Compositions and methods for ameliorating symptoms associated with CLEC16A dysfunction or deficiency

Abstract

Compositions and methods for the treatment of CLEC16A-associated phenotypes and disorders are disclosed.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 374,981, filed September 8, 2022, the entire contents of which are incorporated herein by reference as if set forth in full. FIELD OF THE INVENTION The present invention relates to the field of ameliorating symptoms associated with CLEC16A dysfunction or deficiency. More specifically, the present invention provides agents useful for treating autoimmune disorders, lipodystrophic disorders, and neurodegenerative disorders in patients in need thereof. [Background technology]

[0002] Background of the Invention Several publications and patent documents are cited throughout this specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as if set forth in full.

[0003] CLEC16A is involved in multiple autoimmune diseases. Turning off CLEC16A in adult mice results in dysregulated mitophagy, severe weight loss, a robust autoimmune inflammatory response, severe neurological symptoms associated with neuroinflammation, and progressive neurodegeneration resembling spinocerebellar degeneration. Adult CLEC16A knockout mice (CLEC16A ΔUBC The mouse model exhibits sensory ataxia and lipodystrophy with dystonia, immune and inflammatory components, which are seen in some human cerebellar ataxias and sensory neuropathy.

[0004] Using a drug repurposing approach, we tested the ongoing hypothesis that the observed perturbations in mitophagy / autophagy resulting in the observed phenotypes could be rescued, at least in part, by interventions targeting the mitophagy / autophagy and JAK-STAT pathways, including but not limited to SOCS1 and tofacitinib. Treatment with a JAK / STAT pan-inhibitor (tofacitinib) partially rescued the above phenotypes and improved survival in CLEC16A KO mice by modulating ER stress, lipolysis, mitophagy, and autophagy. However, abnormal partial phenotypic features remained.

[0005] Given the large number of autoimmune and other disorders associated with CLEC16A deficiency or dysfunction, it is clear that new therapies and therapeutic agents are urgently needed to ameliorate the effects of CLEC16A malfunction and alleviate the associated symptoms. Summary of the Invention

[0006] Summary of the Invention According to the present invention, there is provided a method for improving at least one CLEC16A-associated symptom in a subject in need thereof, comprising administering an effective amount of at least one mitophagy-promoting agent, wherein the at least one symptom is selected from the group consisting of a robust autoimmune inflammatory response, severe weight loss, severe neurological symptoms, neuroinflammation, progressive neurodegeneration resembling spinocerebellar degeneration, and fat loss. Also provided herein is a method for treating a CLEC16A-associated disorder in a subject in need thereof, comprising administering an effective amount of at least one mitophagy-promoting agent. In certain embodiments, the CLEC16A-associated disorder is selected from the group consisting of an autoimmune disorder, a lipodystrophic disorder, and a neurodegenerative disorder.

[0007] In certain embodiments of the present invention, at least one mitophagy promoter promotes the removal of mitochondria. Exemplary mitophagy promoters are probucol, quercetin, or acipimox. In certain embodiments, the method further comprises administering one or more agents selected from a JAK-STAT inhibitor, an ER stress regulator, and a SOCS1 inhibitor. Exemplary JAK-STAT inhibitors are selected from tofacitinib, ruxolitinib, baricitinib, peficitinib, decernotinib, filgotinib, solcitinib, itacitinib, SHR0302, upadacitinib, and PF-04965842. Exemplary ER stress modulators are selected from rapamycin, 4-phenylbutyric acid (4-PBA), trimethylamine N-oxide dehydrate (TMAO), dimethyl sulfoxide (DMSO), tauroursodeoxycholic acid (TUDCA), AMPK-activated protein kinase, 5'-aminoimidazole-4-carboximide-1-β-d-ribofuranoside (AICAR), glucagon-like peptide-1 (GLP-1), a DPP4 inhibitor, and n-acetylcysteine ​​(NAC). In certain embodiments, the method further comprises administering a PPARγ inhibitor.

[0008] In certain embodiments, the methods described herein rescue spleen atrophy and / or improve organ weight ratios of thymus, inguinal white adipose tissue (iWAT), and / or gonadal white adipose tissue (gWAT) compared to untreated controls. In certain aspects, the treatment delays the progression of CLEC16A-associated symptoms compared to untreated controls.

[0009] Also provided herein are methods for treating CLEC16A-associated thymic degeneration, comprising administering a mitophagy-promoting agent to alter the thymic weight ratio and ameliorate symptoms associated with thymic degeneration. Also provided herein are methods for treating CLEC16A-associated splenic degeneration, comprising administering a mitophagy-promoting agent to alter the splenic weight ratio, provide a therapeutic benefit, and ameliorate symptoms associated with splenic degeneration.

[0010] In another aspect of the present invention, there is provided a method for treating CLEC16A-associated degeneration of iWAT, comprising administering a mitophagy-promoting agent to alter the weight ratio in iWAT and ameliorate symptoms associated with iWAT degeneration. Also provided herein is a method for treating CLEC16A-associated degeneration of gWAT, comprising administering a mitophagy-promoting agent to alter the weight ratio in gWAT and ameliorate symptoms associated with gWAT degeneration.

[0011] Also provided herein are methods for treating a CLEC16A-associated condition in a subject in need thereof, the methods comprising: a) diagnosing the subject with a CLEC16A-associated disorder; and b) administering an effective amount of a mitophagy-promoting agent. [Brief explanation of the drawings]

[0012] [Figure 1] Figure 1. Body weights of control ± probucol and CLEC16A KO ± probucol mice fed a standard chow diet. [Figure 2] Figures 2A-2L. Organ weight / body weight ratios for control ± probucol and KO ± probucol groups. [Figure 3] Figures 3A-3C. Probucol rescues the response and mitophagy disruption in KO mouse spleen lysates. (Figure 3A) Immunoblot analysis of spleen lysates showing mitophagy disruption and rescue by probucol. (Figure 3B) Quantitative graph showing expression levels of CLEC16A, P62, LC3I / II, PINK1, and Parkin in control ± probucol- and KO ± probucol-treated mice. (Figure 3C) Schematic diagram showing the effect of probucol in removing defective mitochondria during mitophagy. [Figure 4]Figure 4. Probucol slows phenotypic progression in KO mice. Timeline to onset of functional impairment (score 1-4) in cohorts of control ± probucol and KO ± probucol mice treated with tamoxifen for 4 consecutive days. Number of mice / group is shown in each graph. ***P < 0.001. [Figure 5] Figure 5. Probucol partially rescues the lipodystrophic phenotype of CLEC16A KO mice and improves survival. Representative dorsal and ventral anatomical images showing the overall morphology and fat distribution of control, KO, and KO + probucol-treated mice. The bottom panels show the amounts of iWAT, BAT, and gWAT collected from control, KO, and KO + probucol (scores 1, 2, and 3.5) mice. [Figure 6] Figures 6A-6B. Quercetin attenuates the CLEC16a KO phenotype. [Figure 7] Figures 7A-7C. Organ weight / body weight ratios for control ± quercetin and KO ± quercetin groups, and control ± acipimox and KO ± acipimox groups. [Figure 8] Figures 8A-8C. CLEC16A KO phenotype (Figure 8A). Probucol-mediated rescue in eliminating late-stage mitophagy dysregulation (Figure 8B). Study design and probucol dose and administration method during the study period (Figure 8C). [Figure 9] Figure 9. Late-stage mitophagy promoters slow phenotypic progression in a dose-dependent manner. DETAILED DESCRIPTION OF THE INVENTION

[0013] Detailed Description of the Invention CLEC16A deficiency results in several symptoms that result in a specific phenotype. To address this phenotypic feature, we focused on the ultimate endpoint of mitophagy: the removal of damaged mitochondria to mitigate the deleterious effects of mitochondrial damage. As a more personalized approach using a drug repurposing approach, we tested the mitophagy-promoting agent probucol (a lipid-lowering drug) for rescue in our systemically inducible CLEC16A KO. Our findings provide evidence supporting the rescue of the phenotype by targeting mitophagy alone or in conjunction with intervention in the JAK-STAT pathway, supporting the potential for future therapeutic intervention in the treatment of autoimmunity.

