JP2020015678A - Pharmaceutical composition - Google Patents

Abstract

To provide a pharmaceutical composition useful for the treatment or prevention of rheumatoid arthritis.SOLUTION: There is provided a pharmaceutical composition comprising a substance that suppresses or inhibits the function of the RasGRP2 gene as an active ingredient, which is used for treatment or prevention of rheumatoid arthritis. There is provided a method for evaluating the possibility of developing rheumatoid arthritis, in which expression level of the RasGRP2 gene is used as a marker, and in a case where fibroblast-like synoviocytes obtained from a subject animal have the expression level of the RasGRP2 gene at a predetermined threshold or more, the subject animal has a high likelihood of being developing rheumatoid. arthritis. There is also provided a diagnostic marker for detecting rheumatoid arthritis based on the expression level of RasGRP2 gene.SELECTED DRAWING: None

Description

  The present invention relates to a pharmaceutical composition useful for treating or preventing rheumatoid arthritis (RA).

 RA is a chronic polyarthritis due to an autoimmune mechanism, and is characterized by bone and cartilage destruction accompanying the thickening and proliferation of the synovium. In the pathology of RA, tumor-like proliferation is induced by inflammatory cytokines such as T cells activated in the synovial synovium and TNF-α (tumor necrosis factor alpha) and IL-6 (interleukin-6) produced from macrophages. Fibroblast-like synoviocytes (FLS) play a central role. FLS that grows randomly is involved in cartilage destruction through secretion of proteins such as matrix metalloproteinase (MMP) and activates osteoclasts by expressing RANKL (receptor activator of nuclear factor kappa-B ligand) to cause bone destruction. cause. Conventionally, treatment with non-specific oral therapeutic agents or biological agents targeting inflammatory cytokines has been performed, but the remission rate is not always high. Therefore, elucidation of the pathological condition of RA, search for new therapeutic targets, and development of new therapeutic agents are required.

Since RA is a type of immune-related disease, further elucidation of the immune response may lead to the discovery of new therapeutic targets. However, it is difficult to find a promising therapeutic target in practice, because a large number of genes interact in an immune response in a complex manner. For example, Patent Document 1 ^{discloses that} a population of CD4 ⁺ T cells is composed of stimulator cells activated using an anti-CD3 antibody and ICAM-1 or an anti-CD antibody and an anti-CD28 antibody, and resting cells for comparison. It is disclosed that when microarray analysis was performed, a difference was observed in the expression level between stimulated cells and resting cells for about 8,000 genes.

  On the other hand, Non-Patent Document 1 discloses that the expression of RasGRP4 (Ras guanine nucleotide-releasing protein 4) is enhanced in FLS of RA patients, and the mRNA expression level of RasGRP4 in FLS and cell proliferation ability are positively correlated. It has been reported that when RasGRP4 is knocked down using siRNA, the cell proliferation ability of FLS decreases. The document further states that in Collagen-induced arthritis (CIA) rats, intra-ankle administration of RasGRP4 siRNA significantly reduced the ankle arthritis score and ankle joint diameter compared to controls, and It has also been reported to reduce bone and cartilage destruction.

  RasGRP is a kind of GEF (guanine nucleotide exchange factor), also called CalDAG-GEF, and RasGRP family molecules have been identified as RasGRP1 to RasGRP4. Among them, RasGRP2 (CalDAG-GEFI) is not a Ras activator but rather a GEF that controls Rap-1 activation. In platelets, RasGRP2 regulates integrin expression through Rap-1 activation and regulates platelet expression. Has been reported to be involved in adhesion and coagulation (see, for example, Non-Patent Document 2).

WO 2004/047728

Kono, et al., Arthritis & Rheumatology, 2015, vol. 67, p. 396-407. Stefanini, et al., Platelets, 2010, vol.21, p.239-243. Siebuhr, et al., Journal of Translational Medicine, 2012, 10: 195, doi: 10.1186 / 1479-5876-10-195. Pine, et al., Clinical Immunology, 2007, vol.124, p.244-257. Wruck et al., Annals of the Rheumatic Diseases, 2011, vol. 70, p. 844-850.

  An object of the present invention is to provide a pharmaceutical composition useful for treating or preventing RA.

  The present inventors have found that RasGRP2 is highly expressed in FLS derived from RA patients, and that arthritis is improved by suppressing RasGRP2 expression locally in a joint in an arthritis model animal, thereby completing the present invention.

That is, the present invention provides the following pharmaceutical composition, a method for evaluating the possibility of RA onset, and a diagnostic marker.
[1] A pharmaceutical composition comprising, as an active ingredient, a substance that suppresses or inhibits the function of the RasGRP2 gene.
[2] The pharmaceutical composition of the above-mentioned [1], which is used for treatment or prevention of RA.
[3] The pharmaceutical composition of [1] or [2], which suppresses infiltration of synovial tissue.
[4] The pharmaceutical composition according to any one of [1] to [3], further comprising a substance that suppresses or inhibits the function of the RasGRP4 gene as an active ingredient.
[5] The pharmaceutical composition according to any of [1] to [4], wherein the substance that suppresses or inhibits the function of the RasGRP2 gene is an siRNA targeting the RasGRP2 gene.
[6] Using the expression level of RasGRP2 gene as a marker,
When the expression level of the RasGRP2 gene of FLS collected from the test animal is equal to or higher than a predetermined threshold, the test animal is evaluated as having a high possibility of developing RA, and the evaluation of the possibility of developing RA is performed. Method.
[7] Furthermore, the expression level of the RasGRP4 gene is used as a marker,
When the expression level of the RasGRP2 gene in the fibroblast-like synovial cells collected from the test animal is equal to or higher than a predetermined threshold and the expression level of the RasGRP4 gene is equal to or higher than a predetermined threshold, the test animal The method for evaluating the possibility of developing RA according to the above [6], which evaluates that the possibility of developing RA is high.
[8] A diagnostic marker for detecting RA, comprising the expression level of RasGRP2 gene.
[9] A diagnostic marker for detecting RA, comprising the expression levels of RasGRP2 gene and RasGRP4 gene.

