Methods and compositions for treating Wolfram syndrome

Abstract

Methods for treating at least one symptom or treating one or more symptoms of Wolfram syndrome in a subject are provided, comprising administering to the subject a pharmaceutically effective amount of a combination of a bile acid compound and a phenylbutyric acid compound.

Description

[Technical Field]

[0001] The present disclosure relates generally to compositions and methods for treating Wolfram Syndrome. [Background technology]

[0002] Wolfram syndrome is a genetic disorder characterized by early-onset diabetes, progressive blindness, and neurodegeneration. Currently, there are no treatments that can slow, stop, or reverse the progression of the disease. Therefore, there is a need for treatments for individuals with Wolfram syndrome. Summary of the Invention

[0003] Provided herein are methods for treating one or more symptoms of Wolfram syndrome in a subject, comprising administering to the subject a pharmaceutically effective amount of a combination of TURSO and sodium phenylbutyrate. In some embodiments, the subject has or is at risk of developing diabetes. In some embodiments, the diabetes is insulin-dependent diabetes. In some embodiments, the diabetes is early-onset diabetes. In some embodiments, the subject has or is at risk of developing optic atrophy. In some embodiments, the subject has or is at risk of developing hearing impairment. In some embodiments, the subject has one or more mutations in the wolframin (WFS1) gene. In some embodiments, the subject has a c.1672C>T, p.R558C mutation in the WFS1 gene. In some embodiments, the subject has a c.2654C>T, p.P885L mutation in the WFS1 gene. In some embodiments, the subject has one or more mutations in the CDGSH iron-sulfur domain protein 2 (CISD2) gene. In some embodiments, TURSO and sodium phenylbutyrate are administered once daily or twice daily. In some embodiments, TURSO is administered to a subject at a dose of about 5 mg / kg to about 100 mg / kg. In some embodiments, sodium phenylbutyrate is administered to a subject at a dose of about 10 mg / kg to about 400 mg / kg. In some embodiments, TURSO is administered in an amount of about 0.5 to about 5 grams per day. In some embodiments, sodium phenylbutyrate is administered in an amount of about 0.5 grams to about 10 grams per day. In some embodiments, the method comprises administering 1 gram of TURSO and 3 grams of sodium phenylbutyrate to a subject once daily or twice daily. In some embodiments, the method comprises administering to the subject 1 gram of TURSO once daily and 3 grams of sodium phenylbutyrate once daily for about 14 days or more, followed by administering to the subject about 1 gram of TURSO twice daily and 3 grams of sodium phenylbutyrate twice daily. In some embodiments, the TURSO and sodium phenylbutyrate are administered orally.In some embodiments, the TURSO and sodium phenylbutyrate are formulated as a single powder formulation. In some embodiments, the method further comprises administering to the subject one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents are valproic acid, a glucagon-like peptide (GLP)-1 receptor agonist, dantrolene sodium, or an ER Ca2+ stabilizer.

[0004] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice or test the present invention, suitable methods and materials are described below.

[0005] It is understood that certain features of the present disclosure that are described for clarity in the context of separate embodiments can also be provided in combination in a single embodiment. Conversely, various features of the present disclosure that are described for brevity in the context of a single embodiment can also be provided separately or in any suitable subcombination. All combinations of the embodiments of the present disclosure are specifically embraced by the present disclosure and are disclosed herein as if each and every combination were individually and expressly disclosed. Furthermore, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein as if each and every such subcombination were individually and expressly disclosed herein.

[0006] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the present invention will become apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0007] [Figure 1] Panels A and B show the carrier frequency and clinical manifestations of the WFS1 c.1672C>T,p.R558C variant. (A) Carrier frequency of WFS1 c.1672C>T,p.R558C in subjects of Ashkenazi, Ashkenazi / Sephardic, and Sephardic descent. (B) Carrier frequency of WFS1 c.1672C>T,p.R558C by country of origin. [Figure 2] 1 shows classification of pathogenic WFS1 variants.

[0023] FIG. 1 is a schematic diagram of classification of pathogenic WFS1 variants by protein expression. [Figure 3]A–H show that the p.R558C variant of WFS1 is more stable in cells than the p.P885L variant. (A) Diagram of the WFS1 protein showing the locations of the two variants, R558C and P885L. (B) Thermal profiles of WFS1 variants (WT, R558C, and P885L) measured using a SplitLuc-tagged reporter expressed in HEK293T cells (data from three independent experiments). (C) Luminescence intensity of WFS1 variants in cells incubated at 30°C and 37°C for 24 hours (n = 12, ***P < 0.001 and ****P < 0.0001, unpaired t-test). (D) Fold change in luminescence intensity of WFS1 variants treated with the proteasome inhibitor bortezomib for 24 hours (**P < 0.01 and ***P < 0.001, unpaired t-test, compared to the untreated group). (E) (Left) Representative blot images of WFS1(HA) and α-tubulin in a CHX chase assay. The bottom row shows a long-exposure image of WFS1(HA). (Right) Quantification of relative WFS1 protein levels normalized to α-tubulin is shown (n=3, *P<0.05, **P<0.01, ****P<0.0001 by two-way ANOVA). (F) (Top) Representative blot images of WFS1 and α-tubulin in iPS cells are shown. (Bottom) Quantification of relative WFS1 protein levels normalized to α-tubulin is shown (n=3, **P<0.01 and **P<0.0001 by one-way ANOVA compared to BJFF.6, †††P<0.001 and ††††P<0.0001 by one-way ANOVA compared to AN1.1, and #P<0.05 and ##P<0.01 by one-way ANOVA). (G) Relative mRNA levels of WFS1 in iPS cells (n = 7, ****P < 0.0001B by one-way ANOVA compared with JFF.6, ††††P < 0.0001 by one-way ANOVA compared with AN1.1, #P < 0.05 and ##P < 0.01 by one-way ANOVA).(H) Relative mRNA levels of WFS1 in an ActD chase assay (n = 3, *P < 0.05 by one-way ANOVA compared to BJFF.6, ###P < 0.001 by one-way ANOVA compared to AN1.1). [Figure 4]Figures A-H show that combined treatment with 4-PBA and TUDCA ameliorate the deleterious effects of the WFS1 c.1672C>T, p.R558C variant. (A) Schematic diagram of Wolfram syndrome pathogenesis and the targets modulated by combined treatment with 4-PBA and TUDCA (P+T). (B) Expression of HiBiT-tagged WFS1 protein after 24 h of treatment with 500 μM 4-PBA and 50 μM TUDCA (P+T) was shown. NanoLuc levels expressed from the same plasmid backbone were examined (n = 48, P values ​​by unpaired t-test). (C) (Left) Representative blotting images of WFS1 and α-tubulin in iPSCs treated with or without P+T for 48 h. (Right) Quantification of WFS1 protein levels normalized to α-tubulin (n = 3, *P < 0.05 and ***P < 0.01 by unpaired t-test compared to Ctrl (control)). (D) Relative WFS1 mRNA levels in iPSCs treated with or without P+T for 48 hours (n = 5, **P < 0.01 by unpaired t-test compared to Ctrl). (E) Representative immunofluorescence images of NPC markers in NPCs differentiated from patient-derived iPSCs. Scale bar: 100 µm. (F) qPCR analysis of ER stress-related genes in NPCs treated with or without P+T for 48 hours (n = 6, *P < 0.05, **P < 0.01, and ****P < 0.0001 by unpaired t-test compared to Ctrl). (G) Mitochondrial respiration of NPCs treated with or without P+T for 48 h, expressed as a percentage of baseline oxygen consumption rate (OCR) measurements. Respiration was examined by measuring the change in relative OCR after oligomycin (OM), FCCP, and antimycin A (AA) / rotenone (R) injection (n = 3, W024: ***P < 0.001, W392: *P < 0.05, W121: *P < 0.05, compared with the area under the curve (AUC) of Ctrl by unpaired t-test).(H) Caspase 3 / 7 activity normalized to cell viability in NPCs treated with or without 4-PBA, TUDCA, and P+T for 48 h (n=7, ****P<0.001 and ****P<0.0001 by one-way ANOVA compared with Ctrl; #P<0.05, ##P<0.01, and ####P<0.0001 by one-way ANOVA compared with Ctrl). [Figure 5] A–C show a comparison of ER stress levels in NPCs across cell lines. (A) Representative immunofluorescence images of NPC markers in neural progenitor cells (NPCs) differentiated from the control iPS cell line, AN1.1. Scale bar: 100 μm. (B) qPCR analysis of WFS1 expression in NPCs differentiated from each iPS cell line (AN1.1: n = 4, W024 and W392: n = 6, W121: n = 7. *P < 0.05, ***P < 0.001, and ****P < 0.0001 compared to N1.1 by one-way ANOVA; #P < 0.05 and ##P < 0.01 by one-way ANOVA). (C) qPCR analysis of ER stress-related genes in NPCs differentiated from each iPS cell line. (AN1.1: n=4, W024 and W392: n=6, W121: n=7. *P<0.05, ***P<0.001 and ****P<0.0001 compared to AN1.1 by one-way ANOVA, #P<0.05 and ##P<0.01 by one-way ANOVA). [Figure 6]Panels A-B show a comparison of the effects of each treatment on WFS1 protein and ER stress levels. (A) (Top) Representative blot images of WFS1 and α-tubulin in iPSCs treated with or without 4-PBA, TUDCA, or P+T for 48 hours are shown. (Bottom) Quantification of WFS1 protein levels normalized to α-tubulin is shown. (n=3; *P<0.05, **P<0.01, and ****P<0.0001 by one-way ANOVA compared to Ctrl; #P<0.05, ##P<0.01, and ####P<0.0001 by one-way ANOVA). (B) qPCR analysis of ER stress-related genes in NPCs treated with or without 4-PBA, TUDCA, or P+T. (W024: n = 5, W392: n = 4, W121: n = 5. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 by one-way ANOVA compared with Ctrl, #P < 0.05 by one-way ANOVA). [Figure 7]Figures A–C show a comparison of the effects of each treatment on mitochondrial DNA content, mitochondrial membrane potential, and apoptosis. (A) Relative mitochondrial DNA (mtDNA) copy number normalized to nuclear DNA (nDNA) measured by qPCR analysis in NPCs treated with or without 4-PBA, TUDCA, or P+T for 48 hours (W024: n = 3, W392: n = 4, W121: n = 4; *P < 0.05 and **P < 0.01 by one-way ANOVA compared to Ctrl; #P < 0.05, ##P < 0.01, and ###P < 0.001 by one-way ANOVA). (B) Mitochondrial membrane potential measured with the fluorescent probe TMRM in NPCs treated with or without 4-PBA, TUDCA, or P+T for 48 hours (n = 6, ***P < 0.0001 by one-way ANOVA compared to Ctrl, ###P < 0.001 by one-way ANOVA). (C) (Top) Representative blot images of cleaved caspase 3 and α-tubulin in NPCs treated with or without 4-PBA, TUDCA, or P+T for 48 hours (bottom) Quantification of cleaved caspase 3 protein levels normalized to α-tubulin (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by one-way ANOVA compared to Ctrl, #P < 0.05 and ##P < 0.01 by one-way ANOVA). [Figure 8] A-B show that P+T treatment reduced caspase 3 / 7 activity in NPCs derived from patients with typical Wolfram syndrome. (A) Information on four patients with typical Wolfram syndrome is shown, including the genetic location of the autosomal recessive pathogenic variant in WFS1 and the age of symptom onset. Age indicates when the subject was included in the study. DM: diabetes mellitus; OA: optic atrophy. (B) Caspase 3 / 7 activity, normalized by cell viability, in treated and untreated NPCs treated with or without P+T for 24 hours is shown. (n=6, ****P<0.0001 by unpaired t-test compared to Ctrl.) [Figure 9]A-I show that combined treatment with 4-PBA and TUDCA increased insulin secretion in SC islets harboring the WFS1 c.1672C>T, p.R558C variant. (A) Representative flow cytometry dot plots of cells expressing or co-expressing pancreatic beta cell markers or endocrine cell markers in stage 6 SC islets from AN1.1 (n = 4), W024 (n = 3), and W121 (n = 3). (B) Quantification of the percentage of β-cells (FIG) cells (compared to AN1.1 by two-way ANOVA; *P < 0.05 and ****P < 0.0001; #P < 0.05, ##P < 0.01, and ####P < 0.0001 by two-way ANOVA). (C) (Left) Representative blot images of WFS1 and α-tubulin in stage 6 SC islets. (Right) Quantification of relative WFS1 protein levels normalized to α-tubulin (n=3, **P<0.01 and ***P<0.001 by one-way ANOVA compared to AN1.1, #P<0.05 by one-way ANOVA). (D) Relative WFS1 mRNA levels in stage 6 SC islets (n=4, **P<0.01 by one-way ANOVA compared to AN1.1). (E) Static GSIS function evaluation in stage 6 SC islets of AN1.1 (n=7), W024 (n=6), and W121 (n=8) (*P<0.05 and ***P<0.001 by two-way ANOVA compared to 2 mM of each cell line, ##P<0.01 and ####P<0.0001 by two-way ANOVA). (F) Schematic diagram of P+T validation in SC islets. (G) (Top) Representative blot images of WFS1 and α-tubulin in stage 6 SC islets treated with or without P+T for 7 days are shown. (Bottom) Quantification of WFS1 protein levels normalized to α-tubulin is shown (n = 3, *P < 0.05 by unpaired t-test compared to Ctrl). (Bottom) Caspase 3 / 7 activity normalized to cell viability is shown in stage 6 SC islets treated with or without H_P+T for 7 days (n = 3, ***P < 0.001 and ****P < 0.0001 by unpaired t-test compared to Ctrl).(I) Static GSIS function assessment of W024 (n = 5) and W121 (n = 4) treated with or without P+T for 7 days (*P < 0.05 by one-way paired t-test compared with 2 mM of each condition; #P < 0.05 and ##P < 0.01 by two-way unpaired t-test). CP: C-peptide, CHGA: chromogranin A. [Figure 10] Electron microscopy (EM) analysis of SC islets. Representative EM images of stage 6 SC islets from AN1.1, W024, and W121 treated with or without P+T for 7 days are shown. Scale bar: 600 nm. Red arrows indicate dilated endoplasmic reticulum (ER). [Figure 11] A-B show in vivo validation of combined treatment with chemical chaperones. (A) IP-GTT was performed on wild-type (WT) or Wfs1 knockout (KO) mice at baseline and after 1 month of feeding either Ctrl or P+T diets. (B) AUC of IP-GTT is shown (KO, Ctrl: n = 12; KO, P+T: n = 12; WT: n = 7; **P < 0.01 and ***P < 0.001 by one-way ANOVA; ##P < 0.01 and ####P < 0.0001 by one-way ANOVA compared to WT at baseline; ††††P < 0.0001 by one-way ANOVA compared to WT after 1 month). [Figure 12]Figures A-G show additional in vivo validation of combined treatment with chemical chaperones. (A) Serum insulin levels in 5-6 week-old WT and Wfs1 KO mice before feeding either Ctrl or P+T diet (WT: n=5, KO, Ctrl: n=7, KO, P+T: n=9. **P<0.05 and **P<0.01 by two-way ANOVA). (B) Food consumption rate in Wfs1 KO mice fed either Ctrl or P+T diet. (C) Body weights of Wfs1 KO mice before and after feeding either Ctrl or P+T diet (KO, Ctrl: n=14, KO, P+T: n=14). (D) IP-ITT analysis was performed on WT or Wfs1 KO mice before (baseline) and after (1 month) feeding either Ctrl or P+T diet (WT: n = 5; KO, Ctrl: n = 5; KO, P+T: n = 8. *P < 0.05 by two-way ANOVA comparing WT vs. KO vs. Ctrl. #P < 0.05 and ####P < 0.0001 by two-way ANOVA comparing WT vs. KO vs. P+T). (E) IP-ITT analysis was performed on Wfs1 KO mice fed either Ctrl or P+T diet, comparing the samples from each group before (baseline) and after (1 month) feeding. (F) Serum insulin levels in WT and Wfs1 KO mice fed either Ctrl or P+T diets for 1 month (WT: n = 6, KO, Ctrl: n = 7, KO, P+T: n = 9. *P < 0.05, **P < 0.01, and ****P < 0.0001 by two-way ANOVA). (E) Serum insulin levels in Wfs1 KO mice fed either Ctrl or P+T diets were compared before (baseline) and after (1 month) feeding of each group's diet (*P < 0.05 by paired test). DETAILED DESCRIPTION OF THE INVENTION

