JP2019131502A - 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 the treatment or prevention of rheumatoid arthritis (RA).

 RA is chronic polyarthritis due to an autoimmune mechanism, and is characterized by bone and cartilage destruction accompanying synovial thickening and proliferation. In the pathology of RA, tumor-like growth is caused by inflammatory cytokines such as T cells activated locally in the synovial joint, TNF-α (tumor necrosis factor alpha) and IL-6 (interleukin-6) produced from macrophages. Fibroblast-like synoviocytes (FLS) play a central role. Disordered FLS is involved in cartilage destruction through secretion of proteins such as MMP (matrix metalloproteinase), and also activates osteoclasts by expressing RANKL (Receptor activator of nuclear factor kappa-B ligand), thereby causing bone destruction. cause. Conventionally, treatment with a non-specific oral therapeutic agent or a biological product targeting inflammatory cytokines has been performed, but the remission rate is not necessarily high. For this reason, it is required to elucidate the pathology of RA, search for new therapeutic targets, and develop new therapeutic agents.

Since RA is a type of immune-related disease, there is a possibility that new therapeutic targets can be discovered by further elucidating the immune response. However, the immune reaction is complicated by a large number of genes, and it is difficult to actually search for a promising therapeutic target. For example, Patent Document 1 ^{discloses that} a population of CD ⁴⁺ T cells is divided into stimulating cells activated with anti-CD3 antibody and ICAM-1, or anti-CD antibody and anti-CD28 antibody, and resting cells for comparison. When microarray analysis was performed, it was disclosed that a difference in expression level was observed between stimulated cells and resting cells with about 8000 genes.

  On the other hand, Non-Patent Document 1 shows that RasGRP4 (Ras guanine nucleotide-releasing protein 4) expression is increased in FLS of RA patients, and that RasGRP4 mRNA expression level 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. Further, in the literature, in collagen-induced arthritis (CIA) rats, intra-ankle administration of RasGRP4 siRNA significantly reduced the ankle arthritis score and ankle 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 RasGRP1 to 4 are identified as RasGRP family molecules. Among these, RasGRP2 (CalDAG-GEFI) is a GEF that controls activation of two molecules, Ras and Rap-1, and RasGRP2 is expressed in integrin through activation of Rap-1 rather than Ras activator in platelets. Is reported to be involved in platelet adhesion and aggregation (see, for example, Non-Patent Document 2).

International Publication No. 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.

  An object of this invention is to provide the pharmaceutical composition useful for the treatment or prevention of 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 the joint in an arthritis model animal, and completed the present invention.

That is, the present invention provides the following pharmaceutical composition, an evaluation method of RA onset possibility, 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 according to [1], which is used for the treatment or prevention of RA.
[3] The pharmaceutical composition according to [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 one of [1] to [4], wherein the substance that suppresses or inhibits the function of the RasGRP2 gene is siRNA targeting the RasGRP2 gene.
[6] Using the expression level of the RasGRP2 gene as a marker,
Evaluation of the possibility of developing RA when the expression level of RasGRP2 gene of FLS collected from the test animal is equal to or higher than a predetermined threshold value, the test animal is highly likely to develop RA Method.
[7] A diagnostic marker for detecting RA, comprising the expression level of the RasGRP2 gene.

Since the pharmaceutical composition which concerns on this invention uses the substance which suppresses or inhibits the function of RasGRP2 gene as an active ingredient, it can suppress infiltration of a synovial tissue and is useful for the treatment or prevention of RA.
In addition, the expression level of 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 the early detection of RA and the therapeutic agent for RA. Useful for evaluation of therapeutic effects.

