Neutralization of acyl-CoA binding proteins confers autophagy-dependent organ protection

Abstract

Injury to vital organs has serious and even life-threatening consequences. Organ injury has a variety of etiologies, typically including drugs, toxins, and ischemic injury. Acyl-CoA binding protein (ACBP), also known as diazepam-binding inhibitor (DBI), is an extracellular feedback regulator of autophagy. Herein, we report that injection of a monoclonal antibody neutralizing ACBP / DBI (α-DBI) protects mouse livers from ischemia / reperfusion injury, acute intoxication with acetaminophen and concanavalin A, and hepatic fibrosis induced by bile duct ligation or carbon tetrachloride. Notably, the results support the contention that α-DBI mediates a broad organ protective effect against multiple insults.

Description

[Technical field]

[0001] cross reference This application claims priority to European Application No. EP22305137 (filed February 8, 2022), the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present invention is in the field of medicine, and in particular physiology. [Background technology]

[0003] Injury to vital organs (e.g., heart, brain, lungs, kidneys, gastrointestinal tract, or liver) can have serious and even life-threatening consequences. Organ injury has a variety of etiologies, typically including drugs, toxins, and ischemic injury. For example, studies of the cellular pathophysiology of ischemic injury in acute myocardial infarction have consistently shown that a significant portion of tissue injury occurs during reperfusion (the period when blood flow is restored after an ischemic period of more than about 10 minutes). This injury is responsible for paradoxical organ death and tissue injury after the end of the reperfusion period. IRI can be exhibited in almost all organ systems. Mechanisms involved in IRI include reduced high-energy phosphate (ATP) levels for several hours after tissue ischemia, inflammatory cell (neutrophil)-mediated cellular and microvascular injury, no-reflow phenomenon (insufficient reperfusion), microvascular dysfunction due to platelet plugs and endothelial injury due to insufficient tissue perfusion during the reperfusion period, and calcium overload-mediated reperfusion injury.

[0004] A common outcome of organ injury is fibrosis. Fibrosis is, in fact, the formation of fibrous connective tissue in response to injury. It is characterized by the accumulation of extracellular matrix components, especially collagen, at the site of injury. Fibrosis is an adaptive response that is an essential component of wound healing and tissue repair. However, its continued activation is highly detrimental and represents a common final pathway for many pathologies, including neurological, renal, cardiovascular, hepatic, and respiratory diseases.

[0005] Therefore, there remains a need for therapies to protect organs from injury and thus prevent fibrosis.

[0006] Macroautophagy (hereafter referred to as autophagy) is a process in which a portion of the cytoplasm is sequestered in an autophagosome, which then fuses with a lysosome for enzymatic hydrolysis of the autophagy cargo (Morishita and Mizushima, 2019). Although autophagy is often observed in association with cell death, it overwhelmingly serves a cytoprotective function. Thus, excessive autophagy leading to cell death ("autophagic cell death" or "autosis") is a rare phenomenon. Rather, in many cases, cell stress-induced autophagy delays or avoids cell death by promoting cellular adaptation (Kroemer and Levine, 2008; Lopez-Otin and Kroemer, 2021; Schwartz, 2021). This stress-adaptive function of autophagy is due to a combination of factors, including, but not limited to, (i) mobilization of macromolecules, including proteins, mRNA, lipids, and glycogen, to generate energy-rich metabolites and building blocks for anabolic reactions, and (ii) selective removal of damaged cellular structures, including misfolded protein aggregates, unattached or permeabilized mitochondria, and other dysfunctional organelles (Galluzzi et al., 2014; Lopez-Otin and Kroemer, 2021; Mizushima and Klionsky, 2007). As a result, cellular fitness improves in a cell-autonomous manner. Furthermore, activation of pro-inflammatory pathways is blunted by autophagy due to the removal of molecules (e.g., cytoplasmic DNA or reactive oxygen species) that can activate endogenous pattern recognition receptors and the downregulation of downstream signals emanating from such receptors (Deretic, 2021; Galluzzi et al., 2018; Schwartz, 2021). Given the widespread effects of autophagy, its induction has been proposed as a general strategy to combat disease. Recently, an extracellular feedback loop of autophagy has been described, which involves the protein acyl-coenzyme A-binding protein (ACBP), called diazepam-binding inhibitor (DBI) (Bravo-San Pedro et al., 2019a). Indeed, autophagy is coupled to the atypical secretion of this leaderless protein, which is mainly present in the cytoplasm of nucleated cells (Bravo-San Pedro et al., 2019a; Loomis et al., 2010). Once released into the extracellular space, ACBP / DBI then acts on gamma-aminobutyric acid (GABA) receptors to inhibit autophagy via autocrine, paracrine, and neuroendocrine pathways (Bravo-San Pedro et al., 2019a; Joseph et al., 2020). When injected intraperitoneally or intravenously, a monoclonal antibody (mAb) against ACBP / DBI (termed α-DBI) significantly reduced high-fat diet-induced steatosis, diabetes, and hepatic steatosis, while promoting autophagy, lipolysis, and β-oxidation, and simultaneously reducing appetite (Bravo-San Pedro et al., 2019a; Joseph et al., 2020, 2021). These effects were considered to be on-target, since they could be mimicked by inducible whole-body knockout of ACBP / DBI (Bravo-San Pedro et al. 2019a). Thus, methods and pharmaceutical compositions for modulating autophagy based on regulating DBI activity or expression were disclosed (WO2019057742). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2019 / 057742 Summary of the Invention [Means for solving the problem]

[0008] The invention is defined by the claims. In particular, the invention relates to methods and pharmaceutical compositions for protecting organs from injury (i.e., treating or reducing tissue damage due to injury), comprising neutralization of acyl-CoA binding proteins. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Acyl-CoA binding protein (ACBP), also known as diazepam-binding inhibitor (DBI), is an extracellular feedback regulator of autophagy. Herein, we report that injection of a monoclonal antibody that neutralizes ACBP / DBI (α-DBI) (in some embodiments, extracellular DBI) protects mouse livers from (i.e., treats or reduces the incidence of) ischemia / reperfusion injury, acute intoxication with acetaminophen and concanavalin A, and hepatic fibrosis induced by bile duct ligation or carbon tetrachloride. α-DBI administration downregulated inflammatory and profibrotic genes and upregulated antioxidant defense and fatty acid oxidation in the liver. Notably, the results support the contention that α-DBI mediates a broad range of organ protective effects against multiple insults.

[0010] Key definitions: As used herein, the terms "subject," "individual," or "patient" are used interchangeably and refer to any subject for whom diagnosis, treatment, or therapy is desired, particularly humans. Other subjects may include cows, dogs, cats, guinea pigs, rabbits, rats, mice, horses, etc. In some preferred embodiments, the subject is a human.

[0011] As used herein, the term "organ" refers to a solid, vascularized organ that performs a specific function or group of functions within an organism. The term organ includes, but is not limited to, the heart, lungs, kidneys, liver, pancreas, skin, uterus, bone, cartilage, small or large intestine, bladder, brain, breasts, blood vessels, esophagus, fallopian tubes, gallbladder, ovaries, pancreas, prostate, placenta, spinal cord, limbs (including upper and lower limbs), spleen, stomach, testes, thymus, thyroid, trachea, ureters, urethra, and uterus.

[0012] As used herein, the term "organ dysfunction" means and includes any reduction or impairment in the physical structure or function of an organ.

[0013] As used herein, the term "solid organ transplantation" and variations thereof refer to the insertion of a solid organ (also called a graft) into a recipient, whether the transplant is a syngeneic transplant (where the donor and recipient are genetically identical) or an allogeneic transplant (where the donor and recipient are of different genetic origins but are of the same species).

[0014] As used herein, the term "injury" or "injury" refers to any injury that directly or indirectly affects normal function. Injury can have a variety of causes, including but not limited to physiological, chemical, or physical injury. The term encompasses acute and chronic injuries. As used herein, the term "acute injury" includes an injury that occurred recently. For example, an acute injury may have occurred very recently, within an hour, within a day, within a week, or within two weeks. As used herein, the term "chronic injury" refers to an injury that persists for a period of time. For example, a chronic injury may have occurred more than two weeks ago, more than three weeks ago, more than two months ago, or more than three months ago.

[0015] As used herein, the term "ischemia" refers to a restriction of blood supply that results in organ damage or dysfunction. Unlike hypoxia, a more common term that refers to oxygen deficiency (usually the result of a lack of oxygen in the air we breathe), ischemia is an absolute or relative deficiency of blood supply to an organ, i.e., a deficiency of oxygen, glucose, and other blood-derived components. Relative deficiency refers to a mismatch between blood supply (oxygen / fuel supply) and blood demand for adequate metabolism of tissues. As used herein, the term "warm ischemia" has its general meaning in the art and is used to describe ischemia of cells and tissues under normothermic conditions. As used herein, the term "cold ischemia" has its general meaning in the art and refers to organ cooling during reduced blood perfusion or in the absence of blood supply.

[0016] As used herein, the term "reperfusion" has its general meaning in the art and refers to the restoration of blood flow to a tissue after ischemia. Thus, as used herein, the term "ischemia-reperfusion" or "I / R" is intended to encompass an event in which an ischemic episode is followed by an episode of reperfusion.

[0017] As used herein, the term "ischemia-reperfusion injury" or "I / R injury" refers to tissue damage caused by an ischemia-reperfusion event. The absence of oxygen and nutrients from the blood during the ischemic period creates conditions in which inflammation and oxidative damage occur due to the induction of oxidative stress when circulation is restored rather than (or with) the restoration of normal function. As used herein, the term "ischemia-reperfusion injury severity" or "I / R injury severity" refers to a measure of the extent of injury.

[0018] As used herein, the term "fibrosis" refers to the formation of fibrous tissue as a reparative or reactive process rather than as a normal component of an organ or tissue. Fibrosis is characterized by the accumulation of myofibroblasts and collagen deposition in excess of normal deposition in any particular tissue.

[0019] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. These terms also encompass modified amino acid polymers, such as disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with labeling moieties. A polypeptide when considered in the context of gene therapy refers to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof that retains the desired biochemical function of the intact protein.

[0020] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can include modified nucleotides, such as methylated nucleotides and nucleotide analogs, and can be interrupted by non-nucleotide components. Modifications to the nucleotide structure, if present, can be added before or after assembly of the polymer. As used herein, the term polynucleotide refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein (polynucleotide) encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to form a double-stranded form.

[0021] As used herein, the term "encode" refers to the inherent property of a particular nucleotide sequence within a polynucleotide, e.g., a gene, cDNA, or mRNA, to serve as a template for the synthesis of other multimers and macromolecules having either a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence in a biological process, as well as the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA encodes a protein when transcription and translation of the mRNA corresponding to that gene results in the protein in a cell or other biological system. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually shown in the sequence listing) and the non-coding strand (used as a template for transcription of the gene or cDNA) can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "polynucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase "polynucleotide sequence encoding a protein or RNA" can also include introns, to the extent that a nucleotide sequence encoding a protein may in some version include intron(s).

[0022] As used herein, the term "expression inhibitor" refers to a natural or synthetic compound that has the biological effect of inhibiting the expression of a polynucleotide.

