Methods and compositions for treating cellular senescence

Abstract

The present disclosure relates to compositions for use and methods in treating symptoms of senescence in a cell or tissue of a subject, the compositions for use and methods comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression. In some instances, the cell or tissue expresses a GABAA receptor γ2 subunit. In some instances, the cellular senescence characterized by elevated levels of p21 polypeptide in the cell or tissue. The cells or tissues may be selected from a kidney, a liver, or heart. In some instances, the cells are cells that express a senescence marker such as cyclin-dependent kinase inhibitor 2A (CDKN2A) or p21.

Description

[0001] METHODS AND COMPOSITIONS FOR TREATING CELLULAR SENESCENCE

[0002] FIELD OF THE INVENTION

[0003] The present invention isin the field of medicine. In particular, the invention relates to a composition for use in treating symptoms of senescence in a cell or tissue of a subject.

[0004] BACKGROUND OF THE INVENTION

[0005] Diazepam binding inhibitor (DBI), also referred to as acyl-coenzyme A binding protein (ACBP), is a protein that is ubiquitously expressed in all tissues of three human body and can be released into circulation. A dual designation (ACBP / DBI) is sometimes used to reflect the two roles of the protein, and in some cases the terms are used interchangeably. Generally, ACBP is used to refer to an intracellular protein interacting with activated fatty acids as well as with other lipids to facilitate their transport between organelles and DBI is generally used to refer to the protein found in the extracellular space. ACBP / DBI is a leaderless peptide that cannot be secreted by conventional (Golgi-dependent) protein secretion but rather leaves cells through an autophagy-associated pathway. The protein is phylogenetically conserved throughout the eukaryotic radiation, and this mode of unconventional secretion is maintained in unicellular fungi and mammalian cells.

[0006] SUMMARY OF THE INVENTION

[0007] The present invention relates to a method of treating senescence in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent that reduces human diazepam binding inhibitor (DBI) activity or expression in an amount sufficient to treat the symptoms of the senescence in the cell or tissue of the subject upon administration to the subject.

[0008] The present invention also relates to a composition for use in treating symptoms of senescence in a cell or tissue of a subject that expresses a GABAA receptor y2 subunit, the composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression in an amount sufficient to treat the symptoms of the senescence in the cell or tissue of the subject upon administration to the subject. In some embodiments, the tissue is kidney tissue or liver tissue. In some embodiments, the tissue is kidney tissue. In some embodiments, senescence in the kidney tissue occurs upon kidney injury. In some embodiments, the senescence comprises an acute cellular senescence or a chronic cellular senescence. In some embodiments, the agent reduces extracellular DBI activity, relative to an amount of extracellular DBI activity in a comparable cell or tissue of the subject in the absence of the administration. In some embodiments, the agent comprises a DBI-binding polypeptide. In some embodiments, the DBI-binding polypeptide is an anti-DBI antibody. In some embodiments, the anti-DBI antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody comprises a monoclonal chimeric antibody, a monoclonal humanized antibody, or a monoclonal human antibody. In some embodiments, the anti-DBI antibody is a polyclonal antibody. In some embodiments, the DBI-binding polypeptide is an anti-DBI antibody fragment. In some embodiments, the antibody fragment comprises a single chain Fv, Fab’ fragment, or nanobody. In some embodiments, the anti-DBI antibody is an extracellular DBI neutralizing antibody. In some embodiments, the agent comprises a polypeptide antigen that induces production of a neutralizing anti-DBI antibody in the subject. In some embodiments, the agent reduces DBI expression, relative to an amount of DBI expression by administering a composition lacking the agent that reduces DBI expression. In some embodiments, the agent is an siRNA, an endonuclease, an antisense oligonucleotide, a thyroid receptor agonist, or a ribosome. In some embodiments, the agent is the siRNA. In some embodiments, the agent is a thyroid hormone receptor agonist. In some embodiments, the thyroid hormone receptor agonist comprises resmetirom.

[0009] The present invention also relates to a composition for use in treating kidney or liver cellular senescence characterized by elevated levels of p21 polypeptide in a subject, the composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression in an amount sufficient to treat the kidney or liver cellular senescence and reduce the elevated levels of p21 polypeptide upon administration to the subject.

[0010] The present invention also relates to the use of resmetirom in the treatment of a disease characterized by elevated levels of human diazepam binding inhibitor (DBI) in a subject, wherein administration of an effective amount of resmetirom to the subject results in reduced expression of human DBI in the subject, relative to prior to the administration, thereby treating the disease in the subject. The present invention also relates to a method of reducing expression of extracellular human diazepam binding inhibitor (DBI) in a subject, the method comprising administering to the subject a composition that comprises an effective amount of resmetirom that is sufficient to reduce a level of extracellular DBI in the subject, relative to a level of extracellular DBI prior to the administering. In some embodiments, the composition further comprises a second moiety, wherein the second moiety comprises a DBI-binding polypeptide. In some embodiments, the DBI-binding polypeptide is an anti-DBI antibody. In some embodiments, the resmetirom and the DBI-binding polypeptide are linked.

[0011] The present invention also relates to a method of treating senescence in a tissue of a subject, the method comprising administering to the subject a composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression, wherein the administering is sufficient to treat the cellular senescence in the tissue of the subject, as determined in an in vitro assay by a reduction of p21 polypeptide in a tissue sample obtained from the subject, as compared to the amount of p21 polypeptide in the tissue sample prior to the administering. In some embodiments, the agent reduces DBI activity, relative to an activity level prior to the administering. In some embodiments, the agent comprises a DBI-binding polypeptide. In some embodiments, the DBI-binding polypeptide is an anti-DBI antibody. In some embodiments, the anti-DBI antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody comprises a monoclonal chimeric antibody, a monoclonal humanized antibody, or a monoclonal human antibody. In some embodiments, the anti-DBI antibody is a polyclonal antibody. In some embodiments, the DBI-binding polypeptide is an anti-DBI antibody fragment. In some embodiments, the antibody fragment comprises a single chain Fv, Fab’ fragment, or nanobody. In some embodiments, the anti-DBI antibody is an extracellular DBI neutralizing antibody. In some embodiments, the agent comprises a polypeptide antigen that induces production of a neutralizing anti-DBI antibody in the subject. In some embodiments, the agent reduces DBI expression, relative to an expression level prior to the administering. In some embodiments, the agent is an siRNA, an endonuclease, an antisense oligonucleotide, or a ribosome. In some embodiments, the agent is an siRNA that inhibits the expression of DBI.

[0012] The present invention also relates to a method of treating senescence in a population of cells that express senescence marker cyclin-dependent kinase inhibitor 2A (CDKN2A) in a subject, the method comprising administering to the subject a composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression, wherein the administering is sufficient to treat the senescence in the population of cells that express the CDKN2A. In some embodiments, the administering is sufficient to reduce a level of CDKN2A in the population of cells, relative to a level of CDKN2A in the population of cells prior to the administering. In some embodiments, the agent reduces DBI activity, relative to an activity level prior to the administering. In some embodiments, the agent comprises a DBI-binding polypeptide. In some embodiments, the DBI-binding polypeptide is an anti-DBI antibody. In some embodiments, the anti-DBI antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody comprises a monoclonal chimeric antibody, a monoclonal humanized antibody, or a monoclonal human antibody. In some embodiments, the anti-DBI antibody is a polyclonal antibody. In some embodiments, the DBI-binding polypeptide is an anti-DBI antibody fragment. In some embodiments, the antibody fragment comprises a single chain Fv, Fab’ fragment, or nanobody. In some embodiments, the anti-DBI antibody is an extracellular DBI neutralizing antibody. In some embodiments, the agent comprises a polypeptide antigen that induces production of a neutralizing anti-DBI antibody in the subject. In some embodiments, the agent reduces DBI expression, relative to an expression level prior to the administering. In some embodiments, the agent is an siRNA, an endonuclease, an antisense oligonucleotide, or a ribosome. In some embodiments, the agent is an siRNA that inhibits the expression of DBI.

[0013] The present invention also relates to a method of detecting and treating senescence in a tissue of a subject, the method comprising: (a) measuring a level of p21 in a sample obtained from the tissue of the subject using an in vitro ELISA; (b) detecting a level of p21 in the in vitro ELISA that is higher than a reference level of p21 from a healthy subject, thereby detecting the senescence in the tissue of the subject; and (c) administering to the subject a composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression, thereby treating the senescence in the tissue of the subject.

[0014] The present invention is defined by the claims.

[0015] 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.