In the reference example 1, it is the figure which showed the measurement result of the mRNA expression level (relative value) of RasGRP1-4 in FLS extract | collected from the rheumatic patient. In the reference example 1, it is a dyeing | staining image of the tissue section of the synovial tissue extract | collected from the RA patient (upper stage) and the osteoarthritis (osteoarthritis: OA) patient (lower stage). In Reference example 1, it is the immunohistochemical dyeing | staining image by the anti- RasGRP2 antibody of the ankle joint tissue of a CIA mouse and a control mouse. In the reference example 2, it is the figure which showed the measurement result of the mRNA level and protein expression level of RasGRP of FLS stimulated with each cytokine. In the reference example 3, it is the figure which showed the result of the immunoblotting using the anti- RasGRP2 antibody of the RasGRP2 forced expression cell and the control cell, the anti-Rap-1 antibody, and the anti-beta-actin antibody. In the reference example 3, it is the figure which showed the measurement result of the emitted light intensity of the BrdU assay of RasGRP2 forced expression cell and a control cell. In the reference example 3, it is the transmitted light image of a control cell and RasGRP2 forced expression cell immediately after wound formation (after 0 hour), 24 hours after wound formation, and 48 hours after. In Reference Example 3, the measurement results of the relative wound area (%) of control cells and RasGRP2 forced expression cells 24 hours and 48 hours after wound formation, with the wound area immediately after wound formation (after 0 hours) being 100% FIG. In the reference example 3, it is the figure which showed the measurement result of the IL-6 density | concentration in the culture supernatant of a control cell and RasGRP2 forced expression cell. In Example 1, it is the figure which showed the measurement result of the arthritis 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 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. In Example 1, it is the micro CT image 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. 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 has not suppressed the expression of RasGRP2 gene.

  The pharmaceutical composition according to the present invention contains substances that suppress or inhibit the function of the RasGRP2 gene (hereinafter collectively referred to as “RasGRP2 inhibitors”) as active ingredients. RasGRP2 regulates FLS migration and is also involved in IL-6 production. For this reason, 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 the treatment or prevention of RA.

  The pharmaceutical composition according to the present invention targets the invasiveness of the synovium itself. The pharmaceutical composition according to the present invention can be a pharmaceutical for a new therapeutic method, unlike conventional therapeutic methods aiming at suppressing inflammation by targeting immunocompetent cells and 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, since RA treatment with a RasGRP2 inhibitor is considered not to have an immunosuppressive effect, there is a possibility that the problem of infectivity that is inextricably linked to the effect of current RA treatment may be solved. Moreover, since it is generally expected that side effects can be suppressed by selecting treatments with different mechanisms of action, it is also preferable to use the pharmaceutical composition according to the present invention in combination with conventional RA treatment.

  Suppression or inhibition of the function of the RasGRP2 gene can be achieved by inhibiting the expression of the RasGRP2 gene. That is, the RasGRP2 inhibitory substance that is an active ingredient of the pharmaceutical composition according to the present invention includes a substance having an action of suppressing the expression of the RasGRP2 gene itself by RNA interference or the like. Examples of the substance include siRNA (small interfering RNA) and shRNA (short hairpin RNA) having a double-stranded structure consisting of a sense strand and an antisense strand of a partial region of the RasGRP2 gene cDNA (RNAi (RNA interference) target region). ) Or miRNA (micro RNA). Alternatively, it may be an RNAi induction vector capable of producing siRNA or the like in the cells of cells constituting the target FLS or synovial tissue. Production of siRNA, shRNA, miRNA, and RNAi-derived vector can be designed and manufactured by a conventional method from the base sequence information of cDNA of the target RasGRP2 gene. The RNAi induction vector can also be prepared by inserting the base sequence of the RNAi target region into the base sequence of various commercially available RNAi vectors.

  As an active ingredient of the pharmaceutical composition according to the present invention, by binding directly or indirectly to RasGRP2 protein, the function of RasGRP2 protein, in particular, activation of Rap-1 or Ras activation is suppressed or inhibited. It may be a substance. The substance is not particularly limited, and may be any of nucleic acid, peptide, protein, and low molecular weight compound.

