Inhibitor of the transcription factor AP-1 for use in the treatment of age-related diseases

Abstract

The present invention relates to a treatment of Age-related diseases (ARDs). Here, the inventors identified the AP-1 family of transcription factors (TFs) as a novel actionable therapeutic target to reverse the senescence phenotype and to ameliorate age-related chronic lung and cardiac disease outcomes by repurposing a small molecule AP-1 inhibitor (APli) T- 5224. They demonstrated that AP-1 inhibition induces an improve of ARDs such as Emphysema, lung fibrosis or cardiac function. Other cardiac and pulmonary diseases could also benefit of this treatment. Thus, the present invention relates to an AP-1 inhibitor for use in the treatment of ARDs in subjects in need thereof.

Description

[0001] INHIBITOR OF THE TRANSCRIPTION FACTOR AP-1 FOR USE IN THE TREATMENT OF AGE-RELATED DISEASES

[0002] FIELD OF THE INVENTION

[0003] The invention relates to an AP-1 inhibitor for use in treating Age-related diseases in subjects in need thereof.

[0004] BACKGROUND OF THE INVENTION

[0005] Chronological age is the most significant individual risk factor for developing chronic diseases called "Age-Related Diseases" (ARDs). In particular, the risk of pulmonary and cardiovascular diseases increases with age.

[0006] Cellular senescence is a complex stress response that stably arrests the proliferation of damaged and dysfunctional mitotic and postmitotic cells. The senescence process is accompanied by widespread metabolic and gene expression changes, including a notable increase in CDK -inhibitors p21 (CDKN1A) and pl6 (CDKN2A) and a senescence-associated secretory phenotype (SASP), which installs a sterile chronic inflammatory microenvironment perturbing tissue homeostasis and promoting disease development over time.

[0007] Senescent cells (SCs) contribute to inducing ARDs such as chronic obstructive pulmonary disease (COPD), pulmonary fibrosis (PF), and cardiometabolic diseases (CMD) (Gorgoulis V et al., 2019, Cell).

[0008] Activator protein 1 (AP-1) is a transcription factor (TF) family composed of homo- and heterodimers of Jun, FOS, MAF, and ATF proteins. It regulates the expression of many pro- inflammatory cytokines and the expression of instrumental SASP constituents. Its dysregulation involves inflammatory disorders, chronic lung and cardiac diseases, and cancer (Martinez- Zamudio R.I et al., 2020, Nature Cell Biology, Eferl R et al., 2003, Nat Rev Cancer,' Ye N et al., 2014, J Med Cherrr, Wilson N et al., 2003, Am J Physiol Heart Circ Physiol, Frantz S et al., 2003, Cardiovasc Res & Takemoto Y et al., 1999, J Mol Cell Cardiol).

[0009] Methods aiming at reprogramming or eliminating SCs hold great promise for health benefits. While SC ablation in murine models delays ARDs, in the liver, for example, SCs are located among the sinusoidal endothelial cells, which are essential for body detoxification and, thus, removing SCs impacts liver function negatively (Ishida M et al., 2015 J Intensive Care). Therefore, great caution has to be exercised when applying senotherapies, and it is essential to develop new methods to target SCs to prevent or diminish their detrimental impact on tissue and organ function.

[0010] SUMMARY OF THE INVENTION

[0011] The inventors identified the AP-1 family TF as a novel therapeutic target to treat "Age- Related Diseases (ARDs)." Indeed, they discovered that AP-1 inhibition by the small molecule T-5224 reverses the SASP and ameliorates age-related lung and cardiac diseases.

[0012] The present invention relates to an AP-1 inhibitor for treating ARDs in subjects in need thereof.

[0013] The present invention is defined by the claims.

[0014] The following detailed description, figures, and examples do not fall under the scope of the present invention and are present for understanding and illustration purposes only.

[0015] Furthermore, any reference in the description to treatment methods refers to the present invention's compounds, pharmaceutical compositions, and medication for treating the human body by therapy.

[0016] DETAILED DESCRIPTION OF THE INVENTION

[0017] The first object of the invention relates to an AP-1 inhibitor for treating Age-related diseases in subjects in need thereof.

[0018] As used herein, the term "age-related diseases" or "ARDs" refers to diseases often seen with increasing frequency of senescence. Age-related diseases include increased senescence biomarker expression (e.g., pl6 & p21) and the SASP. In particular, "age-related diseases" or "ARDs" refers to any age-related disease involving lung dysfunction. It relates to emphysema, pulmonary fibrosis (PF), or chronic obstructive pulmonary (COPD). In the present invention, the term "age-related diseases" or "ARDs" also refers to cardiometabolic diseases (CMD), such as cardiac remodeling with interstitial myocardial fibrosis and myocardial hypertrophy with or without alteration in systolic and diastolic function. In one embodiment, the AP-1 inhibitor is used to treat COPD. COPD is a chronic inflammatory lung disease characterized by chronic systemic inflammation (Negewo N.A et al., 2015, Respirology).

[0019] In one embodiment, the AP-1 inhibitor is used to treat emphysema. Emphysema is a disease wherein the lung elasticity decreases, and the alveoli are dilated (Sharafkhaneh A et al., 2008, Proc Am Thorac Soc).

[0020] In one embodiment, the AP-1 inhibitor is used to treat pulmonary fibrosis. Fibrosis is a disease wherein the lung tissue becomes thicker and stiffer due to an accumulation of connective tissue (Thannickal V.J. et al., 2004, Annu Rev Med).