[0008] Applicant has discovered that a combination of a bile acid (e.g., taursodiol (TURSO)) and a phenylbutyrate compound (e.g., sodium phenylbutyrate) can be used to treat one or more symptoms of Wolfram syndrome. The present disclosure provides a method of treating at least one symptom of Wolfram syndrome in a subject by administering a bile acid (e.g., TURSO) and a phenylbutyrate compound (e.g., sodium phenylbutyrate). The subject in need of treatment may be suffering from or at risk of developing diabetes, optic atrophy, or hearing impairment.

[0009] Where a range of values ​​is provided, it is understood that, unless the context clearly dictates otherwise, each intervening value between the upper and lower limit of that range, to the tenth of the unit of the lower limit, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, which are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0010] Certain ranges are presented herein with numerical values ​​preceded by the term "about." The term "about" is used herein to provide literal support for the exact number it precedes, as well as for a number that is near or approximately the number it precedes. When determining whether a numerical value is near or approximately a specifically recited numerical value, the near or approximately unrecited numerical value may be a numerical value that, in the context in which it is presented, provides a substantially equivalent numerical value to the specifically recited numerical value.

[0011] Unless otherwise defined, all technical terms, notations, and other scientific terms or terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this application pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art.

[0012] I. Wolfram syndrome Wolfram syndrome (WS) is a rare genetic disorder caused by pathogenic variants in the wolframin (WFS1) gene and, in a small subset of patients, in the CDGSH iron-sulfur domain protein 2 (CISD2) gene. It manifests as diabetes insipidus, diabetes mellitus (e.g., early-onset insulin-dependent diabetes mellitus), optic atrophy, and progressive neurodegeneration. Many patients also develop other symptoms, ranging from hearing loss and endocrine dysfunction to neurological and psychiatric disorders. Thus, recent clinical and genetic findings suggest that Wolfram syndrome is best characterized as a spectrum disorder. Wolfram syndrome (WS) is a progressive neurodegenerative disorder in which patients present with non-autoimmune, non-HLA-linked diabetes mellitus in the first decade, followed by optic nerve atrophy; cranial diabetes insipidus and sensorineural hearing loss in the second decade; early renal tract abnormalities in the third decade; and multiple neurological abnormalities, including cerebellar ataxia, myoclonus, and psychiatric disorders, in the fourth decade. Patients with Wolfram syndrome typically die in the third or fourth decade from central respiratory failure due to brainstem atrophy. The rate of progression varies widely among patients. The clinical phenotype of Wolfram syndrome may share similarities with maternally inherited mitochondrial disorders, including diabetes mellitus, hearing loss, mitochondrial encephalopathy, mitochondrial myopathy, lactic acidosis, stroke-like episodes, and Leber's hereditary optic neuropathy.

[0013] WFS1 variants associated with Wolfram syndrome include missense, nonsense, frameshift, in-frame insertions or deletions, and splice site variants. WFS1 variants are known in the art and are described, for example, in van ven Ouwel and JM, et al. Molecular characterization of WFS1 in patients with Wolfram syndrome. The Journal of molecular diagnostics 2003;5(2):88-95 and Khanim F, et al. WFS1 / wolframin mutations, Wolfram syndrome, and associated diseases. Human mutation. 2001;17(5):357-67. WFS1 encodes an endoplasmic reticulum (ER) transmembrane protein. The ER is a network within all cells involved in protein synthesis, calcium storage and handling, redox regulation, steroidogenesis, and cell signaling, including apoptosis signaling. Given the essential functions of the ER, its dysfunction can trigger various cellular pathologies. Studies have shown that pancreatic beta cells and neurons are particularly sensitive to ER dysfunction, possibly due to their high rates of hormone and neurotransmitter synthesis, respectively. WFS1 can regulate Ca2+ homeostasis within the ER, which is crucial for the synthesis and secretion of neurotransmitters and hormones such as insulin. Loss of WFS1 in the ER leads to disruption of Ca2+ homeostasis, triggering the unfolded protein response (UPR) following chronic ER stress. WFS1 also negatively regulates the UPR molecule ATF6, suppressing its overactivation and resulting in cell apoptosis. Furthermore, WFS1 can affect mitochondrial function by transporting Ca2+ from the ER to mitochondria via the mitochondria-associated endoplasmic reticulum membrane (MAM). In Wolfram syndrome, pancreatic beta cells and neurons can be lost as a result of mutations in the WFS1 gene.In cellular and animal models of Wolfram syndrome, WFS1 mutations cause ER stress, pancreatic β-cell dysfunction, and the initiation of ER-associated cell death.

[0014] A small proportion of patients have mutations in the WFS2 (CISD2) gene, which also encodes an ER transmembrane protein. Diabetes mellitus and hearing impairment have been reported in patients with WFS2 mutations. The clinical phenotype of these patients differs from that of patients with WFS1 mutations, as they lack diabetes insipidus, have upper intestinal ulcers and bleeding, and have defective platelet aggregation.

[0015] The methods described herein can be used to treat a subject exhibiting one or more symptoms associated with Wolfram syndrome or a subject diagnosed with Wolfram syndrome. In some embodiments, the subject may be a subject suspected of having and / or being at risk for developing Wolfram syndrome. The methods described herein may further include determining whether a subject has or is at risk for developing Wolfram syndrome, diagnosing whether a subject has or is at risk for developing Wolfram syndrome, or selecting a subject having or at risk for developing Wolfram syndrome. Many traits, symptoms, and conditions are associated with Wolfram syndrome and can be used for diagnostic purposes. The subject may have or be at risk for developing diabetes mellitus. For example, the subject may have or be at risk for developing early-onset diabetes mellitus (e.g., age of onset less than 15 years). The subject may have or be at risk of developing optic nerve atrophy (e.g., age of onset before 15 years), high-tone sensorineural hearing impairment (e.g., congenital hearing loss), cerebellar ataxia, autonomic dysfunction, dementia or intellectual disability, psychiatric illness, seizures, neurogenic bladder or bladder dysfunction, bowel dysfunction, diabetes insipidus (e.g., central diabetes insipidus), delayed or absent puberty, hypogonadism in males, non-autoimmune hypothyroidism, growth retardation, cardiomyopathy, and structural congenital heart disease. Methods for detecting the above conditions are known in the art. For example, the subject can be diagnosed based on clinical history, family history, physical examination, and neurological examination.

[0016] A subject may also be identified as having or at risk for Wolfram syndrome based on genetic testing. Genetic testing approaches include gene-targeted testing (single-gene testing or multigene panel testing) and comprehensive genomic testing (exome sequencing, exome arrays, and genome sequencing), depending on the phenotype. For example, sequencing of one or more genes involved in Wolfram syndrome (e.g., WFS1, WFS2, or other genes known in the art) can be performed to detect mutations. A multigene panel for hearing loss including WFS1 and other genes of interest can be performed, as described by Tranebjaerg et al. (WFS1 Wolfram Syndrome Spectrum Disorder. February 24, 2009, Editors Adam MP, Everman DB, Mirzaa GM, et al. GeneReviews®. Seattle (WA): University of Washington, Seattle; 1993-2022.) In some embodiments, genetic testing can be used in conjunction with clinical diagnosis to confirm a diagnosis of Wolfram syndrome. Mutations in the WFS1 gene include the H313Y mutation (Hansen L, Eiberg H, Barrett T, et al. Mutation analysis of the WFS1 gene in seven Danish Wolfram syndrome families; four new mutations identified. Eur J Hum Genet. 2005;13(12):1275-84), p.Trp314Arg (Bonnycastle LL, Chines PS, Hara T, et al. Autosomal dominant diabetes arising from a wolfram syndrome mutation. Diabetes. 2013;62(11):3943-50.), c.1672C>T, p.R558C mutation, and p.P885L mutation. Other WFS1 mutations are known in the art.

[0017] The subject may have been exhibiting one or more symptoms of Wolfram syndrome (e.g., any symptom of Wolfram syndrome described herein or known in the art) for about 1 day to about 5 years (e.g., about 1 month to about 6 months, about 7 months to about 18 months, or about 2, 3, or 4 years). The subject may have been diagnosed with Wolfram syndrome for about 1 day to about 5 years (e.g., about 1 to about 6 months, about 7 to about 18 months, or about 2, 3, or 4 years). The subject may be confirmed or identified as having Wolfram syndrome, for example, by a medical professional. The diagnostic process may involve multiple parties. For example, if a sample is collected from the subject as part of the diagnosis, a first party may collect the sample from the subject and a second party may test the sample. In some embodiments, the subject is diagnosed, selected, or referred by a physician (e.g., a general practitioner).

[0018] A skilled physician will recognize that certain factors can affect the bioavailability and metabolism of an administered compound in a subject and will be able to make adjustments accordingly. These include, but are not limited to, liver function (e.g., liver enzyme levels), kidney function, and gallbladder function (e.g., ion absorption and secretion, cholesterol transport protein levels, etc.). Each subject's exposure to an administered compound (e.g., bile acids and phenylbutyrate compounds) will vary, as will excretion levels, and there will be differences in the pharmacokinetics of the compound among the subjects being treated. Any of the factors described herein can affect a subject's drug exposure. For example, decreased compound clearance can increase drug exposure, while improved renal function can decrease actual drug exposure. The degree of drug exposure can correlate with a subject's response to an administered compound and the outcome of treatment.

[0019] The methods described herein can be used for preventative and prophylactic purposes.

[0020] II. Composition The present disclosure provides a method for treating at least one symptom of Wolfram syndrome in a subject, the method comprising administering to the subject a bile acid or a pharmaceutically acceptable salt thereof and a phenylbutyrate compound. In some embodiments, the method comprises administering to the subject a composition comprising TURSO and sodium phenylbutyrate.

[0021] bile acids As used herein, "bile acid" refers to a naturally occurring surfactant having a nucleus derived from cholanic acid, typically substituted with a 3α-hydroxy group at the C6, C7, or C12 position of the sterol nucleus, and optionally with other hydroxy groups. Bile acid derivatives (e.g., water-soluble bile acid derivatives) and amine-conjugated bile acids are also encompassed by the term "bile acid." Bile acid derivatives include, but are not limited to, derivatives in which other functional groups, such as, but not limited to, halogen groups or amino groups, have been introduced at the carbon atoms to which the hydroxy and carboxylic acid groups of the bile acid are attached. Soluble bile acids may include aqueous formulations of the free acid form of bile acids in combination with hydrochloric acid, phosphoric acid, citric acid, acetic acid, ammonia, or arginine. Suitable bile acids include, but are not limited to, taursodiol (TURSO), ursodeoxycholic acid (UDCA), chenodeoxycholic acid (also called "chenodiol" or "kenic acid"), cholic acid, hyodeoxycholic acid, deoxycholic acid, 7-oxolithocholic acid, lithocholic acid, iododeoxycholic acid, iocholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, glycolsodeoxycholic acid, taurocholic acid, glycocholic acid, or analogs, derivatives, or prodrugs thereof.

[0022] In some embodiments, the bile acid of the present disclosure is a hydrophilic bile acid. Hydrophilic bile acids include, but are not limited to, TURSO, UDCA, chenodeoxycholic acid, cholic acid, hyodeoxycholic acid, lithocholic acid, glycolsodeoxycholic acid, etc. Pharmaceutically acceptable salts or solvates of the bile acids disclosed herein are also contemplated. In some embodiments, bases commonly used to form pharmaceutically acceptable salts of bile acids of the present disclosure include hydroxides of alkali metals including sodium, potassium, and lithium, hydroxides of alkaline earth metals such as calcium and magnesium, hydroxides of other metals such as aluminum and zinc, ammonia, organic amines such as unsubstituted or hydroxyl-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine, tributylamine, pyridine, N-methyl, N-ethylamine, diethylamine, triethylamine, mono-, bis-, or tris-(2-OH-(C1-C6)-alkylamines) such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine, N-methyl-D-glucamine, morpholine, thiomorpholine, piperidine, pyrrolidine, and amino acids such as arginine, lysine, and the like.

