ES2710220A1 - Anti-connexin compounds for use in the prevention and/or treatment of degenerative joint diseases (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Anti-connexin compounds for use in the prevention and/or treatment of degenerative joint diseases. The present invention relates to compounds, compositions and anti-connexin formulations comprising at least one anti-connexin compound for use in the prevention or treatment of degenerative joint diseases, such as: arthritis, osteoarthritis, rheumatoid arthritis and osteoporosis . The compounds, compositions and formulations described herein are suitable for the inhibition of gap junctions (GJ) and hemichannels. (Machine-translation by Google Translate, not legally binding)

Description

ANTI-CONEXINE COMPOUNDS FOR USE IN PREVENTION AND / OR

THE TREATMENT OF DEGENERATVAS DISEASES OF THE

ARTICULATIONS

DESCRIPTION

The present disclosure relates to pharmaceutical products which include agents, compounds, compositions and formulations for the modulation, preferably for the inhibition, of gap junctions (GJ) and hemichannels which are useful, for example, for the prevention and / or the treatment of degenerative diseases of the joints, such as arthritis, osteoarthritis, rheumatoid arthritis and osteoporosis. Therefore, the present invention pertains to the field of medicine, in particular to the field of degenerative diseases of the cartilage.

PREVIOUS TECHNIQUE

Osteoarthritis (OA) is a degenerative disease of the joints that mainly affects the cartilage and bone. Osteoarthritis is especially common in older people and usually affects a joint on one side of the body. In osteoarthritis the cartilage is destroyed and wears out, causing pain, swelling and loss of mobility of the joint.

Rheumatoid arthritis (RA) is a systemic disease that, when manifested in the joints, mainly affects the synovial membrane. Rheumatoid arthritis begins at younger ages than osteoarthritis, usually occurs bilaterally in the joints and sometimes produces feelings of discomfort, fatigue and fever.

Although OA and RA have different etiologies, both diseases are characterized by inflammation of the synovial joint, degeneration of the cartilage and loss of joint function. The causes of OA and RA are not fully understood. While RA is an autoimmune joint disease, the risk of developing OA increases substantially with age, through mediators of the inflammation, repeated trauma, diabetes, obesity and congenital and developmental diseases, among other systemic and local factors.

Chondrocytes are highly specialized cells integrated into the extracellular matrix (ECM) of the articular cartilage. These cells give the articular cartilage important mechanical properties and are responsible for the formation and remodeling of the tissue throughout adult life. In the normal cartilage there is a balance between the synthesis and the degradation of the matrix. In the arthritic cartilage, on the other hand, degradation outweighs the synthesis.

Chondrocytes are found in small cavities known as lagoons. This characteristic has given rise to the idea that the chondrocytes are not directly connected to each other and that they communicate with other chondrocytes mainly by diffusion of substances in the matrix. However, adequate intercellular communication is essential for the organization of a timely and uniform response to physical or biological stimuli or cellular damage, or simply to maintain tissue homeostasis. It has been proposed that the poor regenerative capacity of the cartilage is due to the absence of blood and lymphatic vessels and the relative isolation of the cells. This, however, is very probably not the case of joint cartilage, since aging reduces cell density and the thickness of the cartilage, which allows the tissue to maintain its structure and function.

The maintenance of the homeostatic balance and the normal functioning of the tissues are controlled by the interaction between extracellular and intercellular communication networks. Direct intercellular communication, which includes the direct exchange of metabolites, nutrients, second messengers, small RNAs and ions, is achieved through gap junctions ( GJ), which play critical roles in function and development. The GJ are structures of the cell membrane that facilitate direct cell-cell communication. A split junction channel is formed by two hemichannels of connexins (Cx), each composed of six connexin subunits. Each hemichannel of hexameric connexins is coupled with a hemichannel of connexins in the opposite membrane to form a single junction in the slit. It has been reported that the Slit union channels are distributed throughout the body. In general, the connexins constitute a family of proteins usually named according to their molecular weight or classified according to their phylogeny in the alpha, beta and gamma subclasses. At least 21 human connexins and 19 isoforms of murine connexins have been identified. It has been reported that different tissues and cell types have characteristic protein expression patterns of connexins.

In fact, changes in connexin protein levels, assembly status or localization are characteristic of a variety of diseases in which connexins are deregulated, such as degenerative and inflammatory disorders, neuronal damage induced by hypoxia-ischemia, disease chronic kidney or cardiac pathophysiology. There are some studies that suggest that connexins could play a crucial role in the healing and repair of wounds in connective tissues. In this sense, primers from the opposite direction of connexin 43 (Cx43) prevented the increase of protein levels of connexin 43 and the regulation by abnormal increase of connexin 43; Likewise, normal rates of wound closure in the endothelial cells of the skin and cornea were restored.