[0021] In one embodiment, the AP-1 inhibitor treats cardiac remodeling and / or heart hypertrophy induced or not by pulmonary artery dysfunctions or pulmonary diseases (Myers G.B et al., 1948, Am Heart J).

[0022] The AP-1 is well known in the state of the art. AP-1 is a transcription factor composed of homo- and heterodimers of Jun (v-Jun, c-Jun, JunB, and JunD), Fos (v-Fos, c-Fos, FosB, Fral, and Fra2), ATF (ATF2, ATF3 / LRF1, B-ATF, JDP1, and JDP2), and MAF (c-Maf, MafB, MafA, MafG / F / K, and Nrl) proteins (Shaulian E et al., 2001, Oncogene & Chinenov Y et al., 2001, Oncogene). Its Entrez reference number is 843244, and its Uniprot reference number is P05412. AP-1 regulates the expression of different genes involved in stimuli response, such as the expression of cytokines, growth factors, or instrumental SASP constituents (Karakaslar E.O et al., 2022, Aging Cell,' Yadav S et al., 2017, Martinez-Zamudio R.I et al., 2020, Nature Cell Biology; Molecular and Cellular Biochemistry & Samuel S et al., 2005, Biochemical Journal). Many studies demonstrated its involvement in cancer, pulmonary fibrosis, inflammation, transplant rejection, or cardiovascular diseases (Tsuchida K et al., 2006, Medical Chemistry & Hong S.H et al., 2010, Blood). Moreover, it has been shown that AP-1 is strongly involved in regulating heart and lung function. Indeed, studies have shown an increase in Fra2 expression in pulmonary fibrosis and that JunD and Fral play an important role in regulating cardiac hypertrophy (Ye N et al., 2014, J Med Chem). As used herein, the term "AP-1 inhibitor" refers to any molecule or compound, natural or not, capable of inhibiting the function of AP-1, like initiating the proliferation and differentiation of AP-1 or a molecule or compound destabilizing AP-1. The term "AP-1 inhibitor" also denotes an inhibitor of the expression of the gene coding for the protein. The term "AP-1 inhibitor" can also mean an antagonist. "Antagonist" refers to any molecule or compound interacting directly or indirectly with a biological receptor to block or reduce the AP-1 function.

[0023] The term "AP-1 inhibitor" encompasses any AP-1 inhibitor currently known in the art or that will be identified in the future and includes any chemical entity that, upon administration to a subject, results in inhibition or down-regulation of a biological activity associated with activation of the AP-1. Thus, AP-1 inhibitors are well known in the state and comprise small molecules, c-Jun inhibitors, or drugs containing benzophenone derivative (Aikawa Y et al., 2012, US 2012 / 0046326A1 & Ye N et al., 2014, JMedCheni).

[0024] Methods for selecting an appropriate inhibitor are well-known in the art. In particular, the methods for selecting an inhibitor specifically inhibiting AP-1 are known in the prior art (Daschner PJ et al., 1999, Breast Cancer Research and Treatment,' Hocevar B. A et al., 2000, Transforming Growth Factor-Beta Protocols,' Tsuchida K et al., 2006, Medical Chemistry & Martinez-Zamudio R.I et al., 2020, Nature Cell Biology). For example, the AP-1 inhibitors are identified by measurement of the AP-1 concentration in biospecimens (blood, serum, plasma) before and after depletion / inhibition of API. Then, AP-1 is detected using standard protocols such as qRT-PCR, immunoblot, ELISA, or Luminex.

[0025] As used herein, the term "treatment" or "treat" refers to both prophylactic, preventive, and curative or disease-modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from an illness or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disease to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder or to prolong the survival of a subject beyond that expected in the absence of such treatment. "Therapeutic regimen" means the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen," which may include administering a more significant dose of the drug than a physician would use during a maintenance regimen, administering a medication more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for extended periods (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of particular predetermined criteria [e.g., disease manifestation, etc.]).

[0026] In one embodiment, the compound, according to the invention, is a low molecular weight compound, e g., a small organic molecule.

[0027] As used herein, the term "small organic molecule" refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 10000 Da, preferably up to 5000 Da, more preferably up to 2000 Da, and most preferably up to 1000 Da.

[0028] Small organic molecules as inhibitors of AP-1 are well known in the state-of-the-art such as SP 100030 analogs, SPC-839 analogs, K1115A, Curcumin, Mom ordin I, norditerpenes nagilactone, inumakilactone or Citrifolinin A (Ye N et al., 2014, J Med Chem & Tsuchida K et al., 2006, Medical Chemistry). In particular, the 3-5-4-(cyclopentyloxy)-2-hydroxybenzoyl)-2- (3-hydroxy-l,2-benzisoxazol-6-yl)methoxy phenylpropionic acid (T-5224) is known in the prior art and used in many studies (Aikawa Y et al., 2012, US 2012 / 0046326 Al; Izuta S et al., 2012, Biotechnol Lett, Daschner P.J et al., 1999, Breast Cancer Research and Treatment, Aikawa Y et al., 2008, Nature Biotechnology & Ishida M et al., 2015, J Intensive Care). Indeed, it has been shown that this selective inhibitor of c-Fos / AP-1, synthesized by Toyama Chemical Co (Uchihashi S et al., 2011, Drug Metab Dispos), improves survival in lethal lipopolysaccharide (LPS)-induced AKI (Ishida M et al., 2015, J Intensive Care). Moreover, T- 5224 is commonly used in human clinical trials (Ye N et al., 2014, J Med Chem). Methods for selecting an appropriate small organic molecule are well-known in the art. In particular, the procedures for selecting small molecules specifically inhibiting AP-1 are known in the prior art.

