Methods and compositions for treating wolfram syndrome

Abstract

Provided herein are compositions and methods for treating at least one symptom of Wolfram syndrome in a subject in need thereof. The methods disclosed herein comprise administering to the subject a combination of a bile acid or pharmaceutically acceptable salt thereof and a phenylbutyrate compound.

Description

[0001] METHODS AND COMPOSITIONS FOR TREATING WOLFRAM SYNDROME

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims priority to U.S. Provisional Application No. 63 / 709,828, filed on October 21, 2024, U.S. Provisional Application No. 63 / 707,531, filed on October 15, 2024, U.S. Provisional Application No. 63 / 631,878, filed on April 9, 2024, and U.S. Provisional Application No. 63 / 562,618, filed on March 7, 2024, the contents of all of which are herein incorporated by reference in their entirety.

[0004] TECHNICAL FIELD

[0005] The present disclosure generally relates to compositions and methods for treating Wolfram syndrome.

[0006] BACKGROUND

[0007] Wolfram syndrome is a genetic disorder characterized by juvenile onset diabetes, progressive blindness, and neurodegeneration. There are currently no disease-modifying therapies for Wolfram syndrome; treatment strategies focus on use of life-sustaining medications, clinical monitoring, and symptom management. Accordingly, methods for slowing Wolfram syndrome progression, improving endocrinological, neurological, and / or ophthalmological function, and increasing survival time of patients with Wolfram syndrome are needed.

[0008] SUMMARY

[0009] The present disclosure provides combinations of a bile acid (e.g. Taurursodiol (TURSO)) or pharmaceutically acceptable salt thereof and a phenylbutyrate compound (e.g., 4-phenylbutyrate or a pharmaceutically acceptable salt thereof, including, e.g., sodium phenylbutyrate) and demonstrates surprising features of combination therapy with such a bile acid and a phenylbutyrate compound in treating one or more symptoms associated with Wolfram syndrome, and in reversing, preventing, or slowing Wolfram syndrome disease progression. The combination therapy disclosed herein can be useful for improving pancreatic function and alleviating one or more symptoms of diabetes in subjects in need thereof, as well as for improving visual acuity in subjects in need thereof. The combination therapy are also useful for improving overall disease burden for subjects with Wolfram syndrome.

[0010] In a first aspect, provided herein are methods of slowing Wolfram syndrome disease progression in a human subject, the methods comprising administering to the human subject a pharmaceutically effective amount of a combination of taurursodiol (TURSO) and sodium phenylbutyrate, to thereby slow Wolfram syndrome progression in the human subject.

[0011] In a second aspect, provided herein are methods of increasing C-peptide response in a human subject having Wolfram syndrome, the methods comprising administering to the human subject a pharmaceutically effective amount of a combination of TURSO and sodium phenylbutyrate, to thereby increase C-peptide response in the human subject.

[0012] In a third aspect, provided herein are methods of improving endocrinological function in a human subject having Wolfram syndrome, the methods comprising administering to the human subject a pharmaceutically effective amount of a combination of TURSO and sodium phenylbutyrate, to thereby improve endocrinological function in the human subject.

[0013] In a fourth aspect, provided herein are methods of improving neurological function in a human subject having Wolfram syndrome, the methods comprising administering to the human subject a pharmaceutically effective amount of a combination of TURSO and sodium phenylbutyrate, to thereby improve neurological function in the human subject.

[0014] In a fifth aspect, provided herein are methods of improving ophthalmological function in a human subject having Wolfram syndrome, the methods comprising administering to the human subject a pharmaceutically effective amount of a combination of TURSO and sodium phenylbutyrate, to thereby improve ophthalmological function in the human subject.

[0015] In a sixth aspect, provided herein are methods of improving overall disease burden of a human subject having Wolfram syndrome, the methods comprising administering to the subject a combination of TURSO and sodium phenylbutyrate, to thereby improve overall disease burden of the human subject. In some embodiments, overall disease burden is assessed using patient reported global impression of change (PGLC). In some embodiments, overall disease burden is assessed using clinician reported global impression of change (CGLC).

[0016] In a seventh aspect, provided herein are methods of increasing survival time of a human subject having Wolfram syndrome, the methods comprising administering to the human subject a pharmaceutically effective amount of a combination of TURSO and sodium phenylbutyrate, to thereby increase survival time of the human subject.