In fact, there are currently no proven therapies for degenerative joint diseases. There are only a few palliative treatments to control pain and inflammation. Thus, patients suffer from chronic severe progressive pain together with a deterioration of daily function. In current therapy, a class of drugs known as non-steroidal anti-inflammatory drugs (NSAIDs) is used to treat musculoskeletal joint diseases, such as osteoarthritis, but these therapies have a number of drawbacks, including, in particular, gastrointestinal diseases, and can also interfere with the repair and formation of cartflago. Other alternatives resort to surgical interventions, such as arthroscopy and total substitution surgery of the articulation with artificial material, such as prostheses. These palliative treatments, surgeries and physical therapies turn the degenerative diseases of joints into one of the most costly diseases in developed countries. Despite advances in the understanding of the principles of the mechanisms underlying degenerative joint diseases, there continues to be a significant unmet demand for new therapeutic options for the treatment and / or prevention of (for example, the progression of) degenerative joint diseases.

SUMMARY OF THE INVENTION

In order to provide a new therapeutic option for the treatment and / or prevention of degenerative joint diseases, the inventors herein describe anti-connexin compounds for use in the treatment and / or prevention of degenerative diseases. of the joints. Alternatively, the present invention also relates to the use of an anti-connexin compound for the preparation of a medicament or a composition for the prevention and / or treatment of degenerative joint diseases.

As used herein, the term "connexin" refers to a family of structurally related transmembrane proteins that assemble to form slit junctions in vertebrates. Each junction in the slit is made up of two hemichannels, or connexons, which in turn are formed by six connexin molecules. In one aspect, the connexin is a connexin naturally present in a human or animal or naturally occurring in the tissue in which the expression or activity of the connexin is to be reduced. As used herein, it is understood, then, that the reference to a specific connexin is a reference to all variants thereof in the different species, even though their molecular weights are different. Thus, for example, the reference to "connexin 43" refers not only to human connexin 43 but also to the analogous connexin of any other species, regardless of whether it also weighs 43 Kd or not. Similarly, the reference to a non-human "connexin 43" is a reference to the analogue or connexin variant 43 in that species. Thus, for example, the reference to "horse connexin 43" is a reference to the analog or the corresponding variant of human connexin 43 in the horse, even if it does not have a molecular weight of 43 Kd. The connexin gene (including the coding sequence) generally has homology to the coding sequence of one or more of the specific connexins mentioned herein, eg homolog with connexin 43. The connexin is typically a connexion to or p. Preferably, the connexin is a connexin and is expressed in the tissue to be treated. Table 1 shows the nucleotide and polypeptide sequences of different connexins suitable for the present invention.

Table 1. List of human connexins used in the present invention.

As used herein, the term "protein" refers to any polymer of two or more individual amino acids (whether natural or not) bound by peptide bonds. Furthermore, as used herein, it is understood that the term "protein" includes the terms "polypeptide" and "peptide" (which can sometimes be used interchangeably herein). Similarly, fragments, analogs, derivatives and variants of a protein may be referred to as "proteins" herein and shall be considered a "protein" unless otherwise indicated. The term "fragment" of a protein refers to a polypeptide that comprises less than all the amino acid residues of the protein. A "domain" of a protein is also a fragment and comprises the amino acid residues of the protein that are often required to confer the activity or function.

As used herein, the term "anti-connexin compound" refers to those compounds that affect or modulate the activity, expression or formation of a connexin, a connexin hemichannel (link), or a slit junction. These anti-connexin compounds are generally directed to connexins and / or connexin hemichannels (connexins) or to the slit junctions themselves. The hemichannels and the resulting gap junctions that include connexins are independently involved in the release or exchange of small molecules between the cell cytoplasm and extracellular space or tissue when the hemichannels are open, and between the cytoplasm of adjacent cells when the slit junctions are open. The anti-connexin compounds include, without limitation, compounds of the opposite direction (eg, polynucleotides of the opposite direction), antibodies and binding fragments thereof, as well as peptides and polypeptides including "peptidomimetic" and "mimetic" peptides and other compounds , which include connexin phosphorylation compounds, such as inhibitors of the Ser / Thr-phosphatases PP1 and PP2A, such as okadaic acid, caliculin A, dragmacidins, microcystins, nodularins, tautomycin, tautomycetin, cytostatins, phospholine, leustroducsins, foslactomycins, fostriezine and cantharidin, tacrolimus, cyclosporin A, thirsiferyl-23-acetate, nodularin V (motuporin), 2H-microcystin-LA, nodularin R, microcystin LR, dinofisistoxin 1 and dinofisistoxin 2; beonzoylamino-benzopyran compounds such as tonabersat (sb-220453) and carabersat (sb-204269); phenothiazines such as mesoridazine, thioridazine, paroxetine, risperidone, methylphenidate, raloxifene, minoxidil, thioridazine, haloperidol, droperidol, mesoridazine, meperidine, melperone, ditran B (JB-329), N-methyl-3-piperidyl benzylate, promazine, triflupromazine , levomepromazine, methotrimeprazine, fluphenazine, perphenazine, prochlorperazine and trifluperazine; fenamates such as flufenamic acid and derivatives, the quinine derivative mefloquine, meclofenamic acid.

The terms "modulator" or "modulation" or "modulate" of the activity or expression of connexins, as used herein in their various forms, refer to the total or, preferably, partial inhibition of the expression or action or activity of a connexin or connexin hemichannel or connexin slit junction, and may function as anti-connexin agents.