Since the pharmaceutical composition according to the present invention contains a substance that suppresses or inhibits the function of the RasGRP2 gene as an active ingredient, it can suppress infiltration of synovial tissue, and is useful for treating or preventing RA.
In addition, the expression level of the RasGRP2 gene in the cells constituting the synovial tissue is useful as a diagnostic marker for RA. Therefore, the method for evaluating the possibility of RA onset according to the present invention can be used for early detection of RA, Useful for evaluating therapeutic effects.

In Reference Example 1, it is a figure showing the measurement results of mRNA expression levels (relative values) of RasGRP1-4 in FLS collected from rheumatic patients. In Reference Example 1, it is a staining image of a tissue section of synovial tissue collected from an RA patient (upper) and an osteoarthritis (osteoarthritis: OA) patient (lower). 5 is an immunohistochemical staining image of an ankle joint tissue of a CIA mouse and a control mouse with an anti-RasGRP2 antibody in Reference Example 1. In Reference Example 2, it is a figure showing the measurement results of the mRNA amount and protein expression amount of RasGRP of FLS stimulated with each cytokine. In Reference Example 3, it is a figure showing the results of immunoblotting of RasGRP2 forced expression cells and control cells using anti-RasGRP2 antibody, anti-Rap-1 antibody, and anti-β-actin antibody. In Reference Example 3, it is a figure showing the measurement results of the luminescence intensity of the BrdU assay of RasGRP2 forced expression cells and control cells. In Reference Example 3, transmission light images of control cells and RasGRP2 forced expression cells immediately after wound formation (after 0 hour), 24 hours after wound formation, and 48 hours after wound formation. In Reference Example 3, the relative wound area (%) of control cells and RasGRP2 forced expression cells was measured 24 hours and 48 hours after wound formation, where the wound area immediately after wound formation (after 0 hour) was 100%. FIG. In Reference Example 3, it is a figure showing the measurement results of IL-6 concentration in the culture supernatant of control cells and RasGRP2 forced expression cells. FIG. 3 is a view showing the results of measuring arthritis scores of CIA rats in which expression of RasGRP2 gene was suppressed by RNA interference and CIA rats in which expression of RasGRP2 gene was not suppressed in Example 1. In Example 1, it is the figure which showed the measurement result of the diameter of the ankle joint of the CIA rat which suppressed the expression of RasGRP2 gene by RNA interference, and the CIA rat which did not suppress the expression of RasGRP2 gene by RNA interference. 2 is a micro CT image of an ankle joint of a CIA rat in which expression of RasGRP2 gene is suppressed by RNA interference and a CIA rat in which expression of RasGRP2 gene is not suppressed in Example 1. In Example 1, it is the figure which showed the measurement result of the bone erosion score of the CIA rat which suppressed the expression of RasGRP2 gene by RNA interference, and the CIA rat which did not suppress the expression of RasGRP2 gene. In Example 2, CIA rats in which RasGRP2 gene expression was suppressed by RNA interference (RasGRP2 knockdown CIA rats), CIA rats in which RasGRP4 gene expression was suppressed (RasGRP4 knockdown CIA rats), and RasGRP2 gene and RasGRP4 gene expression were suppressed. It is a figure which shows the measurement result of the arthritis score of the suppressed CIA rat (RasGRP2 / RasGRP4 double knockdown CIA rat) and the CIA rat which does not suppress the expression of both the RasGRP2 gene and the RasGRP4 gene (control CIA rat). In Example 2, it is the figure which showed the measurement result of the diameter of the ankle joint of RasGRP2 knockdown CIA rat, RasGRP4 knockdown CIA rat, RasGRP2 / RasGRP4 double knockdown CIA rat, and control CIA rat. In Example 2, it is the micro CT image and HE staining image of the ankle joint of RasGRP2 knockdown CIA rat, RasGRP4 knockdown CIA rat, RasGRP2 / RasGRP4 double knockdown CIA rat, and control CIA rat. In Example 2, it is the figure which showed the measurement result of the bone erosion score of RasGRP2 knockdown CIA rat, RasGRP4 knockdown CIA rat, RasGRP2 / RasGRP4 double knockdown CIA rat, and control CIA rat. In Example 2, it is the figure which showed the measurement result of the Pannus score of RasGRP2 knockdown CIA rat, RasGRP4 knockdown CIA rat, RasGRP2 / RasGRP4 double knockdown CIA rat, and control CIA rat.

  The pharmaceutical composition according to the present invention contains, as an active ingredient, a substance that suppresses or inhibits the function of the RasGRP2 gene (hereinafter, collectively referred to as “RasGRP2 inhibitor”). RasGRP2 controls the migration of FLS and is also involved in the production of IL-6. Therefore, by suppressing or inhibiting the function of the RasGRP2 gene in the synovial tissue, FLS migration and IL-6 production are suppressed, and infiltration and inflammation of the synovial tissue by FLS can be suppressed. Therefore, the pharmaceutical composition according to the present invention is useful for treating or preventing RA.

  The pharmaceutical composition according to the present invention targets the synovial infiltration itself. The pharmaceutical composition according to the present invention can be a drug for a new therapeutic method, unlike the conventional RA treatment, which is different from a therapeutic method aimed at suppressing inflammation targeting immunocompetent cells or cytokines. Therefore, the pharmaceutical composition according to the present invention can be a treatment that can be selected as a treatment for intractable cases of conventional RA treatment. In particular, RA treatment with a RasGRP2 inhibitor is considered not to be mediated by immunosuppressive effects, and thus may possibly solve the problem of infectivity that is inextricably linked to the effects of current RA treatment. In general, suppression of side effects can be expected by selecting a treatment having a different mechanism of action. Therefore, it is also preferable to use the pharmaceutical composition according to the present invention in combination with a conventional RA treatment.