[0023] As used herein, the term "DBI" has its common meaning in the art and refers to diazepam binding inhibitor, acyl-CoA binding protein, encoded by the DBI gene (Gene ID: 1622). This term is also known as EP, ACBP, ACBD1, and CCK-RP. An exemplary amino acid sequence of DBI is represented by SEQ ID NO:1. Accession number 1>sp|P07108|ACBP_HUMAN Acyl-CoA-binding protein OS=Homo sapiens OX=9606 GN=DBI PE=1 SV=2 MSQAEFEKAAEEVRHLKTKPSDEEMLFIYGHYKQATVGDINTERPGMLDFTGKAKWDAWN ELKGTSKEDAMKAYINKVEELKKKYGI

[0024] As used herein, the terms "antibody" and "immunoglobulin" have the same meaning and are used in the present invention. 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 immunospecifically binds to an antigen. Thus, the term antibody includes whole antibody molecules as well as antibody fragments and variants (including derivatives) of antibodies and antibody fragments. In a natural antibody, 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 (l) and kappa (k). There are five main heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain contains a different sequence domain. The light chain contains two domains, the variable domain (VL) and the constant domain (CL). Heavy chains contain three (α, β, γ) to five (μ, ε) domains, a variable domain (VH) and three to four constant domains (CH1, CH2, CH3, and CH4; collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine binding recognition and specificity to antigens. The constant region domains of the light chain (CL) and the heavy chain (CH) confer important biological properties, such as antibody chain association, secretion, transplacental mobility, complement binding, and Fc receptor (FcR) binding. The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of an antibody resides in the structural complementarity between the antibody binding site and an antigenic determinant. The antibody binding site is composed of residues mainly derived from the hypervariable region or complementarity determining region (CDR). In some cases, residues from non-hypervariable or framework regions (FR) may participate in the antibody binding site or may influence the structure of the entire domain and thus the binding site. CDRs refer to amino acid sequences that define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site.The light and heavy chains of immunoglobulins each have three CDRs, which are called L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3, respectively. Thus, typically, an antigen-binding site contains six CDRs, including a set of CDRs from each of the heavy and light chain V regions. The framework region (FR) refers to the amino acid sequence sandwiched between the CDRs. Residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al. This system is described in Kabat et al., 1987 (Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA) (hereinafter "Kabat et al."). This numbering system is used herein. The Kabat residue designations do not necessarily correspond directly to the linear numbering of amino acid residues in sequence order. The actual linear amino acid sequence may contain fewer or more amino acids than the strict Kabat numbering, corresponding to the shortening or insertion of structural components in the basic variable domain structure, whether framework or complementarity determining regions (CDRs). The exact Kabat numbering of residues can be determined for a given antibody by aligning the homologous residues in the antibody sequence with the "standard" Kabat numbering 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.

[0025] 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. Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in minor amounts.

[0026] As used herein, the term "chimeric antibody" refers to an antibody that comprises the VH and VL domains of a non-human antibody and the CH and CL domains of a human antibody. In some embodiments, a "chimeric antibody" refers to an antibody molecule in which (a) the constant regions (i.e., heavy and / or light chains) or portions thereof have been modified, replaced, or exchanged to attach the antigen-binding site (variable region) to a constant region of a different or modified class, effector function, and / or species, or to an entirely different molecule (e.g., an enzyme, toxin, hormone, growth factor, drug, etc.) that confers new properties to the chimeric antibody, or (b) the variable region or portions thereof have been modified, replaced, or exchanged by a variable region with a different or modified antigen specificity. Chimeric antibodies also include primatized antibodies, particularly humanized antibodies. In addition, chimeric antibodies can contain residues that are not found in either the recipient antibody or the donor antibody. Such modifications are made to further refine antibody performance. For details, see Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992) (see U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0027] As used herein, a "humanized antibody" refers to an antibody that has the variable region framework and constant regions of a human antibody but retains the CDRs of a previous non-human antibody. In some embodiments, a humanized antibody contains minimal sequences derived from a non-human immunoglobulin. For the most part, humanized antibodies and antibody fragments thereof can be human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (e.g., mouse, rat, or rabbit) (donor antibody) having the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies / antibody fragments can comprise residues that 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 region is derived, but to avoid immune responses against the non-human antibody. These modifications can further refine and optimize the performance of the antibody or antibody fragment. Typically, a humanized antibody or antibody fragment thereof will contain 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 substantial portion of the FR regions are from a human immunoglobulin sequence. A humanized antibody or antibody fragment may also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For details, see Jones et al., Nature, 321:522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2:593-596, 1992.

[0028] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences. The human antibodies of the invention 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, as used herein, the term "human antibody" 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.

[0029] As used herein, the term "antibody fragment" refers to at least a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody, that retains the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). A "fragment" includes a portion of an intact antibody, generally the antigen-binding site or the variable region. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (herein referred to as "single-chain antibody fragment" or "single-chain polypeptide") (including, but not limited to, (1) a single-chain Fv molecule, (2) a single-chain polypeptide comprising only one light chain variable domain, without associated heavy chain portions, or fragments thereof comprising the three CDRs of the light chain variable domain, and (3) a single-chain polypeptide comprising only one heavy chain variable region, without associated light chain portions, or fragments thereof comprising the three CDRs of the heavy chain variable region); and multispecific antibodies formed from antibody fragments. Fragments of the antibodies of the present invention can be obtained using standard methods.

[0030] As used herein, the term "specificity" refers to the ability of an antibody to detectably bind to a target molecule (e.g., an epitope presented on an antigen) while having relatively little detectable reactivity with other target molecules. Specificity can be relatively determined, for example, by binding assays using a Biacore instrument or competitive binding assays, as described elsewhere herein. Specificity can be demonstrated by an affinity / avidity ratio of, for example, about 10:1, about 20:1, about 50:1, about 100:1, 10.000:1, or more, for binding to a specific antigen versus nonspecific binding to other unrelated molecules.

[0031] As used herein, the term "affinity" refers to the strength of binding between an antibody and a target molecule (such as an epitope). The affinity of a binding protein is given by the dissociation constant Kd. For an antibody, the aforementioned Kd is defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant Ka is defined by 1 / Kd. Preferred methods for determining the affinity of binding proteins can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988; Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993); and Muller, Meth. Enzymol. 92:589-601 (1983), which are incorporated herein by reference in their entireties. One preferred standard method for determining the affinity of binding proteins, well known in the art, is the use of a Biacore instrument.

[0032] As used herein, the term "binding" refers to a direct association between two molecules, for example, by covalent, electrostatic, hydrophobic, and ionic, and / or hydrogen bonding interactions (including interactions such as salt bridges and water bridges). In particular, as used herein, the term "binding" in the context of binding between an antibody and a given target molecule (e.g., an antigen or epitope) typically refers to a binding that occurs between the antibody and the target molecule by a force of about 10 -7 M or less, e.g., about 10 -8 M or less, e.g., about 10 -9 M or less, about 10 -10 M or less, or about 10 -11 K below M or even lower D The binding has an affinity corresponding to

[0033] As used herein, the term "neutralizing anti-DBI monoclonal antibody" refers to an antibody to a monoclonal antibody that has specificity for DBI and inhibits, reduces, or completely inhibits the activity of DBI (e.g., extracellular DBI). Whether an antibody is a neutralizing antibody can be determined by the in vitro assay described in the Examples. Typically, a neutralizing antibody of the invention inhibits the activity of DBI by at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[0034] As used herein, the term "treatment" or "treating" includes both prophylactic or preventive treatment, as well as curative or disease-modifying treatment (including treatment of patients at risk of or suspected of having a disease, as well as patients who are ill or have been diagnosed with a disease or medical condition), and includes suppression of clinical recurrence. Treatment can be administered to patients with a medical disorder, or those who may eventually suffer from a disorder, to prevent, cure, delay onset, reduce the severity, or alleviate one or more symptoms of the disorder or recurring disorder, or to extend the patient's survival beyond that expected in the absence of such treatment. "Treatment regimen" refers to a pattern of treatment of a disease, e.g., a dosing pattern used during therapy. Treatment regimens can include induction regimens and maintenance regimens. The phrase "induction regimen" or "induction period" refers to a treatment regimen (or a portion of a treatment regimen) used in the initial treatment of a disease. The overall purpose of an induction regimen is to provide a high level of drug to the patient during the initial period of the treatment regimen. The induction regimen may use (partially or entirely) a "loading regimen", which may involve the physician administering a larger dose of the drug than he administers during the maintenance regimen, administering the drug more frequently than he administers during the maintenance regimen, or both. The term "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) used to maintain a patient during disease treatment, for example, to keep the patient in remission for an extended period of time (months or years). A maintenance regimen may use continuous therapy (administering a drug at regular intervals (e.g., weekly, monthly, yearly, etc.)) or intermittent therapy (e.g., interruption of treatment, intermittent treatment, treatment upon relapse, or treatment upon the achievement of certain predetermined criteria (e.g., pain, disease manifestation, etc.).

[0035] As used herein, the term "pharmaceutical composition" refers to a composition described herein, or a pharma- ceutical acceptable salt thereof, together with other agents, such as carriers and / or excipients. The pharmaceutical compositions provided herein typically include a pharma- ceutical acceptable carrier.

[0036] As used herein, "consisting essentially of" with respect to a composition means that at least one antibody of the invention as described herein above is the only therapeutic or biologically active agent in the composition.

[0037] As used herein, the term "pharmaceutical acceptable carrier" includes any solvent, diluent, or other liquid vehicle, dispersing or suspending aid, surface active agent, isotonic agent, thickening or emulsifying agent, preservative, solid binder, lubricant, and the like, appropriate for the particular dosage form desired. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for their preparation.

[0038] As used herein, the term "therapeutically effective amount" refers to an amount effective at dosages and for periods of time necessary to obtain a desired therapeutic result. The therapeutically effective amount of an active agent may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the active agent to induce a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the drug are outweighed by the therapeutically beneficial effects. The effective dosage and administration regime of the active agent depends on the disease or condition being treated and can be determined by one of ordinary skill in the art. A physician of ordinary skill in the art can easily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician can begin administration of the active agent used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. In general, a suitable dose of the composition of the present invention is that amount of the compound that is the lowest dose effective to produce a therapeutic effect according to a particular administration regime. Such an effective dose generally depends on the factors described above. For example, a therapeutically effective amount for therapeutic use can be measured by its ability to stabilize the progression of a disease. One of ordinary skill in the art would be able to determine such amounts based on factors such as the size of the patient, the severity of the patient's symptoms, the particular composition or route of administration selected, etc. Exemplary, non-limiting ranges for therapeutically effective amounts of the drugs of the present invention are about 0.1-100 mg / kg, e.g., about 0.1-50 mg / kg, e.g., about 0.1-20 mg / kg, e.g., about 0.1-10 mg / kg, e.g., about 0.5, e.g., about 0.3, about 1, about 3 mg / kg, about 5 mg / kg or about 8 mg / kg. Exemplary, non-limiting ranges for therapeutically effective amounts of the drugs of the present invention are 0.02-100 mg / kg, e.g., about 0.02-30 mg / kg, e.g., about 0.05-10 mg / kg or 0.1-3 mg / kg, e.g., about 0.5-2 mg / kg.

[0039] Methods of conferring organ protection: A first object of the present invention relates to a method of providing organ protection (i.e., treating or reducing tissue damage caused by injury) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent that inhibits the activity or expression of a DBI (e.g., circulating or extracellular DBI). Thus, disclosed herein is a composition for use in treating or reducing tissue damage caused by injury in an organ of a subject, comprising an amount of an agent that inhibits the activity or expression of a diazepam binding inhibitor (DBI), which amount, when administered to a subject, is sufficient to treat or reduce tissue damage caused by injury to an organ of the subject.

[0040] The methods and compositions of the present invention are particularly suited to preventing organ dysfunction.

[0041] The methods and compositions of the present invention are particularly suitable for providing organ protection against any type of injury. In some embodiments, the methods or compositions of the present invention are particularly suitable for providing organ protection against chemical injury (treating or reducing tissue damage caused by chemical injury). In some embodiments, the methods or compositions of the present invention are particularly suitable for providing organ protection against physical injury (treating or reducing tissue damage caused by physical injury). In some embodiments, the methods or compositions of the present invention are particularly suitable for providing organ protection against ischemic injury (treating or reducing tissue damage caused by ischemic injury).