[0029] In one embodiment, the compound, according to the invention, is T-5224.

[0030] In one embodiment, the compound, according to the invention, is an anti-AP-1 antibody.

[0031] The antibodies as inhibitors of AP-1 are well known in the state of the art and commonly sold, such as A5968 by Sigma-Aldrich (Seol D.W et al., 2000, Oncogene & Philips N et al., 1995, Journal of Biological Chemistry).

[0032] Antibodies directed against AP-1 can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against AP-1 can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Methods for production and isolation include but are not limited to the hybridoma technique initially described by Kohler and Milstein (1975), the human B-cell hybridoma technique (Cote et al., 1983), and the EBV-hybridoma technique (Cole et al. 1985). Alternatively, methods for producing single-chain antibodies (e.g., U.S. Pat. No. 4,946,778) can be adapted to produce anti-AP-1 single-chain antibodies. Compounds useful in practicing the present invention also include anti-AP-1 antibody fragments, including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and / or scFv expression libraries can be constructed to rapidly identify fragments with the desired specificity to AP-1.

[0033] Humanized anti-AP-1 antibodies and antibody fragments from there can also be prepared according to known methods. "Humanized antibodies" are nonhuman (e.g., rodent) chimeric antibodies that contain minimal sequence derived from nonhuman immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a nonhuman species (donor antibody) such as a mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, the human immunoglobulin's framework region (FR) residues are replaced by corresponding nonhuman residues. Furthermore, humanized antibodies may comprise residues not found in the recipient or donor antibodies. These modifications are made to refine antibody performance further. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a nonhuman immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Methods for making humanized antibodies are described, for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech, U.S. Pat. No. 4,816,397).

[0034] Thus, methods for selecting an appropriate antibody are well-known in the art. In particular, the procedures for selecting an antibody specifically inhibiting AP-1 are known in the prior art.

[0035] For this invention, neutralizing antibodies of AP-1 are selected.

[0036] In one embodiment, the compound, according to the invention, is an aptamer.

[0037] The aptamers as inhibitors of AP-1 are well known in the state of the art (Walters R.D et al., 2014, PlosOne & Ge M.H et al., 2021, Biomaterials Science).

[0038] Aptamers are a class of molecules representing an alternative to antibodies regarding molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences that can recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through the Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses, and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers are a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A, selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).

[0039] Methods for selecting an aptamer specifically inhibiting AP-1 are known in the prior art. For this invention, neutralizing aptamers of AP-1 are selected. In one embodiment, the compound, according to the invention, is a polypeptide. In a particular embodiment, the polypeptide can prevent the function of AP-1. The polypeptide can be a mutated AP-1 protein or a similar protein without the function of AP-1.

[0040] The polypeptides as inhibitors of AP-1 function are well known in the state of the art.

[0041] The polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. To have sufficient amounts of polypeptide or functional equivalents thereof for use by the present invention, expression may conveniently be achieved by culturing recombinant host cells containing the polypeptide of the invention under appropriate conditions. Preferably, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in various host cells are well known. When expressed in recombinant form, the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria, mammalian, plant, yeast, and baculovirus systems. Mammalian cell lines available in the art for expressing a heterologous polypeptide include Chinese hamster ovary cells: HeLa cells, baby hamster kidney cells, and many others. Bacteria are also preferred hosts for recombinant protein production due to the ease with which bacteria may be manipulated and grown. A standard, preferred bacterial host is E. coli.

[0042] In specific embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified to improve their therapeutic efficacy. Such modification of therapeutic compounds may decrease toxicity, increase circulatory time, or alter biodistribution. For example, the toxicity of potentially important therapeutic compounds can be reduced significantly by combination with a variety of drug carrier vehicles that modify biodistribution. For instance, adding dipeptides can improve the penetration of a circulating agent in the eye through the blood-retinal barrier by using endogenous transporters.

[0043] A strategy for improving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, enhance the mode of cellular uptake, change permeability through physiological barriers, and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water-soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain. Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials that would retain the biocompatibility properties of PEG but would have the added advantage of numerous attachment points per molecule (providing more significant drug loading) and could be synthetically designed to suit a variety of applications. Those with skill in the art are aware of PEGylation techniques for effectively modifying drugs.

[0044] For example, drug delivery polymers that consist of alternating polymers of PEG and tri -functional monomers, such as lysine, have been used by VectraMed (Plainsboro, N.J.). The PEG chains (typically 2000 daltons or less) are linked to lysine's a- and e-amino groups through stable urethane linkages. Such copolymers retain the desirable properties of PEG while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain. The reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules. These polymers are useful in producing stable, long-circulating pro-drugs by varying the polymer's molecular weight, the PEG segments' molecular weight, and the cleavable linkage between the drug and the polymer. The molecular weight of the PEG segments affects the spacing of the drug / linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provide more significant drug loading). In general, increasing the molecular weight of the block copolymer conjugate will increase the circulatory half-life of the conjugate.

[0045] Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomerular filtration (e.g., less than 60 kDa). In addition, the polymer backbone is critical in maintaining circulatory half-life and biodistribution, and linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue. For example, this type of tissue-activated drug delivery is advantageous where delivery to a particular biodistribution site is required, and the therapeutic agent is released at or near the site of pathology. Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, the prevalence of active enzymes, and the cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.