  The suppression or inhibition of the function of the RasGRP2 gene can be achieved by inhibiting the expression of the RasGRP2 gene. That is, examples of the RasGRP2 inhibitor which is an active ingredient of the pharmaceutical composition according to the present invention include substances having an action of suppressing the expression itself of the RasGRP2 gene by RNA interference or the like. As the substance, for example, siRNA (small interfering RNA) having a double-stranded structure composed of a sense strand and an antisense strand of a partial region (target region of RNAi (RNA interference)) of cDNA of RasGRP2 gene, shRNA (short hairpin RNA) ) Or miRNA (micro RNA). Further, it may be an RNAi-inducing vector capable of producing siRNA or the like in the cells of the target cells such as FLS and synovial tissue. The production of siRNA, shRNA, miRNA, and RNAi-inducing vector can be designed and manufactured by a conventional method from the nucleotide sequence information of the target RasGRP2 gene cDNA. The RNAi-inducing vector can also be prepared by inserting the nucleotide sequence of the RNAi target region into the nucleotide sequence of various commercially available RNAi vectors.

  As an active ingredient of the pharmaceutical composition according to the present invention, the function of the RasGRP2 protein, particularly the activation of Rap-1, or the indirect activation of Ras is suppressed by directly or indirectly binding to the RasGRP2 protein. Alternatively, it may be an inhibitory substance. The substance is not particularly limited, and may be any of nucleic acids, peptides, proteins, and low molecular weight compounds.

  The protein that binds to the RasGRP2 protein and inhibits the function of the RasGRP2 protein includes, for example, an antibody that specifically binds to the RasGRP2 protein (anti-RasGRP2 antibody). The antibody may be a monoclonal antibody or a polyclonal antibody. Alternatively, artificially synthesized antibodies such as chimeric antibodies, single-chain antibodies, and humanized antibodies may be used. These antibodies can be produced by a conventional method.

  The active ingredient of the pharmaceutical composition according to the present invention may be a substance that degrades RasGRP2 protein. When the RasGRP2 protein itself is degraded, its function is inhibited. The substance that degrades RasGRP2 protein may be a degrading enzyme that directly degrades RasGRP2 protein, or may be a substance that performs various labels such as polyubiquitin so as to be a substrate for the protease.

  The pharmaceutical composition according to the present invention may contain other active ingredients as long as the action of the RasGRP2 inhibitor is not impaired. Other active ingredients are expected to have therapeutic effects on RA, such as biological agents and immunosuppressants conventionally used for RA treatment, such as TNF-α inhibitors and IL-6 inhibitors. Substances.

  The pharmaceutical composition according to the present invention preferably contains, as an active ingredient, a substance that suppresses or inhibits the function of the RasGRP4 gene together with the RasGRP2 inhibitor (hereinafter, collectively referred to as “RasGRP4 inhibitor”). Since cell growth of FLS can be suppressed by suppressing the expression of the RasGRP4 gene (Non-Patent Document 1), by using a RasGRP2 inhibitor and a substance that suppresses or inhibits the function of the RasGRP4 gene in combination, An additive and synergistic therapeutic effect on RA can be expected. Examples of the RasGRP4 inhibitor include siRNA, shRNA or miRNA having a double-stranded structure composed of a sense strand and an antisense strand of a partial region (target region of RNAi (RNA interference)) of cDNA of RasGRP4 gene, an anti-RasGRP4 antibody, and RasGRP4. Specific examples include low molecular weight compounds that specifically bind.

  The route of administration of the pharmaceutical composition according to the present invention is not particularly limited, and is appropriately determined depending on the target cell and the tissue containing it. For example, the administration route of the pharmaceutical composition according to the present invention includes oral administration, direct administration to a joint, transdermal administration, intravenous administration, intraperitoneal administration, enema administration and the like.

  Pharmaceutical compositions according to the present invention may be prepared by the usual methods, such as powders, granules, capsules, tablets, chewables, oral solids such as sustained-release preparations, solutions, oral liquids such as syrups, and injections. Formulations, enemas, sprays, patches, ointments and the like. Formulation may be carried out, if necessary, by excipients, binders, lubricants, disintegrants, fluidizers, solvents, dissolution aids, buffers, suspending agents, emulsifiers, isotonic agents , A stabilizer, a preservative, an antioxidant, a flavoring agent, a coloring agent, and the like can be compounded by a conventional method.

  By administering the pharmaceutical composition according to the present invention to humans or non-human animals and suppressing or inhibiting the function of the RasGRP2 gene, FLS migration and IL-6 production are suppressed, and joints of these animals are inhibited. It can suppress infiltration and inflammation of synovial tissue. The animal is not particularly limited, and may be a human or an animal other than a human. Non-human animals include mammals such as cows, pigs, horses, sheep, goats, monkeys, dogs, cats, rabbits, mice, rats, hamsters, guinea pigs, and birds such as chickens, quails, ducks, and the like.

  RA FLS has a higher expression level of the RasGRP2 gene than FLS of healthy subjects or OA. Therefore, the expression level of the RasGRP2 gene is useful as a diagnostic marker for detecting RA. For example, when the expression level of the RasGRP2 gene of FLS collected from a test animal is equal to or higher than a predetermined threshold value, it is evaluated that the test animal is highly likely to have developed RA. be able to. Conversely, when the expression level of the RasGRP2 gene of the FLS is smaller than a predetermined threshold value, it can be evaluated that the subject animal has a low possibility of developing RA.

