MX2011008969A - Methods of diagnosis and treatment of osteoarthritis at early stages using interleukin il-1ss as early biomarker of the disease. - Google Patents

Abstract

The present invention relates to methods of detection and treatment of osteoarthritis (OA) at early stages of development of the disease through the detection and neutralization of IL1 ß as an early biomarker of OA. The method of the invention also refers to the relationship of the expression of pro-inflammatory and anti-inflammatory cytokines in healthy cartilage and osteoarthritic cartilage as a mean to assess the role IL-1ß as a potential biomarker of early OA. The present invention also provides efficient methods for treating OA at early stages by using inhibitors of IL-1ß activity in the cartilage, thereby preventing the progression of OA to stages of difficult treatment or to reverse the damage on the cartilage by the presence of IL-1ß.

Description

Methods of diagnosis and treatment of osteoarthritis in early stages through the use of interleukin IL-? ß as early biomarker of the disease Field of the invention.

The present invention relates to methods of diagnosis and treatment of degenerative diseases, particularly to the diagnosis and treatment of osteoarthritis (OA) from early stages through the use of interleukin-ß-ß (IL-? ß) as an early biomarker of OA , specifically through the early detection of IL-? ß in the cartilage and by neutralizing its activity in the articular cartilage in these stages.

BACKGROUND OF THE INVENTION Osteoarthritis (OA) is a chronic-degenerative and incapacitating disease, characterized by the deterioration and gradual destruction of the articular cartilage that covers the bone in all the joints, the limited intra-articular inflammation with synovitis and the alteration in the peri-articular bone. and subchondral1.

Data from the control and disease prevention center of Atlanta in the United States of America indicate that OA is among the diseases of greatest prevalence and incidence in the North American population, where at least 27 million adults have it2, being the OA up to 12 times more frequent than rheumatoid arthritis3. It has been estimated that 26% of the Mexican population has some symptom of rheumatic disease in which OA is the most prevalent4. The economic burden for primary care of patients with OA represented, in the year 2000 for the USA, about 26 billion dollars, representing 2.5% of its GDP5, while a sensitivity study conducted by the OECD found that in Mexico allocates 0.4% of GDP to the attention of rheumatic diseases.

One of the relevant pathological features of OA is the progressive degradation of articular cartilage, which is an avascular and aneural tissue composed of an extracellular matrix (EM), tissue fluid and chondrocytes as the only cell type. The ME is constituted by a network of collagen (collagen II, IX, and XI) and proteoglycans (mainly aggrecan) that together determine the physical-mechanical properties of the cartilage. Cartilage damage occurs due to mechanical stress on the joints and the enzymatic activity of metalloproteinases (MMPs-2, -3, -13) and aggrecanases (ADAMTs-4 and -5) 6 on ME, which are ridiculed by the activity of pro-inflammatory cytokines such as IL-? ß and TNF-a7.

Changes in EM are related to the phenotypic variability described in human cartilage and in an experimental model of OA in rats, 8,9,10 and during the progression of OA several cell populations have been described: a) the type 1 subpopulation consisting of normal chondrocytes located in the superficial zone and the upper middle zone; b) the type 2 subpopulation formed by secretory chondrocytes (with increase in endoplasmic reticulum and Golgi apparatus) and, c) the type 3 subpopulation consisting of chondrocytes that are under a programmed cell death process.

The presence of programmed cell death in the OA of cartilage of patients with OA was first described by the applicants11 and later reported in other studies12, 13, 14. These observations have allowed proposing the hypothesis of activation and "transdifferentiation" of the chondrocyte15 that indicates that when receiving harmful signals, normal chondrocytes present morpho-functional changes to become chondrocytes with reparative capacity but that by continuing the harmful signals, the chondrocytes trigger their death process, which according to the studies carried out by the applicants, involves a combination of apoptosis and autophagy16. Therefore, the loss of the ME of the cartilage and the death of the chondrocytes play a fundamental role in the pathogenesis of OA.

OA is not considered a typical inflammatory disease due to the absence of neutrophils in the synovial fluid and the absence of systemic manifestations of inflammation. Nevertheless, in patients with OA, the presence of synovitis with infiltration of activated mononuclear cells as CD4 + and CD68 + has been described, as well as the overexpression of pro-inflammatory mediators such as IL-? ß and tumor necrosis factor alpha (TNF-a) 17) Therefore, synovial inflammation is a factor that contributes to dysregulation of chondrocyte function, which favors an imbalance in anabolic and catabolic activities during remodeling of ME18. Articular cartilage chondrocytes also synthesize and secrete the pro-inflammatory mediator IL-? ß, which locally contributes to the onset of cartilage damage by inhibiting the expression of ME proteins such as aggrecan and collagen type u19 , 20-21, increases the synthesis of matrix metalloproteases (MMP; -l, -3, -13, and ADAMTs-4, -5) 21,22 and induces the death process of the chondrocyte.

At present there is no treatment that modulates, stops or repairs the progressive degenerative process of cartilage in OA, applying only measures aimed at reducing symptoms or in extreme cases, the surgical replacement of affected joints.

In an attempt to provide effective therapies for OA, various works prior to the present invention have employed the blocking of the activity of IL-? ß to reverse the disease, by gene therapy and the application of various recombinant proteins. In this regard, for example, significant advances have been obtained with the viral transfer of the gene that codes for the receptor antagonist to IL-1 (IL-IRa) 23'24'25.

For its part, Ling Shari26 describes a method for evaluating OA by determining an amount of protein or molecule expression such as soluble vascular adhesion protein 1 (sVAP-1) or interleukin-15 however, no treatment with the use of said molecule. Hannum27 discloses obtaining an inhibitor of IL-1 which suppresses IL-1, having a specific amino acid sequence and which is useful as an immunosuppressant agent, anti-inflammatory agent or similar, for OA therapy.

Tobinick 28 describes cytokine antagonists that are provided for the treatment and prevention of damage to optic nerves, cranial nerves, the spinal cord, nerve roots or muscles affected by OA, bone disorders, etc., including antagonist molecules for the factor of tumor necrosis, IL-1, IL-6 and IL-8.

Despite the fact that with the previous methods, encouraging results have been obtained for the treatment of the disease, it has been observed that with these methods the protection of the cartilage is not total, most likely due to the stage of the disease in which they are applied ( for example in advanced stages). In this sense, the recombinant protein Anakinra® (recombinant form of the antagonist for the human IL-1 receptor) has been used in a clinical trial in patients with advanced OA, however, the results obtained with said therapy showed no relevant benefit in patients probably treated at the stage of disease progression29.

Due to the above, it is necessary to provide and develop OA diagnostic methods that allow the detection of suitable target molecules from early stages of the disease allowing This is its timely treatment, as well as methods of early treatment of OA using these molecules as therapeutic targets, which would increase the opportunities for more efficient treatment of said disease.

Brief description of the figures.

Figure 1. Pro-inflammatory cytokine levels in rat cartilage are shown on the indicated training days (td). The rats were sacrificed and the articular cartilage was processed by Western blot. (A) The data is the average proportion of IL-i / actin + standard deviation of the early OA and NE groups. (B) The data are the average proportion of TNF- / actin ± standard deviation of the early OA and NE groups. Representative experiments of Western blot are shown. The statistical significance is represented as p < 0.05 (*) or P < 0.01 (**) when the values were compared with the normal cartilage and p < 0.05 (#) or p < 0.01 (s) when the NE cartilage data were compared with OA cartilage. Three independent experiments were performed (n = 3). Figure 2. The expression of IL-? Β in the cartilage of rats is shown on the indicated training days (td). The rats were sacrificed and the cartilage, articular, was processed for immunofluorescence of IL-? ß. (A) Representative immunofluorescence experiments are shown for cartilage samples OA and NE. The IL-? ß was labeled with FITC and the nuclei were contrasted with propidium iodide; The samples were examined under a Leica SP5 confocal microscope. (B) The data are the mean of the positive cells for IL-? ß ± standard deviation of the early OA and NE groups. The statistical significance is represented as p < 0.01 (**) when the values were compared with the normal cartilage and p < 0.01 () when the NE cartilage data were compared with the OA cartilage experiments. Three independent experiments were performed (n = 3).