[0042] In particular, the method or composition of the present invention may be used with or without the use of alcohol, 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2-bromoethylamine (BEA), 3-methylcholanthrene, 4-aminophenol (PAP), acetaminophen, adriamycin, allyl alcohol, amiodarone, amphotericin B, Aroclor 1254, Aroclor 1260, arsenic, aspirin, astemizole, benzene, cadmium, carbamedipine, carbon tetrachloride (CCl4), ciprofibrate (Cipro), clofibrate, cobalt chloride, corvastatin, cyclosporine A, diethylnitrosamine, dimethylformamide, dimethylhydrazine (DMH), diquat, ethosuximide, etoposide, famotidine, fluoxetine ... Conazole, gemfibrozil, ganciclovir, hexachloro-1,3-butedien (HCBD), HIV protease inhibitors, hydrazine, indomethacin, ketoconazole, lead acetate (PbAc), lipopolysaccharide (LPS), mercuric chloride (HgCl2), methanol, methapyrilene, methotrexate, metronidazole, miconazole, monocrotaline, nitric oxide, ondansetron, pentamidine, phenobarbital, phenylhydrazine (phenylhyrzn), phenytoin, pravastatin, proprusside, puromycin aminonucleoside (PAN), quinolones, simvastatin, sodium fluoride (NaF), statins, thioacetamide, tocainidine, tricyclic antidepressants, troglitazone, tumor necrosis factor alpha (TNFα), uranyl nitrate, valproic acid, vincristine, Wy-16,463, zidovudine (AZT), α-naphthyl isothiocyanate (ANIT), β-naphthoflavone (BNF), asbestos, radon, tobacco smoke, adhesives, dioxin, nickel, arsenic, mercury, cement (chromium), polychlorinated biphenyls (PCBs), carbon tetrachloride, methylene chloride, vinyl chloride, mercury, chlorinated hydrocarbon solvents, carbon disulfide, cadmium, ozone, tobacco smoke, nitrates, methylene chloride, ethylene dibromide, and polychlorinated biphenyls.

[0043] In some embodiments, the methods or compositions of the invention are particularly suited for providing organ protection (treating or reducing tissue damage caused by chemotherapeutic agents) against chemotherapeutic agents, including, but not limited to, alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethyleneethiophosphoramide and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictine; spongistatins; nitrogen mustards such as chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamol); gammall and calicheamicin omegall); dynemicins (including dynemicin A); bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, authrarnycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and detorubicin). including oxidoxorubicin), mitomycins such as epirubicin, esorubicin, idarubicin, marcelomycin, and mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, keramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); and denopterin, methotrexate, pteropterin, and trimetrexate. Acid analogues; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal corticosteroids such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as floric acid; aceglatone; aldophospha Midoglycosides;Aminolevulinic acid;Eniluracil;Amsacrine;Bestravcil;Bisantrene;Edatraxate;Defofamine;Demecolcine;Diaziquone;Elformitin;Elliptinium acetate;Epothilone;Etoglucide;Gallium nitrate;Hydroxyurea;Lentinan;Lonidynin;Maytansinoids such as maytansine and ansamitocin;Mitoguazone;Mitoxantrone;Mopidammol;Nitraerin;Pentostatin;Fenamet;Pirarubicin;Rosoxantrone;Podophyllic acid;2-Ethylhydrazide;Procarbazine;PSK polysaccharide complex;razoxane;rhizoxin;schizofuran;spirogermanium;tenuazonic acid;triaziquone;2,2',2"-trichlorotriethylamine;trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine);urethane;vindesine;dacarbazine;mannomustine;mitobronitol;mitolactol;pipobroman;gacitosine;arabinosides ("Ara-C");cyclophosphamide;thiotepa;taxoids, e.g. , paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-1 1); the topoisomerase inhibitor RFS 2000; difluoromethylomitin (DMFO); retinoids such as retinoic acid; capecitabine; and pharma- ceutical acceptable salts, acids, or derivatives of any of the above.

[0044] In some embodiments, the methods of the present invention are particularly suitable for conferring organ protection against drug-induced injury (treating or reducing tissue damage caused by drug-induced injury) induced by an anthracycline selected from the list consisting of daunorubicin, doxorubicin, epirubicin, farmorubicin, idarubicin, mitoxantrone, pixantrone, and pharmaceutically acceptable salts thereof.

[0045] In some embodiments, the methods or compositions of the present invention are particularly suited for the treatment of acetaminophen-induced hepatotoxicity, amiodarone-induced pulmonary toxicity, doxorubicin-induced cardiotoxicity, cadmium chloride-induced nephrotoxicity, dimethylnitrosamine-induced splenic toxicity, and O-ethyl-S,S-dipropyl phosphorodithioate (MOCAP)-induced neurotoxicity.

[0046] The method or composition of the present invention can be applied to any ischemic injury or event. Tissues that are particularly sensitive to ischemic events include myocardial, vascular, and neuronal tissues (particularly brain tissue). Other tissues that are sensitive to ischemia include gastrointestinal, liver, kidney, and eye tissues. For example, the need for cardioprotection may arise during trauma or during cardiac arrest due to certain physiological disorders (e.g., unstable angina). In addition, disorders such as stroke, transient ischemic attack, or impending stroke (amarosis fugax) are candidate conditions for treatment using the method of the present invention. In the event of a stroke that creates a risk of a secondary stroke, or another condition that creates a risk of stroke within hours or days, the method can be applied to attenuate such risk. Those skilled in the art will recognize situations associated with increased risk of other ischemic tissue injuries. Such disease states include mesenteric artery insufficiency, renal artery stenosis, hepatic vein thrombosis, peripheral vascular insufficiency, multiple trauma, sepsis, and multiple organ failure. Other ischemic events include angiographic evidence of partial coronary artery occlusion, echocardiographic evidence of myocardial damage, or any other evidence of risk of future or additional ischemic events (e.g., myocardial ischemic events such as myocardial infarction (MI), or neurovascular ischemia such as cerebrovascular accident CVA). Ischemia / reperfusion can damage tissues other than myocardium. The methods or compositions provided herein are particularly suitable for reducing ischemia-reperfusion injury that may occur in brain, liver, gut, kidney, bowel tissue, or any other tissue. Additional applications include blunt or penetrating trauma resulting in the interruption of blood flow to visceral organs, including those resulting from gunshot wounds, stab wounds, or penetrating wounds to the abdomen resulting from deceleration injuries and / or penetrating wounds secondary to motor vehicle accidents or blunt abdominal trauma. Other preferred uses include diseases or procedures that result in systemic hypotension that disrupts or reduces blood flow to visceral organs, including hemorrhagic shock due to blood loss, cardiogenic shock due to myocardial infarction or heart failure, neurogenic shock, or anaphylaxis.

[0047] In some embodiments, the use of the method or composition of the present invention can improve organ protection during surgical procedures that require the cessation of blood supply to an organ and subsequent reperfusion. Examples of surgical procedures that create a risk of ischemia-reperfusion injury include liver resection; revascularization after myocardial infarction (e.g., by thrombolytic therapy, stent placement, or surgical repair); revascularization after stroke (e.g., by thrombolytic therapy or surgical repair); or revascularization after vascular injury (including limb repair or reconnection after ischemic injury or surgical repair of aneurysm). Other examples include upper or lower gastrointestinal tract surgery, including laparoscopic surgery, open heart surgery with or without cardiopulmonary bypass, nose and throat surgery, vascular surgery, neurological (brain) surgery, transplants (liver, heart, lung, kidney, intestine), liver surgery, and Caesarean section. In some embodiments, the surgical procedure is coronary artery bypass surgery (also known as coronary artery bypass graft (CABG) surgery or cardiac bypass surgery or simply bypass surgery), which is a surgical procedure performed to relieve angina and reduce the risk of death from coronary artery disease. An artery or vein from elsewhere in the patient's body is grafted onto the coronary artery to bypass the atherosclerotic narrowing and improve blood supply to the coronary circulation that supplies the myocardium (heart muscle). This surgery is usually performed with the heart stopped, which requires the use of cardiopulmonary bypass, but so-called "off-pump" surgery techniques are also available to perform CABG on a beating heart. In some embodiments, the method of the present invention can be used for any surgical procedure that requires clamping of the blood supply to an organ. In particular, the method of the present invention is applied to all surgical procedures that involve the connection of two blood vessels (e.g., coronary artery bypass, peripheral bypass, hemodialysis access (creation of a fistula), and free flap surgery (breast and facial reconstruction surgery)). More particularly, the method or composition of the present invention can be applied to any surgical procedure that requires anastomosis. As used herein, the term "anastomosis" refers to a surgical connection between tubular structures, such as blood vessels.

[0048] Typically, an effective amount of an agent that inhibits DBI (e.g., extracellular DBI) activity or expression can be administered to a patient before, during, or after reperfusion. In particular, an effective amount of an agent that inhibits DBI activity or expression is administered to a patient during reperfusion of an organ.

[0049] In some embodiments, the methods or compositions of the present invention are particularly suitable for preventing progression to chronic kidney disease (CKD) after acute kidney injury (AKI). As used herein, the term "chronic kidney disease" (CKD) refers to the progressive loss of kidney function over a period of months to years. CKD has a common meaning in the art and is used to classify a number of conditions that affect the kidney, destruction of the renal parenchyma, and loss of functional nephrons or glomeruli. It is further noted that CKD results from a variety of causes, but the ultimate pathway remains renal fibrosis. The terms "acute kidney injury" or "acute renal failure" are typically identified by a rapid deterioration of renal function sufficient to result in the accumulation of nitrogenous waste products in the body (see, e.g., Anderson and Schrier (1994) (Harrison's Principles of Internal Medicine, 13th edition); Isselbacher et al, eds., McGraw Hill Text, New York). An increase in BUN of at least 4-8 mmol / L / day (10-20 mg / dL / day) and an increase in serum creatinine of at least 40-80 μmol / L / day (0.5-1.0 mg / dL / day) are typical in acute renal failure. In patients with acute kidney injury, urine samples may contain remnants of tubular injury. In subjects with catabolism (or hypercatabolism), the increase in BUN may exceed 100 mg / dL / day. The increase in BUN or serum creatinine may be quantified by serial blood tests, preferably at least two blood tests over a period of 6-72 hours, more preferably 12-24 hours. In some cases, "acute" renal failure (worsening over a period of days) is distinguished from "rapidly progressive" renal failure (worsening over a period of weeks). However, as used herein, the term "acute kidney injury" is intended to encompass both syndromes. Acute kidney injury is routinely identified by clinicians as described above.AKI can be due to abnormalities of the vasculature, such as vasoconstrictive diseases (e.g., malignant hypertension, scleroderma, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura) and vasculitis (e.g., polyarteritis nodosa, hypersensitivity vasculitis, serum sickness, Wegener's granulomatosis, giant cell arteritis, mixed cryoglobulinemia, Henoch-Schönlein purpura, systemic lupus erythematosus). AKI can also result from glomerular abnormalities, such as post-infectious (e.g., post-streptococcal, pneumococcal, gonococcal, staphylococcal, enterococcal, viral (e.g., hepatitis B and C, mumps, measles, Epstein-Barr), malarial, or those associated with brucellosis, legionella, listeria, shunt nephritis, leprosy, leptospirosis, or visceral abscess) and non-infectious (e.g., rapidly progressive glomerulonephritis, membranoproliferative glomerulonephritis, Goodpasture's syndrome, systemic lupus erythematosus, Wegener's granulomatosis). In some embodiments, AKI may result from acute interstitial nephritis due to drug-related causes (e.g., penicillins, sulfonamides, carbenicillins, cephalosporins, erythromycin, nafcillin, oxacillin, nonsteroidal anti-inflammatory agents, diuretics (furosemide, ethacrynic acid, thiazides, spironolactone, mercurials), phenytoin, phenobarbital, probenicid, allopurinol, cimetidine), infection-related causes (e.g., acute pyelonephritis, streptococcal, staphylococcal, leptospirosis, malaria, salmonellosis), renal papillary necrosis (e.g., associated with diabetes, sickle cell disease, analgesic abuse, alcoholism), and a variety of other causes (e.g., sarcoidosis, leukemia, lymphoma). In some embodiments, AKI may result from intratubular obstruction due to crystal deposition (eg, uric acid, oxalate, methotrexate) or multiple myeloma and light chain disease.In some embodiments, AKI may result from acute tubular necrosis due to nephrotoxins (e.g., antimicrobial agents such as aminoglycosides, tetracyclines, amphotericin, polymyxins, cephalosporins, etc.), heavy metals (e.g., mercury, lead, arsenic, gold salts, barium), and various other chemical agents (e.g., cisplatin, doxorubicin, streptozocin, methoxyflurane, halothane, ethylene glycol, carbon tetrachloride), ischemia (e.g., hemorrhage, hypotension, sepsis, burns, renal infarction, renal artery dissection, rhabdomyolysis, trauma), or a variety of other causes (e.g., contrast agents, transfusion reactions, myoglobinemia, heat stroke, snake and spider bites).

