Treatment of osteoarthritis using comt inhibitors

Abstract

The invention relates to a catechol O-methyltransferase (COMT) inhibitor for use in the treatment of osteoarthritis, the inhibitor being intended to slow down the progression of osteoarthritis and reduce osteoarthritis pain, as well as to a pharmaceutical composition comprising a COMT inhibitor for the treatment of osteoarthritis.

Description

[0001] TREATMENT OF OSTEOARTHRITIS WITH COMT INHIBITORS

[0002] Technical field

[0003] The present invention aims to provide a new strategy for the treatment of osteoarthritis.

[0004] Technological background

[0005] Osteoarthritis, or chronic degenerative arthropathy, is a chronic joint disease characterized by structural deterioration of articular cartilage. The most commonly affected joints, apart from the spine, are the knee, hand, and hip.

[0006] The symptoms of this pathology can vary depending on the joint affected but are generally characterized by persistent pain associated with functional discomfort, that is to say a limitation of the mobility of the affected joint.

[0007] Articular cartilage is a connective tissue composed of a single type of cell, chondrocytes, which secrete an extracellular matrix, the renewal of which they ensure. This matrix is ​​essentially made up of water, proteoglycans, and collagen.

[0008] Chondrocytes maintain a balance between degradation and restoration of the extracellular matrix. These mechanisms are complex and are regulated, for degradation, by pro-inflammatory cytokines such as TNF-α and IL-1 beta, and for restoration, by modulatory cytokines and growth factors, including IGF-1, TGF-β.

[0009] Arthritis-related cartilage destruction results from a disruption of this balance between degradation and restoration of the extracellular matrix. Several factors can promote this disruption of matrix homeostasis, including mechanical factors, linked for example to hyperpressure at the cartilage level or repeated microtraumas, or metabolic, genetic, hormonal factors, or even aging. The initiation of the arthritic process remains poorly understood to this day.

[0010] Currently, there is no effective and long-lasting treatment for osteoarthritis and its progression. The treatments used are purely symptomatic treatments with immediate or delayed action on the pain felt, most often analgesics, anti-inflammatories and intra-articular infiltrations of hyaluronic acid or corticosteroids.

[0011] The development of new analgesics to relieve osteoarthritis pain in patients intolerant or refractory to standard analgesic treatments is complex. In particular, anti-NGF (Nerve Growth Factor) antibodies, which target a neurotrophin involved in pain signal transduction, have been the subject of numerous clinical studies. These anti-NGFs have demonstrated a very clear pain-relieving effect, which is sometimes, unexpectedly and as yet unexplained, associated with an acceleration of osteoarthritis progression. There is no direct correlation between pain reduction and reduction in the progression of joint destruction.

[0012] The only effective curative treatment for mobility and pain is surgical treatment by implanting a prosthesis. Surgery is most often limited to the hip, knee, or shoulder joints, and very rarely to small joints such as the hands or feet. While hip replacement is the standard surgical treatment because it provides very good results, prosthetic replacement of other joints is not satisfactory in terms of mobility and pain. In addition, the prostheses used have a limited lifespan of between 15 and 20 years, and they must be replaced if they are fitted to young patients.

[0013] Currently, osteoarthritis affects approximately nine to ten million people in France. Given the aging population and the increasing prevalence of obesity in developed countries, a very sharp increase in the number of osteoarthritis patients is expected in the coming years. It is becoming necessary to find new therapeutic strategies capable of slowing the destruction of cartilage in osteoarthritis patients, while reducing joint pain.

[0014] Summary

[0015] The objective of the present invention is to provide novel compounds which can be used in the treatment of osteoarthritis.

[0016] The inventors have shown that, surprisingly, the COMT enzyme is expressed by joint chondrocytes, and that, even more surprisingly, COMT inhibitors administered at the joint are capable of slowing or inhibiting the progression of cartilage destruction, and reducing the pain associated with osteoarthritis via a local and direct action at the joint, through a mechanism that does not involve the central or peripheral nervous system.

[0017] Thus, according to a first subject, the invention relates to a catechol O-methyltransferase (COMT) inhibitor for use in the treatment of osteoarthritis.

[0018] Advantageously, the inhibitor is intended to slow the progression of osteoarthritis and to reduce osteoarthritis-induced pain. The inhibitor may be selected from a nitrocatechol-derived compound, an antibody or an interfering RNA.

[0019] Advantageously, the inhibitor is a nitrocatechol derivative comprising a catechol portion carrying a nitro group in the ortho or para position relative to one of the hydroxy groups of the catechol portion.

[0020] The nitrocatechol derivative is preferably selected from the group consisting of tolcapone, entacapone, opicapone, nitecapone, nebicapone, and mixtures thereof.

[0021] The inhibitor is preferably intended to be administered intra-articularly, orally, or subcutaneously.

[0022] Preferably, the inhibitor is intended to be administered intra-articularly.

[0023] The inhibitor may be administered in combination with one or more active substances selected from the group consisting of analgesics, non-steroidal anti-inflammatory drugs, steroidal anti-inflammatory drugs, and anti-arthritics, typically slow-acting symptomatic anti-arthritics or chondroprotectors.

[0024] The invention also relates to a pharmaceutical composition comprising one or more COMT inhibitors as defined in the first subject of the invention, and a pharmaceutically acceptable excipient, for use in the treatment of osteoarthritis.

[0025] Preferably, the pharmaceutical composition is intended to be administered intra-articularly, orally, or subcutaneously.

[0026] The pharmaceutical composition may further comprise one or more other active substances chosen from the group consisting of analgesics, non-steroidal anti-inflammatories, steroidal anti-inflammatories and anti-arthritics.

[0027] Preferably, the pharmaceutical composition is intended to be administered intra-articularly.

[0028] The pharmaceutical composition may include the inhibitor and anti-arthritic drugs such as platelet-rich plasma (PRP), chondroitin, and glucosamine.

[0029] The pharmaceutical composition may be formulated as a sustained-release composition.

[0030] The pharmaceutical composition may comprise the inhibitor dispersed in a polymeric fraction comprising a biodegradable and / or biocompatible polymer preferably selected from the group consisting of hyaluronic acid, collagen, chitosan, polylactic acid (PLA), glycolic acid, copolymers of lactic acid and glycolic acid (PLGA), polyoxalates, polycaprolactone, polyesters, and mixtures thereof.

[0031] Brief description of the drawings

[0032] [Figure 1]: Study of messenger RNA and proteins in human articular chondrocytes and synoviocytes. The results show that COMT is expressed (both mRNA and protein) in cells present at the joint (human samples)

[0033] [Figure 2]: Histological sections of osteoarthritic mice and normal mice (DMM model). The results show that COMT expression is increased in the joint of osteoarthritic mice compared to normal mice (DMM model).

[0034] [Figure 3]: A. Study of the expression of mRNA coding for MMP13 in chondrocytes. B. Study of the expression of mRNA coding for IL-6 in chondrocytes. The results show that pharmacological inhibition of COMT by tolcapone reduces the expression of genes involved in the osteoarthritic process (inflammation, catabolism) in human articular chondrocytes.

[0035] [Figure 4A]: Schematic illustrating the protocol including induction of osteoarthritis (Day 0) in a murine model of osteoarthritis (MIA mice), repeated injections of the pharmacological COMT inhibitor (tolcapone on Days 7, 14, 21), pain monitoring by the Von Frey technique on Days 12, 29, 37, 49 and 55 and by a posture test on Day 49, and killing on Day 56, for the study of cartilage degradation by histology.