[0017] In some embodiments, the human subject has or is at risk for developing diabetes. In some embodiments, the diabetes is insulin-dependent diabetes. In some embodiments, the diabetes is juvenile onset diabetes. In some embodiments, the human subject has or is at risk for developing optic nerve atrophy. In some embodiments, the human subject has or is at risk for developing a hearing impairment. In some embodiments, the human subject has one or more mutations in the WFS1 gene, which encodes Wolframin. In some embodiments, the human subject has the C.1672OT, p.R558C mutation in the WFS1 gene. In some embodiments, the human subject has the C.2654OT, p.P885L mutation in the WFS1 gene. In some embodiments, the human subject has one or more mutations in the CISD2 gene, which encodes CDGSH iron sulfur domain protein 2. In some embodiments, the TURSO and the sodium phenylbutyrate are administered once a day or twice a day. In some embodiments, TURSO is administered to the human subject at a dose of about 5mg / kg to about 100 mg / kg. In some embodiments, sodium phenylbutyrate is administered to the human subject at a dose of about lOmg / kg to about 400 mg / kg. In some embodiments, the TURSO is administered at an amount of about 0.5 grams to about 5 grams per day. In some embodiments, the sodium phenylbutyrate is administered at an amount of about 0.5 grams to about 10 grams per day. In some embodiments, the methods include administering to the human subject about 1 gram of TURSO and about 3 grams of sodium phenylbutyrate once a day or twice a day. In some embodiments, the methods include administering to the human subject about 1 gram of TURSO once a day and about 3 grams of sodium phenylbutyrate once a day for about 14 days or more, followed by administering to the human subject about 1 gram of TURSO twice a day and 3 grams of sodium phenylbutyrate twice a day. In some embodiments, the TURSO and the sodium phenylbutyrate are administered orally. In some embodiments, the TURSO and the sodium phenylbutyrate are formulated as a single powder formulation. In some embodiments, the methods further include administering one or more additional therapeutic agents to the human subject. In some embodiments, the one or more additional therapeutic agents is valproic acid, glucagon-like peptide (GLP)-l receptor agonists, dantrolene sodium, or ER Ca2+ stabilizers.

[0018] In some embodiments, peak C-peptide levels of the human subject are increased after a meal relative to baseline (i.e., prior to administration of the combination of TURSO and sodium phenylbutyrate). In some embodiments, the peak C-peptide levels are increased by at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more relative to baseline. In some embodiments, the peak C-peptide levels are increased by at least about 5% relative to baseline. In some embodiments, the peak C-peptide levels are measured by a mixed meal tolerance test (MMTT). In some embodiments, the MMTT is used to calculate C-peptide area under the curve (AUC), AC-peptide, and / or time to peak C-peptide of the human subject. In some embodiments, C-peptide levels, C-peptide area under the curve (AUC), AC-peptide, and / or time to peak C-peptide of the human subject are calculated using a method other than MMTT (e.g., using a glucagon stimulation test (GST), oral glucose tolerance test (OGTT), tolbutamide tolerance test (tCP), fasting C-peptide test (fCP), random non-fasting C-peptide test (rCP), urinary C-peptide creatinine ratio (UCPCR), urinary C-peptide (UCP), or 24 h urinary collection (24 h UCP).

[0019] In some embodiments, the C-peptide AUC is increased relative to baseline (i.e., prior to administration of the combination of TURSO and sodium phenylbutyrate). In some embodiments, the C-peptide AUC is increased by at least about 1 min*ng / mL, 5 min*ng / mL, 10 min*ng / mL, 15 min*ng / mL, 20 min*ng / mL, 25 min*ng / mL, 30 min*ng / mL, 35 min*ng / mL, 40 min*ng / mL, 45 min*ng / mL, 50 min*ng / mL, 60 min*ng / mL, 70 min*ng / mL, 80 min*ng / mL, 90 min*ng / mL, 100 min*ng / mL, 110 min*ng / mL, 120 min*ng / mL, 130 min*ng / mL, 140 min*ng / mL, 150 min*ng / mL, 160 min*ng / mL, 170 min*ng / mL, 180 min*ng / mL, 190 min*ng / mL, 200 min*ng / mL, or more relative to baseline when measured at or around the C-peptide peak. In some embodiments, the C-peptide AUC is increased by at least about 10 min*ng / mL relative to baseline when measured at or around the C-peptide peak.