[0050] The method or composition of the present invention is particularly suitable for preventing, reducing the severity of, or reducing the risk of injury in an organ. In some embodiments, the organ is isolated. In some embodiments, the organ is a graft or a transplantable organ.

[0051] In some embodiments, the methods or compositions of the present invention can improve organ transplantation by administering to an isolated (transplanted) organ an effective amount of an agent that inhibits DBI (e.g., extracellular DBI) activity or expression. Thus, in some embodiments, the organ is to be transplanted into a recipient. Thus, the method is performed ex vivo on an isolated organ.

[0052] In some embodiments, the transplant organ is a cadaveric organ, and if the organ is obtained from a cadaveric donor, the agent that inhibits DBI activity or expression can be administered to either the cadaveric organ or the harvested organ. In some embodiments, the transplant organ is a living organ donor, and in such cases, the agent that inhibits DBI activity or expression can be administered to the harvested organ.

[0053] In some embodiments, the organ is isolated and perfused with an effective amount of an agent that inhibits activity or expression of DBI (eg, extracellular DBI).

[0054] In some embodiments, the transplant organ is subject to warm and / or cold ischemia.

[0055] In some embodiments, an effective amount of an agent that inhibits DBI (e.g., extracellular DBI) activity or expression is administered during cold ischemia time. As used herein, the term "cold ischemia time" or "CIT" has its general meaning in the art and refers to the time that extends from the start of cold storage of the recovering organ to the return of warm circulation after transplantation. There is variability depending on the receiving surgeon / institution and on the characteristics of the donor and recipient. Intuitively, the shorter the CIT, the better. For kidney transplantation, the CIT should be less than 24 hours, for pancreatic transplantation, the CIT should be less than 18 hours, and for liver transplantation, the CIT should be less than 8 hours (Bernat JL, D'Alessandro AM, Port FK, Bleck TP, Heard SO, Medina J, et al. Report of a National Conference on Donation after cardiac death. Am J Transplant. 2006; 6: 281-91).

[0056] Ways to prevent fibrosis: The methods and compositions of the invention are also particularly suited to preventing or reducing fibrosis associated with or occurring following organ injury.

[0057] In some embodiments, fibrosis affects at least one organ selected from the group consisting of skin, heart, liver, lung, or kidney. Examples of fibrosis include, but are not limited to, skin scarring, keloid, liver fibrosis, lung fibrosis, renal fibrosis, glomerulosclerosis, pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), liver fibrosis, renal fibrosis, intestinal fibrosis, interstitial fibrosis, pancreatic and pulmonary fibrosis, injection fibrosis, endomyocardial fibrosis, mediastinal fibrosis, bone marrow fibrosis, retroperitoneal fibrosis, progressive macrofibrosis, nephrogenic systemic fibrosis, etc. In some embodiments, fibrosis is caused by surgical implantation of an artificial organ.

[0058] For example, liver (liver) fibrosis occurs as part of the wound healing response of chronic liver injury. Such damage can be the result of viral activity (e.g., chronic hepatitis B or C) or other infectious diseases (e.g., parasites, bacteria), chemicals (e.g., medicines, alcohol, pollutants), immune processes (e.g., autoimmune hepatitis), metabolic disorders (e.g., lipid, glycogen, or metal storage disorders), or cancer growth. Liver fibrosis is characterized by the accumulation of extracellular matrix that can be qualitatively distinguished from normal liver. If liver fibrosis is left unchecked, it progresses to cirrhosis (defined by the presence of encapsulated nodules), liver failure, and death. The methods or compositions described herein can be provided in an amount sufficient to reduce the level of transaminase in a subject when administered, compared to the level of transaminase in the subject before administration. In some embodiments, the transaminase is aspartate transaminase (AST) or alanine transaminase (ALT). The method or composition described herein can be provided in an amount sufficient to reduce fibrosis score by at least 1 / 3 compared to the fibrosis score before administration.The method or composition described herein can be provided in an amount sufficient to reduce the level of hydroxyproline in a subject when administered compared to the level of hydroxyproline in a subject before administration.The method or composition described herein can be provided in an amount sufficient to reduce the NAFLD score in a subject when administered compared to the NAFLD score before administration.

[0059] Renal fibrotic disorders include, but are not limited to, glomerulonephritis (including membranous proliferative, diffuse proliferative, rapidly progressive, post-infectious, and chronic forms), diabetic glomerulosclerosis, focal glomerulosclerosis, diabetic nephropathy, lupus nephritis, tubulointerstitial fibrosis, membranous nephropathy, amyloidosis (affecting the kidney, among other tissues), renal arteriosclerosis, nephrotic syndrome, renal interstitial fibrosis, renal fibrosis in cyclosporine-treated patients, and HIV-associated nephropathy. The glomerulus is the primary target of many types of renal injury, including immunological (e.g., immune complex or T cell mediated), hemodynamic (systemic or renal hypertension), metabolic (e.g., diabetes), "atherosclerosis" (accumulation of lipids in the glomerulus), infiltrative (e.g., amyloid), and toxic (e.g., snake venom). Renal structural changes in patients with diabetic nephropathy include glomerular hypertrophy, thickening of glomerular and tubular membranes (due to matrix accumulation), and increased matrix content in the mesangium and tubulointerstitium. Glomerular hypertension due to altered intrarenal hemodynamics in diabetes may contribute to the progression of diabetic nephropathy. Autoimmune nephritis may also result in altered mesangial cell growth responses. Infection with hepatitis C virus may also result in idiopathic membranoproliferative glomerulonephritis.

[0060] Pulmonary fibrotic disorders include, but are not limited to, silicosis, asbestosis, idiopathic pulmonary fibrosis, bronchiolitis obliterans organizing pneumonia, pulmonary fibrosis associated with high-dose chemotherapy, idiopathic pulmonary fibrosis, and pulmonary hypertension. These diseases are characterized by cell proliferation and increased production of extracellular matrix components such as collagen, elastin, fibronectin, and tenascin-C. The method of the present invention can also be used to treat subjects with asthma and other pulmonary conditions associated with airway remodeling.

[0061] Pancreatic fibrosis occurs in chronic pancreatitis. This condition is characterized by ductal calcification and fibrosis of the pancreatic parenchyma. Like cirrhosis of the liver, chronic pancreatitis is also associated with alcohol abuse.

[0062] The methods of the present invention are also suitable for treating intestinal fibrosis, particularly fibrosis associated with inflammatory bowel disease (eg, Crohn's disease and ulcerative colitis).

[0063] The skin fibrosis conditions that can be treated by the method of the present invention include, but are not limited to, scleroderma, morphea, keloid, hypertrophic scar, familial cutaneous collagenoma, and collagen type connective tissue nevi.Furthermore, the method of the present invention is suitable for inhibiting the overproduction of scars in patients known to form keloids or hypertrophic scars, inhibiting or preventing the overproduction of scars or scars during healing of various types of wounds, including surgical incisions, surgical abdominal wounds, and traumatic lacerations, preventing or inhibiting the scarring and reclosure of arteries after coronary angioplasty, and preventing or inhibiting the excessive scarring or fibrous tissue formation associated with cardiac fibrosis in post-infarction and hypersensitive vasculopathy.

[0064] Fibrotic conditions of the eye include conditions such as diabetic retinopathy, post-surgical scarring (eg, after glaucoma filter surgery and after esotropia surgery), and proliferative vitreoretinopathy.

[0065] Fibroproliferative disorders of bone are characterized by abnormal ectopic bone formation, typically seen as active proliferation of the major cell types involved in bone formation and their synthesis of complex bone matrix. An example of such a bone disorder is the fibrosis that occurs with metastasis of prostate tumors to the axial skeleton. In prostate tumor-associated cancellous bone growth, prostate cancer cells may interact with osteoblasts, resulting in enhanced tumor growth and osteoblast action when deposited in bone. Fibroproliferative responses of bone arising from the skeleton itself include osteopetrosis and hyperostosis. Defects in osteoblast differentiation and function are believed to be the primary cause of osteopetrosis. Osteopetrosis is a genetic disease characterized by bone sclerosis due to reduced bone resorption, extramedullary hematopoiesis due to failure to develop bone marrow cavities, and severe hematological abnormalities with optic atrophy, deafibronectiness, and mental retardation. In osteoarthritis, bone changes are known to occur, and bone collagen metabolism is increased in the osteoarthritic femoral head. The greatest changes occurred in the subchondral region, maintaining a high proportion of osteoid in the diseased tissue.

[0066] Fibroproliferative disorders of the vascular system include, for example, transplant vasculopathy, a major cause of chronic rejection of cardiac transplants, which is a classical fibroproliferative disease characterized by accelerated atherosclerotic plaque formation accompanied by diffuse occlusion of coronary arteries.

[0067] Additional fibrotic conditions that may be treated by the methods of the present invention include rheumatoid arthritis, diseases associated with long-term joint pain and deterioration, progressive systemic sclerosis, polymyositis, dermatomyositis, eosinophilic fasciitis, morphea, Raynaud's syndrome, and nasal polyposis.

[0068] In some embodiments, the method of the present invention is particularly suitable for the treatment of inflammation-induced fibrosis. As used herein, the term "inflammation-induced fibrosis" refers to fibrosis that develops during inflammatory diseases, i.e. diseases associated with or resulting from acute or chronic inflammation (caused by tissue injury, pathogen infection, or toxic agents).

[0069] In some embodiments, the methods of the present invention are particularly suited to the treatment of liver fibrosis.

[0070] Medicament of the invention: In some embodiments, the agent that inhibits the activity of DBI (eg, extracellular DBI) is an antibody directed against DBI.

[0071] In some embodiments, the antibody is directed to a fragment present in the amino acid sequence spanning amino acid residue 43 to amino acid residue 50 of SEQ ID NO:1 (ie, the octapeptide or OP).

[0072] In some embodiments, the antibodies of the invention are chimeric antibodies, typically mouse / human chimeric antibodies.

[0073] In some embodiments, the antibody is a humanized antibody.

[0074] In some embodiments, the antibody is a human antibody. Fully human monoclonal antibodies can also be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Patent Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference.

[0075] In some embodiments, the antibody is a neutralizing antibody.

[0076] In some embodiments, the neutralizing antibodies of the present invention do not mediate antibody-dependent cell-mediated cytotoxicity and therefore do not contain an Fc portion that induces antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the neutralizing antibodies do not contain an Fc domain capable of substantially binding to an FcgRIIIA (CD16) polypeptide. In some embodiments, the neutralizing antibodies lack an Fc domain (e.g., lack a CH2 and / or CH3 domain) or contain an Fc domain of an IgG2 or IgG4 isotype. In some embodiments, the neutralizing antibodies consist of or include a Fab, Fab', Fab'-SH, F(ab')2, Fv, diabody, single chain antibody fragment, or a multispecific antibody comprising multiple different antibody fragments. In some embodiments, the neutralizing antibodies are not conjugated to a toxic moiety. In some embodiments, one or more amino acids selected from the 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). This approach is described in further detail in US Pat. No. 6,194,551 by ldusogie et al.