[0023] The terms "tauroursodeoxycholic acid" (TUDCA) and "taursodiol" (TURSO) are used interchangeably herein.

[0024] Bile acids described herein can be TURSO, as shown in Formula I (carbons are labeled to aid in understanding the sites where substitutions can be made). In some embodiments, the TURSO is a hydrate, such as TURSO dihydrate. [ka]

[0025] The bile acids described herein are UDCAs as shown in Formula II (carbons are labeled to aid in understanding the sites where substitutions can be made): [ka] Or, it may be a pharmaceutically acceptable salt thereof.

[0026] The bile acid derivatives of the present disclosure can be physiologically relevant bile acid derivatives, for example, any combination of substitution of hydrogen at the 3- or 7-position in the formula of TURSO or UDCA, or shift in the stereochemistry of the 3- or 7-position hydroxyl group, is suitable for use in the present compositions.

[0027] "Bile acid" can also be a bile acid conjugated with an amino acid. The amino acid in the conjugate can be, but is not limited to, taurine, glycine, glutamine, asparagine, methionine, or carbocysteine. Other amino acids that can be conjugated with the bile acids of the present disclosure include arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, cysteine, proline, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, and tryptophan, as well as β-alanine and γ-aminobutyric acid. An example of such a bile acid is represented by Formula III: [ka] A compound of the formula During the ceremony, R is —H or C1-C4 alkyl; R1 is -CH2-SO3R3, CH2COOH, or CH2CH2COOH and R2 is -H, or R1 is -COOH and R2 is -CH2-CH2-CONH2, -CH2-CONH2, -CH2-CH2-SCH3, CH2CH2CH2NH(C=NH)NH2, CH2(imidazolyl), CH2CH2CH2CH2NH2, CH2COOH, CH2CH2COOH, CH2OH, CH(OH)CH3, CH2SH, pyrrolidin-2-yl, CH3, 2-propyl, 2-butyl, 2-methylbutyl, CH2(phenyl), CH2(4-OH-phenyl), or -CH2-S-CH2-COOH; R3 is -H or an amino acid residue, or a pharmaceutically acceptable analog, derivative, prodrug, or mixture thereof. An example of an amino acid is a basic amino acid. Other examples of amino acids include glycine, glutamine, asparagine, methionine, carbocysteine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, cysteine, proline, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, and tryptophan, as well as β-alanine and γ-aminobutyric acid.

[0028] Another example of a bile acid of the present disclosure has formula IV: [ka] A compound of the formula During the ceremony, R is —H or C1-C4 alkyl; R1 is -CH2-SO3R3 and R2 is -H, or R1 is -COOH and R2 is -CH2-CH2-CONH2, -CH2-CONH2, -CH2-CH2-SCH3, or -CH2-S-CH2-COOH; R3 is -H or a residue of a basic amino acid, or a pharmaceutically acceptable analog, derivative, prodrug, or mixture thereof. Examples of basic amino acids include lysine, histidine, and arginine.

[0029] In some embodiments, the bile acid is TURSO. TURSO is an amphipathic bile acid and a taurine conjugate of UDCA. TURSO is incorporated into the mitochondrial membrane and restores mitochondrial bioenergetic deficits by reducing Bax translocation to the mitochondrial membrane, decreasing mitochondrial permeability, and increasing the cell's apoptotic threshold (Rodrigues et al. Biochemistry 42, 10:3070-3080, 2003). It is used to treat cholesterol gallstones, and long-term treatment (e.g., 1-2 years) is generally required to achieve complete dissolution. It has been used to treat cholestatic liver diseases, such as primary cirrhosis, pediatric familial intrahepatic cholestasis, primary sclerosing cholangitis, and cystic fibrosis cholestasis. TURSO is contraindicated in patients with biliary tract infections, frequent biliary colic, or problems with bile acid absorption (e.g., ileal disease or resection). Drug interactions include interactions with substances that inhibit bile acid absorption, such as cholestyramine, and with drugs that increase cholesterol excretion in the bile (TURSO reduces biliary cholesterol content). Due to similar physicochemical properties, TURSO and UDCA share similarities in drug toxicity and interactions. The most common adverse reactions (greater than 1%) reported with TURSO use are abdominal discomfort, abdominal pain, diarrhea, nausea, pruritus, and rash. There have been some cases of pruritus, and a few cases of elevated liver enzymes.

[0030] In some embodiments, the bile acid is UDCA. UDCA (ursodiol) is used to treat gallstones and is endogenously produced and secreted by the liver as a taurine (TURSO) or glycine (GUDCA) conjugate. Taurine conjugation increases UDCA's hydrophilicity, thereby increasing its solubility. Because TURSO is actively transported to the distal ileum, it is likely to have a slightly longer intestinal residence time than UDCA, which is transported to the proximal ileum. Ursodiol therapy is not associated with liver damage. Liver enzyme abnormalities have not been associated with Actigall® (Ursodiol USP Capsules) therapy, and Actigall® has been shown to reduce liver enzyme levels in liver disease. However, subjects receiving Actigall® should have their SGOT (AST) and SGPT (ALT) measured at the initiation of treatment and thereafter, depending on the specific clinical situation. Previous studies have shown that bile acid sequestrants, such as cholestyramine and colestipol, may interfere with the effects of ursodiol by reducing its absorption. Aluminum-based antacids have been shown to adsorb bile acids in vitro and are expected to interfere with ursodiol in the same way as bile acid sequestrants. Estrogens, oral contraceptives, clofibrate (and possibly other lipid-lowering drugs) increase hepatic cholesterol secretion and promote cholesterol gallstone formation, potentially counteracting the effectiveness of ursodiol.

[0031] Phenylbutyrate Compounds As used herein, phenylbutyrate compounds are defined to include phenylbutyrate (small aromatic carboxylic acid) as the free acid (4-phenylbutyrate (4-PBA), 4-phenylbutyric acid, or phenylbutyric acid), as well as their pharmaceutically acceptable salts, cocrystals, polymorphs, hydrates, solvates, conjugates, derivatives, or prodrugs. The phenylbutyrate compounds described herein also include analogs of 4-PBA, as well as their pharmaceutically acceptable salts, including, but not limited to, glyceryl tri-(4-phenylbutyrate), phenylacetic acid (the active metabolite of PBA), 2-(4-methoxyphenoxy)acetic acid (2-POAA-OMe), 2-(4-nitrophenoxy)acetic acid (2-POAA-NO), and 2-(2-naphthyloxy)acetic acid (2-NOAA). Phenylbutyrate compounds also encompass physiologically relevant 4-PBA species, including, but not limited to, those in which deuterium is substituted for hydrogen in the 4-PBA structure. Other HDAC2 inhibitors are contemplated herein as substitutes for phenylbutyrate compounds.

[0032] Physiologically acceptable salts of phenylbutyric acid include, for example, sodium, potassium, magnesium, calcium salts, etc. Other examples of salts include ammonium, zinc, lithium salts, or salts of phenylbutyric acid with organic amines such as lysine and arginine.

[0033] In some embodiments of any of the methods described herein, the phenylbutyrate compound is sodium phenylbutyrate, which has the formula: [ka]

[0034] Phenylebutyrate is a pan-HDAC inhibitor that can ameliorate ER stress through upregulation of the master chaperone regulator DJ-1 and recruitment of other chaperone proteins (see, e.g., Zhou et al. J Biol Chem. 286:14941-14951, 2011, and Suaud et al. JBC. 286:21239-21253, 2011). Significant increases in chaperone production have been shown to reduce activation of the canonical ER stress pathway, refold misfolded proteins, and increase survival in in vivo models, including the G93A SOD1 mouse model of ALS (see, e.g., Ryu, H et al. 93:1087-1098, 2005).

[0035] formulation The bile acid and phenylbutyrate compounds described herein can be formulated for use as or in pharmaceutical compositions. For example, the methods described herein can include administering an effective amount of a composition comprising TURSO and sodium phenylbutyrate. The term "effective amount," as used herein, refers to an amount or concentration of one or more drugs administered over a period of time (including acute or chronic administration, regular or continuous administration) that is effective within the context of administration to produce the intended effect or physiological result. The composition can include about 5% to about 15% w / w TURSO (e.g., about 6% to about 14%, about 7% to about 13%, about 8% to about 12%, about 8% to about 11%, about 9% to about 10%, or about 9.7% w / w) and about 15% to about 45% w / w sodium phenylbutyrate (e.g., about 20% to about 40%, about 25% to about 35%, about 28% to about 32%, or about 29% to about 30%, e.g., about 29.2% w / w). In some embodiments, the composition includes about 9.7% w / w TURSO and 29.2% w / w sodium phenylbutyrate.

[0036] The sodium phenylbutyrate and TURSO may be present in the composition in a weight ratio of about 1:1 to about 4:1 (e.g., about 2:1 or about 3:1). In some embodiments, the ratio of sodium phenylbutyrate to TURSO is about 3:1.

[0037] The compositions described herein may contain any pharmaceutically acceptable carrier, adjuvant, and / or vehicle. The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that can be administered to a patient together with a compound disclosed herein, which does not destroy its pharmacological activity and is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the compound. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, and the like, which are compatible with pharmaceutical administration. Pharmaceutical compositions may contain any conventional non-toxic pharmaceutically acceptable carrier, adjuvant, or vehicle. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases, or buffers to enhance the stability of the formulated compound or its delivery form.

[0038] Compositions of the present disclosure may contain about 8% to about 24% w / w dextrates (e.g., about 9% to about 23%, about 10% to about 22%, about 10% to about 20%, about 11% to about 21%, about 12% to about 20%, about 13% to about 19%, about 14% to about 18%, about 14% to about 17%, about 15% to about 16%, or about 15.6% w / w dextrates). Both anhydrous and hydrated dextrates are contemplated herein. Dextrates of the present disclosure can include a mixture of sugars resulting from the controlled enzymatic hydrolysis of starch. Some embodiments of any of the compositions described herein include hydrated dextrates (e.g., NF grade available from JRS Pharma, Colonial Scientific, or Quadra).

[0039] The compositions of the present disclosure may contain about 1% to about 6% w / w sugar alcohol (e.g., about 2% to about 5%, about 3% to about 4%, or about 3.9% w / w sugar alcohol). Sugar alcohols are derived from sugars and contain one hydroxyl group (-OH) attached to each carbon atom. Both disaccharides and monosaccharides can form sugar alcohols. Sugar alcohols may be natural or may be produced by hydrogenation of sugars. Examples of sugar alcohols include, but are not limited to, sorbitol, xylitol, mannitol, and the like. In some embodiments, the compositions contain about 1% to about 6% w / w sorbitol (e.g., about 2% to about 5%, about 3% to about 4%, or about 3.9% w / w).

[0040] The compositions of the present disclosure may contain about 22% to about 35% w / w maltodextrin (e.g., about 22% to about 33%, about 24% to about 31%, about 25% to about 32%, about 26% to about 30%, or about 28% to about 29% w / w, e.g., about 28.3% w / w maltodextrin). Maltodextrin can form a flexible spiral, which, when solubilized in solution, allows for encapsulation of active ingredients (e.g., any of the phenylbutyrate compounds and bile acids described herein), thereby masking the taste of the active ingredients. Maltodextrin produced from any suitable source is contemplated herein, including, but not limited to, pea, rice, tapioca, corn, and potato. In some embodiments, the maltodextrin is pea maltodextrin. In some embodiments, the composition comprises about 28.3% w / w pea maltodextrin. For example, pea maltodextrin (KLEPTOSE® LINECAPS) manufactured by Roquette can be used.

[0041] The compositions described herein may further comprise a sugar substitute (e.g., sucralose). For example, the compositions may comprise about 0.5% to about 5% w / w sucralose (e.g., about 1% to about 4%, about 1% to about 3%, or about 1% to about 2%, e.g., about 1.9% w / w sucralose). Other sugar substitutes contemplated herein include, but are not limited to, aspartame, neotame, acesulfame potassium, saccharin, and advantame.

[0042] In some embodiments, the composition includes one or more flavoring agents. The composition can include about 2% to about 15% w / w (e.g., about 3% to about 13%, about 3% to about 12%, about 4% to about 9%, about 5% to about 10%, or about 5% to about 8%, e.g., about 7.3% w / w) of flavoring agent. Flavoring agents include substances that impart flavor to another substance or that alter the properties of a composition by affecting taste. Flavoring agents can be used to mask unpleasant flavors without affecting physical or chemical stability and can be selected based on the taste of the formulation. Suitable flavoring agents include, but are not limited to, natural flavoring agents, artificial flavoring agents, and imitation flavors. Blends of flavoring agents can also be used. For example, the compositions described herein can include two or more flavoring agents (e.g., two, three, four, five, or more). The flavoring agents can be soluble and stable in water. Selection of a suitable flavoring agent can be based on taste testing. For example, different flavors can be added separately to the composition and taste tested. Examples of flavors include any fruit flavor powder (e.g., peach, strawberry, mango, orange, apple, grape, raspberry, cherry, or mixed berry flavor powder). The compositions described herein can contain about 0.5% to about 1.5% w / w (e.g., about 1% w / w) mixed berry flavor powder and / or about 5% to about 7% w / w (e.g., about 6.3% w / w) masking flavor. Suitable masking flavors are available, for example, from Firmenich.