As used herein, "counter-sense compounds" include different types of molecules that act by inhibiting or decreasing the gene expression, translation or function, including those that are directed to a specific sequence of mRNA for therapeutic applications. Opposite compounds include reverse-sense DNA compounds and reverse-sense RNA compounds. Suitable anti-connexin oligo- and polynucleotides include oligo- and polynucleotides of siRNA and oligo- and siRNA polynucleotides. The The synthesis of oligo- and polynucleotides of the opposite direction and of other anti-connexin oligo- and polynucleotides, such as RNAi, siRNA and oligo- and polynucleotides of ribozymes, as well as of oligo- and polynucleotides with modified skeletons, are known to those skilled in the art. mixed The opposite sense sequences directed to selected targets may or may not bind non-specific to sequences other than the target. Preferential sequences with a minimal non-specific binding, absent or undetectable to sequences other than the target are preferred.

Although counter-sense nucleotides constitute a preferred form of compounds in the opposite direction, the present invention encompasses other oligomeric compounds of the opposite direction including, without limitation, oligonucleotide mimetics. Antisense compounds include ribozymes, oligonucleotides of external glulam sequences (EGS) (oligozymes) and other short catalytic RNAs or catallotic oligonucleotides that hybridize with the target nucleic acid and modulate its expression. They also include phosphorothioate oligodeoxynucleotides (S-ODN). Thus, compounds of the opposite direction include, for example, the main silencing molecules of genes based on nucleic acids, such as, for example, chemically modified oligodeoxyribonucleic acids (UUJNs), ribozymes and siRNAs (Scherer, LJ and Rossi, JJ). Nature Biotechnol 21: 1457-1465 (2003). Counter-sense compounds may also include counter-sense molecules such as, for example, peptide nucleic acids (PNA), morpholino-phosphorodiamidates, DNAzymes and the small nuclear RNAs UI mutated at the end. 5 'The reverse sense polynucleotide is generally in the opposite direction with respect to a connexin mRNA, preferably connexin 43 mRNA, connexin 45 mRNA, connexin 46 mRNA, connexin 26 mRNA or connexin 32 mRNA. More preferably , the polynucleotide of the opposite direction is opposite to the connexin 43 mRNA. A polypeptide of this type can be capable of hybridizing with the mRNA of connexin and, therefore, can inhibit connexin expression by interfering with one or more aspects of connexin mRNA metabolism, including transcription, mRNA processing, mRNA transport from the nucleus, translation or degradation of the connexin. mRNA. The reverse-sense polynucleotide hybridizes typically with the connexin mRNA to form a duplex, which it can produce direct inhibition of the translation and / or destabilization of the mRNA.

The use of an antisense oligonucleotide as a therapeutic agent in order to down-regulate the connexin gene expression by inducing enzyme-dependent degradation of mRNA complementary can be preferably administered with RNAi delivered by nanotechnological and nanoparticle techniques and / or by use of an expression vector, a viral or non-viral vector.

The combined use of polynucleotides directed to separate connexin proteins (for example, targeting 1, 2, 3, 4 or more different connexins) is also contemplated. For example, polynucleotides directed to connexin 43 and one or more members of the connexin family can be used in combination.

Alternatively, reverse-sense polynucleotides can be part of compositions that can comprise polynucleotides against more than one connexin protein. Preferably, one of the connexin proteins to which the polynucleotides are directed is connexin 43. Other connexin proteins to which oligodeoxynucleotides are directed can include, for example, connexins 26, 32, 45 and 46. Although the sequence The polynucleotide of the opposite direction used in the invention depending on the target connexin protein, have been especially suitable for connexin 43 for the polynucleotides having the following sequences (Table 2).

Table 2. Sequences of the Cx43 gene used to design the antisense oligonucleotide sequences of Cx43 RNAi.

Likewise, the use of opligopeptides or polypeptides in the opposite direction against connexin peptides as therapeutic agents is contemplated, in order to regulate by decreasing the activity of the connexin gene. The oligopeptides or reverse-sense polypeptides are generally in the opposite direction with respect to a connexin protein, preferably a connexin protein 43, a connexin protein 45, a connexin protein 46, a connexin 26 protein or a connexin protein. More preferably, oligopeptides or reverse-sense polypeptides are opposite in direction to the connexin 43 protein. The combined use of polypeptides targeting separate connexin proteins (eg, targeting 1, 2, 3) is also contemplated. , 4 or more different connexins). For example, polypeptides directed to connexin 43 and one or more members of the connexin family can be used in combination.

Alternatively, opposite-sense polypeptides can form part of compositions that can comprise polypeptides against more than one connexin protein. Preferably, one of the connexin proteins to which the polypeptides are directed is connexin 43. Other connexin proteins to which polypeptides are directed can include, for example, connexins 26, 32, 45 and 46. Although the sequence If the opposite-sense polypeptide used in the invention is dependent on the target connexin protein, the polynucleotides having the following sequences have been particularly suitable for connexin 43 (tables 2). AVESAWGDEQSAFRCN (SEQ ID NO: 31), TQQPGCENVCY (SEQ ID NO: 32), DKSFPISHVRFWV (SEQ ID NO: 33), YTCKRDPCPHQVDC (SEQ ID NO: 34) and FLSRPTEKTIFIIF (SEQ ID NO: 35).