[0077] Several anti-DBI antibodies that inhibit the activity of DBI (e.g., extracellular DBI) are suitable for use in the methods and compositions described herein. Such anti-DBI antibodies are commercially available and have been described in the literature. For example, an antibody that inhibits the activity of DBI (e.g., extracellular DBI) has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the polypeptide sequence of an antibody selected from the group consisting of ab231910 (rabbit polyclonal; abcam), ab232760 (rabbit polyclonal; abcam), ab16871 (rabbit polyclonal; abcam), sc-30190 (rabbit polyclonal; Santa Cruz Biotechnology), FNab02256 (rabbit polyclonal; Wuhan Fine Biotech Co), PA5-89139 (rabbit polyclonal; Invitrogen), OTI4A8 (mouse monoclonal; OriGene), OTI6E12 (mouse monoclonal; OriGene), or mAb 7A (mouse monoclonal; Fred Hutch Antibody Technology).

[0078] In some embodiments, the agent that inhibits the activity of DBI (e.g., extracellular DBI) is an aptamer directed against DBI. Aptamers are a class of molecules that represent an alternative to antibodies in terms of molecular recognition. Aptamers are oligonucleotide sequences that have the ability to recognize virtually any class of target molecules with high affinity and specificity. Such ligands can be isolated by Systematic Evolution of Ligands by EXponential enrichment (SELEX) of random sequence libraries. Random sequence libraries can be obtained by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer of unique sequence, which is eventually chemically modified. Peptide aptamers consist of conformationally constrained antibody variable regions displayed by platform proteins, such as E. coli thioredoxin A, selected from combinatorial libraries by a two-hybrid method (Colas et al., 1996).

[0079] In some embodiments, the agent that inhibits expression of DBI is an expression inhibitor. In a preferred embodiment of the invention, the gene expression inhibitor is an siRNA, an endonuclease, an antisense oligonucleotide, or a ribozyme.

[0080] In some embodiments, the agent that inhibits the activity of DBI (e.g., extracellular DBI) is present in a vaccine composition suitable for inducing neutralizing autoantibodies against DBI when administered to a subject. For the purposes of the present invention, the term "vaccine composition" is intended to mean a composition that can be administered to a human or animal to induce an immune system response, which 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 that can be specifically bound by an antibody or a T cell receptor (TCR) when processed and presented by an MHC molecule. As used herein, the term "antigen" also encompasses T cell epitopes. Antigens are further capable of being recognized by the immune system and / or inducing a humoral and / or cellular immune response that leads 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 is present in a polypeptide comprising an amino acid sequence having at least 80% identity to the sequence of SEQ ID NO:1 or a fragment thereof (e.g., an epitope). In some embodiments, the antigen is present in a polypeptide comprising i) an amino acid sequence having at least 80% identity to SEQ ID NO:1, or ii) an amino acid sequence having at least 80% identity to the amino acid sequence spanning from amino acid residue 17 to amino acid residue 50 of SEQ ID NO:1, or iii) an amino acid sequence having at least 80% identity to the amino acid sequence spanning 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 the amino acid sequence spanning from amino acid residue 43 to amino acid residue 50 of SEQ ID NO:1. In some embodiments, the polypeptide is conjugated to a carrier protein that is generally sufficiently exogenous to induce a strong immune response to the vaccine. Exemplary carrier proteins are inherently highly immunogenic.Bovine serum albumin (BSA) and limpet hemocyanin (KLH) are commonly used as carriers in the development of conjugate vaccines during animal testing and are contemplated herein as carrier proteins. Proteins that have been used in the preparation of therapeutic conjugate vaccines include, but are not limited to, several toxins and their toxoids from pathogenic bacteria. Suitable carrier molecules are numerous and include, but are not limited to, bacterial toxins or products, such as cholera toxin B-(CTB), diphtheria toxin, tetanus toxoid, and pertussis toxin, as well as fungal hemagglutinin, Shiga toxin, Pseudomonas exotoxin; lectins, such as ricin B subunit, abrin, and sweet pea lectin; subviruses, such as retroviral nucleoprotein (retroNP), rabies ribonucleoprotein (rabies RNP), plant viruses (e.g., TMV, cowpea, and cauliflower mosaic viruses), vesicular stomatitis virus-nucleocapsid protein (VSV-N), poxvirus vectors, and Semliki Forest virus vectors; artificial vehicles, such as multiple antigenic peptides (MAPs), microspheres; yeast virus-like particles (VLPs); malarial protein antigens; and others, such as proteins and peptides, as well as modifications, derivatives, or analogs of any of the above. Other useful carriers include those capable of enhancing mucosal responses, more particularly the LTB family of bacterial toxins, retroviral nucleoprotein (retroNP), rabies ribonucleoprotein (rabies RNP), vesicular stomatitis virus-nucleocapsid protein (VSV-N), and recombinant poxvirus subunits.

[0081] Pharmaceutical Compositions: Typically, the agent that inhibits the activity or expression of DBI (e.g., extracellular DBI) is administered to a patient in the form of a pharmaceutical composition containing a pharma- ceutical acceptable carrier. Pharmaceutically acceptable carriers that can be used in such 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, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat. When used for administration to a patient, the composition is formulated for administration to a patient. The compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or by implantable reservoir. As used herein, includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. Sterile injectable forms of the compositions of the present invention can be aqueous or oily suspensions. Such suspensions can be formulated using suitable dispersing or wetting agents and suspending agents according to techniques known in the art. Sterile injectable preparations can also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents (e.g., as a solution in 1,3-butanediol). Acceptable vehicles and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as solvents or suspending media. For this purpose any bland fixed oil can 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 pharma- ceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose or similar dispersants commonly used in formulating pharma-ceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tween®, Span®, and other emulsifiers or bioavailability enhancers commonly used in the manufacture of pharma-ceutically acceptable solid, liquid, or other dosage forms, may also be used for formulation purposes. The compositions of the present invention may be orally administered in any orally acceptable dosage form, including, but not limited to, capsules, tablets, aqueous suspensions, or solutions. For tablets intended for oral use, commonly used carriers include lactose and corn starch. Lubricants, such as magnesium stearate, are also typically added. When administered orally in capsule form, useful diluents include, for example, lactose. When an aqueous suspension is required for oral use, the agent that inhibits DBI activity or expression is combined with emulsifying and suspending agents. Certain sweetening, flavoring, or coloring agents may be added as desired. Alternatively, the composition of the present invention may be administered in the form of a suppository for rectal administration. Such suppositories 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 melts in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol. The composition of the present invention may be administered topically, especially when the target of treatment includes areas or organs that are easily accessible by topical application, including diseases of the eye, skin, or lower intestinal tract. Suitable topical formulations are easily prepared for each of these areas or organs. For topical application, the composition can be formulated into a suitable ointment containing the active component suspended or dissolved in one or more carriers.Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax and water. Alternatively, the compositions may be formulated into a suitable lotion or cream containing the active components suspended or dissolved in one or more pharma- ceutically 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 to the lower intestinal tract can be in a rectal suppository formulation (see above) or in a suitable enema formulation. A patch may also be used. The compositions of the present invention 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 a saline solution using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents.

[0082] In some embodiments, the agent that inhibits the activity or expression of DBI (e.g., extracellular DBI) of the present invention is administered directly to the subject or isolated organ using an injection, a pump device, and / or any machine (e.g., a bypass machine). In some embodiments, an isolated organ suitable for transplantation is perfused with a preservation solution containing an effective amount of an agent that inhibits the activity or expression of DBI. As used herein, the term "preservation solution" or "organ preservation solution" refers to an aqueous solution having a pH of 6.5 to 7.5 and containing salts, preferably chloride, sulfate, sodium, calcium, magnesium, and potassium; sugars, preferably mannitol, raffinose, sucrose, glucose, fructose, lactobionate (water-resistant), or gluconate; antioxidants, such as glutathione; active agents, such as xanthine oxidase inhibitors, such as allopurinol, amino acids, such as lactate, histidine, glutamic acid (or glutamate), tryptophan, and optionally a colloid, such as hydroxyethyl starch, polyethylene glycol, or dextran. In some embodiments, a device for preserving an organ is used, the device comprising an organ container filled with a preservation solution, the device further comprising one or more means for injecting one or more compounds (e.g., agents that inhibit DBI activity or expression) into the organ container.

[0083] The present invention is further illustrated by the following figures and examples, which should not be construed as limiting the scope of the invention in any way. [Brief description of the drawings]