  Examples of the protein that binds to the RasGRP2 protein and inhibits the function of the RasGRP2 protein include an antibody that specifically binds to the RasGRP2 protein (anti-RasGRP2 antibody). The antibody may be a monoclonal antibody or a polyclonal antibody. Further, it may be an artificially synthesized antibody such as a chimeric antibody, a single chain antibody, a humanized antibody or the like. 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. The function of the RasGRP2 protein is inhibited by being degraded. The substance that degrades the RasGRP2 protein may be a degrading enzyme that directly degrades the RasGRP2 protein, or a substance that performs various labels such as polyubiquitin so as to be a substrate for the proteolytic enzyme.

  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 preparations and immunosuppressants that have been conventionally used for RA treatment, such as TNF-α inhibitors and IL-6 inhibitors. Substances.

  The pharmaceutical composition according to the present invention preferably comprises 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 the cell growth of FLS can be suppressed by suppressing the expression of the RasGRP4 gene (Non-Patent Document 1), by using a RasGRP2 inhibitor in combination with a substance that suppresses or inhibits the function of the RasGRP4 gene, 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 consisting of a sense strand and an antisense strand of a cDNA partial region (RNAi (RNA interference) target region) of RasGRP4 gene, anti-RasGRP4 antibody, RasGRP4. Examples thereof include a low molecular compound that specifically binds.

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

  The pharmaceutical composition according to the present invention is prepared by an ordinary method, such as oral solid preparations such as powders, granules, capsules, tablets, chewable preparations, sustained-release preparations, solution preparations, syrup preparations, and injections. It can be formulated into an agent, enema, spray, patch, ointment and the like. Formulation is carried out according to the formulation needs, such as excipients, binders, lubricants, disintegrants, fluidizers, solvents, solubilizers, buffers, suspending agents, emulsifiers, tonicity agents. Stabilizers, preservatives, antioxidants, flavoring agents, coloring agents, and the like can be blended in a conventional manner.

  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, migration of FLS and production of IL-6 are suppressed, and the joints of these animals are inhibited. Infiltration and inflammation of the synovial tissue can be suppressed. The animal is not particularly limited, and may be a human or an animal other than a human. Examples of non-human animals include mammals such as cows, pigs, horses, sheep, goats, monkeys, dogs, cats, rabbits, mice, rats, hamsters, and guinea pigs, and birds such as chickens, quails, and ducks.

  RA FLS has a higher expression level of RasGRP2 gene than before the onset of RA. For this reason, the expression level of RasGRP2 gene is useful as a diagnostic marker for detecting RA. For example, if the expression level of the RasGRP2 gene of FLS collected from the test animal is equal to or higher than a predetermined threshold value, it is evaluated that the test animal is highly likely to develop RA. be able to. Conversely, if the expression level of the RasGRP2 gene in FLS is less than a predetermined threshold value, it can be evaluated that the test animal is unlikely to develop RA.

  The threshold value can be appropriately set by considering the type of measurement method for the expression level of RasGRP2 gene and performing necessary preliminary tests. For example, from the results of other test methods, the measured value of the expression level of RasGRP2 gene in FLS (non-RA group) collected from a test animal that has been confirmed not to develop RA, and developed RA By comparing the measured value of the expression level of RasGRP2 gene of FLS (RA group) collected from a test animal that has been confirmed to be, a threshold value for discriminating both groups can be set as appropriate it can.

  The expression level of RasGRP2 gene of FLS may be measured at the mRNA level or may be measured at the protein level. The method for measuring the expression level of RasGRP2 gene is not particularly limited as long as it can quantitatively or semi-quantitatively measure the amount of target protein or target mRNA in a cell. It can be appropriately selected from known methods used for protein detection. 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 that can hybridize with the mRNA of the RasGRP2 gene, and a nucleic acid amplification reaction using a primer and a polymerase that can hybridize with the mRNA of the RasGRP2 gene. You may detect by the utilized method. In addition, commercially available detection kits can also be used. For example, after synthesizing cDNA by performing reverse transcription using total RNA extracted from FLS as a template, PCR (Polymerase Chain Reaction) is performed using the obtained cDNA as a template, and the amount of amplification product obtained is measured Thus, the amount of mRNA of the RasGRP2 gene can be quantified. The amount of the amplification product can be quantitatively measured by specifically separating it by gel or capillary electrophoresis and then detecting it. In addition, by performing semi-quantitative PCR such as real-time PCR instead of PCR, the quantification can be easily performed simultaneously with the detection of mRNA of RasGRP2 gene.