[0016] DETAILED DESCRIPTION OF THE INVENTION As used herein, the term “administering” refers to the physical introduction of a composition comprising a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. In some instances, routes of administration for the agents described herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal injection, and infusion, as well as in vivo electroporation. Non-parenteral routes include a topical, epidermal, oral, or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually, or topically. Administering can be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0017] As used herein the term “agent” or grammatical equivalents thereof refers to chemical or biological entities, such as antibodies, antigen binding portions thereof, fragments thereof, aptamers, compounds, small molecules, drugs, etc., or an active portion thereof, that are capable of eliciting a biological action on a biological target of interest, such as anti-DBI agents.

[0018] As used herein the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immune-specifically binds an antigen. As used herein the term “antibody” and “immunoglobulin” have the same meaning, and will be used equally in the present disclosure. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments. In natural antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains, lambda (1) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (V) and a constant domain (C). The heavy chain includes three (a, 5, y) to five (a, 5, y, p, s) domains, a variable domain (V) and three to four constant domains (CHI, CH2, CH3 and CH4 collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. Also encompassed by the term antibody, are heavy-chain antibodies such as camelid antibodies that contain only two heavy chains and lack the two light chains usually found in other mammalian antibodies. Antibodies described herein also include single-domain antibody (sdAb), also known as a nanobody, which is an antibody fragment consisting of a single monomeric variable antibody domain, for instance a VHH which is the antigen binding fragment of a heavy-chain antibody. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FR) can participate with the antibody binding site or influence the overall domain structure and hence the combining site. CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore, typically includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs. The residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NTH, USA (hereafter “Kabat et al”'). The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues in SEQ ID sequences. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31-35B (H-CDR1), residues 50-65 (H-CDR2) and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3) according to the Kabat numbering system.

[0019] The term “antigen” as used herein refers to any known or unknown substance that can be recognized by an antibody, including proteins, glycoproteins and carbohydrates. In some embodiments, these antigens include biologically active proteins, such as hormones, cytokines and their cell surface receptors, bacterial or parasitic cell membranes or purified components thereof, and viral antigens. In one example, the antigens expressed on the surface of said cells are antigens which are difficult to purify or antigens which lose desired epitopes upon biotinylation such as those antigens described above. In another example, the antigen is unknown and the antigen is any material that would provide a source of possible antigens. In some embodiments, that material is of animal origin, e.g., mammalian, plant, yeast, bacterial or viral origin. The material may be a cell or a population of cells for which it would be desirable to isolate antibodies, such as mammalian cells, immunomodulatory cells, lymphocytes, monocytes, polymorphs, T cells, cancer cells, tumor cells, yeast cells, bacterial cells, infectious agents, parasites, and plant cells. In some embodiments, the cell is a tumor cell.

[0020] The term “binding” as used herein refers to an association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, ionic and / or hydrogen-bond interactions, including interactions such as salt bridges and water bridges. As used herein, the term “binding” in the context of the binding of an antibody to a predetermined target molecule (e.g., an antigen or epitope) typically is a binding with an affinity corresponding to a KD of about 10'7M or less, such as about 10'8M or less, such as about 10'9M or less, about 10'10M or less, or about 10'11M or even less.

[0021] As used herein, the term “co-administration” or grammatical equivalents thereof can refer to any of the following: (i) combining two or more agents together and administering them at a single time, (ii) administering a first agent and then administering a second agent a short time later (e.g., 1, 2, 5, 10, 15, 20, 30, and 45 min.; and 1, 2, 4, 6, 8, 16, and 24 hours later), (iii) administering an agent to a subject already undergoing long-term treatment with a first agent and / or with a second agent, (iv) administering two or more agents simultaneously each by a different route of administration.

[0022] As used herein, the term “DBI” has its general meaning in the art and refers to the diazepam binding inhibitor, or acyl-CoA binding protein (ACBP) encoded by the DBI gene (Gene ID: 1622). In some embodiments, DBI refers to extracellular DBI. The term is also known as EP, ACBP, ACBD1, and CCK-RP. An exemplary amino acid sequence for DBI is represented by the NCNI reference sequence NP_001073331.1 (SEQ ID NO:1) (acyl-CoA- binding protein isoform 1). An exemplary human nucleic acid sequence is represented by the NCNI reference sequence NM_001079862.2 (SEQ ID NO:2) (acyl-CoA-binding protein isoform 1).

[0023] SEQ ID NO:1 - MSQAEFEKAA EEVRHLKTKP SDEEMLFIYG HYKQATVGDI NTERPGMLDF TGKAKWDAWN ELKGTSKEDA MKAYINKVEE LKKKYGI

[0024] SEQ ID NO:2 - GCTCGCCCGA GCAGGGTTGG GGCGAGTGGA CCGCGCCTCT AAAGGCGCTT GCCAGTGCAA TCTGGGCGAT CGCTTCCTGG TCCTCGCCTC CTCCGCTGTC TCCCTGGAGT TCTTGCAAGT CGGCCAGGAT GTCTCAGGCT GAGTTTGAGA AAGCTGCAGA GGAGGTTAGG CACCTTAAGA CCAAGCCATC GGATGAGGAG ATGCTGTTCA TCTATGGCCA CTACAAACAA GCAACTGTGG GCGACATAAA TACAGAACGG CCCGGGATGT TGGACTTCAC GGGCAAGGCC AAGTGGGATG CCTGGAATGA GCTGAAAGGG ACTTCCAAGG AAGATGCCAT GAAAGCTTAC ATCAACAAAG TAGAAGAGCT AAAGAAAAAA TACGGGATAT GAGAGACTGG ATTTGGTTAC TGTGCCATGT GTTTATCCTA AACTGAGACA ATGCCTTGTT TTTTTCTAAT ACCGTGGATG GTGGGAATTC GGGAAAATAA CCAGTTAAAC CAGCTACTCA AGGCTGCTCA CCATACGGCT CTAACAGATT AGGGGCTAAA ACGATTACTG ACTTTCCTTG AGTAGTTTTT ATCTGAAATC AATTAAAAGT GTATTTGTTA CTTTAAATAA CTTTAAAAAA AAAA

[0025] As used herein, the term "DBI activity" refers to any biological activity of DBI that includes among others: inhibition of autophagy, induction of hypoglycaemia, stimulation of food intake, stimulation of weight gain, reduction of fatty acid oxidation, upregulation of glucose transporter, upregulation of PPARG, stimulation of glucose uptake, stimulation of glycolysis or stimulation of lipogenesis, or any combinations thereof. As used herein, the terms “enhance,” “increase,” “augment,” “improve,” and grammatical equivalents thereof when in reference to the level of any molecule (e.g., amino acid sequence, and nucleic acid sequence, antibody, etc.), cell (e.g., B cell, T cell, tumor cell), and / or phenomenon (e.g., disease treatment), in a first sample (or in a first subject) relative to a second sample (or relative to a second subject or control), mean that the quantity of molecule, cell and / or phenomenon in the first sample (or in the first subject) is higher than in the second sample (or in the second subject or control) by any amount that is statistically significant using any art-accepted statistical method of analysis. For example, it may refer to a natural, synthetic or engineered compound, agent, or component that has a biological effect to increase a symptom or condition.

[0026] As used herein, the terms “inhibit,” “reduce,” “suppress,” “decrease,” “neutralize,” and grammatical equivalents when in reference to the level of any molecule (e.g., amino acid sequence, and nucleic acid sequence, antibody, etc.), cell (e.g., B cell, T cell, tumor cell), and / or phenomenon (e.g., disease symptom), in a first sample (or in a first subject) relative to a second sample (or relative to a second subject or control), mean that the quantity of molecule, cell and / or phenomenon in the first sample (or in the first subject) is lower than in the second sample (or in the second subject or control) by any amount that is statistically significant using any art- accepted statistical method of analysis. For example, it may refer to a natural, synthetic or engineered compound, agent, or component that has a biological effect to inhibit a protein such as extracellular DBI.

[0027] As used herein, the terms “monoclonal antibody,” “monoclonal Ab,” “monoclonal antibody composition,” “mAb,” and the like, refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody is obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprised in the population are identical except for possible naturally occurring mutations that may be present in minor amounts.

[0028] As used herein, the term “neutralizing anti-DBI monoclonal antibody” refers to an antibody or a monoclonal antibody having specificity for DBI and that inhibits, reduces, or completely neutralizes the activity of DBI (for example, extracellular DBI). Whether an antibody is a neutralizing antibody can be determined by in vitro assays, such as any described in the examples. Typically, the neutralizing antibody of the present disclosure inhibits the activity of extracellular DBI by at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[0029] As used herein, the terms “subject” and “patient” is used interchangeably and refers to any subject for whom diagnosis, treatment, or therapy is desired or has been administered, such as humans. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and the like. In some embodiments the subject is a human.