[0036] [Figure 4B]: Study of pain assessed by the Von Frey technique as a function of time (day 0 = induction of osteoarthritis). The results show that intra-articular injections of a pharmacological COMT inhibitor (tolcapone) reduce pain (here the threshold of paw withdrawal following mechanical stimulation) in a murine model of osteoarthritis (MIA).

[0037] [Figure 5]: Histological sections of mouse knees in a mouse model of osteoarthritis (MIA mouse) with and without tolcapone injection. The results show that intra-articular injections of a pharmacological COMT inhibitor (tolcapone) reduce the progression of osteoarthritis in a mouse model of osteoarthritis (MIA).

[0038] [Figure 6]: Diagram illustrating static weight distribution 7 weeks after osteoarthritis induction (AOI). The results show that intra-articular injections of a pharmacological COMT inhibitor (tolcapone) improve the posture of osteoarthritic mice. Without tolcapone, the mice put less pressure on the osteoarthritic paw than on the normal paw. After intra-articular injection of tolcapone in the osteoarthritic paw, the animals put similar pressure on both hind paws, confirming the reduction in pain.

[0039] [Figure 7]: Diagram illustrating the quantification of the degree of osteoarthritis according to the OARSI score in a mouse model of osteoarthritis (MIA mouse) with and without tolcapone injection. The higher the score, the more severe the osteoarthritis.

[0040] [Figure 8]: Diagram illustrating pain assessed by the Von Frey technique 7 weeks after injection. The results show that intra-articular injections of a pharmacological COMT inhibitor (tolcapone) reduce pain in a murine model of osteoarthritis (MIA) at least as much or more than hyaluronic acid injections.

[0041] [Figure 9]: Study of the expression of mRNA coding for IL-6 in synoviocytes. The results show that pharmacological inhibition of COMT by tolcapone reduces the expression of genes involved in the osteoarthritic process (inflammation) in human joint synoviocytes.

[0042] [Figure 10]: Study of the expression of mRNA coding for COMT and MMP13 in human articular chondrocytes transfected with a control siRNA (SiCtrl) or siRNA directed against COMT (siCOMT) in the presence or absence of IL-1 b. The results show that inhibition of COMT expression by siRNA reduces the expression of genes involved in the osteoarthritic process (catabolism) in human articular chondrocytes.

[0043] Detailed description

[0044] Catechol O-methyltransferase (COMT) is the enzyme responsible for the O-methylation of endogenous neurotransmitters and xenobiotics and hormones incorporating catechol structures. Catecholamines are a family of molecules that act as hormones or neurotransmitters. The main catecholamines present in humans are adrenaline (or epinephrine), noradrenaline (or norepinephrine), and dopamine. They are produced by the adrenal glands and by postganglionic neurons of the parasympathetic nervous system, by an enzyme called tyrosine hydroxylase (TH). Catecholamines are degraded by COMT, which catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to a hydroxyl group on a catechol nucleus.COMT exists in two forms, a long membrane form which allows the cessation of dopaminergic and noradrenergic synaptic neurotransmission and a short soluble form responsible for the elimination of exogenous catechols.

[0045] COMT is a biological drug target for the treatment of various central and peripheral nervous system disorders, including Parkinson's disease, depression, schizophrenia, and other diseases related to dopamine deficiency.

[0046] In particular, drugs in the therapeutic class of catechol-O-methyltransferase (l-COMT) inhibitors are indicated in the management of Parkinson's disease, as an adjuvant to prolong the duration of action of drugs containing dopamine precursors such as L-Dopa. Thus, ICOMTs used in Parkinson's disease are systematically prescribed in combination with L-DOPA, and have no symptomatic effect as monotherapy.

[0047] The inventors have shown that COMT inhibitors can have a therapeutic effect in a completely different class of pathologies, namely osteoarthritis.

[0048] A role of COMT in pain, especially in osteoarthritis pain, has been mentioned by some authors based on genetic association studies (see e.g. Meurs et al., Arthritis & Rheumatism. 60 628-629. 2009; Schutte et al., Research in Gerontological Nursing. Vol. 13, No. 4. 2020; Martire et al., Scandinavian Journal of Pain. 10 6-12. 2016; Govil et al., Eur J Pain. 24 398-412. 2020) but was, however, contested (see e.g. Olesen et al., Pain Pract. 18 587-596. 2018; Neogi et al., Ann Rheum Dis. 73 315-317. 2014). However, it was assumed that the mechanism of COMT inhibitors on pain was related to an effect on pain perception via the nervous system. In other words, COMT inhibitors were suggested as simple analgesics. A direct role of COMT in the joint had never been suggested.Prior to the inventors' work, the COMT enzyme had never been investigated as a target for the treatment of osteoarthritis, and in particular the expression of COMT within the joint had never been investigated.

[0049] The inventors have now demonstrated that, surprisingly, COMT is expressed in articular chondrocytes, and that even more surprisingly, local and direct inhibition of COMT at the joint level makes it possible to reduce, slow down or inhibit the progression of cartilage destruction simultaneously with a reduction in pain. The inventors have thus shown that COMT inhibitors are capable of inhibiting the expression of genes involved in the osteoarthritic destruction process in articular chondrocytes, in particular the gene coding for a pro-inflammatory cytokine such as interleukin-6, and a metalloprotease such as MMP-13.

[0050] The inventors have thus demonstrated a direct inhibitory action of COMT inhibitors on several factors involved in the destruction of cartilage and inflammation during the osteoarthritic process.

[0051] Furthermore, the inventors have shown that COMT inhibitors are also capable of stimulating the expression of genes involved in cartilage biosynthesis, such as COL2A1 which codes for components of type 2 collagen.

[0052] The inventors have thus shown that COMT inhibitors not only reduce cartilage catabolism, but also stimulate its anabolism. Importantly, the inventors have shown that COMT inhibitors administered intra-articularly are capable of slowing the progression of osteoarthritic cartilage degradation and reducing the pain felt by the subject.

[0053] They also showed that COMT inhibitors administered at the joint level are also capable of reducing pain associated with osteoarthritis via a local and direct action at the joint level, by a mechanism that does not involve the central or peripheral nervous system.

[0054] The use of these inhibitors therefore represents a new strategy for treating osteoarthritis. This strategy is all the more advantageous since several COMT inhibitors have already been approved for other therapeutic applications.

[0055] First object of the invention

[0056] According to a first object, the present invention therefore relates to a COMT inhibitor for use in the treatment of osteoarthritis.

[0057] COMT inhibitor

[0058] In the present invention, the expression "COMT inhibitor" or more simply hereinafter "inhibitor" refers to any compound capable of inhibiting COMT enzymatic activity or the expression of the COMT enzyme within articular chondrocytes. Preferably, the COMT inhibitor is also capable of inhibiting COMT enzymatic activity or the expression of the COMT enzyme within articular synoviocytes. According to one embodiment, the COMT inhibitor is capable of slowing down or inhibiting the enzymatic activity of COMT.

[0059] Preferably, the inhibitor used according to the invention is a nitrocatechol derivative.

[0060] A nitrocatechol derivative means a compound comprising a catechol portion bearing a nitro group in the ortho or para position relative to one of the hydroxy groups of the catechol portion.

[0061] The inhibitor used according to the invention is preferably a nitrocatechol derivative chosen from the group consisting of tolcapone, entacapone, opicapone, nitecapone, nebicapone, and mixtures thereof.

[0062] The inhibitor used according to the invention may also be an antibody blocking the activity of COMT.

[0063] The term "antibody" means an immunoglobulin molecule, i.e., a molecule that contains an antigen-binding site capable of immunospecifically binding to an antigen. In the present invention, the term "antibody" encompasses not only whole antibody molecules, but also antibody fragments.