The terms "peptidomimetic" and "mimetic" include natural and synthetic chemical compounds which may have substantially the same structural and functional characteristics as the protein regions they mimic. In the case of the connexins, they can mimic, for example, the extracellular loops of opposing connexins involved in connexin-connexion coupling and the formation of the cell-cell channel and / or the extracellular loops of connexins in the hemichannels. For example, a mimetic composition can be useful as a slit-binding modulator if it is capable of regulating by diminution the actions or biological activities of the connexins, such as, for example, preventing the coupling of connexins to form cell-cell communications. mediated by gap junctions or to prevent the opening of the connexins to expose the cellular cytoplasm to the extracellular medium. The peptidomimetics encompass those described herein as! such as those that may be known in the art, whether those known up to now or those developed subsequently.

In a more preferred embodiment, the peptidomimetic is selected from the group consisting of GAP27 (SRPTEKTIFII SEQ ID NO: 52) and rotigaptide (ZP123).

In another preferred embodiment, the anti-connexin antibody or the antigen-binding fragment thereof is an antibody, an F (v) fragment, a Fab fragment, a Fab 'fragment or an F (ab') 2 fragment.

In a more preferred embodiment, the anti-connexin antibody is selected from the group consisting of anti-connexin 43, anti-connexin 45, anti-connexin 46, anti-connexin 26 or anti-connexin 32. In a more preferred embodiment, the antibody anti-connexin is anti-connexin 43.

In a preferred embodiment, the anti-connexin compounds described herein for use in accordance with the present invention are selected from the group consisting of a gap junction inhibitor and / or a hemichannel inhibitor.

The terms "disorder" or "disease", used interchangeably throughout this document, refer to any pathological condition that will benefit from treatment with a molecule or a compound or composition of the invention, including those described or claimed herein. .

The expression "degenerative disease of the joints" refers to a gradual deterioration of the joint cartilage that covers the joints. A degenerative joint disease is a progressive non-infectious disease of the load joints. The normal joint cartilage is smooth, white and translucent. It consists of cartilaginous cells (chondrocytes) integrated in a spongy matrix made of collagen, protein polysaccharides and water. In the early phase of primary arthritis, the cartilage becomes yellow and opaque with local areas of softening and wrinkling of the surfaces. As the degeneration progresses, the soft areas crack and wear out, exposing the bone beneath the cartilage. The bone then begins to reshape and increase its density while the remaining cartilage begins to twitch. Finally, osteophytes (spurs of new bone) are formed covered by a cartilage at the edge of the articulation. The cartilage needs to be repaired as mechanical wear increases. The cartilaginous cells are unable to produce enough spongy matrix and, therefore, the damaged cartilage can not be repaired on its own. The cartilage lacks a blood supply to promote healing. Most of the degenerative diseases of the joints are due to mechanical instabilities or changes in articulation due to aging. These include the Degenerative arthritis of the elderly and, in younger individuals, may be the result of injuries, contusions, abnormal configuration of the joints (ie, hip dysplasia) or mechanical wear caused by rupture of the anterior cruciate ligament, patellar luxation or osteochondritis dissecans , for example. Degenerative joint disease can appear in any articulation of the body, including without limitation knee, hip, shoulder, hand and spine. The states included in the degenerative diseases of the joints are selected from the group consisting of osteoarthritis, arthritis, physical injury, osteoporosis, Paget's disease and rheumatoid arthritis.

As used herein, "subject" refers to any animal classified as a mammal that includes humans, domestic animals and farm animals, as well like sports animals, zoos or companla animals, such as dogs, horses, cats, sheep, pigs, cows, etc. The preferred mammal in this case is a human being, including adults, children and the elderly.

As used herein, "prevention", "prevent" means to totally or partially prevent, or mitigate or control, or reduce or stop the progress of a pathological condition. As used herein, an "effective amount" or "effective dose" or "therapeutically effective amount" or "therapeutically effective dose" in relation to the molecules, compounds or compositions of the present invention refers to the amount sufficient to induce a desired biological, pharmaceutical or therapeutic result. That result may be the relief of the signs, symptoms or causes of a disease or disease or pathological condition or any other desired alteration of a biological system. In the present invention, the result will involve promoting healing, preventing and / or reducing pain and / or inflammation and degenerative processes. The inventions described and claimed herein may also favor the repair and regeneration of the cartilage in pathological states such as degenerative joint diseases, or after an articular injury.

As used herein, the term "treating" or "treatment" refers to both therapeutic treatment and prophylactic or preventive measures.

In a more preferred embodiment, the degenerative joint disease is selected from the group consisting of arthritis, osteoarthritis, rheumatoid arthritis, physical injury, Paget's disease and osteoporosis.

Another aspect of the present invention relates to a composition for use in the treatment and / or prevention of a degenerative joint disease, the composition comprising at least one anti-connexin compound as described herein. Alternatively, the present invention also describes the use of a composition comprising at least one anti-toxin compound according to the present invention in the production of a medicament for the prevention and / or treatment of a degenerative joint disease.

The term "medicament", "composition" or "pharmaceutical composition" refers to a composition comprising at least one active principle, the composition being susceptible to investigation as to a specified and effective effect in a mammal (for example and without limitation). , a human being). Those of ordinary skill in the art will understand and recognize the appropriate techniques to determine, depending on the needs of the expert, whether an active ingredient has the desired effective effect.