[0014] definition For purposes of the present invention, "a" or "an" entity refers to one or more of that entity; for example, "cDNA" refers to one or more cDNAs or at least one cDNA. Thus, the terms "a" or "an," "one or more," and "at least one" can be used interchangeably herein. It should also be noted that the terms "comprising," "including," and "having" can be used interchangeably. Furthermore, a compound "selected from the group consisting of" refers to one or more compounds in the following list, including a mixture (i.e., combination) of two or more compounds. According to the present invention, an isolated or biologically pure molecule is a compound that has been removed from its natural environment. Thus, "isolated" and "biologically pure" do not necessarily reflect the extent to which a compound has been purified. Isolated compounds of the present invention can be obtained from natural sources, produced using laboratory synthesis techniques, or produced by such chemical synthesis routes.

[0015] As used herein, the terms "ingredient," "composition," "compound composition," "compound," "drug," "pharmaceutical agent," "active agent," "therapeutic," "therapy," "treatment," or "pharmaceutical product" are used interchangeably herein and refer to a compound or composition of compounds or substances that, when administered to a subject (human or animal), induces a desired pharmacological and / or physiological effect through local and / or systemic action. The terms "drug" and "test compound" refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological material such as bacteria, plants, fungi, or animal (especially mammalian) cells or tissues.

[0016] Additionally, the term "compound" or "compounds" refers to the compounds discussed herein and is intended to include precursors and derivatives of the compounds, as well as pharmaceutically acceptable salts of the compounds, precursors and derivatives. The present invention also includes prodrugs of the compounds, pharmaceutical compositions comprising the compounds and a pharmaceutically acceptable carrier, and pharmaceutical compositions comprising prodrugs of the compounds and a pharmaceutically acceptable carrier.

[0017] The phrase "consisting essentially of" when referring to a particular nucleotide or amino acid means a sequence having the characteristics of a given SEQ ID NO. For example, when used in reference to an amino acid sequence, the phrase includes the sequence itself and molecular modifications that do not affect the functional and novel properties of the sequence.

[0018] A "derivative" of a polypeptide, polynucleotide, or fragment thereof refers to a sequence that has been altered by changing the sequence of the construct, for example, by manipulating the nucleic acid encoding the protein or by modifying the protein itself. A "derivative" of a gene or nucleotide sequence refers to an isolated nucleic acid molecule that contains significant sequence similarity to a gene or nucleotide sequence, or a portion thereof. Furthermore, "derivative" includes such isolated nucleic acids that contain modified nucleotides or mimetics of naturally occurring nucleotides.

[0019] As used herein, the term "functional" means that the nucleic acid or amino acid sequence is functional for a stated assay or purpose.

[0020] For purposes of the present invention, the terms "nucleic acid," "nucleotide sequence," or "nucleic acid molecule" as used herein refer to DNA or RNA molecules, either single-stranded or double-stranded, and, if single-stranded, to their complementary sequences, either linear or circular. When discussing nucleic acid molecules, the sequence or structure of a particular nucleic acid molecule may be described herein according to the usual convention of providing the sequence in the 5' to 3' direction. With respect to the nucleic acids of the present invention, the term "isolated nucleic acid" may be used. When applied to DNA, this term refers to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism from which it originates. For example, "isolated nucleic acid" includes a DNA molecule inserted into a vector, such as a plasmid or viral vector, or a DNA molecule integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism. Alternatively, the term may refer to DNA that is sufficiently separated (e.g., substantially free) from other cellular components with which it is naturally associated. "Isolated" does not imply artificial or synthetic admixture with other compounds or substances, or the presence of impurities that do not interfere with essential activity or that may be present due, for example, to incomplete purification. When applied to RNA, the term "isolated nucleic acid" primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term can refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it is associated in its natural state (i.e., in cells or tissues). Isolated nucleic acid (either DNA or RNA) can also refer to a molecule produced directly by biological or synthetic means and separated from other components present during its production.

[0021] A "specific binding pair" refers to a specific binding member (sbm) and a binding partner (bp) that have a particular specificity for each other and that, under normal circumstances, bind preferentially to other molecules. Examples of specific binding pairs include antigens and antibodies, ligands and receptors, and complementary nucleotide sequences. Those skilled in the art will recognize many other examples. Furthermore, the term "specific binding pair" is applicable when either or both of the specific binding member and binding partner form part of a larger molecule. In embodiments where the specific binding pair is comprised of nucleic acid sequences, they are long enough to hybridize to each other under assay conditions, preferably greater than 10 nucleotides in length, more preferably greater than 15 or 20 nucleotides in length.

[0022] According to the present invention, an isolated or biologically pure molecule or cell is a compound that has been removed from its natural environment. Thus, "isolated" and "biologically pure" do not necessarily reflect the extent to which a compound has been purified. The isolated compounds of the present invention can be obtained from natural sources, can be produced using laboratory synthesis techniques, or can be produced by such chemical synthesis routes.

[0023] As used herein, the term "delivery" refers to the introduction of a foreign molecule (i.e., a miRNA-containing nanoparticle) into a cell. As used herein, the term "administration" refers to the introduction of a foreign molecule into a cell. This term is intended to be synonymous with the term "delivery."

[0024] As used herein, the phrase "effective amount" of a compound or pharmaceutical composition refers to an amount sufficient to regulate symptoms associated with abnormal expression of CLEC16A in an animal, particularly a human, including, but not limited to, reducing negative health consequences associated with mitochondrial damage and reducing such consequences by prophylactic administration prior to the onset of symptoms.

[0025] Determining the effective amount of a compound for a particular application and mode of administration is within the capabilities of one skilled in the art. The effective amount can be initially estimated from in vitro activity and metabolic assays. For example, the initial dose of a compound used in animals can be formulated to achieve a specific circulating blood or serum concentration of the metabolically active compound. Calculating the dosage to achieve such a circulating blood or serum concentration, taking into account the bioavailability of a particular compound via the desired route of administration, is within the capabilities of one skilled in the art. The initial dose of a compound can also be estimated from in vivo data, such as animal models. Animal models useful for testing the effectiveness of active metabolites for treating or preventing the various diseases mentioned above are well known in the art. Animal models suitable for testing the bioavailability and / or metabolism of a compound to active metabolites are also well known. One of ordinary skill in the art can appropriately use such information to determine the dosage of a particular compound suitable for administration to humans.