[0046] Methods for selecting a polypeptide specifically inhibiting AP-1 are known in the prior art. In one embodiment, the compound, according to the invention, is an inhibitor of AP-1 gene expression.

[0047] As used herein, the term "inhibitor of the AP-1 gene expression" refers to a natural or synthetic compound that has a biological effect to inhibit gene expression. In some embodiments, said inhibitor of gene expression is a small inhibitory, a ribozyme, or an antisense oligonucleotide.

[0048] The modulation of AP-1 gene expression is well-known in the state-of-the-art (Seol D.W et al., 2000, Oncogene). In particular, the inhibitors of AP-1 gene expression such as siRNA (Comandante-Lou N et al., 2022, Cell Reports & Martinez-Zamudio R.I et al., 2020, Nature Cell Biology) or antisense oligonucleotides (Dean N.M et al., 2009, Antisense & Nucleic acid drug development & Urakami S et al., 1997, Biochem Biophys Res Commun).

[0049] Small inhibitory RNAs (siRNAs) can also function as inhibitors of AP-1 gene expression for use in the present invention. AP-1 gene expression can be reduced by contacting a subject or cell with a small double-stranded RNA (dsRNA) or a vector or construct causing the production of a small double-stranded RNA, such that AP-1 gene expression is specifically inhibited (i.e., RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g., see, for example, Tuchl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01 / 36646, WO 99 / 32619, and WO 01 / 68836). In particular, the methods for selecting small inhibitory, specifically inhibiting AP- 1 gene expression, are known in the prior art.

[0050] Ribozymes can also function as inhibitors of AP-1 gene expression in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of AP-1 mRNA sequences are useful within the present invention's scope. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of about 15 and 20 ribonucleotides corresponding to the target gene region containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be assessed by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays. Methods for selecting ribozymes specifically inhibiting AP-1 gene expression are known in the prior art.

[0051] Antisense oligonucleotides can also function as inhibitors of AP-1 gene expression in the present invention. Antisense oligonucleotides, including antisense RNA molecules and antisense DNA molecules, would act to directly block the translation of AP-1 mRNAby binding to it and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of AP-1, and therefore activity, in a cell. Antisense oligonucleotides can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g., see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). In particular, the methods for selecting antisense oligonucleotides specifically inhibiting AP-1 gene expression are known in the prior art.

[0052] Both antisense oligonucleotides and ribozymes useful as inhibitors of AP-1 gene expression can be prepared by known methods. These include techniques for chemical syntheses, such as, e.g., solid-phase phosphoramidite chemical synthesis. Alternatively, in vitro or in vivo transcription of DNA sequences encoding the RNA molecule can generate antisense RNA molecules. Such DNA sequences can be incorporated into vectors containing suitable RNA polymerase promoters such as the T7 or SP6 polymerase. Various modifications to the invention's oligonucleotides can be introduced to increase intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and / or 3' ends of the molecule or the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

[0053] Antisense oligonucleotides, siRNAs, and ribozymes of the invention may be delivered in vivo alone or association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and, preferably, cells expressing AP-1. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation resulting in the vector's absence. In general, the vectors useful in the invention include but are not limited to, plasmids, phagemids, viruses, and other vehicles derived from viral or bacterial sources that the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences has manipulated. Viral vectors are a preferred type of vector and include, but are not limited to, nucleic acid sequences from the following viruses: retroviruses, such as Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyomaviruses; Epstein-Barr viruses; papillomaviruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

[0054] Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves the reverse transcription of viral genomic RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing the synthesis of the desired proteins but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with a plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles are provided in Kriegler, 1990 and Murry, 1991.

[0055] Preferred viruses for certain applications are the adenoviruses and adeno-associated viruses, double-stranded DNA viruses already approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication-deficient and infect a wide range of cell types and species. It further has advantages such as heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition, thus allowing multiple series of transductions. Reportedly, the adeno- associated virus can integrate into human cellular DNA site-specific, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.

[0056] Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill. See, e.g., Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC / CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art.

[0057] Additionally, plasmids may be custom-designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Various parenteral, mucosal, and topical routes may deliver plasmids. For example, the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppositories, and orally. It may also be administered into the epidermis or a mucosal surface using a gene gun. The plasmids may be given in an aqueous solution, dried onto gold particles, or in association with another DNA delivery system, including but not limited to liposomes, dendrimers, cochleate, and microencapsulation.

[0058] In a particular embodiment, the antisense oligonucleotide, siRNA, shRNA, or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter can also be, e.g., a viral promoter, such as a CMV promoter or any synthetic promoter.

[0059] Another object of the invention relates to a method of treating age-related diseases comprising administrating to a subject in need thereof a therapeutically effective amount of the compounds of the invention.

[0060] Therapeutic composition

[0061] Another object of the invention relates to a therapeutic composition for treating ARDs comprising a therapeutically effective amount of an AP-1 inhibitor for treating age-related diseases in subjects in need thereof.

[0062] The composition of the present invention may, e.g., be formulated for a topical, oral, intranasal, parenteral, intravenous, intramuscular, or subcutaneous administration and the like. The uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). Preferably, the invention's composition may be formulated for any mode of administration suitable for treating age-related diseases. In particular, heart and pulmonary diseases. The form of the composition, the route of administration, the dosage, and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the subject etc.

[0063] According to the invention, the AP-1 inhibitor or the therapeutic composition of the invention is administered in a therapeutically effective amount.