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

EXAMPLES Next, although an Example etc. are shown and this invention is demonstrated further in detail, this invention is not limited to a following example.
Subsequent animal experiments were conducted in accordance with Hokkaido University animal experimentation regulations with the approval of the Hokkaido University Animal Experiment Ethics Committee.

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

<Rheumatic patients>
Among rheumatic patients undergoing artificial knee joint replacement at Hokkaido University Hospital orthopedics from April 2012 to April 2017, 8 RA patients (RA1-8) and 6 OA patients (OA1-6) Then, after giving a written explanation and obtaining consent, synovial tissue was collected. This study was conducted according to the Declaration of Helsinki and the basic philosophy of clinical trials, and was approved by the Ethics Committee of Hokkaido University Graduate School of Medicine (approval number: 008-0103).

<Collecting FLS>
After removing connective tissue and fat, synovial tissue collected from each rheumatic patient was minced and incubated with Hanks' balanced salt solution containing 5 mg / mL Type I collagenase (Sigma) for 2 hours at 37 ° C. 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 bovine serum). Cells separated and cultured by this method are FLS with a purity of 97% or more. FLS can be confirmed by HSP-47 staining.

<Expression analysis based on mRNA of RasGRP>
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 by a Real-time-PCR method using Taq-Man Gene Expression Assay (Applied Biosystems). The expression of GAPDH mRNA was quantified. The mRNA expression levels of RasGRP1 to 4 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) where the ΔΔCT value of RasGPR1 in FLS derived from OA1 patient was 1.

  The measurement result of the mRNA expression level (relative value) of RasGRP1 to 4 in FLS is shown in FIG. In FIG. 1, in the “Biologics” column, “+” indicates that biologics treatment is received, and “−” indicates that no biologic treatment is received. In the FLS of the RA patient group, it was observed that RasGRP1 to 4 all had a tendency to increase the expression of mRNA as compared to the OA patient group. In particular, in some RA patients (RA1 to RA3), a marked increase in the expression of RasGRP2 was observed together with RasGRP4.

<Expression analysis based on immunohistochemical staining of RasGRP>
A portion of the synovial tissue collected from each rheumatic patient was fixed with 4% paraformaldehyde for 24 hours, embedded in paraffin, and then a tissue section having a thickness of 6 μm was prepared. The tissue sections were deparaffinized with xylene, washed with ethanol, and replaced with PBS (phosphate buffered saline). Subsequently, antigen activation liquid High pH (made by DAKO) was added, and it left still at 95 degreeC for 20 minutes, Then, it washed with water. In order to inactivate endogenous peroxidase activity, the tissue sections after washing with water were further immersed in PBS mixed with 0.3% hydrogen peroxide at room temperature for 30 minutes.

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

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

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

  FIG. 3 shows immunohistochemical staining images of an ankle joint tissue of a CIA mouse and a control mouse with an anti-RasGRP2 antibody. RasGRP2 expression was also confirmed in the synovial tissue of CIA mice. It was also confirmed that mouse RasGRP2 was highly expressed in the surface layer portion of the proliferative synovial tissue, and was particularly highly expressed in the site where the proliferative synovium had entered the bone.

[Reference Example 2]
The expression change of RasGRP2 when FLS was stimulated with cytokine 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>
Four to eight passages of FLS were spread 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 medium, and the cells were collected after 24 hours.