  The threshold value can be appropriately set in consideration of the type of the method for measuring the expression level of the RasGRP2 gene and by performing necessary preliminary tests. For example, from the results of other test methods, a measured value of the expression level of the RasGRP2 gene of FLS (non-RA group) collected from a test animal confirmed not to have developed RA, By comparing the measured value of the expression level of the RasGRP2 gene of FLS (RA group) collected from a test animal confirmed to have it can.

  The expression level of the FLS RasGRP2 gene may be measured at the mRNA level or at the protein level. The method for measuring the expression level of the RasGRP2 gene is not particularly limited as long as the method can quantitatively or semi-quantitatively measure the amount of the target protein in the cell or the amount of the target mRNA. It can be appropriately selected from known methods used for protein detection and used. Each method can be performed by a conventional method.

  The amount of mRNA of the RasGRP2 gene may be detected by a hybridization method using a probe capable of hybridizing with the mRNA of the RasGRP2 gene, and a nucleic acid amplification reaction using a primer and a polymerase capable of hybridizing with the mRNA of the RasGRP2 gene may be performed. It may be detected by the used method. In addition, a commercially available detection kit or the like can also be used. For example, a cDNA is synthesized by performing a reverse transcription reaction using the total RNA extracted from the FLS as a template, and then performing PCR (Polymerase Chain Reaction) or the like using the obtained cDNA as a template, and measuring the amount of the obtained amplification product. This makes it possible to quantify the amount of the RasGRP2 gene mRNA. The amount of the amplification product can be quantitatively measured by specifically separating the product by gel or capillary electrophoresis and then detecting the product. In addition, by performing semi-quantitative PCR such as real-time PCR instead of PCR, the quantification of the RasGRP2 gene mRNA can be easily performed simultaneously with the detection thereof.

  The amount of RasGRP2 protein can be measured using an anti-RasGRP2 antibody. For example, immunostaining using an anti-RasGRP2 antibody as a primary antibody is performed, and the presence or absence and expression intensity of RasGRP2 in FLS are examined based on the presence or absence and staining intensity. The immunostaining may be an enzyme antibody staining method using an enzyme-labeled antibody or a fluorescent antibody staining method using a fluorescence-labeled antibody. In addition, the RasGRP2 protein amount of FLS can be quantitatively measured by Western blotting or the like after separating proteins in a cell extract prepared from FLS by SDS-PAGE or the like.

  RA FLS has a higher expression level of the RasGRP4 gene than FLS of healthy individuals or OA (see Non-Patent Document 1). Therefore, the expression level of the RasGRP4 gene is useful as a diagnostic marker for detecting RA. For example, when the expression level of the RasGRP4 gene of FLS collected from a test animal is equal to or higher than a predetermined threshold value, it is evaluated that the test animal is highly likely to have developed RA. be able to. Conversely, when the expression level of the RasGRP4 gene of the FLS is smaller than a predetermined threshold value, it can be evaluated that the subject animal has a low possibility of developing RA. The expression level of the RasGRP4 gene of FLS can be measured in the same manner as the measurement of the expression level of the RasGRP2 gene described above. The threshold for the expression level of the RasGRP4 gene for determining the presence or absence of RA onset can be set in the same manner as the above-described threshold for the expression level of the RasGRP2 gene.

  By using the expression level of the RasGRP2 gene and the expression level of the RasGRP4 gene in combination as a diagnostic marker for detecting RA, RA can be detected more accurately. For example, when the expression level of the RasGRP2 gene of FLS collected from the test animal is equal to or higher than a predetermined threshold, and the expression level of the RasGRP4 gene is equal to or higher than a predetermined threshold, It can be evaluated that the test animal is highly likely to have developed RA. Conversely, when the expression level of the RasGRP2 gene of the FLS is smaller than a predetermined threshold value or when the expression level of the RasGRP4 gene is smaller than a predetermined threshold value, the test animal has RA. It can be evaluated that the likelihood of occurrence is low.

  The test animal to be used as a diagnostic marker for detecting the expression level of the RasGRP2 gene or RasGRP4 gene as a diagnostic marker for detecting RA is not particularly limited, and may be a human or an animal other than a human. It may be. Non-human animals include mammals such as cows, pigs, horses, sheep, goats, monkeys, dogs, cats, rabbits, mice, rats, hamsters, guinea pigs, and birds such as chickens, quails, ducks, and the like.

Next, the present invention will be described in more detail with reference to examples and the like, but the present invention is not limited to the following examples.
In addition, the subsequent animal experiments were performed in accordance with the regulations regarding animal experiments in Hokkaido University under the approval of the Animal Experiment Ethics Committee of Hokkaido University.

[Reference Example 1]
Expression analysis of RasGRP in synovial tissue of rheumatic patients was performed.

<Rheumatic patients>
Among RA patients who underwent total knee arthroplasty at Hokkaido University Hospital Orthopedic Surgery from April 2012 to April 2017, 8 RA patients (RA1-8) and 6 OA patients (OA1-6) After obtaining written informed consent and obtaining consent, synovial tissue was collected. This study was conducted in accordance with the Helsinki Declaration and the basic principles of clinical trials, and was approved by the Hokkaido University Graduate School of Medicine Ethics Committee (approval number: 008-0103).

<Sampling of FLS>
The synovial tissue collected from each rheumatic patient was cut into small pieces after removing connective tissue and fat, and incubated at 37 ° C for 2 hours with Hanks' balanced salt solution containing 5 mg / mL of Type I collagenase (manufactured by Sigma). went. The suspension after the incubation was passed through a mesh, and then centrifuged to collect cell components. The collected cell components were cultured in Iscove's modified Dulbecco's medium (Sigma) containing 10% inactivated FBS (fetal calf serum). Cells separated and cultured by this method are FLS with a purity of 97% or more. In addition, FLS can be confirmed by HSP-47 staining.