The anti-connexin compound may be present in a substantially isolated form. It will be understood that the product can be mixed with carriers or diluents that do not interfere with the purpose for which the product is intended and still considered substantially isolated. The terms "carrier" and "diluent" refer to any pharmaceutical carrier and diluent that does not itself induce the production of antibodies detrimental to the individual receiving the composition and which can be administered without excessive toxicity.

In a preferred embodiment, the composition of the invention also comprises an acceptable excipient, vehicle or pharmaceutical adjuvant.

The term "acceptable pharmaceutical excipient" or "acceptable pharmaceutical vehicle" or "acceptable pharmaceutical adjuvant" as used herein refers to "pharmaceutically acceptable carrier" or "acceptable pharmaceutical carrier" or "acceptable pharmaceutical adjuvant" as used herein. to additives useful for converting pharmacologically active compounds into pharmaceutical forms that are suitable for administration to patients. Suitable excipients, carriers or adjuvants include fillers, binders, disintegrants, surface active agents, lubricants, fluidizers and colorants. Other excipients, vehicles or pharmaceutically acceptable adjuvants can also be included.

Anti-connexin compounds and compositions can be administered alone or in combination with other therapeutic agents or active ingredients in or around the affected site, for example by topical application, instillation or injection, for example peritendinal injection, soft tissue injection, intra-articular injection and periarticular injection. Anti-connexin compounds and compositions may also be administered alone or in combination with other therapeutic agents using interoperative techniques. Administration can also be done by injection or interoperatively in joint capsules or bone cavities. Anti-connexin agent compositions can be administered monotherapy, concomitantly or intraoperatively.

In a more preferred embodiment, the composition of the invention also comprises a second active principle. In a more preferred embodiment, the second active principle is selected from the group consisting of: analgesics, non-steroidal anti-inflammatory drugs (NSAIDs) and intra-articular therapies, such as corticosteroids and hyaluronic acid. In a more preferred embodiment, the second active principle is selected from the list comprising: paracetamol, specific inhibitors of cyclooxygenase-2, tramadol, opiates, NSAIDs, topical NSAIDs, capsaicin, topical salicylate, intraarticular steroids, intraarticular hyaluronic acid and glucosamine and chondroitin.

The anti-connexin compounds and the composition of the invention can be administered in single or divided doses. The doses can be administered once, or the application can be repeated. Typically, the application will be repeated weekly or every two weeks, or monthly or with another frequency that could be determined by the experts. The administration of the anti-toxin agent (s), for example for the regulation by reduction of the expression of connexin or the blocking or inhibition of the opening or activity of the connexin, therefore modulates the communication between cells, or the loss towards the extracellular space in the case of regulation of the connexins, and minimizes the losses or additional injuries of cells or the consequences of injuries.

The routes of administration and the dosages described herein are only intended to be indicative, since an expert physician will determine the optimal route of administration and dosage for each patient and specific pathological condition. In any of the methods for the treatment of a subject with a degenerative joint disease mentioned or described herein, the administration of any of the doses, pharmaceutical forms, formulations and / or compositions described herein can be used.

In another preferred embodiment, the compositions provided herein are preferably in sterile solution or suspension, or in other pharmaceutically acceptable formulations. The composition provided herein may be applied directly to the affected or diseased tissue, or is preferably adapted for intra-articular and / or systemic or parenteral administration. Systemic administrations may include, without limitation, intramuscular, intravenous or subcutaneous injections, or direct adsorption to blood circulation by transmucosal non-gastrointestinal administration, for example sublingual.

In the most preferred embodiment, the compounds or compositions provided herein are adapted for direct injection into a target tissue, for example a tendon, a ligament or a bone, or they can be adapted for direct application on the target tissue. , for example in a gel or a pasty material, to improve and / or prolong contact with the target tissue.

In another preferred embodiment, the composition of the invention is formulated for direct injection into diseased tissue. In a more preferred embodiment, the diseased tissue is selected from the group consisting of: articular cartilage, synovial membrane and subchondral bone.

The therapeutic agent can be administered by intraarticular, periarticular, peritendinal or soft tissue injection. The therapeutic agent can be injected in single dose or in multiple doses. It is contemplated that the methods and composition described herein include administration using various procedures known in the art or developed subsequently, including, for example, laparoscopic techniques. Examples of these techniques may include intraoperative techniques and / or direct injection. These techniques can facilitate the delivery of the drug in the form of prolonged release, achieving a better bioavailability and / or distribution, which improves the therapeutic efficacy.

In another preferred embodiment, the composition of the invention is used for the prevention and / or treatment of degenerative joint disease. In a more preferred embodiment, the degenerative joint disease is selected from the group consisting of arthritis, osteoarthritis, physical injury, Paget's disease, rheumatoid arthritis and osteoporosis.

Another object of the present invention relates to a method for the prevention and / or treatment of a degenerative joint disease, which comprises the administration of a therapeutically effective dose of an anti-connexin compound or of the composition described herein memory to a patient who needs it.

In a preferred embodiment, the method of the invention can preferably be direct administration into diseased tissue. In a more preferred embodiment, the administration is intraarticular, periarticular or peritendinal.