[0043] The compositions described herein may further comprise silicon dioxide (or silica). Adding silica to a composition can prevent or reduce aggregation of the composition's components. Silica functions as an anti-caking agent, adsorbent, disintegrant, or glidant. In some embodiments, the compositions described herein comprise about 0.1% to about 2% w / w of porous silica (e.g., about 0.3% to about 1.5%, about 0.5% to about 1.2%, or about 0.8% to about 1%, e.g., 0.9% w / w). The porous silica may have a higher HO absorption capacity and / or higher porosity than fumed silica at a relative humidity of about 20% or greater (e.g., about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or greater). The porous silica can have an HO absorption capacity of about 5% to about 40% by weight (e.g., about 20% to about 40% by weight, or about 30% to about 40% by weight) at about 50% relative humidity. The porous silica can have a higher porosity at about 20% or greater relative humidity (e.g., about 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) compared to fumed silica. In some embodiments, the porous silica has an average particle size of about 2 μm to about 10 μm (e.g., about 3 μm to about 9 μm, about 4 μm to about 8 μm, about 5 μm to about 8 μm, or about 7.5 μm). In some embodiments, the porous silica has an average pore volume of about 0.1 cc / gm to about 2.0 cc / gm (e.g., about 0.1 cc / gm to about 1.5 cc / gm, about 0.1 cc / gm to about 1 cc / gm, about 0.2 cc / gm to about 0.8 cc / gm, about 0.3 cc / gm to about 0.6 cc / gm, or about 0.4 cc / gm). In some embodiments, the porous silica has a bulk density of about 50 g / L to about 700 g / L (e.g., about 100 g / L to about 600 g / L, about 200 g / L to about 600 g / L, about 400 g / L to about 600 g / L, about 500 g / L to about 600 g / L, about 540 g / L to about 580 g / L, or about 560 g / L).In some embodiments, the compositions described herein comprise about 0.05% to about 2% w / w (e.g., any subrange of this range described herein) of Syloid® 63FP (WR Grace).

[0044] The compositions described herein can further comprise one or more buffering agents. For example, the compositions can comprise about 0.5% to about 5% w / w of buffering agent (e.g., about 1% to about 4% w / w, about 1.5% to about 3.5% w / w, or about 2% to about 3% w / w, e.g., about 2.7% w / w). The buffering agent can include a weak acid or base that maintains the acidity or pH of the composition at or near a selected value after the addition of another acid or base. Suitable buffering agents are known in the art. In some embodiments, the buffering agent in the compositions provided herein is a phosphate, such as sodium phosphate (e.g., dibasic sodium phosphate anhydrous). For example, the composition can comprise about 2.7% w / w of dibasic sodium phosphate.

[0045] The composition can also include one or more lubricants. For example, the composition can include about 0.05% to about 1% w / w of a lubricant (e.g., about 0.1% to about 0.9%, about 0.2% to about 0.8%, about 0.3% to about 0.7%, or about 0.4% to about 0.6%, e.g., about 0.5% w / w of a lubricant). Examples of lubricants include, but are not limited to, sodium stearyl fumarate, magnesium stearate, stearic acid, metal stearates, talc, high-melting point waxes and glycerides, colloidal silica, polyethylene glycol, alkyl sulfates, glyceryl behenate, hydrogenated oils, and the like. Additional lubricants are known in the art. In some embodiments, the composition includes about 0.05% to about 1% w / w of sodium stearyl fumarate (e.g., any subrange of this range described herein). For example, the composition can include about 0.5% w / w of sodium stearyl fumarate.

[0046] In some embodiments, the composition comprises about 29.2% w / w sodium phenylbutyrate, about 9.7% w / w TURSO, about 15.6% w / w dextrates, about 3.9% w / w sorbitol, about 1.9% w / w sucralose, about 28.3% w / w maltodextrin, about 7.3% w / w flavoring, about 0.9% w / w silicon dioxide, about 2.7% w / w sodium phosphate (e.g., dibasic sodium phosphate), and about 0.5% w / w sodium stearyl fumarate.

[0047] The composition can include about 3000 mg sodium phenylbutyrate, about 1000 mg TURSO, about 1600 mg dextrates, about 400 mg sorbitol, about 200 mg sucralose, about 97.2 mg silicon dioxide, about 2916 mg maltodextrin, about 746 mg flavoring (e.g., about 102 mg mixed berry flavor and about 644 mg masking flavor), about 280 mg sodium phosphate (e.g., dibasic sodium phosphate), and about 48.6 mg sodium stearyl fumarate.

[0048] Suitable sweeteners or taste-masking agents can also be included in the composition, such as, but not limited to, xylose, ribose, glucose, mannose, galactose, fructose, dextrose, sucrose, maltose, steviol glycosides, partially hydrolyzed starch, corn syrup solids, etc. Water-soluble artificial sweeteners are contemplated herein, such as soluble saccharin salts (e.g., saccharin sodium salt or saccharin calcium salt), cyclamate salts, acesulfame potassium (acesulfame K), and the free acid form of saccharin, and aspartame-based sweeteners such as L-aspartyl-phenylalanine methyl ester, alitame (Alitame®), or neotame (Neotame®). The amount of sweetener or taste-masking agent can vary depending on the desired amount of sweetener or taste-masking agent selected for a particular final composition.

[0049] In addition to the above, pharmaceutically acceptable binders are also contemplated. Examples include cellulose derivatives such as microcrystalline cellulose, low-substituted hydroxypropyl cellulose (e.g., LH22, LH21, LH20, LH32, LH31, LH30), starches such as potato starch, croscarmellose sodium (i.e., cross-linked carboxymethylcellulose sodium salt, such as Ac-Di-Sol®), alginic acid or alginates, insoluble polyvinylpyrrolidone (e.g., Polyvidon® CL, Polyvidon® CL-M, Kollidon® CL, Polyplasdone® XL, Polyplasdone® XL-10), and sodium carboxymethyl starch (e.g., Primogel® and Explotab®).

[0050] Additional fillers, diluents, or binders can be incorporated, examples of which include polyols, sucrose, sorbitol, mannitol, Erythritol®, Tagatose®, lactose (e.g., spray-dried lactose, α-lactose, β-lactose, Tabletose®, Pharmatose®, Microtose, various grades of Fast-Floc®), microcrystalline cellulose (e.g., various grades of Avicel®, such as Avicel® PH101, Avicel® PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Tai®, Solka-Floc®), hydroxypropyl cellulose, L-hydroxypropyl cellulose (low substituted), (e.g., L-HPC-CH31, L-HPC-LH11, LH 22, LH 21, LH 20, LH 32, LH 31, LH30), dextrin, maltodextrin (e.g., Lodex® 5 and Lodex® 10), starch or modified starch (including potato starch, corn starch, rice starch), sodium chloride, sodium phosphate, calcium sulfate, and calcium carbonate.

[0051] The compositions described herein can be formulated or adapted for administration to a subject via any route (e.g., any route approved by the Food and Drug Administration (FDA)). Exemplary methods are described in the FDA's CDER Data Standards Manual, Version 004 (available at fda.give / cder / dsm / DRG / drg00301.html).

[0052] Pharmaceutical compositions are typically formulated to be compatible with their intended route of administration, which include parenteral (subcutaneous, intradermal, intravenous, intradermal, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques), oral (e.g., via inhalation or feeding tube), transdermal (topical application), transmucosal, and rectal administration.

[0053] The pharmaceutical composition may be in the form of a solution or powder for inhalation and / or nasal administration. In some embodiments, the pharmaceutical composition is formulated as a powder-filled sachet. Suitable powders may include those that are substantially soluble in water. The pharmaceutical composition may be formulated using suitable dispersing or wetting agents (e.g., Tween 80, etc.) and suspending agents according to techniques known in the art. 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 used are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile, fixed oils are conventionally used as solvents or suspending media. For this purpose, any smooth, fixed oil may be used, 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 their polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, or carboxymethylcellulose, or similar dispersing agents commonly used in the preparation of pharmaceutically acceptable dosage forms such as emulsions and / or suspensions. Other commonly used surfactants, such as Tween or Span, and / or other similar emulsifiers or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms, may also be used for formulation purposes.

[0054] The composition may be orally administered in any orally acceptable dosage form, including, but not limited to, powders, capsules, tablets, emulsions, and aqueous suspensions, dispersions, and solutions. For oral powders, the powder can be substantially dissolved in water before administration. For oral tablets, commonly used carriers include lactose and cornstarch. Lubricants such as magnesium stearate may also be added. For oral administration in capsule form, useful diluents include lactose and dried cornstarch. For aqueous suspensions and / or emulsions, the active ingredient may be suspended or dissolved in an oily phase combined with an emulsifying and / or suspending agent. If desired, certain sweeteners and / or flavorings and / or coloring agents may be added.

[0055] Alternatively, or in addition, the compositions can 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 using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art.

[0056] In some embodiments, the therapeutic compositions disclosed herein can be formulated for sale in the United States, imported into the United States, and / or exported from the United States. The pharmaceutical compositions can be included in a container, pack, or dispenser along with instructions for administration. In some embodiments, the present invention provides kits containing bile acids and phenylbutyrate compounds. The kits can also include instructions for physicians and / or patients, syringes, needles, boxes, bottles, vials, etc.

[0057] III. Treatment Methods The present disclosure provides a method for treating one or more symptoms of Wolfram syndrome in a subject, comprising administering to the subject a pharmaceutically effective amount of a combination of a bile acid compound or a pharmaceutically acceptable salt thereof (e.g., TURSO) and a phenylbutyrate compound (e.g., sodium phenylbutyrate). The bile acid or a pharmaceutically acceptable salt thereof and the phenylbutyrate compound can be administered separately or simultaneously, such as as part of a treatment regimen. The compounds can be administered daily (e.g., once daily, twice daily, or three or more times daily), weekly, monthly, or quarterly. The compounds can be administered over a period of weeks, months, or years. For example, the compounds can be administered for a period of at least about 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, or at least about 5 years or longer. The compounds can be administered once or twice daily for 60 days or less (e.g., 55, 50, 45, 40, 35, 30 days or less). Alternatively, the bile acid and phenylbutyrate compounds can be administered once or twice daily for more than 60 days (e.g., 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 180, 200, 250, 300, 400, 500, 600 days or more).

[0058] TURSO is administered in an amount of about 0.5 to about 5 grams per day (e.g., about 0.5 to about 4.5, about 0.5 to about 4, about 0.5 to about 3.5, about 0.5 to about 3, about 0.5 to about 2.5, about 0.5 to about 2, about 0.5 to about 1.5, about 0.5 to about 1, about 1 to about 5, about 1 to about 4.5, about 1 to about 4, about 1 to about 3.5, about 1 to about 3, about 1 to about 2.5, about 1 to about 2, about 1 to about 1.5, about 1.5 to about 5, about 1.5 about 2.5 to about 3.5, about 1.5 to about 3, about 1.5 to about 2.5, about 1.5 to about 2, about 2 to about 5, about 2 to about 4.5, about 2 to about 4, about 2 to about 3.5, about 2 to about 3, about 2 to about 2.5, about 2.5 to about 5, about 2.5 to about 4.5, about 2.5 to about 4, about 2.5 to about 3.5, about 2.5 to about 3, about 3 to about 5, about 3 to about 4.5, about 3 to about 4, about 3 to about 3.5, about 3.5 to about 5, about 3.5 to about 4.5, about 3.5 to about 4, about 4 to about 5, about 4 to about 4.5, or about 4.5 to about 5 grams). In some embodiments, TURSO is administered in an amount of about 1 to about 2 grams per day (e.g., about 1 to about 1.8 grams, about 1 to about 1.6 grams, about 1 to about 1.4 grams, about 1 to about 1.2 grams, about 1.2 to about 2.0 grams, about 1.2 to about 1.8 grams, about 1.2 to about 1.6 grams, about 1.2 to about 1.4 grams, about 1.4 to about 2.0 grams, about 1.4 to about 1.8 grams, about 1.4 to about 1.6 grams, about 1.6 to about 2.0 grams, about 1.6 to about 1.8 grams, or about 1.8 to about 2.0 grams). In some embodiments, TURSO is administered in an amount of about 1 gram per day. For example, TURSO can be administered in an amount of about 1 gram once per day. In some embodiments, TURSO is administered in an amount of about 2 grams per day. For example, TURSO can be administered at about 1 gram twice per day.

[0059] Sodium phenylbutyrate is administered in an amount of about 0.5 to about 10 g per day (e.g., about 1 to about 10, about 1 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 1 to about 2, about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, about 2.5 to about 9.5, about 2.5 to about 8.5, about 2.5 to about 7.5, about 2.5 to about 6 per day). about 5, about 2.5 to about 5.5, about 2.5 to about 4.5, about 3 to about 10, about 3 to about 9, about 3 to about 8, about 3 to about 7, about 3 to about 6.5, about 3 to about 6, about 3 to about 5, about 4 to about 10, about 4 to about 9, about 4 to about 8, about 4 to about 7, about 4 to about 6, about 5 to about 10, about 5 to about 9, about 5 to about 8, about 5 to about 7, about 6 to about 10, about 6 to about 9, about 6 to about 8, about 7 to about 10, about 7 to about 9, or about 8 to about 10 grams). In some embodiments, sodium phenylbutyrate is administered in an amount of about 3 to about 6 grams per day (e.g., about 3 to about 5.5 grams, about 3 to about 5.0 grams, about 3 to about 4.5 grams, about 3 to about 4.0 grams, about 3 to about 3.5 grams, about 3.5 to about 6 grams, about 3.5 to about 5.5 grams, about 3.5 to about 5.0 grams, about 3.5 to about 4.5 grams, about 3.5 to about 4.0 grams, about 4.0 to about 6 grams, about 4.0 to about 5.5 grams, about 4.0 to about 5.0 grams, about 4.0 to about 4.5 grams, about 4.5 to about 6 grams, about 4.5 to about 5.5 grams, about 4.5 to about 5.0 grams, about 5.0 to about 6 grams, about 5.0 to about 5.5 grams, or about 5.5 to about 6.0 grams). In some embodiments, sodium phenylbutyrate is administered in an amount of about 3 grams per day. For example, sodium phenylbutyrate can be administered in an amount of about 3 grams once per day. In some embodiments, sodium phenylbutyrate is administered in an amount of about 6 grams per day. For example, sodium phenylbutyrate can be administered in an amount of about 3 grams twice per day. In some embodiments, the bile acid and the phenylbutyrate compound are administered in a weight ratio of about 2.5:1 to about 3.5:1 (e.g., about 3:1).

