WO2025134073A2

WO2025134073A2 - Methods and compositions for treating diseases or conditions induced by corticosteroids, synovial inflammation, and degenerative joint disease

Info

Publication number: WO2025134073A2
Application number: PCT/IB2024/063072
Authority: WO
Inventors: Flavia LAMBERTUCCI; Hui Pan; Isabelle MARTINS; Guido Kroemer; Uxia NOGUEIRA RECALDE; Oliver Kepp
Original assignee: Institut Gustave Roussy (IGR); Assistance Publique Hopitaux de Paris APHP; Institut National de la Sante et de la Recherche Medicale INSERM; Sorbonne Universite; Universite Paris Cite; Universite Paris Saclay
Current assignee: Institut Gustave Roussy (IGR); Assistance Publique Hopitaux de Paris APHP; Institut National de la Sante et de la Recherche Medicale INSERM; Sorbonne Universite; Universite Paris Cite; Universite Paris Saclay
Priority date: 2023-12-22
Filing date: 2024-12-20
Publication date: 2025-06-26
Anticipated expiration: 2026-06-22
Also published as: WO2025134073A3

Abstract

The present disclosure provides improved methods, therapeutic regimens, and systems for treating a disease associated with a pituitary malignancy in a subject, such as treating Cushing Syndrome, or treating one or more symptoms of Cushing Syndrome in subjects, through the inhibition of extracellular diazepam binding inhibitor (DBI) by various methods. The present disclosure also relates to compositions for use and methods in treating synovial inflammation in a subject, the compositions for use and methods comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression. The present disclosure also relates to compositions for use and methods in treating degenerative joint disease, joint inflammation, and manifestations thereof in a subject, the compositions for use and methods comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression.

Description

METHODS AND COMPOSITIONS FOR TREATING DISEASES OR CONDITIONS INDUCED BY CORTICOSTEROIDS, SYNOVIAL INFLAMMATION, AND DEGENERATIVE JOINT DISEASE

CROSS REFERENCE TO RELATED APPLICATIONS

[001] This application claims priority to European Application No. 23220140.0, filed on 22 December 2023 and European Application No. 24150766.4, filed on 8 January 2024, which are each incorporated herein by reference in their entirety.

TECHNICAL FIELD

[002] The present disclosure relates to compositions, methods, therapeutic regimens, and systems for treating diseases associated with corticosteroids in a subject, such as Cushing syndrome, through the inhibition of diazepam binding inhibitor (DBI), for example by reducing extracellular DBI activity or expression. The present disclosure also relates to compositions, methods, therapeutic regimens, and systems for treating synovial joint inflammation, arthropathy, degenerative joint disorder, and osteoarthritis through the inhibition of diazepam binding inhibitor (DBI), for example by reducing extracellular DBI activity or expression.

BACKGROUND

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

[004] Cushing syndrome results from the chronic hyperactivation of glucocorticoid receptors, usually for several months, and is marked by a characteristic phenotype that allows experienced clinicians to diagnose the condition at a glance. In addition, Cushing syndrome is accompanied by a metabolic syndrome including dyslipidemia (mostly triglyceridemia), insulin resistance, hyperglycemia, and arterial hypertension, sometimes culminating in death due to atherosclerotic disease, cardiac failure, or thromboembolism. Furthermore, immunosuppression may increase the susceptibility to severe infections. Endogenous Cushing syndrome, which is often diagnosed with a significant delay (mean delay to diagnosis: 34 months), can occur as a result of a pituitary adenoma, which may lead to excessive production of endogenous glucocorticoids. Exogenous or iatrogenic Cushing syndrome may result from long-term treatment with synthetic glucocorticoids in a subject to control conditions such as chronic asthma, rheumatoid arthritis, lupus, sarcoidosis, and other severe inflammatory conditions. There remains a need to provide new and improved therapies that treat diseases associated with pituitary malignancies and diseases associated with excess corticosteroid such as Cushing syndrome, including therapies that treat the manifestations of the diseases.

BRIEF SUMMARY

[005] Disclosed herein is a composition for use in treating a disease associated with a pituitary malignancy or treatment with synthetic glucocorticoids in a subject, the composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression in an amount sufficient to treat the disease associated with the pituitary malignancy upon administering to the subject. In some embodiments, the disease is associated with a pituitary adenoma. In some embodiments, the amount is sufficient to treat a symptom of the disease. In some embodiments, the agent reduces DBI activity, relative to an amount of activity by administering a composition lacking the agent that reduces DBI activity. In some embodiments, the agent comprises a DBI- binding polypeptide. In some embodiments, the DBI-binding polypeptide is an anti-DBI antibody. In some embodiments, the anti-DBI antibody is a monoclonal antibody. In some embodiments, the anti-DBI antibody is a polyclonal antibody. In some embodiments, the DBI-binding polypeptide is an anti-DBI antibody fragment. In some embodiments, the DBI-binding polypeptide is an antibody fragment comprising a single chain Fv, Fab’ fragment, or nanobody. In some embodiments, the agent comprises a polypeptide antigen that induces production of a neutralizing anti-DBI antibody. In some embodiments, the agent reduces extracellular DBI expression in the subject, relative to an amount of DBI expression absent the administering. In some embodiments, the agent comprises a thyroid hormone. In some embodiments, the thyroid hormone comprises 3,3',5-triiodo-L-thyronine (T3). In some embodiments, the agent comprises a thyroid hormone receptor agonist. In some embodiments, the thyroid hormone receptor agonist comprises resmetirom or other agonists of the thyroid hormone receptor beta. In some embodiments, the agent is an siRNA, an endonuclease, an antisense oligonucleotide, proteolysis-targeting chimeras (PROTACs), or a ribosome. In some embodiments, the agent is the siRNA.

[006] Disclosed herein is a composition for use in treating symptoms or disorders associated with elevated levels of steroids in a subject, the composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression in the subject in an amount sufficient to treat the symptoms or disorders associated with the elevated levels of steroids in the subject upon administering to the subject, and wherein the symptoms or disorders are selected from the group consisting of: adipocyte hypertrophy, increased liver weight, an increase in circulating liver enzymes, an increase in alanine aminotransferase (ALT), an increases in aspartate aminotransferase (AST), an increase in liver triglycerides (TG), a hypertrophy of white adipose tissue (WAT), a hypertrophy of brown adipose tissue (BAT), a metabolic syndrome, an arterial hypertension dyslipidemia, a triglyceridemia, a round “moon” face that comprises capillary vasodilatation, a skin acne, a facial hirsutism, a cranial alopecia, a skin atrophy, a central obesity, a “buffalo hump” lipodystrophy, a profuse striae, and a sarcopenia. In some embodiments, the elevated levels of steroids are acute elevated levels. In some embodiments, the acute elevated levels are a result of administration of glucocorticoids. In some embodiments, the elevated levels of steroids are chronic elevated levels.

[007] Disclosed herein is a method of treating a disease (or a composition for use in such a method) associated with elevated levels of steroids in a subject, the method comprising administering to the subject a composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression, wherein the administering is sufficient to treat the disease associated with the elevated expression of steroids in the subject. In some embodiments, the disease is associated with elevated levels of cortisol in the subject. In some embodiments, the disease is an endogenous Cushing syndrome. In some embodiments, the endogenous Cushing syndrome is Cushing disease. In some embodiments, the disease is associated with chronic use of steroids by the subject. In some embodiments, the disease is an iatrogenic Cushing syndrome. In some embodiments, the iatrogenic Cushing syndrome is induced by corticosteroids. In some embodiments, the corticosteroids are co-administered with T3 or resmetirom. In some embodiments, the agent reduces DBI activity, relative to an activity level prior to the administering. In some embodiments, agent comprises a DBI-binding polypeptide. In some embodiments, the DBI-binding polypeptide is an anti-DBI antibody. In some embodiments, the anti-DBI antibody is a monoclonal antibody. In some embodiments, the anti-DBI antibody is a polyclonal antibody. In some embodiments, the DBI-binding polypeptide is an anti-DBI antibody fragment. In some embodiments, the DBI-binding polypeptide is an antibody fragment comprising a single chain Fv, Fab’ fragment, or nanobody. In some embodiments, the agent comprises a polypeptide antigen that induces production of a neutralizing anti-DBI antibody. In some embodiments, the agent reduces extracellular DBI expression, relative to an expression level prior to the administering. In some embodiments, the agent comprises a thyroid hormone. In some embodiments, the thyroid hormone comprises 3,3',5-triiodo-L-thyronine (T3). In some embodiments, the agent comprises a thyroid hormone receptor agonist. In some embodiments, the thyroid hormone receptor agonist comprises resmetirom or other agonists of the thyroid hormone receptor beta. In some embodiments, the agent is an siRNA, an endonuclease, an antisense oligonucleotide, proteolysis-targeting chimeras (PROTACs), or a ribosome. In some embodiments, the agent is an siRNA that inhibits the expression of DBI.

[008] Disclosed herein is a method of treating a Cushing syndrome (or a compositions for use in such a method) associated with chronic use of a corticosteroid in a subject, the method comprising administering to the subject a composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression, wherein the administering is sufficient to treat the Cushing syndrome associated with the chronic use of the corticosteroid. In some embodiments, the chronic use comprises use over a period of time. In some embodiments, the period of time comprises one month or longer. In some embodiments, the period of time comprises six months or longer. In some embodiments, the period of time comprises one year or more.

[009] Neutralization/inhibition of extracellular DBI can be achieved in a number of ways, for example by vaccination to induce neutralizing autoantibodies or by administering an agent such as a monoclonal antibody (mAb) that binds extracellular DBI or modulates the activity of extracellular DBI.

[010] Disclosed herein is a composition for use in treating synovial inflammation in a subject, the composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression in an amount sufficient to treat the synovial inflammation upon administering to the subject. In some embodiments, the agent reduces a tibia-femur triangle area upon administering to the subject, relative to a tibia-femur triangle area in the absence of the administering. In some embodiments, the agent reduces an ultrasound biomicroscope (UBM) inflammation score upon administering to a subject, relative to a UBM score in the absence of the administering. In some embodiments, the agent reduces a weight bearing asymmetry percentage caused by the synovial inflammation, relative to a weight bearing asymmetry percentage in absent the administering. In some embodiments, the agent reduces the formation of Bouchard’s nodes and/or Heberden’s nodes in a subject, relative to the formation of Bouchard’s nodes and/or Heberden’s nodes absent the administering. In some embodiments, the agent reduces a joint crepitus in a subject, relative to a joint crepitus absent the administering.

[OH] Also disclosed herein is a composition for use in treating an arthropathy in a subject, the composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression in an amount sufficient to treat the arthropathy upon administering to the subject. In some embodiments, the agent reduces an osteoarthritis cartilage histopathology assessment system (OARSI) grade upon administering to the subject, relative to an OARSI grade absent the administering. In some embodiments, the agent reduces an osteoarthritis cartilage histopathology assessment system (OARSI) stage upon administering to the subject, relative to an OARSI stage absent the administering.

[012] Also disclosed herein is a method of treating degenerative joint disease in a subject in need thereof (or a composition comprising an agent for use in such a method), the method comprising intra-articularly administering to the subject an agent that reduces human diazepam binding inhibitor (DBI) activity or expression in an amount sufficient to treat the degenerative joint disease in the subject. In some embodiments, the administering is sufficient to treat synovial inflammation associated with the degenerative joint disease in the subject. In some embodiments, the administering is sufficient to reduce a tibia-femur triangle area in the subject, relative to a tibiafemur triangle area prior to the administering. In some embodiments, the administering is sufficient to reduce an ultrasound biomicroscope (UBM) inflammation score for the subject, relative to a UBM score prior to the administering. In some embodiments, the administering is sufficient to reduce a weight bearing asymmetry percentage caused by the degenerative joint disease, relative to a weight bearing asymmetry percentage prior to the administering. In some embodiments, the administering is sufficient to reduce the formation of Bouchard’s nodes and/or Heberden’s nodes in a subject, relative to the formation of Bouchard’s nodes and/or Heberden’s nodes prior to administering. In some embodiments, the administering is sufficient to reduce a joint crepitus in a subject, relative to a joint crepitus prior to the administering. In some embodiments, the administering is sufficient to treat arthropathy associated with the degenerative joint disease in the subject. In some embodiments, the administering is sufficient to reduce an osteoarthritis cartilage histopathology assessment system (OARSI) grade for the subject, relative to an OARSI grade prior to the administering. In some embodiments, the administering is sufficient to reduce an osteoarthritis cartilage histopathology assessment system (OARSI) stage for the subject, relative to an OARSI stage prior to the administering.

[013] In some embodiments, the agent reduces extracellular DBI activity, relative to an amount of extracellular DBI activity in a comparable cell or tissue of the subject absent the administering. In some embodiments, the agent reducing extracellular DBI activity comprises nanoparticles. In some embodiments, the agent comprises a DBI-binding polypeptide. In some embodiments, the DBI-binding polypeptide is an anti-DBI antibody. In some embodiments, the anti-DBI antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody comprises a monoclonal chimeric antibody, a monoclonal humanized antibody, or a monoclonal human antibody. In some embodiments, the anti-DBI antibody is a polyclonal antibody. In some embodiments, the DBI- binding polypeptide is an anti-DBI antibody fragment. In some embodiments, the antibody fragment comprises a single chain Fv, Fab’ fragment, or nanobody. In some embodiments, the anti-DBI antibody is an extracellular DBI neutralizing antibody. In some embodiments, the agent comprises a polypeptide antigen that induces production of a neutralizing anti-DBI antibody in the subject. In some embodiments, the agent reduces DBI expression, relative to an amount of DBI expression by administering a composition lacking the agent that reduces DBI expression. In some embodiments, the agent reducing extracellular DBI expression comprises nanoparticles. In some embodiments, the agent is an siRNA, an endonuclease, an antisense oligonucleotide, a thyroid receptor agonist, proteolysis-targeting chimeras (PROTACs), or a ribosome. In some embodiments, the agent is an siRNA. In some embodiments, the agent is a thyroid hormone receptor agonist. In some embodiments, the thyroid hormone receptor agonist comprises resmetirom.

[014] Also disclosed herein is a method of treating osteoarthritis in a subject in need thereof (or a composition comprising an agent for use in such a method), the method comprising intraarticularly administering to the subject an agent that reduces human diazepam binding inhibitor (DBI) activity or expression in an amount sufficient to treat the osteoarthritis in the subject. Also disclosed herein are compositions for use in any of the methods disclosed herein.

FIGURE DESCRIPTIONS

[015] FIG. 1A - FIG. IS show identification of corticosteroids and thyroid hormone as DBI modulators. FIG. 1A depicts scaled ACBP (DBI) fluorescence intensity at 6 and 24 h in human H4 GFP-LC3 cells treated with 720 distinct agonists and antagonists of neurotransmitter and hormone receptors. FIG. IB depicts representative images of the cells treated with agonists and antagonists. FIG. 1C shows a plot of ACBP (DBI)-specific immunofluorescence in cells administered a vehicle or DMSO (Ctrl), rapamycin (RAPA), dexamethasone (DEX), hydrocortisone (HCS), or triiodothyronine (T3). FIG. ID shows a plot of GFP-LC3 puncta formation in cells administered Ctrl, RAPA, DEX, HCS, or T3. FIG. IE shows a plot of ACBP (DBI) production present in the supernatant of cells administered a vehicle or DMSO, RAPA, DEX, HCS, or T3. FIG. IF shows a plot of ACBP (DBI) mRNA produced in cells administered vehicle or DMSO control, RAPA, DEX, HCS, or T3. FIG. 1G shows a plot of ACBP (DBI) levels in plasma of mice treated with HCS (10 mg/kg, 50 mg/kg, and 100 mg/kg; i.p.) for 24 h and no HCS treatment. FIG. 1H shows a plot of ACBP (DBI) mRNA in the liver of mice treated with HCS (10 mg/kg, 50 mg/kg, and 100 mg/kg; i.p.) for 24 h and no HCS treatment. FIG. II shows an immunoblot measuring ACBP (DBI) abundance, LC3 conversion and p62 degradation in liver tissue. FIG. 1 J shows a plot of ACBP (DBI) abundance, relative to P-actin control protein, in liver tissue of mice treated with HCS as compared to controls without HCS. FIG. IK shows a plot of LC3 conversion in liver tissue of mice treated with HCS as compared to controls without HCS. FIG. IL shows a plot of p62, relative to P-actin control protein, in liver tissue of mice treated with HCS as compared to controls without HCS. FIG. IM shows a plot of ACBP (DBI) levels in plasma of mice that were fasted or received HCS (100 mg/kg; i.p.) for 24 h combined with either SaFit2 (40 mg/kg; i.p.) or vehicle. FIG. IN shows a plot of ACBP (DBI) mRNA levels in Human peripheral blood mononuclear cells (PBMCs) that were treated with HCS (0.5 pM) for 16-18 h as compared to controls without HCS. FIG. IO shows a plot of ACBP (DBI) levels in plasma of dermatology patients receiving (n=53) or not receiving (n=39) glucocorticoid treatment. FIG. IP shows a plot of ACBP (DBI) levels in plasma of dermatology patients receiving (n=53) or not receiving (n=39) glucocorticoid treatment, grouped based on sex. FIG. IQ shows a plot of ACBP (DBI) levels in plasma of ACTH-dependent Cushing syndrome patients with hypercortisolemia (n=ll) or in remission (n=13). FIG. 1R shows a plot of body mass index (BMI) versus ACBP (DBI) plasma concentration in plasma of ACTH-dependent Cushing syndrome patient. FIG. IS shows a plot of daily hydrocortisone dose versus ACBP (DBI) plasma concentration in plasma of patients in remission.