[0084] [Figure 1A]Neutralization of ACBP / DBI activates autophagic flux and attenuates organ injury in vivo. A-C: Liver injury caused by 90 min / 4 h ischemia / reperfusion (IR). Mice were pretreated with α-DBI or IgG (2.5 μg / g) and HCQ (50 mg / kg) by intraperitoneal injection 4 h and immediately before IR. Liver injury (A) was assessed by histological examination. Plasma ALT (B) and AST transaminase activities (C) were analyzed by colorimetric assay (n = 5-11 mice per group). [Figure 1B] Neutralization of ACBP / DBI activates autophagic flux and attenuates organ injury in vivo. A-C: Liver injury caused by 90 min / 4 h ischemia / reperfusion (IR). Mice were pretreated with α-DBI or IgG (2.5 μg / g) and HCQ (50 mg / kg) by intraperitoneal injection 4 h and immediately before IR. Liver injury (A) was assessed by histological examination. Plasma ALT (B) and AST transaminase activities (C) were analyzed by colorimetric assay (n = 5-11 mice per group). [Figure 1C] Neutralization of ACBP / DBI activates autophagic flux and attenuates organ injury in vivo. A-C: Liver injury caused by 90 min / 4 h ischemia / reperfusion (IR). Mice were pretreated with α-DBI or IgG (2.5 μg / g) and HCQ (50 mg / kg) by intraperitoneal injection 4 h and immediately before IR. Liver injury (A) was assessed by histological examination. Plasma ALT (B) and AST transaminase activities (C) were analyzed by colorimetric assay (n = 5-11 mice per group). [Figure 2A]α-DBI reduces acetaminophen- and concanavalin A-induced organ toxicity in mice. Injury was induced by acetaminophen (APAP, 300 mg / kg i.p., 16 h) or concanavalin A (ConA, 12 mg / kg i.v., 4 h) in mice pretreated with α-DBI or IgG (2.5 μg / g) and HCQ (50 mg / kg) i.p. injection for 4 h and immediately before liver injury. A-C. Hepatoprotective effect of DBI neutralization after APAP intoxication. Liver injury (A) was measured by histological examination considering the area of ​​cell death, degeneration (ballooning), and inflammation around the central vein. ALT and AST transaminase activity (B and C) from plasma mice (n=4-11 mice per group). D-F. Hepatoprotection from ConA injury by DBI neutralization. Grades of infiltration and hepatocellular necrosis were used to score liver injury (D). Plasma ALT and AST transaminase activities (E and F) (n = 3–11 mice per group). [Figure 2B] α-DBI reduces acetaminophen- and concanavalin A-induced organ toxicity in mice. Injury was induced by acetaminophen (APAP, 300 mg / kg i.p., 16 h) or concanavalin A (ConA, 12 mg / kg i.v., 4 h) in mice pretreated with α-DBI or IgG (2.5 μg / g) and HCQ (50 mg / kg) i.p. injection for 4 h and immediately before liver injury. A-C. Hepatoprotective effect of DBI neutralization after APAP intoxication. Liver injury (A) was measured by histological examination considering the area of ​​cell death, degeneration (ballooning), and inflammation around the central vein. ALT and AST transaminase activity (B and C) from plasma mice (n=4-11 mice per group). D-F. Hepatoprotection from ConA injury by DBI neutralization. Grades of infiltration and hepatocellular necrosis were used to score liver injury (D). Plasma ALT and AST transaminase activities (E and F) (n = 3–11 mice per group). [Figure 2C]α-DBI reduces acetaminophen- and concanavalin A-induced organ toxicity in mice. Injury was induced by acetaminophen (APAP, 300 mg / kg i.p., 16 h) or concanavalin A (ConA, 12 mg / kg i.v., 4 h) in mice pretreated with α-DBI or IgG (2.5 μg / g) and HCQ (50 mg / kg) i.p. injection for 4 h and immediately before liver injury. A-C. Hepatoprotective effect of DBI neutralization after APAP intoxication. Liver injury (A) was measured by histological examination considering the area of ​​cell death, degeneration (ballooning), and inflammation around the central vein. ALT and AST transaminase activity (B and C) from plasma mice (n=4-11 mice per group). D-F. Hepatoprotection from ConA injury by DBI neutralization. Grades of infiltration and hepatocellular necrosis were used to score liver injury (D). Plasma ALT and AST transaminase activities (E and F) (n = 3–11 mice per group). [Figure 2D] α-DBI reduces acetaminophen- and concanavalin A-induced organ toxicity in mice. Injury was induced by acetaminophen (APAP, 300 mg / kg i.p., 16 h) or concanavalin A (ConA, 12 mg / kg i.v., 4 h) in mice pretreated with α-DBI or IgG (2.5 μg / g) and HCQ (50 mg / kg) i.p. injection for 4 h and immediately before liver injury. A-C. Hepatoprotective effect of DBI neutralization after APAP intoxication. Liver injury (A) was measured by histological examination considering the area of ​​cell death, degeneration (ballooning), and inflammation around the central vein. ALT and AST transaminase activity (B and C) from plasma mice (n=4-11 mice per group). D-F. Hepatoprotection from ConA injury by DBI neutralization. Grades of infiltration and hepatocellular necrosis were used to score liver injury (D). Plasma ALT and AST transaminase activities (E and F) (n = 3–11 mice per group). [Figure 2E]α-DBI reduces acetaminophen- and concanavalin A-induced organ toxicity in mice. Injury was induced by acetaminophen (APAP, 300 mg / kg i.p., 16 h) or concanavalin A (ConA, 12 mg / kg i.v., 4 h) in mice pretreated with α-DBI or IgG (2.5 μg / g) and HCQ (50 mg / kg) i.p. injection for 4 h and immediately before liver injury. A-C. Hepatoprotective effect of DBI neutralization after APAP intoxication. Liver injury (A) was measured by histological examination considering the area of ​​cell death, degeneration (ballooning), and inflammation around the central vein. ALT and AST transaminase activity (B and C) from plasma mice (n=4-11 mice per group). D-F. Hepatoprotection from ConA injury by DBI neutralization. Grades of infiltration and hepatocellular necrosis were used to score liver injury (D). Plasma ALT and AST transaminase activities (E and F) (n = 3–11 mice per group). [Figure 2F] α-DBI reduces acetaminophen- and concanavalin A-induced organ toxicity in mice. Injury was induced by acetaminophen (APAP, 300 mg / kg i.p., 16 h) or concanavalin A (ConA, 12 mg / kg i.v., 4 h) in mice pretreated with α-DBI or IgG (2.5 μg / g) and HCQ (50 mg / kg) i.p. injection for 4 h and immediately before liver injury. A-C. Hepatoprotective effect of DBI neutralization after APAP intoxication. Liver injury (A) was measured by histological examination considering the area of ​​cell death, degeneration (ballooning), and inflammation around the central vein. ALT and AST transaminase activity (B and C) from plasma mice (n=4-11 mice per group). D-F. Hepatoprotection from ConA injury by DBI neutralization. Grades of infiltration and hepatocellular necrosis were used to score liver injury (D). Plasma ALT and AST transaminase activities (E and F) (n = 3–11 mice per group). [Figure 3A]ACBP / DBI neutralization attenuates chronic injury-induced fibrosis. A–C. C57BL / 6 mice were subjected to bile duct ligation (BDL) for 2 weeks. Mice were injected intraperitoneally with 2.5 μg / g IgG or α-DBI 4 and 1 h before BDL, and fibrosis scores (A), ALT activity (B), and bilirubin levels (C) were measured twice weekly during BDL (n=5–10 mice per group). D–E. C57BL / 6 mice were injected intraperitoneally with 2.5 μg / g α-DBI or IgG once weekly and 1.6 ml / kg CCl4 twice weekly for 9 weeks. An additional group was treated with HCQ at 50 mg / kg / day for the final 4 weeks of CCl4. Quantification of fibrosis stage (D) and plasma ALT (E) is shown (n=5–14 mice per group). [Figure 3B] ACBP / DBI neutralization attenuates chronic injury-induced fibrosis. A–C. C57BL / 6 mice were subjected to bile duct ligation (BDL) for 2 weeks. Mice were injected intraperitoneally with 2.5 μg / g IgG or α-DBI 4 and 1 h before BDL, and fibrosis scores (A), ALT activity (B), and bilirubin levels (C) were measured twice weekly during BDL (n=5–10 mice per group). D–E. C57BL / 6 mice were injected intraperitoneally with 2.5 μg / g α-DBI or IgG once weekly and 1.6 ml / kg CCl4 twice weekly for 9 weeks. An additional group was treated with HCQ at 50 mg / kg / day for the final 4 weeks of CCl4. Quantification of fibrosis stage (D) and plasma ALT (E) is shown (n=5–14 mice per group). [Figure 3C]ACBP / DBI neutralization attenuates chronic injury-induced fibrosis. A–C. C57BL / 6 mice were subjected to bile duct ligation (BDL) for 2 weeks. Mice were injected intraperitoneally with 2.5 μg / g IgG or α-DBI 4 and 1 h before BDL, and fibrosis scores (A), ALT activity (B), and bilirubin levels (C) were measured twice weekly during BDL (n=5–10 mice per group). D–E. C57BL / 6 mice were injected intraperitoneally with 2.5 μg / g α-DBI or IgG once weekly and 1.6 ml / kg CCl4 twice weekly for 9 weeks. An additional group was treated with HCQ at 50 mg / kg / day for the final 4 weeks of CCl4. Quantification of fibrosis stage (D) and plasma ALT (E) is shown (n=5–14 mice per group). [Figure 3D] ACBP / DBI neutralization attenuates chronic injury-induced fibrosis. A–C. C57BL / 6 mice were subjected to bile duct ligation (BDL) for 2 weeks. Mice were injected intraperitoneally with 2.5 μg / g IgG or α-DBI 4 and 1 h before BDL, and fibrosis scores (A), ALT activity (B), and bilirubin levels (C) were measured twice weekly during BDL (n=5–10 mice per group). D–E. C57BL / 6 mice were injected intraperitoneally with 2.5 μg / g α-DBI or IgG once weekly and 1.6 ml / kg CCl4 twice weekly for 9 weeks. An additional group was treated with HCQ at 50 mg / kg / day for the final 4 weeks of CCl4. Quantification of fibrosis stage (D) and plasma ALT (E) is shown (n=5–14 mice per group). [Figure 3E]ACBP / DBI neutralization attenuates chronic injury-induced fibrosis. A–C. C57BL / 6 mice were subjected to bile duct ligation (BDL) for 2 weeks. Mice were injected intraperitoneally with 2.5 μg / g IgG or α-DBI 4 and 1 h before BDL, and fibrosis scores (A), ALT activity (B), and bilirubin levels (C) were measured twice weekly during BDL (n=5–10 mice per group). D–E. C57BL / 6 mice were injected intraperitoneally with 2.5 μg / g α-DBI or IgG once weekly and 1.6 ml / kg CCl4 twice weekly for 9 weeks. An additional group was treated with HCQ at 50 mg / kg / day for the final 4 weeks of CCl4. Quantification of fibrosis stage (D) and plasma ALT (E) is shown (n=5–14 mice per group). [Figure 4A] Neutralization of ACBP / DBI attenuates fatty liver induced by a methionine-choline-deficient diet. A-D. Male mice aged 2-3 months were fed a normal diet (RCD) or a methionine-choline-deficient diet (MCD) for 4 weeks. Hepatic F4 / 80 macrophages (A), NAFLD activity score (B), ALT and AST activities (C), and p62 levels (D) were measured. [Figure 4B] Neutralization of ACBP / DBI attenuates fatty liver induced by a methionine-choline-deficient diet. A-D. Male mice aged 2-3 months were fed a normal diet (RCD) or a methionine-choline-deficient diet (MCD) for 4 weeks. Hepatic F4 / 80 macrophages (A), NAFLD activity score (B), ALT and AST activities (C), and p62 levels (D) were measured. [Figure 4C] Neutralization of ACBP / DBI attenuates fatty liver induced by a methionine-choline-deficient diet. A-D. Male mice aged 2-3 months were fed a normal diet (RCD) or a methionine-choline-deficient diet (MCD) for 4 weeks. Hepatic F4 / 80 macrophages (A), NAFLD activity score (B), ALT and AST activities (C), and p62 levels (D) were measured. [Figure 4D]Neutralization of ACBP / DBI attenuates fatty liver induced by a methionine-choline-deficient diet. A-D. Male mice aged 2-3 months were fed a normal diet (RCD) or a methionine-choline-deficient diet (MCD) for 4 weeks. Hepatic F4 / 80 macrophages (A), NAFLD activity score (B), ALT and AST activities (C), and p62 levels (D) were measured. [Figure 5A] Neutralization of ACBP / DBI attenuates fatty liver induced by Western diet. A-B. Male mice aged 2-3 months were fed a normal diet (RCD) or a high-fat Western diet + sugar for 4 weeks. NAFLD activity score (A), and ALT and AST activities (B) were measured. [Figure 5B] Neutralization of ACBP / DBI attenuates fatty liver induced by Western diet. A-B. Male mice aged 2-3 months were fed a normal diet (RCD) or a high-fat Western diet + sugar for 4 weeks. NAFLD activity score (A), and ALT and AST activities (B) were measured. [Figure 6A] Neutralization of ACBP / DBI attenuates fatty liver induced by Western diet and CCl4. A-B. Male mice aged 2-3 months were fed a normal diet (RCD) or a high-fat Western diet + sugar + CCl4 for 4 weeks. NAFLD activity score (A), and ALT and AST activities (B) were measured. [Figure 6B] Neutralization of ACBP / DBI attenuates fatty liver induced by Western diet and CCl4. A-B. Male mice aged 2-3 months were fed a normal diet (RCD) or a high-fat Western diet + sugar + CCl4 for 4 weeks. NAFLD activity score (A), and ALT and AST activities (B) were measured. [Figure 7] Neutralization of ACBP / DBI attenuates fibrosis induced by Western diet and CCl4. C57BL / 6 mice were fed a normal diet (RCD) or a high-fat Western diet + sugar + CCl4 for 4 weeks. Mice were injected intraperitoneally with 2.5 μg / g α-DBI or IgG once a week and 1.6 ml / kg CCl4 twice a week for 9 weeks. Quantification of fibrosis scores is shown (n=5-14 mice per group). [Figure 8A]ACBP / DBI neutralization reverses CCl4-induced fibrosis. Mice were administered CCl4 for 9 weeks, then treated with vehicle (oil) and allowed to recover for 4 weeks (R). 2.5 μg / g IgG or α-DBI was injected intraperitoneally 1 day before recovery and once a week during R. Quantification of fibrosis stage (A), plasma ALT activity (B), and hydroxyproline levels (C) were measured (n = 4-12 mice per group). [Figure 8B] ACBP / DBI neutralization reverses CCl4-induced fibrosis. Mice were administered CCl4 for 9 weeks, then treated with vehicle (oil) and allowed to recover for 4 weeks (R). 2.5 μg / g IgG or α-DBI was injected intraperitoneally 1 day before recovery and once a week during R. Quantification of fibrosis stage (A), plasma ALT activity (B), and hydroxyproline levels (C) were measured (n = 4-12 mice per group). [Figure 8C] ACBP / DBI neutralization reverses CCl4-induced fibrosis. Mice were administered CCl4 for 9 weeks, then treated with vehicle (oil) and allowed to recover for 4 weeks (R). 2.5 μg / g IgG or α-DBI was injected intraperitoneally 1 day before recovery and once a week during R. Quantification of fibrosis stage (A), plasma ALT activity (B), and hydroxyproline levels (C) were measured (n = 4-12 mice per group). EXAMPLES

[0085] Example 1: Materials and Methods Chemicals and Reagents Reagents were obtained from Axon Medchem BV (Groningen, Netherlands), Qiagen (Hilden, Germany), Millipore (MA, USA), Randox (Antrim, UK), Roche Applied Science (Upper Bavaria, Germany), and Sigma Aldrich (MO, USA). Electrophoresis reagents were obtained from Thermo Fisher Scientific (MA, USA) and BioRad (CA, USA). Antibodies were obtained from Abcam (TX, USA), Abnova (Taipei, Taiwan), Cell Signaling (MA, USA), and Sigma Aldrich.

