WO2012007632A9 - Use of edg2 inhibitors for treating rheumatoid arthritis - Google Patents

Abstract

The present invention relates to the novel use of EDG2 inhibitors for treating rheumatoid arthritis, particularly in those patients exhibiting elevated levels of pro-inflammatory molecules despite having been treated with anti-inflammatories, i.e. patients not responding to said treatments.

Description

 Use of EDG2 inhibitors for the treatment of rheumatoid arthritis

The present invention is framed in the field of Molecular Biology, Cell Biology, Biotechnology and Biomedicine. The present invention relates to the use of EDG2 inhibitors for the treatment of rheumatoid arthritis, particularly in those patients who have elevated levels of proinflammatory molecules despite being treated with anti-inflammatories. STATE OF THE PREVIOUS TECHNIQUE

 Rheumatoid Arthritis (RA) is a chronic inflammatory disease that affects the peripheral joints and involves pain, inflammation and, in many cases, functional disability, due to the progressive destruction of the cartilage and bone of the affected joints (Firestein GS; Nature 2003, 423: 356-61; Huber LC et al. Rheumatology 2006, 45: 669-675; Pope RM. Nature Rev Immunol 2002, 2: 527-535).

 RA is characterized by hyperplasia of resident synoviocytes and by infiltration of inflammatory cells from the bloodstream. Synoviocytes and inflammatory cells secrete interleukins, chemokines, adhesion molecules and metalloproteases that perpetuate inflammation. Synovial hyperplasia is a decisive factor in joint destruction and there is evidence that seems to indicate that the mechanism responsible for said hyperplasia is the resistance to apoptosis of synovial cells. In vitro and in vivo studies have shown that only a small percentage of synovial cells undergo fasting ligand-dependent apoptosis, TNFa (from English "tumor necrosis factor a") and TRAIL (from English "TNF-related apoptosis-inducing ligand ') , despite expressing cell death receptors (Baier A et al. Curr Opin Rheumatol 2003, 15: 274-279; Korb A et al. Apoptosis 2009, 14: 447-454).

Apoptosis is a very regulated and fundamental process in many physiological situations. Two main pathways can be distinguished, the extrinsic pathway, through the activation of death receptors, and the intrinsic or mitochondrial pathway. In the extrinsic pathway, the union of FasL, TNFa and TRAIL to their receptors leads to the recruitment of FADD (from the English "Fas associated Death Domain") and pro-caspasa-8, which form the DISC (from English "Death-Inducing Signaling Comp / ex "), where caspase-8 is activated. In turn, caspase-8 activates caspase-3, which ultimately causes DNA fragmentation and cell death. The mitochondrial pathway is induced in situations of hypoxia, deprivation of growth factors and exposure to cytotoxic drugs, leading to the release of cytochrome c and activation of caspase-9 mediated by Apaf-1 (Apoptosis protease-activating factor-1) ") (Ashkenazi A. et al. Science 1998, 281: 1305-1308; Green DR et al. Science 2004, 305: 626-629; Wajant H. Science 2002, 296: 1635-1636).

 In recent years, new biological treatments have been developed based on the inhibition of inflammatory cytokines such as TNFa, interleukin 1 (IL-1) and interleukin 6 (IL-6), which have allowed in many cases to prevent disease progression towards destruction of cartilage and bone. However, about 40% of patients do not respond to these therapies and, in an even larger percentage, the response is partial (Chen YF et al. Health Technol Assess, 2006, 10 (42): iii-iv, xi -xiii, 1-229 .; Rubbert-Roth A et al. Arthritis Res Ther 2009, 1 1 Sup 1: S1).

 Lysophosphatidic acid receptor 1 (LPAi, LPAR1, EDG2, Gpcr26,

GPR26, Mrec1 .3, network .3, vzg-1, VZG1) is a receptor coupled to G proteins that in humans is encoded by the gene LPAR1 or EDG2. Nochi et al. (J. Immunology, 2008, 181 (7): 511 1-9) have described that the lysophosphatidic acid receptor 1 (LPAR1 or EDG2) is expressed at high levels in the synoviocytes of patients with RA and that the union of lysophosphatidic acid (LPA) to this receptor activates the expression of cyclooxygenase 2, a molecule involved in inflammation. In addition, Zhao C et al. (Mol Pharmacol, 2008; 73: 587-600) have shown that the binding of LPA to this receptor also induces, in synoviocytes of patients with RA, the secretion of other inflammatory cytokines such as IL-6 and IL -8. Mototani et al. (Hum Mol Genet. 2008, 15; 17 (12): 1790-7) have described that certain polymorphisms in the EDG2 gene increase the likelihood of suffering from knee osteoarthritis. Ikegawa and Mototani (EP2153847A1) have described the use of EDG2 inhibitory agents for the treatment of a variety of conditions that involve destruction of cartilage and bone, among which rheumatoid arthritis is mentioned. These authors describe the anti-inflammatory effect of EDG2 inhibitory agents and propose their use to reduce inflammation in all patients suffering from cartilage and bone diseases. Most of the RA treatments, both pharmacological and biological, are aimed at reducing inflammation, so those patients who do not respond to anti-inflammatory treatments have no solution. Therefore, a new therapeutic approach is needed to control the disease in all patients with RA, including those who do not respond to anti-inflammatory treatments.

DESCRIPTION OF THE INVENTION

 The present invention relates to the new use of EDG2 inhibitors for the treatment of rheumatoid arthritis (RA), particularly in those patients who have elevated levels of proinflammatory molecules after treatment with anti-inflammatories, that is, who do not respond to such treatments.

 The authors of the present invention have shown that inhibition of EDG2 corrects synovial hyperplasia in an inflammatory environment, with elevated TNFa, which mediates the apoptosis of said cells. Therefore, the authors of the present invention have shown that inhibition of EDG2 is effective in improving the clinical evolution of RA in an inflammatory environment where there are high levels of inflammatory molecules and, more specifically, where there are elevated levels of TNFa.