[0064] In one embodiment, the antibody is a monoclonal anti-COMT antibody.

[0065] According to another embodiment, the inhibitor is capable of slowing or inhibiting the expression of COMT. Such an inhibitor may be an interfering RNA or an antisense oligonucleotide complementary to the COMT messenger RNA sequence.

[0066] The RNA interference strategy is well known to those skilled in the art. This technique uses a process that occurs naturally in animal cells, including the reduction or silencing of targeted gene expression. The formation of double-stranded RNA leads to the specific degradation by the host's cellular machinery of transcripts complementary to this double-stranded RNA. Thus, by targeting COMT by RNA interference, it is possible to reduce the level of expression or completely suppress the expression of the COMT gene in the targeted cells.

[0067] A small interfering RNA (hereinafter siRNA) is characterized in that it has a region with a double-stranded (ds) structure. The inhibition is specific to the target sequence, the nucleotide sequence of one strand of the ds region of the RNA comprising 15 to 25 nucleotides and being identical to the portion of the target gene. The nucleotide sequence of the other strand of the ds region of the RNA is complementary to that of the first strand and to the portion of the target gene. The person skilled in the art knows how to prepare siRNAs targeting COMT. In particular, numerous programs are available for the design of siRNAs:

[0068] - “siSearch Program” (Improved and automated prediction of effective siRNA”, Chalk AM, Wahlesdelt C, and Sonnhammer ELL, Biochemical and Biophysical Research Communications, 2004).

[0069] - “SiDirect” (Direct: highly effective, target-specific siRNA design software for mammalian RNA interference, Yuki Naito et al, Nucleic Acids Res, Vol. 32, No. Web Server issue (C) Oxford University Press 2004).

[0070] According to one embodiment, the inhibitor comprises a double-stranded RNA of approximately 15 to 30 nucleotides, preferably 19 to 25, more preferably 19, 20, 21, 22 or 23 nucleotides in length, complementary (strand 1) and identical (strand 2) to a portion of the COMT sequence.

[0071] Preferably, the gene encoding COMT has a sequence having the identification number NG_011526 in the GenBank database.

[0072] Preferably, the COMT messenger RNA has a sequence having the identification number NM_000754 in the GenBank database.

[0073] Preferably, the COMT protein has a sequence having the identification number NP_000745 (MB-COMT isoform) or NP_009294 (S-COMT isoform) in the GenBank database.

[0074] These siRNAs may additionally contain a TT or UU dinucleotide at the 3' end.

[0075] In a particular embodiment, the nitrocatechol derivative, antibody, siRNA or antisense oligonucleotide described above is capable of reducing or even specifically blocking the expression of COMT. For example, the invention relates to a nitrocatechol derivative, an antibody targeting COMT, a siRNA targeting COMT or an antisense oligonucleotide targeting COMT allowing a reduction of greater than 80%, 90%, 95% or 99% of the expression of COMT, in which this capacity is preferably tested in vitro on chondrocytes stimulated with interleukin-1 b (IL-1 b).

[0076] I L-1 b is classically used to model osteoarthritis in vitro, and the person skilled in the art knows how to perform this type of test.

[0077] In a particular embodiment, the nitrocatechol derivative, antibody, siRNA or antisense oligonucleotide described above, in addition to its ability to reduce the expression of COMT as described above, is also capable of reducing the expression of matrix metalloprotease 13 (MMP13) and / or interleukin-6 (IL-6). For example, the invention relates to a nitrocatechol derivative, an antibody, a siRNA or an antisense oligonucleotide allowing a reduction of at least 10%, 15%, 20%; 30%, 40% or 50% of the expression of MMP13 and / or IL-6, in which this ability is preferably tested in vitro on chondrocytes stimulated with interleukin-1 b (IL-1 b).

[0078] An antisense oligonucleotide (hereinafter OAS) is a single-stranded DNA fragment that can specifically bind to a target messenger RNA. The nucleotide sequence of the antisense oligonucleotide is complementary to that of the messenger RNA it targets and generally comprises 15 to 25 nucleotides. By hybridizing to their target mRNA, OAS induces its degradation by ribonuclease H (RNAse H).

[0079] The inhibitor used according to the invention is preferably chosen from the group consisting of tolcapone, entacapone, opicapone and their mixtures.

[0080] Treatment methods

[0081] The term "treatment" refers to any action that reduces, eliminates or delays the symptoms associated with a pathology in a subject to be treated. It includes both curative and prophylactic treatment of a disease. A curative treatment is defined as a treatment resulting in a cure or a treatment alleviating, improving and / or eliminating, reducing and / or stabilizing the symptoms of a disease or the suffering it causes. A prophylactic treatment includes both a treatment resulting in the prevention of a disease and a treatment reducing and / or delaying the incidence of a disease or the risk of its occurrence. In the context of the present invention, the term "treatment" refers more particularly to the slowing down or inhibition of cartilage destruction in the osteoarthritic process, which is accompanied by a reduction in joint pain.

[0082] Thus, the inhibitor used according to the invention allows the slowing down or inhibition of the destruction of cartilage in the context of the arthritic process.

[0083] In the present invention, a “chondroprotector” or “background antiarthritic” designates a drug making it possible to slow down or inhibit the process of destruction of cartilage.

[0084] Advantageously, the inhibitor used according to the invention also allows the reduction of pain caused by osteoarthritis, in particular without action on the central or peripheral nervous system. Advantageously, the inhibitor used according to the invention allows a reduction of pain caused by osteoarthritis without direct action on the central nervous system. Advantageously, the inhibitor used according to the invention can therefore be used both as a chondroprotective agent and as a pain-relieving agent.

[0085] In one embodiment, the inhibitor is used for the treatment of osteoarthritis, wherein the treatment comprises slowing the destruction of cartilage and reducing the pain associated with osteoarthritis. The treatment includes, in particular, reducing the expression of genes involved in catabolism such as MMP13, reducing the expression of genes involved in inflammation such as IL-6, and stimulating the expression and secretion of collagen such as type II collagen (COL2A1), at the joint.

[0086] As will be seen below, the inhibitor according to the invention is preferably formulated so as to be administered directly to the joint, that is to say intra-articularly.

[0087] The present invention also relates to the use of an inhibitor according to the invention, preferably a nitrocatechol derivative chosen in particular from the group consisting of tolcapone, entacapone, opicapone and their mixtures, for the manufacture of a medicament intended for the treatment of osteoarthritis, in which the medicament is preferably administered intra-articularly.

[0088] The present invention further relates to a method for treating osteoarthritis in a subject, said method comprising administering a therapeutically effective dose of an inhibitor according to the invention to said subject, wherein the inhibitor according to the invention is preferably a nitrocatechol derivative in particular chosen from the group consisting of tolcapone, entacapone, opicapone and mixtures thereof, and wherein the inhibitor according to the invention is preferably administered to said subject intra-articularly.

[0089] The present invention also relates to the use of an inhibitor according to the invention for manufacturing a medicament intended to treat osteoarthritis, in which the inhibitor according to the invention is preferably a nitrocatechol derivative notably chosen from the group consisting of tolcapone, entacapone, opicapone and their mixtures, and in which the inhibitor according to the invention is preferably administered to said subject by intra-articular route.