In another preferred embodiment of the method of the invention, the degenerative joint disease is selected from the group consisting of arthritis, osteoarthritis, physical injury, Paget's disease, rheumatoid arthritis and osteoporosis.

In another aspect, the invention provides a kit for the treatment and / or prevention of degenerative joint diseases. The kit may include one or more compositions described herein. For example, the kit may include a composition comprising at least a therapeutically effective amount of an anti-connexin compound as described herein or combinations thereof.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly understood by one skilled in the art to which this invention pertains. In the practice of the present invention, methods and materials similar or equivalent to those described herein can be used. Throughout the description and the claims, the word "comprises" and its variations, does not intend to exclude other technical characteristics, additives, components or steps. Objects, advantages and additional features of the invention will become apparent to those skilled in the art upon examination of the description, or may be learned by practicing the invention. The following examples, drawings and sequence listing are provided by way of illustration and are not intended to be limiting of the present invention.

Brief description of the drawings

Figure 1. Photographs of frozen cartilage slices of healthy donors and subjects affected by OA, showing that the Cx43 protein is expressed in excess in the articular cartilage of patients with OA. The expression of Cx43 was analyzed by immunohistochemical tests (IHC).

Figure 2. Photograph of a cartilage sample of a patient with OA treated with safranin O / solid green. The graph shows the RNA expression of Col2AIIB, Col2AIIA, MMP3 and MMP9 in cartilage samples from the patient with OA. It is shown that the inhibition of connexin channels in the cartilage of the patient with OA leads to the expression of the anabolic type II collagen gene and to the decrease in the expression of catabolic factors such as MMP3 and MMP9.

Figure 3 shows that the inhibition of connexin channels regulates the phenotype of osteoarthritic chondrocytes by inhibiting cell proliferation and reducing the expression of catabolic mediators (A), and causes a decrease in the expression of cyclin D1 (CCND1) and a increased expression of p21 cell cycle inhibitors (Cip1) of osteoarthritic chondrocytes (B and C). In addition, the treatment of the human primary chondrocytes with 1-octanol increases the expression of type II collagen and reduces the expression of MMP3 (B and C).

EXAMPLES

The present invention will be described in more detail by examples. The examples are intended to be illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.

Materials and methods

1.1 Obtaining and processing cartilage

Samples of articular cartilage of the knee and the head of the femur were obtained from adult subjects undergoing joint surgery. All the subjects signed an informed consent form and the ethics committee of the institution approved the study (registration code of the CAEIG of Galicia: 2012/094-PI13 / 00591 and 2016/349-PI16 / 00035). The cartilage samples were immediately frozen in situ in Cryomold® Standard using the Tissue-Tek® OCT ™ compound (Sakura Finetec, The Netherlands) and isopentane in liquid nitrogen and stored at -80 ° C. The cartilage of healthy subjects was obtained in situ from individuals without a history of joint disease who had suffered a fracture of the knee or hip. The healthy samples were assigned based on the data of their medical histories and the histological study. The histological samples (healthy and OA with radiological diagnosis) were classified according to the modified Mankin scale. The samples (patients with OA and healthy subjects of the same age) were divided into four groups: normal / healthy (score = 0-1); OA early (score 2-3), called grade I; OA mild (score = 4-5), called grade II; Moderate OA (score = 6-7), called grade III; and severe OA, called grade IV (score = 8-13). 4 mm cartilage samples were prepared from cartilage explants that were cut in the operating room immediately after the surgery. The samples were grown overnight in 48-well culture plates in DMEM with 15% FCS containing 1 mM 1-octanol (Sigma) as an anti-connexin channel compound and incubated for 17 hours. For the isolation and cultivation of chondrocytes The primary cells were washed with a cartilage freshly extracted with saline solution and the cells were isolated. Briefly, the cells (2.5 million) were seeded in 162 cm2 flasks and incubated at 37 ° C in 5% CO2 and air in DMEM supplemented with 100 pg / ml Primicin ™ (InvivoGen, San Diego, USA). UU.) And 15% FCS until reaching a confluence of approximately 80-90%. The chondrocytes were used in the experiments during the third or fourth week of primary culture.

1-octanol is a long-chain alkanol and a known connexin channel blocker that reduces intercellular conductance and rapidly inhibits the channels and hemichannels of the slit junctions.

1.2 Tissue processing and immunohistochemical assays (IHC)

The chip cuts were cut in series (4 mm) at -20 ° C in a cryostat (Leica CM 1510). The tissue sections were fixed with acetone for 10 min at 4 ° C, dried at room temperature (RT) and washed for 10 min with PBST (PBS with 0.1% Tween 20, pH 7.6). Before the staining, the endogenous peroxidase activity was inhibited by incubation with H2O2 and methanol for 10 min. Next, the cells were washed once with PBST and the anti-Cx43 primary antibody (sc-6560) obtained from Santa Cruz Biotechnology was applied for 1 h at RT. After washing them three times with PBST for 10 min each time, the sections were incubated for 1 h with a polymer labeled with peroxidase and conjugated with a goat Ig directed against mouse / rabbit Ig (Dako, Denmark). After three washes with PBST the peroxidase activity was revealed using a freshly prepared solution of chromogen as substrate containing 3,3-diaminobenzidine tetrahydrochloride and H2O2 (Dako, Denmark). The sections were washed in distilled water, counter-stained with Gill's hematoxylin, gradually dehydrated with alcohol of different grades and mounted in xylene with Depex (Serva, Germany). Negative controls were made (omitting the primary antibody) to test the specificity of each antibody. The tissue sections were also contratineron with Gill's hematoxylin. The slides were visualized using an Olympus BX61 microscope and a DP71 digital camera (Olympus). The calibration of the images was done with the AnalySISD V.5.0 software (Olympus Biosystems, Hamburg, Germany).