 In addition, the authors of the present invention have observed that inhibition of EDG2 does not correct inflammation, and therefore does not decrease levels of inflammatory molecules.

 Therefore, the present invention is intended to provide a solution to the treatment of RA specifically in those patients who do not respond to conventional therapies, known in the state of the art, and meet the requirements specified throughout the invention related with the levels of proinflammatory molecules, thereby contributing to the field of technology a very useful tool in solving a problem that has not yet been resolved. Thus, the use of EDG2 inhibitors in this type of patients implies an improvement in their quality of life, an aspect that has remained unattended to date.

In this regard, the present invention relates to the use of a pharmaceutical composition (hereinafter pharmaceutical composition of the invention) comprising at least one inhibitor of a protein whose sequence has at least 95% identity with SEQ ID NO: 1 for prepare a medicine for RA treatment. Preferably, the pharmaceutical composition comprises at least one inhibitor of a protein whose sequence has at least 97%, more preferably 98%, even more preferably 99% identity with SEQ ID NO: 1. Preferably, the pharmaceutical composition comprises the less an inhibitor of a protein whose sequence is SEQ ID NO: 1.

 SEQ ID NO: 1 is the amino acid sequence of the human lysophosphatic acid receptor 1 or EDG2. The amino acid sequences of EDG2 of chimpanzee, cow, dog, mouse, rat and rooster have a high homology with the human sequence, as shown in Table 1.

 Table 1 . % sequence identity for the gene and EDG2 protein of humans with different species.

The term "inhibitor" refers to a substance or molecule capable of reducing or eliminating the activity of the EDG2 receptor, either by blocking its function or by reducing its presence. Numerous EDG2 inhibitor molecules have been described to date, both drugs and antibodies blocking the receptor signaling pathway, or as nucleotide sequences that interfere in the translation of the receptor itself from the messenger RNA (mRNA) that encodes it. One skilled in the art knows how to recognize an inhibitor of the EDG2 receptor for example, but not limited to, using the assay described in McAllister et al. 2000. Molecular Pharmacology 58: 407 ^ 112.

The expression "% sequence identity" refers to the percentage of amino acids in a candidate sequence that is identical to the amino acids of SEQ ID NO: 1, after aligning the sequences to achieve maximum percentage of sequence identity. The% sequence identity can be determined by any of the procedures or algorithms established in the art, such as ALIGN or BLAST. Here, the% sequence identity is calculated by dividing the number of amino acids that are identical after aligning SEQ ID NO: 1 and the candidate sequence, by the total number of amino acids of SEQ ID NO: 1, and multiplying the 100 result.

 In a preferred embodiment, the pharmaceutical composition of the invention is used for the treatment of RA of patients presenting more than at least one pro-inflammatory molecule, with respect to a control, despite having been treated with at least one anti-inflammatory agent. Preferably, the proinflammatory molecule is TNF. Even more preferably, it is TNFa.

 The term "quantity", as used herein, refers to the absolute or relative value that is obtained by detecting and quantifying a pro-inflammatory molecule in an isolated sample of an RA patient. This data can be compared with that obtained for healthy individuals, which serve as control.

 An anti-inflammatory agent can be any known molecule that has an anti-inflammatory effect. Multiple anti-inflammatory agents are known, among which are NSAIDs (non-steroidal anti-inflammatory drugs), corticosteroids, methotrexate, anti-TNF or anti-interleukin 6 (anti-IL-6). The last two are therapies based on blocking TNF or IL-6 through the use of antibodies.

In the present description, proinflammatory molecules are understood as those molecules that participate in inflammation, promoting said process. There is a wide variety of proinflammatory molecules, including interleukin 1 (IL-1), interleukin 8 (IL-8), prostaglandins, nitric oxide, interferon and and TNF. In addition, there are some pro-inflammatory enzymes, such as phospholipase type II or PLA ₂ , cyclooxygenase 2 (COX-2) or inducible nitric oxide synthase (¡NOS), which activate the synthesis of platelet activating factor, leukotrienes, prostanoids or nitric oxide. Chemokines are among the inflammatory mediators involved in the pathogenesis of RA. These molecules are potent chemoattractants of the cells involved in the inflammatory response and the expression of chemokines and their receptors has been detected in the synovial fluid and in the synoviocytes of patients with RA. Tumor necrosis factor or TNFa (from the English "tumor necrosis factor alpha") is the first member of the TNF superfamily, to which some cytokines involved in the acute phase of the inflammatory process belong. The members of this superfamily have a common phylogenetic origin and most also have a common function: they promote apoptosis and the activation of the transcription factor NF-KB.

 The authors of the present invention have studied the levels of some proinflammatory molecules, such as IL-6, IL-8, monocyte chemoattractant protein 1 (MCP-1 or CCL2), COX-2, collagenase 1 ( MMP-1) and stromelysin-1 (MMP-3).

 IL-6 is a glycoprotein secreted by macrophages, T cells, endothelial cells and fibroblasts. Its release is induced by interleukin 1 and is increased in response to TNFa. It is a cytokine with anti-inflammatory and pro-inflammatory activity.

 IL-8 is a cytokine of the family of chemokines, of a pro-inflammatory nature. Its synthesis is carried out in fibroblasts, endothelial cells, monocytes and macrophages. It is a potent chemotactic factor of neutrophils, regulates the production of adhesion proteins, the formation of bioactive lipids and amplifies the local inflammatory response.

 MCP-1 induces monocytes to enter tissue from the bloodstream to become tissue macrophages.

 COX-2 is responsible for the biosynthesis of prostanoids involved in inflammatory response.

 MMP-1 and MMP-3 are proteolytic enzymes responsible for the degradation of the extracellular matrix in RA. MMP-3 plays an important role as a mediator of the inflammatory response in the joint.