[0020] In some embodiments, AC-peptide is increased relative to baseline (i.e., prior to administration of the combination of TURSO and sodium phenylbutyrate). In some embodiments, the AC-peptide is increased by at least about 0.05 ng / mL, 0.06 ng / mL, 0.07 ng / mL, 0.08 ng / mL, 0.09 ng / mL, 0.1 ng / mL, 0.15 ng / mL, 0.2 ng / mL, 0.25 ng / mL, 0.3 ng / mL, 0.35 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.6 ng / mL, 0.7 ng / mL, 0.8 ng / mL, 0.9 ng / mL, 1.0 ng / mL, 1.5 ng / mL, 2.0 ng / mL, 2.5 ng / mL, 3.0 ng / mL, or more relative to baseline when measured at or around the C-peptide peak. In some embodiments, the AC-peptide is increased by at least about 0.08 ng / mL relative to baseline when measured at or around the C-peptide peak.

[0021] In some embodiments, time to peak C-peptide of the human subject is shortened relative to baseline (i.e., prior to administration of the combination of TURSO and sodium phenylbutyrate). In some embodiments, the time to peak C-peptide of the human subject is shortened by at least about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or more relative to baseline. In some embodiments, the time to peak C-peptide of the human subject is shortened by at least about 10% relative to baseline. In some embodiments, the time to peak C-peptide is shortened by about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, or more relative to baseline.

[0022] In some embodiments, hemoglobin Ale (HbAlc) levels of the human subject are decreased relative to baseline (i.e., prior to administration of the combination of TURSO and sodium phenylbutyrate). In some embodiments, the HbAlc levels of the human subject are decreased by at least about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, or more relative to baseline. In some embodiments, the HbAlc levels of the human subject are decreased by at least about 0.1% relative to baseline.

[0023] In some embodiments, time in target glucose range of the human subject is increased relative to baseline (i.e., prior to administration of the combination of TURSO and sodium phenylbutyrate). In some embodiments, the time in target glucose range of the human subject is increased by at least about 1%, 1.5%, 2% , 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, or more relative to baseline. In some embodiments, the time in target glucose range of the human subject is increased by at least about 2% relative to baseline.

[0024] In some embodiments, the human subject exhibits improved or stabilized best-corrected visual acuity (BCVA) relative to baseline (i.e., prior to administration of the combination of TURSO and sodium phenylbutyrate). In some embodiments, BCVA is measured on the LogMAR scale by sight tests. In some embodiments, the sight tests are performed using the Snellen chart. In some embodiments, a LogMAR scale reading of the human subject decreases by at least about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 1.0, or more units in one or both eyes.

[0025] 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 in the practice or testing of the present invention, suitable methods and materials are described below.

[0026] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub -combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0027] 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. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1 shows the overall study design for an open-label study to assess the safety and efficacy of AMX0035 in participants living with Wolfram syndrome.

[0030] FIG. 2A and FIG. 2B show mean C-peptide levels (ng / mL) for patients at the screening visit and after receiving AMX0035 for 12, 24, 36, or 48 weeks, as assessed during a 0-240 minute mixed-meal tolerance test (MMTT). FIG. 2A shows the mean C-peptide levels for the “Intent to Treat” (ITT) group, and FIG. 2B shows the mean C-peptide levels for the “Per Protocol” group.

[0031] FIG. 3 shows the C-peptide response to the MMTT for the ITT group and Per Protocol group. Trough-adjusted C-peptide AUC (min*ng / mL) mean change from baseline at the 120- min timepoint of the MMTT is depicted following AMX0035 treatment for 24, 36, and 48 weeks.

[0032] FIG. 4A and FIG. 4B show mean change from baseline of AC-peptide following AMX0035 treatment of the ITT group and Per Protocol group for 12, 24, 36, or 48 weeks. FIG. 4A shows AC-peptide mean change from baseline at the peak of the MMTT, and FIG. 4B shows AC-peptide mean change from baseline at the 120-min timepoint of the MMTT.