<Expression analysis based on RasGRP mRNA>
The FLS subcultured for 4 to 8 passages was collected, and mRNA was extracted using TRIzol RNA reagent. The extracted mRNA is converted into cDNA using SuperScript VILO, and human RasGRP1 mRNA, human RasGRP2 mRNA, human RasGRP3 mRNA, human RasGRP4 mRNA and human GAPDH mRNA expression was quantified. The RasGRP1 to 4 mRNA expression levels in each FLS were compared by the ΔΔCT value method. The mRNA expression level of RasGRP1 to 4 was determined as a relative amount (RQ) when the ΔΔCT value of RasGPR1 in FLS derived from OA1 patient was 1.

  FIG. 1 shows the measurement results of the mRNA expression levels (relative values) of RasGRP1 to 4 in FLS. In FIG. 1, in the column of “Biologics”, “+” indicates that the patient is receiving the biologic treatment, and “−” indicates that the patient is not receiving the biologic treatment. In the FLS of the RA patient group, a tendency was observed in which RasGRP1 to 4 all had enhanced mRNA expression compared to the OA patient group. In particular, in some RA patients (RA1 to RA3), RasGRP4 and RasGRP2 were significantly increased in expression.

<Expression analysis based on immunohistochemical staining of RasGRP>
A part of the synovial tissue collected from each rheumatic patient was fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, and then a 6 μm-thick tissue section was prepared. The tissue section was deparaffinized with xylene, washed with ethanol, and replaced with PBS (phosphate buffered saline). Subsequently, an antigen activating solution High pH (manufactured by DAKO) was added, the mixture was allowed to stand at 95 ° C. for 20 minutes, and then washed with water. The tissue section after washing with water was further immersed in PBS mixed with 0.3% hydrogen peroxide at room temperature for 30 minutes to inactivate endogenous peroxidase activity.

  Subsequently, to each tissue section, an anti-Cadherin-11 antibody (manufactured by R & D Systems) and an anti-RasGRP2 antibody (manufactured by Abcam) were added, incubated at 4 ° C. overnight, and subjected to immunohistochemical staining. Co-expression of these proteins was examined by immunohistochemical staining using serial sections.

  FIG. 2 shows a stained image of a synovial tissue section derived from an RA1 patient and a synovial tissue section derived from an OA4 patient. In immunohistochemical staining, expression of RasGRP2 was confirmed in vascular endothelial cells of RA patients and FLS identified by Cadherin-11. On the other hand, in OA patients, expression of RasGRP2 in vascular endothelial cells was observed, but expression in FLS was not confirmed.

<Immunohistochemical staining of CIA mouse ankle tissue>
CIA mice were prepared by the method described in Non-Patent Document 1, and RasGRP2 expression analysis was performed by immunohistochemical staining. Specifically, in the same manner as the synovial tissue collected from each rheumatic patient, a 6 μm-thick tissue section was prepared from the ankle joint tissue of the CIA mouse, and immunohistochemical staining with an anti-RasGRP2 antibody was performed. As a control, immunohistochemical staining of the ankle joint tissue of a healthy mouse was also performed in the same manner.

  FIG. 3 shows immunohistochemical staining images of the ankle joint tissue of the CIA mouse and the control mouse with the anti-RasGRP2 antibody. Expression of RasGRP2 was also confirmed in synovial tissue of CIA mice. Further, it was also confirmed that mouse RasGRP2 was highly expressed in the surface layer of the proliferating synovial tissue, and was particularly highly expressed in the site where the proliferating synovium invaded the bone.

[Reference Example 2]
The expression change of RasGRP2 when FLS was stimulated with cytokines was examined. Cytokines include TNF-α, IL-6, IL-1β, IFN-γ (interferon gamma), IL-17A, IL-22, PDGF (platelet-derived growth factor), VEGF (vascular endothelial growth factor), and TGF -Β (transforming growth factor beta) was used.

<Cytokine stimulation>
The FLS of passages 4 to 8 were sprayed on a 12-well plate, and cultured in Iscove's modified Dulbecco's medium containing 10% inactivated FBS. 1 to 10 ng / mL of each cytokine was added to the culture solution, and the cells were collected 24 hours later.

<RasGRP expression analysis>
MRNA was extracted from a portion of the collected cells in the same manner as in Reference Example 1, and the expression of human RasGRP2 mRNA and human GAPDH mRNA was quantified by Real-time-PCR. The RasGRP2 mRNA expression level upon cytokine stimulation was compared by the ΔΔCT value method. The ΔΔCT value was defined as the expression level of RasGPR1 in control cells to which no cytokine was added, being 1.

  The rest of the collected cells was treated with a cell lysis buffer (manufactured by Wako) containing a protease inhibitor (manufactured by Sigma), left on ice for 15 minutes, centrifuged for 10 minutes, and the supernatant was collected. Sodium dodecyl sulfate (SDS) was added to the concentration-adjusted supernatant, and the mixture was heated at 95 ° C for 10 minutes. Next, a 9% acrylamide gel was prepared, and proteins in the sample were electrophoresed. Thereafter, the protein was transferred to a PVDF (polyvinylidene difluoride) membrane, and blocking was performed with skim milk. An anti-RasGRP2 antibody diluted 1000-fold with Solution 1 (Toyobo) was reacted at room temperature overnight, washed, and reacted with a secondary antibody. Amerisham (registered trademark) ECL (registered trademark) Western Blotting Detection Reagents (GE Healthcare) was used as a detection reagent.