 In a preferred embodiment, the use of the invention comprises an increase in apoptosis of synovial cells. Preferably, said apoptosis is mediated by TNFa. When apoptosis is mediated by TNFa, the TNFa ligand binds to its receptor and thus activates cell signaling that leads to controlled cell death.

In a preferred embodiment of the invention, the inhibitor is a nucleic acid (hereinafter called the nucleic acid of the invention). Preferably, the Nucleic acid comprises a nucleotide sequence encoding an interference RNA of a protein whose sequence has at least 95% identity with SEQ ID NO: 1. Preferably, the nucleotide sequence encoding the interfering RNA has as target sequences SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and / or SEQ ID NO: 21. In another preferred embodiment of the invention, the inhibitor is an interference RNA of a protein whose sequence has at least 95% identity with SEQ ID NO: 1. Preferably, the inhibitor is an interference RNA whose target sequences are SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and / or SEQ ID NO: 21.

 An interference RNA is a small molecule of about 20-25 nucleotides of double stranded RNA that interferes with the translation of an mRNA and therefore prevents the production of a peptide or protein, causing its deficiency. Here, siRNA ("small interfering RNA") refers to an interfering RNA.

 A target sequence of an interfering RNA or a DNA encoding an interfering RNA is a sequence of the mRNA of the gene whose expression is interfered, where the interfering RNAs are to be bound to prevent the production of the peptide or protein encoded by said mRNA.

 In a preferred embodiment of the invention, the inhibitor is a vector comprising the nucleic acid of the invention. In a preferred embodiment of the invention, the nucleic acid is an appropriate vector for gene therapy. A vector is a nucleic acid molecule used to transfer genetic material to a cell. Apart from said genetic material, a vector may also contain different functional elements that include transcription control elements, such as promoters or operators, regions or enhancers of binding to transcription factors, and control elements to initiate and terminate translation. Vectors include, but are not limited to: plasmids, cosmids, viruses, phages, recombinant expression cassettes and transposons.

The term "gene therapy", as used herein, refers to the transfer of a genetic material of interest (DNA or RNA) to a host to treat or prevent a genetic or acquired disease or condition. The genetic material of interest encodes a product (a polypeptide or protein, a peptide or a functional RNA) whose in vivo production is desired. By For example, the genetic material of interest can encode an interference RNA of therapeutic value.

 In another preferred embodiment of the invention, the inhibitor is an EDG2 antagonist. Preferably, the antagonist is ki 6425.

 The term "antagonist", as used herein, refers to a substance that binds to a receptor preventing agonist binding of said receptor, without causing any effect. In this case, the antagonist prevents the binding of lysophosphatidic acid (LPA) to its receptor, EDG2.

 In another preferred embodiment of the invention, the EDG2 inhibitor is an antibody or a fragment of an antibody.

 The term "antibody", as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain a specifically binding antigen binding and binding site ( immunoreacts) with a protein. There are five major isotypes or classes of immunoglobulins: immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin E (IgE).

 In a preferred embodiment, the medicament is presented in a form adapted to oral administration. In another preferred embodiment, the medicament is presented in a form adapted to parenteral administration. Preferably, the medicament is presented in a form adapted to intravenous administration. Preferably, the medicament is presented in a form adapted to intra-articular administration. Oral administration is one that is done by mouth. Parenteral administration is one that is done through an injection or infusion. Some examples of parenteral administration are subcutaneous, intravenous, intraarticular or intramuscular injection.

In a preferred embodiment, the pharmaceutical composition of the invention is administered in a therapeutically effective amount. The term "effective amount" refers to an amount of a substance sufficient to achieve the intended purpose. For example, an effective amount of an EDG2 inhibitor is an amount sufficient to reduce the activation of said receptor signaling, which can be extrapolated from the data obtained in in vitro assays as described in Gülden and Seibert, 2003. Toxicology, 189: 21 1-222. A "therapeutically effective amount" of an inhibitor to treat a disease or disorder is an amount of the inhibitor sufficient to reduce or eliminate the symptoms of the disease or disorder. The effective amount of a given substance will vary with factors such as the nature of the substance, the route of administration, the size and species of the animal that will receive the substance and the purpose for which the substance is given. The effective amount in each individual case can be determined empirically by the person skilled in the art according to the procedures established in the art.

 In a preferred embodiment, the medicament further comprises a pharmacologically acceptable excipient. The term "excipient" refers to a substance that helps the absorption of the inhibitor, stabilizes it or helps the preparation of the drug in the sense of giving it consistency or providing flavors that make it more pleasant. Thus, the excipients could have the function of keeping the ingredients together such as starches, sugars or cellulose, sweetening function, dye function, drug protection function such as to isolate it from air and / or moisture, function filling a tablet, capsule or any other form of presentation such as dibasic calcium phosphate, a disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph.

 The term "pharmacologically acceptable" refers to the fact that the compound to which it refers is permitted and evaluated so that it is safe, non-toxic and does not cause adverse effects to the organisms to which it is administered.

Another preferred embodiment relates to the use of the pharmaceutical composition where the medicament further comprises a pharmaceutically acceptable carrier. In addition, the vehicle must be pharmacologically acceptable. A "pharmaceutically acceptable carrier" refers to those substances, or combination of substances, known in the pharmaceutical sector, used in the preparation of pharmaceutical forms of administration and includes, but are not limited to, solids, liquids, solvents or surfactants. The vehicle can be an inert substance or action analogous to any of the inhibitors of the present invention. The function of the vehicle is to facilitate the incorporation of the inhibitor, as well as other compounds, to allow a better dosage and administration or to give consistency and form to the pharmaceutical composition. When the presentation form is liquid, the vehicle is the diluent. In a preferred embodiment, the medicament further comprises another active substance. In a preferred embodiment, the medicament is administered as a combination therapy. A combination therapy refers to the medication being given along with another medication. Preferably, this other medication is an immunomodulatory substance, that is, a substance capable of altering the immune system's response by activating, suppressing or modifying some of its mechanisms. More preferably, the immunomodulatory substance is the anti-CD20 antibody (Rituximab) or the CTLA4-immunoglobulin fusion protein (Orencia, abatacept, belatacept).

 Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

FIG 1. EDG2 deficiency increases synoviocyte apoptosis in the presence of TNFa. It shows the activity of caspase 3/7, which is significantly higher in synoviocytes deficient in EDG2 and especially in the presence of TNFa (p = 0.00058, Wilcoxon test). Activated caspase 3/7 hydrolyzes the luminous substrate DEVD-aminoluciferin and generates a luminescent signal directly proportional to caspase activity 3/7, so that this activity is expressed in RLU (relative luminescence units).

FIG 2. Shows the change in gene expression in 7 synoviocyte lines with EDG2 siRNA relative to expression in the same lines with non-specific siRNA (control). EDG2 deficiency increases TNFa-induced transcription of IL-6, IL-8, MCP-1, COX-2 and MMP-1, and reduces MMP-3 transcription.

FIG 3. Shows the amount of IL-6, IL-8 and MCP-1 in culture supernatants of four lines of rheumatoid synoviocytes transfected with EDG2 siRNA or with nonspecific control siRNA. EDG2 deficiency increases the TNFa-mediated production of these inflammatory mediators. FIG 4. Shows a significant decrease in the clinical score of rheumatoid arthritis in mice treated with KM 6425 compared to control mice, treated with vehicle (p = 0.03, day 3; p = 0.0068, day 6; p = 0.0018, day 7; p = 0.00072, day 8 and p = 0.00072, day 9).

FIG 5. The suppression of EDG2 increases the protein expression of TRAIL (A), TRADD (B) and PYCARD (C) in synoviocytes with RA. RA synoviocytes transfected with control siRNA or with siRNA for EDG2 were treated with 10 ng / ml of TNF for 12 h and total proteins were extracted for western blot analysis. Densitometric quantification of the normalized band intensity with respect to an actin control is shown, and the data are expressed as mean (SEM) of synoviocytes from 6-8 patients with RA. Representative blots are shown. ^* indicates p <0.05 and ^*** indicates p <0.001. FIG 6. TRAIL, TRADD and PYCARD are necessary for the increase of TNF-induced apoptosis in synoviocytes with RA deficient in EDG2. Synoviocytes with RA were either not transfected (NT) or transfected with: control siRNA, siRNA for EDG2, siRNA for EDG2-TRADD, siRNA for EDG2-TRAIL, or siRNA for EDG2-PYCARD. Apoptosis was determined by quantification of nucleosome release after 48 h with 10ng / ml of TNF. The graphs show the release of nucleosomes as a number of times with respect to the untransfected control. Data are expressed as the mean (SEM) of synoviocytes in 6-8 patients with RA. ^* indicates p <0.05 and ^** indicates p <0.01.

FIG 7. Shows the increase in the TNF-induced inflammatory response in synoviocytes with RA with EDG2 suppressed. RA synoviocytes transfected with control siRNA, siRNA for EDG2 or siRNA for EDG2-TRADD were stimulated with 10 ng / ml of TNF for 12 h and the secretion of IL-6, IL-8 and MCP-1 was analyzed in the culture supernatant (A) and activation of MAP kinases was analyzed by western blot (B). Data are expressed as the mean ± SEM of 6-9 different synoviocyte lines with RA. ^* and # indicate p <0.05, Wilcoxon test. A representative blot is shown.

EXAMPLES

 The invention will now be illustrated by tests carried out by the inventors, which show the specificity and effectiveness of the use of EDG2 inhibitors for the treatment of rheumatoid arthritis (RA).

EXAMPLE 1: EDG2 deficiency in synoviocytes of patients with RA in vitro.

 7 cell lines of fibroblast-like synoviocytes (FLS) were transfected with EDG2 siRNA or with a nonspecific control siRNA. They were stimulated with 10 ng / ml of TNFa and apoptosis was analyzed after 48 hours of treatment. Using quantitative or real-time PCR, the efficiency of EDG2 suppression was assessed, which was greater than 90% in all transfected cell lines.

 The apoptosis of these cells was analyzed by determining the activity of caspase 3/7 and quantifying oligonucleosomes. A significant increase in TNFa-induced apoptosis was observed in synoviocytes deficient in EDG2 compared to controls (Figure 1, p = 0.00058, Wilcoxon test). Similar results were obtained in the nucleosome release analysis.

 To analyze the effect of EDG2 suppression on the inflammatory response induced by TNFa in rheumatoid synoviocytes, the levels of messenger RNA (mRNA) of IL-6, IL8 / CXCL8, MCP-1 / CCL2, COX-2, MMP were determined -1 and MMP-3 (stromielisin 1) after stimulation with TNFa, by quantitative real-time PCR.

 As seen in Figure 2, EDG2 suppression induced an increase in TNFa-induced transcription of IL-6, IL-8, MCP-1, COX-2 and MMP-1. However, a reduction in MMP-3 transcription was observed.

 The result obtained by quantitative PCR was confirmed by ELISA analyzing the production of IL-6, IL-8 and MCP-1 in culture supernatants of four lines of rheumatoid synoviocytes transfected with EDG2 siRNA or with non-specific siRNA. As seen in Figure 3, EDG2 suppression increased TNFa-mediated production of these inflammatory mediators.

 All these results indicate that TNFa-mediated apoptosis sensitization after suppression of EDG2 in rheumatoid synoviocytes is not accompanied by a reduction in the TNFa-mediated inflammatory response.

FLS cell lines.