[016] FIG. 2A - FIG. 2P show prevention of Cushing syndrome by autoantibody-mediated neutralization of DBI. FIG. 2A shows plasma ACBP (DBI) levels in mice treated with CORT (100 pg/ml) or vehicle control (Ctrl) in drinking water, p.o. for 5 weeks together with KLH-ACBP for autoimmunization or KLH alone (administered i.p., n=10/group). FIG. 2B shows corticosterone (CORT) levels of the treated mice. FIG. 2C shows an immunoblot measuring hepatic ACBP (DBI) and NR3C1 and FIG. 2D shows a plot of ACBP (DBI) relative to P-actin control protein and FIG. 2E shows a plot of NR3C1 relative to P-actin control protein. FIG. 2F shows average food intake (n=4x5/group) of the treated mice. FIG. 2G shows body weight (n=10) of the treated groups of mice. FIG. 2H shows representative frontal photographs of one mouse of each treated group at the end of week 5 (n=3/group). FIG. 21 shows measured face angles of the treated groups of mice. FIG. 2J shows a heatmap illustrating tissue weights (with visceral, inguinal, and perigonadal white adipose tissue (vWAT, iWAT, and pWAT) and interscapular brown adipose, liver, thymus, adrenal, erector spinae and gastrocnemius tissue (iBAT)) relative to body weight in the indicated treatment groups (n=10/group). FIG. 2K shows plasma alanine aminotransferase (ALT) in mice treated with CORT or vehicle only (control, Ctrl), together with KLH-ACBP (DBI) for 5 weeks (n=10/group). FIG. 2L shows aspartate aminotransferase (AST) of the treated mice. FIG. 2M shows liver triglycerides (TG) of the treated mice. FIG. 2N shows plasma TG of the treated mice. FIG. 20 shows plasma insulin of the treated mice. FIG. 2P shows fasting (16 h) glycemia level of the treated mice.

[017] FIG. 3A - FIG. 3 J show prevention of Cushing syndrome by genetic inhibition of DBI. FIG. 3A shows plasma ACBP (DBI) of mice (Gabrg^2F77I/F771) or wild type controls (Gabrg2^+/+) treated with CORT (100 pg/ml) or Vehicle (Ctrl) in drinking water (p.o. for 5 weeks, n=7- 10/group). FIG. 3B shows hepatic Acbp (DBI) mRNA of the treated mice. FIG. 3C shows average food intake (n=3x3-4/group) of the treated mice. FIG. 3D shows body weight (n=9-10/group) of the treated mice. FIG. 3E shows a heatmap illustrating the indicated tissue weights (with visceral, inguinal, and perigonadal white adipose tissue (vWAT, iWAT, and pWAT) and interscapular brown adipose tissue (iBAT)), liver thymus, adrenal, erector spinae and gastrocnemius tissues relative to body weight in the treated mice (w= 7-70/group). FIG. 3F shows plasma ACBP (DBI) of mice (Dbi'¹') or wild type controls (Dbi^+/+) treated with CORT (100 pg/ml) or Vehicle (Ctrl) in drinking water, (p.o., for 5 weeks, «=7-S/group). FIG. 3G shows hepatic Acbp (DBI) mRNA in the liver of the treated mice (n=6/group). FIG. 3H shows average food intake (n 3x4 group) of the treated groups of mice. FIG. 31 shows the body weight (n=8/group) of the treated mice. FIG. 3J shows a heatmap illustrating the vWAT, iWAT, pWAT, liver, thymus, adrenal, erector spinae and gastrocnenius tissue weights relative to body weight in the indicated treatment groups (// 7-<7group).

[018] FIG. 4A - FIG. 4Q show prevention of the manifestation of Cushing syndrome by passive immunization of mice by neutralizing monoclonal anti-DBI monoclonal antibody. FIG. 4A shows the experimental schedule for passive immunization including a forced swim test (FST) in the fifth week. FIG. 4B shows the plasma ACBP (DBI) levels of mice treated with CORT (100 pg/ml) or vehicle control (Ctrl) in drinking water, (p.o. for 5 weeks) together with ACBP/DBI mAb (aACBP, 5 mg/kg body weight, i.p., semiweekly) using an isotype as a control. FIG. 4C shows the CORT levels of the treated groups at the end of week 5. FIG. 4D shows average food intake (n=4x5/group) of the treated groups at the end of week 5. FIG. 4E shows the body weight of the treated groups at the end of week 5. FIG. 4F shows the measured facial angles of treated groups at the end of week 5. FIG. 4G shows the plasma alanine aminotransferase (ALT) of the treated groups at the end of week 5. FIG. 4H shows the aspartate aminotransferase (AST) levels of the treated groups at the end of week 5. FIG. 41 shows the liver triglycerides (TG) levels of the treated groups at the end of week 5. FIG. 4 J shows the plasma TG levels of the treated groups at the end of week 5. FIG. 4K shows the plasma insulin of the treated groups at the end of week 5. FIG. 4L shows the 16 h fasting glycemia levels of the treated groups at the end of week 5. FIG. 4M shows the postprandial glycemia levels of the treated groups at the end of week 5. FIG. 4N shows the results of intraperitoneal glucose tolerance tests (GTT; D-glucose (2 g/kg body weight)) immediately after 6 h fasting during week 5. FIG. 40 shows the results of normalized intraperitoneal insulin tolerance tests (ITT; insuline asparte (0.5 Ul/kg body weight)) immediately after 2 h fasting during week 5. FIG. 4P shows the results of immobility time assessed by forced swim test (w=70/group). FIG. 4Q shows a heatmap illustrating tissue weights (with visceral, inguinal, and perigonadal white adipose tissue (vWAT, iWAT, and pWAT) and interscapular brown adipose tissue (iBAT)) relative to body weight in the indicated treatment groups (// /O/group).

[019] FIG. 5A - FIG. 51 shows prevention of Cushing syndrome by inhibition of DBI by selective thyroid hormone receptor P (THR-P) agonist resmetirom. FIG. 5A shows the experimental schedule for treatment of mice with resmetirom (RES; 0.033 mg/mL) or vehicle control (Ctrl) per gavage. FIG. 5B shows normalized plasma ACBP levels of the treated mice. FIG. 5C shows liver Acbp (DBI) mRNA of the treated mice. FIG. 5D shows the experimental schedule for treatment of female mice with corticosterone (CORT, 100 pg/ml, p.o.) and RES (0.033 mg/mL in drinking water, p.o.) for 5 weeks. FIG. 5E shows white adipose tissue (WAT) Acbp (DBI) mRNA (n=5/group) of the treated mice at the end of the fifth week. FIG. 5F shows plasma ACBP (n=9- 10/group) of the treated mice at the end of the fifth week. FIG. 5G shows average food intake (n=9-12) of the treated mice. FIG. 5H shows the body weight (n=9-lff) of the treated mice. FIG. 51 shows a heatmap illustrating the vWAT, iWAT, pWAT, iBAT, liver, thymus, adrenal, erector spinae and gastrocnemius tissue weights relative to body weight in the indicated treatment groups (w=9-70/group).

[020] FIG. 6A - FIG. 6G shows identification as corticosteroids and thyroid hormones as DBI modulators. FIG. 6A shows LC3 conversion in human glioma H4 cells treated with DEX, HCS, or CORT (1 pM) for 6 h with/without bafilomycin Al (BafAl, 100 nM) for the last 2 h, using RAPA (10 pM) as a positive control for autophagic flux and P-actin was used as a loading control. FIG. 6B shows quantifications of RAPA, DEX, HCS, and CORT in the treated groups (n=3/group). FIG. 6C shows ACBP (DBI) levels in cells treated with siRNA targeting the essential autophagy effectors ATG5 and ATG7 or control siRNA (siCtrl) (n 3 group; AU, arbitrary units). FIG. 6D is a western blot measuring ACBP (DBI) and NR3C1 levels in cell culture supernatants of NR3C1 knock down with/without DEX (1 pM, 24 h) and P-actin as a positive control. FIG. 6E shows a plot of NR3C1 relative to P-actin control protein in the related mice. FIG. 6F shows ratio of ACBP (DBI)/p-actin in the treated mice. FIG. 6G shows the ACBP (DBI) level in culture supernatant from the treated cells.

[021] FIG. 7A - FIG. 7E shown the time-dependent induction of DBI secretion by hydrocortisone. FIG. 7A shows the experimental schedule of hydrocortisone (HCS, 100 mg/kg, i.p.) administration in female mice for 12, h 24 h, 48 h, and 72 h. FIG. 7B shows the percentage of mouse thymus relative to body weight following HCS treatment at various time points (n=5/time point). FIG. 7C shows normalized plasma ACBP (DBI) levels under HCS treatments at various time points (n=5/time point). FIG. 7D shows western blot images measuring ACBP (DBI) levels of mice treated with HCS at various time points (n=5/group), using P-actin as a loading control. FIG. 7E shows the ratio of ACBP/p-actin in the treated mice.

[022] FIG. 8A - FIG. 8E shows the effects of short-term triiodothyronine administration on DBI expression. FIG. 8A shows the experimental schedule of triiodothyronine (T3; i.p 3) administration in female mice for 16 h. FIG. 8B shows plasma ACBP (DBI) levels under different doses (0 mg/kg, 0.1 mg/kg, 0.2 mg/kg, and 0.5 mg/kg) of T3 treatment (w=6/group). FIG. 8C shows liver Acbp (DBI) mRNA levels (n=6/group) of mice treated with different doses of T3. FIG. 8D shows a western blot image measuring ACBP (DBI) levels in mice treated with different doses of T3 (n=3/group) and using P-actin as a loading control. FIG. 8E shows the ratio of ACBP/p-actin in liver.

[023] FIG. 9A - FIG. 9F show attenuation of corticosterone-induced changes in hepatic morphology and adipose tissue by autoantibody-mediated neutralization of DBI. FIG. 9A shows representative hematoxylin and eosin stains of liver, visceral, inguinal, and perigonadal white adipose tissue (vWAT, iWAT, and pWAT), and interscapular brown adipose tissue (iBAT) of female mice treated with corticosterone (CORT; 100 pg/ml) or vehicle control (Ctrl) in drinking water, p.o. for 5 weeks together with KLH-ACBP for autoimmunization or KLH alone both administered (i.p.). FIG. 9B shows medium area of visceral WAT (vWAT) levels in the treated mice. FIG. 9C shows medium area of inguinal WAT (iWAT) levels in the treated mice. FIG. 9D shows medium area of perigonadal WAT (pWAT) levels in the treated mice. FIG. 9E shows medium area of iBAT levels in the treated mice. FIG. 9F shows medium area of hepatocyte levels in the treated mice.

[024] FIG. 10A - FIG. 10F show attenuation of corticosterone-induced changes in hepatic morphology and adipose tissue by DBI mAb-mediated neutralization of DBI. FIG. 10A shows representative hematoxylin and eosin stains of liver, visceral, inguinal, and perigonadal white adipose tissue (vWAT, iWAT, and pWAT), and interscapular brown adipose tissue (iBAT) of female mice treated with CORT (100 pg/ml) or vehicle (Ctrl) in drinking water, p.o.) with or without aACBP (5 mg/kg body weight, i.p.) semiweekly for 5 weeks. FIG. 10B shows medium area of visceral WAT (vWAT) levels in the treated mice. FIG. 10C shows medium area of inguinal WAT (iWAT) levels in the treated mice. FIG. 10D shows medium area of perigonadal WAT (pWAT) levels in the treated mice. FIG. 10E shows medium area of iBAT levels in the treated mice. FIG. 10F shows medium area of hepatocytes levels in the treated mice.

[025] FIG. 11 shows the effect of corticosterone and anti-DBI mAh on plasma hormone concentrations. A heatmap shows the metabolic plasma hormones of female mice treated with CORT (100 pg/ml) or vehicle (Ctrl) in drinking water, p.o. with or without aACBP (5 mg/kg body weight, /./?.) semiweekly for 5 weeks.

[026] FIG. 12A - FIG. 12G sh ows l iver RNA sequencing analysis of an anti-DBI mAb model. FIG. 12 A is a heatmap showing genes with differential expression in the liver in a CORT and anti-ACBP/DBI (aACBP) mAb experiment. FIG. 12B shows a volcano plot of differential genes between isotype + CORT and isotype +Ctrl groups. FIG. 12C shows a volcano plot of differential genes between aACBP + CORT and isotype + CORT groups. FIG. 12D show an overlap of 58 genes in a Venn diagram illustrating overlaps of the transcriptomic CORT effects on isotype and aACBP mAb neutralization. FIG. 12E show an overlap of 442 genes in a Venn diagram illustrating overlaps of the transcriptomic CORT effects on isotype and aACBP mAb neutralization. FIG. 12F shows GO enrichment analysis of the genes obtained from the overlap in FIG. 12D. FIG. 12G shows GO enrichment analysis of the genes obtained from the overlap in FIG. 12E

[027] FIG. 13A - FIG. 13C show liver and plasma metabolomics of Cushing syndrome treated with anti-DBI mAb. FIG. 13A shows a heatmap illustrating metabolite profiling of liver from a corticosterone (CORT) and anti-ACBP/DBI (aACBP) mAb experiment. FIG. 13B shows a heatmap illustrating metabolite profiling of plasma from from the CORT and anti-ACBP/DBO (aACBP) mAb experiment. FIG. 13C shows a Venn Diagram displaying the repartition across liver and plasma of the metabolites shown in FIG. 13A and FIG. 13B.

[028] FIG. 14A - FIG. 141 shows Citalopram effects on DBI. FIG. 14A shows the experimental set up for passive immunization of female mice with ACBP/DBI mAb (aACBP, 5 mg/kg body weight, injection i.p. semiweekly) and co-administrated with citalopram (CTP, 0.15 mg/ml or vehicle control (Ctrl) in drinking water, p.o.) for 8 weeks., using isotype (5 mg/kg body weight, injection i.p. semiweekly) as control. FIG. 14B shows average food intake (// 3x3— //group) in the treated groups. FIG. 14C shows body weight (// /0/group) measurements in different treatment groups. FIG. 14D shows experimental set up administration with corticosterone (CORT, 100 pg/ml solution, p.o.) or citalopram (CTP, 0.15 mg/ml in drinking water, p.o.) for 24 h in female 57BL/6 mice. FIG. 14E shows normalized plasma ACBP level under CORT and CTP treatments (w=6/group). FIG. 14F shows a representative western blot image measuring ACBP levels in liver treated with CORT or CTP (randomly selected w=3/group), using P-actin as a loading control. FIG. 14G shows the ratio of ACBP/p-actin in the treated mice. FIG. 14H shows representative western blot images measuring ACBP levels in white adipose tissue (WAT) treated with CORT or CTP (randomly selected w=3/group), using P-actin as a loading control. FIG. 141 shows the ratio of ACBP/p-actin in the treated mice.

[029] FIG. 15A - FIG. 15R show prevention of Cushing syndrome by endocrine inhibition of DBI. FIG. 15A shows experimental set up for female mice co-administrated with corticosterone (CORT, 100 pg/ml, p.o.) or triiodothyronine (T3, 3.3pg/ml, p.o.) for 5 weeks. FIG. 15B shows liver Acbp (DBI) mRNA levels (n=5/group) of mice (a) treated with CORT or vehicle (Ctrl), or (b) co-treated with CORT and T3 or Ctrl an T3 for 5 weeks. FIG. 15C shows white adipose tissue (WAT) Acbp (DBI) mRNA levels of the treated mice. FIG. 15D shows a representative western blot image measuring ACBP (DBI) levels in liver and WAT, using P-actin as a loading control. FIG. 15E shows the ratio of ACBP/p-actin in liver of the treated mice. FIG. 15F shows the ratio of ACBP/ P-actin in WAT of the treated mice. FIG. 15G shows the plasma ACBP (DBI) levels in the treated mice. FIG. 15H shows average food intake (n=3x3-4/group) of the treated mice. FIG. 151 shows the body weight (n=10/group) of the treated mice. FIG. 15J shows the relative weight of thymus in the treated mice. FIG. 15K shows the relative weight of the adrenal in the treated mice. FIG. 15L shows the relative weight of the erector spinae in the treated mice. FIG. 15M shows the relative weight of the gastrocnemius in the treated mice. FIG. 15N shows the relative weight of the visceral fat in the treated mice. FIG. 150 shows the relative weight of the inguinal fat in the treated mice. FIG. 15P shows the relative weight of the perigonadal fat in the treated mice. FIG. 15Q shows the relative weight of the interscapular brown adipose tissue (iB AT) in the treated mice. FIG. 15R shows the relative weight of the liver in the treated mice.

[030] FIG. 16A - FIG. 16B provides an overview of the mouse joint inflammation experiments. FIG. 16A is a schematic representation of the experimental design for joint inflammation induction. FIG. 16B is a schematic representation of the experimental timeline for intraarticular (i.a.) injections of a-DBI and preclinical monitoring of C57BI/6 mice.

[031] FIG. 17A - FIG. 17C show an inflammation-induced increased in plasma ACBP/DBI levels in wild-type mice. Joint inflammation was induced in 15-week-old male mice. Blood samples were collected both before and one week after joint inflammation induction to assess plasma ACBP/DBI levels. (FIG. 17A) Plasma ACBP/DBI results are displayed as box-and- whisker plots, with each dot representing an individual mouse (n = 20 mice per condition). For statistical analysis, p-values were calculated using a Mann-Whitney test. (FIG. 17B) Schematic representation of the experimental design for joint inflammation induction in ACBP/DBI-deficient mice. (FIG. 17A) Plasma ACBP/DBI results are displayed as box-and-whisker plots, with each dot representing an individual mouse (n = 8 to 11 mice per condition). For statistical analysis, p- values were calculated using a Kruskal-Wallis test. Schematics in (FIG. 17C ) were generated using BioRender.

[032] FIG. 18A - FIG. 18D illustrate the effects of DBI neutralization on degenerative joint disease (DJD). FIG. 18A shows representative Safranin O-Fast Green stained knee sections from mice treated with IgG2a (control) or anti-ACBP/DBI antibody (7G4a mAh) (1.25 pg/pl, intraarticular) for 12 weeks. FIG. 18B is a plot of OARSI grade to evaluate cartilage damage in mice administered the IgG2a control or anti-ACBP/DBI antibody. FIG. 18C is a plot of OARSI stage to evaluate cartilage damage in mice administered the IgG2a control or anti-ACBP/DBI antibody. FIG. 18D is a representative index of combined OARSI grades and OARSI stages in mice administered the IgG2a control or anti-ACBP/DBI antibody. Results are displayed as box-and- whisker plots with each dot representing one single mouse (n=10-13 mice per condition). . The black arrows indicate areas of cartilage perturbations For statistical analysis, p-values were extracted from 2-way linear models, testing treatment significance within different genotypes and testing genotype significance within different treatments.

[033] FIG. 19A - FIG. 19E ACBP/DBI inhibition via monoclonal antibody treatment mitigated joint inflammation severity. (FIG. 19A) Schematic representation of the experimental timeline. (FIG. 19B) Representative Safranin O-Fast Green stained knee sections from different treatment groups, illustrating cartilage integrity are shown. Scale bars, 250 pm and 100 pm. (FIG. 19B - FIG. 19E) Histological evaluation of joint inflammation severity. (FIG. 19C) OARSI semiquantitative scoring for cartilage degradation (grade), (FIG. 19D) OARSI semiquantitative scoring for cartilage damage (stage), and (FIG. 19E) a combined index of grade and stage were used to evaluate the extent of cartilage damage. Results are displayed as box-and-whisker plots, with each dot representing an individual mouse (n = 2 to 6 mice per condition). For statistical analysis, p-values were extracted from 2-way linear models, testing treatment significance within different genotypes.