[0090] Osteoarthritis

[0091] For the purposes of the present invention, the term "osteoarthritis" refers to any chronic joint pathology resulting in structural deterioration of the articular cartilage. The term "osteoarthritis" thus includes in particular primary or primitive osteoarthritis, in which the subject suffers from osteoarthritis without an identifiable cause or particular predisposition, in particular without a traumatic cause or associated inflammatory disease. The term "osteoarthritis" also includes secondary osteoarthritis, in which the subject has suffered trauma to a joint (the osteoarthritis is then referred to as "traumatic osteoarthritis"), for example during a road accident, or suffers from a disease predisposing to osteoarthritis, for example chronic inflammatory rheumatism such as rheumatoid arthritis, spondyloarthropathy, microcrystalline rheumatism, or metabolic diseases such as obesity, diabetes, or hemochromatosis.More generally, in the present invention, the term "osteoarthritis" also includes all joint pathologies in which there is structural deterioration of the articular cartilage such as rheumatoid arthritis, gout or psoriatic rheumatism, as well as diseases which affect the joints and generate pain such as hemochromatosis, joint dysplasias and rare skeletal diseases.

[0092] Osteoarthritis is suspected during the interview and clinical examination. The patient describes mechanical joint pain of specific location. This pain is most often reproduced during the clinical examination.

[0093] The diagnosis of osteoarthritis is based on imaging, particularly standard X-rays, CT scans, or MRIs.

[0094] Standard X-rays can reveal the main characteristic of osteoarthritis, namely a reduction in the quantity of cartilage (the most commonly used radiological term is joint narrowing), and depending on the stage of osteoarthritis, bone damage such as osteosclerosis (hardening of the bone immediately under the cartilage, whitened image on X-ray), the presence of osteophytes (bone growths on X-ray), and / or geodes (cysts in the bone under the cartilage visible on X-ray).

[0095] A radiological definition is based on the Kellgren and Lawrence classification (Kellgren JH, Lawrence JS. Radiological assessment of osteoarthritis. Ann Rheum Dis, 1957, 16: 494-502). The Kellgren and Lawrence score is a composite index taking into account osteophytes and joint space narrowing. This classification has 4 stages: doubtful, minimal, certain, and advanced osteoarthritis. Stage 2 is usually used as the diagnostic threshold. The radiological stages of knee osteoarthritis (gonarthrosis) are as follows: Stage 0 normal radiograph; Stage 1 osteophyte of doubtful significance; Stage 2 clear osteophyte without modification of the joint space; Stage 3 clear osteophyte and reduction of the joint space; Stage 4 severe narrowing of the joint space and sclerosis of the subchondral bone.The radiological stages of hip osteoarthritis (coxarthrosis) according to the Kellgren and Laurence classification are as follows: Stage 0: normal radiograph; Stage 1: joint narrowing, doubtful pericapital osteophytosis; Stage 2: joint narrowing, osteophytosis, moderate bone sclerosis; Stage 3: clear joint narrowing with slight osteophytosis, bone sclerosis with cyst, minimal deformation of the femoral head and acetabulum; Stage 4: disappearance of the joint space with bone sclerosis and cyst, significant deformation of the femoral head and acetabulum, with major osteophytosis.

[0096] The use of CT or MRI is interesting and often performed in cases of clinical symptoms strongly suggestive of osteoarthritis when X-rays are normal. These imaging examinations are more sensitive and allow for the detection of earlier abnormalities not visible on standard X-rays.

[0097] In the present invention, osteoarthritis can affect any joint, including the joints of the spine and peripheral joints, in particular those of the knee (gonarthrosis), hip (coxarthrosis), ankle, foot, hand, wrist, elbow or even shoulder.

[0098] According to one embodiment, the osteoarthritis to be treated is osteoarthritis of a peripheral joint, preferably osteoarthritis of the knee.

[0099] According to one embodiment, the osteoarthritis to be treated is primary or secondary osteoarthritis, preferably primary osteoarthritis.

[0100] The inhibitor according to the invention is preferably used in the treatment of osteoarthritis of a peripheral joint, in particular of a knee joint.

[0101] According to one embodiment, the inhibitor according to the invention is used in the treatment of osteoarthritis which is not primary osteoarthritis.

[0102] Topic to be addressed

[0103] The subject to be treated, or patient, is an animal, preferably a mammal.

[0104] According to one embodiment, the subject to be treated is a human.

[0105] The human subject is preferably an adult over 40 years of age, preferably over 50 years of age.

[0106] The subject is preferably a subject having stage 2 or 3 osteoarthritis according to the Kellgren and Lawrence classification. In another embodiment, the subject is a domestic animal, preferably a companion animal, an animal intended for sporting use, or a production animal.

[0107] The pet is preferably chosen from a cat or a dog.

[0108] The animal intended for sporting use is preferably the horse, typically a riding horse.

[0109] The production animal is preferably chosen from a bovine such as a cow, bull or ox, a sheep such as a mutton or a pig.

[0110] According to one embodiment, the subject to be treated does not have disorders of the central and / or peripheral nervous system, such as Parkinson's disease, schizophrenia and other diseases linked to dopamine deficiency.

[0111] According to one embodiment, the subject to be treated does not present any pain other than that linked to osteoarthritis.

[0112] Mode of administration

[0113] The inhibitor used according to the invention can be administered by any known route of administration, including in particular intra-articular, oral, or subcutaneous.

[0114] According to a particularly preferred embodiment, the inhibitor is administered by intra-articular injection, in particular at the level of the arthritic joint.

[0115] The term "intra-articular", relating to the method of administration, designates any route of administration intended for the implantation or injection of a compound or composition within or near the intra-articular space of a joint, in particular an arthritic joint. Preferably, the term "intra-articular" designates a method of administration in which the inhibitor is administered within the intra-articular space of an arthritic joint.

[0116] The inventors have shown that, particularly surprisingly, the intra-articular administration of a COMT inhibitor makes it possible to limit the progression of cartilage degradation.

[0117] Equally surprisingly, the inventors have shown that intra-articular administration of a COMT inhibitor also makes it possible to sustainably reduce pain for the subject, even when the COMT inhibitor is administered locally and not systemically. The inventors consider that intra-articular administration of the inhibitor according to the invention has the advantage of maximizing the effectiveness of the inhibitor both in terms of limiting cartilage degradation and reducing pain. In other words, without wishing to be bound by a particular theory, the inventors consider that intra-articular administration of the inhibitor according to the invention has the advantage of making it possible to reduce the therapeutically effective dose relative to other routes of administration such as the oral route.Furthermore, as mentioned previously, the inventors consider that the inhibitor administered at the intra-articular level allows a reduction of pain according to a local and direct mechanism at the level of the joint which does not involve the central or peripheral nervous system.

[0118] In the case of intra-articular administration, as will be detailed below, the inhibitor according to the invention may be administered in combination with other substances, in particular substances allowing the prolonged release of the inhibitor, such as the use of hydrogels, for example hyaluronic acid.

[0119] The inhibitor used according to the invention can also be administered orally. Oral administration is particularly preferred when the subject is a domestic animal, in particular a cat or a dog or a horse. According to one embodiment, the subject is a domestic animal and the administration is intra-articular.

[0120] The inhibitor used according to the invention is preferably administered to the subject at a therapeutically effective dose. The term "therapeutically effective dose" as used herein refers to the amount necessary to observe a therapeutic or preventive activity on osteoarthritis, in particular the amount necessary to observe an inhibition or a slowing down of the destruction of osteoarthritic cartilage. The amount of inhibitor to be administered as well as the duration of the treatment can be evaluated by a person skilled in the art according to criteria such as the physiological state, sex, age, weight of the subject to be treated, the nature of the osteoarthritic joint(s) to be treated, the inhibitor chosen as well as the route of administration used. The therapeutically effective dose for each treatment can be administered in the form of a single dose or divided doses.

[0121] The inhibitor used according to the invention can be administered in the form of a single dose or in divided doses.