This work was carried out on 7 primary lines of FLS from patients with RA. In the laboratory, synoviocytes were isolated from synovial tissue of patients with RA undergoing joint replacement surgery or synovial biopsy. All patients met the criteria of the College American Rheumatology (ACR) 1987 for the diagnosis of RA (Arnett FC et al. 1988 Arthritis Rheum. 31 (3): 315-24). Synoviocytes were used between passes 4 and 8 in all experiments, grown in DMEM medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin and 1% L-glutamine at 37 C and 5% CO2 for later use

 Transfection with interfering RNA (siRNA) in rheumatoid synoviocytes.

This technique was used to suppress the expression of the LPA Receptor associated with G proteins: EDG2 or LPA1, in synoviocytes of patients with RA. 8.5 x 10 ⁴ cells / well were seeded in wells of P6 plates in DMEM-10% FBS and incubated overnight at 37 ° C. Then the medium was removed and DMEM 10% FBS was added without antibiotic for 6 hours. . After this time, the cells were transfected with 20 nM of EDG2 siRNA or with nonspecific control siRNA (Dharmacon, Fisher Bioblock Scientific, Cedex, France) in 1.25 μg / ml of Dharmafect transfection reagent (Dharmacon), diluted in medium OPTI-MEM I (Gibco). After 24 hours, the transfection medium was removed and DMEM-10% FBS was added. The silencing efficiency for EDG2 was verified by real-time PCR and in all cases it was greater than 90%. The experiments were performed 48-96 hours after transfection.

TNF-α treatment of rheumatoid synoviocytes with EDG2 siRNA or with a nonspecific siRNA.

 Rheumatoid synoviocts were deprived of serum for 16 hours in DMEM-1% FBS. After this period of time, they were stimulated with TNF-α at a concentration of 10 ng / ml. The treatment time was adjusted according to the test to be performed. Thus, the expression and production assays of the cytokines IL-6 and IL-8, the chemokine MCP-1 and the metalloproteases MMP-3 and MMP1, and the enzyme COX-2 (from English "prostaglandin-endoperoxide synthase 2" ), were performed at 12 hours after starting treatment, and apoptosis assays were carried out at 48 hours of stimulation with TNF-α (Figure 2).

Polymerase chain reaction (PCR) in real time

 Using this technique, mRNA expression levels of different genes in human rheumatoid synoviocytes were analyzed.

First, the level of silencing for LPA1 was verified in synoviocytes treated with the EDG2 siRNA versus baseline expression in Control synoviocytes For this, the total RNA of the synoviocytes, cultured in wells of P6 plates (8.5 x 10 ⁴ cells / well), was extracted by the Rneasy Kit and the RNase-Free DNase Set (Qiagen GmbH), following the instructions manufacturer.

 Real-time PCR was performed on a Mx3005P Real-Time thermal cycler

PCR System (Strategene, La Jolla, CA, USA) using the Brilliant II SYBR Green Single Step QRT-PCR Master Mix kit (Stratagene). PCR reactions were carried out in a total volume of 25 μΙ containing 2-5 ng of RNA; 0.5 μΜ of each primer; 1.5 mM MgC ^; 30 nM of the ROX reference dye; 0.04x of StrataScript RT / RNase block enzyme mixture and 1x of SYBR QRT-PCR master mix. The samples were analyzed in duplicate and the primers that were used are shown in Table 2.

 Table 2. Primers used for real-time PCR and amplified fragment size.

The amplification conditions for EDG2 were the following: Synthesis of the copy DNA (cDNA) at 50 ° C for 30 minutes; denaturation at 95 ° C for 10 minutes followed by 40 cycles of denaturation at 95 ° C for 30 seconds, hybridization at 63 ° C for 1 minute and elongation at 72 ° C for 30 seconds. Finally, a dissociation curve of 55 ° C to 95 ° C was performed increasing 1 ° C every second. Β-actin was used as a normalizer.

 The mRNA levels of IL-6, IL-8, MCP-1, COX-2, MMP-3 and MMP1 were also analyzed in synoviocyte cultures of patients with RA treated with the EDG2 siRNA or with a nonspecific siRNA, submitted to stimulation with TNF-a, by real-time PCR in a single step.

 The amplification conditions for IL-6, IL-8 and MCP-1 were: cDNA synthesis at 50 ° C for 30 minutes; denaturation at 95 ° C for 10 minutes followed by 40 cycles of denaturation at 95 ° C for 30 seconds and hybridization at 63 ° C for 1 minute. Finally, a dissociation curve of 55 ° to 95 ° C was performed increasing 1 ° C every second.

 The amplification conditions for COX-2 were as follows: cDNA synthesis at 50 ° C for 30 minutes; denaturation at 95 ° C for 10 minutes followed by 40 cycles of denaturation at 95 ° C for 30 seconds, hybridization at 60 ° C for 1 minute and elongation at 72 ° C for 1 minute. Finally, a dissociation curve of 55 ° to 95 ° C was performed increasing 1 ° C every second.

 The amplification conditions for MMP-3 were the following: cDNA synthesis at 50 ° C for 30 minutes; denaturation at 95 ° C for 10 minutes followed by 40 cycles of denaturation at 95 ° C for 30 seconds, hybridization at 63 ° C for 1 minute and elongation at 72 ° C for 1 minute. Finally, a dissociation curve of 55 ° to 95 ° C was performed increasing 1 ° C every second.

 The amplification conditions for MMP-1 were as follows: cDNA synthesis at 50 ° C for 30 minutes; denaturation at 95 ° C for 10 minutes followed by 40 cycles of denaturation at 95 ° C for 30 seconds, hybridization and elongation at 57 ° C for 1 minute. Finally, a dissociation curve of 55 ° to 95 ° C was performed, increasing 1 ° C every second.