[0060] The methods described herein can include administering about 1 g of TURSO once daily and about 3 g of sodium phenylbutyrate once daily, or about 1 g of TURSO twice daily and about 3 g of sodium phenylbutyrate twice daily. The method can include administering about 1 gram of TURSO once daily and about 3 grams of sodium phenylbutyrate once daily for at least about 14 days (e.g., at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 27, 30, 35, or 40 days), followed by administering about 1 gram of TURSO twice daily and about 3 grams of sodium phenylbutyrate twice daily for at least one day (e.g., at least 30, 40, 50, 60, 80, 100, 120, 150, 180, 250, 300, or 400 days). For example, the method can include administering about 1 gram of TURSO once daily and about 3 grams of sodium phenylbutyrate once daily for about 14 to 21 days, followed by administering about 1 gram of TURSO twice daily and about 3 grams of sodium phenylbutyrate twice daily.

[0061] In some embodiments, the methods described herein involve administering TURSO to a subject in a range of about 5 mg to about 100 mg per kg of body weight (e.g., about 10 to about 50 mg / kg, about 5 to about 10 mg / kg, about 10 to about 15 mg / kg, about 15 to about 20 mg / kg, about 20 to about 25 mg / kg, about 25 to about 30 mg / kg, about 30 to about 35 mg / kg, about 35 to about 40 mg / kg, about 40 to about 45 mg / kg, about 45 to about 50 mg / kg, about 50 to about 55 mg / kg, about 55 to about 60 mg / kg, about 60 to about 65 mg / kg, about 65 to about 70 mg / kg, about 70 to about 75 mg / kg, about 75 to about 80 mg / kg, about 80 to about 85 mg / kg, about 85 to about 90 mg / kg, about 90 to about 95 mg / kg, or about 95 to about 100 mg / kg, etc.).

[0062] In some embodiments, the methods described herein involve administering sodium phenylbutyrate to a subject in the range of about 10 mg to about 400 mg per kg of body weight (e.g., about 10 to about 15 mg / kg, about 15 to about 20 mg / kg, about 20 to about 25 mg / kg, about 25 to about 30 mg / kg, about 30 to about 35 mg / kg, about 35 to about 40 mg / kg, about 40 to about 45 mg / kg, about 45 to about 50 mg / kg, about 50 to about 55 mg / kg, about 55 to about 60 mg / kg, about 60 to about 65 mg / kg, about 65 to about 70 mg / kg, about 70 to about 100 mg / kg, about 100 to about 150 mg / kg, about 150 to about 200 mg / kg, about 200 to about 300 mg / kg, about 300 to about 400 mg / kg, etc.).

[0063] In some embodiments, TURSO is administered in an amount of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg / kg of body weight. In some embodiments, sodium phenylbutyrate is administered in an amount of about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, or 150 mg / kg of body weight.

[0064] The methods described herein can be used to treat or ameliorate at least one symptom of Wolfram syndrome. The methods can also be used to prophylactically treat subjects at risk of developing Wolfram syndrome. The methods can be used to treat subjects exhibiting one or more symptoms, such as diabetes insipidus, diabetes mellitus (e.g., early-onset diabetes), optic atrophy, progressive neurodegeneration, hearing loss, endocrine deficiency and neuropsychiatric disorders, cerebellar ataxia, autonomic dysfunction, dementia or intellectual disability, psychiatric disorders, seizures, neurogenic bladder or bladder dysfunction, bowel dysfunction, delayed / absent puberty, male hypogonadism, non-autoimmune hypothyroidism, growth retardation, cardiomyopathy, or structural congenital heart disease. In some embodiments, the subject has or is at risk of developing diabetes, e.g., insulin-dependent diabetes mellitus or early-onset diabetes. In some embodiments, the subject has or is at risk of developing optic atrophy or hearing impairment.

[0065] In some embodiments, the subject has one or more mutations in the WFS1 gene. For example, the subject may have a c.1672C>T, p.R558C mutation in the WFS1 gene or a c.2654C>T, p.P885L mutation in the WFS1 gene. In some embodiments, the subject has one or more mutations in the CDGSH iron-sulfur domain protein 2 (CISD2) gene.

[0066] In some embodiments, administration of a combination of a bile acid compound (e.g., TURSO) and a phenylbutyrate compound (e.g., sodium phenylbutyrate) improves treatment of one or more symptoms of Wolfram syndrome compared to each compound alone. For example, treatment with a combination of TURSO and sodium phenylbutyrate may alleviate the symptoms of a subject described herein to a greater extent or at a faster rate than administration of each compound alone.

[0067] The methods described herein may include treatment of one or more symptoms of Wolfram syndrome, as well as treatment of Wolfram syndrome itself. "Treating" Wolfram syndrome does not require 100% elimination of the disease or symptoms of the disease in a subject. Alleviating or reducing the severity of symptoms or characteristics of the disease is contemplated. "Treating" Wolfram syndrome can also refer to delaying the onset of symptoms (e.g., prophylactic treatment) or slowing the progression of symptoms or loss of function associated with the disease. "Treating" Wolfram syndrome can also refer to eliminating or reducing one or more side effects of treatment (e.g., side effects caused by any of the therapeutic agents for treating Wolfram syndrome disclosed herein or known in the art). "Treating" Wolfram syndrome can also refer to eliminating or reducing one or more direct or indirect effects of the disease progression of Wolfram syndrome. A subject may not exhibit symptoms of Wolfram syndrome, but may be at risk for Wolfram syndrome. For example, a subject may have a mutation in a gene associated with Wolfram syndrome or may have a family history of Wolfram syndrome. The subject may exhibit early signs of disease or may exhibit symptoms of established or progressive disease. The present disclosure contemplates any degree of delay in the onset of symptoms, alleviation of one or more symptoms of disease, or delay in the progression of one or more disease symptoms.

[0068] The treatments provided herein can be initiated at any stage during the progression of the disease. For example, treatment can be initiated pre-symptomatically (e.g., in subjects at risk of developing Wolfram syndrome), at symptom onset, or shortly after detection of Wolfram syndrome symptoms, upon the observation of one or more symptoms that would lead a skilled physician to suspect that the subject may be developing Wolfram syndrome. Treatment can also be initiated at a later stage. For example, treatment can be initiated during the progression of the disease.

[0069] Treatment methods include single doses, multiple doses, and repeated doses as needed to prevent or treat Wolfram syndrome or at least one symptom of Wolfram syndrome. The duration of preventative treatment can be a single dose, or treatment can continue indefinitely (e.g., multiple doses), for example, several years or even the lifespan of the subject. For example, a subject at risk for Wolfram syndrome may be treated with the methods provided herein for days, weeks, months, or even years to prevent the onset or progression of the disease. In some embodiments, treatment methods can include assessing the level of disease in the subject before, during, and / or after treatment. The treatments provided herein can be administered one or more times daily, or can be administered weekly or monthly. In some embodiments, treatment can be continued until a reduction in the level of disease in the subject is detected.

[0070] As used herein, the terms "administer," "administering," or "administration" refer to administering a drug described herein, regardless of form, to a subject using any art-known method, such as, for example, ingestion, injection, implantation, absorption, or inhalation. In some embodiments, one or more compounds disclosed herein can be administered to a subject by oral ingestion and / or topical administration (e.g., nasal administration). For example, the methods herein include administering an effective amount of a compound or compound composition to achieve a desired or described effect. The specific dosage and treatment regimen for any particular subject will depend on a variety of factors, including the activity of the particular compound used, age, weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition, or symptom, the subject's disposition to the disease, condition, or symptom, and the judgment of the treating physician.

[0071] After administration of the bile acid or a pharmaceutically acceptable salt thereof and the phenylbutyrate compound, the subject can be evaluated and the level of Wolfram syndrome disease can be detected, assessed, or determined. In some embodiments, treatment can be continued until a change (e.g., a decrease) in the subject's disease level is detected.

[0072] Upon improvement of the patient's condition (e.g., a change (e.g., a decrease) in the level of disease in the subject), a maintenance dose of a compound, composition, or combination of the present disclosure may be administered, if necessary. Thereafter, the dosage or frequency of administration, or both, can be reduced as a function of symptoms, to a level at which the improved condition is maintained. However, the patient may require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.

[0073] The methods described herein may further include administering one or more additional therapeutic agents to the subject, for example, in an amount effective to achieve treatment or regulation of at least one symptom of Wolfram syndrome. Any agent known in the art for treating Wolfram syndrome can be used as the additional therapeutic agent. Examples of therapeutic agents include valproic acid, glucagon-like peptide (GLP)-1 receptor agonists, dantrolene sodium, ER Ca2+ stabilizers, etc.

[0074] The bile acid or its pharmaceutically acceptable salt and phenylbutyrate compound can be administered immediately after a meal (e.g., within 2 hours after a meal) or under fasting conditions. The subject may have eaten food (e.g., solid or liquid food) less than 2 hours before administering the bile acid or its pharmaceutically acceptable salt and / or phenylbutyrate compound, or will eat food less than 2 hours after administering one or both of the compounds. Food can affect the rate and extent of absorption of the bile acid or its pharmaceutically acceptable salt and / or phenylbutyrate compound. For example, food can alter the bioavailability of a compound by delaying gastric emptying, stimulating bile flow, changing gastrointestinal pH, increasing splanchnic blood flow, altering the intraluminal metabolism of a substance, or physically or chemically interacting with a dosage form or substance. The nutritional composition, calories, amount, and temperature of a meal can induce physiological changes in the gastrointestinal tract that affect drug transit time, luminal dissolution, drug permeability, and systemic availability. Generally, meals high in total calories and fat content are more likely to affect gastrointestinal physiology, thereby significantly impacting drug bioavailability. The methods provided herein may further include administering multiple foods to the subject, for example, less than 2 hours (e.g., less than 1.5 hours, less than 1 hour, or less than 0.5 hours) before or after administering the bile acid or a pharmaceutically acceptable salt thereof and / or a phenylbutyrate compound. [Example]

[0075] Additional embodiments are disclosed in more detail in the following examples, which are provided by way of illustration and are not intended to limit the scope of the disclosure or the claims in any way.

[0076] Example 1. Multidimensional analysis and treatment development using a disease model derived from iPS cells from Wolfram syndrome patients To generate induced pluripotent stem cell (iPSC) lines, peripheral blood mononuclear cells (PBMCs) were obtained from Wolfram syndrome patients. For in vivo studies, 129S6 whole-body Wfs1 knockout mice, aged 5–6 weeks, were treated with either control food or food containing AMX0035 (PB: 0.338%, TURSO: 0.225%; see AMX0035 diet) for 1 month.

[0077] overview Cellular functions of neural progenitor cells Using a HiBiT-tagged reporter, we performed an in vivo assay to assess whether AMX0035 stabilized WFS1 protein and restored organelle function in NPCs derived from three patient-derived iPS cell lines. Incubation with AMX0035 significantly increased steady-state levels of WFS1 p.R558C protein. AMX0035 treatment significantly increased WFS1 protein levels in iPS cells derived from all three patient-derived cell lines evaluated. WFS1 mRNA levels were also increased by AMX0035 treatment. Expression of the ER stress marker genes BiP and spliced ​​XBP1 (sXBP1) was unaffected by AMX0035, whereas expression of the ER stress-induced apoptotic genes CHOP and TXNIP was significantly reduced in each of the three patient-derived cell lines. Increased oxygen consumption rates (OCRs) were observed throughout the assay in each of the three patient-derived cell lines treated with AMX0035. Furthermore, AMX0035 inhibited apoptosis, as indicated by caspase 3 / 7 activity, in each of the three patient-derived cell lines.

[0078] Insulin secretion and viability in SC-β cells harboring the WFS1 c.1672C>T, p.R558C variant In vivo evaluation was performed to determine whether AMX0035 was effective in ameliorating the dysfunction of W024 and W121 SC islets. WFS1 protein expression was restored in treated SC islets, as observed in both W024 and W121 iPS cells. AMX0035 significantly suppressed cell death in W024 and W121 SC islets.

[0079] Attenuation of cellular stress in SC islets harboring the WFS1 c.1672C>T, p.R558C variant In vivo evaluations were performed to assess whether AMX0035 quells cellular stress. Untreated SC-β cells were enriched for gene sets related to apoptosis and ER stress. AMX0035-treated SC-β cell populations were enriched for gene sets related to insulin secretion and β-cell development. Furthermore, cytosolic K, which plays an important role in β-cell differentiation and function, was upregulated. + Levels and Ca 2+ The gene set associated with regulating levels was increased.

[0080] Progression of diabetic phenotype in Wfs1-deficient mice We used a mouse model of 129S6 whole-body Wfs1-KO mice, which develop progressive glucose intolerance during adolescence, to determine the effects of AMX0035 on the Wolfram diabetic phenotype in vivo. After one month of feeding, Wfs1KO mice developed more severe glucose intolerance than mice fed a control diet. In contrast, blood glucose curves from intraperitoneal glucose tolerance tests (IP-GTTs) were similar to baseline results in Wfs1KO mice fed the AMX0035 diet, indicating that the AMX0035 diet slowed the progression of the diabetic phenotype.

[0081] This study utilized multiple iPS cell-derived in vitro disease models to demonstrate the efficacy of AMX0035 in vivo. AMX0035 increased WFS1 expression and suppressed apoptosis by mitigating ER stress and mitochondrial dysfunction. AMX0035 restored WFS1 expression and increased insulin secretion capacity in W024 and W121 SC islets. AMX0035 treatment mitigated the increased cellular stress caused by pathogenic WFS1 variants without altering β-cell identity, resulting in increased β-cell function and insulin secretion in W024 and W121 SC β-cells. In vivo, a delay in Wolfram diabetes phenotype was observed in Wfs1KO mice fed an AMX0035 diet.