[0086] Animal testing Wild-type (Wt) C57BL / 6 mice (Envigo, Gannat, France), homozygous Atg4b - / - Mice (kindly provided by Dr. Carlos Lopez-Otin; University of Oviedo, Spain), tamoxifen-inducible whole-body knockout floxed Acbp / Dbi - / - Mouse (UBC-cre / ERT2::Acbp / Dbi fl / fl , Control: Acbp / Dbi fl / fl (no CRE) (Bravo-San Pedro et al., 2019a), homozygous Gabrg2 mut / mutMice (harboring a point mutation F77I within the binding site of ACBP / DBI of the gamma-aminobutyric acid A receptor γ2 subunit) (Wulff et al., 2007) and transgenic mice expressing LC3 conjugated to green fluorescent protein (GFP-LC3-Tg) (Mizushima et al., 2004) were bred and maintained in accordance with FELASA guidelines and local guidelines from the Animal Experimental Ethics Committee (permission numbers 25000, 31411, 34537, 34538, and 34539). Mice were kept in a temperature-controlled environment with a 12-h light / dark cycle and had free access to food and water. All animals were sacrificed and organs were snap frozen in liquid nitrogen and stored at −80°C or fixed overnight in 4% buffered paraformaldehyde at 4°C and embedded in paraffin. Plasma was obtained by cardiac puncture.

[0087] Neutralization of DBI by passive or active immunization Monoclonal antibodies against DBI (passive immunization) or isotype IgG (Bioxcell, NH, USA) were used in vivo (2.5 μg / g body weight (BW), intraperitoneally (ip), in 200 μL) in single or multiple doses. In some experiments, leupeptin (Leu, 30 mg / kg body weight) was injected intraperitoneally 2 h before the end of the experiment.

[0088] Autoantibody production (active immunization) was induced by conjugation of limpet hemocyanin (KLH; Thermo) and mouse recACBP (KLH-DBI) as described by Montegut et al. (Montegut et al., 2022). Briefly, KLH and DBI were mixed in a 1:20 molar ratio and gradually adjusted to 0.25% (v / v) glutaraldehyde. The reaction was then terminated by adding glycine solution and ultrafiltration using a 100 KDa membrane (Millipore). Formaldehyde solution was added to a final concentration of 0.2% (v / v) and the reaction was quenched by adding glycine solution followed by ultrafiltration with 70 mM pH 7.8 phosphate buffer. Eight-week-old male C57BL / 6 mice were immunized by intraperitoneal injection with 30, 30, 30, or 10 μg of KLH-DBI or KLH alone as an adjuvant emulsion (1:1) along with Montanide ISA-51vg (Seppic, Paris, France) on days 0, 7, 14, and 21, respectively.

[0089] Acute liver injury in mice To induce hepatic ischemia-reperfusion injury, 12-week-old male C57BL / 6 mice were anesthetized with 2% isofluorane to evaluate a model of segmental (70%) warm hepatic I / R protocol (Motino et al., 2019). Briefly, hepatic ischemia was induced for 90 min and reperfusion was initiated by removing the clamp for 4 h. To induce hepatotoxicity, 12-week-old male C57BL / 6 mice were treated with 12 mg / kg concanavalin A (ConA; Sigma Aldrich) or 300 mg / kg acetaminophen (APAP; Sigma Aldrich) for 4 h or 16 h, respectively. To inhibit autophagic flux, animals were injected intraperitoneally with two doses of 50 mg / kg hydroxychloroquine (HCQ, in PBS; Axon Medchem BV) 4 h before and immediately before liver injury.

[0090] In vivo liver fibrosis model To induce liver fibrosis, CCl4 (Sigma Aldrich) was administered intraperitoneally to 2-month-old male C57BL / 6 mice at a dose of 1.6 ml / kg twice weekly for 9 weeks (Motino et al., 2016). Control animals were injected intraperitoneally with the vehicle olive oil (Sigma Aldrich). An additional group received 50 mg / kg HCQ intraperitoneally daily for the final 4 weeks of CCl4. Another approach to induce liver fibrosis involves bile duct ligation (BDL) for 2 weeks (Tag et al., 2015).

[0091] Biochemical assays Serum ALT and AST activities were quantified using colorimetric kits (Randox) according to the manufacturer's instructions. To quantify collagen, liver hydroxyproline content was assayed by a commercial kit (Sigma Aldrich).

[0092] Histopathological examination Paraffin-embedded sections (5 μm) were stained with hematoxylin-eosin-safranin (HES) or Sirius Red and evaluated by an experienced pathologist blinded to the characteristics of the animal groups. All slides were scanned with an AxioScan Z1 (Carl Zeiss, Jena, Germany). NAFLD activity scores were assessed using the NAFLD scoring system for mouse models validated by Liang et al. (Liang et al., 2014). Briefly, steatosis grades were classified as follows: grade 0: <5% steatotic hepatocytes; grade 1: 5–33%; grade 2: 33–66%; grade 3: >66%. Lobular inflammation was scored as follows: 0: no foci; 1: <2 foci; 2: 2–4 foci; 3: >4 foci. Ballooning was classified as 0: none, 1: few balloon cells, and 2: many balloon cells. NAFLD activity scores were calculated for each liver biopsy based on the sum of the scores of steatosis, inflammation, and ballooning. In addition, liver fibrosis staging (Metavir score) was defined as 0: absent, 1: perisinusoidal and / or pericentral, 2: incomplete central / central bridging fibrosis, 3: complete central / central bridging fibrosis, and 4: overt cirrhosis (Bedossa and Poyard, 1996). The severity of hepatic IR was graded on a scale from 0 to 4 according to Suzuki's criteria. For necrosis, congestion, or centro-lobular ballooning, none (0%), minimal (10%), mild (11-30%), moderate (30-60%), and severe (>60%) were assigned grades 0, 1, 2, 3, and 4, respectively (Suzuki et al., 1993). To measure the hepatotoxicity of APAP, liver samples were classified into none (0: 0%), mild (1: less than 20%), moderate (2: 20-70%), and severe (3: more than 70% of the hepatic lobule) taking into account areas of cell death, ballooning, and inflammation around the central vein (Naiki-Ito et al., 2010). The following grades were used to score the liver injury induced by ConA:0: no necrotic infiltration, 1: small necrotic cell foci between hepatocytes or surrounding individual hepatocytes, 2: large necrotic cell foci containing 100 necrotic cells or 30 hepatocytes, 3: involving 10% of the liver cross section, and 4: involving 30% of the liver cross section (Zhao et al., 2020). To quantify the abundance of hepatic macrophages and Kupffer cells, liver sections from fixed paraffin blocks were immunohistochemically stained using anti-mouse F4 / 80 according to standard procedures.

[0093] Liver extract For protein or RNA extraction, tissues were homogenized in 20 mM Tris buffer (pH 7.4) containing 150 mM NaCl, 1% Triton® X-100, 10 mM EDTA, and Complete® protease inhibitor cocktail (Roche Applied Science), or QIAzol (Qiagen), respectively, using a Precellys 24 tissue homogenator (Bertin Technologies, Montigny-le-Bretonneux, France) at 5,500 rpm for 20 s for two cycles. Protein extracts were then centrifuged at 12,000 g (4°C) for 15 min, and the supernatants were collected. Protein concentrations in the supernatants were assessed by the bicinchoninic acid technique (BCA protein assay kit; Thermo Fisher Scientific). Homogenate RNA was purified using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. RNA purity and concentration were determined by NanoDrop™ (Thermo Fisher Scientific).

[0094] Data analysis Data are expressed as mean ± SEM. For statistical analysis, normal distribution of results was first assessed by D'Agostino & Pearson normality test and Shapiro-Wilk normality. Statistical significance was analyzed using Student's unpaired two-tailed t-test or unpaired two-tailed Mann-Whitney test to evaluate differences between treated and untreated mice within a single genotype and between genotypes. Analyses were performed by using the statistical software GraphPad Prism 5. Statistical analysis of whole transcriptome sequencing and GEO datasets was tested by Fisher's exact test. For metabolomic statistical analysis, p-values ​​were calculated by Mann-Whitney test. All target treatment data were merged and cleaned by a dedicated R (version 3.4) package (@Github / Kroemerlab / GRMeta). p<0.05 was considered statistically significant.

[0095] Example 2: Organ protective effect of ACBP / DBI neutralization against acute injury. Injection of a monoclonal antibody (mAb) neutralizing ACBP / DBI (α-DBI) (2.5 μg / g, intraperitoneally (i.p.), 6 and 2 h before sacrifice) enhanced hepatic lipidation of the autophagy marker microtubule-associated protein 1A / 1B light chain 3B (hereafter referred to as LC3B), resulting in the electrophoretically more mobile LC3-II form (data not shown) (Mizushima et al., 2004). This effect was further enhanced by injection of the lysosomal protease inhibitor leupeptin (30 mg / kg, i.p., 2 h before sacrifice), confirming the increase in autophagic flux (Haspel et al., 2011) (data not shown). Correspondingly, a single injection of α-DBI (2.5 μg / g, i.p., 4 h before sacrifice) induced the formation of autophagic puncta in hepatocytes of mice expressing a transgene encoding a green fluorescent protein (GFP)-LC3 fusion protein (Mizushima et al., 2004) (data not shown), and two injections of α-DBI reduced histological signs of hepatic ischemia / reperfusion (congestion, ballooning, and necrosis summarized by the Suzuki score) (Suzuki et al., 1993) (Fig. 1A) and also reduced the increase in plasma concentrations of two transaminases, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (Fig. 1B, C). Combining α-DBI injection with hydroxychloroquine (50 mg / kg), a lysosomotropic agent that inhibits autophagy in vivo (Cook et al., 2014), abolished the hepatoprotective effect of ACBP / DBI neutralization on ischemia / reperfusion (Figure 1A-C). A similar hydroxychloroquine-inhibitory hepatoprotective effect of α-DBI was obtained in two models of pharmacological hepatotoxicity induced by acetaminophen (APAP, trade name paracetamol) and the lectin concanavalin A (ConA) (Figure 2). In both models, α-DBI reduced histological signs of liver injury and circulating transaminase levels (Figure 2).

[0096] Example 3: ACBP / DBI neutralization inhibits fibrosis. We used a model in which liver fibrosis was induced by bile duct ligation (BLD) (Brea et al., 2018). Two weeks after BLD, liver injury and fibrosis were prominent in mice treated with isotype control antibody, but were significantly attenuated after biweekly injections of α-DBI (Figure 3A-C). Similar results were obtained in a well-established model of carbon tetrachloride (CCl4)-induced liver fibrosis, which was modulated by weekly administration of α-DBI (or isotype IgG control for 9 weeks) and / or daily injections of hydroxychloroquine (or vehicle control for the final 4 weeks of the experiment). In this model, α-DBI attenuated weight loss, signs of fibrosis detectable by Sirius Red staining (Fig. 3D) or quantification of the collagen-rich amino acid hydroxyproline (data not shown), as well as liver injury reflected by transaminase activity (Fig. 3E) and immunoblot detection of the profibrotic markers collagen 1A1 and α-smooth muscle actin (α-SMA) (data not shown). The beneficial effects of α-DBI on liver injury and fibrosis were lost when autophagy was inhibited by hydroxychloroquine (Fig. 3D, E). CCl4-induced alterations in p62 and LC3-II were restored by α-DBI, but only in the absence, but not in the presence, of hydroxychloroquine (data not shown). α-DBI also restored CCl4-induced elevations in circulating transaminases, again only in the absence, of hydroxychloroquine (Fig. 3E). Moreover, α-DBI reversed most, if not all, of the transcriptional effects of chronic CCl4 intoxication, reducing the expression of profibrotic, proinflammatory, macrophage-associated, or transforming growth factor-β (TGF-β)-associated genes, but enhancing the expression of antioxidant enzymes. These transcriptional effects of α-DBI were abolished when hydroxychloroquine was coadministered (data not shown). In a further series of experiments, we determined whether CCl4-induced liver fibrosis could be reversed more efficiently when CCl4 withdrawal was combined with weekly α-DBI injections for 4 weeks (data not shown). In this curative setting, too, α-DBI reduced signs of liver fibrosis (data not shown).Taken together, these data indicate that ACBP / DBI neutralization has beneficial effects on liver fibrosis, and that these effects are critically dependent on autophagy.