Figure 3. The expression of TNF-a in the cartilage of rats is shown on the indicated training days (td). The rats were sacrificed and the articular cartilage was processed for indirect immunofluorescence of TNF-α. (A) Representative immunofluorescence experiments are shown for cartilage samples OA and NE. The TNF-a in the OA cartilage was marked with FITC and the nuclei were contrasted with propidium iodide, whereas in the NE cartilage the TNF-ct was labeled with TRIC and the nuclei with DAPI. The samples were examined under a Leica SP5 confocal microscope. (B) The data are the mean of the cells positive for TNF-a ± standard deviation of the early OA and NE groups. The statistical significance is represented as p < 0.01 (**) when the values were compared with the normal cartilage and p < 0.01 () when the cartilage data NE were compared with the OA cartilage experiments. Three independent experiments were performed (n = 3).

Figure 4. The levels of anti-inflammatory cytokines in the cartilage of rats are shown on the indicated training days (td). The rats were sacrificed and the articular cartilage was processed by Western blot. (A) The data is the average proportion of TGF ^ l / actin ± standard deviation of the early OA and NE groups. (B) The data are the average proportion of IL-10 / actin ± standard deviation of the early OA and NE groups. Representative experiments of Western blot are shown. The statistical significance is represented as p < 0.05 (*) or p < 0.01 (**) when the values were compared with the normal cartilage and p < 0.05 (") when the NE cartilage data were compared with the OA cartilage Three independent experiments were performed (n = 3) Figure 5. The expression of TGF-ββ in the cartilage of rats, in the days of The rats were sacrificed and the articular cartilage was processed for indirect immunofluorescence of TGF-β. (A) Representative immunofluorescence experiments were shown for cartilage samples OA and NE. FITC and nuclei were contrasted with propidium iodide Samples were examined under a Leica SP5 confocal microscope (B) The data are the average of cells positive for TGF-ββ ± standard deviation of the early and NE OA groups. The statistical significance is represented as p <0.01 (**) when the values were compared with the normal cartilage and p <0.01 (##) when the NE cartilage data were compared with OA cartilage. , experiment s independent (n = 3).

Figure 6. The expression of IL-10 in rat cartilage is shown on the indicated training days (td). The rats were sacrificed and the articular cartilage was processed for indirect immunofluorescence of IL-10. (A) Representative experiments are shown of immunofluorescence for cartilage samples OA and NE. IL-10 was labeled with FITC and the nuclei were contrasted with propidium iodide. The samples were examined under a Leica SP5 confocal microscope. (B) The data are the mean of positive cells for IL-10 ± standard deviation of the early OA and NE groups. The statistical significance is represented as p < 0.01 (**) when the values were compared with normal cartilage and p < 0.01 (#) when the NE cartilage data were compared with OA cartilage. Three independent experiments were performed (n = 3). Figure 7. The effect of culture time on the content of proteoglycans in the cartilage is shown. Oestochondral explants were cultured at the indicated times and then processed for staining with fast Safranin-O-green. The figure is representative of three independent experiments. Scale bar 50 μ? T ?.

Figure 8. The dose-response of IL-? Β in the cartilage is shown. The osteochondral explants were cultured for 24 hours and then the IL-? ß was added at 0, 10, 20 and 50 ng / ml. After 24 hours of treatment the explants were processed for staining with Safranina-O- fast green ( left panel) and TUNEL (right panel). The figure is representative of three independent experiments. Scale bar 50 μp ?.

Figure 9. The dose-response of the neutralizing antibody anti-IL-? Β in the cartilage is shown. Oestochondral explants were cultured for 24 hours and then the antibody was added at doses of 0, 25, 50 and 100 μg / ml. After 24 hours of treatment the explants were processed for staining with Safranin-O-green fast (left panel) and TUNEL (right panel). The figure is representative of three independent experiments. Scale bar 50 μ ??.

Figure 10. The effect of the neutralization of exogenously added IL-? Β on the integrity of the cartilage is shown. The ostéochondral explants were cultured for 24 hours and then IL-ßβ at 20 ng / ml preincubated 1 hour with the anti-IL-αβ neutralizing antibody at doses of 0, 25 and 50 μg / ml. After 24 hours of treatment, the explants were processed for staining with fast Safranin-O-green (left panel) and TUNEL (right panel). The figure is representative of three independent experiments. Scale bar 50 pm.

Figure 11. The effect of the neutralization of exogenously added IL-? Β on the integrity of the cartilage is shown. The osteochondral explants were cultured for 24 hours and then the IL-ββ was added at 20 ng / ml. After two hours of incubation, the anti-IL-αβ neutralizing antibody was added at doses of 0, 25 and 50 μg / ml. After 24 hours of treatment the explants were processed for staining with fast Safranin-O-green (left panel) and TUNEL (right panel). The figure is representative of three independent experiments. Scale bar 50 μ ??.

Detailed description of the invention.

The present invention provides a method of detecting IL-αβ interleukin for the diagnosis of OA in early stages. The validity of the method involves the relationship of the expression of pro-inflammatory and anti-inflammatory cytokines in a healthy cartilage and cartilage! osteoarthritic, inducing OA in animal models through a partial menisectomy and a high-impact exercise regimen, removing samples of cartilage under aseptic conditions.

The present invention also provides the determination of the changes of expression of proteins in the cartilage during the progression of OA from early stages, performing studies of Western blot and immunohistochemistry to determine the expression of IL-? Β, TNF-a, TGF- H.H? and IL-10 in articular cartilage in an early OA rat model, induced by partial menisectomy followed by high-impact exercise training. In this case, the protein profiles are compared with those of normal rats that received the training with high impact exercises (NE), observing that the levels of IL-? ß showed an ascending expression in both cases, early OA and NE, although in the last stages the increase was lower in the NE groups. On the other hand, TNF-a levels increased in both groups, but in the NE group the ascending expression started up to the later stages, whereas the levels of TGF-β? they were regulated ascending to the same level in the LO and in the NE model. On the other hand, in the OA model, the IL-10 showed a transitory increase in its expression that later fell to its baseline levels, while in the NE model its levels increased at each hourly point analyzed. The number of chondrocytes that expressed the aforementioned cytokines was associated with protein levels determined by Western blot. The detection method OA early of the invention, suggests an accurate regulation in the cartilage between anti-inflammatory and pro-inflammatory molecules, in order to maintain the balance and integrity in the tissue. The present invention also refers to the relationship of the performance of high-impact exercises in the formation of the patterns of expression of pro-inflammatory and anti-inflammatory cytokines in healthy cartilage and in cartilage with early OA, determining that in healthy cartilage , the anabolic proteins effectively counteract the action of the catabolic proteins induced by exercise, while in the OA cartilage, the principles of the activity of catabolic proteins are stronger than the anabolism, hence the progress of OA.

The present invention also seeks the detection of the expression of IL-? Β and TNF-a in early stages of OA helping to determine the appearance of cartilage deterioration in the advanced stages of OA, since these cytokines directly inhibit the expression of specific genes of the cartilage extracellular matrix, induce the activity of proteases that degrade the cartilage matrix and induce the death of chondrocytes by apoptosis.