[0082] result Recent genetic and clinical findings have revealed that Wolfram syndrome is a spectrum disorder. Therefore, genotype-phenotype correlation analysis is necessary for diagnosis and treatment development. Here, we focus on the WFS1 c.1672C>T, p.R558C variant, which is highly prevalent in the Ashkenazi Jewish population. Clinical investigations have shown that subjects with the homozygous WFS1 c.1672C>T, p.R558C variant exhibit a mild Wolfram syndrome phenotype. Expression of WFS1 p.R558C is stable compared with other known recessive pathogenic variants associated with Wolfram syndrome. Human induced pluripotent stem cell (iPSC)-derived pancreatic islets (SC islets) homozygous for the WFS1 c.1672C>T variant recapitulate the genotype-associated Wolfram syndrome phenotype. Enhancement of the remaining WFS1 function by combined treatment with chemical chaperones alleviates the deleterious effects of the WFS1 c.1672C>T,p.R558C variant and increases insulin secretion in SC islets. Thus, the WFS1 c.1672C>T,p.R558C variant causes a mild Wolfram syndrome phenotype that can be ameliorated by combined treatment with chemical chaperones.

[0083] WFS1 c.1672C>T, p.R558C is common in the Ashkenazi Jewish population and causes a mild Wolfram phenotype. To determine the carrier frequency of the WFS1 c.1672C>T, p.R558C variant in Jewish populations, we genotyped 87,093 subjects from several Jewish populations. In the original dataset, subjects were classified by self-identification as Ashkenazi, Sephardi, Ashkenazi / Sephardi, convert, or unknown. Samples from converts and individuals of unknown origin totaled 773 and were excluded from the analysis. The observed carrier frequencies of WFS1 c.1672C>T, p.R558C reached 2.32% (1:43) in Ashkenazi Jewish subjects, 1.32% (1:76) in Ashkenazi / Sephardi Jewish subjects, and 0.04% (1:2,268) in Sephardi Jewish subjects (Figure 1A). To determine whether the WFS1 c.1672C>T, p.R558C mutation is prevalent among Jewish populations in various countries, the data were categorized based on the self-reported ancestry of four grandparents. Subjects reporting mixed origins from two or more countries were excluded from the analysis. Because South African Jews are primarily of Lithuanian descent, if a subject listed South Africa as their country of origin, the sample was redefined as Lithuanian. Subjects whose ancestry included Israel or the United States were also excluded, as Jews in these countries are often mixed with Ashkenazi ancestry. Subjects who did not provide information about their grandparental origins or who stated it was unknown were excluded. Subjects of Ukrainian origin were combined with the Russian group. Subjects of Belarusian and Czech origins were excluded from the analysis because their total number of individuals was less than 100, potentially generating spurious signals from small sample sizes. The frequencies of the data categorized by country of origin are as follows: Romania 3.50% (1:29), Poland 2.57% (1:39), Russia 2.07% (1:48), Hungary 1.63% (1:61), Germany 1.60% (1:63), Lithuania 0.87% (1:116) (Figure 1B).Clinical investigation revealed that most subjects with the homozygous WFS1 c.1672C>T, p.R558C variant developed diabetes mellitus, but the age of diagnosis was older than that of typical Wolfram syndrome (approximately 6 years) (Barrett TG, Bundey SE. Wolfram (DIDMOAD) syndrome. J Med Genet. 1997;34(10):838-41.) (Table 1). Only four subjects were clinically diagnosed with optic atrophy. The optic atrophy was mild, and no cases were diagnosed with legal blindness (Table 1). Furthermore, no subjects developed hearing loss or diabetes insipidus (Table 1). Taken together, the WFS1 c.1672C>T, p.R558C variant is common in Ashkenazi Jewish populations, particularly those of Romanian origin, and this variant causes a milder or less severe phenotype of Wolfram syndrome.

number

[0084] The p.R558C variant of WFS1 is degraded more than the wild type but less than the p.P885L variant of WFS1 Pathogenic WFS1 variants are classified based on their effect on WFS1 expression: Class A is a defective protein that results in depletion or reduced WFS1 protein, leading to loss or incomplete function, and Class B is a defective WFS1 protein that results in gain of function. Class A is further divided into three subclasses: Class A1, WFS1 depletion due to WFS1 mRNA degradation (nonsense-mediated decay, NMD); Class A2, WFS1 depletion due to WFS1 protein degradation; and Class A3, WFS1 depletion due to both mRNA and protein degradation (de Heredia ML, et al. Genotypic classification of patients with Wolfram syndrome: insights into the natural history of the disease and correlation with phenotype. Genet Med. 2013;15(7):497-506; Rigoli L, et al. Genetic and clinical aspects of Wolfram syndrome 1, a severe neurodegenerative disease. Pediatr Res. 2018;83(5):921-9.) (Figure 2). To determine the class specification of the WFS1 c.1672C>T, p.R558C variant, we investigated the thermal stability of WFS1 p.R558C and p.P885L by adding a HiBiT-based tag to detect the variant in cells.The p.R558C variant is less thermostable than wild-type WFS1, suggesting an altered folding state, but is more stable than the known autosomal recessive variant p.P885L, which is pathogenic and associated with Wolfram syndrome phenotypes (Hardy C, et al. Clinical and molecular genetic analysis of 19 Wolfram syndrome kindreds demonstrating a wide spectrum of mutations in WFS1. American Journal of Human Genetics. 1999;65(5):1279-90.; Qian X, et al. Phenotype Prediction of Pathogenic Nonsynonymous Single Nucleotide Polymorphisms in WFS1. Scientific Reports. 2015;5:14731.) (Figures 3A and 3B). Expression of both p.R558C and p.P885L can be restored by incubating cells at low temperatures, confirming the folding defect conferred by this variant (Figure 3C). Treatment with the proteasome inhibitor bortezomib increased WFS1 protein levels from both variants, with the fold change of p.P885L being greater than that of p.R558C (Figure 3D). This indicates that p.R558C is less susceptible to proteasomal degradation than p.P885L. To confirm this observation, we performed a cycloheximide (CHX) chase assay using HA-tagged WFS1 variants. After inhibiting nascent protein translation by CHX treatment, the protein levels of p.R558C and p.P885L rapidly decreased within 2 h (Figure 3E). However, the decay rate of p.P885L was higher than that of p.R558C (Figure 3E). Furthermore, the basal expression of p.P885L was reduced before CHX treatment compared to WT and p.R558C, all of which were consistent with the more rapid degradation of p.P885L (Figure 3E).

[0085] Next, we investigated whether endogenously expressed WFS1 variants in cells exhibit similar post-translational stability. Peripheral blood mononuclear cells (PBMCs) were obtained from three subjects with pathogenic variants in the WFS1 gene (W024: c.1672C>T, c.1672C>T; W392: c.1672C>T, c.1672C>T; W121: c.1672C>T, c.2654C>T) and iPSCs were generated (Table 2). Consistent with our clinical findings, subjects W024, W392, and W121 exhibited a mild Wolfram syndrome phenotype (Table 2). Western blot (WB) analysis revealed reduced WFS1 protein levels in W024, W392, and W121 compared with two control iPSC lines (BJFF.6 and AN1.1) (Figure 3F). Among the three patient-derived cell lines, W121 had lower WFS1 protein levels than W024 and W392 (Figure 3F). WFS1 mRNA expression was not significantly reduced in W024 and W392 compared to the control lines, but was reduced in W121 (Figure 3G). We also performed an actinomycin D (ActD) chase assay to determine the stability of WFS1 mRNA in each iPS cell line. WFS1 mRNA decay was higher than that of one control line, AN1.1, but similar to that of another control line, BJFF.6 (Figure 3H). Meanwhile, WFS1 mRNA in W121 was less stable than both control lines, consistent with endogenous WFS1 expression (Figure 3F). Taken together, the WFS1 c.1672C>T,p.R558C variant reduced the expression of defective WFS1 protein caused by post-translational proteolysis rather than mRNA alterations, and we designated this variant as class A2.

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[0086] Combined treatment with 4-PBA and TUDCA improves cellular function of neural progenitor cells carrying the c.1672C>T, p.R558C variant We first tested whether combined treatment with 4-PBA and TUDCA (P+T) stabilizes WFS1 protein using a HiBiT-tagged reporter. Incubation with P+T significantly increased the steady-state levels of WFS1 p.R558C protein, but not WT or NanoLuc control expressed from the same plasmid backbone (Figure 4B). Treatment also slightly increased the steady-state levels of WFS1 p.P885L, but this was not statistically significant (P = 0.0697). Furthermore, screening the NCATS Pharmaceutical Collection (approximately 2000 compounds) found approved drugs as well as 4-PBA, but not TUDCA. A small number of compounds were found to increase WFS1 p.R558C protein levels, with disulfiram being the most significant, although the magnitude of the effect was similar to that of P+T. Next, we compared the endogenous WFS1 protein levels in iPSCs treated with P+T. P+T treatment significantly increased WFS1 protein levels in iPSCs derived from all three patient-derived cell lines (Figure 4C). Notably, WFS1 protein levels in W024 and W392 were restored to levels comparable to those in the control line (Figure 4C). Furthermore, WFS1 mRNA levels were increased by P+T treatment (Figure 4D). We previously reported organelle dysfunction and subsequent cell death in neural progenitor cells (NPCs) differentiated from iPSCs derived from a typical Wolfram syndrome patient (Lu S, et al. A calcium-dependent protease as a potential therapeutic target for Wolfram syndrome. Proceedings of the National Academy of Sciences of the United States of America. 2014;111(49):E5292-301). NPCs differentiated from the three patient-derived cell lines and the control line (AN1.1) express the NPC markers NESTIN and SOX1 (Figure 4E and Figure 5A).WFS1 expression showed a similar pattern in iPS cells from these cell lines (Figure 5B). Interestingly, the expression of the ER stress marker genes BiP and spliced ​​XBP1 (sXBP1) did not change significantly between the lines, whereas the ER stress-induced apoptotic gene CHOP was significantly increased in each of the three patient-derived cell lines (Figure 5C). TXNIP, another ER stress-induced apoptotic gene, was increased in W392 and W121 compared to AN1.1 (Figure 5A).

[0087] Next, we investigated whether combined treatment with 4-PBA and TUDCA could restore organelle function in NPCs derived from three patient iPS cell lines. While BiP and sXBP1 expression was unaffected by P+T treatment, CHOP and TXNIP expression were significantly reduced in each of the three patient-derived cell lines (Figure 4F). To further assess mitochondrial function, we also measured the oxygen consumption rate (OCR) of NPCs. Increases in OCR were observed throughout the assay in each of the three patient-derived cell lines treated with P+T. To determine whether the increase in OCR was due to increased mitochondrial number or improved mitochondrial function, we measured the mitochondrial DNA content and mitochondrial membrane potential of NPCs treated with or without P+T. Interestingly, mitochondrial DNA increased in W024 and W392 NPCs upon treatment (Figure 7A), whereas mitochondrial membrane potential was unaffected (Figure 7B). In W121 NPCs, both mitochondrial DNA and membrane potential increased upon treatment (Figures 7A and 7B). In addition to these improvements, P+T treatment suppressed apoptosis in each of the three patient-derived cell lines, as indicated by caspase 3 / 7 activity and cleaved caspase 3 protein levels (Figure 4H and Figure 7C).

[0088] To determine whether combined treatment with 4-PBA and TUDCA was beneficial, we compared the efficacy of P + T with treatment with either 4-PBA or TUDCA alone. The effect of P + T on endogenous WFS1 protein levels was greatest in each of the three patient iPSC lines (Figure 6A). Notably, in W392 and W121, P + T treatment significantly increased WFS1 protein levels compared with single treatment with each compound (Figure 6A). Expression of ER stress-induced apoptotic genes was similar in W024 and W392 NPCs, regardless of single or combined treatment, whereas in W121, P + T treatment alone significantly reduced ER stress-induced apoptotic gene expression (Figure 6B). Mitochondrial DNA was not significantly altered by any single treatment in each of the three patient-derived cell lines. In W121 NPCs, P + T treatment increased mitochondrial membrane potential, but this was not observed with treatment with either compound alone (Figure 7B). Finally, single treatment with 4-PBA inhibited apoptosis in all three patient-derived cell lines, and TUDCA also reduced apoptosis in W392 (Figures 4H and 7C). However, the magnitude of inhibition was greatest with P+T treatment in each of the three patient-derived cell lines (Figures 4H and 7C). Furthermore, P+T treatment also reduced caspase 3 / 7 activity in NPCs derived from typical Wolfram syndrome patients (Figures 8A and 8B). Collectively, combined treatment with 4-PBA and TUDCA inhibited apoptosis by increasing WFS1 expression and mitochondrial dysfunction and mitochondrial stress. This was more beneficial than treatment with either 4-PBA or TUDCA alone, although there was some variability across cell lines.