[0033] FIG. 5 shows the time to peak C-peptide (min) for the ITT group and Per Protocol group at the screening visit and after receiving AMX0035 for 12, 24, 36 or 48 weeks.

[0034] FIG. 6A shows mean change from baseline of HbAlc (%) for the ITT group and Per Protocol group after receiving AMX0035 for 12, 24, 36 or 48 weeks. FIG. 6B shows mean exogenous insulin dose through week 24 for the ITT group and Per Protocol group.

[0035] FIG. 7 shows time in target glucose range change from baseline (%) for the ITT group and Per Protocol group following treatment with AMX0035 for 12, 24, 36 or 48 weeks.

[0036] FIG. 8 show changes in visual acuity for the ITT group and Per Protocol group following AMX0035 treatment for 24 or 48 weeks. Best-Corrected Visual Acuity (BCVA) for best eye (LogMAR) change from baseline is depicted.

[0037] FIG. 9A shows the effect of AMX0035 treatment on Patient-Reported Global Impression of Change (PGI-C) after 24, 36, or 48 weeks. FIG. 9B shows the effect of AMX0035 treatment on Clinician-Reported Global Impression of Change (CGI-C) after 24, 36, or 48 weeks.

[0038] FIG. 10 shows change from baseline in study participants’ Most Bothersome Symptom (MBS) following AMX0035 treatment for 24, 36, or 48 weeks.

[0039] DETAILED DESCRIPTION

[0040] Wolfram syndrome is a rare genetic disorder with many different possible clinical manifestations, including diabetes insipidus, diabetes mellitus (e.g. juvenile-onset insulindependent diabetes), optic nerve atrophy, and progressive neurodegeneration.

[0041] Applicant has surprisingly discovered that a combination of a bile acid (e.g. Taurursodiol (TURSO)) and a phenylbutyrate compound (e.g., 4-phenylbutyrate or a pharmaceutically acceptable salt thereof, including, e.g., sodium phenylbutyrate) is able to stabilize Wolfram syndrome disease or reverse disease progression and can be used for treating one or more symptoms associated with Wolfram syndrome. Accordingly, the present disclosure provides compositions and methods for treating at least one symptom associated with Wolfram syndrome in subjects in need thereof. The subjects in need of treatment can have or be at risk for developing diabetes, optic nerve atrophy, or a hearing impairment. The subjects in need of treatment also include those diagnosed with Wolfram syndrome, suspected as having Wolfram syndrome, or at risk for developing Wolfram syndrome. The present disclosure contemplates treatment of at least one symptom associated with Wolfram syndrome by administering a bile acid (e.g. TURSO) and a phenylbutyrate compound (e.g., 4-phenylbutyrate or a pharmaceutically acceptable salt thereof, including, e.g., sodium phenylbutyrate).

[0042] Pancreatic beta cell function generally deteriorates over time during natural Wolfram syndrome disease progression. Applicant discovered that administration of a combination of TURSO and sodium phenylbutyrate, for example, is able to improve pancreatic function and glycemic control, which can be assessed using methods known in the art, including via markers of glucose metabolism and various beta cell function tests. Treatment with a combination of TURSO and sodium phenylbutyrate surprisingly resulted in an improved C-peptide response (e.g., higher peak C-peptide levels, higher mean C-peptide levels, shorter time to peak C- peptide levels, and increased Area Under the Curve (AUC) for C-peptide). C-peptide response can be assessed using methods known in the art, including, e.g., the mixed-meal tolerance test (MMTT). Treatment with a combination of TURSO and sodium phenylbutyrate also reduced the subjects’ HbAlc level. Moreover, the time in target glucose range also improved following treatment with a combination of TURSO and sodium phenylbutyrate. Further, Wolfram syndrome is associated with vision loss which deteriorates over time. Surprisingly, Applicant has discovered that a combination treatment of TURSO and sodium phenylbutyrate, for example, is able to improve visual acuity. Additionally, treatment with a combination of TURSO and sodium phenylbutyrate led to improvements in symptom burden and stabilization of the disease, for example, as assessed using questionnaires including, e.g., the Clinician Reported Global Impression of Change (CGIC) or the Patient Reported Global Impression of Change (PGIC), a surprising result given the progressive nature of Wolfram syndrome.