By means of the method of the present invention it is possible to also detect the ratio of the expression of pro-inflammatory and anti-inflammatory cytokines in a healthy cartilage and osteoarthritic cartilage, which allows to determine the type of inflammatory molecules involved in the establishment of the disease from early stages.

The present invention also provides an efficient treatment method for OA in early stages by blocking and / or neutralizing the activity of IL-? Β in the articular cartilage, which allows to avoid the progression of OA to difficult or impossible stages treatment, as well as avoid the degradation of the cartilage, allowing its recovery in a short time.

The functionality of the OA diagnostic method in early stages of the present invention is evaluated by the induction of osteoarthritis in cellular and animal models by a partial menisectomy and a high impact exercise regimen, removing cartilage samples under aseptic conditions, allowing then the detection of the levels of IL-? ß generated from early stages by said conditions, while the treatment method of the present invention is based on the blocking and / or neutralization of the IL-? ß produced from early stages of the disease, which allows to efficiently reverse the adverse effects observed by said cytokine during the course of the disease.

The present invention also relates to the determination of changes in protein expression in the cartilage during the progression of same, one of the objects of the present invention early diagnosis of osteoarthritis, and the blocking and / or neutralization of said intereleucine from early stages to provide an effective treatment of the disease. | In accordance with the present invention, the applicants demonstrate that IL-? ß is expressed in early stages of OA in a rat model, IL-ß ß being an early biomarker of OA and that blocking or neutralizing its activity in the articular cartilage from early stages allows the reversal of the progress of OA, IL-ß ß also being a suitable therapeutic target for OA, whereby the present invention provides effective methods of diagnosis and treatment of OA since early stages. j Based on the information described in the present invention, it is possible to suggest that although IL-1β is a molecule that plays an important role during the initiation and progression of OA, its detection and use in a local intraarticular anti-IL therapy. -? ß in early stages is essential to stop or delay OA. Therefore, the present invention is focused in part to determine if in an in vitro model the IL-? ß induces changes in the articular cartilage j related to OA, such as the loss of proteoglycans and death of chondrocytes, and the application of molecules that inhibit the activity of IL-? β in the articular cartilage, such as, for example, by neutralizing antibodies in early stages of OA or during the progression of Harmful stimulation in OA are able to stop damage to the articular cartilage.

As can be seen in the following examples and in accordance with the present invention, the early detection of IL-? ß is associated with the generation of OA in early stages, while the blocking and / or neutralization of its activity in the articular cartilage from Early stages by anti-IL-? β inhibitors, such as anti-JIL-? ß antibodies, allow to reverse and / or stop the progress of OA.

Therefore, the present invention also provides methods for the efficient treatment of OA in early stages through the use of inhibitors of the activity of IL-? Β in the articular cartilage, such as, for example, through the administration of anti-inflammatory antibodies. 11-? ß.

Articular cartilage degradation and osteoarthritis (OA) have been associated with; the inadequate i activation of cytokine signaling pathways. In addition, high impact exercise is a factor in i risk for OA, usually after the joint injury. However, even before the present invention, the relationship between the expression of cytokines after a high-impact exercise training in osteoarthritic cartilage and healthy cartilage, as a way to monitor and evaluate the natural progress of the disease, had not been analyzed.

There are several works that illustrate the chondroprotective effect of several molecules, such as IL-10 and transforming growth factor β (TGF-β), which even antagonize the intrinsic pathways of apoptosis induced by TNF-a or IL-? ß in the chondrocytes or, they can counteract the harmful effects of the IL-? ß and TNF-ct through the stimulation of the synthesis of the proteins of the cartilage matrix and the inhibition of the expression of metalloproteinases.

TGF-β, which is expressed in its three isoforms (1, 2 and 3) in the cartilage, appears as one of the main elements in the tissue repair potential. The remarkable decrease of type II receptor for TGF-β in fibrillar cartilage chondrocytes in the experimental OA model in rabbits, the presence of OA-type lesions in transgenic mice expressing non-functional type II receptors and repair of cartilage in arthritis induced by zymosan by the local administration of TGF-β? in the joints of the knee of mice, strongly suggest that the TGF-β system plays an important role in the repair of articular cartilage. Most studies of OA pathogenesis have been induced by surgery of animal models of OA, which closely resemble post-traumatic OA in humans. Some OA models employ high-impact exercise in order to accelerate (from weeks to days) the OA process. It is generally accepted that some forms of exercise are beneficial for human OA, for example, mild to moderate walking or resistance training exercises relieve pain and restore joint function.

Although the inflammatory and anti-inflammatory elements are undoubtedly involved in the pathogenesis of OA, these have not been thoroughly studied due, on the one hand, to the difficulty to obtain the cartilage of humans in the early stages of OA. for your study. In fact, little is known about the activation of inflammatory pathways of OA in early stages, when the therapeutic intervention may be more useful. In addition, it is not known yet how the inflammatory elements adapt to healthy cartilage during high-impact exercise training to overcome the onset of OA.

One embodiment of the present invention is to provide a method of early diagnosis of OA by detecting, for example by means of Western blot and immunohistochemistry, the expression of IL-β, TNF-α, TGF-β? and that of IL-10, where the detection of IL-? ß in early stages of OA is indicative of the onset and development of the disease.

Another embodiment of the present invention is to determine the changes of expression of proteins in the cartilage during the progression of OA and with a regime of high impact exercises, for example by means of Western blot studies.

An additional modality is to determine, for example through immunohistotymic, whether changes in cartilage protein levels are associated with changes in the number of cells expressing the protein or whether there has been a redistribution of expressing cells. to the cytokines, within the different areas of the cartilage.

Another of the embodiments of the present invention is to provide an efficient therapeutic method for OA from early stages by the use of inhibitors of IL-αβ activity in the articular cartilage, for example by the use of anti-IL-α antibodies. H.H.

Still another embodiment of the present invention is the use of IL-? ß as an early biomarker of OA, which allows using said molecule according to the present invention, in the timely and early detection of the development of OA, as well as for the treatment of said disease in early stages.

One of the objectives of the present invention is to compare the protein expression profile of cytokines in OA and in normal rats subjected to a high impact exercise (NE) regimen, in order to determine the pro-inflammatory factors involved in the development of the OA. Currently, OA is diagnosed in advanced stages through different cabinet studies that include x-rays of the affected joint. On an X-ray, the reduction of the joint space, the loss of cartilage, the wear of the ends of the bone and the formation of spurs can be clearly seen. Until prior to the present invention, there was no medication or treatment that cures the OA or could even be detected from early stages, so that only measures are applied to reduce the associated symptoms such as pain and inflammation, while in cases extremes, a surgical replacement of the joint affected by a prosthesis is performed that does not replace the total functionality of the joint, besides being very expensive.

There are a number of risk factors for the development of OA, among which age, obesity and joint injuries stand out. Joint injuries can be caused by the practice of high impact exercises and may include, for example, fractures, transection of the anterior cruciate ligament (TLCA) and damage to the meniscus. For example, in a 14-year follow-up study, it has been estimated that 40% of soccer players who suffered from TLCA presented symptoms of advanced OA30. Therefore, early and timely diagnosis in subjects at risk of OA will facilitate their treatment.