[0122] According to one embodiment, the inhibitor used according to the invention is preferably a nitrocatechol derivative chosen in particular from the group consisting of tolcapone, entacapone, opicapone and their mixtures, and administered to the subject by intra-articular route. According to one embodiment, the inhibitor used according to the invention is preferably a nitrocatechol derivative chosen in particular from the group consisting of tolcapone, entacapone, opicapone and their mixtures, and administered to the subject by oral route.

[0123] The inhibitor used according to the invention may be administered in combination with other active substances. The inhibitor according to the invention and the other active substance(s) may be administered simultaneously (at the same time) or sequentially (at different times), by the same route of administration or different routes of administration.

[0124] Within the scope of the present invention, the following are particularly distinguished among the active substances useful in the treatment of osteoarthritis:

[0125] -fast-acting symptomatic anti-arthritics, which are effective against the painful symptoms of osteoarthritis. This category includes various products including analgesics such as paracetamol, non-steroidal anti-inflammatory drugs such as aceclofenac, niflumic acid, tiaprofenic acid, celecoxib, diclofenac, etodolac, etoricoxib, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meloxicam, nabumetone, naproxen, piroxicam, sulindac tenoxicam, and steroidal anti-inflammatory drugs, particularly corticosteroids, particularly immediate- or long-acting injectable corticosteroids such as betamethasone;

[0126] - slow-acting symptomatic anti-arthritics, or AASAL, which are effective on the painful symptoms of osteoarthritis. Various products are classified in this category, including platelet-rich plasma (PRP), chondroitin, for example chondroitin sulfate, glucosamine, for example glucosamine phosphate or sulfate, and hyaluronic acid. Glucosamine and chondroitin are usually administered orally. Hyaluronic acid and PRP are usually administered intra-articularly;

[0127] - chondroprotectors or basic anti-arthritic drugs. As defined above, chondroprotectors are effective in slowing down or inhibiting the process of cartilage destruction and degradation. Some AASALs, such as PRP, are sometimes considered to have chondroprotective activity; however, this remains controversial.

[0128] Thus, according to one embodiment, the inhibitor used according to the invention can in particular be administered in combination with one or more active substances chosen from the group consisting of analgesics such as paracetamol, non-steroidal anti-inflammatory drugs such as aceclofenac, niflumic acid, tiaprofenic acid, celecoxib, diclofenac, etodolac, etoricoxib, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meloxicam, nabumetone, naproxen, piroxicam, sulindac tenoxicam; steroidal anti-inflammatory drugs, in particular of the corticosteroid type, in particular injectable corticosteroids with immediate or prolonged action such as betamethasone; slow-acting symptomatic anti-arthritics such as platelet-rich plasma (PRP), chondroitin, for example chondroitin sulfate, and glucosamine, for example glucosamine phosphate or sulfate; chondroprotectors.

[0129] Second object of the invention

[0130] The inhibitor of the present disclosure is preferably administered in the form of a pharmaceutical composition.

[0131] Thus, according to a second subject, the invention relates to a pharmaceutical composition comprising one or more COMT inhibitors according to the first subject of the invention, for the treatment of osteoarthritis and a pharmaceutically acceptable excipient.

[0132] “Pharmaceutically acceptable” means a substance that is not biologically or otherwise undesirable, i.e., can be incorporated into a pharmaceutical composition administered to a patient or animal without causing undesirable biological effects or without interacting deleteriously with any of the other components of the composition in which it is contained, for example by inhibiting or diminishing the anti-inflammatory properties of the COMT inhibitor.

[0133] Typically, the pharmaceutically acceptable excipient may be selected from a diluent, a disintegrant, a binder, a glidant, a lubricating agent, a wetting agent, a buffering agent, a suspending agent, an adjuvant, an emulsifier, an absorbent, a preservative, a surfactant, a sweetening agent, an antioxidant, or a mixture thereof. These excipients are for example described in "The Science and Practice of Pharmacy 1995, edited by EW Martin, Mack Publishing Company, 19th edition, Easton, Pa."

[0134] The amount of COMT inhibitor according to the invention in the compositions can vary so as to administer an effective amount of inhibitor to obtain the desired therapeutic response for a particular patient or pet.

[0135] The composition may further comprise one or more other active substances as defined above, preferably chosen from the group of immediate or prolonged-acting injectable corticosteroids such as betamethasone; slow-acting symptomatic antiarthrosis agents such as platelet-rich plasma (PRP), chondroitin, for example chondroitin sulfate, and glucosamine, for example glucosamine phosphate or sulfate; chondroprotectors.

[0136] The inhibitor(s) are preferably one or more nitrocatechol derivatives chosen in particular from the group consisting of tolcapone, entacapone, opicapone and mixtures thereof.

[0137] The pharmaceutical composition used according to the invention is preferably in the form of an ingestible or injectable composition, preferably in the form of a composition intended to be administered intra-articularly or orally.

[0138] In one embodiment, the pharmaceutical composition used according to the invention is a composition in the form of an injectable composition intended to be administered intra-articularly.

[0139] In this embodiment, the composition is advantageously in the form of a prolonged-release composition.

[0140] The term "extended release" in relation to the formulation means a formulation for reducing the clearance of the inhibitor of the invention, or in other words for increasing the half-life of the inhibitor in the joint after its administration. The terms "extended release"; "controlled release", "delayed release", or "long-acting" are interchangeable in the present invention.

[0141] A prolonged-release pharmaceutical composition has the advantage of increasing the therapeutic efficacy of each dose, and in the context of fractional administration, of extending the time interval between two administrations.

[0142] In this embodiment, the pharmaceutical composition used according to the invention may in particular comprise the inhibitor according to the invention and a polymeric fraction, the inhibitor preferably being dispersed in the polymeric fraction.

[0143] The polymeric fraction may in particular comprise a polymer, preferably a biodegradable and / or biocompatible polymer, chosen from the group including but not limited to hyaluronic acid, collagen, chitosan, polylactic acid (PLA), glycolic acid, copolymers of lactic acid and glycolic acid (PLGA), polyoxalates, polycaprolactone, polyesters, and mixtures thereof.

[0144] In one embodiment, the polymeric moiety is in the form of a gel or capable of forming a gel. The term "gel" refers to a non-fluid polymer network swollen by a solvent.

[0145] The term "gel-forming polymeric fraction" refers to a polymeric fraction capable of gelling in situ, i.e., of forming a gel once placed in the biological environment in which it has been administered, typically the intra-articular space.

[0146] The pharmaceutical composition of this embodiment may, for example, be in the form of a solution comprising the inhibitor(s) and the polymeric fraction or a suspension of particles, typically microparticles, in which the particles are formed from the polymeric fraction in the form of a gel in which the inhibitor(s) are encapsulated.

[0147] In a particular embodiment, the polymer, polymeric fraction or gel is biocompatible and / or biodegradable.

[0148] The term "biodegradable," in relation to the polymer, polymeric fraction, or gel, means that in situ, i.e., in the joint, the polymer, polymeric fraction, or gel is capable of degrading naturally, under physiological conditions, thus allowing the release of the inhibitor in a prolonged manner. The reactions involved in biodegradation may include hydrolysis reactions, i.e., the breaking of covalent bonds by reaction with water. These reactions may be catalyzed by the action of enzymes naturally present at the injection site, typically in the joint.

[0149] As used herein, the term "biocompatible" as used in the polymer, polymeric fraction or gel means a polymer, polymeric fraction or gel having the ability to not degrade the biological environment in which it is placed.