[034] FIG. 20A - FIG. 20B illustrate the effects of DBI neutralization on synovial inflammation. FIG. 20A shows representative Safranin O-Fast Green stained sections from mice treated with IgG2a (control) or anti-ACBP/DBI antibody (7G4a mAb) (1.25 pg/pl; intra-articular) for 12 weeks. FIG. 20B is a plot analysing inflammation in the synovial membrane using a semiquantitative scoring system. Results are displayed as box-and-whisker polys, with each dot representing an individual mouse (n=10-13 mice per condition) For statistical analysis, p-values were extracted from 2-way linear models, testing treatment significance within different genotypes and testing genotype significance within different treatments. [035] FIG. 21A - FIG. 21E show the effect of a-DBI intraarticular injections on cartilage integrity and weight-bearing asymmetry in mice with joint inflammation. (FIG. 21A). In 15-week- old male C57B1/6 mice, mechanical destabilization of the right knee joint was induced via MCL/DMM surgery, while the left knee underwent sham surgery. (FIG. 21B, FIG. 21D) Representative knee sections stained for ACBP/DBI (FIG. 21B) and LC3 (FIG. 21D). Scale bar, 50 pm. (FIG. 21C, FIG. 21E) Quantification of ACBP/DBI-positive cells (FIG. 21C) and LC3- positive cells (FIG. 21E) in the medial femoral condyle (MFC) and medial tibial plateau (MTP). Results are displayed as box-and-whisker plots, with each dot representing an individual mouse (n = 5 mice per condition). For statistical analysis, p-values were extracted from 2-way linear models, testing treatment significance within surgery status and testing surgery significance within different treatments.

[036] FIG. 22A - FIG. 22B illustrate improvement in weight bearing symmetry percentage by ACBP/DBI neutralization, as determined by pain-behavior outcomes measures. FIG. 22A is a plot showing weight-bearing asymmetry percentage evaluated over a three-second period in mice during a twelve-week study with surgical destabilization of the medial meniscus + medial collateral ligament (DMM+MCL) surgery and IgG2a versus anti-ACBP/DBI 7G4a mAb treatment (1.25 pg/pl; intra-articular), represented as the percentage ratio between weight-bearing on the right and left limbs. FIG. 22B is a plot of weight bearing distribution deviation evaluated over a three-second period in mice during a twelve-week study with DMM+MCL surgery and IgG2a versus anti-ACBP/DBI 7G4a mAb treatment (1.25 pg/pl; intra-articular), represented as the deviation of the ratio between weight on the right knee and weight on the left knee. Ten measurements were performed for each mouse. Mean ± SEM values (n=10-13 mice per condition) are presented, and statistical analysis employed the area under the curve (AUC) for calculation of p-values by ANOVA with Tukey test for multiple comparison.

[037] FIG. 23A - FIG. 23F depict characterization of monoclonal antibodies against ACBP/DBI. FIG. 23A is a representative ACBP immunoblot from human and mouse recACBP/DBI (10, 25, 50, 100 ng) using the 82B2G9 antibody. FIG. 23B depicts putative epitopes onto a modelled structure for mouse ACBP for the anti-ACBP/DBI antibody 82B2G9. FIG. 23C depicts putative epitopes onto a modelled structure for mouse ACBP for the anti- ACBP/DBI antibody 7G4a. FIG. 23D illustrates an experimental procedure of the damage induced by acetaminophen (APAP, i.p. 300 mg/kg for 16 hours) in mice pre-treated with i.p. injection of a-DBI or IgG (2.5 pg/g) for 4 hours and just before hepatic injury. FIG. 23E is a plot of ALT transaminases activity from plasma obtained from each mouse (n=5-16 mice per group). FIG. 23F is a plot of AST transaminases activity from plasma obtained from each mouse (n=5-16 mice per group). Results for FIG. 23E and FIG. 23F are displayed as whisker plots (with each dot representing one single mouse). Statistical analyses (p values) were calculated by means of ANOVA (uncorrected fisher’s LSD).

[038] FIG. 24A - FIG. 24C illustrate that ACBP/DBI neutralization using an exemplary anti- ACBP/DBI antibody 7G4a improves radiological signs of degenerative joint disease when administered intra-articularly, as determined by ultrasound biomicroscope B-mode. FIG. 24A depicts echographies of knees after 12 weeks of treatment with IgG2a and anti-ACBP/DBI mAb clone 7G4a (1.25 pg/pl; intra-articular). Dots indicate hypoechogenic zones. FIG. 24B is a plot quantifying the tibial-femur triangle area in mice administered the anti-ACBP/DBI mAb clone 7G4a or IgG2a control. FIG. 24C is a plot of ultrasound biomicroscopy score for inflammation in mice administered the anti-ACBP/DBI mAb clone 7G4a or IgG2a control. Results are displayed as box-and-whisker polys, with each dot representing an individual mouse (n=10-13), with p- values calculated by means of two-way ANOVA with Tukey test for multiple comparison.

[039] FIG. 25A - FIG. 25C illustrate that ACBP/DBI neutralization using an exemplary anti- ACBP/DBI antibody 82B2G9 improves radiological signs of degenerative joint disease (DJD) when administered intra-articularly, as determined by ultrasound biomicroscope B-mode. FIG. 25A depicts echographies of knees after 12 weeks of treatment with IgG2a and anti-ACBP/DBI mAb clone 82B2G9 (1.25 pg/pl; intra-articular). Dots indicate hypoechogenic zones. FIG. 25B is a plot quantifying the tibial-femur triangle area in mice administered the anti-ACBP/DBI mAb clone 82B2G9 or IgG2a control. FIG. 25C is a plot of ultrasound biomicroscopy score for inflammation in mice administered the anti-ACBP/DBI mAb clone 82B2G9 or IgG2a control. Results are displayed as box-and-whisker plots, with each dot representing an individual mouse (n=10-13), with p-values calculated by means of two-way ANOVA with Tukey test for multiple comparison.

[040] FIG. 26A - FIG. 26C illustrate that ACBP/DBI neutralization using an exemplary anti- ACBP/DBI antibody 7G4a improves radiological signs of degenerative joint disease (DJD) when administered systemically, as determined by ultrasound biomicroscope B-mode. FIG. 26A depicts echographies of knees after 12 weeks of treatment with IgG2a and anti-ACBP/DBI mAb clone 7G4a (5 mg/kg body weight; i.p). Dots indicate hypoechogenic zones. FIG. 26B is a plot quantifying the tibial-femur triangle area in mice administered the anti-ACBP/DBI mAb clone 7G4a or IgG2a control. FIG. 26C is a plot of ultrasound biomicroscopy score for inflammation in mice administered the anti-ACBP/DBI mAb clone 7G4a or IgG2a control. Results are displayed as box-and-whisker polys, with each dot representing an individual mouse (n=10-13), with p- values calculated by means of two-way ANOVA with Tukey test for multiple comparison. [041] FIG. 27A - FIG. 27D shows intraarticular injection of anti-ACBP/DBI monoclonal antibody reduced cartilage destruction in joint inflammation. (FIG. 27A) Representative Safranin O-Fast Green stained knee sections from mice treated with a-DBI or isotype control antibody. (FIG. 27B) Semiquantitative scoring system about histological changes. The minimum value 0 corresponds to normal cartilage and the maximum value 12 represents the sum of the destroyed cartilage in MFC and MTP. (FIG. 27C) Represents the area occupied by the damage in the cartilage, where the value 0 is intact cartilage and the value 8 is the maximum, corresponding to the sum MFC+MTP. (FIG. 27D) Represents the relationship of the damage and the occupied area. The value 0 is the minimum and 48 the maximum. (Results are displayed as box-and-whisker plots, with each dot representing an individual mouse (n = 9 to 14 mice per condition). For statistical analysis, p-values were extracted from 2-way linear models, testing treatment significance within surgery status and testing surgery significance within different treatments.

[042] FIG. 28A - FIG. 28C shows ACBP/DBI neutralization reduces synovial inflammation in mice with joint inflammation. (FIG. 28A) Representative images of knee synovial membrane from mice treated with a-DBI or isotype control antibody, stained with Safranin-O. Scale bar 250 pm and 100 pm. (FIG. 28B) Quantification of Kreen score to determine synovitis. Data for each mouse range from 0 (no synovitis) to 9 (maximal inflammation) (n = 12 - 16 mice per group). For statistical analysis, p-values were extracted from 2-way linear models, testing treatment significance within surgery status and testing surgery significance within different treatments. (FIG. 28C) Plasma ACBP/DBI levels in Sham and joint inflammation mice treated by i.a. injections. Results are displayed as box-and-whisker plots, with each dot representing an individual mouse (n = 9 to 26 mice per condition). For statistical analysis, p-values were calculated using two-way ANOVA corrected for multiple comparisons.

[043] FIG. 29A - FIG. 29D: ACBP/DBI neutralization reduces pro-inflammatory cytokines in mice with joint inflammation. (FIG. 29A) Heatmap representation of the pro-inflammatory cytokine panel in mouse plasma treated with a-DBI or isotype control antibody. The normality of log2-transformed values was tested using the Shapiro-Wilk test. Cytokines with a normal distribution were analyzed by two-way ANOVA with Tukey’s HSD for pairwise comparisons. Non-normally distributed cytokines were analyzed using the Kruskal-Wallis test, followed by Dunn's post-hoc test with Benjamini -Hochberg correction for multiple comparisons. (FIG. 29B- FIG. 29D) Individual representation of cytokine levels for (FIG. 29B) IL-la, (FIG. 29C) IL-33, and (FIG. 29D) TNF-a. Results are displayed as column plots, with each dot representing an individual mouse (n = 10 mice per condition), shown as mean ± SEM. For statistical analysis, p- values were calculated using two-way ANOVA followed by Tukey’s test for multiple comparisons.

DETAILED DESCRIPTION

[044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure relates. Any methods, systems, and materials similar or equivalent to those described herein can be used in the practice of embodiments described herein. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

Definitions

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

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

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

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

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

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

[051] As used herein, the term “DBI” has its general meaning in the art and refers to the diazepam binding inhibitor, or acyl-CoA binding protein (ACBP) encoded by the DBI gene (Gene ID: 1622). In some embodiments, DBI refers to extracellular DBI. The term is also known as EP; ACBP; ACBD1; and CCK-RP. An exemplary amino acid sequence for DBI is represented by the NCNI reference sequence NP_001073331.1 (SEQ ID NO:1) (acyl-CoA-binding protein isoform 1). An exemplary human nucleic acid sequence is represented by the NCNI reference sequence NM_00 1079862.2 (SEQ ID NO:2) (acyl-CoA-binding protein isoform 1).

[052] SEQ ID NO: 1:

MSQAEFEKAAEEVRHLKTKPSDEEMLFIYGHYKQATVGDINTERPGMLDF

TGKAKWDAWNELKGTSKEDAMKAYINKVEE LKKKYGI

[053] SEQ ID NO:2:

GCTCGCCCGAGCAGGGTTGGGGCGAGTGGACCGCGCCTCTAAAGGCGCTTGCCAGT GCAATCTGGGCGATCGCTTCCTGGTCCTCGCCTCCTCCGCTGTCTCCCTGGAGTTCTT GCAAGTCGGCCAGGATGTCTCAGGCTGAGTTTGAGAAAGCTGCAGAGGAGGTTAGG CACCTTAAGACCAAGCCATCGGATGAGGAGATGCTGTTCATCTATGGCCACTACAA ACAAGCAACTGTGGGCGACATAAATACAGAACGGCCCGGGATGTTGGACTTCACGG GCAAGGCCAAGTGGGATGCCTGGAATGAGCTGAAAGGGACTTCCAAGGAAGATGCC ATGAAAGCTTACATCAACAAAGTAGAAGAGCTAAAGAAAAAATACGGGATATGAG AGACTGGATTTGGTTACTGTGCCATGTGTTTATCCTAAACTGAGACAATGCCTTGTTT TTTTCTAATACCGTGGATGGTGGGAATTCGGGAAAATAACCAGTTAAACCAGCTACT CAAGGCTGCTCACCATACGGCTCTAACAGATTAGGGGCTAAAACGATTACTGACTTT CCTTGAGTAGTTTTTATCTGAAATCAATTAAAAGTGTATTTGTTACTTTAAATAACTT TAAAAAAAAAA

[054] As used herein, the term “DBI activity” refers to any biological activity of DBI that includes among others: inhibition of autophagy, induction of hypoglycaemia, stimulation of food intake, stimulation of weight gain, reduction of fatty acid oxidation, upregulation of glucose transporter, upregulation of PPARG, stimulation of glucose uptake, stimulation of glycolysis or stimulation of lipogenesis, or any combinations thereof.

[055] As used herein, the terms “enhance,” “increase,” “augment,” “improve, ’’and grammatical equivalents thereof when in reference to the level of any molecule (e.g., amino acid sequence, and nucleic acid sequence, antibody, etc.), cell (e.g., B cell, T cell, tumor cell), and/or phenomenon (e.g., disease treatment), in a first sample (or in a first subject) relative to a second sample (or relative to a second subj ect or control), mean that the quantity of molecule, cell and/or phenomenon in the first sample (or in the first subject) is higher than in the second sample (or in the second subject or control) by any amount that is statistically significant using any art-accepted statistical method of analysis. For example, it may refer to a natural, synthetic or engineered compound, agent, or component that has a biological effect to increase a symptom or condition.

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

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

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

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

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

[061] As used herein, the term “treatment” or “treat” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of a subject at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. [062] As used herein, the term “pharmaceutical composition” refers to a composition described herein, or pharmaceutically acceptable salts thereof, with other agents such as carriers and/or excipients. The pharmaceutical compositions as provided herewith typically include a pharmaceutically acceptable carrier.

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

[064] As used herein, the term “elderly patient” refers to an adult patient sixty-five years of age or older. [065] As used herein, the term “obesity” refers to a condition characterized by an excess of body fat. The operational definition of obesity is based on the Body Mass Index (BMI), which is calculated as body weight per height in meters squared (kg/m²). Obesity refers to a condition whereby an otherwise healthy subject has a BMI greater than or equal to 30 kg/m², or a condition whereby a subject with at least one co-morbidity has a BMI greater than or equal to 27 kg/m². An “obese subject” is an otherwise healthy subject with a BMI greater than or equal to 30 kg/m² or a subject with at least one co-morbidity with a BMI greater than or equal 27 kg/m². A “subject at risk of obesity” is an otherwise healthy subject with a BMI of 25 kg/m² to less than 30 kg/m² or a subject with at least one co-morbidity with a BMI of 25 kg/m² to less than 27 kg/m². The increased risks associated with obesity may occur at a lower BMI in people of Asian descent. In Asian and Asian-Pacific countries, including Japan, “obesity” refers to a condition whereby a subject has a BMI greater than or equal to 25 kg/m². An “obese subject” in these countries refers to a subject with at least one obesity -induced or obesity -related co-morbidity that requires weight reduction or that would be improved by weight reduction, with a BMI greater than or equal to 25 kg/m². In these countries, a “subject at risk of obesity” is a person with a BMI of greater than 23 kg/m² to less than 25 kg/m².

[066] Disclosed herein is the use of an agent that inhibits extracellular diazepam binding inhibitor (DBI), such as an anti-DBI agent, for example, in a sample, an organism, or a subject, such as a human subject. Such uses may be useful in the therapeutic treatment of diseases associated with pituitary malignancies, such as Cushing syndrome. The present disclosure and embodiments described herein are based, at least in part, on compelling evidence of a therapeutic effect in various models upon inhibition of extracellular DBI by various methods.

[067] The present disclosure provides for compositions for use in treating a disease associated with a pituitary malignancy in a subject. In some embodiments, the composition comprises an agent that reduces human diazepam binding inhibitor (DBI) activity or expression. In some embodiments, the agent reduces human DBI activity. In some embodiments, the reduction in human DBI activity is sufficient to treat the disease associated with the pituitary malignancy. In some embodiments, the agent reduces human DBI expression. In some embodiments, the reduction in human DBI expression is sufficient to treat the disease associated with the pituitary malignancy. In some embodiments, the disease is associated with a pituitary adenoma. In some embodiments, the amount is sufficient to treat a symptom of the disease. In some embodiments, the amount is sufficient to treat the disease. In some embodiments, the agent reduces DBI activity, relative to an amount of activity by administering a composition lacking the agent that reduces DBI activity. In some embodiments, the agent reduces DBI expression, relative to an amount of expression by administering a composition lacking the agent that reduces DBI expression.

[068] Acyl coenzyme A (CoA) binding protein (ACBP), which is also called diazepam binding inhibitor (DBI), is constitutively expressed by most if not all nucleated cells, where it is present in the cytoplasm and the nucleus. Stressed and dying cells can release ACBP/DBI protein into the extracellular space. Extracellular ACBP/DBI then acts on gamma-aminobutyric acid (GABA) receptors of the A type (GABAAR) containing the gamma-2 subunit (GABRG2) to inhibit autophagy in various cell types. Of note, DBI mRNA and ACBP/DBI plasma levels are elevated in patients who are suffering from joint inflammatory diseases, such as those with synovial inflammation, arthropathy, and degenerative joint disease. Thus, the present application demonstrates that neutralization of DBI activity or expression can be used to treat joint inflammatory diseases arthropathy, degenerative joint diseases (DJD) and manifestations thereof. [069] Accordingly, disclosed herein is a composition for use in treating synovial inflammation in a subject, where the composition comprises an agent that reduces human diazepam binding inhibitor (DBI) activity or expression. Also disclosed herein is a composition for use in treating degenerative joint disease in a subject, where the composition comprises an agent that reduces human diazepam binding inhibitor (DBI) activity or expression.

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

[071] In some embodiments, the agent reducing extracellular DBI activity comprises nanoparticles. In some embodiments, the nanoparticles provide enhanced paracellular permeability, bioavailability, and sustained release. In some embodiments, the nanoparticles biodegrade to biocompatible byproducts in situ. In some embodiments, the nanoparticles have a mean particle size between about 50 and 400 nanometers. In some embodiments, the nanoparticles have a mean particle size of between about 100 and 300 nanometers. In some embodiments, the nanoparticles have a mean particle size of between about 100 and 200 nanometers. In some embodiments, the agent comprises a DBI-binding polypeptide. In some embodiments, the DBI- binding polypeptide is an anti-DBI antibody. In some embodiments, the anti-DBI antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody comprises a monoclonal chimeric antibody, a monoclonal humanized antibody, or a monoclonal human antibody. In some embodiments, the monoclonal antibody comprises a monoclonal chimeric antibody. In some embodiments, the monoclonal antibody comprises a monoclonal humanized antibody. In some embodiments, the monoclonal antibody comprises a monoclonal human antibody.