In accordance with the present invention, a method of detecting IL-αβ for the early diagnosis of OA in patients with risk factors is proposed, which comprises the steps of: a) Obtain a sample of cartilage from the suspected subject to be diagnosed under aseptic conditions, for example through a small biopsy, b) Extract and obtain total proteins from said sample, and c) Detecting the presence of IL-? ß in the proteins obtained in b), for example by means of Western blot and immunohistochemistry where the presence of IL-? ß is indicative of the development of OA in early stages in the patient. On the other hand and according to the present invention, in the case of at-risk patients subjected to high-impact exercise regimens, the method of the invention additionally includes: a) Obtain a sample of normal cartilage from the patient as a control, for example through a small biopsy, and obtain total proteins from it, b) Detect and quantify the presence of IL-? ß in the proteins obtained in the control sample, for example by means of Western blot and immunohistochemistry and c) Determine the relationship of the concentration of IL-? ß in the sample of cartilage suspected of presenting OA between the concentration of IL-? ß in the control sample, where a value of at least 1.5 in the relationship of part c) is indicative of the development of OA from early stages in the patient.

According to the animal model used in the present invention, the detection of IL- [beta] in the cartilage of rats exposed to high impact exercise as a way of demonstrating the production and detection of said interleukin from early stages of OA, is carried out as follow: a) Induce OA in the animal model through a partial menisectomy and a high-impact exercise regimen, b) Obtain cartilage samples from the animal exposed to the conditions of part a), removed under aseptic conditions, for example from male Wistar rats, and extract their proteins, as well as cartilage samples from animals not exposed to the conditions of the subsection a) as it shows control, c) Detect and quantify the presence of IL-? ß in the proteins obtained in b), for example by means of Western blot and immunohistochemistry, and d) Determine the ratio of the concentration of IL-? ß in the sample of the animal exposed to the conditions of part a) between the concentration of IL-? ß in the sample of normal cartilage, where a value of at least 1.5 in the relation of subsection d), is indicative of the development of OA. The following examples show significant differences in the expression profiles of cytokines between OA cartilage and NE cartilage and suggest that, in healthy cartilage, anabolic proteins effectively counteract the action of catabolic proteins, while in cartilage OA activity of catabolic proteins is more intense than anabolic activity, which induces OA.

Once the presence of IL-? ß is detected in early stages of OA, inhibitors of IL-ßβ activity, such as anti-IL-ßß antibodies or other molecules that can be applied directly to the articular cartilage, can be applied directly to the cartilage. To be used for this purpose, in such quantities as to allow the reversal of the OA to be observed or to stop the progress of the OA at more advanced stages.

Inhibitors of IL-αβ activity in articular cartilage that can be used in accordance with the present invention are selected from the group comprising polyclonal anti-IL-βß antibodies, anti-IL-αβ monoclonal antibodies, nucleic acid aptamers. anti-IL-? ß, antagonists of the interleukin-converting enzyme, or mixtures thereof, which can form part of pharmaceutical compositions as active ingredients for the treatment of OA in early stages. Said compositions can be made according to the knowledge previously known in the art and can be applied to subjects under treatment of OA via the intra-articular route. The administration regime of said composition containing inhibitors of the activity of IL-? Β in the articular cartilage will depend on the stage in which the progression of OA is detected until the disease is reversed or, if appropriate, stop its progression to more advanced stages, where other treatment methods can be applied as complementary treatment. However, in general terms and for the purposes of this j invention, the inhibitor of IL-αβ activity in the articular cartilage can be preferably administered at a concentration of at least 25μ / τt? according to the therapeutic regimen that is treated and according to the type of IL-ß-β inhibitor used.

As can be observed in the results obtained in the present invention, the inhibition of the activity of IL- [beta] in the articular cartilage by blocking and / or neutralization by anti-IL-ββ antibodies stops the progression of cartilage degradation and in some cases it reverses the damage caused by said IL-? ß in the cartilage, which makes it possible to use the method of the present invention as an efficient therapy to prevent the progression of OA and favor the recovery of the damaged joint due to OA.

The following examples are merely illustrative and describe a way to carry out the object of the invention and are not intended to be limiting of its scope.

Example 1. Detection of IL-? ß in early stages of OA. 1. 1. Experimental model of OA.

Male Wistar rats weighing 130-150 g that were housed during a 12-h light, 12-h dark program allowing free access to food were used. and water. All surgical procedures were performed on anesthetized rats with an intraperitoneal injection of 60 mg / Kg of ketamine and 4 mg / Kg of xylazine solution. The animals were sacrificed by inhalation of C02 and the cartilage samples were removed under aseptic conditions. All procedures for the care and use of animals were approved by an ad hoc institutional commission (IACUC) and were carried out following the official Mexican standard NOM-062-ZOO-1999 for the use and care of experimental animals.

The induction of OA was achieved through a partial menisectomy and a high-impact exercise regimen.

The training began 2 days after the surgery and was performed daily for 15 minutes during 3, 6, 8, and 10 days, called training days (td). Normal rats without surgical intervention were subjected to the same protocol of high impact exercises and were designated as NE. As a control group, rats were included with surgery, but without partial menisectomy (sham operation) and normal rats without surgery both without high impact exercises. 1. 2. Processing of cartilage samples for Western blot studies. : For the Western blot studies, the articular cartilages were removed from the right femoral condyles of 10 rats of each experimental group, therefore 10 rats were i considered n = l. The collected cartilages were immediately frozen and stored at -80 ° C until they were processed to extract the proteins.

The cartilage samples were homogenized in Polytron (Kinematica Inc., Bohemia, NY, USA) in a lysis buffer [25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.2 mm EDTA, 0.5 rhm dithiothreitol, 1 % Triton X-100 and the cocktail of enzyme inhibitors (Complete, Roche Applied Scie'nce, Manheim, Germany)] and clarified by centrifugation for 5 minutes at 10,000 g. The concentration of proteins was determined by the Bradford procedure. The SDS-PAGE was performed with 40 mg of protein per lane (30 mg for TGF-β?) In 15% polyacrylamide gels. The proteins were transferred by wet transfer for 2.5 hours at 350 mA, to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5% nonfat dry milk and 0.5% BSA in Tris-saline buffer, pH 7.5, with 0.19% of Tween 20 i.

(TBS-T) for 2 hours at 37 ° C with gentle shaking and then incubated overnight at 4 ° C with the following antibodies: rabbit polyclonal anti-IL-? ß (1: 800; Santa Cruz Biotechnology Inc. Santa Cruz , CA, USA), polyclonal goat anti-TNF-a (1: 600, Santa Cruz Biotechnology Inc.), polyclonal rabbit anti-TGF-β? (1: 400, Santa Cruz Biotechnology Inc.) and polyclonal anti-IL-10 (1: 600, Santa Cruz Biotechnology Inc.). The signals were observed, after 1 hour of incubation with the secondary anti-goat or anti-rabbit antibodies (1: 40000, Jackson Irrimunoresearch Laboratories Inc. West Grove, PA, USA), labeled with horseradish peroxidase and using the ECL Plus Chemiluminescence Detection System (GE Healthcare, Buckinghamshire, United Kingdom). The expression of β-actin (1: 1000, Santa Cruz Biotechnology Inc.) was used as an internal control. The protein bands were quantified by densitometry with the Image J software. ; 1. 3. Statistical analysis for Western blot studies. ' The expression values of the protein are presented as the ratio of cytocihas / actin. The data are shown as the mean ± standard deviation. The statistical significance is represented as p < 0.05 (*) or p < 0.01 (**) when the values were compared with the normal cartilage and p < 0.05 i (#) or p < 0.01 ("**) when NE cartilage data were compared with OA cartilage Three independent studies were performed (n = 3) All statistical analyzes were performed with the Graph Pad INSTAT program (Graph Pad Software Inc., San Diego , CA, USA) An analysis of variance (ANOVA) was used using the Dunnet test for the comparison of means between the experimental groups. 1. 4. Processing of cartilage samples for immunohistochemistry studies.

For immunohistochemical studies, the right femoral condyles were removed and fixed for 2 hours at room temperature in 4% paraformaldehyde in PBS, pH 7.2. After 3 washes with PBS, the samples were incubated in 10% sucrose in PBS, pH 7.2, for 12 hours at 4 ° C. Subsequently, the samples were imbibed in a tissue freezing medium and immediately frozen at -20"C.