[0150] According to one embodiment, the pharmaceutical composition used according to the invention comprises one or more inhibitors according to the first subject of the invention, preferably one or more nitrocatechol derivatives notably chosen from the group consisting of tolcapone, entacapone, opicapone and their mixtures, and hyaluronic acid. EXAMPLES

[0151] Example 1: In vitro tests on human cartilage (tolcapone)

[0152] Materials and methods:

[0153] Cell culture:

[0154] The cells were obtained from surgical waste according to current regulations (protocol approved by the North-West III Personal Protection Committee (CPP). After the patients signed the free and informed consent, femoral heads, synovial membranes and bone marrow are recovered after hip arthroplasty operations in osteoarthritic patients.

[0155] The femoral heads are dissected and cartilage chips are made. Chondrocytes are released by digestion with pronase type XIV (2 mg / ml for 15 minutes; Sigma), followed by digestion with collagenase type II (2 mg / ml overnight; Gibco). The cells are then seeded at 4x10 4 cells / cm 2in Dulbecco's modified Eagle's medium (DM EM) supplemented with 10% fetal calf serum (FCS) with a cocktail of antibiotics. They are then incubated at 37°C in a humidified atmosphere containing 5% CO2.

[0156] Synoviocytes were obtained from synovial membranes of patients undergoing hip replacement surgery. Synoviocytes were released by digestion with type I collagenase (2 mg / ml overnight; Gibco). After rinsing with PBS, the recovered cells were seeded with DMEM medium supplemented with 10% FCS and an antibiotic cocktail.

[0157] Bone marrow cells were fractionated on a Hypaque-Ficoll density gradient. Mononuclear cells were isolated, seeded at a density of 5.10 4 cells / cm 2and cultured in alpha-MEM supplemented with 10% fetal bovine serum (Invitrogen, Cergy-Pontoise, France), 2 mM L-glutamine, 1 ng / ml FGF-2 (Sigma Chemical Co), and antibiotics. At nearly 80% confluence, cells were harvested by trypsinization (0.25% trypsin / 1 mM EDTA, Invitrogen) and seeded at 10 3 cells / cm 2 After 5 passages, the disappearance of hematopoietic markers CD34 and CD45 was verified by RT-PCR and the cells were used for experiments.

[0158] For all three cell types, cells were amplified in a humid chamber at 37°C under 5% CO2. Culture media were changed 2 to 3 times per week. A mycoplasma test was performed to verify that they were not contaminated. A portion was treated with IL-113. 1 ng / mL (Sigma) diluted in DMEM medium with 10% FCS and 0.1% penicillin and streptomycin for 48 hours, in the presence or absence of tolcapone (HY-17406, CliniSciences).

[0159] mRNA extraction and RT-PCR:

[0160] Total RNA is extracted using the RNeasy mini kit (Qiagen) according to the manufacturer's instructions. 1 pg of RNA is then treated with DNAse I (Sigma-Aldrich) and reverse-transcribed into cDNA using Moloney murine leukemia virus reverse transcriptase (Invitrogen) according to the supplier's protocol. The cDNA is diluted 1 / 100 and stored at -20°C until use in real-time PCR.

[0161] Real-time PCR primers routinely used in the laboratory or designed using the Primer-BLAST tool (NCBI) against RPL13A, IL6, MMP13 and COMT are used.

[0162] Forward and reverse primers were diluted to 130 nM, with 5 μl of cDNA diluted 1 / 100th and 7.5 μl of Power SYBR Green master mix in a 15 μL reaction volume (Applied Biosystems). Samples were denatured at 95°C for 10 seconds, followed by primer annealing and polymerization for 1 minute at 60°C for 40 cycles. These cycles were performed using the Step One Plus Real Time PCR system (Applied Biosystems). Relative mRNA expression levels were calculated using the 2-AACT method and normalized to the expression of RPL13A, a housekeeping gene classically used as a reference for gene expression normalization in human joint cells. To visualize amplification, samples after RT-PCR were loaded onto a 2% agarose gel. The signals were revealed using a Bio-Rad imager.

[0163] Protein extraction and Western-Blot:

[0164] After treatment, the cell lawns are rinsed with PBS, then the cell lawn is scraped into a lysis buffer called "radioimmunoprecipitation" (RIPA: Tris-HCl 50 mM; IGEPAL 1%; NaCl 150 mM; EGTA 1 mM; NaF 1 mM) supplemented with protease inhibitors (leupeptin, aprotinin, pepstatin, phenylmethylsulfonyl fluoride (PMSF), and phosphatases (sodium orthovanadate NasVO^, in order to lyse the cells and extract the proteins. The cell lysates obtained are incubated for 30 minutes at 4°C, then centrifuged for 30 min at 10,000g at 4°C. The supernatants containing the proteins are recovered and stored at -20°C until their use in western blotting. The protein concentration of each sample is then determined according to the Bradford method with the Bradford Protein Assay reagent (Biorad). A standard range is produced in duplicate and the samples in triplicate.

[0165] 30 pg of protein per sample migrated by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and were transferred to polyvinylidene difluoride membranes (Bio-Rad). After blocking nonspecific sites, the membrane was incubated with primary antibodies against COMT (PA5-76864; Thermo-Fisher) diluted 1 / 1000 overnight at 4°C. After rinsing, the membranes were incubated with secondary antibodies (SC-516087, Santa-Cruz) coupled to peroxidase for 1 h at room temperature. After incubation with the oxidizing reagent and luminol (1 minute with shaking, Western Lightning ECL Pro, PerkinElmer. Actin (sc-47778, Santa-Cruz) was used to verify the homogeneous deposition of the samples. The signals were revealed using an imager (Bio-Rad).

[0166] Proteomics:

[0167] The proteomics experiments were performed as a service on the Proteogen platform of the University of Caen Normandy. The analyses were carried out using protein samples from the cell layer (RIPA extraction) or directly on the culture media, according to the procedure described in the following article (Brochard S, Pontin J, Bernay B, Boumediene K, Conrozier T, Baugé C. The benefit of combining curcumin, bromelain and harpagophytum to reduce inflammation in osteoarthritic synovial cells. BMC Complement Med Ther. 2021 Oct 14;21(1):261. doi: 10.1186 / s12906-021-03435-7. PMID: 34649531 ; PMCID: PMC8515758.).

[0168] Results :

[0169] COMT expression in different cell types present in the joint (chondrocytes, synoviocytes, mesenchymal stem cells) was analyzed by RT-PCR and Western blotting (Figure 1). These analyses revealed that COMT is expressed at both mRNA and protein levels in the 3 cell types studied. Thus, RT-PCR results show that human chondrocytes, human synoviocytes and mesenchymal stem cells from human bone marrow express COMT mRNA. Furthermore, Western blotting demonstrates production of soluble (24 kDa) and membrane (30 kDa) forms of COMT protein by these 3 cell types.

[0170] Having demonstrated that COMT was expressed in chondrocytes, we investigated the effect of inhibiting COMT activity in these cells stimulated with interleukin-1b (IL-1b). IL-1b is indeed classically used to model osteoarthritis in in vitro studies. This pro-inflammatory cytokine is present in the osteoarthritic joint and is involved in the pathophysiology of osteoarthritis (inflammation, catabolism). Thus, human articular chondrocytes were treated with increasing doses of a pharmacological COMT inhibitor, tolcapone.

[0171] Our experiments (Figure 3) clearly show that COMT inhibition by tolcapone opposes the harmful action of I L-1 b and notably reduces the expression of genes involved in catabolism (such as MMP13) and inflammation (such as IL-6) in chondrocytes.

[0172] To gain a more comprehensive understanding of the effect of COMT inhibitors on chondrocyte phenotype, proteomics experiments were performed. The results of these experiments (not shown) show that pharmacological inhibition of COMT affects the expression of a large number of proteins involved in the osteoarthritic process in human articular chondrocytes. In particular, pharmacological inhibition of COMT stimulates the expression and secretion of collagens, including type 2 collagen (COL2A1).