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

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

[074] In some embodiments, the agent reducing extracellular DBI expression comprises nanoparticles. In some embodiments, the agent is an siRNA, an endonuclease, an antisense oligonucleotide, a thyroid receptor agonist, proteolysis-trageting chimeras (PROTACs), or a ribosome. In some embodiments, the agent is the siRNA. In some embodiments, the agent is an endonuclease. In some embodiments, the agent is an antisense oligonucleotide. In some embodiments, the agents are proteolysis-targeting chimeras (PROTACs). In some embodiments, the agent is a ribosome. In some embodiments, the agent is a thyroid hormone receptor agonist. In some embodiments, the thyroid hormone receptor agonist comprises resmetirom.

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

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

[077] In some embodiments, the present disclosure provides for a method of treating degenerative joint disease in a subject (or a composition for use in such a method). In some embodiments, the method comprises intra-articularly administering to the subject a composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity. In some embodiments, the method comprises intra-articularly administering to the subject a composition comprising an agent that reduces human DBI expression. In some embodiments, the administering is sufficient to treat the degenerative joint disease in the subject.

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

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

Anti-DBI agents

[080] Systems, methods and compositions described herein comprise an agent that inhibits DBI activity or uses thereof. In some embodiments, an agent that inhibits extracellular DBI activity is an anti-DBI agent. In some embodiments, the agent that inhibits DBI activity inhibits extracellular DBI. In some embodiments, the agent that inhibits extracellular DBI activity inhibits extracellular DBI expression.

[081] In some embodiments, the agent comprises a DBI-binding polypeptide. In some embodiments, the DBI-binding polypeptide is an anti-DBI antibody. In some embodiments, the anti-DBI antibody is a monoclonal antibody. In some embodiments, the anti-DBI antibody is a polyclonal antibody. In some embodiments, the DBI-binding polypeptide is an anti-DBI antibody fragment. In some embodiments, the DBI-binding polypeptide is an antibody fragment comprising a single chain Fv. In some embodiments, the DBI-binding polypeptide is an antibody fragment comprising a Fab’ fragment. In some embodiments, the DBI-binding polypeptide is an antibody fragment comprising a nanobody. In some embodiments, the agent comprises a polypeptide antigen that induces production of a neutralizing anti-DBI antibody.

[082] In some embodiments, the agent reduces extracellular DBI expression, relative to an amount of DBI expression by administering a composition lacking the agent that reduces DBI expression. In some embodiments, the agent comprises a thyroid hormone. In some embodiments, the thyroid hormone comprises 3,3',5-triiodo-L-thyronine (T3). In some embodiments, the agent comprises a thyroid hormone receptor agonist. In some embodiments, the thyroid hormone receptor agonist comprises resmetirom. In some embodiments, the agent is an siRNA. In some embodiments, the agent is an endonuclease. In some embodiments, the agent is an antisense oligonucleotide. In some embodiment, the agents are proteolysis-trageting chimeras (PROTACs). In some embodiments, the agent is a ribosome.

[083] In some embodiments, the anti-DBI agent is an agent that reduces extracellular human diazepam binding inhibitor (DBI) activity. In some embodiments, the agent reduces extracellular human DBI activity, relative to an activity level prior to the administering.

[084] In some embodiments, the agent is an antisense oligonucleotide. In some embodiments, the agents are proteolysis-targeting chimeras (PROTACs). In some embodiments, the agent is a ribosome. In some embodiments, the agent is an siRNA that inhibits the expression of DBI.

[085] In some embodiments, extracellular DBI may be mammalian DBI, such as extracellular human DBI. In some embodiments, the anti-DBI agent described herein is a biologic molecule or a chemical molecule. In some embodiments, a biologic molecule is an antibody, and antigenbinding portion thereof, or an aptamer. In some embodiments, chemical molecule is a small molecule, drug, or compound. In some embodiments, the anti-DBI agent described herein is an antibody, and antigen-binding portion thereof, or an aptamer directed against extracellular DBI.

[086] In some embodiments, an antibody or aptamer described herein is directed against the fragment consisting in the amino acid sequence ranging from the amino acid residue at position 43 to the amino acid residue at position 50 in SEQ ID NO: 1 (i.e., the octapeptide or OP). In some embodiments, the agent that inhibits the activity of DBI (e.g., extracellular DBI) is an aptamer directed against extracellular DBI.

[087] In some embodiments, the agent that inhibits the activity of DBI (e.g., extracellular DBI) is an antibody directed against extracellular DBI. In some embodiments, an antibody of the present disclosure is a chimeric antibody, typically a chimeric mouse/human antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. 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.

[088] In some embodiments, an antibody described herein is a neutralizing antibody. In some embodiments, the neutralizing antibody of the present disclosure does not mediate antibodydependent cell-mediated cytotoxicity and thus does not comprise an Fc portion that induces antibody dependent cellular cytotoxicity (ADCC). In some embodiments, the neutralizing antibody does not comprise an Fc domain capable of substantially binding to a FcgRIIIA (CD16) polypeptide. In some embodiments, the neutralizing antibody lacks an Fc domain (e.g., lacks a CH2 and/or CH3 domain) or comprises an Fc domain of IgG2 or IgG4 isotype. In some embodiments, the neutralizing antibody consists of or comprises a Fab, Fab', Fab'-SH, F (ab¹) 2, Fv, a diabody, single-chain antibody fragment, or a multi-specific antibody comprising multiple different antibody fragments. In some embodiments, the neutralizing antibody is not linked to a toxic moiety. In some embodiments, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered C2q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6, 194,551 by Idusogie et al.

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

[090] Antibodies described herein may be obtained commercially or synthesized through any suitable method. For example, anti-DBI human monoclonal antibodies may be synthesized using peptides derived from the full length human ACBP and the phage display technology. In some embodiments, antibodies described herein are mutated antibodies. In some embodiments, antibodies described herein are selected based on favorable kinetic parameters, such as specificity for or affinity against human ACBP. Specificities of antibodies described herein can be validated by western blot, immunofluorescence and flow cytometry on human ACBP/DBI knock out cell lines. In some embodiments, antibodies described herein react only with human ACBP and not with mouse ACBP. KD measurements may also be performed where KD is the equilibrium dissociation constant (ratio of kd/ka between the antibody and its antigen) and KD and affinity are inversely related. In some embodiments, antibodies described herein have a high affinity against human ACBP. In some embodiments, antibodies described herein have favorable kinetics parameters, wherein favorable kinetic parameters are indicative of a faster association and slower dissociation. For example, Sensorgram shapes of antibodies described herein demonstrate a clear concentration-response relationship at lower concentrations.

[091] In some embodiments, the agent that inhibits the expression of extracellular DBI is an inhibitor of expression. In some embodiments, said inhibitor of gene expression is a siRNA, an endonuclease, an antisense oligonucleotide or a ribozyme.

[092] In some embodiments, the agent that inhibits the activity of DBI (e.g., extracellular DBI) consists of a vaccine composition suitable for eliciting neutralizing autoantibodies against extracellular DBI when administered to the subject. For the purpose of the present disclosure, the term "vaccine composition" is intended to mean a composition which can be administered to humans or to animals in order to induce an immune system response; this immune system response can result in the production of antibodies against extracellular DBI. Typically, the vaccine composition comprises at least one antigen derived from DBI. As used herein the term “antigen” refers to a molecule capable of being specifically bound by an antibody or by a T cell receptor (TCR) if processed and presented by MHC molecules. The term "antigen", as used herein, also encompasses T-cell epitopes. An antigen is additionally capable of being recognized by the immune system and/or being capable of inducing a humoral immune response and/or cellular immune response leading to the activation of B- and/or T-lymphocytes. An antigen can have one or more epitopes or antigenic sites (B- and T- epitopes). In some embodiments, an antigen of the present disclosure consists of a polypeptide.

[093] In some embodiments, an antigen of the present disclosure consists of a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 80% of identity with the sequence of SEQ ID NO:1 or a fragment thereof (e.g., an epitope). In some embodiments, the polypeptide comprises (i) an amino acid sequence having at least 80% of identity with SEQ ID NO:1, or (ii) an amino acid sequence having at least 80% of identity with the amino acid sequence ranging from the amino acid residue at position 17 to the amino acid residue at position 50 in SEQ ID NO:1, or (iii) an amino acid sequence having at least 80% of identity with the amino acid sequence ranging from the amino acid residue at position 33 to the amino acid residue at position 50 in SEQ ID NO:1, or (iv) an amino acid sequence having at least 80% of identity with the amino acid sequence ranging from the amino acid residue at position 43 to the amino acid residue at position 50 in SEQ ID NO:1.

[094] In some embodiments, the polypeptide is conjugated to a carrier protein which is generally sufficiently foreign to elicit a strong immune response to the vaccine. Illustrative carrier proteins are inherently highly immunogenic. Both bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) have commonly been used as carriers in the development of conjugate vaccines when experimenting with animals and are contemplated herein as carrier proteins. Proteins which have been used in the preparation of therapeutic conjugate vaccines include, but are not limited to, a number of toxins of pathogenic bacteria and their toxoids. Suitable carrier molecules are numerous and include, but are not limited to: bacterial toxins or products, for example, cholera toxin B-(CTB), diphtheria toxin, tetanus toxoid, and pertussis toxin and filamentous hemagglutinin, shiga toxin, pseudomonas exotoxin; lectins, for example, ricin-B subunit, abrin and sweet pea lectin; sub virals, for example, retrovirus nucleoprotein (retro NP), rabies ribonucleoprotein (rabies RNP), plant viruses e.g., TMV, cow pea and cauliflower mosaic viruses), vesicular stomatitis virus-nucleocapsid protein (VSV-N), poxvirus vectors and Semliki forest virus vectors; artificial vehicles, for example, multiantigenic peptides (MAP), microspheres; Yeast virus-like particles (VLPs); malarial protein antigen; and others such as proteins and peptides as well as any modifications, derivatives or analogues of the above. Other useful carriers include those with the ability to enhance a mucosal response, such as, LTB family of bacterial toxins, retrovirus nucleoprotein (retro NP), rabies ribonucleoprotein (rabies RNP), vesicular stomatitis virus-nucleocapsid protein (VSV-N), and recombinant pox virus subunits.

[095] In some embodiments, the antibody of the present disclosure is a chimeric antibody, typically a chimeric mouse/human antibody. [096] As used herein, the term “chimeric antibody” refers to an antibody which comprises a VH domain and a VL domain of a non-human antibody, and a CH domain and a CL domain of a human antibody. In some embodiments, a “chimeric antibody” is an antibody molecule in which (a) the constant region (/.< ., the heavy and/or light chain), or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity. Chimeric antibodies also include primatized and in particular humanized antibodies. Furthermore, chimeric antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.

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

[098] As used hereon, the term “humanized antibody” refers to an antibody having variable region framework and constant regions from a human antibody but retains the CDRs of a previous non-human antibody. In some embodiments, a humanized antibody contains minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies and antibody fragments thereof may be human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antib ody/antibody fragment can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Such antibodies are designed to maintain the binding specificity of the non-human antibody from which the binding regions are derived, but to avoid an immune reaction against the non-human antibody. These modifications can further refine and optimize antibody or antibody fragment performance. In general, the humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a significant portion of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. [099] In some embodiments, the antibody is a human antibody. As used herein the term “human antibody” as used herein, is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody” as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

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

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

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

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

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

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

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

Pharmaceutical compositions

[107] The present disclosure provides for compositions for use in treating symptoms of associated with elevated levels of steroids in a subject. In some embodiments, the composition comprises an agent that reduces human diazepam binding inhibitor (DBI) activity. In some embodiments, the composition comprises an agent that reduces human DBI expression. In some embodiments, the human DBI activity is reduced in an amount sufficient to treat the symptoms of associated with the elevated levels of steroids in the subject upon administration to the subject. In some embodiments, the symptoms are selected from the group consisting of: adipocyte hypertrophy, increased liver weight, an increase in circulating liver enzymes, an increase in alanine aminotransferase (ALT), an increases in aspartate aminotransferase (AST), an increase in liver triglycerides (TG), a hypertrophy of white adipose tissue (WAT), a hypertrophy of brown adipose tissue (BAT), a metabolic syndrome, an arterial hypertension dyslipidemia, a triglyceridemia, a round “moon” face that comprises capillary vasodilatation, a skin acne, a facial hirsutism, a cranial alopecia, a skin atrophy, a central obesity, a “buffalo hump” lipodystrophy, a profuse striae, and a sarcopenia. In some embodiments, the symptom is a adipocyte hypertrophy. In some embodiments, the symptom is an increased liver weight. In some embodiments, the symptom is an increase in circulating liver enzymes. In some embodiments, the symptom is an increase in alanine aminotransferase (ALT). In some embodiments, the symptom is an increase in aspartate aminotransferase (AST). In some embodiments, the symptom is, an increase in liver triglycerides (TG). In some embodiments, the symptom is a hypertrophy of white adipose tissue (WAT). In some embodiments, the symptom is a hypertrophy of brown adipose tissue (BAT). In some embodiments, the symptom is a metabolic syndrome. In some embodiments, the symptom is an arterial hypertension dyslipidemia. In some embodiments, the symptom is a triglyceridemia. In some embodiments, the symptom is a round “moon” face that comprises capillary vasodilatation. In some embodiments, the symptom is a skin acne. In some embodiments, the symptom is a facial hirsutism. In some embodiments, the symptom is a cranial alopecia. In some embodiments, the symptom is a skin atrophy. In some embodiments, the symptom is a central obesity. In some embodiments, the symptom is a “buffalo hump” lipodystrophy. In some embodiments, the symptom is a profuse striae. In some embodiments, the symptom is a sarcopenia.

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

[109] In some embodiments, the one or more agents that inhibit the activity or expression of DBI (e.g., extracellular DBI) of the present disclosure are administered directly into the subject or isolated organ using injection, pump device and/or any machine (e.g., bypass machine). In some embodiments, an isolated organ suitable for transplantation is perfused with a preservation solution which comprises the effective amount of the agent that inhibits the activity or expression of extracellular DBI. As used herein, the terms “preservation solution” or “organ preservation solution” refer to an aqueous solution having a pH between 6.5 and 7.5, including salts, such as chloride, sulfate, sodium, calcium, magnesium and potassium; sugars, mannitol, raffinose, sucrose, glucose, fructose, lactobionate (which is a water resistant), or gluconate; antioxidants, for instance glutathione; active agents, for instance xanthine oxidase inhibitors such as allopurinol, lactates, amino acids such as histidine, glutamic acid (or glutamate), tryptophan; and optionally colloids such as hydroxyethyl starch, polyethylene glycol or dextran. In some embodiments, a device for preserving an organ is used wherein said device comprises an organ container filled with a preservation solution, characterized in that said device further comprises one or more mean for injecting one or more agents (e.g., the agent that inhibits the activity or expression of extracellular DBI) into the organ container.

Methods of treatment

[HO] Disclosed herein, amongst other disclosures are methods of treating a disease associated with elevated levels of steroids in a subject. In some embodiments, the methods comprise administering to the subject a composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression. In some embodiments, the administering is sufficient to treat the disease associated with the elevated expression of steroids in the subject. In some embodiments, the disease is associated with elevated levels of cortisol in the subject. In some embodiments, the disease is Cushing syndrome. In some embodiments, the disesase is an endogenous Cushing syndrome. In some embodiments, the endogenous Cushing syndrome is Cushing disease. In some embodiments, the disease is associated chronic use of steroids by the subject. In some embodiments, the disease is an iatrogenic Cushing syndrome. In some embodiments, the iatrogenic Cushing syndrome is induced by corticosteroids. In some embodiments, the corticosteroids are co-administered with T3. In some embodiments, the corticosteroids are co-administered with resmetirom.

[Hl] The present disclosure also provides for methods of treating a Cushing syndrome in a subject. In some embodiments, the Cushing syndrome is associated with chronic use of a corticosteroid by the subject. In some embodiments, the method comprising administering to the subject a composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity. In some embodiments, the method comprising administering to the subject a composition comprising an agent that reduces human DBI expression. In some embodiments, the administering of the agent is sufficient to treat the Cushing syndrome. In some embodiments, the administering of the agent is sufficient to treat the Cushing syndrome associated with the chronic use of the corticosteroid.

[112] In some embodiments, the Cushing syndrome is endogenous Cushing syndrome or iatrogenic Cushing syndrome. In some embodiments, the Cushing syndrome is endogenous Cushing syndrome. In some embodiments, the Cushing syndrome is iatrogenic Cushing syndrome. In some embodiments, the iatrogenic Cushing syndrome is induced by corticosteroids. In some embodiments, the corticosteroids are co-administered with T3. In some embodiments, the corticosteroids are co-administered with resmetirom.

[113] In some embodiments, the method treats a symptom of the Cushing syndrome. In some embodiments, the symptom comprises: increased food intake, increased appetite, weight gain, excessive adiposity, adipocyte hypertrophy, increased liver weight, an increase in circulating liver enzymes, an increase in alanine aminotransferase (ALT), an increases in aspartate aminotransferase (AST), an increase in liver triglycerides (TG), a liver damage, an increase in plasma triglycerides, a hypertrophy of white adipose tissue (WAT), a hypertrophy of brown adipose tissue (BAT), a metabolic syndrome, an arterial hypertension dyslipidemia, a sign of type- 2 diabetes (T2D), a hyperinsulinemia, a hyperglycemia, a triglyceridemia, a hypertriglyceridemia, an insulin resistance, a round “moon” face that comprises capillary vasodilatation, a skin acne, a facial hirsutism, a cranial alopecia, a skin atrophy, a central obesity, a “buffalo hump” lipodystrophy, a profuse striae, or a sarcopenia.

EXAMPLES

EXAMPLE A: METHODS AND COMPOSITIONS FOR TREATING DISEASES OR CONDITIONS INDUCED BY CORTICOSTEROIDS

[114] Applicants demonstrated herein that glucocorticoids induced DBI secretion by cultured cells, while triiodothyronine (T3) inhibited the transcription of the DBI gene. Cushing syndrome may be mediated by an elevation of endogenous or pharmacologically administered glucocorticoids. Applicants observed that patients under corticotherapy exhibit higher plasma DBI concentrations, and that clinical remission from Cushing syndrome was accompanied by a reduction of circulating DBI levels. In a mouse model of Cushing syndrome, plasma DBI levels were elevated as well.