1.3 Cell proliferation assay

The progression of the cell cycle was measured by flow cytometry using propidium iodide (PI) staining to detect the total DNA content. The cells were harvested with trypsin, fixed in cold 70% ethanol and stored at 4 ° C for 12-24 hours. The cells were washed by centrifugation (2000 rpm, 15 minutes) in cold PBS. After centrifugation, the supernatant was discarded and the pellet resuspended in a solution containing 4 mg / ml RNase, 1 mg / ml PI and incubated at room temperature for 20 minutes. 20 x 104 cells were analyzed by BD FACScalibur ™ from BD biosciences.

1.4 RNA isolation and real-time PCR assay

The cultured primary chondrocytes were recovered by trypsinization. On the other hand, articular cartilage samples were digested in digestion buffer (200 mmol / l of Tris-HCl, 200 mmol / l of NaCl, 5% of SDS and 100 mmol / l of sodium citrate) containing proteinase K. The TRIzol reagent was added to the sample according to the manufacturer's instructions. Total RNA was treated with DNase (RNase-free DNase, Invitrogen) according to the manufacturer's instructions to ensure complete degradation of the DNA in the sample. CDNA was synthesized from 1 qg of RNA using the Superscript® VILO ™ cDNA synthesis kit (Invitrogen). The cDNA was diluted 10 times and the real-time PCR was performed with 5 ql of cDNA with the primers listed in Table 3 and SyBR-Green (Roche) in the LightCycler 480 Instrument Roche Applied Science. The cDNA was amplified at 95 ° C for 10 minutes, at 95 ° C for 10 seconds, at 60 ° C for 10 seconds and at 72 ° C for 12 seconds over 45 cycles, and then at 72 ° C for 5 minutes .

Table 3. Primers used in real-time PCR assays

Example 1. Identification of Cx43 as a factor inducing cartilage degeneration.

Cx43 is normally expressed in several normal tissues, which include osteocytes, osteoblasts, chondrocytes and synovial cells. As shown in Figure 1, Cx43 was located around chondrocytes and in all layers of articular cartilage. The samples of cartilage with different degrees of OA were analyzed. The IHC tests of the OA cartilage showed that the Cx43 is located in the membrane (surrounding area) and throughout the cytoplasm and the nucleus. OA cartilage showed levels of the Cx43 protein greater than healthy cartilage. The slides were visualized under an Olympus BX61 microscope with a DP71 digital camera (Olympus). The calibration of the images was made with the AnalySIS 5.0 software (Olympus Biosystems, Hamburg, Germany). The stain was examined under the optical microscope at 100x magnification with scale bars at 50 p.m.

Example 2. Inhibition of intercellular communication through cleft junctions (GJIC) exerts a protective effect against the production of catabolic factors in articular chondrocytes.

The progressive loss of articular cartilage includes the regulation by increase of the enzymes that degrade the extracellular matrix, such as metalloproteinases (MMP). MMP9 and MMP3, by degrading collagen and other extracellular matrix proteins, play a critical role in ECM homeostasis.

In order to demonstrate that the regulation by reduction of Cx43 or GJ protects against catabolic factors in the OA cartilage, 4 mm cartilage samples were prepared from explants obtained from patients with OA that were cut in the operating room immediately after the surgery. The samples were grown overnight in 48-well tissue culture plates with 1 ml of DMEM with 15% FCS containing 1 mM 1-octanol (Sigma) as anti-toxin compound 43 and incubated for 17 hours. The image comes from an explant of joint cartilage of a patient with grade 1 OA. The results presented in Figure 2 show that the inhibition of connexin channels induces a decrease in the expression of MMP3 and MMP9 and an increase in expression. of collagen type II more than double. The damaged surface area, affected by the loss of proteoglycans, is green due to the safranin O / solid green absorption.

In the analysis of gene expression by RT-PCR in real time of RNA isolated from cartilage explants, it is observed that 1-octanol increases the expression of type II collagen and reduces the expression of MMP3 and MMP9. These results show the beneficial effects of the anti-connexin compound in the articular chondrocytes of patients with OA.

Example 3. Inhibition of slit junction channels reduces cell proliferation of chondrocytes.

The cells were isolated from cartilage explants of patients with OA and cultured for 3-4 weeks at 37 ° C as described in materials and methods. The chondrocytes were cultured for 48 hours without serum to increase the proportion of cells in the G0 / G1 phase (70-80%). After 48 hours, 1 mM 1-octanol and serum (15% fetal bovine serum, FCS) were added to the medium as shown in Figure 3.