  FIG. 4 shows the measurement results of the mRNA level and protein expression level of RasGRP2 of FLS stimulated with each cytokine. Data are shown as mean ± standard error (*: p <0.05, **: p <0.01, t-test). Stimulation of VEGF and TGF-β increased the expression level of RasGRP2 mRNA. In addition, although a statistically significant difference could not be confirmed, expression of RasGRP2 mRNA also tended to increase by stimulation of IL-6, IL-1β, IFN-γ, IL-17A, IL-22, and PDGF. Admitted. In immunoblotting, an increase in the protein expression level of RasGRP2 due to the stimulation of IL-22, PDGF, VEGF, and TGF-β was confirmed.

[Reference Example 3]
RasGRP2 was forcibly expressed in FLS and its effect was examined.

<Preparation of RasGRP2 expression vector>
A RasGRP2 reference sequence (accession number: NM_001098670.1, Homo sapiens RAS guanyl releasing protein 2, transcript variant 3) was obtained from PubMed, and primers were used to contain the entire open reading frame (ORF) using Primer-BLAST. Was designed as shown in Table 1.

  Using the cDNA of Jurkat cells as a template, the ORF sequence of RasGRP2 was amplified by PCR using KOD plus Neo (Toyobo). The obtained PCR product was subjected to agarose gel electrophoresis, and it was confirmed that a DNA fragment of a desired size was amplified. The target DNA was extracted from the gel pieces using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega). The obtained DNA was subjected to a T addition reaction using a Taq enzyme (Toyobo), and then ligated to pcDNA3.1 / V5-His-TOPO expression vector (Thermo Fisher Scientific).

  Then, E. coli competent cells were transformed and cultured. Colony PCR was performed on the obtained colonies using the primers shown in Table 2. The PCR product was subjected to agarose gel electrophoresis, the colony in which the target band was obtained was further cultured in a liquid medium, and the plasmid vector was isolated using the Wizard (registered trademark) Plus SV Minipreps DNA Purification System (manufactured by Promega). Purification was performed.

<Forced expression of RasGRP2>
First, transfection was performed using a green fluorescent protein (GFP) expression vector, and conditions for maximizing transfection efficiency were examined by fluorescence microscopy.
Next, the RasGRP2 expression vector is transfected under the conditions where the transfection efficiency is maximized, and the Real-time-PCR method and immunoblotting are performed in the same manner as in Reference Example 2 to increase the expression levels of RasGRP2 mRNA and protein. It was confirmed.

<Immunoblotting of signaling-related factors>
Signal transduction-related factors were subjected to immunoblotting to examine changes in signal transduction pathways due to forced expression of RasGRP2.
The RasGRP2 expression vector was transfected into FLS, and cells were collected 72 hours later. An empty vector was transfected into FLS in the same manner as a control.
Cell lysates of each FLS were prepared in the same manner as in Reference Example 2. Some of them were incubated with GST (Glutathione S-transferase) fusion protein and glutathione agarose beads (manufactured by Cell signaling technology), and GTP-bound with SDS solution. Rap-1 was sedimented (pull-down assay). The obtained lysate and eluate were subjected to anti-RasGRP2 antibody, anti-Erk1 / 2 antibody, anti-P-Erk antibody, anti-p38MAPK antibody, anti-P-p38MAPK antibody, anti-JNK antibody, anti-P-JNK after polyacrylamide gel electrophoresis. Immunoblotting was performed using an antibody, an anti-m-TOR antibody, an anti-Rap-1 antibody, and an anti-β-actin antibody.

  As a result, the expression levels of Erk1 / 2, P-Erk, p38MAPK, P-p38MAPK, JNK, P-JNK, and m-TOR were determined by the RasGRP2 forced expression cells into which the RasGRP2 expression vector was introduced and the control cells into which the empty vector was introduced. There was no difference. That is, no apparent change in the Erk pathway, the p38 MAPK pathway, the JNK pathway, and the m-TOR pathway was observed by the forced expression of RasGRP2. On the other hand, in the RasGRP2 forced expression cells, the total amount of Rap-1 protein was not changed as compared to the control cells, but the amount of GTP-bound Rap-1 protein was clearly increased. FIG. 5 shows the results of immunoblotting using an anti-RasGRP2 antibody, an anti-Rap-1 antibody, and an anti-β-actin antibody. In the figure, “Control” indicates the results of control cells, and “RasGRP2 +” indicates the results of RasGRP2 forced expression cells.

<BrdU assay>
The relationship between RasGRP2 forced expression and cell proliferation ability was examined by BrdU assay.
RasGRP2 expression vector or empty vector was transfected into FLS, respectively, and 48 hours later, BrdU (bromodeoxyuridine) was added to the cell culture medium. BrdU, a thymidine analog, is incorporated into newly synthesized DNA during the S phase of the cell cycle and reflects cell proliferation. Twenty-four hours after the addition, the cells were fixed, and reacted with a peroxidase-labeled anti-BrdU antibody (Roche). Thereafter, a chemiluminescent substrate was added, and the luminescence intensity was measured with an ELISA reader.

  FIG. 6 shows the measurement results of the emission intensity. Data are shown as mean ± standard error. The luminescence intensity was not significantly different between control cells ("Control" in the figure) and RasGRP2 forced-expressing cells ("RasGRP2 +" in the figure), and the forced expression of RasGRP2 was apparent in the evaluation of the cell proliferation ability in the BrdU assay. Had no effect.

<Wound healing assay>
A wound healing assay was performed to examine the relationship between RasGRP2 forced expression and cell migration ability.
The RasGRP2 expression vector or the empty vector was transfected into FLS, respectively, and cultured in a 12-well plate so as to become confluent 48 hours later. The confluent 12-well plate was scratched with the tip of a 1000 μL pipette tip to create a blank zone of cells (wound). Thereafter, the state in which the cells migrated and the blanks were filled (the wound healed) was observed over time with an optical microscope, and the size of the blank (wound) area was quantified using Image J software Image J.