The frozen condyles were cut in a cryostat to obtain slices of 6 μ? T? of thickness that were mounted on gelatin coated slides. Sections were hydrated for 15 minutes in PBS, pH 7.2 and permeabilized with 0.2% Tween 20 in PBS for 10 min at room temperature. They were then incubated with 0.2% BSA for 20 min at room temperature. To detect the proteins, the tissues were incubated overnight at 4 ° C with the following antibodies: polyclonal rabbit anti-IL-? ß (1: 100); polyclonal goat anti-TNF-a (1: 100); polyclonal rabbit anti-TGF-β? (1: 100) and polyclonal goat anti-IL-10 (1: 100), (all from Santa Cruz Biotechnology Inc.). The rescent signal was observed when the tissues were incubated for 1 hour at room temperature with the anti-rabbit antibodies coupled with FITC or anti-goat coupled to FITC or TRITC (1:60, Jackson Immunoresearch, Inc.). For each of the cytokines, macrophages induced by LPS were used as positive controls. The cartilage and macrophage samples induced by LPS, where the corresponding primary antibody was omitted, were used as negative controls. The nuclei were contrasted with propidium iodide (10 μg / ml, Sigma-Aldrich Inc., St. Louis, MO, USA) for 5 minutes or with DAPI (10 μg / ml, Sigma-Aldrich, Inc.) for 5 min. 1. 5. Statistical analysis for immunohistochemical studies.

The cell count was performed on the images taken with the confocal microscope; for each protein, nine microscopic fields chosen at random (for each animal) obtained from the cartilage of three rats with OA induced with exercise for 3, 6, and 10 days, three NE rats with the same exercise regimen, three rats were analyzed. with sham surgery and without exercise, but sacrificed at the same times as rats with induced OA and, finally, three normal rats without any treatment. The total count of the chondrocytes in the cartilage photographic images was considered as 100%. The LAS AF software of the confocal microscope (Leica Microsystems) was used to obtain the data of the positive cells. The data are shown as the mean ± standard deviation of the mean. The statistical significance was represented as p < 0.05 (*) or p < 0.01 (**) when the values were compared with the normal cartilage and p < 0.05 (#) or p < 0.01 (**) when NE cartilage data were compared with OA cartilage. Three independent experiments were performed (n = 3). All the statistical analyzes were carried out with the Graph Pad INSTAT program. An analysis of variance (ANOVA) was used using the Dunnet test for the comparison of means between the experimental groups. i 1. 6. Confocal microscopy. rescence studies were performed with an inverted confocal microscope (Leica LSM-SPC-5Mo, Leica Microsystems) with the objective 40X neor immersion in oil. The rochromes were excited with the argon laser at 488 nm and the Helium neon laser lines at 543 nm. Using the LAS AF program of the confocal microscope (Leica Microsystems), images from three different fields of each tissue section were captured and processed. 1. 7. Expression of pro-inflammatory atocins in early OA and in rats exercised with high impact.

Compared to normal cartilage, the levels of IL-? ß and TNF-a in rat cartilage with simulated operation without exercise did not change at any point in the time evaluated. During OA development, IL-? ß levels were over expressed in a time-dependent manner, up to 2.3 times at 10 td (see figure 1A). In the NE cartilage, IL-? ß levels were also up-regulated throughout the experiment; however, at 8 and 10 td their levels were 2.1 and 1.6 times lower, respectively, than in the OA cartilage (see figure 1A). The levels of the TNF-a protein increased during the progression of the OA about 2.4 times (figure IB). In NE cartilage, TNF-ct was maintained at baseline levels up to 6 td and then increased by almost 2.3 times, at 8 and 10 td (Figure IB).

Compared to normal cartilage, the number of chondrocytes that expressed IL-? ß and TNF-a in rats with sham operation without exercise did not change significantly at the points of i time evaluated. During the progression of OA, the number of chondrocytes that expressed IL-? ß increased by 59% at 6 td and 66% at 10 td (figure 2), and they were distributed in all the cartilage zones (figure 2A) . In NE cartilage, cells positive for IL-? ß also increased in all cartilage zones at 6 td and 10 td (Figure 2), although, in comparison with OA cartilage, chondrocytes positive for IL-? ß they were around 40% less at 10 td (Figure 2B). The chondrocytes positive for TNF-a increased in a time-dependent manner in both cartilage conditions OA and NE, up to 70% and 60% respectively at 10 td (figure 3). The cells positive for TNF-a appeared in all the areas of the cartilage OA and lNE, but in the cartilage of the OA model the presence of positive signal was more evident in the chondrocytes of the superficial zone of (figure 3A), whereas in the NE cartilage the positive signal was more obvious in the chondrocytes of the middle and deep zone (Figure 3A). 1. 8. Expression of TGF-β? and IL-10 in early OA and in normal rats subjected to high-impact exercise.

Compared with normal cartilage, the levels of TGF-β? and IL-10 in the rat cartilage with simulated operation without exercise did not change at any time point evaluated. Levels of TGF-β? they were regulated positively in all the time points of the experiment; both in OA cartilage and in NE cartilage, no significant differences were found in TGF-β levels. between both models (figure 4A). On the other hand, IL-10 protein levels were transiently regulated positively during the progression of OA by about 4.9 times at 3 td which fell to basal levels of 6 to 10 td (Figure 4B). Surprisingly, in it NE cartilage, the levels of IL-10 were strongly overexpressed during the time of; duration of the experiment and were clearly superior to the levels in the OA cartilage (figure 4B).

In comparison with normal cartilage, the number of chondrocytes that expressed TGF-β? and IL-10 in rats with simulated operation without exercise did not change significantly at the time points evaluated. In the OA model, cells positive for TGF-β? were up transitorily by 70% at 3 td. Later, the positive cells TGF-β? they were reduced by 59% to 6 td and at baseline levels to 10 td (figure 5). In contrast, in the NE group, the cells positive for TGF-β? they were increased in a time-dependent manner, by up to 39% at 10 td compared to normal cartilage (FIG. 5B). However, compared to OA cartilage, the number of cells positive for TGF-β? was significantly lower in NE cartilage, except at 10 td, where there was 46% more cells positive for TGF-β? (figure 5B). Cells that expressed TGF-β? they were located mainly in the superficial zone, both in the OA cartilage and in the NE cartilage, although at 3 td in the OA cartilage a positive signal was detected in all the cartilage zones (figure 5A). On the other hand, the cells positive for IL-10 remained unchanged (approximately 10-24%) during the course of the time of induction of the OA (figure 6), the positive signal was located mainly in the chondrocytes of the superficial zone and a half (figure 6A). In contrast, IL-10 positive cells increased significantly by 79% at 3, 8 and 10 td in NE cartilage (FIG. 6). The positive signal for IL-10 was detected in all areas of the NE cartilage (Figure 6A).

The present invention reveals the role of high-impact exercise in the formation of the patterns of expression of pro-inflammatory and anti-inflammatory cytokines in healthy cartilage and in cartilage with early OA. The results of the present invention show that in healthy cartilage, anabolic proteins effectively counteract the action of catabolic proteins induced by exercise, while in OA cartilage, the activity of catabolic proteins is stronger than anabolism, hence the progress of OA.