[115] Six different methods were employed for DBI inhibition in mice, namely, (i) vaccination with DBI to induce autoantibodies, (ii) injection of a neutralizing monoclonal anti -DBI antibody, (iii) knockout of the Dbi gene, (iv) mutation of the DBI receptor Gabrg2^F77I/F77F) to abrogate its interaction with DBI, as well as (v) injections of T3 or (vi) the thyroid hormone receptor beta agonist resmetirom to block Dbi transcription. These six approaches abolished phenotypic manifestations of corticosterone-induced Cushing syndrome such as increased food intake, weight gain, excessive adiposity, liver damage, hypertriglyceridemia and signs of type 2 diabetes including hyperinsulinemia and insulin resistance. Without wishing to be bound to any theory, Applicants surprisingly found that DBI constitutes an actionable target that is causally involved in the development of Cushing syndrome.

[116] Applicants discovered herein that glucocorticoid receptor activation stimulates the release of DBI from cultured cells and increases plasma DBI concentrations in mice. Moreover, thyroid hormone transcriptionally downregulates DBI. In a mouse model of iatrogenic Cushing syndrome, knockout of the Dbi gene, mutation of Gabrg, antibody-mediated neutralization of DBI, or transcriptional downregulation of DBI by thyroid hormone, all prevent the metabolic consequences of chronic glucocorticoid administration. Accordingly, this example demonstrates that important facets of Cushing syndrome are mediated by extracellular DBI levels.

Results

Discovery of corticosteroids and thyroid hormones as ACBP/DBI modulators

[117] Applicants designed a screen to evaluate the impact of 710 distinct agonists and antagonists of neurotransmitter and hormone receptors on DBI expression by H4 human neuroglioma cells. For this, H4 cells expressing an autophagy biosensor (microtubule-associated protein 1 A/lB-light chain fused to green-fluorescent protein, GFP-LC3) were cultured for 6 or 24 h in the absence or presence of 5 pM of each of the agents assembled in a custom arrayed compound library. The cells were then subjected to an immunofluorescence detection of DBI. Automated fluorescence microscopy followed by image analyses confirmed that autophagy inducers used as positive controls (rapamycin (RAPA), torin-1) reduced the fluorescent signal corresponding to DBI while they caused the aggregation of GFP-LC3 in cytoplasmic dots (FIG. 1A - FIG. ID). Glucocorticoids exemplified by hydrocortisone (HCS, the natural human hormone) and dexamethasone (DEX, a synthetic analogue) induced a similar pattern of DBI reduction and GFP- LC3 puncta (FIG. 1A - FIG. ID). In contrast, the thyroid hormone 3,3',5-triiodo-L-thyronine (triiodothyronine, T3) attenuated DBI expression without induction of GFP-LC3 puncta (FIG. 1A - FIG. ID). These effects, also detected in the HepG2 cell line, were dose-dependent (FIG. 1C and FIG. ID) and were accompanied by the secretion of DBI into culture supernatants for rapamycin, DEX and HCS but not for T3 (FIG. IE). Both HCS and DEX upregulated, whereas T3 downregulated, the mRNA coding for DBI (FIG. IF). HCS and DEX induced the autophagy- associated lipidation of LC3, giving rise to the electrophoretically more mobile LC3-II band, and this was found both in the absence and in the presence of bafilomycin Al (BafAl), indicating that corticosteroids induce autophagic flux (FIG. 6A and FIG. 6B). Moreover, DBI secretion in response to glucocorticoids was inhibited by the knockdown of autophagy related 5 (ATG5) and autophagy related 7 (ATG 7) (FIG. 6C). The capacity of corticosteroids to attenuate intracellular ACBP expression and to increase secretion of DBI into culture supernatants was inhibited by knockdown of the glucocorticoid receptor NR3C1 (FIG. 6D - FIG. 6G). In mice, HCS induced a dose and time-dependent thymolysis (FIG. 7A and FIG. 7B), accompanied by a surge in plasma ACBP/DBI concentrations (FIG. 1G and FIG. 7C), an increase in hepatic Acbp/Dbi mRNA (FIG. 1H), but a decrease in liver DBI protein (FIG. II, FIG. 1J, and FIG. 7D - FIG. 7E) accompanied by signs of autophagy such as LC3 lipidation and depletion of SQSTMl/p62 (FIG. II, FIG. IK, and FIG. IL). Of note, the fasting and HCS-induced surge in plasma ACBP/DBI was abrogated by treatment with SAFit2 (FIG. IM), which is an inhibitor of autophagy-dependent secretion. All effects caused by HCS in vivo were mimicked by DEX and were counteracted by the glucocorticoid receptor antagonist mifepristone (Mif), indicating that they act on-target. Short-term (16 h) administration of T3 had no effect on plasmaDBI protein, but reduced Acbp/Dbi mRNA and protein in the liver (FIG. 8 A - FIG. 8E).

[118] In sum, this example indicates that corticosteroids reduce the intracellular content of DBI, while T3 downregulates ACBP/DBI expression at the mRNA level.

Glucocorticoid use and hypercortisolemia are accompanied by elevated plasma DBI in humans

[119] Hydrocortisone significantly enhanced ACBP/DBI expression in peripheral blood mononuclear cells (PBMC) from healthy human donors (FIG. IN). The expression of DBI in two human cohorts were analyzed. Results from the first cohort indicated that plasma DBI concentrations in both female and male patients receiving glucocorticoids (together n=53) were significantly higher than those in the control group (n=39) (FIG. IO and FIG. IP). The second cohort comprised patients with ACTH-dependent Cushing syndrome. Plasma DBI concentrations were significantly higher in patients with active disease (n=l l) compared to patients in remission (n=13) (FIG. IQ). In patients with active disease, DBI concentration exhibited a significant and robust positive correlation with BMI, which is a proxy of Cushing syndrome severity (FIG. 1R). Moreover, in patients in remission, supplemental glucocorticoid doses correlated with DBI (FIG. IS).

Auto-antibody-mediated neutralization of DBI prevents the metabolic consequences of Cushing syndrome

[120] It was speculated that (part of) the Cushing syndrome phenotype including increased food intake, weight gain, adiposity and type 2 diabetes (T2D) might be related to the increase in DBI. To explore this possibility, C57BL/6 mice were repeatedly immunized with DBI protein coupled to the potent immunogen keyhole limpet hemocyanine (KLH) using a protocol that breaks selftolerance against DBI and hence induces neutralizing autoantibodies. Immunization with KLH alone was performed as a control. Then the mice received high-dose corticosterone (CORT, which is the primary adrenal corticosteroid in laboratory rodents) for 5 weeks in the drinking water (or 0.66% ethanol in water as a control). Immunization with KLH-ACBP reduced the plasma DBI concentration in both control and CORT-treated mice (FIG. 2A), but did not affect CORT levels (FIG. 2B) In KLH-only immunized mice, CORT induced an increase in DBI expression in the liver (FIG. 2C and FIG. 2D) and enhanced the expression of the glucocorticoid receptor NR3C1 (FIG. 2C - FIG. 2E). All these CORT effects were abolished upon vaccination with KLH-ACBP (FIG. 2C - FIG. 2E). Concomitantly, KLH-ACBP vaccination prevented the CORT- induced surge in food intake (FIG. 2F) and body weight gain (FIG. 2G). CORT administration to KLH-only immunized mice induced a major increase in body mass with an increase in the face angle reminiscent of the ‘moon face’ found in patients with Cushing syndrome (FIG. 2H - FIG. 21). At necropsy, signs of CORT-induced thymolysis, atrophy of the adrenal gland as well as sarcopenia affecting the erector spinae and gastrocnemius muscles were not prevented by KLH- ACBP vaccination (FIG. 2J). However, the increase of liver weight, visceral, inguinal, perigonadal white adipose tissue (WAT) and interscapular brown adipose tissue (iBAT), which was accompanied by an increase in median adipocyte diameter (FIG. 9A - FIG. 9F), was attenuated by ACBP/DBI autoantibodies (FIG. 2J). In addition, circulating liver enzymes including alanine aminotransferase (ALT) (FIG. 2K) and aspartate aminotransferase (AST) (FIG. 2L) and liver triglycerides (TG) (FIG. 2M) were prevented by KLH-ACBP vaccination. Concomitantly, an elevation of circulating TG (FIG. 2N) and signs of T2D such as hyperinsulinemia (FIG. 20) and altered fasting plasma glucose (FIG. 2P) were found in KLH- only vaccinated mice treated with CORT, but not after KLH-ACBP vaccination.

[121] In sum, autoantibodies neutralizing ACBP/DBI blunt major phenotypic and metabolic manifestations of Cushing syndrome including an increase in appetite, weight gain, hypertrophy of WAT and iBAT, liver damage, dyslipidemia and insulin resistance.

Genetic inhibition of DBI prevents Cushing syndrome.

[122] Autoantibodies against DBI might mediate off-target and side effects due to immune complex disease. To rule out this possibility, Applicants attempted to inhibit Cushing syndrome by two alternative methods, namely, (i) a constitutive point mutation (F77I in subunit GABRG2) in the ACBP/DBI receptor, GAB AAR that abolishes binding to DBI, or (ii) tamoxifen-inducible expression of a CRE recombinase that excises the floxed intron 2 of the gene encoding ACBP/DBI, thus leading to its conditional ablation.

[123] Mice homozygous for the Gabrg2 F77I mutation (genotype: Gabrg2^F77I/F771') were compared to their wild type (WT) controls, while tamoxifen was injected into mice bearing Cre alone or in combination with floxed Acbp/Dbi (genotype: Db ⁷D). Then, the animals were treated with CORT in the drinking water for 5 weeks. The Gabrg2 F77I mutation blunted the CORT- induced augmentation of plasma ACBP/DBI (FIG. 3A), reduced the CORT-elicited increase in hepatic Acbp/Dbi mRNA expression (FIG. 3B), and prevented the surge in food intake (FIG. 3C) and weight gain (FIG. 3D). The Gabrg2 F77I mutation attenuated the CORT-induced atrophy of adrenal and skeleton muscle as it reversed the expansion of WAT and iBAT, as well as liver hypertrophy, but failed to prevent thymic atrophy (FIG. 3E). The conditional Acbp/Dbi knockout (Dbi'¹') (which rendered circulating ACBP/DBI and hepatic Dbi mRNA undetectable, (FIG. 3F - FIG. 3G) suppressed CORT-induced appetite (FIG. 3H) and weight gain (FIG. 31) and concomitantly prevented hypertrophy of WAT, BAT and liver, but did not prevent adrenal or thymic atrophy (FIG. 3J).

[124] Thus, this example demonstrates that genetic or endocrine inhibition of the DBI system, for example with the use of autoantibodies causing on-target effects, prevents weight gain and other metabolic manifestations of Cushing syndrome induced by corticosterone.

Monoclonal antibody-mediated neutralization of ACBP/DBI prevents Cushing syndrome

[125] It was next determined whether passive immunization of mice using a monoclonal antibody (mAb) specific for DBI (injected twice weekly at a dose of 5 mg/kg body weight) would be capable of preventing the Cushing phenotype (FIG. 4A). This protocol succeeded in neutralizing the increase of circulating DBI induced by CORT (FIG. 4B). Neutralization of ACBP/DBI did not interfere with hypercorticosteronemia (FIG. 4C), but prevented the increase in food intake (FIG. 4D), weight gain (FIG. 4E), adipocyte hypertrophy (FIG. 10A - FIG. 10F), increase in face angle (FIG. 4F), increases in circulating ALT (FIG. 4G) and AST (FIG. 4H), liver (FIG. 41) and plasma (FIG. 4J) triglycerides, hyperinsulinemia (FIG. 4K) when compared to the isotype control antibody. A decreased fasting hyperglycemia (FIG. 4L) and postprandial glycemia (FIG. 4M) were observed and might indicate an insulin resistance. Accordingly, anti-ACBP/DBI mAb (aACBP) normalized the CORT-induced alteration in glucose tolerance (GTT; FIG. 4N) and insulin tolerance tests (ITT; FIG. 40). A forced swimming experiment with mice revealed that, compared to the control group, mice receiving CORT exhibited a longer immobility, while ACBP/DBI neutralization resulted in a shorter time of immobility. Reduced time spent immobile indicates diminished depressive-like responses, indicating that the DBI neutralization can alleviate CORT -induced depressive-like behaviors in mice (FIG. 4P). Inhibition of ACBP/DBI by aACBP reversed the hypertrophy of adrenal, liver and adipose tissues, but failed to prevent CORT -induced muscular and thymic atrophy (FIG. 4Q), Multi-omics analyses supported the idea that DBI neutralization normalized most metabolic alterations induced by CORT. Thus, aACBP attenuated the CORT-induced hyperleptinemia, as well as the increase in peptide tyrosine tyrosine (PYY), C-peptide, glucose-dependent insulinotropic polypeptide (GIP), glucagon and resistin (FIG. 11). RNAseq-based transcriptomic analyses of liver tissues indicated that aACBP reversed most of the transcriptional changes induced by CORT (FIG. 12A-12G). Finally, mass spectrometric metabolomics of the liver and plasma revealed a surge in triglyceride metabolites induced by CORT that was prevented by DBI neutralization (FIG. 13A - FIG. 13C).

[126] As shown in the data provided (FIG. 14A - FIG. 14C), the prevention of weight gain and food intake was more significant in subjects that received CORT than in the subjects receiving the antidepressant citalopram (CTP), which, in contrast to CORT, failed to increase DBI in the plasma (FIG. 14D - FIG. 14E), ACBP/DBI depletion in the liver (and to upregulate ACBP/DBI in WAT) (FIG. 14F - FIG. 141) This finding indicates that appetite reduction by anti-DBI is greater under conditions where DBI is enhanced in the plasma, thus demonstrating that the effects seen by administration of anti-DBI are a result of DBI neutralization in the circulation.

[127] All the aforementioned results have been obtained in female mice. To exclude any possible sexual dimorphism, Applicants performed experiments in male C57BL/6J mice to demonstrate that CORT-induced hyperphagy, weight gain and metabolic syndrome are largely abolished by aACBP. Applicants also performed pair feeding experiments in female mice to investigate whether aACBP solely interferes with CORT-induced metabolic syndrome by suppressing hyperphagy. The CORT induced increase of circulating ACBP/DBI was attenuated by aACBP. Even when pair-feeding was performed in a way that the bodyweight of the animals treated with vehicle only, corticosterone, alone or in combination with the aACBP antibody, was undistinguishable, the metabolic effects of corticosterone. Thus, under these conditions, corticosterone caused dyslipidemia (enhanced TG and FFA), hyperinsulinemia, a shift in body composition from lean mass to fat mass determined by nuclear magnetic resonance relaxometry, an increase in adiposity and a reduction of muscle mass. Most of these signs of corticosterone- induced metabolic syndrome were attenuated by ACBP/DBI.

[128] aACBP mAb failed to prevent weight gain and food intake induced by the antidepressant citalopram (CTP), which, in contrast to CORT, failed to increase ACBP/DBI in the plasma, ACBP/DBI depletion in the liver (and to upregulate ACBP/DBI in WAT). This finding suggests that ACBP/DBI neutralization is only reducing appetite when ACBP/DBI is elevated in the circulation.

[129] Without wishing to be bound by theory, an mAb neutralizing DBI phenocopies the effects of autoantibody -mediated or genetic inhibition of DBI in thus far that it prevents the metabolic manifestations of Cushing syndrome.

Endocrine inhibition of DBI avoids Cushing syndrome

[130] As indicated above, T3 downregulates Acbp/Dbi Dbi mRNA expression. CORT and T3 were co-administered over 5 weeks (FIG. 15A), finding that this treatment led to a reduction in Acbp/Dbi mRNA in the liver and WAT, especially if combined with CORT (FIG. 15B - FIG. 15C). Similarly, the level of DBI protein detectable in liver and WAT were lower in mice treated with CORT plus T3 than in animals receiving CORT alone (FIG. 15D - FIG. 15F). Similarly, coadministration of T3 reduced CORT-induced ACBP/DBI in plasma to normal levels (FIG. 15G). T3 alone stimulated appetite, and T3 was unable to prevent appetite stimulation by CORT (FIG. 15H) However, T3 prevented weight gain induced by CORT, as measurable at the whole-body level (FIG. 151). The co-administration did not attenuate adrenal, muscular or thymic atrophy. However, the increase in fat mass and the increase in liver weight due to CORT were suppressed when co-administered with T3 (FIG. 15J - FIG. 15R). Treating mice with the selective thyroid hormone receptor P (THR-P) agonist resmetirom (RES) led to a reduction of ACBP/DBI in plasma (FIG. 5A - FIG. 5B) and decreased Acbp/Dbi mRNA levels in the liver (FIG. 5C). Coadministration of CORT with RES (FIG. 5D), similarly reduced Acbp/Dbi mRNA in WAT (FIG. 5E) and DBI protein in plasma (FIG. 5F). In contrast to T3, RES was able to prevent appetite stimulation by CORT (FIG. 5G). Accordingly, RES prevented weight gain induced by CORT (FIG. 5H). RES did not attenuate adrenal and thymic atrophy, but fully reversed the increase in body fat and liver weight induced by CORT and mediated partial effects on sarcopenia (FIG. 51).

[131] In conclusion, T3 as well as the selective THR-P agonist resmetirom decreased Dbi expression and reversed metabolic signs of Cushing syndrome.

Discussion

[132] Classical and tissue hormones as well as neurotransmitters are long- or short-distance messengers embedded in complex communication networks. For this reason, Applicants were intrigued by the possibility that DBI would be connected to other neuroendocrine factors. In a screen (which was based on neuroblastoma and hepatoma cells), Applicants found two other neuroendocrine factors, (i) glucocorticoids and (ii) thyroid hormone, that negatively affected the cellular content of DBI, although due to rather distinct mechanisms. Applicants demonstrate herein that glucocorticoid receptor agonists increase ACBP/DBI mRNA, induce the release of DBI into circulation, resulting in accumulation in the supernatant of neuroblastoma or hepatoma cells. In accordance with the in vitro results, mice treated with glucocorticoids manifested an increase in circulating DBI protein levels, and patients under corti cotherapy or with endogenous Cushing syndrome exhibited elevated DBI levels as well. T3 administration also blocked the corticotherapy-induced surge in ACBP/DBI plasma concentrations.

