but is 0.001 to 100 mg/kg, preferably 0.01 to 10 mg/kg, administered once or several times a day. can In addition, the dosage of the composition according to the present invention may be increased or decreased depending on the route of administration, the degree of disease, sex, weight, age, and the like. Accordingly, the above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, and humans by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, endobronchial inhalation, intrauterine dural or intracerebroventricular injection.

The present invention provides a first step of treating a test substance in a biological sample isolated from the human body; a second step of measuring the expression or activity level of CD83 or CD83 mRNA in the sample treated with the test substance; and a third step of selecting a test substance in which the expression or activity level of the CD83 or the CD83 mRNA has decreased compared to the control sample; provides a method for screening a drug for preventing or treating Behcet's disease, including.

The biological sample isolated from the human body may include, but is not limited to, a tissue, cell, serum, etc. sample that is different from the control in the expression or activity level of the CD83 or CD83 mRNA.

The test substance is a substance used in screening to examine whether the CD83 or the CD83 mRNA expression or activity is affected, and may be a CD83 inhibitor according to the present invention, for example, CD83 siRNA.

The protein expression or activity level was determined by Western blot, Enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), Radioimmunodiffusion, Oukteroni immune diffusion method, rocket Immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, flow cytometry (FACS) and protein chip can be measured by any one or more methods selected from the group consisting of, but limited thereto it's not going to be

The expression level of the gene was determined by RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), Northern blotting. blotting) and DNA microarray chip may be measured by any one or more methods selected from the group consisting of, but is not limited thereto.

The present invention provides a biomarker composition for diagnosing Behcet's disease comprising CD83 or CD83 mRNA as an active ingredient.

In addition, the present invention provides a composition for diagnosing Behcet's disease, comprising an agent for measuring the activity or expression level of CD83 or CD83 mRNA. The agent may be an antibody, peptide, peptide mimetics, aptamer, or compound that specifically binds to the CD83, or a primer or probe that specifically binds to the CD83 mRNA, but is not limited thereto.

In the present invention, "primer" refers to a nucleic acid sequence having a short free 3'hydroxl group, a short nucleic acid sequence capable of forming a base pair with a complementary template and serving as a starting point for template strand copying. it means. Primers are capable of initiating DNA synthesis in the presence of different 4 nucleotide triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) in an appropriate buffer and temperature. In the composition according to the present invention, the primer may be a sense and antisense nucleic acid having a sequence of 7 to 50 nucleotides as the gene-specific primer, and does not change the basic properties of the primer serving as the starting point of DNA synthesis. As long as it is possible, additional features may be incorporated.

In the present invention, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to several bases to several hundred bases in length that can specifically bind to mRNA, and is labeled to determine the presence or absence of a specific mRNA, expression quantity can be checked. The probe may be formed in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, or the like. Appropriate probes and hybridization conditions may be appropriately selected according to techniques known in the art.

In addition, the present invention provides a kit for diagnosing Behcet's disease comprising the diagnostic composition.

The diagnostic kit according to the present invention may include an antibody specific for CD83, or a sense, antisense primer, or complementary probe of CD83 mRNA.

Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are provided to more completely explain the present invention to those with average knowledge in the art, and are only illustrative of the contents of the present invention, so that the scope of the present invention is limited to the following examples no.

[Experimental example]

Behcet's disease-like mouse model

ICR strain 5-week-old mice were inoculated with herpes simplex virus type 1 into the wounded auricle. Among the mice, a mouse showing Behcet's disease characteristics along with inflammatory symptoms such as skin ulceration and erythema was selected as a Behcet's disease-like mouse model and used in this experiment.

Administration method

1.0 μmol of CD83 siRNA was intraperitoneally injected three times with an interval of 4 days mixed with a transfection reagent. Improvement was confirmed 14 days after the start of administration.

CD83 siRNA sequence:

5'-GUGCUUUUCAGUCAUCUACAAGCTA -3'

3'-CUCACGAAAAGUCAGUAGAUGUUCGAU -5'

Experiment result

1 shows the change in symptoms before and after CD83 siRNA treatment in a Behcet's disease-like mouse model according to an experimental example of the present invention.

Referring to FIG. 1 , in a Behcet's disease-like mouse model, symptoms were photographed before CD83 siRNA intraperitoneal injection and 14 days after the start of administration to compare whether improvement was achieved. As a result, skin rash and skin edema 14 days after CD83 siRNA intraperitoneal injection administration. Symptoms showed clear improvement, and skin ulcer symptoms improved.

When the frequency of CD83-expressing cells was compared in Behcet's disease mice and asymptomatic control mice, it was found to be significantly higher in Behcet's disease mice, and CD83 siRNA was found on the cell membrane of peripheral blood cells of normal mice. also decreased the frequency of CD83-expressing cells in the peripheral blood cells of Behcet's disease mice.

2 is a graph showing the frequency of CD83-expressing (CD83+) cells in the intraperitoneal cavity of a Behcet's disease-like mouse model according to an experimental example of the present invention.

Referring to FIG. 2 , after injecting 0.5 μmol or 1.0 μmol of CD83 siRNA into the abdominal cavity of a Behcet's disease mouse, intraperitoneal macrophages were isolated and the CD83+ cell frequency was investigated by flow cytometry (FACS). As a result, in mice injected with CD83 siRNA, It was found that the frequency of CD83+ cells was reduced compared to the control group. Through this, it can be confirmed that there is a relationship between the increase in CD83-expressing cells and the symptoms of Behcet's disease.

3 shows changes when CD83 siRNA treatment is stopped in a Behcet's disease-like mouse model according to an experimental example of the present invention.

Referring to FIG. 3 , after CD83 siRNA injection into Behçet's disease mice improved symptoms, the symptoms worsened again when CD83 siRNA treatment was stopped in the mice. Through this, it can be confirmed that the improvement of the symptoms in the mice is a therapeutic effect according to the administration of CD83 siRNA.

4 is an image observed with a transmission electron microscope after CD83 siRNA treatment in dendritic cells isolated from a Behcet's disease-like mouse model according to an experimental example of the present invention.

4, in the process of in vitro culture of monocytes isolated from the bone marrow of Behcet's disease mice and differentiating them into dendritic cells, 0.5 μmol of CD83 siRNA was treated and observed with a transmission electron microscope. As a result, it was confirmed that the dendrite of the cell membrane was reduced compared to the control (Scramble siRNA treatment).

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

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Claims (10)

A pharmaceutical composition for the prevention or treatment of Behcet's disease comprising CD83 siRNA comprising the nucleotide sequence represented by SEQ ID NO: 1 or 2 as an active ingredient. delete delete delete delete delete delete delete delete delete

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