Β-actin was used as a normalizer. The purity of the amplified products was analyzed by agarose gel electrophoresis and determination of the dissociation curve of each sample. The analysis of the results was performed using the comparative method Ct. 2, in which: ¿_ICt = [Ctg _en - Ct _{ac t na} ] synoviocytes transfected with nonspecific siRNA "[Ctgen-Ct _{ac t na} ] synoviocytes transfected with siRNA EDG2-

For synoviocytes transected with nonspecific siRNA AACt = 0 and 2 ^o = 1. For synoviocytes transfected with EDG2 siRNA the value 2 ^" indicates the increase or decrease in the levels of gene expression compared to those transfected with non-specific siRNA.

ELISA (Enzyme-Linked Immunosorbent Assay).

Using this technique, the expression levels of the IL-6, IL-8 cytokines and MCP-1 chemokine present in the synoviocyte culture supernatant of patients with RA treated with EDG2 siRNA or with a non-specific siRNA were analyzed. Rheumatoid synoviocytes (10 ⁴ cells / well) were seeded in 96-well plates in DMEM-10% FBS and deprived of serum for 16 hours in DMEM-1% FBS. Synoviocytes treated with EDG2 siRNA or with a nonspecific siRNA were stimulated with 10 ng / ml of TNF-α for 12 hours, at which time the supernatant was collected for later analysis.

 The cytokine determination was carried out by ELISA kits for IL-6, IL-8 and MCP-1 (OptEIA ELISA Sets, BD Pharmingen), following the manufacturer's instructions.

Apoptosis Assays

 The percentage of apoptosis of rheumatoid synoviocytes was analyzed by two different techniques, apoptosis ELISA and determination of caspase activity 3/7 (FIG 1).

After suppressing EDG2 expression in rheumatoid synoviocytes by siRNA, 3x10 ³ cells / well were seeded in P96 plates in DMEM-10% FBS and serum deprived for 16 hours in DMEM-1% FBS. Subsequently, the medium was removed and stimulated with 1 μg / ml TNF-α for 48 hours in DMEM-1% FBS.

 The percentage of apoptosis was determined by the Cell Death Detection ELISA Kit (Roche Diagnostics, Spain), which detects the mono and oligonucleosomes released to the cytoplasm during apoptosis.

To quantify the activity of caspase 3/7, 10x10 ³ cells / well were seeded in P96 plates in DMEM-10% FBS and deprived of serum for 16 hours in DMEM-1% FBS. Subsequently, the medium was removed and stimulated with 1 μ9 / ηιΙ of TNF-α for 48 hours in DMEM-1% FBS. Caspase 3/7 activity in synoviocytes treated with the EDG2 siRNA or with a non-specific siRNA was determined by the Caspase-Glo ^R 3/7 Assay kit (Promega), which determines the presence of caspase 3/7 activated by processing of a luminogenic substrate (DEVD-aminoluciferin). For this, the cells were incubated for 1 h with the reconstituted reagent of the kit and the activated caspase 3/7 of the cells generated the luminescent signal after the hydrolysis of the DEVD-aminoluciferin luminogenic substrate. This signal was, therefore, proportional to caspase activity 3/7 and was measured in a Fluostar OPTIMA microplate reader (BMG Labtech, Offenburg, Germany).

 EXAMPLE 2: Treatment of RA in vivo with an EDG2 antagonist.

 Arthritis was induced in a total of 41 C57BL / 6 mice of 8-10 weeks of age by injecting 2 doses of 100 μΙ of a serum from K / BxN arthritic mice at day 0 and day 2.

 22 mice were treated with 4 doses of 20 mg / kg of KM 6425 inhibitor on days 0, 1, 2 and 3 and 19 mice were treated with vehicle following the same therapeutic guideline. The clinical evolution of arthritis until day 9 was analyzed.

 The clinical evolution of arthritis (FIG 4) showed a significant decrease in the clinical score of arthritis in mice treated with KM 6425 compared to vehicle-treated controls (p = 0.03, day 3; p = 0.0068, day 6; p = 0.0018, day 7; p = 0.00072, day 8 and p = 0.00072, day 9), indicating an improvement in clinical outcomes in animals treated with the EDG2 antagonist. Induction of passive arthritis in mice and clinical evaluation of arthritis

 Arthritis was induced in male and female C57BL / 6 mice aged 8-10 weeks using the passive arthritis model K BxN. K / BxN mice were obtained by crossing KRN transgenic mice in B6 background with NOD mice. These transgenic mice develop severe arthritis, which begins early, at 2-3 weeks of age. Injection of serum obtained from K / BxN mice 4-8 weeks of age in C57BL / 6 mice causes the development of arthritis with characteristics similar to human RA.

C57BL / 6 mice were treated with the competitive EDG receptor inhibitor, KM 6425 (Cayman Chemicals, Ann Arbor, Michigan, USA), dissolved in DMSO / PBS (1: 3) or injected only with the vehicle: DMSO- PBS (1: 3). The compound KM 6425 (3 - [[[4- [4 - [[[1 - (2-chorophenyl) ethoxy] carbonyl] amino] -3-methyl-5 isoxazoly] phenyl] methyl] thio] -propanoic acid) is a potent inhibitor of the EDG2 / LPA1 receptors (Ki = 0.35 μΜ) and EDG-7 / LPA3 (Ki = 0.93 μΜ) and, to a lesser extent, inhibits the EDG4 / LPA2 receptor (Ki = 6.5 μΜ) .

 A total of 22 mice were injected with Ki 16425 and 19 mice with vehicle, in 5 different experiments. The therapeutic scheme was as follows: arthritis was induced by intraperitoneal injection of 100 μΙ of serum K / BxN on days 0 and 2. Mice were treated for 4 days with KM 6425 at a dose of 20 mg / kg mouse / day. The first dose was administered on day 30, 30 minutes before the injection of serum K / BxN. The second dose was administered on day 1; the third, on day 2, 30 minutes before the injection of serum K / BxN and the last one was administered on day 3. Control mice were injected with DMSO / PBS (1: 3) following the same therapeutic scheme.