[0026] Dosages typically range from about 0.0001 mg / kg / day, 0.001 mg / kg / day, or 0.01 mg / kg / day to about 100 mg / kg / day, but may be higher or lower depending on, among other factors, the activity of the active metabolite compound, the bioavailability of the compound, its metabolic kinetics and other pharmacokinetic properties, the mode of administration, and various other factors discussed above. Dosage amounts and administration intervals can be individually adjusted to provide plasma concentrations of the compound(s) and / or active metabolite compound(s) sufficient to maintain therapeutic or prophylactic effect. For example, compounds can be administered once a week, several times a week (e.g., every other day), once a day, or multiple times a day, depending, among other factors, on the mode of administration, the particular indication being treated, and the judgment of the prescribing physician. In cases of selective uptake, such as local administration or topical administration, the effective local concentration of the compound(s) and / or active metabolite compound(s) may not be related to plasma concentration. A skilled artisan will be able to optimize an effective topical dosage without undue experimentation.

[0027] Generally, the initial therapeutically effective amount of a compound described herein or a pharmaceutically acceptable salt thereof administered ranges from about 0.01 to about 200 mg / kg or about 0.1 to about 20 mg / kg of patient body weight per day, with a typical initial range being about 0.3 to about 15 mg / kg / day. Oral unit dosage forms, such as tablets and capsules, can contain from about 0.1 mg to about 1000 mg of the compound or a pharmaceutically acceptable salt thereof. In another embodiment, such dosage forms contain from about 50 mg to about 500 mg of the compound or a pharmaceutically acceptable salt thereof. In yet another embodiment, such dosage forms contain from about 25 mg to about 200 mg of the compound or a pharmaceutically acceptable salt thereof. In yet another embodiment, such dosage forms contain from about 10 mg to about 100 mg of the compound or a pharmaceutically acceptable salt thereof. In a further embodiment, such dosage forms contain from about 5 mg to about 50 mg of the compound or a pharmaceutically acceptable salt thereof.

[0028] "CLEC16A-associated immune disorders" include autoimmune lipodystrophy and neurodegenerative disorders. Examples of such disorders include, but are not limited to, AD: Alzheimer's disease; ADPD: Alzheimer's disease and Parkinson's disease; AMDF: ataxia, myoclonus, and hearing loss; CIPO: chronic intestinal pseudo-obstruction with myopathy and ophthalmoplegia; CPEO: chronic progressive external ophthalmoplegia; DEAF: maternally inherited or aminoglycoside-induced hearing loss; DEMCHO: dementia and dementia; DMDF: diabetes and hearing loss; exercise intolerance; ESOC: epilepsy, stroke, optic atrophy, and cognitive decline. FBSN: Familial bilateral striatal necrosis; FICP: Fatal infantile cardiomyopathy plus, MELAS-associated cardiomyopathy; GER: Gastroesophageal reflux; KSS: Kearns-Sayre syndrome; LDYT: Leber hereditary optic neuropathy and dystonia; LHON: Leber hereditary optic neuropathy; LIMM: Fatal childhood mitochondrial myopathy; MDM: Myopathy and diabetes; MELAS: Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes; MEPR: Myoclonic epilepsy and psychomotor regression; MERME: 5 MERRF / MELAS overlap disorder; MERRF: myoclonic epilepsy and ragged-red fibers; MHCM: maternally inherited hypertrophic cardiomyopathy; MICM: maternally inherited cardiomyopathy; MILS: maternally inherited Leigh syndrome; mitochondrial encephalocardiopathy; mitochondrial encephalomyopathy; MM: mitochondrial myopathy; MMC: maternal myopathy and cardiomyopathy; multisystem mitochondrial disorders (myopathy, encephalopathy, blindness, deafness, peripheral neuropathy); NARP: neurogenic muscle weakness, motor Ataxia, retinitis pigmentosa; another phenotype at this locus has been reported as Leigh syndrome; NIDDM: non-insulin-dependent diabetes mellitus; PEM: progressive encephalopathy; PME: progressive myoclonic epilepsy; RTT: Rett syndrome; SIDS: sudden infant death syndrome, multiple sclerosis, primary adrenal insufficiency, Crohn's disease, primary biliary cirrhosis, juvenile idiopathic arthritis, rheumatoid arthritis, alopecia areata, uveitis, and lupus.

[0029] The phrase "CLEC16A-associated phenotype" or "CLEC16A-associated symptom" includes at least one symptom of a CLEC16A disorder. A patient may experience a CLEC16A-associated phenotype without being diagnosed with a CLEC16A-associated immune disorder. Examples of a CLEC16A-associated phenotype include, but are not limited to, a robust autoimmune inflammatory response, severe weight loss, severe neurological symptoms, neuroinflammation, progressive neurodegeneration resembling spinocerebellar degeneration, and fat loss.

[0030] A "sample" or "patient sample" or "biological sample" generally refers to a sample that can be tested for a particular molecule, such as a marker described herein. Samples include, but are not limited to, cells, and bodily fluids such as blood, serum, plasma, cerebrospinal fluid, urine, saliva, tears, and pleural effusions.

[0031] As used herein, the terms "agent" and "compound" are used interchangeably to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal (especially mammalian) cells or tissues. Biological macromolecules include siRNA, shRNA, antisense oligonucleotides, peptides, peptide / DNA complexes, and the like. Agents and compounds may also be referred to as "test agents" or "test compounds," which are evaluated for potential biological activity by inclusion in screening assays described herein below.

[0032] "Antilipidemia agents" or "antidyslipidemic drugs" refer to compounds that lower the levels of any or all lipids or lipoproteins, including fats, cholesterol, or triglycerides, in the blood. In certain embodiments, antilipidemia agents are administered to patients with elevated levels of any or all lipids or lipoproteins. Certain antilipidemia agents inhibit cholesterol synthesis and / or delay cholesterol absorption. In certain embodiments, antilipidemia agents inhibit the oxidation of cholesterol in LDL and delay foam cell formation. Such inhibitors are known in the art and include, but are not limited to, siRNA molecules, peptidomimetics, and small molecules. Examples of antilipidemia agents include, but are not limited to, probucol, atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin.

[0033] Mitophagy refers to a mitochondrial quality control mechanism that allows damaged and redundant mitochondria to be degraded to prevent mitochondrial dysfunction. A "mitophagy promoter" refers to a compound that increases the degradation of damaged and redundant mitochondria. In certain embodiments, the mitophagy promoter is also an antilipidemic agent. In certain embodiments, the mitophagy promoter targets the final endpoint of mitophagy—the removal of damaged mitochondria. Such inhibitors are known in the art and include, but are not limited to, siRNA molecules, peptidomimetics, and small molecules. Examples of mitophagy promoters that promote the removal of damaged mitochondria include, but are not limited to, probucol, quercetin, and acipimox.

[0034] The inhibitor of JAK-STAT pathway is also useful in the present invention.In certain embodiments, the inhibitor is a JAK-STAT pan-inhibitor.Such inhibitor is known in the art, and includes but is not limited to siRNA molecule, peptidomimetic and small molecule.These include but are not limited to tofacitinib, ruxolitinib, baricitinib, peficitinib, decernotinib, filgotinib, solcitinib, itacitinib, SHR0302, upadacitinib and PF-04965842.