[0064] As used herein, the term "therapeutically effective amount" refers to an amount effective of the compounds of the invention, at dosages and for periods necessary, to achieve a desired therapeutic result. The therapeutically effective amount of the compound or the composition of the present invention, the time of administration, route of administration, and the duration of the treatment may vary according to factors well known in the medical art, such as the disease state, age, sex, and weight of the individual, and the ability of the compound of the invention to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound or the composition of the present invention or the composition are outweighed by the therapeutically beneficial effects. The efficient dosages and dosage regimens for the compound or the composition of the present invention depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the compound or the composition of the invention required. For example, the physician could start doses of the compound or the composition of the present invention employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired result is achieved.

[0065] The suitable dose of the compound or the composition of the present invention will be the amount of the compound which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend upon the factors described above. For example, a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize disease progression. Alternatively, this property of a composition may be evaluated by examining the ability of the compound to induce cytotoxicity by in vitro assays known to the skilled practitioner. One of the ordinary skills in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.

[0066] An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is about 0.1-100 mg / kg, such as about 0.1-50 mg / kg, for example, about 0.1-20 mg / kg, such as about 0.1-10 mg / kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg / kg, about 5 mg / kg or about 8 mg / kg. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is 0.02-100 mg / kg, such as about 0.02-30 mg / kg, such as about 0.05-10 mg / kg or 0.1-3 mg / kg, for example about 0.5-2 mg / kg.

[0067] Administration of the compound may, e.g., be intravenous, intramuscular, intraperitoneal, or subcutaneous, and, for instance, administered proximal to the target site. Dosage regimens in the above treatment methods and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the necessities of the therapeutic situation.

[0068] An effective dose of the compound of the present invention may also be administered using a weekly, biweekly, or triweekly dosing period. The dosing period may be restricted to, e.g., 8 weeks, 12 weeks, or until clinical progression has been established. As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of the compound of the present invention in an amount of about 0.1-100 mg / kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof. If desired, an effective daily dose of the compound or the composition of the present invention may be administered as two, three, four, five, six, or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

[0069] In some embodiments, the efficacy of the treatment is monitored during the therapy, e.g., at predefined points in time.

[0070] In some embodiments, the efficacy may be monitored by visualization of the disease area or other diagnostic methods.

[0071] In some embodiments, the compound or the composition of the present invention is administered by slow continuous infusion over a long period, such as more than 24 hours, to minimize any unwanted side effects. In one embodiment, the compound of the invention may be combined with pharmaceutically acceptable excipients and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

[0072] As used herein, the term "pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administrated to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type.

[0073] Particularly, the pharmaceutical compositions contain pharmaceutically acceptable vehicles for a formulation capable of being injected. These may be in isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts) or dry, especially freeze-dried compositions. In particular, these may be in organic solvents such as DMSO and ethanol, which, upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

[0074] Combination and kit of parts

[0075] Another object of the invention relates to a kit comprising an inhibitor according to the invention and at least one further therapeutic active agent.

[0076] In one embodiment, agents may be added to the pharmaceutical composition or used in combination with the compound of the invention in the case of treating pulmonary or heart diseases.

[0077] Agents include bronchodilators, steroids, P-blockings, calcic inhibitors, or combinations thereof. For example, further agents may be selected in the group consisting of bronchodilators like P2 agonists and anticholinergics, corticosteroids, P2 -adrenoceptor agonists like salbutamol, anticholinergic like ipratropium bromide or adrenergic agonists like epinephrine. A further agent may also be an antiviral compound like amantadine, rimantadine, or pleconaril.

[0078] The following figures and examples will further illustrate the invention. However, the examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0079] FIGURES Figure 1: Degree of emphysema in COPD-mouse model treated with APli T-5224

[0080] 2 months old mice (strain C57bl / 6) are treated with elastase (PPE) inducing emphysema. Then, they are treated with APli T-5224 for 21 days. Then, the emphysema is measured by microscopy and quantified by the stereological metric of the mean linear intercept emphysema (pM) (Fig. 1A). Gene expression of senescence biomarkers (p21 and SASP-factor ILip) are measured at days 8 and 21 (Fig. IB).

[0081] Figure 2: Degree of emphysema in old mice treated with APli T-5224 or ABT263

[0082] 15 months old mice (strain C57bl / 6) are treated with APli T-5224 or ABT263 for 21 days. Then, systolic pressure (A), right ventricular hypertrophy weight ratio (B), degree of emphysema (C), Ashcroft score (D), and senescence markers (pl 6, p21, and phospho-yH2Ax) (E) are measured. ABT263 is an established senolytic, and phospho-yH2Ax is a marker of DNA damage.

[0083] Figure 3: Degree of emphysema in old mice treated with APli T-5224

[0084] 26 months old mice (strain C57bl / 6) are treated with APli T-5224 for 21 days. Then, emphysema is measured.

[0085] Figure 4: Measurement of heart function in old mice treated with APli T-5224

[0086] 26 months old mice (strain C57bl / 6) are treated with APli T-5224 for 21 days. Then, heart function is measured by quantifying anterior and posterior strain rate, a sensitive parameter of systolic function.

[0087] Figure 5: Degree of pulmonary fibrosis induced by bleomycin in mice treated with APli T-5224

[0088] 2 months old mice (strain C57bl / 6) or bleomycin-exposed 2-month-old mice (strain C57bl / 6) are treated with APli T-5224 for 21 days. Then, pulmonary compliance (A), pulmonary elastance (B), Ashcroft score (C), and degree of emphysema (D) are measured. Bleomycin mice represent a lung fibrosis model after a single dose of bleomycin (2-6 U / Kg). Figure 6 : Measurement of p!6 and aSMA gene expression in mice undergoing bleomycin- induced pulmonary fibrosis and treated with APli T-5224

[0089] Bleomycin 2-month-old mice (strain C57bl / 6) are treated with APli T-5224 for 21 days. Then, the expression of pl6 and aSMA are measured. aSMAis a marker of pulmonary fibrosis.