[0030] As used herein, the term "therapeutically effective amount" refers to a sufficient amount of one or more of agents of the present disclosure for reaching a therapeutic effect. It will be understood, however, that the total daily usage of the compounds / agents and compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound / agent employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound / agent employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound / agent employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound / agent at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 4,000 mg per adult per day. In some instances, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, 500 or 1000 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament may contain from about 0.01 mg to about 1000 mg of the active ingredient. An effective amount of the compound / agent, such as a therapeutically effective amount, may be supplied at a dosage level from 0.0002 mg / kg to about 50 mg / kg of body weight per day, especially from about 0.001 mg / kg to 10 mg / kg of body weight per day.

[0031] As used herein, the term “treatment” or “treat” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of a subject 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 a disease 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 disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

[0032] As used herein, the term “pharmaceutical composition” refers to a composition described herein, or pharmaceutically acceptable salts thereof, with other agents such as carriers and / or excipients. The pharmaceutical compositions as provided herewith typically include a pharmaceutically acceptable carrier.

[0033] As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.

[0034] As used herein, the term “elderly patient” refers to an adult patient sixty -five years of age or older.

[0035] As used herein, the term “obesity” refers to a condition characterized by an excess of body fat. The operational definition of obesity is based on the Body Mass Index (BMI), which is calculated as body weight per height in meters squared (kg / m2). Obesity refers to a condition whereby an otherwise healthy subject has a BMI greater than or equal to 30 kg / m2, or a condition whereby a subject with at least one co-morbidity has a BMI greater than or equal to 27 kg / m2. An “obese subject” is an otherwise healthy subject with a BMI greater than or equal to 30 kg / m2or a subject with at least one co-morbidity with a BMI greater than or equal 27 kg / m2. A “subject at risk of obesity” is an otherwise healthy subject with a BMI of 25 kg / m2to less than 30 kg / m2or a subject with at least one co-morbidity with a BMI of 25 kg / m2to less than 27 kg / m2. The increased risks associated with obesity may occur at a lower BMI in people of Asian descent. In Asian and Asian-Pacific countries, including Japan, “obesity” refers to a condition whereby a subject has a BMI greater than or equal to 25 kg / m2. An “obese subject” in these countries refers to a subject with at least one obesity -induced or obesity -related comorbidity that requires weight reduction or that would be improved by weight reduction, with a BMI greater than or equal to 25 kg / m2In these countries, a “subject at risk of obesity” is a person with a BMI of greater than 23 kg / m2to less than 25 kg / m2.

[0036] In some embodiments, the present disclosure relates to a composition for use in treating symptoms of senescence in a cell or tissue of a subject. In some embodiments, the cell or tissue of the subject expresses a GABAA receptor y2 subunit. In some embodiments, the composition comprises an agent that reduces human diazepam binding inhibitor (DBI) activity. In some embodiments, the composition comprises an agent that reduces human diazepam binding inhibitor (DBI) expression. In some embodiments, the reduction is in an amount sufficient to treat the symptoms of the senescence in the cell or tissue of the subject upon administration to the subject.

[0037] In some embodiments, the tissue is kidney tissue, liver tissue, or heart tissue. In some embodiments, the tissue is kidney tissue. In some embodiments, the tissue is a liver tissue. In some embodiments, the tissue is a heart tissue. In some embodiments, senescence in the kidney tissue occurs upon kidney injury. In some embodiments, senescence in the liver tissue occurs upon kidney injury. In some embodiments, senescence in the heart tissue occurs upon kidney injury.

[0038] In some embodiments, the senescence comprises an acute cellular senescence or a chronic cellular senescence. In some embodiments, the senescence is an acute cellular senescence. In some embodiments, the senescence is a chronic senescence.

[0039] In some embodiments, the agent reduces extracellular DBI activity, relative to an amount of extracellular DBI activity in a comparable cell of the subject in the absence of the administration. In some embodiments, the agent reduces extracellular DBI activity, relative to an amount of extracellular DBI activity in a comparable tissue of the subject in the absence of the administration.

[0040] In some embodiments, the agent comprises a DBI-binding polypeptide. In some embodiments, the DBI-binding polypeptide is an anti-DBI antibody. In some embodiments, the anti-DBI antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody comprises a monoclonal chimeric antibody, a monoclonal humanized antibody, or a monoclonal human antibody. In some embodiments, the monoclonal antibody comprises a monoclonal chimeric antibody. In some embodiments, the monoclonal antibody comprises a monoclonal humanized antibody. In some embodiments, the monoclonal antibody comprises a monoclonal human antibody.

[0041] In some embodiments, the anti-DBI antibody is a polyclonal antibody. In some embodiments, the DBI-binding polypeptide is an anti-DBI antibody fragment. In some embodiments, the antibody fragment comprises a single chain Fv, Fab’ fragment, or nanobody.

[0042] In some embodiments, the antibody fragment comprises a single chain Fv. In some embodiments, the antibody fragment comprises a Fab’ fragment. In some embodiments, the antibody fragment comprises a nanobody.

[0043] In some embodiments, the anti-DBI antibody is an extracellular DBI neutralizing antibody. In some embodiments, the agent comprises a polypeptide antigen that induces production of a neutralizing anti-DBI antibody in the subject. In some embodiments, the agent reduces DBI expression, relative to an amount of DBI expression by administering a composition lacking the agent that reduces DBI expression.

[0044] In some embodiments, the agent is an siRNA, an endonuclease, an antisense oligonucleotide, a thyroid receptor agonist, or a ribosome. In some embodiments, the agent is the siRNA. In some embodiments, the agent is an endonuclease. In some embodiments, the agent is an antisense oligonucleotide. In some embodiments, the agent is a ribosome. In some embodiments, the agent is a thyroid hormone receptor agonist. In some embodiments, the thyroid hormone receptor agonist comprises resmetirom.

[0045] As disclosed herein, administration of resmetirom can be used to reduce or inhibit the expression of DBI in circulation. Without wishing to be bound by theory, Applicants have discovered that administration of resmetirom to a subject, which is a selective thyroid hormone receptor P (THR-P) agonist, results in transcriptional downregulation of Acbp / Dbi mRNA in the subject. Accordingly, resmetirom can be used to treat a disease characterized by elevated levels of DBI in a subject. Thus, disclosed herein is a method of treating a disease characterized by elevated levels of human diazepam binding inhibitor (DBI) in a subject, the method comprising administering to the subject an effective amount of resmetirom, wherein the administering results in reduced expression of human DBI in the subject, relative to prior to the administration, thereby treating the disease in the subject. Also disclosed herein in the use of resmetirom in the treatment of a disease characterized by elevated levels of human diazepam binding inhibitor (DBI) in a subject, wherein administration of an effective amount of resmetirom to the subject results in reduced expression of human DBI in the subject, relative to prior to the administration, thereby treating the disease in the subject. Also disclosed herein is a method of reducing expression of extracellular human diazepam binding inhibitor (DBI) in a subject, the method comprising administering to the subject a composition that comprises an effective amount of resmetirom that is sufficient to reduce a level of extracellular DBI in the subject, relative to a level of extracellular DBI prior to the administering.

[0046] In some embodiments, resmetirom can be administered in a composition to reduce expression of extracellular DBI in the subject, and / or to treat a disease characterized by elevated levels of DBI in the subject. In some cases, the composition can comprise a second moiety. The second moiety can include any agent described herein that reduces human diazepam binding inhibitor (DBI) activity or expression. In some embodiments, the second moiety can be a DBI-binding polypeptide such as an anti-DBI antibody described herein or a DBI-binding fragment thereof. In some embodiments, the resmetirom and the second moiety can be coadministered in the same composition. In some embodiments, the resmetirom and the second moiety can be administered sequentially in different compositions. In some embodiments, the resmetirom can be linked to the second moiety. For example, an anti-DBI antibody as described herein, or a DBI-binding fragment thereof, can be linked to resmetirom to form a single agent that (a) can reduce the activity of extracellular DBI in a subject, and (b) can reduce the expression of extracellular DBI in the subject.

[0047] In some embodiments, the present disclosure relates to a composition for use in treating kidney or liver cellular senescence. In some embodiments the kidney or liver cellular senescence is characterized by elevated levels of p21 polypeptide in a subject. In some embodiments, the composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression in an amount sufficient to treat the kidney or liver cellular senescence. In some embodiments, the composition is in an amount sufficient to reduce the elevated levels of p21 polypeptide upon administration to the subject.

[0048] In some embodiments, the present disclosure provides for a method of treating senescence in a tissue of a subject. In some embodiments, the method comprises administering to the subject a composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity. In some embodiments, the method comprises administering to the subject a composition comprising an agent that reduces human DBI expression. In some embodiments, the administering is sufficient to treat the cellular senescence in the tissue of the subject. In some embodiments, the treating of the cellular senescence is determined in an in vitro assay by a reduction of p21 polypeptide in a tissue sample obtained from the subject, as compared to the amount of p21 polypeptide in the tissue sample prior to the administering.