[0043] Also provided herein are methods of treating diabetes, methods of improving pancreatic function, and methods of increasing beta cell function in a subject in need thereof. In some aspects, provided herein are methods of shortening the time to peak C-peptide, increasing the area under the curve (AUC) of C-peptide, and / or increasing the peak C-peptide level in a subject in need thereof. In some aspects, provided herein are methods of decreasing HbAlc in a subject in need thereof. The methods disclosed herein can include administration of a combination of a bile acid (e.g., TURSO) and a phenylbutyrate compound (e.g. sodium phenylbutyrate). Subjects in need of such treatment can be subjects with diabetes or subjects at risk for developing diabetes. Subjects in need of such treatment can also include subjects diagnosed with Wolfram syndrome, suspected as having Wolfram syndrome, or at risk for developing Wolfram syndrome.

[0044] The present disclosure further provides methods of improving visual acuity in a subject in need thereof. Subjects in need of such treatment can also include subjects diagnosed with Wolfram syndrome, suspected as having Wolfram syndrome, or at risk for developing Wolfram syndrome.

[0045] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range 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, and 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.

[0046] Certain ranges are presented herein with numerical values being preceded by the term “about”. The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0047] Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0048] I. Wolfram syndrome

[0049] Wolfram syndrome is a rare genetic disorder that can be caused by pathogenic variants in the WFS1 gene, which encodes the Wolframin protein, or, in a small fraction of patients, pathogenic variants in the CISD2 gene, which encodes CDGSH iron sulfur domain protein 2. Wolfram syndrome is manifested by diabetes insipidus, diabetes mellitus (e.g. juvenile-onset insulin-dependent diabetes), loss of vision (e.g., due to optic nerve atrophy), and progressive neurodegeneration. Many patients also develop other symptoms, ranging from hearing loss, difficulty breathing, and endocrine deficiencies to neurological and psychiatric conditions. Accordingly, recent clinical and genetic findings have revealed that Wolfram syndrome is best characterized as a spectrum disorder. Symptoms associated with Wolfram syndrome include those related to the central nervous system, including, e.g., hearing loss, balance & coordination problems, irregular breathing, difficulty breathing (e.g., due to damage of the brain stem), loss of sense of smell, loss of gag reflex, muscle spasms, seizures, behavior problems, and depression; those related to the gastrointestinal system, including e.g., ulcers (e.g., Wolfram syndrome type 2) and bleeding; those related to the peripheral nervous system, including e.g., peripheral neuropathy; those related to the ophthalmologic system, including e.g., vision loss; those related to the endocrine system, including e.g., insulin shortage (diabetes mellitus), pituitary gland dysfunction

[0050] (diabetes insipidus); and those related to the urogenital system, including e.g., reduced testosterone (males), duct obstruction, disrupted urination, incontinence, and high-capacity atonal bladder.

[0051] Wolfram syndrome is a progressive neurodegenerative disorder in which patients can present with nonautoimmune and non-HLA-linked diabetes mellitus followed by optic atrophy in the first decade; cranial diabetes insipidus and sensorineural deafness in the second decade; renal tract abnormalities early in the third decade; and multiple neurological abnormalities, such as cerebellar ataxia, myoclonus, and psychiatric illness early in the fourth decade. Wolfram syndrome patients usually die from central respiratory failure as a result of brainstem atrophy in their third or fourth decade. A large degree of inter-subject variability exists in the rate of progression. The clinical phenotype of Wolfram syndrome can show resemblance with mitochondrial disorders, such as maternally inherited diabetes and deafness, mitochondrial encephalopathy, mitochondrial myopathy, lactic acidosis and stroke-like episodes, or Leber’s hereditary optic neuropathy.

[0052] The median age of diabetes mellitus onset for patients with Wolfram syndrome is about age 6. Diabetes mellitus is generally managed using treatments known in the art, including, for example insulin, GLP-1R agonists, or metformin. The median age of onset of optic nerve atrophy is around age 11. The median age of onset of deafness is around age 14, which is generally managed using treatments known in the art, including, for example, hearing aids or cochlear implants. The median age of onset of diabetes insipidus is around age 13. Diabetes insipidus is generally managed using treatments known in the art, including Desmopressin (DDAVP). Neurodegeneration in Wolfram syndrome patients generally occurs during the later years of adolescence. In some cases, females have milder symptoms than males.