[0090] Figure 7 : Elimination of senescent cells treated with T5224-iAPl. Twenty-two-month-old INK4a-ATTAC mice (n=5) were treated once daily for 21 days with vehicle (vhc), T5224 iAPl (oral, 30 mg / kg / day) or twice a week with AP20187 (IP 0,5 mg / kg) to eliminate pl6 Ink4a senescent cells. Mean linear intercept and lung fibrosis were quantified using Picrosirius red (Psr) staining and Ashcroft score.

[0091] Figure 8 : Measure of senescence biomarkers in lung . Mice that were 1.8 months old (C57bl / 6 strain) were treated with 2U elastase for the indicated number of days (D) to induce emphysema. The study measured senescence biomarkers in lung slices using indirect immunofluorescence staining. These biomarkers included an increase in senescence-associated beta-galactosidase (SABG) and p-gH2Ax positivity, as well as a decrease in Ki67 and Lamin Bl positivity. The results, showing the percentage of positive cells, are displayed as box plots.

[0092] EXAMPLES

[0093] Material & Methods

[0094] Mice.

[0095] Adult male mice (C57Bl / 6j) were used according to institutional guidelines, which complied with national and international regulations. All animal experiments were approved by the Institutional Animal Care and Use Committee of the French National Institute of Health and Medical Research (INSERM)-Unit 955, Creteil, France. To evaluate the in vivo effect of the API inhibitor, we treated mice preventively or curatively by gavage with T5224 (AP-1 inhibitor) at 30 mg / kg / day 5 days a week and in drinking water the remaining 2 days of the week. The solvent is composed of 0.5% of the final DMSO diluted in water (this composition served as a control to test the effects of the vehicle). Force-feeding of 50 pL per day was carried out using a 1 mL syringe and a suitable gavage probe without inducing suffering or distress in the mice.

[0096] Animal preparation, lung emphysema and pulmonary hypertension assessment, and lung tissue analysis. Mice were anesthetized with an i.p. injection of ketamine (60 mg / kg) and xylazine (10 mg / kg). Right ventricular systolic pressure (RVSP) was measured as described previously. The RV was dissected from the left ventricle plus septum (LV+S) and weighed to determine the Fulton index (RV / [LV+S]). The right lung was quickly removed and divided into 2 parts, immediately snap-frozen in liquid nitrogen, and then stored at -80°C until total RNA and protein extraction for biological measurements (real-time PCR, Western blot, and ELISA). Immunostaining, Western blot, ELISA, and real-time PCR were performed as described previously. The left lungs were fixed by intratracheal infusion of 4% paraformaldehyde aqueous solution (MilliporeSigma) at a transpleural pressure of 30 cmH2 O. For morphometry studies, 5 pm-thick sagittal sections along the greatest axis of the left lung were cut systematically and stained with H&E. Alveolar morphometry studies were performed by an observer blinded to treatment.

[0097] Procedure for intratracheal administration of elastase or bleomycin

[0098] After a week of quarantine in our animal facility, mice undergo intratracheal injection of elastase or bleomycin. For this, the mice received an intraperitoneal injection of xylazine (10 mg / kg); they were then exposed to 4% isoflurane in an induction chamber and maintained throughout the surgery under 2% isoflurane through the nose. An incision in the skin of the neck is made. The trachea is exposed using forceps and without cutting any tissue, and the surrounding tissues are kept in place with retractors. A 26G Hamilton syringe needle with a capacity of 50 pl is inserted between two trachea rings. The solution of elastase (2U), bleomycin (2-6 U / kg), or physiological serum is slowly injected into the trachea. Once instillation is complete, a stitch using absorbable thread is made, then the awakening of the mice on a heating mat is monitored. Preventive treatments are carried out by force-feeding until the 21st day.

[0099] Lung emphysema assessment

[0100] To assess the effects of the API inhibitor on lung emphysema, mice (2 months) were investigated after intratracheal injection of saline or elastase (2UI) and studied at 21 days postinstillation. Lung emphysema was assessed using the point-counting method for alveolar surface density and the mean-linear-intercept (MLI) method described by Knudsen et al. (56). Light microscope fields were quantitated at an overall magnification of 400, using a 42-point and 21-line grid. We examined 20 fields / animals (10 per lung) using a systematic sampling method from a random starting point. To correct area values for shrinkage associated with fixation and paraffin processing, we used the factor of 1.22 calculated during a previous study. MLI was also determined by computer-assisted morphometry using a macro designed with ImageJ software (Molecular Devices). Five black and white digital photomicrographs were acquired from each slide's cranial, medial, and caudal regions at 400 x magnification, excluding areas where large bronchi or vessels predominated, to obtain 15 images per lung. Emphysema was then quantified by measuring the mean alveolar chord length with image analysis software (ImageJ System) at 5-pm intervals. This automated analysis was made vertically and horizontally on each photomicrograph. The mean alveolar chord length was obtained by averaging the measurements. All morphometric data were expressed as absolute values.