[0049] In some embodiments, the present disclosure further provides for method of treating senescence in a population of cells that express senescence marker cyclin-dependent kinase inhibitor 2A (CDKN2A) in a subject. In some embodiments, the method comprises administering to the subject a composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity. In some embodiments, the method comprises administering to the subject a composition comprising an agent that reduces human DBI expression. In some embodiments, the administering is sufficient to treat the senescence in the population of cells that express the CDKN2A. In some embodiments, the administering is sufficient to reduce a level of CDKN2A in the population of cells, relative to a level of CDKN2A in the population of cells prior to the administering. In some embodiments, the agent reduces DBI activity, relative to an activity level prior to the administering. In some embodiments, the agent comprises a DBI-binding polypeptide. In some embodiments, the DBI-binding polypeptide is an anti-DBI antibody. In some embodiments, the anti-DBI antibody is a monoclonal antibody.

[0050] In some embodiments, the monoclonal antibody comprises a monoclonal chimeric antibody. In some embodiments, the monoclonal antibody comprises a monoclonal humanized antibody. In some embodiments, the monoclonal antibody comprises a monoclonal human antibody. In some embodiments, the anti-DBI antibody is a polyclonal antibody. In some embodiments, the DBI-binding polypeptide is an anti-DBI antibody fragment. In some embodiments, the antibody fragment comprises a single chain Fv. In some embodiments, the antibody fragment comprises a Fab’ fragment. In some embodiments, the antibody fragment comprises a nanobody. In some embodiments, the anti-DBI antibody is an extracellular DBI neutralizing antibody. In some embodiments, the agent comprises a polypeptide antigen that induces production of a neutralizing anti-DBI antibody in the subject. In some embodiments, the agent reduces DBI expression, relative to an expression level prior to the administering. In some embodiments, the agent is an siRNA. In some embodiments, the agent is an endonuclease. In some embodiments, the agent is an antisense oligonucleotide. In some embodiments, the agent is a ribosome. In some embodiments, the agent is an siRNA that inhibits the expression of DBI.

[0051] In some embodiments, the present disclosure relates to a method of detecting and treating senescence in a tissue of a subject. In some embodiments, the method of detecting and treating senescence in a tissue of a subject comprises measuring a level of p21 in a sample obtained from the tissue of the subject using an in vitro ELISA. In some embodiments, the method of detecting and treating senescence in a tissue of a subject comprises detecting a level of p21 in the in vitro ELISA that is higher than a reference level of p21 from a healthy subject, thereby detecting the senescence in the tissue of the subject. In some embodiments, the method of detecting and treating senescence in a tissue of a subject comprises administering to the subject a composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression, thereby treating the senescence in the tissue of the subject.

[0052] As described herein, an agent that reduces the activity or expression of DBI includes an agent that binds to or neutralizes DBI, and thus disrupts the function of DBI when administered. The specific agent that is utilized can be substituted without departing from the present disclosure. Indeed, the present disclosure provides for the use of any agent that can bind to and inhibit the activity or expression of DBI (such as extracellular DBI) in order to treat a disease, symptom, or dysfunction described herein. Such agents can include small molecules, polypeptides, or other agents that bind to and inhibit the activity or expression of extracellular DBI. Without wishing to be bound by theory, such agents when administered can bind to and block interaction with a binding partner of DBI. For example, the agent can be provided in circulation in order to bind to extracellular DBI present in circulation. The extracellular DBI, when bound by the agent, is thus blocked from performing its biological function. For example, the extracellular DBI, when present in circulation and bound by an agent as described herein, can no longer repress an autophagic state in neighbouring cells. Furthermore, the extracellular DBI, when present in circulation and bound by an agent as described herein, may be prohibited from binding to a biological binding partner such as a gamma-aminobutyric acid type A receptor (GABR) (e.g., a GABR that expresses a GAB AA receptor y2 subunit). Without wishing to be bound by theory, disrupting interaction of extracellular DBI to a biological binding partner using an agent described herein results in the treatment of the diseases, symptoms and / or dysfunctions described herein. Furthermore, such agents when bound to extracellular DBI may indirectly result in reduction in levels of extracellular DBI in circulation. For example, extracellular DBI levels can be elevated in response to extracellular release of intracellular ACBP (for example, due to starvation-induced autophagy), resulting in inhibition of autophagy by the extracellular DBI. Without wishing to be bound by theory, an agent as described herein that reduces extracellular release of intracellular ACPB (for example, through binding to and inhibiting the activity of extracellular DBI) thus indirectly reduces the levels of extracellular DBI in circulation by preventing further extracellular export of intracellular ACBP. Alternatively, an agent can directly reduce the expression of DBI (including extracellular DBI) by silencing the expression of the DBI gene. Such agents include, for example, siRNAs, endonucleases, antisense oligonucleotides, or ribozymes as described herein.

[0053] Thus, while the present application provides exemplary agents that reduce the activity or expression of DBI, these agents are merely exemplary.

[0054] In some embodiments, the agent that reduces the activity or expression of DBI is an antibody or an aptamer directed against DBI. In some embodiments, the agent is an antibody directed against DBI. In some embodiments, the agent is an aptamer directed against DBI. In some embodiments, the agent that reduces the expression of DBI is an inhibitor of expression. In some embodiments, the agent that inhibits the activity of DBI is an antibody directed against DBI. In some embodiments, the agent is thus a neutralizing anti-DBI monoclonal antibody. In some embodiments, the antibody is directed against a fragment of DBI consisting of amino acid residue 43 to the amino acid residue 50 of SEQ ID NO: 1

[0055] In some embodiments, the antibody is a monoclonal chimeric antibody, a monoclonal humanized antibody, or a monoclonal human antibody.

[0056] Any anti-DBI antibody that inhibits the activity of DBI (e.g., extracellular DBI) is suitable for use in the methods and compositions described herein. Such anti-DBI antibodies are commercially available and described in literature, and the sequences of such antibodies are known or can be derived. For instance, an antibody that inhibits the activity of DBI (e.g., extracellular DBI) and suitable for use as described herein has at least 80%, at least 85%, at least 90%, at least 95%, or has 100% sequence identity to a polypeptide sequence of an antibody selected from the group consisting of: ab232760 (Rabbit polyclonal, abeam); ab 16871 (Rabbit polyclonal, abeam); sc-30190 (Rabbit polyclonal, Santa Cruz Biotechnology); FNabO2256 (Rabbit polyclonal, Wuhan Fine Biotech Co); PA5-89139 (Rabbit polyclonal, Invitrogen); OTI4A8 (Mouse monoclonal, OriGene); OTI6E12 (Mouse monoclonal, OriGene), mAb 7A (Mouse monoclonal, Fred Hutch Antibody Technology); Abeam (catalogue no. abl6871; RRID: AB 302557); DBI human or mouse FL-87 monoclonal antibodies from Santa Cruz (catalogue number sc-30190; RRID: AB 2211046); DBI human C-9 polyclonal antibodies from Santa Cruz (catalogue number sc-376853; RRID: AB 2722761) DBI mouse polyclonal antibodies from Abeam (catalogue number ab231910); DBI mouse 7a monoclonal antibodies from Fred Hutch Antibody Technology, DBI polyclonal antibodies from Invitrogen (catalogue numbers PA5-89139, PA5-79138, PA5-40659, PA5-102751, PA5-84066, PA5-76729 and PA5-92426); DBI monoclonal antibodies from OriGene (catalogue numbers CF813069, CF813070, CF813117, TA813069, TA81370, and TA813117); DBI polyclonal antibodies from Proteintech (catalogue number 14490-1-AP); DBI polyclonal antibodies from Abnova (catalogue number H00001622-D01P); or more than one of the foregoing.

[0057] Antibodies described herein may be obtained commercially or synthesized through any suitable method. For example, anti -DBI human monoclonal antibodies may be synthesized using peptides derived from the full length human DBI and the phage display technology. In some embodiments, antibodies described herein are mutated antibodies. In some embodiments, antibodies described herein are selected based on favorable kinetic parameters, such as specificity for or affinity against human DBI. Specificities of antibodies described herein can be validated by western blot, immunofluorescence and flow cytometry on human ACBP / DBI knock out cell lines.

[0058] In some embodiments, the antibody is directed against the fragment consisting in the amino acid sequence ranging from the amino acid residue at position 43 to the amino acid residue at position 50 in SEQ ID NO: 1 (z.e., the octapeptide or OP).

[0059] In some embodiments, the antibody of the present disclosure is a chimeric antibody, typically a chimeric mouse / human antibody.

[0060] As used herein, the term “chimeric antibody” refers to an antibody which comprises a VH domain and a VL domain of a non-human antibody, and a CH domain and a CL domain of a human antibody. In some embodiments, a “chimeric antibody” is an antibody molecule in which (a) the constant region (z.e., the heavy and / or light chain), or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and / or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity. Chimeric antibodies also include primatized and in particular humanized antibodies. Furthermore, chimeric antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In some embodiments, the antibody is a humanized antibody.