[0053] Neurogenic bladder is another symptom associated with Wolfram syndrome and is generally managed using treatments known in the art, including urodynamic testing, anticholinergic medications, botox injections, antibiotics, neural stimulator, or catheterization.

[0054] WFS1 variants associated with Wolfram syndrome include missense, nonsense, frameshift, in-frame insertion or deletions, and splice-site variants. Variants of WFS1 are known in the art and described in e.g., van ven Ouweland 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. WFSl / wolframin mutations, Wolfram syndrome, and associated diseases. Human mutation. 2001;17(5):357-67.

[0055] Pathogenic mutations in WFS1 can lead to endoplasmic reticulum (ER) stress and / or impaired mitochondrial dynamics. 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, steroid synthesis, and cell signaling, including apoptotic signaling. Given the vital functions of the ER, its dysfunction can trigger a range of cellular pathologies. Studies have shown that pancreatic beta cells and neurons are particularly sensitive to ER dysfunction, potentially due to high rates of hormone and neurotransmitter synthesis, respectively. WFS1 can regulate Ca2+ homeostasis in the ER, which is crucial in the synthesis and secretion of neurotransmitters and hormones such as insulin. WFS1 deficiency in the ER causes Ca2+ homeostasis disruption, leading to chronic ER stress followed by the unfolded protein response (UPR). WFS1 also negatively regulates ATF6, a UPR molecule, inhibiting hyperactivation of ATF6 and consequent cell apoptosis. Furthermore, WFS1 can impact mitochondrial function by transporting Ca2+ from the ER to the mitochondria via the mitochondria-associated ER membrane (MAM). In Wolfram syndrome, pancreatic P cells and neuronal cells can be lost as a consequence of mutations in the WFS1 gene. In cell and animal models of Wolfram syndrome, WFS1 mutations lead to ER stress, pancreatic P cell dysfunction, and the initiation of ER-associated cell death. In some cases, having one or two missense WFS1 variants is correlated with milder symptoms, as compared to the more severe symptoms seen with two frameshift / nonsense WFS1 variants. One exemplary mutation in the WFS1 gene associated with mild manifestations is C.1672OT (p.Arg558Cys).

[0056] A small portion of patients have mutations in the WFS2 (CISD2) gene. WFS2 also encodes an ER transmembrane protein. In patients with WFS2 mutations, diabetes mellitus and hearing impairment are reported. The clinical phenotype of these patients may differ from patients carrying WFS1 mutations with the absence of diabetes insipidus, the presence of upper intestinal ulcers and bleeding events, and defective platelet aggregation.

[0057] Wolfram syndrome Type 1 is generally associated with two autosomal recessive WFS1 alleles, while Wolfram syndrome Type 2 is generally associated with two autosomal recessive CISD2 alleles.

[0058] The compositions and methods described herein can be useful for treating a subject that exhibits one or more symptoms associated with Wolfram syndrome, or for treating a subject that has been diagnosed with Wolfram syndrome. In some embodiments, the subject is suspected as having Wolfram syndrome, or at risk for developing Wolfram syndrome. The methods described herein can further include determining that a subject has or is at risk for developing Wolfram syndrome, diagnosing a subject as having or at risk for developing Wolfram syndrome, or selecting a subject having or at risk for developing Wolfram syndrome. A number of features, symptoms, and conditions are associated with Wolfram syndrome and can be used for diagnostic purposes. The subject can have or be at risk for developing diabetes mellitus. For example, the subject can have or be at risk for developing juvenile-onset diabetes mellitus (e.g. with an onset age of < 15 years). The subject can have or be at risk for developing optic atrophy (e.g. onset age < 15 years), high-tone sensorineural hearing impairment (e.g., congenital hearing impairment), cerebellar ataxia, autonomic dysfunction, dementia or intellectual disability, psychiatric disease, seizures, neurogenic bladder orbladder dyssynergia, bowel dysfunction, diabetes insipidus (e.g., central diabetes insipidus), delayed / absent puberty, hypogonadism in males, non-autoimmune hypothyroidism, growth retardation, cardiomyopathy and structural congenital heart defects. Methods of detecting the above conditions are known in the art. For example, the subject can be diagnosed based on clinical history, family history, physical or neurological examinations.