The distribution during the various phases of the cell cycle (DNA content) was measured after 17 hours of treatment by flow cytometry using 1 mg / ml of propidium iodide (IP). The number of cells in each phase of the cell cycle is shown in percentages (Figure 3A). In the absence of 1-octanol, the cells cross the cell cycle. However, ~ 60% of the cells treated with octanol remained for 17 hours in a state of arrest of the cell cycle in G0 / G1. The results shown in Figure 3 demonstrate that inhibition of GJIC using 1 mM 1-octanol for 17 hours inhibits the cell cycle progression of chondrocytes in primary culture.

In addition, the analysis of the expression of cell cycle regulators in G1 revealed that 1-octanol increases the expression of the p21 inhibitor of cdk and reduces the expression of the nuclear protein necessary for the progression of the cell cycle, cyclin D1 (CCND1) . The analysis of the expression of anabolic / catabolic factors confirmed that 1-octanol increases the expression of type II collagen and reduces the expression of the MMP3 gene (Figure 2B).

Figure 3C also shows the results obtained when the cells were previously cultured in the absence of serum. Figure 3C shows the analysis of the gene expression of the cells cultured for 17 hours in the presence of 15% FCS and 1-octanol.

In summary, the data shown herein have shown that individuals suffering from arthritis or presenting a joint injury and / or damage to the cartilage experience a continuous increase in Cx43 expression that triggers phenotypic changes, cell proliferation and an increase in the synthesis of proteins, accompanied by an increase in MMPs and other enzymes that degrade the extracellular matrix. The continued action of MMP, ADAMTS and other proteases results in degradation and / or loss of the cartilage. The treatment of patients susceptible to arthritis and / or degeneration of the cartilage with anti-connexin compounds, preferably with anti-connexin 43 compounds, induces the recovery of the original phenotype and the activation of the mechanisms involved in the regeneration of the cartilage and the repair of the tissue, inactivating cellular proliferation, the synthesis of catabolic factors and the propagation and transmission of mediators of inflammation and other toxic signals through the GJ channels.

Claims (15)

1. Anti-connexin compound for use in the treatment and / or prevention of a degenerative joint disease. 2. The anti-connexin compound for use according to claim 1, wherein the anti-connexin compound is selected from the group consisting of a gap junction inhibitor and / or a hemichannel inhibitor. The anti-connexin compound for use according to any of claims 1 to 2, wherein the anti-connexin compound is selected from the group consisting of an anti-connexin polynucleotide, an anti-connexin polypeptide, an anti-connexin peptide, an anti-connexin antibody or an antigen binding fragment thereof. The anti-connexin compound for use according to claim 3, wherein the anti-connexin polynucleotide is selected from the group consisting of antisense oligonucleotides, antisense polynucleotides, deoxyribozymes, morpholino-oligonucleotides, RNAi molecules, siRNA molecules, PNA molecules, DNAzymes and small nuclear RNAs UI mutated at the 5 'end and analogous thereof. The anti-connexin compound for use according to any of claims 3 to 4, wherein the anti-connexin polynucleotide is selected from any of the group comprising SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28 or SEQ ID NO: 30; the anti-connexin polypeptide is selected from the group comprising AVESAWGDEQSAFRCN (SEQ ID NO: 31), TQQPGCENVCY (SEQ ID NO: 32), DKSFPISHVRFWV (SEQ ID NO: 33), YTCKRDPCPHQVDC (SEQ ID NO: 34) and FLSRPTEKTIFIIF (SEQ ID NO. : 35); the anti-connexin peptide is selected from any of the group comprising a peptidomimetic, a mimetic peptide, a connexin phosphorylation compound, a benzoylaminobenzopyran compound, phenothiazides and fenamates; the anti-connexin antibody or the antigen-binding fragment thereof is an antibody, an F (v) fragment, a Fab fragment, a Fab 'fragment or an F (ab') 2 fragment. The anti-connexin compound for use according to any one of claims 1 to 5, wherein the anti-connexin compound blocks or reduces intercellular communication and / or the opening of the hemichannels. The anti-connexin compound for use according to any one of claims 1 to 5, wherein the anti-connexin compound inhibits the expression, formation and / or activity of a connexin, a connexin hemichannel or a slit junction. connexins. The anti-connexin compound for use according to any of claims 1 to 7, wherein the connexin is selected from the group comprising: connexin 43, connexin 45, connexin 46, connexin 26 or connexin 32. The anti-connexin compound for use according to any of claims 1 to 8, wherein the connexin is connexin 43. The anti-connexin compound for use according to any one of claims 1 to 9, wherein the degenerative joint disease is selected from the group consisting of arthritis, osteoarthritis, physical injury, Paget's disease, rheumatoid arthritis and osteoporosis . 11. Composition for use in the treatment and / or prevention of a degenerative joint disease comprising at least one anti-connexin compound according to any of claims 1 to 10. 12. The composition for use according to claim 11, further comprising a second active ingredient and pharmaceutically acceptable excipients, adjuvants or vehicles. The composition for use according to any of claims 11 to 12, wherein the composition is formulated for direct injection into diseased tissue. The composition for use according to claim 13, wherein the diseased tissue is selected from the group consisting of: articular cartilage, synovial membrane and subchondral bone. 15. The composition for use according to any of claims 11 to 14, wherein the degenerative joint disease is selected from the group consisting of arthritis, osteoarthritis, physical injury, Paget's disease, rheumatoid arthritis and osteoporosis.