[0061] As used hereon, the term “humanized antibody” refers to an antibody having variable region framework and constant regions from a human antibody but retains the CDRs of a previous non-human antibody. In some embodiments, a humanized antibody contains minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies and antibody fragments thereof may be human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody / antibody fragment can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Such antibodies are designed to maintain the binding specificity of the non-human antibody from which the binding regions are derived, but to avoid an immune reaction against the non-human antibody. These modifications can further refine and optimize antibody or antibody fragment performance. In general, the humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non- human immunoglobulin and all or a significant portion of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0062] In some embodiments, the antibody is a human antibody. As used herein the term “human antibody” as used herein, is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody” as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. In some embodiments, the neutralizing antibody of the present disclosure does not mediate antibody-dependent cell-mediated cytotoxicity and thus does not comprise an Fc portion that induces antibody dependent cellular cytotoxicity (ADCC). In some embodiments, the neutralizing antibody does not comprise an Fc domain capable of substantially binding to a FcgRIIIA (CD 16) polypeptide. In some embodiments, the neutralizing antibody lacks an Fc domain (e.g., lacks a CH2 and / or CH3 domain) or comprises an Fc domain of IgG2 or IgG4 isotype. In some embodiments, the neutralizing antibody consists of or comprises a Fab, Fab', Fab'-SH, F (ab1) 2, Fv, a diabody, single-chain antibody fragment, or a multispecific antibody comprising multiple different antibody fragments. In some embodiments, the neutralizing antibody is not linked to a toxic moiety. In some embodiments, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered C2q binding and / or reduced or abolished complement dependent cytotoxicity (CDC).

[0063] In some embodiments, the agent that inhibits the activity of DBI is an aptamer directed against DBI. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library. 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. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods.

[0064] In some embodiments, the agent that inhibits the expression of DBI is an inhibitor of expression. In a preferred embodiment of the present disclosure, said inhibitor of gene expression is an siRNA, an endonuclease, an antisense oligonucleotide or a ribozyme. In some embodiments, the agent that reduces the expression of DBI is an inhibitor of expression. In some embodiments, the inhibitor of expression is an siRNA, an endonuclease, an antisense oligonucleotide or a ribozyme. In some embodiments, the inhibitor of expression is an siRNA. In some embodiments, the inhibitor of expression is an endonuclease. In some embodiments, the inhibitor of expression is an antisense oligonucleotide. In some embodiments, the inhibitor of expression is a ribozyme.

[0065] In some embodiments, the agent that inhibits the activity of DBI consists of a vaccine composition suitable for eliciting neutralizing autoantibodies against DBI when administered to the subject.

[0066] In some embodiments, the agent that reduces the activity of DBI is a vaccine composition suitable for eliciting neutralizing autoantibodies against DBI when administered to the patient. In some embodiments, the vaccine composition comprises a polypeptide antigen comprising (i) an amino acid sequence having at least 80% identity with SEQ ID NO: 1; (ii) an amino acid sequence having at least 80% identity to a polypeptide fragment consisting of the amino acid sequence ranging from amino acid residue 17 to amino acid residue 50 of SEQ ID NO: 1; (iii) an amino acid sequence having at least 80% identity to a polypeptide fragment consisting of the amino acid sequence ranging from amino acid residue 33 to amino acid residue 50 of SEQ ID NO: 1; or (iv) an amino acid sequence having at least 80% identity to a polypeptide fragment consisting of the amino acid sequence ranging from amino acid residue 43 to amino acid residue 50 of SEQ ID NO: 1 In some embodiments, the amino acid sequence has at least 80% identity with SEQ ID NO: 1. In some embodiments, the amino acid sequence has at least 80% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 98% identity with SEQ ID NO: 1. In some embodiments, the polypeptide antigen comprises an amino acid sequence having at least 80% identity to a polypeptide fragment consisting of the amino acid sequence ranging from amino acid residue 17 to amino acid residue 50 of SEQ ID NO: 1 In some embodiments, the polypeptide antigen comprises an amino acid sequence having at least 80% identity to a polypeptide fragment consisting of the amino acid sequence ranging from amino acid residue 33 to amino acid residue 50 of SEQ ID NO: 1. In some embodiments, the polypeptide antigen comprises an amino acid sequence having at least 80% identity to a polypeptide fragment consisting of the amino acid sequence ranging from amino acid residue 43 to amino acid residue 50 of SEQ ID NO: 1. In some embodiments, the amino acid sequence has at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 98% identity with any of the foregoing polypeptide fragments of SEQ ID NO: 1.

[0067] For the purpose of the present disclosure, the term “vaccine composition” is intended to mean a composition which can be administered to humans or to animals in order to induce an immune system response; this immune system response can result in the production of antibodies against DBI. Typically, the vaccine composition comprises at least one antigen derived from DBI. As used herein the term “antigen” refers to a molecule capable of being specifically bound by an antibody or by a T cell receptor (TCR) if processed and presented by MHC molecules. The term "antigen", as used herein, also encompasses T-cell epitopes. An antigen is additionally capable of being recognized by the immune system and / or being capable of inducing a humoral immune response and / or cellular immune response leading to the activation of B- and / or T-lymphocytes. An antigen can have one or more epitopes or antigenic sites (B- and T- epitopes). In some embodiments, the antigen consists of a polypeptide comprising an amino acid sequence having at least 80% of identity with the sequence of SEQ ID NO: 1 or a fragment thereof (e.g., an epitope). In some embodiments, the antigen consists in a polypeptide comprising (i) an amino acid sequence having at least 80% of identity with SEQ ID NO: 1 or (ii) an amino acid sequence having at least 80% of identity with the amino acid sequence ranging from the amino acid residue at position 17 to the amino acid residue at position 50 in SEQ ID NO: 1; or (iii) an amino acid sequence having at least 80% of identity with the amino acid sequence ranging from the amino acid residue at position 33 to the amino acid residue at position 50 in SEQ ID NO: 1; or (iv) an amino acid sequence having at least 80% of identity with the amino acid sequence ranging from the amino acid residue at position 43 to the amino acid residue at position 50 in SEQ ID NO: 1. In some embodiments, the polypeptide is conjugated to a carrier protein which is generally sufficiently foreign to elicit a strong immune response to the vaccine. Illustrative carrier proteins are inherently highly immunogenic. Both bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) have commonly been used as carriers in the development of conjugate vaccines when experimenting with animals and are contemplated herein as carrier proteins. Proteins which have been used in the preparation of therapeutic conjugate vaccines include, but are not limited to, a number of toxins of pathogenic bacteria and their toxoids. Suitable carrier molecules are numerous and include, but are not limited to: Bacterial toxins or products, for example, cholera toxin B-(CTB), diphtheria toxin, tetanus toxoid, pertussis toxin, filamentous hemagglutinin, shiga toxin and pseudomonas exotoxin; Lectins, for example, ricin-B subunit, abrin and sweet pea lectin; Sub virals, for example, retrovirus nucleoprotein (retro NP), rabies ribonucleoprotein (rabies RNP), plant viruses (e.g., TMV, cow pea and cauliflower mosaic viruses), vesicular stomatitis virus- nucleocapsid protein (VSV-N), poxvirus vectors and Semliki forest virus vectors; Artificial vehicles, for example, multiantigenic peptides (MAP), microspheres; Yeast virus-like particles (VLPs); Malarial protein antigen; and others such as proteins and peptides as well as any modifications, derivatives or analogs of the foregoing. Other useful carriers include those with the ability to enhance a mucosal response, more particularly, LTB family of bacterial toxins, retrovirus nucleoprotein (retro NP), rabies ribonucleoprotein (rabies RNP), vesicular stomatitis virus-nucleocapsid protein (VSV-N), and recombinant .pox virus subunits.

[0068] DBI is known to be released from cells through an unconventional (Golgi-independent) pathway, in an autophagy-dependent fashion, as was first demonstrated for several fungal species. In some embodiments of the present disclosure, the agent that reduces the activity or expression of DBI reduces the activity or expression of extracellular DBI. In some embodiments, the agent reduces the activity of extracellular DBI by binding to extracellular DBI. In some embodiments, the binding to the extracellular DBI disrupts binding of the extracellular DBI to a binding partner naturally present in a human cell. In some embodiments, the binding partner is a gamma-aminobutyric acid type A receptor (GABR). In some embodiments, the agent as disclosed herein binds to a surface of the extracellular DBI that is responsible for binding of the extracellular DBI to the GABR. In some embodiments, the agent reduces levels of extracellular DBI in circulation. In some embodiments, the agent reduces the activity of extracellular DBI, thereby resulting in induction of autophagy. In some embodiments, the induction of autophagy reduces levels of extracellular DBI in circulation. In some embodiments, the agent that reduces the levels of extracellular DBI in circulation does not reduce levels of intracellular DBI when administered to the patient.