The presence of pro-inflammatory cytokines in the articular cartilage is an accepted fact in the pathogenesis of OA; however, the present invention discloses that the expression of IL-? β and TNF-a begins as early as 3 days after the induction of OA. Likewise, our results show that the most superficial zone of chondrocytes expresses TNF- in the OA cartilage, while in the NE cartilage, the positive signals are almost not evident, supporting the phenotypic variability of chondrocytes described above. Likewise, the expression of IL-? ß and TNF-a in early stages of OA could explain the appearance of cartilage deterioration in the advanced stages of OA, since these cytokines directly inhibit the expression of specific protein genes. the cartilage matrix, at the same time as they induce the expression of proteases that degrade it or induce the death of chondrocytes by apoptosis.

The pro-inflammatory cytokines were also positively regulated in NE cartilage; however, in some stages their levels of expression were lower than in OA cartilage. These results suggest that physical training stimulates the expression of pro-inflammatory cytokines by articular chondrocytes. However, when there is a joint injury, the activity of pro-inflammatory cytokines can not be regulated and OA develops. In healthy cartilage, the high expression of pro-inflammatory cytokines is adequately regulated by anti-inflammatory elements to prevent the onset of OA. Anti-inflammatory IL-10 acts as a chondroprotective agent because it stimulates type II collagen and proteoglycan expression, inhibits proteases that degrade the matrix, inhibits the production of nitric oxide and antagonizes the apoptosis pathways induced by TNF-a or IL -?H.H. However, the low levels of IL-10 after the experimental induction of OA, are not sufficient to overcome the pro-inflammatory activity and could be related to the increase of pro-inflammatory molecules.

In the groups with normal physical training and without menisectomy, the levels of IL-10 showed an intense positive regulation, an increase that could be associated with the homeostatic maintenance in the tissue since IL-10 can reduce the effects of IL-? ß and TNF-a by decreasing the damage and increasing the repair of the joint. In cultures of human chondrocytes, the overexpression of IL-10 counteracts the increase in MMP-13 expression induced by TNF-α. In addition, IL-10 is able to decrease caspase activity and the bax / bcl-2 ratio in chondrocytes in alginate bead culture, modulating the pro-apoptotic capacity of TNF-ot. The transient increase in IL-10 levels in the OA group could be an attempt to antagonize anti-inflammatory activity. On the other hand, TGF-β, which is an anabolic growth factor present in normal cartilage, had a significant increase in both the OA group and the NE group, which could constitute an attempt to repair the OA lesion.

A difference was found between the number of cells expressing TGF-β and the expression levels of cytokines in the cartilage of both OA and NE, which can be explained by differences in the production of cytokines by individual chondrocytes. In healthy cartilage, TGF-ß could counteract the activity of IL-? ß by reducing the appearance of catabolic molecules such as nitric oxide and prostaglandins (PG). However, in the OA cartilage the activity of IL-? ß could deteriorate the signaling of TGF-ß? through a decrease in receptor levels (TGF-PRI I) that is mediated primarily by the p65 / NF-kappa pathways and the activating protein 1 / JNK and secondly, by decreasing Smad7, which is an inhibitor of the Smad protein that blocks the transduction of the TGF-pi / Smad pathway.

The early changes in the concentration of OA cytokines in the experimental OA show a significant imbalance in the anabolic / catabolic balance. The characterization of the molecules involved in this process is very important in the search for target molecules for new therapeutic purposes and early diagnosis for OA, where IL-? ß turns out to be useful as an early biomarker of OA. In addition, normal rats subjected to high impact exercises without partial menisectomy maintained the anabolic / catabolic balance despite the fact that both types of cytokines were substantially elevated. This could support the genetic root of a deterioration in the ability to repair cartilage damage in certain circumstances.

Example 2. Treatment of OA in early stages by blocking and / or neutralizing the activity of IL-? Β in cartilage. 2. 1. Animals Male Wistar rats with a weight of 130 to 150 grams were used, kept under controlled conditions of a bioterium with a cycle of 12 light hours and 12 hours of darkness and fed ad libitum. The animals were sacrificed by CO 2 overdose in accordance with the Official Mexican Standard NOM-062-ZOO-1999 that meets the criteria of the institutional ethics committee. 2. 2. Osteochondral explants.

In conditions of sterility, the joints of both knees were carefully dissected. Surgical specimens were placed in sterile PBS, and with a razor the osteochondral explants were extracted from the loading areas of the femoral condyles, which were submerged in sterile PBS to avoid desiccation. The explants of approximately 1.5 X 3 mm were cultivated in 96-well plates (Jet Biofil, Guangzhou, China), one explant per well with 250μ? of DMEM medium (Dulbecco's Modified Eagle Medium; Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (PAA Laboratories; Pasching, Austria), 50 μg / ml L-ascorbic acid (Sigma-Aldrich Inc. , St. Louis, MO, USA), 1% antibiotic-antifungal (PAA Laboratories) and 1% glutamine (Invitrogen corporation) (DMEM-S). The cultures were kept at rest for 24 hours in an incubation chamber at 37 ° C and 5% C02. At the end of the 24 The DMEM-S was changed by the medium containing the following treatments: 1) rat recombinant IL-? ß (R & D systems; Minneapolis, MN, USA), 2) neutralizing monoclonal anti-IL-? b antibody. of rat (R & D systems) and 3) a combination of recombinant IL-? ß and neutralizing anti-IL-? β antibody. All treatments were incubated for 24 hours at 37 ° C and 5% C02. Each treatment was carried out in three independent experiments (n = 3).

To evaluate the viability of the osteochondral explants, in our culture conditions, these were cultured without any treatment for 96 hours, changing the DMEM-S every 24 hours. i Every 24 hours the samples that were processed to analyze the integrity of the cartilage were obtained. ! i 2. 3. Treatments. ! 2. 3.1. Dosage response with IL-? ß. | DMEM-S was prepared with IL-? ß at concentrations of 0, 10, 20 and 50 ng / ml. At the end of the 24-hour rest period the explants were changed to the culture medium containing the IL-ββ in the different doses. The cultures were incubated for 24 hours at 37 ° C and at term; They were processed for histological analysis. ! ! i I 2. 3.2. Dosage response with neutralizing anti-IL-? Β antibody.

DMEM-S was prepared with the antibody in concentrations of 0, 25, 50 and 100 μg / ml. At the end of the 24-hour rest period, the explants were changed to the culture medium containing the antibody at the different doses. The explants were incubated for 24 hours and at the end they were processed for histological analysis. i 2. 3.3. Neutralization of IL-? ß.

Two strategies were used, which were called start and progression; in the first l (start) the IL-? ß at 20 ng / ml was preincubated for one hour at 37 ° C in DMEM-S medium with the concentrations of 0.25 and 50 μg / ml neutralizing antibody. They were then added to the explants and were: incubated by i 24 hours at 37 ° C; At the end of the incubation, the samples were processed for histological analysis. In the second strategy (progression) the osteochondral explants were treated for 2 hours with 20 ng / ml of the IL-? ß and then the neutralizing antibody anti-IL-? ß was added to I i I i concentrations of 0, 25 and 50 ng / ml. The cultures were incubated for 24 hours and then processed for histological analysis. 2. 4. Fixation and histological sections of the osteochondral explants.

At the end of the treatments the explants were washed twice for 5 minutes with PBS IX, pH 7.2 and fixed for 24 hours at 4 ° C with paraformaldehyde (SPI Supplies; West Chester, PA, USA) at 4% in PBS IX, then washed twice with PBS IX and incubated for 24 hours in 10% sucrose in PBS IX. They were then immersed in the tissue freezing medium (Leica Microsystems, Wetzlar, Germany) and cut into a cryostat (Leica CM1100, Leica Microsystems). The 8 pm slices were placed on slides previously gelatinized with 0.5% porcine skin gelatin (Ted Pella, Redding, CA, USA) and 0.05% potassium chromium sulfate (Merk, Darmstadt, Germany) in water. The lamellae with the cuts were stored at -20 ° C until processing. 2. 5. Staining with Safranin O-Green Fast.