[0173] In particular, we observe, in human articular chondrocytes:

[0174] - a decrease in the secretion of MMP13 and IL-6 in the presence of tolcapone, confirming the anti-catabolic and anti-inflammatory effect of tolcapone,

[0175] - an increase in COL2A1 secretion, suggesting an anabolic effect of tolcapone.

[0176] These results are presented in the Table below:

[0177] Table 1: Secretome study: comparison of proteins secreted by IL-1-stimulated chondrocytes in the presence or absence of tolcapone. These results confirm the anti-inflammatory and anti-catabolic action of COMT inhibition in IL-1-stimulated human articular chondrocytes and also show a complementary action on anabolism since we identify an increase in the expression and secretion of the type 2 collagen protein (an essential matrix protein of cartilage). These results show that targeting COMT could have an anti-catabolic, anti-inflammatory and chondroprotective action, and suggest a direct action on the cells present at the joint level.

[0178] Example 2: In vivo tests in a mouse model

[0179] Materials and methods:

[0180] Animals, osteoarthritis induction and treatments

[0181] This study was authorized by the Ministry of Higher Education, Research and Innovation after favorable opinion from the local ethics committee, CENOMEXA (approval n°B14118015). The study is carried out in an approved animal facility (UMS-CYCERON). Male C57BL7 mice, 10 weeks old, were used. The mice were placed in cages of 8, in a reverse cycle, with food and water available ad libitum. The health status of each mouse was assessed daily.

[0182] The traumatic osteoarthritis model, reproduced by destabilization of the medial meniscus (DMM) in mice, is induced under general anesthesia. After disinfection of the skin with 70% alcohol, the skin and subcutaneous fascia are cut with sterile scissors. Then, the medial lateral ligament was cut transversely and the fibers of the vastus medialis muscle were sectioned longitudinally to allow external dislocation of the patella. Then, the meniscectomy of the medial meniscus of the right knee was performed under a magnifying glass. Finally, the vastus medialis muscle and the skin were sutured. These mice constitute an osteoarthritis group (DMM). A control group, called "Sham", consisting of mice undergoing the same intervention without meniscectomy, was established.

[0183] For the inflammatory osteoarthritis (MIA) model, an intra-articular injection of Monosodium lodoAcetate (0.75 mg in 10 μl) was performed under general anesthesia. After clinical identification of the patellar ligament, injections were made intra-articularly in the knee after passing the patellar ligament with a 27-gauge needle. These mice constituted an osteoarthritis group (MIA). A control group, called "sham", consisted of mice undergoing an injection of physiological saline. Tolcapone injections were performed according to the same protocol at 7, 14 and 21 days after the induction of osteoarthritis according to the experimental design shown in Figure 4A.

[0184] Immunohistology

[0185] Mouse knees were fixed in 4% PFA for 24 h and decalcified in 0.5 M EDTA pH 8.0 for 10 days before being embedded in paraffin. Sagittal sections of 5 μm thickness were made and mounted on SuperFrost® Silanized microscope slides. Before use, the slides were deparaffinized by 3 x 15 min baths of xylene. Then rehydrated in successive baths of 100%, 70% and 40% ethanol and then two successive baths of water and PBS. Antigenic remasking was performed by heat (70°) in a citrate buffer (pH 6) for 4 h, followed by digestion with hyaluronidase (1 mg / ml in PBS). After 2 rinses with PBS, endogenous peroxidases are inhibited by incubation with 3% H2O2 at room temperature for 10 minutes. Non-specific sites are blocked by incubation at room temperature for 30 minutes with a blocking solution (2.5% horse serum, 2.5% BSA in PBS).The rabbit polyclonal antibody against COMT (PA5-76864; Thermo-Fisher) is used at 1 / 500 diluted in blocking solution diluted to half, its incubation is done overnight at 4°. Detection is done using the ImmPRESS staining reagent kit (Vector Laboratories, France) with an anti-rabbit antibody. Visualization is carried out using the diaminobenzidine substrate kit for peroxidase (Vector Laboratories, France). Finally, a counterstaining with blue water (three drops of 1% toluidine blue in 200ml of water) before mounting with ENTELAN.

[0186] Histology

[0187] The joints were placed in a fixative solution (37% formaldehyde, PBS) for 3 days, then decalcified using Osteosoft for 48 hours. Then, the joints were stored at -80°C in the Optimal Cutting Temperature compound (OCT). 10 μm thick sections were made at -25°C using a Cryostat (CM 3050 S, Leica BIOSYSTEMS, France), then mounted on SuperFrost® microscope slides. The slides were then stained with Fast Green and then with safranin-O. Safranin-O highlights the components of the articular cartilage. Glycosaminoglycans and proteoglycans appear in red. Fast Green serves as a counterstain and stains mineralized bone green.

[0188] Von Frey: Mechanical allodynia (pain triggered by a stimulus that is normally painless) is assessed by the Von Frey filament test (BIOSEB In Vivo Research Instruments®). This test is based on the principle that when the end of the filament (of given length and diameter) is applied perpendicularly, the force exerted by the filament increases until the filament flexes. Beyond this, the force exerted by the filament remains constant. For this test, mice are placed in an individual cage, on a grid. A 30-minute habituation period was required before the start of the test. Filaments exerting increasingly greater force are applied under the mouse's paw, from the bottom up, perpendicular to the plantar surface of the mouse until the filament begins to flex. Withdrawal of the paw, startle, or licking of the paw are interpreted as positive responses, synonymous with pain or discomfort.The results are presented as the value obtained on the arthritic paw relative to the sensitivity of the contralateral (non-arthritic) paw.

[0189] Results :

[0190] We continued our study by investigating COMT protein expression in normal and osteoarthritic mouse joints (DMM traumatic model) (Figure 2). Immunohistochemistry results show that COMT expression is increased in osteoarthritic joints compared to healthy joints and that COMT is mainly expressed by chondrocytes.

[0191] The effect of tolcapone was then tested in vivo. For this, intra-articular injections of tolcapone were performed 7 days, 14 days and 21 days after the induction of osteoarthritis in mice (MIA inflammatory model). Pain was assessed throughout the experiment by Von Frey analysis. The histological study was done at 8 weeks (see experimental design, Figure 4A).

[0192] Our results clearly show a beneficial effect of tolcapone on the progression of osteoarthritis. On the one hand, we observe that intra-articular injection of tolcapone at the osteoarthritic joint significantly reduces the induced pain (Figure 4B), while improving the structure of the joint (Figure 5). Thus, the histological study clearly shows that intra-articular injections of the COMT inhibitor at the osteoarthritic joint slowed the progression of osteoarthritis since it reduced joint damage and very significantly limited cartilage degradation in these mice ("MIA+tolcapone") compared to untreated osteoarthritic mice ("MIA+vehicle"). These results demonstrate that COMT inhibition at the joint is useful in the treatment of osteoarthritis. Not only does it slow the progression of osteoarthritis (chondroprotective effect) but it also simultaneously reduces the induced pain.

[0193] Example 3: In vivo test in a murine model: additional characterizations (OARSI / SWB)

[0194] The study of Example 2 was continued by investigating the effects of tolcapone on static incapacitance (posture test) and OARSI score (quantification of osteoarthritis progression).

[0195] Materials and methods

[0196] Animals, osteoarthritis induction and treatments

[0197] The animals, the induction of osteoarthritis and the treatments are as described in Example 2.