[0101] Lung fibrosis assessment

[0102] To assess the effects of the API inhibitor on lung fibrosis, mice were studied after intratracheal injection of bleomycin (2-6 U / kg) or saline and sacrificed at 21 days postinstillation. Lung fibrosis was evidenced on lung sections stained with Masson's trichrome (HT15-1KT, Sigma-Aldrich) and quantified on lung sections stained with Sirius Red (Piero Sirius Red Stain Kit, ab 150681, Abeam, Cambridge, UK) using the modified Ashcroft scale and on lung sections stained with Masson's trichrome (HT15-1KT, Sigma-Aldrich). Briefly, the lungs were scanned microscopically with a 20-fold objective, which allowed the evaluation of fine structures while providing a sufficiently broad view. The entire section was examined by inspecting each field in a raster pattern. Areas with dominating tracheal or bronchial tissue were omitted. The grades were summarized and divided by the number of fields to obtain a fibrotic index for the lung.

[0103] Cardiac function assessment

[0104] To assess cardiac function, transthoracic echocardiography was performed in nonsedated mice to avoid any depression effect of anesthetic agents, as previously described (Sawaki D et al., 2018, Circulation). Images were acquired from a parasternal position at the level of papillary muscles using a 13-MHz linear-array transducer with a digital ultrasound system (Vivid 7, GE Medical System, Chicago, IL). Left ventricular diameters, ejection fraction, and anterior and posterior wall thicknesses were obtained from gray-scale M-mode acquisition. Peak systolic strain rate values in the anterior and posterior walls were obtained using Tissue Doppler Imaging. Tissue Doppler Imaging loops were acquired from the same parasternal view at a mean frame rate of 450 i / s and a depth of 1 cm. The Nyquist velocity limit was set around 12 cm / s. Strain rate analysis was performed offline by an observer blinded to the groups using the EchoPac Software (GE Medical System). The region of interest size was set at 0.2 mm, and temporal smoothing filters were turned off for all measurements. Because slight respiratory variations exist, we averaged peak systolic strain rate on 8 consecutive cardiac cycles.

[0105] Protein extraction and imnuinoblotting

[0106] Total proteins were extracted using RIPA lysis buffer (10 mM sodium phosphate pH 8, 150 mM NaCl, 1% sodium deoxycholate, 1% NP40, 0.5% SDS, 1 mM PMSF, 10 mM NaF, 1 mM sodium orthovanadate, and protease inhibitor cocktail [Roche, Meylan, France]). Immunoblots were carried out using the indicated antibodies and detected using an enhanced chemiluminescence detection system (GE Healthcare, Little Chalfont, UK). Densitometric quantification was normalized for the beta-actin or GAPDH level using GeneTools software (Ozyme, Montigny le Bretonneux, France).

[0107] Real-time quantitative PCR (RT-qPCR)

[0108] Total RNAfrom tissues and cells was extracted using the RNeasy Plus Mini Kit (Qiagen, Courtaboeuf, France) according to the manufacturer's instructions. The RNA concentration was assessed using a Nanodrop ND- 1000 spectrophotometer (Thermo Fisher Scientific, Les Ulis, France), and RNA integrity was confirmed by electrophoresis. One pg of total RNA was used for cDNA synthesis using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific). The quantitative PCR was performed in triplicate with TaqMan Probes (TaqMan Gene Expression assays, Applied Biosystems, Illkirch, France) and TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) in the QS6, QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems, Illkirch, France). All data were analyzed using QuantStudio Real-Time PCR software (Applied Biosystems). The relative expression level of target genes was calculated by the ACt method and normalized to the reference genes 18S rRNA and SF3A1.

[0109] Materials.

[0110] The API inhibitor (T-5224, Purity 98%) was purchased from MedChemexpress and dissolved in 0.5% DMSO. Bleomycin N-acetylcysteine was obtained from Clinisciences (Nanterre - France) and dissolved in physiological serum at 2-6 U / kg concentration. Elastase was purchased from Elastin Products (Owensville, MO, USA) and dissolved in physiological serum at a 2U concentration. Results

[0111] The AP-1 inhibitor T-5224 reduces and ameliorates emphysema

[0112] Two month-old mice were investigated after intratracheal injection of elastase (PPE) or saline (PBS) (2UI). They were studied either time-resolved at the indicated time points (Fig.8) or at 21 days post-instillation (Fig.l), during which the mice were treated with T-5224 (APli+) or without T-5224 (APli-). The PPE / APli- mice develop alveoli altered compared to PBS / APli- mice and thus emphysema (Fig.lA). Emphysema is associated with an increase of senescence biomarker expression p21, pl 6, an increase in senescence-associated betagalactosidase (SABG) and y-pH2Ax (DNA damage marker) positivy and a decrease in Ki67 (proliferative capacity marker) and lamin Bl positivy (Fig. IB & Fig.8). These data therefore attest to the coincidence of cellular senescence in emphysema. The treatment with the specific AP-1 inhibitor T-5224 reduces the alveolar alteration close to the alveolar sizes of PBS / APli- control mice. Indeed, this treatment on PPE / APli+ mice protects lungs from emphysema observed in PPE / APli- mice (Fig.lA). Thus, T-5224 is an AP-1 inhibitor highly effective for reducing emphysema (Fig.lA) and p21 and pl6 senescence biomarker expression (Fig. IB). Therefore, in preventive treatment, the inhibition of AP-1 by T-5224 prevents the senescence arrest inducing ARDs and reduces emphysema.