To evaluate the content of proteoglycans in the articular cartilage, the 8-μm cuts of the explants of each treatment were hydrated in PBS IX and stained with the Safranina O-green rapid technique, where the presence of proteoglycans is evidenced by a color red while the absence of proteoglycans is evidenced with a green color, according to the following: The lamellae were hydrated in PBS IX pH 7.2 for 5 min and subsequently incubated for 2 min. in distilled H20. Later it was added to the green samples 0.05% fast in water for 10 min, then washed with 1% acetic acid for 15 sec .; The treated samples were incubated with 0.5% safranin-0 in water for 40 min., then dehydrated in 50%, 70%, 95% and 100% ethanol for 30 sec. to finally incubate the samples stained twice with xylene 1 min each and mount them with Polymount resin for later microscopic observation.

The resulting images of the stained samples were obtained with a digital camera (DFC320, Leica Microsystems, Wetzlar, Germany) coupled to a light microscope (DMLS, Leica Microsystems) with the 40x objective. 2. 6. TUNEL staining (Terminal deoxynucleotidyl transferase dUTP nick end labeling).

To determine if different treatments induce death of chondrocytes, the TUNEL technique was performed following the instructions of the manufacturer of the DeadEnd Fluorometric TUNEL System kit (Promega Corporation, Madison, WI, USA). The images were obtained with a digital camera (DFC320, Leica Microsystem) coupled to an epifluorescent microscope (DMLS, Leica Microsystems) using a filter for fluorescein and with the 40x objective. 2. 7. Neutralization of the activity of IL-? ß in cartilage.

In order to have a system that allows us to determine the damage to articular cartilage, we evaluated an in vitro system with osteochondral explants.

As can be seen in figure 7, in our culture conditions the explants maintain their proteoglycan content for up to 72 hours, a content that decreases perceptibly at 96 hours; thus, cultures of osteochondral explants can be used without affecting the proteoglycan content for up to 72 hours. Based on these results, the following experiments were carried out within the first 48 hours in culture.

To determine the concentration of IL-? ß that affects the integrity of the cartilage, we carried out dose-response experiments with concentrations of IL-? ß of 0, 10, 20 and 50 ng / ml. Our results showed that with 24 hours of treatment, the doses of 20 and 50 ng / ml decrease the levels of proteoglycans that correlated with the death of the chondrocytes (figure 8). Based on these results, we selected the dose of 20 ng / ml for the following experiments.

To determine whether the anti-IL-? Β neutralizing antibody affects the integrity of the cartilage we perform a dose response curve with concentrations of 0, 25, 50 and 100 μ? /? \ Of the anti-IL-? Β antibody. The results showed that the doses of 25 and 50 are innocuous but the dose of 100 μg / ml induces death of the chondrocytes with the consequent loss of proteoglycans, reason why this concentration was discarded for the rest of the experiments (figure 9).

With the results obtained, we evaluated the effect of neutralizing IL-? ß in two conditions that represent the beginning and progression of cartilage damage. In the initial model, the I -? ß at 20 ng / ml was pre-incubated with two concentrations of the anti-IL-? Β antibody for one hour and the complex formed was added to the explant in culture (figure 10). In the progression model the explants in culture were treated for two hours with IL-? ß and after the noxious stimulus the neutralizing anti-IL-? Β antibody was added (figure 11). As can be observed in figures 10 and 11, in both situations the neutralizing anti-IL-βß antibody at concentrations of 25 and 50 μg / ml was able to adequately arrest the loss of proteoglycans and the death of the chondrocytes.

Osteoarthritis is the most common form of arthritis and the most important cause of discouragement in the elderly. It remains incurable and there is no treatment at present. Therefore, it is urgent to develop new approaches for the diagnosis and treatment of this disease.

According to the results described here (see example 1), it is observed that the expression of IL-? ß during OA in an animal model begins from the first days of induction, with which it is suggested that the IL-? ß it is an early biomarker of OA. To deepen the role of this pro-inflammatory cytokine in the initiation and progression of OA, we evaluate the effect of blocking IL-? ß with a neutralizing antibody in the onset and progression of articular cartilage degeneration (see example 2). ). Our results suggest that blocking IL-? ß in early stages stops the progression of cartilage degradation, which supports its role as an early biomarker of OA.

For a long time, IL-? ß has been considered as a proinflammatory cytokine that plays a role in the pathogenesis of OA, making it a natural target for the treatment of this disease. However, since the anti-IL-? ß treatments are not able to control the disease completely, perhaps because it is applied in advanced stages or once the damage is irreversible23,24,25,29, it is necessary to use early diagnosis strategies of the disease as the method of the invention described herein for then applying therapies that can stop or delay cartilage damage, such as the OA treatment method of the present invention.

According to the present invention, the detection of IL-? ß in early stages of OA development, as well as the neutralization of its activity in cartilage by anti-IL-? ß inhibitors in early stages allow the timely detection and treatment of OA , thus avoiding the progression of the disease to later stages where any treatment is inefficient and increasing the opportunities to provide efficient treatments of OA.

References. 1. Martel-Pelletier J, Boileau C, Pelletier JP, Roughley PJ (2008). Cartilage ¡h normal and osteoarthritis conditions. Best Pract Res Clin Rheumatol. 22: 351-84. www.cdc.gov.

Wieland HA, Michaelis M, Kirschbaum BJ, Rudolphi KA. 2005. Osteoarthritis - untreatable disease? Nat Rev Drug Discov. 4: 331-344.

I Espinosa R, Hernández L, Arroyo C. 2005. Prevalence of musculoskeletal manifestations in Mexico. Rev Mex Reumatol. 20: 5 From Pavia-Mota E, Larios-González MG, Briceño-Cortés G. 2005. Management of osteoarthrosis in Family Medicine and Orthopedics. Archives in Family Medicine. 7: 93-98.

Burrage PS, Mix KS, Brinckerhoff CE. 2006. Matrix metalloproteinases: role in arthritis. Front i Biosci. 11: 529-543. 1 Kapoor M, Martel-Pelletier J, Lajeunesse D, Pelletier JP, Fahmi H. 2011. Role of proinflammatory cytokines in the pathophysiology of osteoarthritis. Nat Rev Rrjeumatol. 7: 33-42.

Kouri JB, Jiménez S, Quintero M, Chico A. 1996. ultrastructure of the chondrocytes from the fibrillated and non-fibrillated human osteoarthritic cartilage. Osteoarthritis Cartilage; 4: 111-125. ! Kouri JB, Argüello C, Luna J, Mena R. 1998. Use of microscopical techniques in the study of human chondrocytes from osteoarthritic cartilage: an overview. Microsc Res Tech. 40: 22-36. Kourí-Flores JB, Abbud-Lozoya KA, Roja-Morales L. 2002. Kinetics of the ultrastructural changes in apoptotic chondrocytes from an osteoarthrosis rat model: a windowj of comparison j to the cellular mechanism of apoptosis in human chondrocytes. Ultrastruct Pathol. 26: 33-40. Kouri JB, Rosales-Encina JL, Chaudhuri PP, Luna J, Mena R. 1997. apoptosis in human osteoarthritic cartilage: a microscopy report. J Med Sci Res. 25: 245-248.