[0097] Example 4: ACBP / DBI attenuates diet-induced hepatic steatosis. Diet-induced hepatic steatosis was used to model the treatment of nonalcoholic fatty liver disease (NAFLD). First, the effect of ACBP / DBI neutralization on a NASH model that occurs in association with weight loss as a result of a methionine-choline-deficient diet (MCD) (control: normal diet (RCD)). NASH features were evaluated after MCD administration over the course of 4 weeks in mice injected once a week with α-DBI (control: isotype immunoglobulin G [IgG] mAb). α-DBI reduced inflammation associated with NASH, as measured by reduction in F4 / 80 macrophages in the liver (Figure 4A), and largely prevented histological signs (quantified by NAFLD activity score measured as the sum of steatosis, inflammation, and ballooning: Figure 4B) as well as enzymatic signs (ALT and AST) of NASH induced by MCD (Figure 4C). Furthermore, α-DBI restored the inhibition of autophagic flux associated with NASH. This is evidenced by the reduction of the autophagy substrate p62 in α-DBI-treated mice compared with controls.

[0098] Next, the effect of ACBP / DBI neutralization on a NASH model occurring in subjects consuming a high-fat Western diet was quantified. NASH characteristics were evaluated after a 4-week course of Western diet in mice administered weekly injections of α-DBI (control: isotype immunoglobulin G [IgG] mAb). In addition, another group of mice fed the Western diet received intraperitoneal administration of CCl4 to induce fibrosis. Similar to MCD-induced NASH, α-DBI largely prevented the histological (quantified by NAFLD activity score: Fig. 5A) and enzymatic (ALT and AST) signs of NASH induced by the Western diet (Fig. 5B). Furthermore, administration of α-DBI reduced the NAFLD activity score (Fig. 6A) and enzymatic (ALT and AST: Fig. 6B) signs of NASH in Western diet-fed CCl4-treated mice. Furthermore, administration of α-DBI reduced CCl4-associated fibrosis in Western diet-fed mice, as evidenced by reduced fibrosis scores (Figure 7).

[0099] Finally, the ability of α-DBI to reverse liver fibrosis induced by CCl4 was determined. To induce liver fibrosis, 2-month-old male C57BL / 6 mice were intraperitoneally administered CCl4 (Sigma Aldrich) at a dose of 1.6 ml / kg twice weekly for 9 weeks, followed by treatment with vehicle (oil) for 4 weeks of recovery (R). 2.5 μg / g IgG or α-DBI was intraperitoneally injected 1 day before recovery and once weekly during R. Control animals were intraperitoneally injected with the vehicle olive oil (Sigma Aldrich). In this curative setting, α-DBI also reduced signs of liver fibrosis injury, as determined by reductions in fibrosis score (Figure 8A), ALT activity (Figure 8B), and hydroxyproline content (Figure 8C).

[0100] Taken together, ACBP / DBI neutralization by α-DBI reduced NASH-associated hepatic steatosis and liver fibrosis in various models and could even reverse post-formation liver fibrosis, further demonstrating the therapeutic potential of ACBP / DBI neutralization by α-DBI.

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Claims

1. A composition for use in treating tissue damage caused by chemical, physical, or ischemic injury in an organ of a subject, comprising an agent that inhibits the activity or expression of extracellular diazepam binding inhibitor (DBI), the agent being in an amount sufficient, when administered to the subject, to treat the tissue damage caused by chemical, physical, or ischemic injury in the organ of the subject.

2. Alcohol, 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2-bromoethylamine (BEA), 3-methylcholanthrene, 4-aminophenol (PAP), acetaminophen, adriamycin, allyl alcohol, amiodarone, amphotericin B, Aroclor 1254, Aroclor 1260, arsenic, aspirin, astemizole, benzene, cadmium, carbamedipine, carbon tetrachloride (CCl4), ciprofibrate (Cip (b), clofibrate, cobalt chloride, colvastatin, cyclosporin A, diethylnitrosamine, dimethylformamide, dimethylhydrazine (DMH), diquat, ethosuximide, etoposide, famotidine, fluconazole, gemfibrozil, ganciclovir, hexachloro-1,3-butedien (HCBD), HIV protease inhibitors, hydrazine, indomethacin, ketoconazole, lead acetate (PbAc), lipopolysaccharide (LPS), mercury(II) chloride (HgCl2), methanol, methapyrilene, methotrexate, metronidazoline ethanol, miconazole, monocrotaline, nitric oxide, ondansetron, pentamidine, phenobarbital, phenylhydrazine (phenylhyrzn), phenytoin, pravastatin, proprusside, puromycin aminonucleoside (PAN), quinolones, simvastatin, sodium fluoride (NaF), statins, thioacetamide, tocainidine, tricyclic antidepressants, troglitazone, tumor necrosis factor alpha (TNFα), uranyl nitrate, valproic acid, vincristine, Wy-16,463, zidovudine (AZT), α-naphthylisothiazolinone 10. The composition for use according to claim 1, for use in treating tissue damage caused by chemical insult induced by a toxin selected from the group consisting of nitrates (ANIT), β-naphthoflavone (BNF), asbestos, radon, tobacco smoke, adhesives, dioxin, nickel, arsenic, mercury, cement (chromium), polychlorinated biphenyls (PCBs), carbon tetrachloride, methylene chloride, vinyl chloride, mercury, chlorinated hydrocarbon solvents, carbon disulfide, cadmium, ozone, tobacco smoke, nitrates, methylene chloride, ethylene dibromide, and polychlorinated biphenyls.

3. 2. The composition for use according to claim 1 for use in treating tissue damage caused by chemotherapeutic agents.

4. 4. The composition for use of claim 3, wherein the chemotherapeutic agent is an anthracycline selected from the group consisting of daunorubicin, doxorubicin, epirubicin, farmorubicin, idarubicin, mitoxantrone, pixantrone, and pharmaceutically acceptable salts thereof.

5. 2. The composition for use of claim 1, wherein the agent that inhibits DBI activity or expression is present in an amount sufficient to treat acetaminophen-induced hepatotoxicity, amiodarone-induced pulmonary toxicity, doxorubicin-induced cardiotoxicity, cadmium chloride-induced nephrotoxicity, dimethylnitrosamine-induced splenotoxicity, and O-ethyl-S,S-dipropylphosphorodithioate (MOCAP)-induced neurotoxicity.

6. 2. The composition for use according to claim 1, wherein the ischemic injury is a myocardial infarction or the subject is suffering from ischemia-reperfusion injury.

7. 2. The composition of claim 1, wherein the agent that inhibits DBI activity or expression is present in an amount sufficient to reduce or delay the progression of acute kidney injury (AKI) to chronic kidney disease (CKD).

8. A composition for use in reducing the severity of injury in an isolated organ, comprising an agent that inhibits the activity or expression of extracellular diazepam binding inhibitor (DBI) in an amount sufficient to reduce the severity of the injury in the isolated organ when contacted with the isolated organ.

9. 9. The composition for use according to claim 8, wherein the isolated organ is a transplantable organ.

10. A composition for use in reducing fibrosis associated with organ injury in a subject in need thereof, comprising an agent that inhibits the activity or expression of extracellular diazepam binding inhibitor (DBI), the agent being in an amount sufficient, upon administration, to reduce the amount of fibrosis associated with organ injury in the subject's organ compared to the amount of fibrosis in the organ in the absence of said administration.

11. 11. The composition for use according to claim 10, wherein the fibrosis occurs in an organ selected from the group consisting of the skin, heart, liver, lungs, and kidneys.

12. The composition for use according to claim 11, wherein the organ is the liver.

13. A composition for use as described in claim 12, wherein the agent that inhibits the activity or expression of extracellular DBI is present in an amount sufficient to reduce the level of transaminase in the subject when administered compared to the level of transaminase in the subject before administration.

14. 14. The composition for use according to claim 13, wherein the transaminase is aspartate transaminase (AST) or alanine transaminase (ALT).

15. The composition for use of claim 12, wherein the agent that inhibits the activity or expression of extracellular DBI is present in an amount sufficient to reduce the fibrosis score by at least one-third of the fibrosis score before administration.

16. The composition for use of claim 12, wherein the agent that inhibits the activity or expression of extracellular DBI is present in an amount sufficient to reduce the level of hydroxyproline in the subject when administered compared to the level of hydroxyproline in the subject prior to said administration.

17. 13. The composition for use according to claim 12, wherein the fibrosis is associated with non-alcoholic fatty liver disease (NAFLD).

18. A composition for use as described in claim 17, wherein the agent that inhibits the activity or expression of extracellular DBI is present in an amount sufficient, when administered, to reduce the NAFLD score in the subject relative to the NAFLD score before administration.

19. The composition for use of claim 1, wherein the agent that inhibits the activity of extracellular DBI is an antibody or aptamer directed against DBI.

20. 20. The composition for use according to claim 19, wherein the antibody is directed against a polypeptide fragment of DBI comprising the amino acid sequence of positions 43 to 50 of SEQ ID NO:

1.

21. 20. The composition for use of claim 19, wherein the antibody is a monoclonal chimeric antibody, a monoclonal humanized antibody, or a monoclonal human antibody.

22. The composition for use of claim 1, wherein the agent that inhibits the expression of extracellular DBI is an expression inhibitor selected from the group consisting of siRNA, endonucleases, antisense oligonucleotides, and ribozymes.

23. A composition for use as described in claim 1, wherein the agent that inhibits the activity of extracellular DBI is present as a vaccine composition suitable for inducing neutralizing autoantibodies against DBI when administered to the subject.

24. 24. The composition for use of claim 23, wherein the vaccine composition comprises an antigen comprising: (i) an amino acid sequence having at least 80% identity to SEQ ID NO: 1; (ii) an amino acid sequence having at least 80% identity to the amino acid sequence of positions 17 to 50 of SEQ ID NO: 1; (iii) an amino acid sequence having at least 80% identity to the amino acid sequence of positions 33 to 50 of SEQ ID NO: 1; or (iv) an amino acid sequence having at least 80% identity to the amino acid sequence of positions 43 to 50 of SEQ ID NO:

1.

25. A composition comprising an agent that inhibits the activity or expression of extracellular diazepam binding inhibitor (DBI), wherein when administered to a subject, the composition contains the agent in an amount sufficient to treat tissue damage in the liver of the subject caused by chemical injury, physical injury, or ischemic injury.

26. A composition comprising an agent that inhibits the activity or expression of extracellular diazepam binding inhibitor (DBI), wherein when administered to a subject, the composition contains the agent in an amount sufficient to treat tissue damage in the subject's heart caused by chemical injury, physical injury, or ischemic injury.

27. ​​A composition comprising an agent that inhibits the activity or expression of extracellular diazepam binding inhibitor (DBI), wherein the composition, when administered, contains an amount of the agent sufficient to reduce the amount of fibrosis associated with organ injury in a subject compared to the amount of fibrosis in the absence of said administration.

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