[0113] The curative effects of T-5224 treatment and an established senolytic ABT (ABT263) have been studied. 15- and 26-month-old mice were treated for 21 days, with or without T-5224 (APli+ or APli-) or with or without ABT (ABT+ or ABT-). Mice were studied after 21 days. Both treatments significantly ameliorated emphysema (Fig.2C & Fig.3). However, T-5224 treatment induces a better effect than ABT treatment (Fig.2C). Therefore, data demonstrate that the inhibition of AP-1 by T-5224 is an effective curative treatment for alveolar alteration diseases such as emphysema.

[0114] The AP-1 inhibitor T-5224 reduces and ameliorates pulmonary fibrosis

[0115] To study the T-5224 preventive treatment on pulmonary fibrosis, 2-month-old mice were treated by intratracheal injection with bleomycin (2-6 U / kg) inducing pulmonary fibrosis. Then, mice were treated for 21 days, with or without T-5224 (APli+ or APli-). The T-5224 treatment on bleomycin mice reduces the compliance and elastance alterations relating to pulmonary fibrosis in bleomycin mice (Fig.5 . Moreover, the expressions of fibrosis markers pl6 and aSMA are lower than those of untreated bleomycin mice (Fig.6) and they are close to the rate of control mice. Therefore, inhibiting AP-1 by T-5224 prevents the senescence arrest-inducing ARDs in preventive treatment and reduces pulmonary fibrosis. The curative effects of the AP-1 inhibition by T-5224 have been studied and compared with the impact of the established senolytic ABT (ABT263). 15-month-old mice have been treated for 21 days, with or without T-5224 (APli+ or APli-) or with or without ABT (ABT+ or ABT-). Then, mice were studied after 21 days. The study demonstrates a decrease in the Ashcroft score on ABT+ mice and APli+ mice and, thus, an amelioration of pulmonary fibrosis. Moreover, the results show that the T-5224 treatment is more efficient than the ABT treatment (Fig.2D). Therefore, the data demonstrate that the inhibition of AP-1 by T-5224 is an effective curative treatment for ARDs such as pulmonary fibrosis.

[0116] The AP-1 inhibitor T-5224 ameliorates right heart hypertrophy and left ventricular systolic dysfunction induced by aging

[0117] The curative effects of the AP-1 inhibition by T-5224 on correct heart function have been studied and compared with the impact of the established senolytic ABT (ABT263). 15- month-old mice have been treated for 21 days, with or without T-5224 (APli+ or APli-) or with or without ABT (ABT+ or ABT-). Then, mice were studied after 21 days. The study demonstrates that the T-5224 treatment ameliorates the systolic pressure and reduces the right ventricle hypertrophy much better than the ABT treatment (Fig.2A & Fig.2B).

[0118] Moreover, measures of anterior and posterior strain rate underline a significant improvement in the systolic myocardial function in APli+ 26-month-old mice (Fig.4). Therefore, the data demonstrate that the inhibition of AP-1 by T-5224 is an effective curative treatment for age-related cardiac remodeling and dysfunction.

[0119] The use of the AP-1 inhibitor T-5224 is more advantageous for the treatment of ARDs

[0120] First, the data demonstrate that the T-5224 treatment does not induce dysregulations in control mice and, thus, has no detectable adverse effects (Fig.lA & Fig.5).

[0121] Then, the data underline a more significant impact of the T-5224 treatment than the available treatment for ARDs. Indeed, the application shows that the T-5224 curative treatment is more efficient than the ABT treatment (Fig.2C, Fig.2D, Fig.2A & Fig.2B).

[0122] Moreover, the T-5224 treatment decreases systolic pressure and does not increase the pl6 expression, contrary to the ABT treatment (Fig.2A & Fig.2E).

[0123] Therefore, T-5224 inhibits AP-1 and it is an effective treatment for ARDs without any or minor adverse effects.

[0124] T5224-iAPl improves mechanical lung and fibrotic parameters INK4a-ATTAC mouse is a transgenic mouse model in which apoptosis of cells highly expressing the senescence biomarker pl6 Ink4a can be induced upon administration of the drug AP20187 (AP), leading to their elimination (Baker et al., 2011). Notably, the specific elimination of pl6-positive senescent cells or treatment with T5224-iAPl caused similar improvements in mechanical lung and fibrotic parameters underscoring the efficacy of T5224- iAPl treatment of emphysema (Fig. 7).

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Claims

CLAIMS1. An AP-1 inhibitor for use in the treatment of Age-related diseases (ARDs) in subjects in need thereof.

2. An AP-1 inhibitor for use according to the claim 1 wherein Age-related disease is emphysema, pulmonary fibrosis, COPD and / or heart hypertrophy.

3. An AP-1 inhibitor for use according to the claims 1 or 2 wherein the AP-1 inhibitor is an inhibitor of AP-1 activity and / or an inhibitor of AP-1 expression.

4. An AP-1 inhibitor according to the claim 3 wherein the AP-1 inhibitor is a small molecule, an anti-AP-1 antibody, an aptamer, a polypeptide, a RNA, a nuclease, a ribozyme or an antisense oligonucleotide.

5. An AP-1 inhibitor according to the claims 3 or 3 wherein the AP-1 inhibitor is T-5224.

6. A method for treating ARDs comprising administrating to a subject in need thereof a therapeutically effective amount of an AP-1 inhibitor according to the claims 1 to 5.

7. A pharmaceutical composition for treating ARDs comprising a therapeutically effective amount of an AP-1 inhibitor according to the claims 1 to 5.

8. Akit comprising an AP-1 inhibitor according to the claims 1 to 7 and at least one further therapeutic active agent.

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