[0035] "ER stress regulator" refers to a compound that increases or decreases the incomplete folding of endoplasmic reticulum folding. Such inhibitors are known in the art and include, but are not limited to, siRNA molecules, peptidomimetics, and small molecules. These include, but are not limited to, mTOR inhibitors, rapamycin, 4-phenylbutyric acid (4-PBA), trimethylamine N-oxide dehydrate (TMAO), dimethyl sulfoxide (DMSO), tauroursodeoxycholic acid (TUDCA), AMPK-activated protein kinase, 5'-aminoimidazole-4-carboximide-1-β-d-ribofuranoside (AICAR), glucagon-like peptide-1 (GLP-1), DPP4 inhibitors, and n-acetylcysteine ​​(NAC). Additional regulators are known to those skilled in the art. See, e.g., Jung TW, Choi KM. Pharmacological Modulators of Endoplasmic Reticulum Stress in Metabolic Diseases. Int J Mol Sci. 2016 Feb 1;17(2):192. Doi: 10.3390 / ijms17020192. PMID: 26840310; PMCID: PMC4783926 (incorporated herein by reference).

[0036] Inhibitors of SOCS1 are also useful in the present invention. Such inhibitors are known in the art and include, but are not limited to, siRNA molecules, peptidomimetics, and small molecules.

[0037] Modulators of the PPAR pathway, including PPARα and PPARγ, are also useful in the present invention. Such inhibitors are known in the art and include, but are not limited to, siRNA molecules, peptidomimetics, and small molecules such as fenofibrate.

[0038] The term "inhibit" means to decrease or reduce activity or expression. This can be complete or partial inhibition of activity or expression. Inhibition can be compared to a control or standard level. Inhibition can be any of the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 11 3, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.

[0039] An "inhibitor" (interchangeably "antagonist") of a polypeptide of interest is an agent that inhibits the activity or function of the polypeptide of interest, e.g., partially or completely blocks, inhibits, or neutralizes biological activity mediated by the polypeptide of interest. For example, an antagonist of polypeptide X may refer to any molecule that partially or completely blocks, inhibits, or neutralizes biological activity mediated by polypeptide X. Examples of inhibitors include antibodies; ligand antibodies; small molecule antagonists; and antisense and inhibitory RNA (e.g., siRNA) molecules. Inhibition is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 119, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 3, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.

[0040] As used herein, the terms "modulate," "modulation," or "regulation" refer to changing the rate at which a particular process occurs, inhibiting a particular process, reversing a particular process, and / or preventing the initiation of a particular process. As used herein, the term "modulate" refers to increasing / promoting or decreasing / inhibiting a particular cellular, biological, or signaling function associated with the normal activity of the CLEC16A molecule described herein. For example, the term modulate refers to the ability of a test compound or test agent to rescue the CLEC16A phenotype by interfering with the removal of damaged mitochondria.

[0041] As used herein, a "condition in need of treatment" refers to a determination by a caregiver (e.g., a doctor, nurse, nurse practitioner, or individual in the case of a human, or a veterinarian in the case of an animal, including a non-human mammal) that a subject is in need of or would benefit from treatment, based on a variety of factors within the caregiver's expertise, including knowledge that the subject is ill or will become ill as a result of a condition treatable by the disclosed compounds.

[0042] "Treatment" and "treating" refer to the medical management of a subject with the intent of curing, ameliorating, or stabilizing a pathological condition or disorder. The term includes active treatment, i.e., treatment specifically directed at ameliorating a disease, pathological condition, or disorder, and also includes causal treatment, i.e., treatment directed at eliminating the cause of the associated disease, pathological condition, or disorder. Additionally, the term includes palliative treatment, i.e., treatment aimed at alleviating symptoms rather than curing the disease, pathological condition, or disorder, and supportive treatment, i.e., treatment used to complement another specific therapy directed at ameliorating the associated disease, pathological condition, or disorder. It is understood that a treatment is intended to cure, ameliorate, or stabilize a disease, pathological condition, or disorder, but need not actually result in a cure, amelioration, or stabilization. The effectiveness of treatment can be measured or assessed as described herein and as known in the art as appropriate for the disease, pathological condition, or disorder involved. Such measurement and assessment can be qualitative and / or quantitative. Thus, for example, a property or characteristic of a disease, pathological condition, or disorder, and / or a symptom of a disease, pathological condition, or disorder can be reduced to any effect or by any amount.

[0043] Drug therapy The elucidation of the role played by CLEC16A facilitates the development of pharmaceutical compositions useful for the treatment and diagnosis of phenotypes associated with CLEC16A dysfunction. These compositions may contain, in addition to one of the above-mentioned substances, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not inhibit the effectiveness of the active ingredient. The exact nature of the carrier or other materials may depend on the route of administration, for example, oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal.

[0044] The compounds described herein can be formulated for enteral, parenteral, topical, or systemic administration. The compounds can be combined with one or more pharmaceutically acceptable carriers and / or excipients that are deemed safe and effective and can be administered to individuals without causing undesired biological side effects or undesired interactions. A carrier refers to any component present in a pharmaceutical formulation other than the active ingredient or components. Typical carriers that can be used in conjunction with the preparation of compound formulations and conventional methods for preparing pharmaceutical compositions are known to those skilled in the art. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc.

[0045] Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers, for example, to a diluent, adjuvant, excipient, adjuvant, or vehicle with which an active agent of the present invention is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or aqueous solutions of saline, dextrose, and glycerol are preferred carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E.W. Martin.

[0046] The pharmaceutical compositions of the present invention can be administered by any suitable route, such as injection, oral, pulmonary, nasal, or other administration modes. In general, pharmaceutical compositions contemplated within the scope of the present invention contain, inter alia, pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such compositions may contain diluents of various buffer contents (e.g., Tris hydrochloride, acetate, phosphate), pH, and ionic strength; additives such as detergents and solubilizers (e.g., Tween 80, polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol), and bulking agents (e.g., lactose, mannitol); particulate formulations of polymeric compounds such as polylactic acid, polyglycolic acid, or incorporation of materials into liposomes. Such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the components of the pharmaceutical compositions of the present invention. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pp. 1435-1712 (incorporated herein by reference). Pharmaceutical compositions of the present invention can be prepared, for example, in liquid form or can be a dry powder, such as a lyophilized form. Specific methods for administering such compositions are described below.

[0047] In yet another embodiment, the pharmaceutical compositions of the present invention can be delivered in a controlled release system, such as by intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In certain embodiments, a pump can be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Biomed. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press: Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley. Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Macromol. 23:61 (1983); see also, Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in the vicinity of the target tissue in an animal, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138 (1984)). Other controlled release systems are discussed in the review by Langer [Science 249:1527 1533 (1990)].

[0048] Treatment method As described above, the present invention also includes methods for treating CLEC16A-related conditions and disorders. CLEC16A-related disorders include, but are not limited to, autoimmune disorders, lipodystrophic disorders, and neurodegenerative disorders. An exemplary method involves administering a pharmaceutically effective amount of a mitophagy-promoting agent to a subject in need thereof. In certain embodiments, the treatment may also include administering a JAK-STAT inhibitor, an ER stress regulator, and / or a SOCS1 inhibitor.