<RasGRP expression analysis>
From a part of the collected cells, mRNA was extracted 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 method. The expression level of RasGRP2 mRNA in cytokine stimulation was compared by the ΔΔCT value method. As for ΔΔCT value, the expression level of RasGPR1 in control cells without addition of cytokine was taken as 1.

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

  FIG. 4 shows the measurement results of RasGRP2 mRNA level and protein expression level of FLS stimulated with each cytokine. Data are shown as mean ± standard error (*: p <0.05, **: p <0.01, t-test). RasGRP2 mRNA expression level was increased by stimulation with VEGF and TGF-β. In addition, although no statistically significant difference could be confirmed, the expression level of RasGRP2 mRNA tends to increase by stimulation with IL-6, IL-1β, IFN-γ, IL-17A, IL-22, and PDGF. Admitted. In immunoblotting, it was confirmed that the protein expression level of RasGRP2 was increased by stimulation with IL-22, PDGF, VEGF, and TGF-β.

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

<Preparation of RasGRP2 expression vector>
RasGRP2 reference sequence (accession number: NM_0010988670.1, Homo sapiens RAS guanyl releasing protein 2, transcript variant 3) was obtained from PubMed, and primers were used to include the entire open reading frame (ORF) using Primer-BLAST. Was designed as shown in Table 1.

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

  Thereafter, the cells were transformed into E. coli competent cells 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 from which the target band was obtained was further cultured in a liquid medium, and plasmid vector isolation was performed using Wizard (registered trademark) Plus SV Minipreps DNA Purification System (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 observation with a fluorescence microscope. Next, the RasGRP2 expression vector was transfected under conditions for maximizing transfection efficiency. In the same manner as in Reference Example 2, Real-time-PCR and immunoblotting were performed, and it was confirmed that the expression level of RasGRP2 mRNA and protein increased.

<Immunoblotting of signaling related factors>
Immunoblotting of signal transduction-related factors was performed, and changes in the signal transduction pathway due to RasGRP2 forced expression were examined.
RasGRP2 expression vector was transfected into FLS and cells were harvested 72 hours later. As a control, an empty vector was transfected into FLS in the same manner.
Cell lysates of each FLS were prepared in the same manner as in Reference Example 2, and 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 precipitated (pull-down assay). The obtained lysate and eluate were subjected to polyacrylamide electrophoresis, followed by anti-RasGRP2 antibody, anti-Erk1 / 2 antibody, anti-P-Erk antibody, anti-p38MAPK antibody, anti-P-p38MAPK antibody, anti-JNK antibody, anti-P-JNK. Immunoblotting was performed using an antibody, anti-m-TOR antibody, anti-Rap-1 antibody, and 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 expressed as a RasGRP2 forced expression cell into which a RasGRP2 expression vector was introduced and a control cell into which an empty vector was introduced. There was no difference. That is, by the forced expression of RasGRP2, no obvious change was observed in the Erk pathway, p38 MAPK pathway, JNK pathway, and m-TOR pathway. On the other hand, in the RasGRP2 forced expression cells, the total amount of Rap-1 protein was not changed as compared with the control cells, but the GTP-bound Rap-1 protein was clearly increased. The results of immunoblotting using anti-RasGRP2 antibody, anti-Rap-1 antibody, and anti-β-actin antibody are shown in FIG. In the figure, “Control” shows the result of the control cell, and “RasGRP2 +” shows the result of the RasGRP2 forced expression cell.

<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 BrdU (bromodeoxyuridine) was added to the cell culture solution 48 hours later. BrdU, a thymidine analog, is taken up by newly synthesized DNA in the S phase of the cell cycle and reflects cell growth. 24 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.

  The measurement results of the emission intensity are shown in FIG. Data are shown as mean ± standard error. Luminescent intensity is not significantly different between control cells (“Control” in the figure) and RasGRP2 forced expression cells (“RasGRP2 +” in the figure), and the forced expression of RasGRP2 is evident in the evaluation of cell proliferation ability in the BrdU assay. Did not have a significant impact.