[0069] Typically, the agent that inhibits the activity or expression of DBI is administered to the patient in the form of a pharmaceutical composition which comprises a pharmaceutically acceptable carrier.

[0070] Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. For use in administration to a patient, the composition will be formulated for administration to the patient. The compositions of the present disclosure may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The administrations of the present disclosure include subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Sterile injectable forms of the compositions of this disclosure may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solids, liquids, or other dosage forms may also be used for the purposes of formulation. The compositions of this disclosure may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include, e.g., lactose. When aqueous suspensions are required for oral use, the agent that inhibits the activity or expression of DBI is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. Alternatively, the compositions of this disclosure may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols. The compositions of this disclosure may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs. For topical applications, the compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this disclosure include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Patches may also be used. The compositions of this disclosure may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents.

[0071] The present disclosure will be further illustrated by the accompanying figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present disclosure.

[0072] FIGURES

[0073] Figure 1: Scheme of doxorubicin-induced accelerated aging experiments.

[0074] Figure 2: Effects of DBI neutralization on doxorubicin-induced heart muscle senescence.

[0075] Each point on the graph represents one mouse (resulting from the quantification of 10 regions of interest) and the statistical analyses have been performed by two-way ANOVA. Figure 3: Effects of DBI neutralization on doxorubicin-induced senescence in kidney glomeruli.

[0076] Each point on the graph represents one mouse (resulting from the quantification of 10 regions of interest) and the statistical analyses have been performed by two-way ANOVA.

[0077] Figure 4: Effects of ACBP / DBI neutralization on doxorubicin-induced senescence in kidney tubuli.

[0078] Each point on the graph represents one mouse (resulting from the quantification of 10 regions of interest) and the statistical analyses have been performed by two-way ANOVA.

[0079] Figure 5: Scheme of high-fat diet (HFD) / CC14-induced liver senescence experiments.

[0080] Figure 6: Effects of DBI neutralization high-fat diet (HFD) / CC14-induced liver senescence. Each point on the graph represents one mouse (resulting from the quantification of 10 regions of interest) and the statistical analyses have been performed by two-way ANOVA.

[0081] Figure 7: Experimental procedure of repeated low-dose cisplatin injection treatment. Cisplatin (CDDP) was injected at 8 mg / kg weekly for 4 consecutive weeks in mice treated with i.p. injection of anti-DBI or IgG (5 pg / g B.W.) weekly for 8 weeks.

[0082] Figure 8: Blood Urea Nitrogen (BUN) levels in plasma of mice submitted to CDDP repeated low-dose injections and treated with IgG or anti-DBI for 7 weeks.

[0083] 4 injections CDDP (8 mg / kg); IgG2a rat isotype (5 ug / g); anti-DBI: Murine clone 7G4a (5 ug / g); Envigo C57BL / 6J. Males 12 weeks-old; Time point sacrifice: 7 weeks of treatment). Results are displayed as means ± SEM. Statistical analyses (p value) were calculated by ANOVA test (n=3-8 mice per group).

[0084] Figure 9: Experimental procedure of septic shock model.

[0085] Mice were pre-treated with i.p. injection of anti-DBI or IgG (2.5 pg / g B.W.) and DEX (10 mg / kg B.W.) 4 h and / or 1 h before CLP procedure as indicated in the scheme (A). CLP procedure was performed with a 75% ligation of the cecum, which corresponds with a highgrade sepsis. The illustration of the CLP model was created with BioRender.com with number of agreement BK246PS4V0 (B). Figure 10: Prevention of CLP-induced kidney injury by anti-DBI antibody. pCCL values (A), Ca++values (B), pCL values (C), Na+values (D), bicarbonate (cHCCL ) values (E), base excess of extracellular fluid (BE (ecf)) values (F), base excess (BE (b))values (G), total carbon dioxide (TCO2) values (H), creatinine values (I), and blood urea nitrogene (BUN) values (J) were measured in blood from C57BL / 6 mice treated with anti-DBI or IgG and / or DEX, 24 h after the CLP procedure. Data are expressed as means ± SEM. Statistical analyses (p values) were performed by pairwise t-test with a False Discovery Rate correction procedure (FDR) (n=4-10 mice per group).

[0086] Figure 11: Senolytic activity of the DBI-neutralizing antibody - Immunostaining of p21 in cardiac sections of doxorubicin-treated mice.

[0087] (A) Schematic representation of DBI-neutralizing antibody (anti-DBI) administration to doxorubicin (DOX)-treated C57B1 / 6J female mice. (B) Representative images of the left ventricular areas stained for the nuclear senescence marker p21. (C) Quantification of p21 in the left ventricular areas. Statistical significance was tested by two-way ANOVA with Tukey’s correction for multiple comparisons.

[0088] Figure 12: Inhibition of ACBP / DBI expression using resmetirom. (A) Schematic representation of resmetirom administration to C57B1 / 6J female mice. (B) Quantification of ACBP / DBI protein levels in mouse plasma using ELISA 24 hours after administration of resmetirom. (C) Quantitation of Acbp / Dbi mRNA levels in mouse tissue 24 hours after administration of resmetirom.

[0089] EXAMPLES

[0090] Example 1: Anti-senescence effects of DBI neutralization

[0091] Aging is accompanied by senescence, an irreversible cell cycle arrest characterized by the upregulation of cyclin dependent kinase inhibitor 1A (CDKN1A, also known as p21). In several paradigms of accelerated aging and organ damage, Applicants surprisingly found that an antibody specific for acyl coenzyme A binding protein (ACBP, encoded by the gene diazepam binding inhibitor, DBI) can prevent cellular senescence determined by the immunohistochemically detectable upregulation of p21. This applies to doxorubicin-induced accelerated aging of the heart muscle and renal damage, as well as to liver damage induced by a combination of high-fat diet and CCh. The senescence of cardiomyocytes, various renal cell types (glomerular podocytes and peritubular capillaries) and hepatocytes was inhibited by DBI neutralization, demonstrating a broad antiaging effect of DBI neutralization across different organ systems.

[0092] Material and Methods

[0093] Anthracycline-induced cardiac aging and kidney damage.

[0094] 8-week-old C57B1 / 6J female mice were used in this example. Following one week of acclimatization, mice were randomized (1 :1) to receive either mouse monoclonal anti-DBI- neutralizing antibody (5 mg / kg body weight, injected i.p. once per week, Fred Hutch Antibody Technology clone 7a) or its isotype IgG2a (negative control; 5 mg / kg body weight, BioXCell, clone 2A3, #BE0089). Each group was further randomized (1 :2) to receive a weekly intraperitoneal injection of saline or doxorubicin (DOX, 5 mg / kg body weight; a cumulative dose of 20 mg / kg body weight over 4 weeks. Anti-DBI was administered 24 hours prior to doxorubicin injection. All mice used in this study were housed in a temperature-controlled environment with 12 h light / dark cycles and ad libitum access to water and food (standard chow diet).

[0095] Model of liver damage.

[0096] 8-week-old male C57BL / 6 mice received a regular chow diet (RCD) or high-fat chow diet (HFD) and water ad libitum. CCh was i.p. administered at a dose of 1.6 ml / kg twice weekly for 4 weeks. Control animals were i.p. injected with the vehicle olive oil.

[0097] Neutralization of DBI by passive immunization

[0098] The monoclonal antibody against DBI (anti-DBI, passive immunization) or isotype IgG was used in vivo (5 pg / g body weight, B.W., intraperitoneally, i.pf as indicated in the schemes illustrating the experimental setup.

[0099] Immunohistochemistry

[0100] On the day of euthanasia, left ventricles from the heart or other tissues were briefly rinsed with PBS and fixed for 16-24 h in a 4 % formaldehyde solution (diluted in PBS, pH = 7.4). After fixation, tissues were transferred to 70 % ethanol until inclusion in paraffin. Paraffin- embedded tissue were cut to 2-3 pm sections, air dried and let to dry completely at 60 °C overnight. Cell Conditioning buffer was used for antigen retrieval, and casein was used as blocking agent. The antibody directed against p21 (clone HUGO 291H / B5, CNIO) was diluted 1 :50 in antibody diluent. Staining was performed for 60 minutes (ready -to-use). Secondary staining was done with the anti-rat HRP. Slides were counterstained with hematoxylin and mounted with toluene-free mounting medium. The specificity of the staining was assessed in each organ by using rat IgG isotype control (6-001-F, R&D Systems) as primary antibody.