[0089] Combined treatment with 4-PBA and TUDCA improves insulin secretion and viability in SC-β cells carrying the WFS1 c.1672C>T, p.R558C variant. The majority of patients with Wolfram syndrome develop diabetes mellitus due to the deleterious effects of pathogenic WFS1 variants on pancreatic beta cells (Fonseca SG, et al. WFS1 Is a Novel Component of the Unfolded Protein Response and Maintains Homeostasis of the Endoplasmic Reticulum in Pancreatic Beta Cells. The Journal of Biological Chemistry. 2005;280(47):39609-15; Fonseca SG, et al. Wolfram syndrome 1 gene negatively regulates ER stress signaling in rodent and human cells. The Journal of Clinical Investigation. 2010;120(3):744-55; Abreu D, et al. Wolfram syndrome 1 gene regulates pathways maintaining beta-cell health and survival. Laboratory Investigation; a Journal of Technical Methods and Pathology. 2020;100(6):849-62). To evaluate the effect of the WFS1 c.1672C>T, p.R558C variant on β cells, we generated stem cell-derived islets (SC islets) from iPSCs from W024 and W121 strains, as well as from AN1.1 iPSCs as a control. We previously developed a six-stage differentiation strategy incorporating cytoskeleton modulation to generate SC islets containing hormone-secreting endocrine cell types, including insulin-positive stem cell-derived β (SC-β), glucagon-positive stem cell-derived α (SC-α), and somatostatin-positive stem cell-derived δ (SC-δ) cells. Stage 6 SC islets from W024 and W121 strains produced C-peptide+ cells that co-expressed the β cell differentiation marker (NKX6.1) and the differentiated endocrine cell marker (CHGA).The β-cell population was similar in W024 and control SC islets but decreased in the W121 line (Figures 9A and 9B). WFS1 protein was expressed in SC islets from all three cell lines, with higher expression detected in control SC islets (Figure 9C). Notably, WFS1 protein levels were significantly higher in W024 SC islets compared to W121 (Figure 9C). However, both patient-derived SC islets (W024 and W121) showed a significant decrease in WFS1 mRNA levels (Figure 9D), which was not observed in W024 iPSCs or NPCs (Figures 3G and 5B). We previously demonstrated that WFS1 expression significantly increases during the differentiation of SC islets from stage 5 to stage 6, suggesting that WFS1 expression in SC islets may be much higher than that in iPSCs or NPCs. Previous studies have shown that WFS1 deficiency causes mild dilation of the ER in beta cells (Riggs AC, et al. Mice conditionally lacking the Wolfram gene in pancreatic islet beta cells exhibit diabetes as a result of enhanced endoplasmic reticulum stress and apoptosis. Diabetologia. 2005;48(11):2313-21; Akiyama M, et al. Increased insulin demand promotes while pioglitazone prevents pancreatic beta cell apoptosis in Wfs1 knockout mice. Diabetologia. 2009;52(4):653-63; Hatanaka M, et al. Wolfram syndrome 1 gene (WFS1) product localizes to secretory granules and determines granule acidification in pancreatic beta cells. Hum Mol Genet. 2011;20(7):1274-84).Electron microscopy analysis showed well-formed ER structures in AN1.1 SC islets (Figure 10). In contrast, the ER in W024 and W121 SC islets was distorted, fragmented, and dilated (Figure 10). We tested the functional capacity of SC islets in response to high glucose (20 mM) using a glucose-stimulated insulin secretion (GSIS) assay. Throughout the GSIS period, W024 and W121 SC islets secreted less insulin than control SC islets. W024 SC islets were able to increase insulin secretion in response to glucose stimulation, whereas W121 SC islets were unable to do so (Figure 9E). These data suggest that the WFS1 c.1672C>T, p.R558C variant has a milder effect on β-cell insulin secretion than the WFS1 c.2654C>T, p.P885L variant.

[0090] Next, we tested whether P+T treatment was effective in ameliorating the dysfunction of W024 and W121 SC islets (Figure 9F). WFS1 protein expression was restored in the treated SC islets, as observed in both W024 and W121 iPS cells (Figure 9G). We observed that dilated ERs were ameliorated in a portion of P+T-treated W024 and W121 SC islets (Figure 10). Furthermore, P+T treatment significantly suppressed cell death in W024 and W121 SC islets (Figure 9H). As expected from the increase in WFS1 protein, insulin secretion from W024 and W121 SC islets in low and high glucose conditions was increased by P+T treatment (Figure 9I). Collectively, P+T treatment restored WFS1 expression and increased the insulin secretory capacity of W024 and W121 SC islets.

[0091] Combined treatment with 4-PBA and TUDCA attenuates cellular stress in SC islets carrying the WFS1 c.1672C>T,p.R558C variant. We performed multiplexed single-cell RNA sequencing (scRNA-seq) using the 10x Genomics platform to more precisely investigate genotype-phenotype correlations and the efficacy of combined P+T treatment in SC-β cells. We utilized cell hashing, which applies oligo-tagged antibodies to cell surface proteins of individual samples, allowing for the detection of individual samples within the pooled cell population. Four biological replicates per cell line were sequenced from independent differentiated cell lines treated with or without P+T for 7 days. In total, 16 samples were sequenced, including eight samples within each pooled population submitted individually based on cell line. In total, we sequenced 13,951 stage 6 SC islet cells differentiated from W024 and W121 iPSCs to examine the effects of P+T treatment (W024: 2,619 cells, W024, P+T: 3,158 cells, W121: 3,625 cells, and W121, P+T: 4,549 cells, four biological replicates per sample). The scRNA-seq data were analyzed using dimensionality reduction and unsupervised clustering to classify individual cells into cell populations based on the similarity of their transcriptome profiles. Cell types were identified by matching the most up-regulated genes within each cell cluster population with published pancreatic transcriptome data. After identifying the β-cell population for each sample, we combined 2,329 SC-β cells from four experimental conditions (W024: 377 cells, W024, P+T: 220 cells, W121: 749 cells, and W121, P+T: 680 cells) and performed principal component analysis (PCA) and unsupervised clustering. β cells clustered based on genetic background, regardless of the combination treatment, suggesting that the transcriptional profile of β cells did not significantly change in response to P+T. MT1X and ERO1B were expressed more highly in SC-β cells from W121 treated with P+T than in untreated controls.

[0092] Gene set enrichment analysis (GSEA) was performed on SC-β cells. Gene sets related to NMD, ubiquitination-mediated protein degradation, and oxidative stress were enriched in untreated SC-β cells compared with P + T-treated SC-β cells. Interestingly, inflammation and selective mitophagy pathways were also found to be enriched in untreated SC-β cell populations. Gene sets related to apoptosis and ER stress were enriched in untreated SC-β cells. Notably, gene sets related to insulin secretion and β-cell development were enriched in P + T-treated SC-β cell populations. Furthermore, gene sets related to the regulation of cytoplasmic K+ and Ca2+ levels, which play important roles in β-cell differentiation and function, were increased. Taken together, P + T treatment quelled the increased cellular stress caused by the pathogenic WFS1 variant without altering β-cell identity, resulting in increased β-cell function and insulin secretion in W024 and W121 SC-β cells.

[0093] Combined treatment with 4-PBA and TUDCA delays the progression of the diabetic phenotype in Wfs1-deficient mice Finally, we validated the efficacy of our combination treatment through in vivo studies. There is currently no mouse model of the c.1672C>T, p.R558C WFS1 mutation. Therefore, we used 129S6 whole-body Wfs1 knockout (Wfs1KO) mice. This mouse model is a mouse model of Wolfram syndrome, as it develops progressive glucose intolerance during adolescence (Abreu D, et al. Wolfram syndrome 1 gene regulates pathways maintaining beta-cell health and survival. Laboratory investigation; a journal of technical methods and pathology. 2020;100(6):849-62.). We confirmed that Wfs1KO mice developed glucose intolerance at 5–6 weeks of age (Figures 11A and 11B). Furthermore, Wfs1KO mice did not exhibit glucose-stimulated increases in serum insulin levels, and their serum insulin levels were lower than those of WT mice (Figure 12A). Five- to six-week-old mice were treated with a diet containing 4-PBA and TUDCA (4-PBA: 0.338% and TUDCA: 0.225%, see P+T diet) for one month. Both groups of Wfs1KO mice consumed similar amounts of diet. After one month of dietary intake, Wfs1KO mice fed the control diet developed more severe glucose intolerance (Figures 11A and 11B). In contrast, the blood glucose curves from intraperitoneal glucose tolerance tests (IP-GTT) were similar to the baseline results of Wfs1KO mice fed the P+T diet (Figures 11A and 11B), indicating that the P+T diet delayed the progression of the diabetic phenotype. Body weight and insulin sensitivity were not significantly altered by the P+T diet. Basal serum insulin levels (0 min) were higher in Wfs1KO mice fed the P+T diet compared to mice fed the Ctrl diet (Figure 12F). Compared to baseline, serum insulin levels 30 min after glucose injection were reduced in Wfs1KO mice fed the Ctrl diet but were comparable in mice fed the P+T diet (Figure 12G).Taken together, we observed a delay in the Wolfram diabetic phenotype in Wfs1KO mice using P+T treatment, and therefore expect that this combined treatment will be effective in vivo against the diabetic Wolfram phenotype caused by the WFS1 c.1672C>T,p.R558C variant.

[0094] Example 2: A Phase II Study of the Safety and Efficacy of AMX0035 in Adult Patients with Wolfram Syndrome Study Design: A single-center, open-label study in which up to 12 participants will be treated with AMX0035 for up to 24 weeks.

[0095] The study will consist of a screening period of up to four weeks, a 24-week open-label treatment period, and a post-treatment follow-up visit at week 28. Upon completion of screening and baseline procedures, eligible participants will receive standard of care plus a pre-defined dose of AMX0035.

[0096] Eligible participants will be enrolled in the open-label treatment period of the study on Day 1 and receive their first dose of study medication. During the first three weeks of treatment, participants will take one sachet of AMX0035 daily, increasing to one sachet twice daily (morning and evening) if tolerated. Participants will return to the study site approximately every 12 weeks for study procedures, assessments, and blood draws.

[0097] Test Purpose Main purpose To evaluate the effect of AMX0035 on residual beta-cell function by monitoring C-peptide levels during a 0-240 minute mixed meal tolerance test (MMTT). To evaluate the safety and tolerability of oral AMX0035 administered for up to 24 weeks in adult patients with diabetes mellitus due to Wolfram syndrome.

[0098] Secondary Objectives -To estimate the magnitude of the treatment effect of AMX0035 on best-corrected visual acuity in both eyes, measured using the LogMar scale, using office-based visual acuity testing using Snellen charts. Evaluate the effect of AMX0035 by tracking changes in total daily insulin dose (including basal vs. bolus percentage). Evaluate the effect of AMX0035 by tracking time in a good blood glucose range (70-180 mg / dL), time below range (54-69 mg / dL), and time above range (181-250 mg / dL) as measured by continuous glucose monitoring (CGM). -To evaluate the effect of AMX0035 by tracking the reduction in HbA1c.

[0099] exploratory purpose The effects of AMX0035 will be evaluated on the following items: Wolfram Unified Rating Scale (WURS) Scale for the Assessment of Ataxia (SARA) Visual function using the Visual Functioning Questionnaire-25 (VFQ-25) ·Diabetes measurement Blood biomarker (panel) levels of neurodegeneration and neuroinflammation Changes in retinal pathology based on optical coherence tomography (OCT) (including OCT angiography) Global Impression Scales, Patient-Reported Global Impression of Change (PGIC) and Clinician-Reported Global Impression of Change (CGIC) Most bothersome symptoms (MBS)

[0100] Evaluation items: Safety evaluation items Incidence and severity of adverse events (AEs) and serious adverse events (SAEs) -Incidence of abnormal laboratory test values

[0101] Efficacy endpoints Primary efficacy endpoint Area under the curve (AUC) response of C-peptide to 0-240 min MMTT at 24 weeks (and additional time points) Change in ΔC peptide from baseline (using 0-240 min MMTT) at week 24 (and additional time points)

[0102] Secondary efficacy endpoints Change from baseline (to week 24) in best-corrected visual acuity in both eyes, measured on the LogMar scale. Visual acuity tests were performed in the clinic using a Snellen chart. Values ​​were obtained for each eye after correction. Values ​​range from 0, representing normal vision (values ​​of -0.1 or -0.2, representing better than normal vision, are also possible), to +2, representing near-blindness. An increase in LogMAR represents deterioration. Change in exogenous insulin dose per kg body weight per 24 hours (including basal vs. bolus dose ratio) from baseline (through week 12 and week 24), and change between baseline and subsequent visits Change in total time blood glucose measured by CGM was in range (70-180 mg / dL) from baseline to week 24 Time in Range (TIR), the percentage and time of readings in the 70-180 mg / dL range Time Below Range (TBR), the percentage and time of readings in the 54-69 mg / dL range Time Over Range (TAR), the percentage and time of readings in the 181-250 mg / dL range Change in HbA1c levels from baseline to week 24

[0103] Exploratory efficacy endpoints -Evaluate changes from baseline in the following items: oWURS oSARA oVFQ-25 oOCT measurements oCGI-C oPGI-C oMBS Blood biomarker (panel) levels of neurodegeneration and neuroinflammation Diabetes measurements including fasting glucose, fasting proinsulin, AUC c-peptide / AUC glucose, and delta-proinsulin Changes in C-peptide levels from week 24 to week 28

[0104] Study population: Inclusion criteria 1. Participants have a confirmed diagnosis of Wolfram syndrome based on the following items: At the time of screening, evidence of functionally relevant recessive mutations in both alleles of the WFS1 gene based on previous test results (if available) or test results from an accredited laboratory. 2. Stimulated C-peptide level ≥ 0.2ng / mL during the screening visit. 3. Insulin-dependent diabetes mellitus due to Wolfram syndrome 4. Be 17 years of age or older at the time of written informed consent 5. Women of childbearing potential (e.g., not postmenopausal for more than 1 year or surgically sterile) must agree to use adequate contraception* during the study and for 6 months after the final dose of study drug. Women must not plan to become pregnant during the study and for 6 months after the final dose of study drug. 6. Men must agree to use contraception* during the study and for at least 6 months after the final dose of study drug. Men must not plan to father children or donate sperm during the study and for 6 months after the final dose of study drug.