[0049] In certain embodiments, the treatment method improves the symptoms associated with CLEC16A-associated conditions and disorders. Symptoms can vary according to the type of CLEC16A-associated phenotype or disorder. In certain embodiments, the symptoms of CLEC16A-associated phenotypes and disorders include, but are not limited to, a robust autoimmune inflammatory response, severe weight loss, severe neurological symptoms, neuroinflammation, progressive neurodegeneration resembling spinocerebellar degeneration, and fat loss. [Example]

[0050] The following examples are provided to illustrate certain embodiments of the present invention and are not intended to limit the invention in any way.

[0051] Example I Antilipidemic agents suppress fat and body weight loss in CLEC16A KO mice Loss of CLEC16A in adult mice results in dysregulated mitophagy, a robust autoimmune inflammatory response [1], severe weight loss [2], severe neurological symptoms associated with neuroinflammation, and progressive neurodegeneration resembling spinocerebellar degeneration [3]. Removal of damaged mitochondria is essential for the survival of dopaminergic neurons, the loss of which is responsible for the phenotype observed in CLEC16A KO mice. Current treatments for the majority of neurodegenerative disorders aim simply to ameliorate symptoms without affecting disease progression. Therefore, disease-modifying therapies remain a significant unmet need. Therefore, drug repurposing, combined with existing pharmacological information on candidate compounds, offers an opportunity to accelerate the clinical trial pipeline and efficiently address this unmet need for neurodegeneration modulation.

[0052] Based on previously published results and reports of mitophagy dysregulation leading to the accumulation of defective mitochondria, we treated CLEC16A KO mice with probucol. Probucol is a lipid-lowering drug that has been characterized as an effective mitophagy promoter [4]. Probucol is known to function downstream of PINK and Parkin, modulating the final endpoint of mitophagy removal of damaged mitochondria. Improvement of this process can mitigate the negative consequences of mitochondrial damage in dopaminergic neurons, resulting in rescue of sensory neurodegeneration resembling spinocerebellar degeneration in KO mice. CLEC16A KO mice showed significant weight loss as early as day 9 compared to both control mice. This loss became more significant throughout the study period (Figure 1). As expected, probucol treatment significantly reduced weight loss and improved survival in CLEC16A KO mice (Figure 1). Probucol-treated control mice remained healthy and gained significantly more weight throughout the study period compared to control mice.

[0053] Furthermore, CLEC16A KO mice were treated with quercetin and acipimox. CLEC16A KO mice showed significant weight loss as early as day 9 compared to both control mice. This loss became more significant throughout the study period (Figure 6). As expected, quercetin and acipimox treatment significantly reduced weight loss and improved the survival rate of CLEC16A KO mice (Figure 6). Control mice treated with quercetin and acipimox remained healthy and gained significantly more weight throughout the study period compared to control mice.

[0054] Example II The antilipidemic agent probucol rescues atrophy of spleen, thymus, inguinal white adipose tissue (iWAT), brown adipose tissue (BAT), and gonadal white adipose tissue (gWAT) In our CLEC16A knockout mice, we have generated inducible atrophy of the spleen, thymus, and white fat. Here, we evaluate the effect of probucol on organ weight in our whole-body inducible CLEC16A knockout mice. Figure 2 shows the ratio graphs for the spleen (Figure 2A), thymus (Figure 2B), liver (Figure 2C), kidney (Figure 2D), inguinal white adipose tissue (iWAT) (Figure 2E), brown adipose tissue (BAT) (Figure 2F), gonadal white adipose tissue (gWAT) (Figure 2G), pancreas (Figure 2H), muscle (Figure 2I), heart (Figure 2J), cerebellum (Figure 2K), and glucose (Figure 2L). In addition to weight loss and severe atrophy of the spleen (Figure 2A), thymus (Figure 2B), and inguinal white adipose tissue (IWAT) (Figure 2E), we report severe atrophy of brown adipose tissue (BAT) (Figure 2F) and gonadal white adipose tissue (gWAT) (Figure 2G). KO mice exhibit a significant decrease in glucose (Figure 2L).

[0055] Probucol treatment rescued splenic atrophy (Figure 2A). Probucol significantly improved the organ weight ratios of the thymus (Figure 2B), iWAT (Figure 2E), and gWAT (Figure 2G). CLEC16A KO mice showed significant decreases in the weights and body weight ratios of the spleen (Figure 2A), thymus (Figure 2B), inguinal white adipose tissue (iWAT) (Figure 2E), brown adipose tissue (BAT) (Figure 2F), and gonadal white adipose tissue (gWAT) (Figure 2G) compared to controls over the study period.

[0056] The weight / body weight ratios of the spleen, thymus, iWAT, BAT, and gWAT in CLEC16A KO mice were significantly reduced compared to controls (Figure 2). The liver, kidney, pancreas, muscle, heart, and cerebellum showed no significant changes compared to control littermates. Tamoxifen- and probucol-treated littermates showed no adverse effects on organ weights and their ratios throughout the study period. As expected, probucol treatment significantly rescued spleen atrophy. Probucol significantly improved the organ weight ratios of the thymus, iWAT, and gWAT. Overall, probucol, a mitophagy-promoting agent, had a significant rescue effect on organ weights in our systemically inducible CLEC16A knockout mice.

[0057] Example III The antilipidemic drugs quercetin and acipimox rescue atrophy of spleen, thymus, inguinal white adipose tissue (iWAT), brown adipose tissue (BAT), and gonadal white adipose tissue (gWAT) In our CLEC16A knockout mice, we have generated inducible atrophy of the spleen, thymus, and white adipose tissue. Here, we evaluate the effects of quercetin or acipimox on organ weight in our whole-body inducible CLEC16A knockout mice. Figure 7 shows the ratio graphs for the spleen (Figure 7A), thymus (Figure 7A), liver (Figure 7A), kidney (Figure 7A), inguinal white adipose tissue (iWAT) (Figure 7B), brown adipose tissue (BAT) (Figure 7B), gonadal white adipose tissue (gWAT) (Figure 7B), pancreas (Figure 7C), muscle (Figure 7C), heart (Figure 7C), cerebellum (Figure 7C), and glucose (Figure 7B). We report weight loss and severe atrophy of the spleen, thymus, and inguinal white adipose tissue (iWAT), as well as severe atrophy of brown adipose tissue (BAT) and gonadal white adipose tissue (gWAT). KO mice also showed a significant decrease in glucose.

[0058] Treatment with quercetin or acipimox rescued spleen atrophy. Quercetin or acipimox significantly improved the organ weight ratios of the thymus, iWAT, and gWAT. CLEC16A KO mice showed significant decreases in the weights and body weight ratios of the spleen, thymus, inguinal white adipose tissue (iWAT), brown adipose tissue (BAT), and gonadal white adipose tissue (gWAT) compared with controls over the study period.