[133] As disclosed above, to demonstrate that DBI is involved in Cushing syndrome, Applicants used six different approaches for ACBP/DBI inhibition, namely, (i) induction of DBI neutralizing antibodies, (ii) intraperitoneal injection of a neutralizing mAb, (iii) conditional whole- body knockout of the Dbi gene, (iv) mutation of the DBI receptor Gabrg2^F77I/F77F) and (v) treatment with T3 or (vi) RES to block Dbi transcription. These six methods convergently abolished important facets of CORT -induced Cushing syndrome, particularly increased food intake, weight gain, adiposity affecting all WAT subtypes and iBAT with hypertrophy of adipocytes, as well as signs of T2D such as hyperinsulinemia and insulin resistance.

[134] Surprisingly and unexpectedly, these major characteristics of Cushing syndrome manifest through the action of extracellular DBI acting on GABAAR receptors containing the GABRG2 subunit, rather than being directly mediated by the action of glucocorticoids on metabolically active cell types (such as adipocytes, hepatocytes and muscle cells). Thus, inhibition of extracellular DBI directly treats Cushing syndrome. Such GABAAR receptors are indeed expressed by multiple metabolically active cell types. Further, ACBP/DBI neutralization demonstrated appetite and weight gain-inhibitory effects. Thus, in a heterogeneous population of overweight or obese individuals, elevated plasma DBI concentrations constitute a biomarker that predicts sensitivity to DBI inhibition.

[135] It is noteworthy that ACBP/DBI neutralization apparently has appetite and weight gain- inhibitory effects. Thus, as shown here, ACBP/DBI inhibition prevented hyperphagia and adiposity induced by glucocorticoids but not by citalopram. Of note glucocorticoids, rosiglitazone and starvation increase circulating ACBP/DBI levels, while citalopram and ghrelin fail to do so. These findings suggest that in a heterogeneous population of overweight or obese individuals, elevated plasma ACBP/DBI concentrations might constitute a biomarker that predicts sensitivity to ACBP/DBI inhibition. Moreover, pair-feeding experiments in which caloric intake (and, as a result, body weight) are kept constant indicate that glucocorticoids can induce signs of metabolic syndrome (such as dyslipidemia and insulin resistance) in the absence of weight gain and that ACBP/DBI neutralization reverses such effects independently from its effects on food intake. Cell culture and reagents

[136] Human neuroglioma H4 cells wild type or stably expressing green fluorescent protein (GFP)-LC3 were cultured in a basal medium for support growth of the cells, supplemented with 10% (v/v) o f a fetal bovine serum, 100 U/mL penicillin and 10 pg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO2. Human hepatocellular carcinoma HepG2 cells wild type or stably expressing GFP-LC3 were cultured in basal medium supplemented as above.

High-throughput screening

[137] Two thousand H4 GFP-LC3 cells/well were seeded in 384-well pciear imaging plates 24 h before experimentation. Following compound treatment, cells were fixed with 4% paraformaldehyde containing 10 mM Hoechst dye in PBS for 30 min at room temperature (RT), permeabilized with 0.1% Triton X-100 for 10 min and blocked with 5% bovine serum albumin (BSA) in PBS for 1-2 h, followed by overnight incubation with anti-ACBP antibody (Cat. sc- 376853, Santa Cruz Biotechnology, Santa Cruz, CA, US) at 4 °C. After 2x washing with PBS, cells were incubated with a fluorescence-conjugated secondary antibody for 2 h at RT. Cells were washed 3x before acquisition using a bioimager. Briefly, cells were segmented into cytoplasmic and nuclear regions according to fluorescent signal. Cytoplasmic GFP- LC3 puncta were detected and quantified by applying a top hat filter and ACBP/DBI fluorescence was assessed.

Human cohort

[138] Ninety -five dermatology patients with skin disease were included in cohort I. Subjects in the treatment group (n=56) received glucocorticoid therapy and patients (n=3) who developed resistance to synthetic glucocorticoids were excluded. For cohort II patients with ACTH- dependent Cushing syndrome (21 with ACTH dependent pituitary Cushing syndrome, i.e., Cushing disease, and 3 with ectopic ACTH secretion due to a bronchial carcinoid) (median age 56.5 years; range, 22-73) were prospectively recruited from 2014 to 2017 at Marseille University Hospital, France. The active group consisted of 11 newly diagnosed patients. The « remission » group consisted of 13 patients in remission for at least 2 years, but no more than 6 years, regardless of the treatment modality.

Animal experimentation

[139] Six-week-old male and female C57BL/6L mice were maintained in 12-h light/dark cycles, under temperature-controlled SPF conditions with food and water ad libitum. Food intake/day/mouse was calculated by measuring chow weight semiweekly. Mice were kept one week to acclimate upon arrival before commencing experiments. ACBP/DBI autoimmunization

[140] Autoantibody production via active immunization was initiated by conjugating a keyhole limpet hemocyanin and a mouse recombinant ACBP/DBI (KLH-ACBP). Wild-type 6- week-old female C57BL/6 mice were immunized via intraperitoneal (i.p.) injections of 30, 30, 30, and 10 pg of KLH-ACBP, or KLH alone, emulsified (1 : 1) with Montanide ISA-5 Ivg on days 0, 7, 14, and 21, respectively. After 4 weeks ACBP autoantibodies were assessed by subjecting plasma to immunoblotting against recombinant target protein. For further validation ELISA was used to assess circulating ACBP levels. Starting from week 6, Cushing syndrome was induced by administration for 5 weeks of a corticosterone dissolved ethanol (EtOH)), at 100 pg/mL in drinking water. Final EtOH concentration was 0.66%. Water consumption was measured semiweekly, and corticosterone concentration was adjusted to maintain an average daily corticosterone exposure of approximately 500 pg/mouse. The control group received 0.66% vehicle in drinking water. Gabrg2^F77I/F771 mutation. C57BL/6 Gabrg2^tmlWul A Gabra flox mice came from the Jackson Laboratory. All mice used for experimentation were female. Dbi gene knockout. C57BL/6 Acbp^ with loxP sites flanking Acbp exon 2 were generated. All mice used for experiments were female, mice. Crerecombinase was activated by administration of tamoxifen (i.p. 75 mg/kg body weight/mouse/day for 5 days). Tamoxifen was diluted in 90% com oil, 10% EtOH (v/v) at a concentration of 20 mg/mL under agitation overnight at 37 °C. Following the procedure, mice were kept for at least a week (wash out) before starting the treatment.

Neutralization of DBI by anti-ACBP mAh

[141] Experiments were conducted with 8-week- old female C57BL/6 mice. Passive immunization was performed by semiweekly i.p. injections of 5 mg/kg body weight anti-ACBP mAb or isotype control (IgG2a, clone 2A3).

Resmetirom treatment

[142] Experiments were conducted with 8-week-old female C57BL/6 mice. Resmetirom was prepared in 10% DMSO, 40% PEG300, 5% Tween 80, and 45% drinking water (v/v), at a final concentration of 0.033 mg/mL.

Triiodothyronine (T3) system

[143] Experiments were conducted with 8-week-old female C57BL/6 mice. The initial concentration of T3 was 3.3 pg/mL and was adjusted to maintain an average daily exposure of approximately 10 pg/mouse. Citalopram treatment

[144] Experiments were conducted with 8-week-old female C57BL/6 mice. Citalopram was administered at a concentration of 0.15 mg/ml (diluted in water). For all experiments described above corticosterone was administered as described earlier. Water consumption, body weight and food intake were measured semiweekly. At the end of the 5th week, mice were sacrificed, and tissues were collected and weighed.

Co-administration of dexamethasone and mifepristone

[145] Experiments were conducted with 8-week-old female C57BL/6 mice. Dexamethasone was diluted in 10% DMSO, 90% corn oil (v/v) and administered i.p. (5 mg/kg body weight). Mifepristone was diluted in drinking water containing 1% carboxymethyl cellulose with 0.20% Tween 80 (v/v) and administrated by oral gavage (120 mg/kg body weight). Dexamethasone was injected daily for two weeks. Mifepristone was administered from day 7 to day 14.

SAFit2 and fasting experiment

[146] Experiments were conducted with 8- week-old female C57BL/6 mice. SAFit2 was solubilized in vehicle (4% EtOH, 5% Tween80, and 5% PEG400 (v/v) in 0.9% saline (Veh-1)). Corticosterone (CORT) was dissolved in 100% EtOH, to a final EtOH concentration of 0.66% (Veh-2). Fasting was performed by removing food for 24 h. SAFit2 was injected i.p. at 40 mg/kg/day and corticosterone (500 pg/mouse) was given by oral gavage.

[147] Pair-Feeding: C57BL/6J female mice were housed under standard conditions with a 12 h light/dark cycle and ad libitum access to water. Mice were randomly assigned to four treatment groups: vehicle, corticosterone, anti-ACBP/DBI antibody, and corticosterone plus anti-ACBP/DBI antibody. To ensure controlled food intake, a pair feeding protocol was implemented. Initially, baseline body weights and food consumption were measured over a 3 day period to establish average intake. The vehicle-treated group served as the control for food intake. The average daily food intake of the vehicle-treated group was calculated and used to determine the amount of food provided to the other groups. Mice in the corticosterone, anti-ACBP/DBI antibody, and corticosterone plus anti-ACBP/DBI antibody groups were given the same amount of food consumed by the control group on the previous day. Food intake and body weights were recorded daily to ensure precise matching of food quantities across groups. Adjustments in food allocation were made based on the control groups' consumption. Daily corticosterone exposure was adjusted to approximately 500 pg/mouse. Indirect calorimetry measurements

[148] Indirect calorimetry was conducted using automated metabolic cages, in which mice were individually housed for consecutive seven-day periods over four weeks. Each cage was equipped with bedding and provided unrestricted access to food and water. Food and water consumption were continuously monitored. Measurements included oxygen (O2) consumption, carbon dioxide (CO2) production, the respiratory exchange rate (RER = VCO2/VO2), and heat production (H). Locomotor activity, including ambulatory and fine movements as well as speed, was tracked using an infrared light beam-based system. O2 and CO2 volumes were assessed at the inlet ports of each cage and periodically calibrated against a reference empty cage. All measurements were conducted at four-minute intervals throughout the experiment, ensuring continuous recording during both light and dark phases.

Forced swim test

[149] For forced swim test (FST) mice were placed in a vertical glass cylinder filled with water and behavior was observed for 5 min. The water temperature was maintained at 25 °C. Distinct phases of active swimming and immobility were documented. The time spent immobile during the test was considered an indicator of behavioral despair. Conversely, less time spent immobile suggested potential antidepressant effects.

Face angle assessment

[150] Mice were anesthetized and placed on a scaled matrix with a protractor. Birds view images were taken and analyzed to measure the angle between the edges of the two cheeks considering the tip of the nose as the vertex.

Western blots

[151] Cells were collected after being washed twice with PBS. A radioimmunoprecipitation assay (RIP A) buffer was used for protein extraction. Samples were subjected to ultrasonication for 3 pulses of 10 sec on ice and then centrifuged for 10 min at 13,000 x g. Analogously 30 pg liver tissues and 60 pg adipose tissue were collected in Precellys lysing kits with RIPA buffer and protease/phosphatase inhibitors, followed by 2 cycles of homogenization for 20 sec at 5,500 rpm using a homogenizer. Then samples were centrifuged at 13,000 x g for 30 min and supernatants were collected. A BCA assay was used for protein concentration assessment. Loading buffer and reducing agent were added before denaturation (100 °C for 15 min). After sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransfer to PVDF membranes, unspecific binding sites were blocked for 1 to 2 h with 5% BSA at RT, followed by overnight incubation at 4 °C with primary anti-human ACBP/DBI antibody (Cat. sc-376853, Santa Cruz Biotechnology), anti -mouse ACBP/DBI antibody (Cat. ab231910, Abeam, Cambridge, UK), anti-LC3B antibody, anti-SQSTMl/p62 antibody, anti-glucocorticoid receptor (D6H2L) XP antibody or HRP coupled anti-P-actin antibody. Membranes were washed and processed by incubation with HRP-coupled secondary antibody for 1 h at RT. Imaging and quantification were conducted.

Mouse and human ACBP/DBI ELISA

[152] Cells were treated and culture supernatants were collected, centrifuged at 500 x g for 5 min and stored at -80 °C until analysis. For in vivo experiments, mouse plasma was collected with lithium heparin separator, then centrifuged at 1,500 x g for 10 min and ACBP/DBI levels were measured by enzyme-linked immunosorbent assay (ELISA). Human anti- ACBP/DBI (Cat. MBS768488, MyBioSource, San Diego, CA, US) and murine anti- ACBP/DBI capture antibodies (Cat. ab231910, Abeam) diluted 1 pg/mL in PBS were used for coating high-binding 96-well plates with 100 pL/well overnight at 4 °C. Subsequently, plates were washed twice with washing solution (0.05% Tween 20 (v/v) in PBS) and unspecific binding was blocked with 100 pL sterile blocking buffer (1% BSA, 0.05% Tween 20 (v/v) in PBS) for 2 h at RT. For sample assessment, 100 pL/well of either sample or standard (human serum at 1 :50-1 :75, murine plasma at 1 :20, and cell culture supernatant at 1 :4 dilution, with the flexibility to adjust as dictated by experimental requirements) were incubated for 2 h at RT and subsequently being rinsed 3x with washing buffer. Then 100 pL/well human anti-ACBP/DBI (LS-C299614, Lifespan Biosciences, Linwood, W A, US) and murine anti-ACBP/DBI detection antibodies (Cat. MB S2005521, MyBioSource) diluted 1 pg/mL in PBS were added for one hour at RT followed by 3x rinsing with washing buffer. Subsequently, plates were incubated 30 min at RT with 100 pL of HRP-coupled avidin diluted in PBS (1/5000 for human and 1/1000 for murine samples). Subsequently plates were rinsed 4x with washing buffer. To visualize bound protein, 100 pL of ELISA substrate solution was added and incubated 10- 30 min at RT in the dark. Following 50 pL of stop solution (2N H2SO4) was added and absorbance was measured at 450 nm using a microplate reader.

RNA interference

[153] Two siRNAs were used to knockdown the expression of NR3C1 in H4 cells employing the following siRNA oligos:

Biochemical assays

[154] Several ELISA kits were used for detecting biochemical indices, such as mouse ALT ELISA kit, mouse AST ELISA kit, mouse insulin ELISA kit, triglyceride assay kit, free Fatty Acid Assay Kit, and plasma corticosterone ELISA kit. Plasma CORT samples were collected during the first hour of light at 08:00 h, and the collection process was performed under general anesthesia using isoflurane inhalation. All procedures strictly followed the manufacturer's protocol. For Luminex multiplex assays, plasma was collected in an EDTA anti-coagulant collecting tube with additional DPPIV inhibitor, protease inhibitor cocktail, aprotinin and serine protease inhibitor, then centrifuged for 10 min at 1000 x g within 30 min of collection. Then immediately aliquoted and stored at -80 °C. Mouse hormones were detected by using the mouse metabolic hormone magnetic bead panel and adiponectin single kit following the manufacturer’s protocol.

Glucose tolerance test and insulin tolerance test

[155] Mice were trained for tail pinch adaptation one week in advance. Animals were fasted for 6 h to perform a glucose tolerance test (GTT). Blood for glycemia measurement was collected from tail vein incisions 0, 15, 30, 60, 90, and 120 min after the injection of D- glucose (2 g/kg body weight, i.p. For insulin tolerance test (ITT), animals were fasted for 2 to 4 h before injection of insulin (0.5 U/kg body weight, /./?.). Blood was collected from tail cuts at 0, 15, 30, 60, 90, and 120 min and glucose was measured using a precision glucometer. Mice were monitored frequently and hypoglycemic shock was avoided by administration of 20% glucose solution. The homeostatic model assessment for insulin resistance (HOMA-IR) was calculated using the following formula: fasting plasma glucose (measured after 16 h of fasting, in mM) multiplied by fasting plasma insulin (measured after 16 h of fasting, in pU/L), divided by 22.5.

Histopathology

[156] Fresh tissue was harvested and fixed in 4% PF A (or 10% formalin) for a maximum of 24 h at RT, then processed by serial paraffin-embedment. Ten micron thick slices were obtained with a microtome. Standard hematoxylin/eosin staining was performed and slides were scanned by a semi-automatic slide scanner equipped with 20x and 40x objectives. The images were analyzed and quantified using software.

Gene expression analysis

[157] For RNA extraction, a kit was used. About 25-30 mg of tissue was collected in a lysis buffer. The tissue was homogenized in two cycles using a homogenizer for 20 sec at 5,500 rpm. The lysate was centrifuged and subjected to further purification procedures as necessary. About 1 pg total RNA was reversed transcribed using a cDNA Synthesis Kit. Quantitative real- time PCR (qRT-PCR) was conducted by using a master mix with a real-time PCR System.

The 2'^AACT method was used for analysis of real-time PCR data with the following primers:

Bulk RNA-sequencing

[158] Total RNA was isolated from murine liver tissue. Subsequent to sample preparation, sequencing was conducted on a sequencing instrument, yielding paired-end reads of 2 * 150 base pairs, with a total of 40 million reads obtained per sample. Alignment and mapping to the GRCm39 (mm39) genome assembly were accomplished using HISAT2. The resulting SAM file was subsequently processed by HTSeq-count utilizing the union mode and including non-unique features for the generation of gene count tables.

Differential gene expression analysis

[159] For gene expression comparison, volcano plots using the Enhanced Volcano package, Venn diagrams using the Venn package, and heatmaps using the Complex Heatmap package were generated. Differentially expressed genes (E- value < 0.05 and |log2FoldChange| > 1.0) were selected and further assessed by functional enrichment analysis, employing various R-packages, including clusterProfiler, tidyverse, ggplot, forcats, biomaRt, stringr, org.Mm.eg.db. The gene background was defined using all sequenced genes.

Liver sample preparation and metabolite analysis

[160] Approximately 30 mg of liver sample was homogenized. For extraction of endogenous metabolites, samples were mixed with 1 mL of ice-cold 90% methanol, 10% water (v/v) at -20 °C, along with a cocktail of internal standards and thoroughly homogenized using a tissue homogenizer, applying 3 cycles of 20 sec at 6500 rpm. After centrifugation (10 min at 15,000 x g, 4 °C) supernatants were divided into fractions and processed following established protocols. One fraction was used for short chain fatty acids analysis (derivatization prior to injection). Another fraction was allocated to LC/MS analysis, while the third fraction was employed for GC/MS analysis. Following the analysis fractions were collected, dried at 40 °C, and subsequently kept at -80 °C. Widely targeted analysis was conducted using a gas chromatography system coupled to a quadrupole mass spectrometer for GC-MS/MS. For the analysis of polyamines, short-chain fatty acids, and bile acids, LC-MS/MS was employed. Furthermore, a pseudo-targeted analysis was performed.