[0101] Quantification of immunohistochemistry

[0102] Quantification of the p21 staining was performed after blinding the samples names. For the heart, 250 pm-wide squares were chosen to be representative of longitudinally cut cardiomyocytes, and five others were chosen in the transversally-cut regions for each mouse. For kidneys, 12 glomeruli and 3 tubular regions of interest (500 pm x 500 mp were analyzed per mouse. For livers, the number of p21 -positive hepatocytes was determined on 5 randomly chosen fields (with a 20x objective) per mouse. Non-relevant regions were excluded from the area of analysis and, after color deconvolution, a threshold was applied to automatically quantify the p21+areas. The average value in the 10 regions of interest was plotted for each animal, and analyzed by two-way ANOVA to determine the effect of doxorubicin treatment, DBI neutralization, and their interaction.

[0103] Results

[0104] Antisenescence effects of anti-ACBP / DBI antibody on the heart.

[0105] Mice were treated with doxorubicin to induce accelerated aging by subjecting the mice to chronic doxorubicin (DOXO) treatment (cumulative dose: 20 mg / kg body weight, injected intraperitoneally (i.p.) over 4 weeks) or the mice were injected with a vehicle. The mice were also injected with a murine monoclonal DBI-neutralizing antibody (anti -DBI; 5 mg / kg body weight, injected i.p. weekly, 24 hours before DOXO or vehicle) or an isotype IgG control, as shown in Fig. 1. At the end of the experiment, the mice were sacrificed and their organs were extracted and subjected to paraformaldehyde fixation. Immunohistochemical detection of the senescence marker cyclin dependent kinase inhibitor 1A (CDKN1A, also known as p21) revealed that DOXO induced a significant increase in the frequency of cardiomyocytes that expressed p21 in the left ventricle of the heart. This increase was totally inhibited by the anti- DBI antibody. The antibody also reduced spontaneous senescence (occurring in the absence of DOXO, with vehicle) to close-to-undetectable levels (as shown in Fig. 2).

[0106] Antisenescence effects of anti-ACBP / DBI antibody on the kidney.

[0107] The kidneys of the animals described above were also examined, to identify senescence of renal cells. It was found that both the glomeruli and the tubular areas of kidneys from D0X0- treated mice contained more p21 -positive cells than vehicle-treated controls and that this surge in cellular senescence was fully blocked by treatment with the anti-DBI antibody. The affected cell types in glomeruli are likely podocytes, while those in the tubular area are probably peritubular capillary cells (see Figs. 3 and 4).

[0108] Antisenescence effects of anti-ACBP / DBI antibody on the liver.

[0109] A model of hepatocyte senescence induced by a combination of high-fat diet (HFD) and liver damage by repeated CCh injections was also investigated. This model was also treated with anti-DBI antibody injections (or IgG isotype controls) as depicted in Fig. 5. HFD combined with CCI4 induced a significant surge in the frequency of p21 -positive hepatocytes that was strongly inhibited by DBI neutralization (see Fig. 6).

[0110] Without wishing to be bound by any theory, Applicant submits that the neutralization of DBI prevents senescence, which is one of the principal mechanisms of aging, in various organ systems and surprising found that anti-DBI agents can be used for treating aging and age-related diseases.

[0111] Example 2 : Kidney-protective effects of DBI neutralization

[0112] Kidney failure can be induced by numerous conditions and can be chronic or acute. Applicants chose a model for chronic kidney failure induced by the chemotherapeutic agent cisplatin (also called cis-diamminedichloroplatine (II), abbreviated CDDP), as well as one induced by acute septic shock induced by cecal ligation and puncture (CLP). In both models, Applicants surprisingly observed that neutralization of acyl coenzyme A binding protein (ACBP, encoded by the gene diazepam binding inhibitor, DBI) with a suitable monoclonal antibody ameliorated signs of renal failure. Based on this observation, and without wishing to be bound by theory, Applicants surprising found that neutralization of DBI can prevent and treat acute and chronic kidney failure. Material and Methods

[0113] Animal experimentation

[0114] Wild-type (Wt) C57BL / 6 mice, 9-12 weeks old, were bred and maintained according to the FELASA guidelines and local guidelines from the Animal Experimental Ethics Committee. Mice were housed in a temperature-controlled environment with 12 h light / dark cycles and were fed with diet and water ad libitum.

[0115] Neutralization ofDBI by passive immunization and dexamethasone treatment

[0116] The monoclonal antibody against DBI (anti-DBI, passive immunization) or isotype IgG) was used in vivo (2.5 pg / g body weight, B.W., intraperitoneally, i.p., except if otherwise stated), as indicated in the schemes illustrating the experimental setup. Dexamethasone (DEX) was injected i.p. (10 mg / kg, dissolved in PBS), and control animals were i.p. injected with vehicle (PBS IX).

[0117] Repeated low-dose cisplatin treatment to induce kidney injury

[0118] 12-weeks-old male C57BL / 6 mice were given four consecutive weekly injections of 8 mg / kg cisplatin (CDDP). Control animals were i.p. injected with the vehicle (PBS lx). Mice received a weekly injection of IgG or anti-DBI (5 pg / g B.W., i.p) during the 7 weeks of treatment. Mice were sacrificed by cervical dislocation 7 weeks after the first dose of CDDP and kidney functional analysis were performed in blood samples.

[0119] Cecal Ligation and Puncture (CLP) procedure

[0120] 9-weeks-old C57BL / 6 male mice were operated under general isoflurane (2%) anesthesia. Buprenorphine (0.05 mg / kg B.W. s.c.) was injected to achieve analgesia. Before surgery intervention mice were injected with IgG or anti-DBI (2 doses, 4 h and 1 h before CLP) or DEX (1 dose, 1 h before CLP). The abdomen of the mouse was opened via a midline incision, the cecum was exposed and ligated 75% above its distal end with a 4-0 silk suture. Following ligation, the cecum was perforated through-and-through with a 21 -gauge needle. Perforation of the cecum was followed by the release of fecal material into the peritoneal cavity. Subsequently, the cecum was returned to the peritoneal cavity and 0.1 mL of prewarmed saline was administered to the peritoneal cavity, which was subsequently sutured with a 4-0 suture. The skin was then closed with 9 mm clips and mice were placed under a heating lamp to recuperate. Sham animals undergo the same surgical procedure except for the CLP (no ligation and no puncture). Mice that died during the first 24 h after surgery were considered as perioperative mortality and were immediately excluded from the experiment, as their death was due not to sepsis but to post-surgery complications. The animals receive an injection of IgG, anti-DBI or DEX 90 min before sacrifice. Mice were sacrificed 24 h after CLP procedure by cervical dislocation, blood was collected, and renal function analysis was performed.

[0121] Renal and biochemical studies pH, carbon dioxide (pCCL), oxygen (pCL), bicarbonate (cHCCL ), base excess of extracellular fluid (BE (ecf)), base excess (BE(b)), sulfur dioxide (SO2), sodium (Na+), potassium (K+), calcium (Ca++), chloride (CP), total carbon dioxide (TCO2), Hematocrit (Het), calculated hemoglobin (cHgb) and creatinine were measured in blood and analyzed. Samples with creatinine values below the detection limit were given an arbitrary value of 0.30 mg / dL. For other measurements, blood was sampled by tail tip bleeding. Blood glucose (Glu) concentrations were measured using a glucometer, and lactate (Lac) levels were measured with the use of lactate meter. Plasma Blood Urea Nitrogen (BUN) was determined by means of colorimetric kits.

[0122] Results

[0123] Kidney protection against chronic damage.

[0124] Young adult mice received repeated weekly injections of cisplatin (also called cis- diamminedichloroplatine (II), abbreviated CDDP) for 4 weeks (see Fig. 7). During this period as well as during the subsequent 4 week (total 8 weeks), the mice were injected intraperitoneally ( / . / ?.) with a monoclonal antibody against DBI or an isotype-matched control antibody (IgG). At the end of experiment (after 8 weeks), blood was drawn and kidney functions were assessed by measuring multiple blood parameters. Blood urea nitrogen (which is typically elevated in kidney failure) were enhanced by the cisplatin treatment and this effect was blunted by treatment with the anti-DBI antibody, indicating the avoidance of renal failure (see Fig. 8).

[0125] Kidney protection against acute damage.

[0126] For the induction of acute renal failure, mice were subjected to cecal ligation puncture (CLP), which causes the release of cecal bacteria into the peritoneal cavity resulting in bacterial infection and septic shock within 24 hours (see Fig. 9B). Mice were injected i.p. 4 hours and 1 hour before and 22.5 hours after CLP with a monoclonal antibody against DBI or an isotype- matched control antibody (IgG). In addition, mice receive injections of dexamethasone (DEX) or vehicle one hour before or 22.5 hours after CLP (see Fig. 9B). Then, the effects of anti-DBI alone, DEX alone or their combination on renal function was assessed by measuring multiple blood parameters. Anti-DBI antibody was able to reduce kidney damage alone (Data not shown) since it attenuated the CLP -induced increase in pCCL and Na+ as well as the CLP- induced decrease of pCL and Ca++(see Figs. 10A-10D). In addition, anti-DBI antibody synergized with DEX (Data not shown) to correct CLP -induced effects on bicarbonate (CHCO3 ), base excess of extracellular fluid (BE (ecf)), base excess (BE (b)), total carbon dioxide (TCO2) and creatinine values (see Fig. 10E-10I). Such an improved effect of the combination of anti-ACBP / DBI antibody plus DEX was also observed for BUN, that became close-to-normal in all mice treated with the combination, but only in a fraction of mice treated with anti-DBI antibody or DEX alone (see Fig. 10J).