[0059] The weight / body weight ratios of the spleen, thymus, iWAT, BAT, and gWAT in CLEC16A KO mice were significantly reduced compared to controls (Figure 7). The liver, kidney, pancreas, muscle, heart, and cerebellum showed no significant changes compared to control littermates. Quercetin or acipimox control littermates showed no adverse effects on organ weights or their ratios throughout the study period. As expected, treatment with quercetin or acipimox significantly rescued spleen atrophy. Quercetin or acipimox significantly improved the organ weight ratios of the thymus, iWAT, and gWAT. Overall, the mitophagy-promoting agents, quercetin or acipimox, had a significant rescue effect on organ weights in our systemically inducible CLEC16A knockout mice.

[0060] Example IV Antilipidemic drugs rescue PINK-Parkin-dependent mitophagy dysregulation in our systemically inducible CLEC16A knockout mice Defective mitophagy. Knockout of CLEC16A disrupts the Nrdp1 / PINK / Parkin-dependent mitophagy pathway in vivo, resulting in the aggregation of fragmented mitochondria in splenic immune cells and predisposing mice to a cascade of altered signaling function, resulting in pathogenic inflammation, lipodystrophy, and sensory neurodegeneration resembling spinocerebellar degeneration [2,3,5].

[0061] We next performed immunoblot analysis of whole splenocyte lysates from control (untreated / vehicle-tamoxifen) ± probucol and CLEC16A KO ± probucol-treated mice to investigate possible defects in mitophagy / autophagy signaling and their rescue by probucol (Figure 3). Representative immunoblot images demonstrating Nrdp1 / PINK1 / Parkin-dependent defective mitophagy in CLEC16A KO mouse spleen lysates are shown in Figure 3A. As expected, CLEC16A KO mouse splenocyte lysates showed increased expression of PINK1 and Parkin. Splenocyte lysates from untreated and vehicle-tamoxifen-treated mice showed similar detectable levels of PINK, Parkin, and p62. The significant accumulation of p62 and decreased LC3-II expression in CLEC16A KO mice compared with both controls indicate impaired mitophagy. Furthermore, p62 is degraded by autophagy, and inhibition of autophagy increases its abundance.

[0062] In probucol-treated KO mice, levels of p62, PINK, and Parkin were significantly decreased, and LC3II was increased, indicating successful rescue of mitophagy disruption. Control, probucol-treated mice showed further enhancement of basal mitophagy / autophagy. The percentages in Figure 3 represent the levels of CLEC16A remaining after ablation. Untreated and tamoxifen-treated mice were scored based on their functional impairment (Figure 4). CLEC1A KO ± probucol mice received a score of 3.5, indicating severe functional impairment and phenotype. Probucol control mice received a score of 0, indicating no functional impairment. Therefore, probucol 1) functions downstream of PINK and Parkin, 2) regulates the final endpoint of mitophagy removal of damaged mitochondria, and 3) improves overall survival in KO mice. Quantitative graphs show the expression levels of CLEC16A, p62, LC3I / II, PINK1, and Parkin in control ± probucol-treated and KO ± probucol-treated mice, normalized to β-actin. Data are presented as the mean ± SE of three independent experiments (Figure 4).

[0063] Example V Antilipidemic drugs slow phenotypic progression, rescue fat loss, and slow the progression of lipodystrophy After 4 consecutive days of tamoxifen treatment, the timeline to onset of functional impairment (scores 1-4) was determined in cohorts of control ± probucol mice and KO ± probucol mice (Figure 4). Ten mice per group were used. Both males and females were included in this study. Young adult UBC-Cre-ER-Clec16a mice were used. loxP / loxP Mice were treated with tamoxifen for 4 consecutive days to induce adult CLEC16A knockout (CLEC16A ΔUBC Before treatment, CLEC16A loxP / loxP and UBC-Cre-ERT2-Clec16a- loxP / loxP was indistinguishable.

[0064] Approximately 8 days after tamoxifen induction, UBC-Cre-ERT2-CLEC16A loxP / loxP (CLEC16A ΔUBC) mice showed rapidly progressive weight loss over the course of days [2]. Tremors and other mild neurological behaviors (impairment score = 1; Figure 4) began after approximately 10 days and progressed rapidly. Approximately 60% of these mice showed the highest level of impairment with dystonic posture after 25 days (impairment score = 4; Figure 4).

[0065] Survival curves show the severity of the CLEC16A-associated phenotype. KO mice with a severity score of 3.5 exhibit the highest level of functional impairment with dystonic posture after 25 days and do not survive. Probucol treatment slows the progression of the phenotype in KO mice; only 25% of probucol-treated KO mice achieve a severity score of 3 or higher (Figure 4). Thus, probucol functions downstream of PINK and Parkin to regulate the final endpoint of mitophagy removal of damaged mitochondria and improve the survival rate of CLEC16A KO mice.

[0066] CLEC16A KO mice exhibit impaired mitophagy, a systemic inflammatory response involving multiple cytokines / chemokines via inflammatory and classical lipolytic pathways, and a significant reduction in body fat despite the absence of a reduction in food intake [2].

[0067] Next, we evaluated the effect of probucol on fat loss and rescue of the lipodystrophic phenotype. Control, KO, and KO ± probucol mice (scores 1, 2, and 3.5) were dissected to assess fat distribution. Inguinal WAT (iWAT), brown WAT (BAT), and gonadal WAT (gWAT) were collected (Figure 5). Compared to control mice, KO mice lacked fat in the inguinal and gonadal regions. Brown WAT also showed a significant reduction. Treatment with probucol inhibited this fat loss. Probucol-treated KO mice with severity scores of 1, 2, and 3 retained significantly more iWAT, BAT, and gWAT than KO mice (score 3.5). Thus, probucol rescued fat loss and delayed the progression of lipodystrophy in KO mice over the study period. Probucol may promote more efficient transport of lipid species and proteins for the mitophagy process downstream of Parkin, for example, by expanding autophagosomes, which facilitates the removal of accumulated defective mitochondria. After mitochondrial damage, swelling of robust lipid vesicles occurs. Recent studies have reported increased accumulation of lipid droplets in dopaminergic neurons in histological specimens from Parkinson's disease (PD) patients [6]. Similarly, epidemiological studies of Japanese and Korean populations who continue to safely use probucol for its anti-atherogenic activity may reveal a reduced incidence of PD.

[0068] Taken together, probucol exerts pleiotropic effects by 1) modulating PINK1 / Parkin-mediated mitophagy disruption, 2) improving survival, and 3) inhibiting CLEC16A. ΔUBCProbucol delays sensory neurodegeneration, resembling lipodystrophy and spinocerebellar degeneration phenotypes in CLEC16A knockout (KO) mice. We conclude that in patient populations harboring loss-of-function mutations in CLEC16A, drugs with modulatory effects on mitophagy / SOCS1-JAK-STAT signaling may compensate for the loss of CLEC16A activity and represent promising candidates for targeted intervention. Thus, our mouse model and the current results with probucol and interventions in the SOCS1-JAK-STAT pathway provide a valuable tool for evaluating therapeutic interventions in patients with diseases caused by CLEC16A mutations. This therapeutic agent may impact multiple autoimmune diseases by modulating and rescuing the observed mitophagy / autophagy dysregulation.