  Transmitted light images of control cells (“Control” in the figure) and RasGRP2 forced-expressing cells (“RasGRP2 +” in the figure) are shown immediately after wound formation (0 hour), 24 hours and 48 hours after wound formation. FIG. FIG. 8 shows the measurement results of the relative wound area (%) at 24 hours and 48 hours after the wound formation, where the wound area immediately after the wound formation (after 0 hour) was 100%. At 24 hours and 48 hours later, the wound area was smaller in the RasGRP2 forced expression cells ("RasGRP2 +" in the figure) than in the control cells ("Control" in the figure). In other words, significant promotion of wound healing (migration) was confirmed by forced expression of RasGRP2.

<Measurement of IL-6 production amount>
The relationship between RasGRP2 forced expression and IL-6 production was examined.
The RasGRP2 expression vector or the empty vector was transfected into FLS, respectively, and the medium was exchanged 48 hours later, and the cell culture supernatant was collected 24 hours later. The IL-6 concentration in the culture supernatant was quantified using an ELISA kit (manufactured by R & D System).

  FIG. 9 shows the quantification results. In the RasGRP2 forced cells ("RasGRP2 +" in the figure), the IL-6 concentration in the culture supernatant was significantly increased as compared to the control cells ("Control" in the figure). From these results, it was found that forced expression of RasGRP2 promoted IL-6 secretion of FLS.

  From these findings, it is clear that RasGRP2 is deeply involved in the pathology of RA. More specifically, RasGRP2 is involved in migration and IL-6 production during the process in which the FLS of RA proliferates like a tumor and migrates and infiltrates bone and cartilage to form pannus.

[Example 1]
The effect of RasGRP2 knockdown in RA model rats was examined. CIA rats were used as RA model rats. In addition, knockdown of RasGRP2 was carried out by using siRNA (RasGRP2-1) (S1676787: Applied Biosystems) targeting a region in the sixth exon of the RasGRP2 gene and siRNA (RasGRP2) targeting a region in the seventh exon. -2) (S167865: manufactured by Applied Biosystems) was used. As a control siRNA, siRNA (Control) (manufactured by Applied Biosystems) was used.

<Preparation of CIA rat>
Seven week old LEW rats (females) were immunized by subcutaneously administering 200 μg of bovine Type II collagen (Chondrex) dissolved in 200 μL of incomplete Freund's adjuvant (Chondrex) to the tail (Day 0). ).
On day 7 after collagen administration, booster immunization was performed by subcutaneously administering 100 μg of bovine Type II collagen (Chondrex) dissolved in 100 μL of incomplete Freund's adjuvant (Chondrex) to the tail. .
From the 7th day after the collagen administration, the arthritis score and the diameter of the ankle joint were measured once a week for the induced arthritis.

<Arthritis score>
The arthritis score was 0: no redness and swelling of the joints, 1 point: slight redness and swelling of the tarsal bone or ankle joint, 2 points: slight redness and swelling from the ankle joint to the tarsal bone 3 points: moderate redness and swelling from the ankle to metatarsal joint; 4 points: severe redness and swelling extending to the ankle, foot and toes, or toe joint ankylosis (See Non-Patent Document 3).

<Knockdown of RasGRP2 by RNA interference>
On the 14th day from collagen administration (Day 14), in a joint of a CIA rat, siRNA (RasGRP2-1), siRNA (RasGRP2-2), or siRNA (Control) was added to atelocollagen (manufactured by Koken) at 10 μM. 50 μL of the mixed siRNA solution was administered.

  FIG. 10 shows the measurement results of the arthritis score of each CIA rat, and FIG. 11 shows the measurement results of the diameter of the ankle joint of each CIA rat as the mean ± standard error (*: p <0.05, **: p <0.01, t-test). As a result, in the CIA rat to which siRNA (RasGRP2-1) was administered (“siRNA RasGRP2-1” in the figure) or the CIA rat to which siRNA (RasGRP2-2) was administered (“siRNA RasGRP2-2” in the figure), Both the arthritis score and the diameter of the ankle joint were clearly smaller than those of CIA rats to which control siRNA (Control) was administered ("Control" in the figure).

<Micro CT imaging of ankle joint tissue and bone erosion score>
CIA rats were euthanized by anesthesia whole blood sampling on day 35 after collagen administration. The ankle joint tissue of each CIA rat was fixed with 4% paraformaldehyde for 24 hours. Next, after imaging the micro CT of the ankle joint, it was decalcified and embedded in paraffin.

  The bone erosion score based on microCT findings was 0 point: normal, 1 point: only soft tissue swelling, 2 points: soft tissue swelling and mild bone erosion, 3 points: severe bone erosion (non-patent literature) 4).

  FIG. 12 shows a micro CT image of the ankle joint of each CIA rat, and FIG. 13 (*: p <0.05, t-test) shows the results of the bone erosion score. In the CIA rats to which siRNA (Control) was administered ("Control" in the figure), severe bone erosion was observed (arrows in the left figure in FIG. 12), but siRNA (RasGRP2-1) or siRNA (RasGRP2- In the CIA rats to which 2) was administered (in the figure, "SiRNA RasGRP2"), although soft tissue swelling was observed, no severe bone erosion occurred (the right arrow in FIG. 12), and bone erosion was observed. The score was also small.

  These results indicate that the pathology of RA is improved by suppressing the expression of the RasGRP2 gene by RNA interference, that is, the RasGRP2 inhibitor is useful as an active ingredient of a pharmaceutical composition for treating RA. Was confirmed.