[0105] *Appropriate contraceptive methods that may be used in this study are as follows: a. Hormonal methods such as birth control pills, patches, injections, vaginal rings, and implants b. Barrier methods (such as condoms or diaphragms) combined with spermicides (foams, creams, or gels that kill sperm) c. Intrauterine device (IUD) d. Abstinence (no heterosexual intercourse) Only one partner who is surgically sterile (male) or non-fertile (female)

[0106] Exclusion criteria 1. Clinically significant, non-Wolfram-related central nervous system (CNS) lesions that, in the opinion of the investigator, may interfere with the accurate performance and interpretation of protocol evaluations. 2. Any clinically significant unstable medical condition (excluding Wolfram syndrome) that, in the opinion of the investigator, may pose a risk to the participant if they participate in the study. 3. If the investigator determines that the participant has a clinically significant infection or inflammation at the time of screening or admission. If the infection or inflammation resolves, the participant may be re-screened. 4. Acute gastrointestinal symptoms (e.g., nausea, vomiting, diarrhea) at screening or on admission. 5. In the investigator's judgment, the participant has unstable psychiatric illness, cognitive impairment, dementia, or substance abuse that impairs their ability to provide informed consent or follow instructions. 6. Major surgery has been performed within 4 weeks of screening. 7. Anyone who, in the opinion of the investigator, is unable to comply with the protocol (e.g., has a clinically relevant medical condition that makes it difficult to carry out the protocol, has an unstable social situation, is known to have clinically significant psychiatric / behavioral instability, is unable to attend the study site for the period required for study evaluation, or is otherwise unlikely to complete the study). 8. Known allergy to phenylbutyrate (PB) or bile salts 9. Abnormal liver function defined as aspartate transaminase (AST) and / or alanine transaminase (ALT) >3 times the upper limit of normal (ULN). Renal failure defined as having an estimated glomerular filtration rate (eGFR) greater than 10.60 mL / min / 1.73 m2. 11. Anemia with hemoglobin (Hgb) concentration less than 10.0 g / dL at screening. 12. Pregnant or breastfeeding women 13. You currently have biliary tract disease that may cause biliary obstruction or obstruct biliary flow, such as active cholecystitis, primary biliary cirrhosis, sclerosing cholangitis, gallbladder cancer, gallbladder polyps, gallbladder gangrene, or gallbladder abscess. 14. History of heart failure according to the New York Heart Association (NYHA). 15. Personal or family history of breast and / or ovarian cancer. 16. Participants who are under severe salt restriction and whose treatment-related additional salt intake is deemed by the investigator to be endangering the patient. 17. Received treatment with an investigational drug or device within 30 days prior to screening / study entry. 18.Subjects who have received a blood product transfusion within 90 days prior to screening. 19. Subjects who have received gene therapy or cell therapy. 20. Evidence of organ dysfunction or clinically significant deviations from normal values ​​in physical examination, vital signs, or clinical laboratory values ​​beyond the range consistent with the target population in the opinion of the investigator. 21. There was a clinically significant abnormality in the 12-lead electrocardiogram before administration of the study drug, which was confirmed by repeat measurement. 22. History of clinically significant suicidal ideation and / or behavior within 1 year of screening, as determined by the investigator. 23. Any condition that, in the opinion of the investigator, would prevent the participant from fully complying with or completing the study. 24. If you are currently receiving, have received within 30 days prior to screening, or are scheduled to receive treatment with any of the following prohibited substances:

[0107] Investigational drugs, excluding the study drug, being used or being evaluated for the treatment of Wolfram syndrome are prohibited from use for the duration of the study, beginning 30 days (or 5 half-lives, whichever is longer) before the first dose (or screening, if at the same visit).

[0108] Participants will be excluded from enrollment if they have used gene or cell therapy (e.g., NurOwn, Q-Cells, T-modulating therapy) prior to this study, which is also prohibited during the trial.

[0109] Unless approved by the investigator in consultation with the sponsor, participants should not receive the following medications shown in Table 1 (this is not a comprehensive list): [Table 1]

[0110] Study treatment: All participants will receive oral AMX0035 treatment. For the first three weeks, participants will take one sachet daily, increasing to one sachet twice daily (morning and evening) if tolerated.

[0111] AMX0035 will be supplied by the sponsor to the site pharmacy in a cardboard box containing single-use sachets. Each sachet of AMX0035 contains the active ingredients (3 g PB and 1 g taursodiol [TURSO]) and excipients in a powder formulation. The study drug will be mixed with approximately one glass of water and taken orally.

[0112] Study and treatment duration: Treatment duration will be up to 24 weeks. For participants who complete the study, the planned overall study duration is up to 32 weeks.

[0113] Individual stopping criteria: All AEs, safety test results, and concomitant medication use will be closely monitored by the investigator throughout the study. If clinically significant laboratory or clinical abnormalities occur, participants will be closely monitored until the abnormality resolves or they are clinically stable.

[0114] Management of dose-limiting treatment-emergent adverse events (TEAEs) that are not tolerated by the participant and that the investigator determines may be related to the study drug may be managed by gradual dose reduction to a lower dose of one sachet of study drug per day. If this first level of dose reduction does not produce improvement within 7 to 14 days, the dose may be reduced to one sachet of study drug every other day. The investigator may decide to discontinue treatment at any time.

[0115] If a participant exhibits treatment-emergent signs of neurotoxicity that the investigator believes may be related to the study drug, including, but not limited to, vomiting, nausea, headache, dizziness, somnolence, loss of taste, hearing loss, disorientation, confusion, memory loss, or neuropathy, the investigator should consider reducing or interrupting the dose.

[0116] Dose adjustments, including the reason and date of adjustment, will be documented on the source document and electronic case report form (eCRF). Dose changes may be discussed with the medical monitor.

[0117] The new regimen, dose reduction, or discontinuation can be continued for as long as necessary until symptoms improve. The investigator may then choose to resume the higher dose or continue the participant on the reduced dose. If discontinuing the drug, the medical monitor should be consulted.

[0118] If dose-limiting TEAEs recur upon rechallenge with study drug, treatment will be permanently discontinued.

[0119] If any of the following AEs occur, treatment will be temporarily discontinued. Persistent diarrhea: Multiple (more than 5) loose, watery stools requiring fluid replacement therapy persist for more than 3 days after initiation of treatment. Treatment-emergent elevations in serum creatinine or liver enzymes according to the following guidance: A confirmed increase in serum creatinine of more than 50% from baseline oALT or AST >8x ULN ALT or AST >5x ULN for ≥2 weeks oALT or AST >3x ULN AND serum total bilirubin >2x ULN OR international normalized ratio >1.5 ALT or AST >3x ULN AND any of the following occurs: fatigue, nausea, vomiting, right upper quadrant pain or tenderness, fever, rash, and / or eosinophilia (>5%) Treatment-emergent Grade 3 AEs based on the NCI Common Terminology Criteria for Adverse Events (CTCAE) version 5.0, unless otherwise specified.

[0120] Diagnostic Tools and Rating Scales Diagnostic tools and assessment scales include: 0-240 minute MMTT (primary efficacy endpoint)

[0121] The MMTT test measures residual β-cell function (Bus 1982). The night before the MMTT, participants received an evening dose of Lantus insulin and fasted from midnight until the test at 8:00 a.m. The mixed meal consisted of Boost Original (Societe des Produits Nestlé SA, Vevey, Switzerland), 6 mL / kg (maximum 360 mL).

[0122] The following instructions must be followed: Prior to the MMTT, participants should not take short-acting insulin, short-acting GLP-1 receptor agonists, metformin, or SGLT inhibitors. Boost Original can be consumed within 5 minutes. Blood samples for glucose and C-peptide measurements were taken at -10, 0, 15, 30, 60, 90, 120, 180, and 240 minutes, with a sampling time of ±5 minutes. If the participant's fasting glucose is above 11.1 mmol / L, the test will not be performed, but fasting glucose and C-peptide will be obtained. Participants using continuous subcutaneous insulin infusion (CSII) for diabetes management should have their insulin adjusted appropriately before the MMTT test. Appropriate clinical safety measures should be implemented to ensure participant safety during the 4-hour MMTT. The investigator and study staff are responsible for appropriate clinical monitoring throughout the study, including safety monitoring during the MMTT (e.g., ketone monitoring). In the investigator's opinion, appropriate clinical safety measures should be in place to ensure participant safety during the 4-hour MMTT.

[0123] ·Diabetes measurement The primary responsibility for diabetes management rests with the treating diabetes specialist or referring care provider, but the investigator's study team will provide close additional support through telephone interactions as needed. Diabetes management will be monitored by the investigator's study staff by telephone between study visits as needed.

[0124] Diabetes measurements include: Fasting blood glucose, fasting proinsulin, AUC c-peptide / AUC blood glucose, delta-proinsulin

[0125] Diabetes measurement allows you to: Continuous glucose monitoring (CGM) Track changes in total daily insulin dose Tracking HbA1c reduction

[0126] WURS The Wolfram Syndrome Review Scale (WURS) (Nguyen 2012) is a clinical scale for measuring the severity and progression of Wolfram Syndrome. The scale consists of three domains: Area A = Physical - Doctor's evaluation Area B = Physical - Parental Assessment Domain C = Behavior - Parent Rating The physical domain is rated from 0 to 4, with 0 corresponding to no symptoms and 4 corresponding to the most severe symptoms. The behavioral domain is rated from 0 to 3, with 0 corresponding to normal behavior and 3 indicating the presence of more severe impairment.

[0127] SARA The Scale for the Rating and Assessment of Ataxia (Subramony 2007) is an eight-item performance-based scale with a total score ranging from 0 (no ataxia) to 40 (most severe ataxia). Scores are based on participants' performance on the following: 1) gait, 2) posture, 3) sitting, 4) speech impairment, 5) finger tracking, 6) finger-nose test, 7) rapid alternating hand movements, and 8) heel-shin slides.

[0128] VFQ-25 The National Eye Institute Visual Function Questionnaire-25 (VFQ-25) is designed to measure vision-related function and the impact of vision-related problems. The VFQ-25 represents 11 vision-related constructs, with a maximum of 39 items, and an additional single-item general health question. For scoring, raw scores are converted to a 100-point scale, with higher scores indicating poorer performance.

[0129] CGI-C The CGI-C assesses improvement in seven categories: very improved, much improved, slightly improved, no change, slightly worse, much worse, and very worse. These assessments are administered to participants by the site (investigator).

[0130] PGI-C Participants will rate the change in Wolfram syndrome-related symptoms since starting study medication by selecting one of seven responses. The PGI-C is a 7-point response scale. Participants will be asked by the investigator or qualified designee to rate the change in their condition using the following 7-point scale: 1 = very improved, 2 = very much improved, 3 = slightly improved, 4 = no change 5 = slightly worse, 6 = much worse, 7 = very worse. Respondents were defined using the PGI-C responses of "very improved," "much improved," "slightly improved," and "no change."

[0131] MBS Patient-identified most bothersome symptoms (MBS) will be identified at screening, and participants will describe their MBS as associated with Wolfram syndrome. At follow-up visits, participants will be asked to rate the overall change in their symptoms from baseline using a 7-item Likert scale ranging from "much improved" to "much worse." 1 = very improved, 2 = very much improved, 3 = slightly improved, 4 = no change 5 = slightly worse, 6 = much worse, 7 = very worse. This will be administered to participants by a clinician.

[0132] OCT Optical coherence tomography (OCT) is a non-invasive eye imaging test. OCT uses light waves to take cross-sectional pictures of a participant's retina for the purpose of diagnosing and researching eye diseases. OCT measurement tests also include angiography and visual acuity testing. A detailed OCT manual is included as a separate document.

[0133] C-SSRS The C-SSRS is a systematically administered assessment tool developed to track suicidal AEs throughout a treatment study. The tool is designed to assess suicidal behavior and ideation and to track and assess the lethality of all suicidal acts and attempts. Additional features assessed include frequency, duration, controllability, reasons for ideation, and deterrence. The C-SSRS is considered a low-burden assessment tool because it takes less than five minutes to administer. It is administered to participants by the investigator or a qualified designee.

[0134] Participants who answer "yes" to questions 4 or 5 on the C-SSRS regarding suicidal ideation or who, through the clinical interview, are identified as having had suicidal thoughts or plans within the past month will be immediately evaluated by the investigator. This will also be reported to the medical monitor and sponsor. Appropriate measures will be taken to protect the participant, including, but not limited to, possible withdrawal from the study (as determined by the investigator or medical monitor) and referral to appropriate psychiatric care. Such participants at screening or Day 1 will also be excluded from the study.

Claims

1. 1. A method of treating one or more symptoms of Wolfram syndrome in a subject, comprising administering to the subject a pharmaceutically effective amount of a combination of TURSO and sodium phenylbutyrate.

2. 10. The method of claim 1, wherein the subject has or is at risk of developing diabetes.

3. 3. The method of claim 2, wherein the diabetes is insulin-dependent diabetes.

4. 3. The method of claim 2, wherein the diabetes is juvenile-onset diabetes.

5. 10. The method of any one of the above claims, wherein the subject is suffering from or at risk of developing optic atrophy.

6. 10. The method of any one of the preceding claims, wherein the subject suffers from or is at risk of developing a hearing impairment.

7. 10. The method of any one of the above claims, wherein the subject has one or more mutations in the wolframin (WFS1) gene.

8. 8. The method of claim 7, wherein the subject has a c.1672C>T, p.R558C mutation in the WFS1 gene.

9. 9. The method of claim 7 or 8, wherein the subject has a c.2654C>T, p.P885L mutation in the WFS1 gene.

10. 10. The method of any one of the above claims, wherein the subject has one or more mutations in the CDGSH iron-sulfur domain protein 2 (CISD2) gene.

11. 10. The method of any one of the preceding claims, wherein the TURSO and the sodium phenylbutyrate are administered once daily or twice daily.

12. 10. The method of any one of the preceding claims, wherein TURSO is administered to the subject at a dose of about 5 mg / kg to about 100 mg / kg.

13. 10. The method of any one of the preceding claims, wherein sodium phenylbutyrate is administered to the subject at a dose of about 10 mg / kg to about 400 mg / kg.

14. 10. The method of any one of the preceding claims, wherein the TURSO is administered in an amount of about 0.5 to about 5 grams per day.

15. 10. The method of any one of the preceding claims, wherein the sodium phenylbutyrate is administered in an amount of about 0.5 grams to about 10 grams per day.

16. 10. The method of any one of the preceding claims, comprising administering to the subject 1 gram of TURSO and 3 grams of sodium phenylbutyrate once daily or twice daily.

17. 10. The method of any one of the preceding claims, comprising administering to the subject 1 gram of TURSO once daily and 3 grams of sodium phenylbutyrate once daily for about 14 days or more, followed by administering to the subject about 1 gram of TURSO twice daily and 3 grams of sodium phenylbutyrate twice daily.

18. 10. The method of any one of the preceding claims, wherein the TURSO and the sodium phenylbutyrate are administered orally.

19. 10. The method of any one of the preceding claims, wherein the TURSO and the sodium phenylbutyrate are formulated as a single powder formulation.

20. 10. The method of any one of the above claims, further comprising administering to the subject one or more additional therapeutic agents.

21. 21. The method of claim 20, wherein the one or more additional therapeutic agents are valproic acid, a glucagon-like peptide (GLP)-1 receptor agonist, dantrolene sodium, or an ER Ca2+ stabilizer.

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