[0069] References 1. Pandey, R., et al., The Autoimmune Disorder Susceptibility Gene CLEC16A Restrains NK Cell Function in YTS NK Cell Line and Clec16a Knockout Mice. Frontiers in Immunology, 2019. 10(68). 2. Pandey, R., et al., JAK / STAT inhibitor therapy partially rescues the lipodystrophic autoimmune phenotype in Clec16a KO mice. Sci Rep, 2021. 11(1): p. 7372. 3. Hain, HS, et al., Inducible knockout of Clec16a in mice results in sensory neurodegeneration. Sci Rep, 2021. 11(1): p. 9319. 4. Georgakopoulos, ND, G. Wells, and M. Campanella, The pharmacological regulation of cellular mitophagy. Nat Chem Biol, 2017. 13(2): p. 136-146. 5. Pandey, R., et al., CLEC16A regulates splenocyte and NK cell function in part through MEK signaling. PLoS One, 2018. 13(9): p. e0203952. 6. Brekk, OR, et al., Cell type-specific lipid storage changes in Parkinson's disease patient brains are recapitulated by experimental glycolipid disturbance. Proc Natl Acad Sci USA, 2020. 117(44): p. 27646-27654.

[0070] Example IV Probucol dose response for rescue of Clec16aΔUBC phenotype CLEC16A is involved in many autoimmune diseases, and its dysfunction leads to dysregulated mitophagy, severe weight loss, inflammatory responses, and progressive neurodegeneration in Clec16aΔUBC mice. Our findings support the role of dysregulated mitophagy, UPR, and ER stress in danger sensing and their contribution to abnormal immune responses in autoimmune and autoinflammatory diseases. This mouse model mimics human sensory ataxia, dystonia, and lipodystrophy. To address these issues, we tested whether the mitophagy-promoting drugs probucol (a lipid-lowering drug) and quercetin (an ER stress inhibitor) rescue the phenotypes caused by Clec16a loss in our systemically inducible Clec16a knockout model.

[0071] Our findings provide evidence that targeting mitophagy and ER stress pathways, combined with intervention in the JAK-STAT pathway, rescues the phenotype. Current treatments for many autoimmune and neurodegenerative diseases only address symptom management without affecting disease progression. This lack of disease-modifying options highlights a significant gap in available treatments.

[0072] Probucol treatment A schematic of the treatment protocol is shown in Figure 8. Probucol was purchased from Cayman Chemical (cat#15043) and formulated in saline (0.9% NaCl) with 2% DMSO, 2.5% PEG300, and 2.5% Tween 80. This solution was administered intraperitoneally (IP) daily for 16 days at three doses: 3.5 mg / kg, 10 mg / kg, and 50 mg / kg (Figure 8C). Vehicle-treated mice received saline with 2% DMSO, 2.5% PEG300, and 2.5% Tween 80. Fresh solutions were made daily before injection. All animals were sacrificed according to approved IACUC protocols and humane endpoints.

[0073] Probucol, a late-stage mitophagy promoter, dose-dependently slows phenotypic progression . We performed a comprehensive dose-response study of probucol and carefully evaluated its effect on the observed phenotypes (Figure 9). Our evaluation included measuring the impact of varying concentrations of probucol on the observable phenotypes. A clear trend emerged, revealing increasingly pronounced phenotypic rescue with increasing doses of probucol (3.5 mg / kg, 10 mg / kg, 50 mg / kg) (Figure 9). By day 11, significant improvement was observed with the 10 mg / kg dose. Probucol at 50 mg / kg demonstrated a robust rescue response as early as day 8, which remained significant throughout the study. Overall, probucol significantly rescued organ weights in Clec16aΔUBC mice compared with vehicle-treated KO mice. Tamoxifen control and probucol control littermates showed no adverse effects on organ weights or ratios throughout the study. In general, probucol at doses of 3.5 mg / kg, 10 mg / kg, and 50 mg / kg substantially significantly prolonged the survival of Clec16a KO mice and slowed the progression of the observed phenotype (Fig. 9 ).

[0074] This pattern indicates that higher doses of probucol significantly enhanced the investigated phenotype. Clearly, there is a direct correlation between the degree of phenotypic rescue and the dose of probucol. This finding highlights probucol as a strong candidate to ameliorate or even reverse the observed phenotype, further highlighting its potential therapeutic application in our study setting. This result reinforces the therapeutic potential of probucol as an effective promoter of late-stage mitophagy for the treatment of autoimmunity and neurodegeneration.

[0075] The observed positive relationship between the dose of probucol and the degree of phenotypic rescue indicates that this compound may be involved in the specific biological mechanism under investigation. Our study delved deep into the complex mechanisms underlying probucol-promoted mitophagy rescue. Mitophagy is a critical process responsible for the selective removal of damaged mitochondria and is paramount in maintaining cellular health. Experimental administration of probucol yielded compelling results: a significant enhancement of the mitophagy process, aptly termed "mitophagy rescue." This remarkable finding demonstrates probucol's ability to amplify the removal of damaged mitochondria. Removal of damaged mitochondria is crucial for maintaining cellular function and preventing the accumulation of defective organelles, all of which may be beneficial for the treatment and / or prevention of autoimmune diseases and neurodegenerative tendencies in humans.

[0076] Taken together, we conclude that probucol, a late-stage mitophagy promoter, exerts pleiotropic effects by modulating PINK1 / Parkin-mediated mitophagy disruption, improving survival and dose-dependently delaying the lipodystrophy and sensory neurodegeneration resembling the spinocerebellar degeneration phenotype in Clec16aΔUBC(KO) mice. We conclude that in patient populations harboring CLEC16A loss-of-function mutations, drugs modulating mitophagy, ER stress, and SOCS1-JAK-STAT signaling may compensate for the attenuation of CLEC16A activity and be useful for targeted intervention.

[0077] Example V Tests and treatments to improve symptoms associated with CLEC16A deficiency The information herein can be clinically applied to patients for therapeutic intervention, particularly for the treatment of symptoms associated with CLEC16A deficiency. A preferred embodiment of the present invention encompasses the clinical application of the information described herein to patients. In some embodiments, a CLEC16A-related disease or condition is evaluated, monitored, or diagnosed by a method comprising the following steps: (i) measuring one or more clinical symptoms or signs of CLEC16A deficiency in a subject; (ii) combining the obtained measurements into a single composite measurement; and (iii) comparing the composite measurement with a baseline value or another composite measurement in the same subject to assess the overall severity of, or change in, CLEC16A deficiency in the subject. (i) one or more composite measurements are employed to measure the clinical effect of a diagnostic, therapeutic, or other type of medical intervention on a subject; (ii) for each of the measurements tested, the subject is classified as: (a) a responder or non-responder, (b) a member of a clinical category, or (c) a member of a measurement range based on the change in said one or more clinical symptoms measured using the particular clinical symptom or metabolic pathway being evaluated; and (iii) the measurements obtained are combined into a single composite measurement by either: (a) individually evaluating the change in each measurement obtained from each assay performed before combining the measurements into a single composite measurement, or (b) combining measurements obtained from a first time point to generate a single composite measurement for said first time point, and then comparing the single composite measurement for the first time point to a single composite measurement generated from the same assays for a second time point.