 The clinical evolution of arthritis in the back and front legs was evaluated by two independent observers on days 3, 6, 7, 8 and 9, following a semi-quantitative scale from 0 to 4: grade 0, without inflammation; grade 1, slight edema and joint erythema; grade 2, moderate edema and erythema; grade 3, severe edema and erythema; Grade 4, maximum inflammation with stiffness gives the joint. Following this scale, the "Clinical Score" or clinical graduation of the severity of arthritis is obtained. The maximum possible value of this "Score" for each mouse is 16 and corresponds to the sum of the maximum values obtained in each of the 4 legs. Mice were sacrificed on day 9 for joint removal.

 EXAMPLE 3: Overexpression of genes related to apoptosis in synoviocytes deficient in EDG2.

 The expression of 84 genes related to apoptosis was analyzed using a PCR array of human apoptosis. Synoviocytes from 6 patients with RA were analyzed after 12 hours of stimulation with 10 ng / ml of TNF.

PCR Array of Human Apoptosis

The relative expression of 84 genes related to apoptosis was analyzed using the human apoptosis PCR array (SABiosciences). Synoviocytes from patients with RA (4x10 ⁵ cells / well) were cultured with 10 ng / ml of TNF for 12 hours. Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Venlo, Holland). The cDNAs were subjected to reverse transcription from ^ g of RNA using the ReactionReady First Strand cDNA Synthesis Kit (SABiosciences, Tebu-Bio, Boechout, Belgium). Twenty μΙ cDNA from each sample was mixed with RT ² real-time SYBR Green / Rox PCR Master Mix (SABiosciences) and real-time PCR was performed with reverse transcriptase (qRT-PCR). Relative levels of gene expression were normalized with respect to 5 reference genes using the comparative method of Ct. The raw data in the array was processed and analyzed using the PCR Array Data Analysis System through the website http://sabiosciences.com/pcrarraydataanalysis.php with the threshold set at 2.0. Western blot

FLS with AR (8.5 10 ⁴ cells / well) were cultured in six-well plates, treated or not with 10 ng / ml of TNF-α for 12 hours, washed twice with cold PBS, and the proteins extracted using a buffer of lysis (50 mM Tris HCI pH 7.5, 250 mM NaCI, 1% Triton X-100, 30 mM NaPO, 5 mM EDTA pH 8.0, 100 mM NaF, 1 mM Na ₃ V0 ₄ , 10 μg / ml aprotinin , 10 μg / ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Protein concentrations in the extracts were determined using the Qubit fluorometer (Invitrogen, Prat de Llobregat, Barcelona, Spain) according to the manufacturer's protocol. Lysates of whole cells (10-30μg of protein) were fractionated by SDS-PAGE with 10% sodium, transferred to polyvinylidene fluoride (PVDF) membranes (Hybond-P, Amersham Biosciences, Little Chalfont, United Kingdom) and incubated with antibodies against P-p38, p38, P-ERK, ERK, β-actin (Sigma-Aldrich) P-JNK, JNK, PYCARD (Santa Cruz Biotechnology, CA, USA), TRAIL (Cell Signaling Technology, New England BioLabs ) and TRADD (BD Pharmingen, San José, California, USA). The bound antibody was revealed with secondary antibodies conjugated with horseradish peroxidase (Santa Cruz) and blot with ECL Plus Detection System (Amersham Biosciences, UK) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific Inc).

 A total of 17 genes showed significant overexpression in synoviocytes

FLS transfected with EDG2 siRNA compared to synoviocytes transfected with control siRNA (those genes whose expression changed 2 or more times that of the control and with p <0.05) were considered. Among the 17 genes that showed overexpression in synoviocytes from patients with RA lacking EDG2, 9 were pro-apoptotic genes (BCL2L11, CASP1, CASPIO, CASP3, CASP7, PYCARD, TNFRSF25, TNFSF10, TRADD), 4 anti-apoptotic genes (BCL2, BIRC3, XIAP, CFLAR) and 4 were genes with function both pro and anti-apoptotic depending on the stimulus and tissue analyzed (CD40, RIPK2, TNF, CD70). Three genes with pro-apoptotic function (TRAIL, TRADD and PYCARD; Table 3) were chosen for confirmatory experiments given the significant change in their level of expression (p <0.01) and given their location downstream in the apoptotic pathway. Its overexpression was confirmed by western blot analysis in synoviocytes of patients with RA. As shown in Figure 5, the levels of TRAIL, TRADD and PYCARD proteins are higher when EGD2 is suppressed by siRNA than when it is not suppressed (Figure 5; p <0.001, p <0.015, p <0.015, respectively, test of Wilcoxon).

Table 3: The 3 pro-apoptotic genes (PYCARD, TRAIL and TRADD) selected for confirmation by western blot analysis with statistically significant overexpression, equal to or greater than 2 times the expression observed in synoviocytes transfected with control siRNA.

EXAMPLE 4: Awareness to apoptosis by suppression of EDG2 is dependent on TRAIL, TRADD and PYCARD.

The role of TRAIL, TRADD and PYCARD in the sensitization to TNF-induced apoptosis in synoviocytes from patients with RA without EDG2 was studied by suppressing these genes by siRNA. Synoviocytes with RA were either not transfected or transfected with control siRNA, siRNA for EDG2 alone, or combined with siRNAs for TRAIL, TRADD or PYCARD. The Suppression efficiency was verified by western blot of total synoviocyte proteins.