Data analysis

[161] Unless otherwise specified, data are presented as means ± SEM. Prior to conducting statistical analysis, normal distribution of the results was assessed using the D'Agostino& Pearson normality test, Shapiro-Wilk normality test, and the Kolmogorov- Smirnov test. For data that exhibited a Gaussian distribution, unpaired two-tailed Student's t-test or one-way analysis of variance (ANOVA) or two-way ANOVA was employed. In the case of data with non-Gaussian distributions, the Mann-Whitney U test was used for two-group comparisons, while the Kruskal- Wallis test followed by Dunn's post hoc test was used for comparisons involving multiple groups. Body weight curves and food intake were longitudinally analyzed with type II ANOVA.

EXAMPLE B: METHODS AND COMPOSITIONS FOR TREATING DISEASES OR CONDITIONS INDUCED BY CORTICOSTEROIDS, SYNOVIAL INFLAMMATION, AND DEGENERATIVE JOINT DISEASE

Materials and Methods

Human cartilage samples

[162] Articular cartilage explants from subjects without joint imnflammation (mean ± SD: 78.57 ± 18.42 years old, Mankin Score 0, n = 7), and subjects with joint inflammation (mean ± SD: 84 ± 7.31 years old, Mankin Score 8, n = 5; mean ± SD: 77.75 ± 10.5 years old, Mankin Score 12, n = 4) were employed to quantify the expression of both ACBP/DBI and GABRG2 proteins. Joint inflammation was diagnosed according to Mankin Score. These samples were obtained from the collection of samples for the investigation of Rheumatic Diseases. This collection was registered in the National Registry of Biobanks, with registration code: C.0000424 and approved by the Ethics Committee of Galicia with registration code: 2013/107.

Immunohistochemistry

[163] Human cartilage samples from donors with and without joint inflammation were fixed in 10% zinc-buffered formalin for 24h. Knee joints from C57B1/6J mice with joint inflammation induced by DMM+MCL were fixed in 4% buffered paraformaldehyde overnight at 4 °C and decalcified in TBD-2 for 48 hours. Then, the two types of samples were embedded in paraffin, sliced to 4 pm thickness deparaffinized in xylene and rehydrated in graded ethanol and water. For antigen unmasking, lOmM sodium citrate (pH = 6.0) at 95 °C was added on the sections for 15 minutes and renewed every 3min. Next, slides were washed with water for 15 minutes at room temperature (RT) and a Bloxall® pre-block solution was added for 10 minutes. Afterward, samples were washed with 1% TBS-T and blocked with 10% goat serum for 1 hour at RT. Then, sections were incubated with primary antibodies for human DBI (1 :50, Santa Cruz #sc376853), mouse DBI (1 :500, Abeam #Ab231910), human GABRG2 (1 :200, Antibodies Online #ABIN754003) or mouse LC3 (1 :750, MBL #PM036) overnight at 4 °C. After washing with 1% TBS-T, sections were incubated with horseradish peroxidase rabbit/mouse secondary antibody for 30 minutes at RT and washed with 1% TBS-T. The signal was developed with Diaminobenzidine (DAB)- Peroxidase substrate kit. Finally, sections were mounted with DePex. Multiple images of each slide were taken using an Olympus BX61 microscope. The number of DBI, GABRG2 and LC3- positive cells was quantified using Image J, and the quantifications consisted of counting the positive cells from several images and establishing the total sum.

Induction of ACBP/DBI knockout

[164] Male C57B1/6J mice bearing a floxed exon 2 oiAcbp/Dbi gene in homozygosity as well as a ubiquitously expressed tamoxifen-inducible transgene coding for the Cre recombinase in homozygosity (genotype: UBC- cre/ERT2 Acbp/Dbiⁱ abbreviated as Dbi

or 'wild type' (WT) control mice (genotype: Acbp/Db f without CRE) were injected with tamoxifen (i.p. 75mg/KG BW tamoxifen/mouse daily during 5 days). Prior to injection, tamoxifen was diluted in corn oil (90%) + ethanol (10%) at a concentration of 20 mg/ml and shaken overnight at 37 °C.

Induction and treatment of degenerative joint disease

[165] Fifteen-weeks-old male C57B1/6 mice were subjected to destabilization of the knee joint of the right posterior limb by cutting the medial collateral ligament (MCL) and surgical destabilization of the medial meniscus (DMM). Sham procedures consisted in subjecting the control left knee to an operation involving only the opening of the joint capsule without ligament transection and meniscectomy. Two weeks later, mice were subjected to right intra-articular (i.a.) injections of anti-ACBP/DBI mAbs (either clone 7G4a or clone 82B2G9) or their respective isotype control antibodies (IgG2a for clone 7G4a or IgGl for clone 82B2G9) at concentrations of 1.25 pg/pl PBS buffer and an injection volume of 8 pL (z.e., 10 pg of antibody per joint) twice per week for 12 weeks, when mice were sacrificed and the knee articulations were excised and subjected to paraformaldehyde fixation for 24 hours and subsequently decalcified for 48 hours. In some experiments, 7G4a mAb and isotype control IgG2a were not injected intra-articularly but intraperitoneally (i.p.) at a dose of 5 mg/kg body weight. All animal experimentation procedures applied to institutional rules and guidelines. Mice were housed in a temperature-controlled environment with 12 h light/dark cycles and were fed with diet and water ad libitum. Functional assessment of degenerative joint disease

[166] A static weight bearing incapacitance test was used to monitor the distribution of weight between the left and the right extremities in mice. Mice were placed into a size-adjustable holder specially designed to maintain the animal comfortably and naturally positioned on two separated sensor plates. These sensors allow the measurement of the weight distribution (in grams) in each hind paw reflecting spontaneous postural changes. Each measurement had a duration of 3 seconds and a total of 10 measurements were made per mouse. The recorded data were displayed in a unit that shows real-time weighing curves for left and right paws, as well as the static values. This unit assesses fast postural changes over the test period due to its 1000 Hz sampling frequency. In the absence of hind paw injury, animals applied equal weight to both hind paws, indicating a postural equilibrium. After unilateral hind paw tissue injury, a change in the weight distribution on the sensor occurs, depending on the level of discomfort. These measurements were performed on a weekly basis.

Ultrasound biomicroscope B-mode analysis

[167] An ultrasound biomicroscope (UBM) Imaging System was employed to measure the surface area occupied by the tibia-femur triangle as well as hypoechographic zones, which both increase in subjects with joint inflammation, to determine the so-called UBM score. These measurements were performed on live anesthetized mice using 2% isoflurane in air, allowing for monthly examinations of the same mice. To monitor the development of disease, each mouse was placed on a heat platform in a supine position and the hair was removed from the paws with depilatory cream. To perform an ultrasound examination the knee was placed at 90°. A high frequency transducer (40 MHz) was used in a sagittal and transversal position to obtain a better anatomical observation of the knee. Several images were acquired and image acquisition was applied to frame-based modes based on B-mode data. After examining the joint, mice were deprived of isoflurane and a warm light was provided to facilitate awakening. To visualize morphological changes in the joint of this degenerative joint disease model, three ultrasound images of each knee were analyzed.

Histological assessment of degenerative joint disease

[168] Saf O-Fast Green staining was performed on paraffin-embedded sagittal knee slices (thickness: 4 pm) to determine the following parameters in the femoral condyle and in the tibial plateau: OARSI grade (which determines severity of cartilage damage by attributing a score to erosion, destruction and calcification), OARSI stage (which determines the percentage of cartilage damage), as well as the composite OARSI score (grade multiplied by stage). In addition, the synovial inflammation score was determined, which results from the addition of three scores measuring (i) enlargement of the synovial lining layer; (ii) the reduction of the cellularity of synovial stroma with increase in multicellularity, as well as the later formation of pannus and rheumatoid granulomas; and (iii) the density of the synovial leukocyte infiltrate culminating in the formation of follicle-like aggregates.

Biochemical characterization of monoclonal antibodies 7G4a and 82B2G9

[169] The development of the monoclonal antibody (mAh) 82B2G9 against recombinant mouse ACBP/DBI protein was conducted using hybridoma technology. This antibody was chosen based on its capability to detect the ACBP/DBI protein in both ELISA and Western blot assays. Furthermore, its specificity was validated through Western blot analyses using human and mouse ACBP/DBI knockout cell lines.

[170] A linear and conformational epitope mapping was employed. The linear and Chemically Linked Peptides on Scaffolds (CLIPS) peptides were synthesized based on the amino acid sequence of the target protein using standard Fmoc-chemistry and deprotected using trifluoric acid with scavengers. For conformational mapping, the constrained peptides were synthesized on chemical scaffolds in order to reconstruct conformational epitopes, using CLIPS technology. The binding of antibody to each peptide was tested in a PEPSCAN-based ELISA. The 82B2G9 antibody exhibited strong signals on both human and mouse sequences of ACBP/DBI, with similar putative epitopes identified. For the human ACBP/DBI protein sequence, the core epitope was determined as PSDEEMLFIYG, and for the mouse ACBP/DBI protein sequence, it was PTDEEMLFIYS. 7G4a mAb did not show strong binding to peptides derived from human ACBP/DBI, but exhibited strong binding to some peptides derived from mouse ACBP/DBI protein. The core epitope in mouse ACBP/DBI recognized by 7G4a was identified as DRPGLLDL.

Measurement of ACBP/DBI concentrations by ELISA

[171] Mouse plasma was obtained from blood samples collected in lithium heparin tubes and centrifuged at 8,500 rpm for 10 minutes at 4 °C. ACBP/DBI concentrations were measured using an ELISA assay. Briefly, high-binding 96-well plates were coated with 100 pL/well of anti- ACBP/DBI capture antibody (1 pg/mL, diluted in PBS) and incubated overnight at 4°C. After washing, plates were blocked with 1% BSA in PBS-Tween 20 for 2 hours at room temperature (RT). Samples (murine plasma 1/20) and standards were added in 100 pL volumes and incubated for 2 hours at RT. Plates were washed and incubated with 100 pL of detection antibody (1 pg/mL) for 1 hour at RT, followed by incubation with HRP-conjugated avidin (1/1000 for murine) for 30 minutes. After washing, 100 pL of TMB substrate was added and incubated in the dark for 10-30 minutes, followed by 50 pL of stop solution (2M H2SO4). Absorbance was read at 450 nm using a microplate reader.

Plasma Cytokine Multiplex Analysis

[172] Plasma cytokine concentrations were determined using a proximity extension assay with the Target 48 Mouse Cytokine panel. Briefly, 1 pL of plasma from fresh aliquots stored at -80°C was thawed and incubated for 16 hours at 4°C in an incubation mix containing cytokine-specific antibody pairs, each coupled to forward and reverse probes. Extension of the complementary probes occurred on a thermal cycler and was possible only when both antibodies corresponding to a single cytokine were in close proximity, binding to neighboring epitopes on the target cytokine. For detection, a microfluidic chip was primed and loaded with the samples and probes using an MX controller, and real-time PCR was performed. PCR data analysis was conducted using Real- Time PCR Analysis software, with automatic (global) Ct threshold determination set using the following parameters: quality threshold = 0.5, linear baseline correction. Data processing, quality control, and determination of absolute concentrations were performed using the Olink® NPX Signature software (v 1.13.0).

[173] The absolute concentrations were imported into R (version 4.3.3) and log2-fold change- transformed data were visualized using the ComplexHeatmap package (version 2.16.0). The distribution of log2 -transformed values was tested for normality using the Shapiro-Wilk test. Cytokines that followed a normal distribution across the four groups were analyzed using two-way ANOVA, followed by Tukey’s HSD test for pairwise comparisons. Cytokines that did not follow a normal distribution were analyzed using the Kruskal -Wallis test, followed by Dunn's post-hoc test, with Benjamini-Hochberg correction applied for multiple comparisons.

Acute liver damage in mice

[174] To induce hepatotoxicity, male 12-week-old C57BL/6 mice were treated with 300 mg/kg acetaminophen for 16 hours Mice received intraperitoneal injections of anti-ACBP/DBI monoclonal antibodies (either clone 7G4a or clone 82B2G9) or their respective isotype control antibodies at a concentration of 2.5 pg/g body weight. The a-ACBP/DBI or IgG injections were administered 4 hours before and immediately prior to the induction of hepatic injury. Elevations of transaminases (aspartate transaminase, AST, and alanine transaminase, ALT) were determined in the plasma.

Immunodetection of proteins

[175] For Western blot analyses, human and mouse recombinant ACBP/DBI protein were boiled for 5 min in Laemmli sample buffer, and 10 ng, 25 ng , 50 ng and 100 ng amounts of protein were separated on 4-12% Bis-Tris acrylamide precast gels and electro-transferred to nitrocellulose membrane at a constant voltage of 100V at 4°C for 1.5 h. Unspecific binding sites of the membranes were saturated by incubating for 1 h in 0.05% Tween 20 (v:v in TBS) supplemented with 5% non-fat powdered milk (w:v in TBS). Subsequently, proteins were determined by overnight incubation of membranes with 82B2G9 antibody. Red ponceau was used to control equal loading of lanes. The blots were revealed using appropriate horseradish peroxidase (HRP)-labelled secondary antibodies plus ECL prime chemiluminescent substrate. Different exposure times were utilized for each blot with a charged coupling device camera in a luminescent image analyser LAS 4000 to ensure the linearity of the band intensities. Quantification of proteins was carried out by densitometric analysis of the bands using ImageJ software and was expressed as relative expression levels.

Statistical Analyses

[176] For all figures, except where otherwise specified, a linear model was fitted using robust regression; p-values were derived from the significance of model coefficients.

Treatment of arthropathy using anti-DBI agents

Mitigation of histological signs of joint inflammation and/or degenerative joint disease (DJD) by ACBP/DBI neutralization

[177] In 15-week-old mice, joint inflammation was induced by mechanical destabilization of the knee joint of the right posterior limb by cutting the medial collateral ligament (MCL) and surgical destabilization of the medial meniscus (DMM) (FIG. 16A). The left knee was subjected to sham surgery. After a post-surgical recovery phase of 2 weeks, the affected joint was subjected to repeated intraarticular (i.a.) injections of anti-ACBP/DBI mAb (10 pg of antibody in 8 pl of volume) or isotype control antibody twice weekly for 12 weeks until 28 weeks of age and euthanized at 29 weeks of age (FIG. 16B).

[178] Fifteen to 16 weeks after surgery, ACBP/DBI plasma concentrations were approximately 3 times higher in mice with joint inflammation than in healthy controls (FIG. 17A). Then, we reproduced this approach in mice that can be subjected to tamoxifen-inducible whole-body knockout of floxed Acbp/Dbi (genotype: UBC- cre/ERT2. cbp/Dbi^iK, abbreviated as Dbi or 'wild type' (WT) control littermates (Acbp/Dbi^f/f without CRE). Mice of both genotypes were treated with tamoxifen, subjected to joint inflammation-inducing or sham surgery, and ACBP/DBI plasma levels were measured (FIG. 17B). As expected, the joint inflammation-induced surge in circulating ACBP/DBI was observed in WT mice but prevented in Dbi animals (FIG. 17C). Moreover, the percentage of cells staining positively for ACBP/DBI in joints were reduced in Dbi ■^/_ compared to WT mice (FIG. 18A and FIG. 18B), suggesting that the tamoxifen-induced knockout was also efficient in this location. Interestingly, a decrease in intracellular ACBP levels was observed in WT mice under joint inflammation conditions. This aligns with the increase in ACBP/DBI levels in the plasma of the same mice. These findings suggest that ACBP may be secreted from tissues into the bloodstream in response to the pathological changes associated with joint inflammation.

[179] Human joint inflammation specimens were also subjected to immunohistochemical detection of ACBP/DBI and its receptor gamma-aminobutyric acid receptor subunit gamma-2 (GABRG2), indicating that both proteins, the ligand and the receptor, are present in the joints of patients with joint inflammation.

[180] Histological analyses of the affected and contralateral knees revealed that ACBP/DBI neutralization significantly (p<0.05, two-way ANOVA) reduced cartilage destruction, as quantified at the levels of Osteoarthritis Research Society International (OARSI) grade, stage and score, when compared to the isotype control antibody (FIG. 18A - FIG. 18D). These findings indicate that local ACBP/DBI neutralization mitigates joint inflammation and/or degenerative joint disease.

As an alternative strategy of ACBP/DBI inhibition, mice were intraperitoneally with a monoclonal antibody (7G4a) that neutralizes extracellular ACBP/DBI (abbreviated oc-DBI) or an isotype control IgG antibody. This treatment was optionally combined with the synthetic glucocorticoid dexamethasone (DEX) (FIG. 19A). While DEX alone did not inhibit joint inflammation severity, DBI alone or in combination with DEX did mitigate the histological signs of joint inflammation (FIG 19B - FIG. 19E)

[181] Experimental joint inflammation is linked to the systemic upregulation of ACBP/DBI. In addition, knockout of ACBP/DBI or antibody-mediated neutralization of extracellular ACBP/DBI reduces the severity of joint inflammation, indicating a bidirectional crosstalk between joint inflammation and the ACBP/DBI system.

Treatment of synovial inflammation using anti-DBI agents

[182] In addition, treatment of the mice with anti-ACBP/DBI mAb, as described above, caused a significant diminution of synovial inflammation, as quantified by means of a standardized histopathological scoring system (FIG. 20A and FIG. 20B). These findings indicate that local ACBP/DBI neutralization mitigates synovial inflammation.

Use of anti-DBI agents for treating deviating weight distribution caused by joint inflammation and/or degenerative joint disease

Functional improvement of weight distribution symmetry by ACBP/DBI neutralization [183] Intraarticular injections of anti-ACBP/DBI antibody mitigate joint inflammation at the functional level. In the next series of experiments, Applicants explored the possibility of treating joint inflammation locally, by intraarticular (i.a.) injections of oc-DBI. The right knee joints of 15-week-old male C57B1/6 mice were subjected to mechanical destabilization by MCL+DMM surgery, while the left knees underwent sham surgery. After a post-surgical recovery phase of 2 weeks, the affected joint was subjected to repeated i.a. injections of oc-DBI (10 ng of antibody in 8 pl of vehicle) or isotype control antibody twice weekly for 12 weeks until 28 weeks of age and then were euthanized (FIG. 21A). At endpoint, oc-DBI induced a reduction of immunohistochemically detectable DBI-positive cells in joints with inflammation (FIG. 21B and FIG. 21C). Applicants also observed that inflammed joints injected with isotype control antibody exhibited a reduction in autophagic (LC3B-positive) cells. This LC3B reduction was suppressed by oc-DBI (FIG. 21D and FIG. 21E), indicating that local ACBP/DBI neutralization may restore normal levels of autophagy.