[0078] Important clinical evaluations for CLEC16A-related diseases or conditions include improvement or slowing of progression of one or more of: robust autoimmune inflammatory response, severe weight loss, severe neurological symptoms, neuroinflammation, progressive neurodegeneration resembling spinocerebellar degeneration, and fat loss. Other clinical evaluations include enhanced mitochondrial removal, rescue of splenic atrophy, and / or improved organ weight ratios of thymus, inguinal white adipose tissue (iWAT), and / or gonadal white adipose tissue (gWAT) compared to untreated controls.

[0079] The therapeutic dose of a mitophagy-promoting agent for human induction can be determined by one skilled in the art based on the response rate. The mitophagy-promoting agent, or a pharmaceutically acceptable composition comprising the mitophagy-promoting agent, can be administered at a dose of 0.0001 mg / kg / day, 0.001 mg / kg / day, or 0.01 mg / kg / day to about 100 mg / kg / day, but may be higher or lower depending, inter alia, on the activity of the active metabolite compound, the bioavailability, metabolic kinetics and other pharmacokinetic properties of the compound, the mode of administration, and various other factors discussed above.

[0080] Generally, the initial therapeutically effective amount of a compound described herein or a pharmaceutically acceptable salt thereof administered ranges from about 0.01 to about 200 mg / kg or about 0.1 to about 20 mg / kg of patient body weight per day, with a typical initial range being about 0.3 to about 15 mg / kg / day. Oral unit dosage forms, such as tablets and capsules, can contain from about 0.1 mg to about 1000 mg of the compound or a pharmaceutically acceptable salt thereof. In another embodiment, such dosage forms contain from about 50 mg to about 500 mg of the compound or a pharmaceutically acceptable salt thereof. In yet another embodiment, such dosage forms contain from about 25 mg to about 200 mg of the compound or a pharmaceutically acceptable salt thereof. In yet another embodiment, such dosage forms contain from about 10 mg to about 100 mg of the compound or a pharmaceutically acceptable salt thereof. In a further embodiment, such dosage forms contain from about 5 mg to about 50 mg of the compound or a pharmaceutically acceptable salt thereof.

[0081] In certain embodiments, the mitophagy-promoting agent can be administered with one or more agents selected from a JAK-STAT inhibitor, an ER stress regulator, and a SOCS1 inhibitor. Exemplary JAK-STAT inhibitors include, but are not limited to, tofacitinib, ruxolitinib, baricitinib, peficitinib, decernotinib, filgotinib, solcitinib, itacitinib, SHR0302, upadacitinib, and PF-04965842. Exemplary ER stress modulators include, but are not limited to, rapamycin, 4-phenylbutyric acid (4-PBA), trimethylamine N-oxide dehydrate (TMAO), dimethyl sulfoxide (DMSO), taursodeoxycholic acid (TUDCA), AMPK-activated protein kinase, 5'-aminoimidazole-4-carboximide-1-β-d-ribofuranoside (AICAR), glucagon-like peptide-1 (GLP-1), DPP4 inhibitors, and N-acetylcysteine ​​(NAC).

[0082] Treatment may be administered after patients present with CLEC16A-related diseases or symptoms. Mitophagy-promoting drugs such as probucol, quercetin, and acipimox have been shown to be well tolerated, and symptoms were assessed using a clinical scoring system.

[0083] While certain preferred embodiments of the present invention have been described and specifically exemplified, it is not intended that the invention be limited to such embodiments. Various modifications may be made without departing from the scope and spirit of the invention as defined in the following claims.

Claims

1. A pharmaceutical composition for improving at least one CLEC16A-related symptom, comprising an effective amount of at least one mitophagy promoter, wherein the at least one symptom is selected from a severe autoimmune inflammatory response, severe weight loss, severe neurological symptoms, neuroinflammation, progressive neurodegeneration similar to spinocerebellar degeneration, and fat loss. A pharmaceutical composition in which the mitophagy promoter is selected from probucol, quercetin, or acipimox.

2. The pharmaceutical composition according to claim 1, wherein the mitophagy promoter is probucol.

3. a) The mitophagy promoter rescues splenic atrophy and / or improves the organ weight ratio of the thymus, inguinal white adipose tissue (iWAT), and / or gonadal white adipose tissue (gWAT) compared to an untreated control, or b) The pharmaceutical composition according to claim 1, wherein the mitophagy promoter delays the progression of CLEC16A-related symptoms compared to an untreated control.

4. A pharmaceutical composition for treating CLEC16A-related degeneration of the thymus or spleen, comprising a mitophagy promoter, which alters the weight ratio of the thymus or spleen and improves symptoms associated with the degeneration of the thymus or spleen.

5. The pharmaceutical composition according to claim 4 for treating thymic degeneration associated with CLEC16A.

6. The pharmaceutical composition according to claim 4 for treating splenic degeneration associated with CLEC16A.

7. A pharmaceutical composition for treating white adipose tissue (WAT) degeneration associated with CLEC16A, comprising a mitophagy promoter, which alters the weight ratio of white adipose tissue and improves symptoms associated with white adipose tissue degeneration.

8. The pharmaceutical composition according to claim 7, wherein the white adipose tissue is inguinal white adipose tissue (iWAT).

9. The pharmaceutical composition according to claim 7, wherein the white adipose tissue is gonadal white adipose tissue (gWAT).

10. The pharmaceutical composition according to any one of claims 1 to 9, further comprising a PPARγ inhibitor.

11. The pharmaceutical composition according to any one of claims 4 to 9, wherein the CLEC16A-related degeneration is selected from autoimmune disorders, lipodystrophy disorders, and neurodegenerative disorders.

12. The pharmaceutical composition according to any one of claims 1 to 9, wherein at least one mitophagy promoter promotes the removal of mitochondria.

13. The pharmaceutical composition according to any one of claims 4 to 9, wherein the mitophagy promoter is selected from probucol, quercetin, or acipimox.

14. The pharmaceutical composition according to any one of claims 4 to 9, wherein the mitophagy promoter is probucol.

15. A pharmaceutical composition according to any one of claims 1 to 9, further comprising one or more agents selected from JAK-STAT inhibitors, ER stress modifiers, and SOCS1 inhibitors.

16. a) The drug is a JAK-STAT inhibitor selected from tofacitinib, ruxolitinib, baricitinib, peficitinib, desernotinib, filgotinib, sorucitinib, itacitinib, SHR0302, upadacitinib, and PF-04965842, or b) The pharmaceutical composition according to claim 15, wherein the agent is an ER stress modulator selected from rapamycin, 4-phenylbutyric acid (4-PBA), trimethylamine N-oxide dehydrated product (TMAO), dimethyl sulfoxide (DMSO), taursodeoxycholic acid (TUDCA), AMPK-activated protein kinase, 5'-aminoimidazole-4-carboxymide-1-β-d-ribofuranoside (AICAR), glucagon-like peptide-1 (GLP-1), DPP4 inhibitor, and N-acetylcysteine ​​(NAC).

17. The pharmaceutical composition according to claim 15, wherein the drug is tofacitinib.

18. A mitophagy promoter is a) Rescuing splenic atrophy and / or improving the organ weight ratio of the thymus, inguinal white adipose tissue (iWAT), and / or gonadal white adipose tissue (gWAT), compared to untreated controls, or b) Slows the progression of CLEC16A-related symptoms compared to untreated controls. A pharmaceutical composition according to any one of claims 1 to 9.

Images