[0198] Static incapacitance

[0199] The static weight bearing test (SWB; BioSeb, France) allows the evaluation of the weight applied by the animal under each hind paw in a static position. For this, the animal is placed in a transparent restraint box so that its hind paws rest on two weight sensors connected to a recorder. This recorder is connected to a foot pedal to start the acquisitions. Zero is achieved between each animal and verified using a 2 g standard weight. The recording parameters are 2 seconds per acquisition. The value retained is an average of the weight applied during these 2 seconds. Ten successive acquisitions are carried out. The acquisition values ​​are retained only if the animal has not changed position during the recording and if its hind paws are the only elements resting on the sensors.For analysis, the average weight applied under the right paw is reported to the average weight applied to both paws (right + left).

[0200] OARSI score:

[0201] Joint samples after safranin-0 staining were scored according to the Osteoarthritis Research Society International (OARSI) scale as detailed in Pritzker et al., 2006 (Pritzker, KP, et al., Osteoarthritis cartilage histopathology: grading and staging. Osteoarthritis Cartilage, 2006. 14(1): p. 13-29). This score is separated into 6 grades, including for the last two, changes in the subchondral bone. The different grades of the OARSI score according to Pritzker allow for precise quantification of cartilage degradation, with a score ranging from 0 to 6.5. The higher the score, the more severe the osteoarthritis.

[0202] Results

[0203] Static incapacitance

[0204] The results are presented in Figure 6.

[0205] The results clearly show a beneficial effect of tolcapone on static weight distribution. These results demonstrate that tolcapone restores the animal's posture, i.e., similar support on both hind legs (no difference in support between the arthritic leg and the normal leg), confirming the absence of pain in the arthritic leg after treatment with tolcapone.

[0206] OARSI score

[0207] The results are presented in Figure 7.

[0208] The results show a beneficial effect of tolcapone on the progression of osteoarthritis.

[0209] Example 4: In vivo test in a murine model: tolcapone / hyaluronic acid comparison

[0210] The study presented in Example 2 is continued by comparing the effects of tolcapone with those of hyaluronic acid (HA).

[0211] Materials and methods:

[0212] Animals, osteoarthritis induction and treatments

[0213] The animals, osteoarthritis induction and treatments are as described in Example 2, except that three groups of mice are tested:

[0214] - MIA: MIA mice subjected to injections of physiological serum,

[0215] - MIA HA: MIA mice subjected to hyaluronic acid injections

[0216] - MIA Tolcapone: MIA mice subjected to tolcapone injections.

[0217] Von Frey:

[0218] Pain is assessed by the Von Frey technique as detailed in Example 2, 7 weeks after injection. Results:

[0219] The results are presented in Figure 8.

[0220] The results show that intra-articular injection of tolcapone into the arthritic joint reduces the induced pain at least as much or more than hyaluronic acid.

[0221] Example 5: In vitro test on human cartilage (synoviocytes)

[0222] The study of Example 1 was continued by investigating the effect of tolcapone on synoviocytes.

[0223] Materials and methods:

[0224] The materials and methods are the same as those outlined in Example 1, except that the study is conducted on synoviocytes.

[0225] Results :

[0226] The results are shown in Figure 9.

[0227] The results show that, similarly to articular chondrocytes, pharmacological inhibition of COMT by tolcapone reduces the expression of genes involved in the osteoarthritic process (inflammation) in human articular synoviocytes such as IL-6.

[0228] Example 6: In vitro test on human cartilage (SiRNA)

[0229] The study of Example 1 was continued by investigating the effect of siRNA directed against COMT.

[0230] Materials and methods:

[0231] The materials and methods are the same as those set out in Example 1, except that the cells are placed in the presence of SiRNA.

[0232] Control or COMT-directed siRNA (ON-TARGETplus Human COMT (1312) siRNA - SMARTpool, Dharmacon) were transfected into chondrocytes by nucleofection (Amaxa), according to the manufacturer's protocol. Briefly, chondrocytes were harvested. Then, 4 million cells were mixed with 200nmol of siRNA (control or COMT siRNA) in P3 Primary Cell 4D-NucleofectorX solution and placed in 1 cm transfection cuvettes. 2 to be electroporated using the 4D-Nucleofector X unit. The predefined program ER-100 was used. After nucleofection, the cells were plated in appropriate dishes and incubated in culture medium.

[0233] Results :

[0234] The results are shown in Figure 10. The results show that COMT knockdown by RNA interference reduces MMP13 expression in IL1-stimulated chondrocytes. This shows that pharmacological inhibition of COMT reduces the expression of genes involved in the osteoarthritic process (catabolism) in human articular chondrocytes, regardless of the structure of the COMT inhibitor used: nitrocatechol derivative such as tolcapone (see Example 1) or small interfering RNA as in the present example.

Claims

CLAIMS 1. Catechol O-methyltransferase (COMT) inhibitor for use in the treatment of osteoarthritis.

2. An inhibitor for use according to claim 1, wherein the inhibitor is intended to slow the progression of osteoarthritis and to reduce osteoarthritis-induced pain.

3. An inhibitor for use according to any one of claims 1 or 2, wherein the inhibitor is selected from a nitrocatechol-derived compound, an antibody or an interfering RNA.

4. An inhibitor for use according to any one of claims 1 to 3 wherein the inhibitor is a nitrocatechol derivative comprising a catechol moiety carrying a nitro group in the ortho or para position relative to one of the hydroxy groups of the catechol moiety.

5. An inhibitor for use according to any one of claims 3 or 4, wherein the nitrocatechol derivative is selected from the group consisting of tolcapone, entacapone, opicapone, nitecapone, nebicapone, and mixtures thereof.

6. An inhibitor for use according to any one of claims 1 to 5, wherein the inhibitor is intended to be administered intra-articularly, orally, or subcutaneously.

7. An inhibitor for use according to any one of claims 1 to 6, wherein the inhibitor is intended to be administered intra-articularly.

8. An inhibitor for use according to any one of claims 1 to 7, wherein the inhibitor is administered in combination with one or more active substances selected from the group consisting of analgesics, non-steroidal anti-inflammatories, steroidal anti-inflammatories, and anti-arthrosis drugs, typically slow-acting symptomatic anti-arthrosis drugs or chondroprotectors.

9. A pharmaceutical composition comprising one or more COMT inhibitors as defined in any one of claims 1 to 7, and a pharmaceutically acceptable excipient, for use in the treatment of osteoarthritis.

10. A pharmaceutical composition for use according to claim 9, wherein the composition is intended to be administered intra-articularly, orally, or subcutaneously.

11. Pharmaceutical composition for use according to any one of claims 9 or 10, further comprising one or more other active substances selected from the group consisting of analgesics, non-steroidal anti-inflammatories, steroidal anti-inflammatories and anti-arthrosis drugs, typically slow-acting symptomatic anti-arthrosis drugs or chondroprotectors.

12. Pharmaceutical composition for use according to any one of claims 9 to 11, intended to be administered intra-articularly.

13. A pharmaceutical composition for use according to claim 12, comprising the inhibitor and anti-arthritics such as platelet-rich plasma (PRP), chondroitin, and glucosamine.

14. A pharmaceutical composition for use according to any one of claims 9 to 13, formulated as a sustained release composition.

15. A pharmaceutical composition for use according to any one of claims 9 to 13, comprising the inhibitor dispersed in a polymeric fraction comprising a biodegradable and / or biocompatible polymer preferably selected from the group consisting of hyaluronic acid, collagen, chitosan, polylactic acid (PLA), glycolic acid, copolymers of lactic acid and glycolic acid (PLGA), polyoxalates, polycaprolactone, polyesters, and mixtures thereof.

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