[184] The combination of pain and mechanical joint failure resulting from joint inflammation and/or degenerative joint disease causes mice to asymmetrically distribute their weight between their legs. Quantitation of the asymmetry of weight distribution by means of a dynamic balance demonstrated that mice subjected to joint destabilization followed by intra-articular injections of anti-ACBP/DBI mAb exhibited less imbalance in their bodyweight distribution than control animals treated with the isotype control antibody (FIG. 22A and FIG. 22B). These findings support the conclusion that treatment of joint inflammation and/or degenerative joint disease with ACBP/DBI neutralization improves functional outcome.

Improvement of echographic signs of joint inflammation by ACBP/DBI neutralization

[185] The evolution of degenerative joint disease was determined by monthly non-invasive assessments ofjoint inflammation using an ultrasound biomicroscope (UBM). It was observed that the areas occupied by the tibia-femur triangle, as well as hypoechogenic zones corresponding to inflamed tissue with a high content of fat, liquid or semi-solid material expanded in knees of subjects with joint inflammation as compared to sham-operated controls. Both these joint inflammation-associated alterations were mitigated by injection of anti-ACBP/DBI mAb clone 7G4a (FIG. 24A - FIG. 24C)

[186] Antibody mAb 7G4a detects recombinant mouse (but not human) ACBP/DBI protein in immunoblots. Another mAb, 82B2G9, recognized both human and mouse ACBP/DBI (FIG. 23A). Of note, both mAbs recognize different epitopes in mouse ACBP/DBI, likely explaining their differential cross-reactivity with respect to human ACBP/DBI (FIG. 23B and FIG. 23C). However, both antibodies similarly reduced acetaminophen-induced hepatotoxicity (FIG. 23D - FIG. 23F), indicating that they effectively inhibit endogenous mouse ACBP/DBI in vivo.

[187] Importantly, mAb 82B2G9 was as efficient as mAb 7G4a in mitigating degenerative joint disease (DJD) in the mouse model (FIG. 25A - FIG. 25C). Hence, distinct anti-ACBP/DBI mAbs, including a cross-species-reactive mAb can be used for treating degenerative joint disease.

[188] The aforementioned experiments have been based on the use of local, intra-articular (i.a.) injections of anti-ACBP/DBI mAb. Importantly, systemic (intraperitoneal, i.p.) injections of anti- ACBP/DBI mAb also led to the attenuation of degenerative joint disease and joint inflammation at the level of tibia-femur triangle area (FIG. 26A - FIG. 26C). Hence, both local and system administration of anti-ACBP/DBI mAb can be effective against degenerative joint disease (DJD).

[189] Administration of anti-ACBP/DBI mAb convergently attenuated the echographic, histological and functional manifestations of joint inflammation, showing that inhibition of ACBP/DBI has therapeutic effects against degenerative joint disease. This effect is observed when neutralizing mAbs are injected into affected joints but is also found after systemic injection of anti- ACBP/DBI mAb.

Mitigation of histological joint inflammation and systemic inflammation by intra-articular ACBP/DBI neutralization

[190] Histological analyses performed at endpoint confirmed that ACBP/DBI neutralization significantly (p<0.05, two-way ANOVA) reduced cartilage destruction, as quantified at the levels of OARSI grade, stage and score, when compared to the isotype control antibody (FIG. 27A - FIG. 27D). In addition, anti-ACBP/DBI mAb caused a significant (p<0.05, two-way ANOVA) diminution of synovial inflammation, as quantified by means of a standardized histopathological scoring system (FIG. 28A and FIG. 28B). Thus, local ACBP/DBI neutralization by i.a. injection of oc-DBI mitigates histological signs of joint inflammation-associated synovitis.

[191] Additionally, the local (i.a.) injection of oc-DBI reduced the joint inflammation-associated surge in systemic plasma ACBP/DBI concentrations (FIG. 28C), suggesting that the observed effect are primarily due to the local anti-inflammatory (and anti -joint inflammatory) effects of oc- DBI rather than to systemic spillover of the antibody. Consistent with this, Applicants observed that in control mice receiving IgG control antibody, the induction of joint inflammation led to an increase in the plasma level of several pro-inflammatory cytokines (Il-loc, IL-33, TNF-oc) in the IgG control group that was abolished by i.a. oc-DBI (FIG. 29A - FIG. 29D). Local inhibition of ACBP/DBI reduces both local and systemic signs of joint inflammation-associated inflammation.

[192] While embodiments of the present disclosure have been shown and described herein, such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur without departing from the present disclosure. It should be understood that various alternatives to the embodiments described herein may be employed. It is intended that the following claims define the scope of the present disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. A composition for use in treating a disease associated with a pituitary malignancy in a subject, the composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression in the subject in an amount sufficient to treat the disease associated with the pituitary malignancy upon administering to the subject.

2. The composition for use of claim 1, wherein the disease is associated with a pituitary adenoma.

3. The composition for use of claim 2, wherein the amount is sufficient to treat a symptom of the disease.

4. The composition for use of claim 1, wherein the agent reduces DBI activity, relative to an amount of DBI activity in the subject absent the administering.

5. The composition for use of claim 4, wherein the agent comprises a DBI-binding polypeptide.

6. The composition for use of claim 5, wherein the DBI-binding polypeptide is an anti-DBI antibody.

7. The composition for use of claim 6, wherein the anti-DBI antibody is a monoclonal antibody.

8. The composition for use of claim 6, wherein the anti-DBI antibody is a polyclonal antibody.

9. The composition for use of claim 5, wherein the DBI-binding polypeptide is an anti-DBI antibody fragment.

10. The composition for use of claim 9, wherein the DBI-binding polypeptide is an antibody fragment comprising a single chain Fv, Fab’ fragment, or nanobody.

11. The composition for use of claim 4, wherein the agent comprises a polypeptide antigen that induces production of a neutralizing anti-DBI antibody.

12. The composition for use of claim 1, wherein the agent reduces extracellular DBI expression, relative to an amount of DBI expression in the subject absent the administering.

13. The composition for use of claim 12, wherein the agent comprises a thyroid hormone.

14. The composition for use of claim 13, wherein the thyroid hormone comprises 3,3',5- triiodo-L-thyronine (T3).

15. The composition for use of claim 12, wherein the agent comprises a thyroid hormone receptor agonist.

16. The composition for use of claim 15, wherein the thyroid hormone receptor agonist comprises resmetirom.

17. The composition for use of claim 12, wherein the agent is an siRNA, an endonuclease, an antisense oligonucleotide, proteolysis-targeting chimeras (PROTACs), or a ribosome.

18. The composition for use of claim 17, wherein the agent is the siRNA.

19. A composition for use in treating symptoms associated with elevated levels of steroids in a subject, the composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression in the subject in an amount sufficient to treat the symptoms of associated with the elevated levels of steroids in the subject upon administering to the subject, and wherein the symptoms are selected from the group consisting of: adipocyte hypertrophy, increased liver weight, an increase in circulating liver enzymes, an increase in alanine aminotransferase (ALT), an increases in aspartate aminotransferase (AST), an increase in liver triglycerides (TG), a hypertrophy of white adipose tissue (WAT), a hypertrophy of brown adipose tissue (BAT), a metabolic syndrome, an arterial hypertension dyslipidemia, a triglyceridemia, a round “moon” face that comprises capillary vasodilatation, a skin acne, a facial hirsutism, a cranial alopecia, a skin atrophy, a central obesity, a “buffalo hump” lipodystrophy, a profuse striae, and a sarcopenia.

20. The composition of claim 19, wherein the elevated levels of steroids are acute elevated levels.

21. The composition of claim 20, wherein the acute elevated levels are a result of steroid administration of a synthetic glucocorticoid.

22. The composition of claim 19, wherein the elevated levels of steroids are chronic elevated levels.

23. A method of treating a disease associated with elevated levels of steroids in a subject, the method comprising administering to the subject a composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression in the subject, wherein the administering is sufficient to treat the disease associated with the elevated levels of steroids in the subject.

24. The method of claim 23, wherein the disease is associated with elevated levels of cortisol in the subject.

25. The method of claim 24, wherein the disease is an endogenous Cushing syndrome.

26. The method of claim 25, wherein the endogenous Cushing syndrome is Cushing disease.

27. The method of claim 23, wherein the disease is associated chronic use of steroids by the subject.

28. The method of claim 27, wherein the disease is an iatrogenic Cushing syndrome.

29. The method of claim 28, wherein the iatrogenic Cushing syndrome is induced by corticosteroids.

30. The method of claim 29, wherein the corticosteroids are co-administered with T3 or resmetirom.

31. The method of claim 23, wherein the agent reduces DBI activity, relative to an activity level prior to the administering.

32. The method of claim 31, wherein the agent comprises a DBI-binding polypeptide.

33. The method of claim 32, wherein the DBI-binding polypeptide is an anti-DBI antibody.

34. The method of claim 33, wherein the anti-DBI antibody is a monoclonal antibody.

35. The method of claim 33, wherein the anti-DBI antibody is a polyclonal antibody.

36. The method of claim 32, wherein the DBI-binding polypeptide is an anti-DBI antibody fragment.

37. The method of claim 36, wherein the DBI-binding polypeptide is an antibody fragment comprising a single chain Fv, Fab’ fragment, or nanobody.

38. The method of claim 31, wherein the agent comprises a polypeptide antigen that induces production of a neutralizing anti-DBI antibody.

39. The method of claim 23, wherein the agent reduces extracellular DBI expression, relative to an expression level prior to the administering.

40. The method of claim 39, wherein the agent comprises a thyroid hormone.

41. The method of claim 40, wherein the thyroid hormone comprises 3,3',5-triiodo-L- thyronine (T3).

42. The method of claim 39, wherein the agent comprises a thyroid hormone receptor agonist.

43. The method of claim 42, wherein the thyroid hormone receptor agonist comprises resmetirom.

44. The method of claim 39, wherein the agent is an siRNA, an endonuclease, an antisense oligonucleotide, proteolysis-targeting chimeras (PROTACs), or a ribosome.

45. The method of claim 44, wherein the agent is an siRNA that inhibits the expression of DBI.

46. A method of treating a Cushing syndrome associated with chronic use of a corticosteroid in a subject, the method comprising administering to the subject a composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression, wherein the administering is sufficient to treat the Cushing syndrome associated with the chronic use of the corticosteroid.

47. The method of claim 46, wherein the chronic use comprise use over a period of time.

48. The method of claim 47, wherein the period of time comprises a month or longer, 6 months or longer, or one year or longer.

49. A composition for use in the method of any one of claims 23-48.

50. A composition for use in treating synovial inflammation in a subject, the composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression in the subject in an amount sufficient to treat the synovial inflammation upon administering to the subject.

51. The composition for use of claim 50, wherein the agent reduces a tibia-femur triangle area upon administering to the subject, relative to a tibia-femur triangle area in absence of the administering.

52. The composition for use of claim 50, wherein the agent reduces an ultrasound biomicroscope (UBM) inflammation score upon administering to a subject, relative to a UBM score absent the administering.

53. The composition for use of claim 50, wherein the agent reduces a weight bearing asymmetry percentage caused by the synovial inflammation, relative to a weight bearing asymmetry percentage absent the administering.

54. The composition for use of claim 50, wherein the agent reduces formation of Bouchard’s nodes and/or Heberden’s nodes in a subject, relative to formation of Bouchard’s nodes and/or Heberden’s nodes absent the administering.

55. The composition for use of claim 50, wherein the agent reduces a joint crepitus in a subject, relative to a joint crepitus absent the administering.

56. A composition for use in treating an arthropathy in a subject, the composition comprising an agent that reduces human diazepam binding inhibitor (DBI) activity or expression in an amount sufficient to treat the arthropathy upon administering to the subject.

57. The composition for use of claim 56, wherein the agent reduces an osteoarthritis cartilage histopathology assessment system (OARSI) grade upon administering to the subject, relative to an OARSI grade absent the administering.

58. The composition for use of claim 56, wherein the agent reduces an osteoarthritis cartilage histopathology assessment system (OARSI) stage upon administering to the subject, relative to an OARSI stage absent the administering.

59. The composition for use of any one of claims 50-58, wherein the agent reduces extracellular DBI activity, relative to an amount of extracellular DBI activity in a comparable cell or tissue of the subject absent the administering.

60. The composition for use of claim 59, wherein the agent comprises nanoparticles.

61. The composition for use of claim 59 or 60, wherein the agent comprises a DBI-binding polypeptide.

62. The composition for use of claim 61, wherein the DBI-binding polypeptide is an anti-DBI antibody.

63. The composition for use of claim 62, wherein the anti-DBI antibody is a monoclonal antibody.

64. The composition for use of claim 63, wherein the monoclonal antibody comprises a monoclonal chimeric antibody, a monoclonal humanized antibody, or a monoclonal human antibody.

65. The composition for use of claim 62, wherein the anti-DBI antibody is a polyclonal antibody.

66. The composition for use of claim 61, wherein the DBI-binding polypeptide is an anti-DBI antibody fragment.

67. The composition for use of claim 66, wherein the anti-DBI antibody fragment comprises a single chain Fv, Fab’ fragment, or nanobody.

68. The composition for use of claim 62, wherein the anti-DBI antibody is an extracellular DBI neutralizing antibody.

69. The composition for use of claim 59 or 60, wherein the agent comprises a polypeptide antigen that induces production of a neutralizing anti-DBI antibody in the subject.

70. The composition for use of any one of claims 50-58, wherein the agent reduces DBI expression, relative to an amount of DBI expression by administering a composition lacking the agent that reduces DBI expression.

71. The composition for use of claim 70, wherein the agent comprises nanoparticles.

72. The composition for use of claim 70 or 71, wherein the agent is an siRNA, an endonuclease, an antisense oligonucleotide, a thyroid receptor agonist, proteolysis-targeting chimeras (PROTACs), or a ribosome.

73. The composition for use of claim 70 or 71, wherein the agent is an siRNA.

74. The composition for use of claim 70 or 71, wherein the agent is a thyroid hormone receptor agonist.

75. The composition for use of claim 74, wherein the thyroid hormone receptor agonist comprises resmetirom.

76. A method of treating degenerative joint disease in a subject in need thereof, the method comprising intra-articularly administering to the subject an agent that reduces human diazepam binding inhibitor (DBI) activity or expression in an amount sufficient to treat the degenerative joint disease in the subject.

77. The method of claim 76, wherein the administering is sufficient to treat synovial inflammation associated with the degenerative joint disease in the subject.

78. The method of claim 76, wherein the administering is sufficient to reduce a tibia-femur triangle area in the subject, relative to a tibia-femur triangle area prior to the administering.

79. The method of claim 76, wherein the administering is sufficient to reduce an ultrasound biomicroscope (UBM) inflammation score for the subject, relative to a UBM score prior to the administering.

80. The method of claim 76, wherein the administering is sufficient to reduce a weight bearing asymmetry percentage caused by the degenerative joint disease, relative to a weight bearing asymmetry percentage prior to the administering.

81. The method of claim 76, wherein the administering is sufficient to reduce formation of Bouchard’s nodes and/or Heberden’s nodes in a subject, relative to formation of the Bouchard’s nodes and/or Heberden’s nodes prior to the administering.

82. The method of claim 76, wherein the administering is sufficient to reduce a joint crepitus in a subject, relative to a joint crepitus prior to the administering.

83. The method of claim 76, wherein the administering is sufficient to treat arthropathy associated with the degenerative joint disease in the subject.

84. The method of claim 83, wherein the administering is sufficient to reduce an osteoarthritis cartilage histopathology assessment system (OARSI) grade for the subject, relative to an OARSI grade prior to the administering.

85. The method of claim 83, wherein the administering is sufficient to reduce an osteoarthritis cartilage histopathology assessment system (OARSI) stage for the subject, relative to an OARSI stage prior to the administering.

86. The method of any one of claims 76-85, wherein the agent reduces extracellular DBI activity, relative to an amount of extracellular DBI activity in a comparable cell or tissue of the subject in absence of the administering.

87. The method of claim 86, wherein the agent comprises nanoparticles.

88. The method of claim 86 or 87, wherein the agent comprises a DBI-binding polypeptide.

89. The method of claim 88, wherein the DBI-binding polypeptide is an anti-DBI antibody.

90. The method of claim 89, wherein the anti-DBI antibody is a monoclonal antibody.

91. The method of claim 90, wherein the monoclonal antibody comprises a monoclonal chimeric antibody, a monoclonal humanized antibody, or a monoclonal human antibody.

92. The method of claim 89, wherein the anti-DBI antibody is a polyclonal antibody.

93. The method of claim 88, wherein the DBI-binding polypeptide is an anti-DBI antibody fragment.

94. The method of claim 93, wherein the anti-DBI antibody fragment comprises a single chain Fv, Fab’ fragment, or nanobody.

95. The method of claim 89, wherein the anti-DBI antibody is an extracellular DBI neutralizing antibody.

96. The method of claim 86 or 87, wherein the agent comprises a polypeptide antigen that induces production of a neutralizing anti-DBI antibody in the subject.

97. The method of any one of claims 76-85, wherein the agent reduces DBI expression, relative to an amount of DBI expression by administering a composition lacking the agent that reduces DBI expression.

98. The method of claim 97, wherein the agent comprises nanoparticles.

99. The method of claim 97 or 98, wherein the agent is an siRNA, an endonuclease, an antisense oligonucleotide, a thyroid receptor agonist, proteolysis-targeting chimeras (PROTACs), or a ribosome.

100. The method of claim 97 or 98, wherein the agent is an siRNA.

101. The method of claim 97 or 98, wherein the agent is a thyroid hormone receptor agonist.

102. The method of claim 101, wherein the thyroid hormone receptor agonist comprises resmetirom.

103. A method of treating osteoarthritis in a subject in need thereof, the method comprising intra-articularly administering to the subject an agent that reduces human diazepam binding inhibitor (DBI) activity or expression in the subject in an amount sufficient to treat the osteoarthritis in the subject.

104. A composition for use in any of the methods of claims 76-103.