Blanco FJ., Guitian R, Vázquez-Martul E, of Toro FJ., Galdo F. 1998. j Osteoarthritis chondrocytes die by apoptosis. a possible pathway for osteoarthritis pathplogy. Arthritis Rheum. 4: 284-289. j Hashimoto S, Ochs Rl., Komiya S, Lotz M. 1998. Linkage of chondrocyte apoptosis and cartilage degradation in human osteoarthritis. Arthritis Rheum. 41: 1632-1638.; i Roach Hl, Aigner T, Kouri JB. 2004. Chondroptosis: a variant of apoptotic cell death in chondrocytes ?. Apoptosis 9: 265-277. j j Kouri JB and Lavalle C. 2006. Do chondrocytes undergo "activation" and "transdifferentiation" during the pathogenesis of osteoarthritis? A review of the ultrastructural and Í immunohistochemical evidence. Histol Histopathol. 21: 793-802. ? Almonte-Becerril M, Navarro-Garcia F, Gonzalez-Robles A, Vega-Lopez MA, Lavalle C, Kouri JB. 2010. Cell death of chondrocytes a combination between apoptosis and autophagy during the pathogenesis of Osteoarthritis within an experimental model. Apoptosis 15: 631-638. Benito MJ, Véale DJ, FitzGerald O, van den Berg WB, Bresnihan B. 2005. Synovial tissue inflammation in early and late osteoarthritis. Ann Rheum Dis. 64: 1263-1267. j Pelletier JP, Martel-Pelletier J, Abramson SB. 2001. Osteoarthritis, an inflammatory disease: potential implication for the selection of new therapeutic targets. Arthritis 44: 1237-1247.

Lefebvre V, Peeters-Joris C, Vaes G. 1990. Modulation by interleukin 1 and tumor necrosis I factor-a of production of collagenase, tissue inhibitor of metalloproteinases and collagen types in differentiated and dedifferentiated articular chondrocytes. Biochirri Biophys Acta 1052: 366-378.; Goldring MB, Birkhead JR, Sandell U, Kimura T, Krane SM. 1988. Interleukin 1 suppresses expression of cartilage-specific types II and IX collagens and increments types I and III collagens in human chondrocytes. J Clin Invest. 82: 2026-2037. i j Richardson DW, Dodge GR. 2000. Effects of interleukin-1 and tumor necrosis factor-a on expression of matrix-related genes by cultured equine articular chondrocytes. Am J Vet Res. 61: 624-630.

Mengshol JA, Vincenti MP, Coon Cl, Barchowsky A, Brinckerhoff CE. 200?'. Interleukin-1 induction of collagenase 3 (matrix metalloproteinase 13) gene expression in chondrocytes requires p38, c-Jun N-terminal kinase, and nuclear factor ??: differential: regulation of collagenase 1 and collagenase 3. Arthritis Rheum. 43: 801-811.

Pelletier JP, Caron JP, Evans C, Robbins PD, Georgescu Hl, Jovanovic D, Fernandes JC, Martel-Pelletier J. 1997. In vivo suppression of early experimental osteoarthritis by interleukin-1 receptor antagonist using gene therapy. Arthritis Rheum. 40: 1012-1019. ! Frisbie DD, Ghivizzani SC, Robbins PD, Evans CH, Mcllwraith CW. 2002. Treatment of experimental equine osteoarthritis by in vivo delivery of the equine interleukin-1 receptor antagonist gene. Gene Ther. 9: 12-20.

Wang HJ, Yu CL, Kishi H, Motoki K, Mao ZB, Uraguchi A. 2006. Suppression of experimental osteoarthritis by adenovirus-mediated double gene transfer. Chínese Medical Journal. 119: 1365-1373.

Ling Shari M., et.al. 2006. Biomarkers for osteoarthritis. WO2006023412.

Hannum Charles H., et.al. 2001. Interleukin-1 inhibitor. JP2001029093.

Tobinick Edward L. 2001. Cytokine antagonists for the treatment of localized disorders. US2001016195 (Al).

Chevalier X, Goupille P, Beaulieu AD, Burch FX, Bensen WG, Conrozier T, Loeuille D, Kivitz AJ, Silver D, Appleton BE. 2009. Intraarticular injection of anakinra in osteoarthritis of the knee: a multicenter, randomized, double-blind, placebo-controlled study. Arthritis Rheum 15: 344-352. von Porat A, Roos EM, Roos H. 2004. High prevalence of osteoarthritis 14 years after an anterior cruciate ligament tear in male soccer players: a study of radiographic and patient relevant outcomes. Ann Rheum Dis; 63: 269-273.

Claims (17)

Claims

1. A method for the detection or diagnosis of osteoarthritis from early stages, characterized in that it comprises the steps of: a) Obtain a cartilage sample from the patient, b) Obtain total proteins from the sample obtained in a), and c) Detect the presence of IL-? ß in the proteins obtained in b) where the presence of IL-? ß is indicative of the development of osteoarthritis in early stages.

2. The method of claim 1, characterized in that obtaining the sample in step a) is done under aseptic conditions and by biopsy.

3. The method of claim 1, characterized in that the detection and concentration of IL-? Β in step c) is carried out by means of Western blot.

4. The method of claim 1, characterized in that the detection and concentration of IL-? ß in step c) is carried out by immunohistochemistry.

5. The method of claim 1, characterized in that it further comprises: a) Obtain a sample of normal cartilage from the patient as a control and obtain total proteins from said sample, b) Detect and quantify the presence of IL-? ß in the proteins obtained in the cartilage sample of the patient and the control sample, and c) Determine the relationship of the concentration of IL-? ß in the sample of cartilage of the patient between the concentration of IL-? ß in the control sample, where a value of at least 1.5 in the relationship of part c) is indicative of the development of osteoarthritis in the patient's sample from early stages.

6. A method for the treatment of osteoarthritis from early stages, characterized in that it comprises administering in the joint of a patient with osteoarthritis in early stages a pharmaceutical composition containing an inhibitor of the activity of IL-? Β in the articular cartilage.

7. The method for the treatment of osteoarthritis of claim 6, characterized in that the inhibitor of the activity of IL-? ß is selected from the group comprising anti-IL-? ß monoclonal antibodies, polyclonal anti-IL-? Β antibodies, aptamers of anti-IL-? β nucleic acids, interleukin-converting enzyme antagonists and mixtures thereof.

8. The method for the treatment of osteoarthritis of claim 6, characterized in that the pharmaceutical composition containing the inhibitor of the activity of IL-? Β is administered intra-articularly.

9. The method for the treatment of osteoarthritis of claim 6, characterized in that the pharmaceutical composition has an inhibitor concentration of at least 25 μg per mL.

10. The use of an inhibitor of the activity of IL-? Β in the cartilage for the manufacture of a pharmaceutical composition for the treatment of osteoarthritis from early stages.

11. The use of claim 10, characterized in that the inhibitor of the activity of IL-? ß is selected from the group comprising anti-IL-? Β monoclonal antibodies, polyclonal anti-IL-? Β antibodies, anti-IL nucleic acid aptamers. -? ß, antagonists of the interleukin-converting enzyme and mixtures thereof. ! i

12. The use of claim 10, characterized in that the pharmaceutical composition containing i the inhibitor of IL-β-activity is administered intra-articularly. !

13. The use of claim 12, characterized in that the administration is made directly in the joint affected by osteoarthritis.

14. The use of IL-? ß as an early biomarker of osteoarthritis.

15. A pharmaceutical composition for the treatment of osteoarthritis, characterized in that it comprises an inhibitor of the activity of IL-? Β in the cartilage in a pharmaceutically acceptable vehicle.

16. The pharmaceutical composition of claim 15, characterized in that the inhibitor of IL-? Β activity is selected from the group comprising anti-IL-1β monoclonal antibodies, polyclonal anti-IL-? Β antibodies, anti-IL nucleic acid aptamers. -? ß, antagonists of the interleukin-converting enzyme and mixtures thereof.

17. The pharmaceutical composition of claim 15, characterized in that the inhibitor of IL-ββ activity is at a concentration of at least 25 μg per mL.