[0127] Example 3 : Effects of DBI neutralization of cardiomyocyte senescence

[0128] Applicants used an animal model of anthracycline-induced cardiomyocyte senescence to determine whether DBI is causally involved in cardiac disease.

[0129] Material and Methods

[0130] Cardiac toxicity experiments

[0131] Eight-week-old C57B1 / 6J female mice were used in this example. Following 2 weeks of acclimatization, mice were randomized (1 : 1) to receive either mouse monoclonal anti-DBL neutralizing antibody (5 mg / kg body weight, injected intraperitoneally once per week) or mouse isotype IgG (negative control; 5 mg / kg body weight). Each group was further randomized (1 : 1) to receive a weekly intraperitoneal injection of saline or doxorubicin (5 mg / kg body weight; cumulative dose of 20 mg / kg body weight over 4 weeks). Of note, anti-DBI was always administered 24 hours prior to doxorubicin injection. All mice used in this study were housed in a temperature-controlled environment with 12 h light / dark cycles and ad libitum access to food and water.

[0132] Immunohistochemistry of p21

[0133] On the day of euthanasia, left ventricles were briefly rinsed with PBS and fixed for 16- 24 h in a 4 % formaldehyde solution (diluted in PBS, pH = 7.4). After fixation, tissues were transferred to 70 % ethanol until inclusion in paraffin. Paraffin-embedded tissue were cut to 2- 3 pm sections, air dried and let to dry completely at 60 °C overnight. Cell Conditioning buffer was used for antigen retrieval, and casein was used as blocking agent. The antibody directed against p21 (clone HUGO 291H / B5, CNIO) was diluted 1:50 in antibody diluent. Staining was performed for 60 minutes. Secondary staining was performed with anti-rat HRP. Slides were counterstained with hematoxylin and mounted with toluene-free mounting medium. The specificity of the staining was assessed in each organ by using rat IgG isotype control as primary antibody.

[0134] Quantification of the p21 staining was performed after blinding the samples names. Five 250 pm-wide squares were chosen to be representative of longitudinally cut cardiomyocytes, and five others were chosen in the transversally-cut regions. Non-cardiomyocytes regions were excluded from the area of analysis and, after color deconvolution, a threshold was applied to automatically quantify the p21+ cardiomyocytes area. The average value in the 10 regions of interest was plotted for each animal, and analyzed by two-way ANOVA to determine the effect of doxorubicin treatment, ACBP / DBI neutralization, and their interaction.

[0135] Results

[0136] DBI neutralization reduces anthracycline-accelerated cardiac aging.

[0137] An animal model of anthracy cline-induced cardiomyocyte senescence (Fig. 11 A) was used to understand the impact of the DBI antibody on the induction of senescence by doxorubicin by studying one of the most widely used markers, the nuclear p21 protein. DOX damage increased the presence of p21 staining in cardiomyocytes, a phenomenon that was completely abrogated by concomitant treatment with anti -DBI (Figs. 11B and 11C). Mice were subjected to chronic doxorubicin (DOX) treatment (cumulative dose: 20 mg / kg body weight, injected intraperitoneally over 4 weeks). Taken together, DBI neutralization reduces the cardiotoxicity of elevated anthracycline doses and shows a senolytic activity.

[0138] Without wishing to be bound by theory, Applicants surprising found that since anthracycline- induced heart failure and anthracycline-induced senescence are a model of accelerated cardiac aging, it is plausible to use DBI inhibition or neutralization as a method to prevent or treat aging of the cardiovascular system.

[0139] Example 4 : Inhibition of DBI expression using resmetirom

[0140] Experiments were conducted with 8-week-old female C57BL / 6 mice. Resmetirom was prepared in 10% DMSO, 40% PEG300, 5% Tween 80, and 45% drinking water (v / v), and was administered to the C57BL / 6 mice p.o. at a final concentration of 0.033 mg / mL. Fig. 12A is a schematic representation of resmetirom administration to the C57B1 / 6J female mice.

[0141] Mouse plasma was collected 24 hours after resmetirom administration, was treated with lithium heparin separator, and was centrifuged at 1,500 xgfor 10 min. ACBP / DBI levels were measured by enzyme-linked immunosorbent assay (ELISA). Murine anti- ACBP / DBI capture antibodies (Cat. ab231910, Abeam) diluted 1 pg / mL in PBS were used for coating high-binding 96-well plates with 100 pL / well overnight at 4 °C. Subsequently, plates were washed twice with washing solution (0.05% Tween 20 (v / v) in PBS) and unspecific binding was blocked with 100 pL sterile blocking buffer (1% BSA, 0.05% Tween 20 (v / v) in PBS) for 2 h at RT. For sample assessment, 100 pL / well of either sample or standard (murine plasma at 1 :20, and cell culture supernatant at 1 :4 dilution, with the flexibility to adjust as dictated by experimental requirements) were incubated for 2 h at RT and subsequently rinsed 3x with washing buffer. Then 100 pL / well murine anti -ACBP / DBI detection antibodies (Cat. MBS2005521, MyBioSource) diluted 1 pg / mL in PBS were added for one hour at RT followed by 3x rinsing with washing buffer. Subsequently, plates were incubated 30 min at RT with 100 pL of HRP- coupled avidin diluted in PBS (1 / 1000). Subsequently plates were rinsed 4x with washing buffer. To visualize bound protein, 100 pL of TMB-ELISA substrate solution was added and incubated 10- 30 min at RT in the dark. Following 50 pL of stop solution (2N H2SO4) was added and absorbance was measured at 450 nm using a microplate reader. Fig. 12B shows that treatment of mice with resmetirom led to a reduction of ACBP / DBI protein expression in mouse plasma.

[0142] The effect of resmetirom administration on ACBP / DBI mRNA transcripts was then determined for the C57BL / 6 mice administered resmetirom. About 25-30 mg of tissue from each mouse was collected in lysis buffer. The tissue was homogenized in two cycles for 20 sec at 5,500 rpm. The lysate was centrifuged and subjected to further purification procedures. About 1 pg total RNA was reversed transcribed. Quantitative real-time PCR (qRT-PCR) was conducted. As shown in Fig. 12C, treatment of mice with resmetirom resulted in a reduction Acbp / Dbi mRNA levels in the mouse liver.

[0143] Accordingly, this example demonstrates that administration of resmetirom results in reduced expression of DBI in circulation, along with reduced levels of Acbp / Dbi mRNA in the liver of the mouse. Thus, this example validates the use of resmetirom for treating conditions characterized by increased expression of DBI in circulation, such as treating cellular senescence. While embodiments of the present disclosure have been shown and described herein, such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur without departing from the present disclosure. It should be understood that various alternatives to the embodiments described herein may be employed. It is intended that the following claims define the scope of the present disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0144] REFERENCES Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS1. A method of treating senescence in a subject in need thereof comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression in an amount sufficient to treat the symptoms of the senescence in the cell or tissue of the subject upon administration to the subject.

2. A method of treating senescence in a tissue of a subject, the method comprising administering to the subject a composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression, wherein the administering is sufficient to treat the cellular senescence in the tissue of the subject, as determined in an in vitro assay by a reduction of p21 polypeptide in a tissue sample obtained from the subject, as compared to the amount of p21 polypeptide in the tissue sample prior to the administering.

3. A method of treating senescence in a population of cells that express senescence marker cyclin-dependent kinase inhibitor 2A (CDKN2A) in a subject, the method comprising administering to the subject a composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression, wherein the administering is sufficient to treat the senescence in the population of cells that express the CDKN2A.

4. The method according to claims 1 or 2, wherein the tissue is kidney tissue or liver tissue.

5. The method according to claims 1 to 4 wherein the agent that reduces DBI activity is an anti-DBI antibody or an anti-DBI antibody fragment.

6. The method according to claim 5 wherein the anti-DBI antibody is a monoclonal antibody or a polyclonal antibody.

7. The method according to claim 6, wherein the monoclonal antibody comprises a monoclonal chimeric antibody, a monoclonal humanized antibody, or a monoclonal human antibody.

8. The method according to claim 5 wherein the antibody fragment comprises a single chain Fv, Fab’ fragment, or nanobody.

9. The method according to claims 1 to 4 wherein the agent is an siRNA, an endonuclease, an antisense oligonucleotide, a thyroid receptor agonist, or a ribosome.

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