[Example 2]
The effect of RasGRP2 and RasGRP4 knockdown in RA model rats was examined. CIA rats produced in the same manner as in Example 1 were used as RA model rats. Knockdown of RasGRP2 was performed using siRNA (RasGRP2-1) (S167678: manufactured by Applied Biosystems) targeting a region in the sixth exon of the RasGRP2 gene, and knockdown of RasGRP4 was performed in the sixth exon of the RasGRP4 gene. SiRNA (RasGRP4-1) (S139320: manufactured by Applied Biosystems) targeting the region was used. As a control siRNA, siRNA (Control) (manufactured by Applied Biosystems) was used.

<Knockdown of RasGRP2 and / or RasGRP4 by RNA interference>
RasGRP2 knockdown CIA rats, RasGRP4 knockdown CIA rats, and control CIA rats were each prepared by using siRNA (RasGRP2-1), siRNA (RasGRP4-1), or siRNA (Control) atelocollagen (manufactured by Koken) at 10 μM. ) Was administered into the joints of CIA rats on day 14 after the administration of collagen (Day 14) (n = 3, respectively). RasGRP2 / RasGRP4 double knockdown CIA rats were prepared by mixing 25 μL of an siRNA solution obtained by mixing siRNA (RasGRP2-1) with atelocollagen (manufactured by Koken) at 10 μM, and atelocollagen with siRNA (RasGRP4-1) at 10 μM. 25 μL of the siRNA solution mixed with Koken Co., Ltd.) was administered into the joints of CIA rats on day 14 after collagen administration (Day 14) (n = 3).

<Arthritis score and ankle diameter>
For each CIA rat, the arthritis score and the diameter of the ankle joint were measured in the same manner as in Example 1.

<Micro CT imaging of ankle joint tissue and bone erosion score>
Each CIA rat was euthanized by anesthesia whole blood sampling 35 days after collagen administration (Day 35). The ankle joint tissue of each CIA rat was fixed with 4% paraformaldehyde for 24 hours. Next, after imaging the micro CT of the ankle joint, it was decalcified and embedded in paraffin. The bone erosion score was measured in the same manner as in Example 1 based on the micro CT image of the ankle joint.

<HE staining and Pannus score>
HE (hematoxylin-eosin) staining was performed on a section prepared from paraffin embedding, and pannus formation was evaluated from the HE-stained image. Pannus formation was scored as 0 points: normal, 1 point: mild pannus formation, 2 points: moderate pannus formation, and 3 points: severe pannus formation (see Non-Patent Document 5).

  14 shows the measurement results of the arthritis score of each CIA rat, FIG. 15 shows the measurement results of the diameter of the ankle joint of each CIA rat, FIG. 17 shows the measurement results of the bone erosion score of each CIA rat, and FIG. The results of measuring the pannus score of the rat are shown as mean ± standard error (*: p <0.05, **: p <0.01, t-test). FIG. 16 shows a micro CT image and an HE-stained image of the ankle joint of each CIA rat. In each figure, “Control” is the result of control CIA rat, “siRNA RasGRP2” is the result of RasGRP2 knockdown CIA rat, “siRNA RasGRP4” is the result of RasGRP4 knockdown CIA rat, and “siRNA RasGRP2 + 4” is the result of RasGRP2 / RasGRP4 double knockdown CIA rat. Represents In FIG. 16, an arrow indicates an ankle joint, and an arrowhead indicates a proliferative synovial tissue.

  As a result, in the RasGRP2 knockdown CIA rats and the RasGRP4 knockdown CIA rats, all of the arthritis score, the diameter of the ankle joint, the bone erosion score, and the Pannus score were clearly smaller than the control CIA rats. The improvement effect on the arthritis score, ankle joint diameter, bone erosion score, and pannus score was larger in RasGRP4 knockdown CIA rats than in RasGRP2 knockdown CIA rats. Further, for bone erosion and pannus formation, RasGRP2 / RasGRP4 double knockdown CIA rats showed a higher improvement effect than RasGRP4 knockdown CIA rats, even though only half of the administered siRNA was used. . From these results, it was confirmed that suppressing the functions of both RasGRP2 and RasGRP4 has a synergistic effect on the therapeutic effect on various symptoms of arthritis such as infiltration of synovial tissue.

Claims (9)

  A pharmaceutical composition comprising, as an active ingredient, a substance that suppresses or inhibits the function of RasGRP2 gene.   The pharmaceutical composition according to claim 1, which is used for treating or preventing rheumatoid arthritis.   The pharmaceutical composition according to claim 1 or 2, which suppresses infiltration of synovial tissue.   The pharmaceutical composition according to any one of claims 1 to 3, further comprising a substance that suppresses or inhibits the function of the RasGRP4 gene as an active ingredient.   The pharmaceutical composition according to any one of claims 1 to 4, wherein the substance that suppresses or inhibits the function of the RasGRP2 gene is an siRNA that targets the RasGRP2 gene. Using the expression level of RasGRP2 gene as a marker,
When the expression level of the RasGRP2 gene in the fibroblast-like synovial cells collected from the test animal is equal to or higher than a predetermined threshold, it is evaluated that the test animal is highly likely to have rheumatoid arthritis. A method for evaluating the possibility of developing rheumatoid arthritis. Furthermore, the expression level of RasGRP4 gene is used as a marker,
When the expression level of the RasGRP2 gene in the fibroblast-like synovial cells collected from the test animal is equal to or higher than a predetermined threshold and the expression level of the RasGRP4 gene is equal to or higher than a predetermined threshold, the test animal The method for evaluating the possibility of developing rheumatoid arthritis according to claim 6, wherein the method is evaluated as having a high possibility of developing rheumatoid arthritis.   A diagnostic marker for detecting rheumatoid arthritis, comprising a RasGRP2 gene expression level.   A diagnostic marker for detecting rheumatoid arthritis, comprising a RasGRP2 gene expression level and a RasGRP4 gene expression level.