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

  Transmitted light images of control cells ("Control" in the figure) and RasGRP2 forced expression cells ("RasGRP2 +" in the figure) immediately after wound formation (after 0 hours), 24 hours and 48 hours after wound formation 7 shows. Further, FIG. 8 shows the measurement results of the relative wound area (%) 24 hours and 48 hours after the wound formation, with the wound area immediately after wound formation (after 0 hours) being 100%. At both 24 hours and 48 hours, RasGRP2 forced expression cells (“RasGRP2 +” in the figure) had a smaller wound area than control cells (“Control” in the figure). That is, 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.
RasGRP2 expression vector or empty vector was transfected into FLS, medium was changed 48 hours later, and cell culture supernatant was collected 24 hours later. The IL-6 concentration in the culture supernatant was quantified with an ELISA kit (R & D System).

  The quantitative results are shown in FIG. In the RasGRP2 forced cells ("RasGRP2 +" in the figure), the IL-6 concentration in the culture supernatant was significantly increased compared to the control cells ("Control" in the figure). From this result, it was found that IL-6 secretion of FLS was promoted by forced expression of RasGRP2.

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

[Example 1]
The effect of RasGRP2 knockdown in rheumatoid arthritis model rats was examined. CIA rats were used as rheumatoid arthritis model rats. In addition, RasGRP2 knockdown involves siRNA targeting the region in the 6th exon of the RasGRP2 gene (RasGRP2-1) (S167867: manufactured by Applied Biosystems) and siRNA targeting the region in the 7th exon (RasGRP2). -2) (S167865: Applied Biosystems) was used. As a control siRNA, siRNA (Control) (Applied Biosystems) was used.

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

<Arthritis score>
Arthritis score: 0 points: no redness and swelling in the joint, 1 point: mild redness and swelling in the tarsal bone or ankle joint, 2 points: mild redness and swelling from the ankle joint to the tarsal bone 3 points: moderate redness and swelling from the ankle to metatarsal joints, 4 points: severe redness and swelling extending to the ankles, feet, and toes, or toe joint stiffness (See Non-Patent Document 3).

<RasGRP2 knockdown by RNA interference>
10 μM siRNA (RasGRP2-1), siRNA (RasGRP2-2), or siRNA (Control) was mixed with 50 μL atelocollagen (manufactured by Koken) in the joints of CIA rats on day 14 after collagen administration. The siRNA solution was administered.

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

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

  The bone erosion score according to micro CT findings was as follows: 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).

  The micro CT image of the ankle joint of each CIA rat is shown in FIG. 12, and the result of the bone erosion score is shown in FIG. 13 (*: p <0.05, t-test). Severe bone erosion was observed in CIA rats administered with siRNA (Control) (“Control” in the figure), but CIA rats administered with siRNA (RasGRP2-1) or siRNA (RasGRP2-2) (shown in the figure) In “siRNA RasGRP2”), although soft tissue swelling was observed, severe bone erosion did not occur (indicated by an arrow in FIG. 12), and the bone erosion score was small.

  From these results, suppression of RasGRP2 gene expression by RNA interference improves the pathology of RA, that is, RasGRP2 inhibitor is useful as an active ingredient in a pharmaceutical composition for RA treatment. Was confirmed.

Claims (7)

  A pharmaceutical composition comprising a substance that suppresses or inhibits the function of the RasGRP2 gene as an active ingredient.   The pharmaceutical composition according to claim 1, which is used for treatment or prevention of rheumatoid arthritis.   The pharmaceutical composition according to claim 1 or 2, which suppresses infiltration of synovial tissue.   Furthermore, the pharmaceutical composition as described in any one of Claims 1-3 which uses the substance which suppresses or inhibits the function of 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 siRNA targeting 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 synoviocytes 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 rheumatoid arthritis Evaluation method of the possibility of developing rheumatoid arthritis.   A diagnostic marker for detecting rheumatoid arthritis, comprising the expression level of the RasGRP2 gene.