 The expression of TRAIL, TRADD and PYCARD proteins was suppressed by 76.6 ± 0.06%, 95.2 ± 0.03% and 74.9 ± 0.09%, respectively. TNF-induced apoptosis was analyzed in synoviocytes with RA in which EDG2 and TRAIL, TRADD or PYCARD were suppressed (Figure 6). Suppression of any of the three pro-apoptotic genes reversed apoptosis sensitivity (p = 0.03, p = 0.03, p = 0.008; for TRAIL, TRADD and PYCARD, respectively, Wilcoxon test). Specifically, TNF-mediated apoptosis was suppressed when EDG2 suppression was combined with that of TRAIL or PYCARD, since, in both cases, the level of apoptosis was similar to that observed in synoviocytes with RA not transfected or transfected with control siRNA. The suppression of TRADD led to a decrease in apoptosis, but still the levels of apoptosis were significantly higher than in synoviocytes with RA transfected only with control siRNA (Figure 6).

 Transfection of synoviocytes from patients with RA with siRNA from TRAIL, TRADD and PYCARD.

siRNAs for Apo2L / TRAIL, TRADD and PYCARD / TMS1 / ASC, as well as control siRNAs were obtained from Dharmacon (Fisher Bioblock Scientific, Strasbourg, FR). Synoviocytes with RA (8.5 x 10 ⁴ cells / well in 6-well plates) were transiently transfected with siRNA for TRAIL (50nM), siRNA for TRADD (100nM), siRNA for PYCARD (100 nM) or control siRNA in medium reduced serum Opti-MEM I (Gibco) using 1.25 mg / ml DharmaFECT 1 (Dharmacon). Suppression was determined by Western blot or quantitative real-time PCR. The experiments were carried out at 72-112 hours after transfection.

 EXAMPLE 5: The suppression of EDG2 increases the TNF response of synoviocytes with RA.

The effects of EDG2 suppression on the TNF response of synoviocytes with RA were analyzed by the production of inflammatory mediators and the activation of the three MAPKinases: p38, JNK and ERK, which are downstream in the signaling pathway. TNF Synoviocytes with RA were stimulated with TNF, and the secretion of IL-6, IL-8 and MCP-1 was determined in the culture supernatant by ELISA. As shown in Figure 7A, EDG2 suppression increases TNF-induced production of the three mediators analyzed. This increased inflammatory response to TNF was accompanied by an increase in MAP kinase activation since the expression of p38, pERK and pJNK was significantly higher in synoviocytes with RA deficient in EDG2 compared to synoviocytes transfected with control siRNA (Figure 7B).

 Since TRAIL, TRADD and PYCARD are also involved in inflammation, the impact of their suppression on the increase in the inflammatory response to TNF observed in synoviocytes with RA deficient in EDG2 was analyzed. As shown in Figure 7A, the absence of TRADD in synoviocytes without EDG2 completely suppressed the increase in the inflammatory response to TNF. However, the absence of TRAIL or PYCARD did not change the production levels of IL-6, IL-8 and MCP-1 in synoviocytes deficient in EDG2.

ELISA

The synoviocytes of patients with RA (1 χ 10 ⁴ cells / well) were stimulated with TNF-a (10 ng / ml, Sigma Aldrich) for 12 hours. Supernatants were collected at 12, 24, and 36 hours of treatment, and analyzed for IL6, IL8 / CXCL8 and monocyte chemoattractant protein (MCP) -1 / CCL2 by ELISA as described in Example 1.

Claims

1. Use of a pharmaceutical composition comprising at least one inhibitor of a protein whose sequence has at least 95% identity with SEQ ID NO: 1 to prepare a medicament for the treatment of rheumatoid arthritis.

2. Use of the pharmaceutical composition according to claim 1 for the treatment of rheumatoid arthritis of patients presenting more than at least one pro-inflammatory molecule, with respect to a control, despite having been treated with at least one anti-inflammatory agent.

3. Use according to claim 2 wherein the proinflammatory molecule is TNF.

4. Use according to claim 3 wherein the proinflammatory molecule is TNFa.

5. Use according to any of claims 1 to 4 wherein the inhibitor of a protein whose sequence has at least 95% identity with SEQ ID NO: 1 is a nucleic acid.

6. Use according to claim 5 wherein the nucleic acid comprises a nucleotide sequence encoding an interfering RNA of a protein whose sequence has at least 95% identity with SEQ ID NO: 1.

7. Use according to claim 6 wherein the nucleotide sequence encoding the interfering RNA has as target sequences SEQ ID NO: 18, SEQ ID

NO: 19, SEQ ID NO: 20 and / or SEQ ID NO: 21.

8. Use according to any of claims 1 to 4, wherein the inhibitor is a vector comprising the nucleic acid described in any of claims 5 to 7.

9. Use according to claim 5 wherein the nucleic acid is an interfering RNA of a protein whose sequence has at least 95% identity with SEQ ID NO: 1.

10. Use according to claim 9 wherein the nucleic acid is an interfering RNA whose target sequences are SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and / or SEQ ID NO: 21.

eleven . Use according to any one of claims 1 to 4 wherein the inhibitor of a protein whose sequence has at least 95% identity with SEQ ID NO: 1 is an EDG2 antagonist.

12. Use according to claim 11 wherein the antagonist is ki 6425.

13. Use according to any of claims 1 to 4 wherein the inhibitor of a protein whose sequence has at least 95% identity with SEQ ID NO: 1 is an antibody or a fragment of an antibody.

14. Use according to any of claims 1 to 13 wherein the medicament is presented in a form adapted to oral or parenteral administration.

15. Use according to claim 14 wherein the medicament is presented in a form adapted to intravenous administration.

16. Use according to claim 15 wherein the medicament is presented in a form adapted to intra-articular administration.

17. Use according to any of claims 1 to 16 wherein the medicament further comprises a pharmacologically acceptable excipient.

18. Use according to any of claims 1 to 17 wherein the medicament further comprises a pharmaceutically acceptable carrier.

19. Use according to any of claims 1 to 18 wherein the medicament further comprises another active substance.