[0059] A subject may also be identified as having or at risk for developing Wolfram syndrome based on genetic testing. Genetic testing approaches can include gene-targeted testing (singlegene testing or multigene panel) and comprehensive genomic testing (exome sequencing, exome array, genome sequencing) depending on the phenotype. For example, sequence analysis 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 deafness multigene panel that includes WFS1 and other genes of interest can be performed as described in Tranebjserg et al. (WFS1 Wolfram Syndrome Spectrum Disorder. 2009 Feb 24. In: Adam MP, Everman DB, Mirzaa GM, et al., editors. GeneReviews®. Seattle (WA): University of Washington, Seattle; 1993-2022.) In some embodiments, genetic testing can be used in combination with clinical diagnosis to confirm Wolfram syndrome diagnosis. Mutations in the WFS1 gene can 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(l 1):3943— 50.), the c, 1672C>T, p.R558C mutation, and the p.P885L mutation. Other mutations of WFS1 are known in the art.

[0060] The subject may have shown one or more symptoms of Wolfram syndrome (e.g. any symptoms of Wolfram syndrome described herein or known in the art) for about 1 day to about 40 years (e.g. about 1 to about 6 months, about 7 to about 18 months, or about 2, 3, 4, or 5 years or more). The subject may have been diagnosed with Wolfram syndrome for about 1 day to about 40 years (e.g. about 1 to about 6 months, about 7 to about 18 months, or about 2, 3, 4, or 5 years or more). The subject can be confirmed or identified, e.g. by a healthcare professional, as having Wolfram syndrome. Multiple parties may be included in the process of diagnosis. For example, where samples are obtained from a subject as part of a diagnosis, a first party can obtain a sample from a subject and a second party can test the sample. In some embodiments, the subject is diagnosed, selected, or referred by a medical practitioner (e.g., a general practitioner).

[0061] Skilled practitioners will appreciate that certain factors can affect the bioavailability and metabolism of the administered compounds for a subject and can make adjustments accordingly. These include but are not limited to liver function (e.g. levels of liver enzymes), renal function, and gallbladder function (e.g., ion absorption and secretion, levels of cholesterol transport proteins). There can be variability in the levels of exposure each subject has for the administered compounds (e.g., bile acid and a phenylbutyrate compound), differences in the levels of excretion, and in the pharmacokinetics of the compounds in the subjects being treated. Any of the factors described herein may affect drug exposure by the subject. For instance, decreased clearance of the compounds can result in increased drug exposure, while improved renal function can reduce the actual drug exposure. The extent of drug exposure may be correlated with the subject’s response to the administered compounds and the outcome of the treatment.

[0062] The methods described herein can also be useful for preventative and prophylaxis purposes.

[0063] II. Composition

[0064] The present disclosure provides compositions and methods useful for treating at least one symptom of Wolfram syndrome or slowing, preventing, or reversing Wolfram syndrome progression in a subject. Compositions provided herein include bile acids or pharmaceutically acceptable salts thereof and phenylbutyrate compounds, for example, as a combination treatment administered in the same or separate dosage forms. In some embodiments, the present disclosure contemplates the administration of a composition comprising a taurursodiol (TURSO) and a 4-phenylbutyrate or pharmaceutically acceptable salts thereof (e.g., sodium phenylbutyrate) to a subject in need thereof.

[0065] Bile Acid

[0066] As used herein, “bile acid” refers to naturally occurring surfactants having a nucleus derived from cholanic acid substituted with a 3a-hydroxyl group and optionally with other hydroxyl groups as well, typically at the C6, C7 or C12 position of the sterol nucleus. Bile acid derivatives (e.g., aqueous soluble bile acid derivatives) and bile acids conjugated with an amine are also encompassed by the term “bile acid”. Bile acid derivatives include, but are not limited to, derivatives formed at the carbon atoms to which hydroxyl and carboxylic acid groups of the bile acid are attached with other functional groups, including but not limited to halogens and amino groups. Soluble bile acids may include an aqueous preparation of a free acid form of bile acids combined with one of HC1, phosphoric acid, citric acid, acetic acid, ammonia, or arginine. Suitable bile acids include but are not limited to, taurursodiol (TURSO), ursodeoxycholic acid (UDCA), chenodeoxycholic acid (also referred to as “chenodiol” or “chenic acid”), cholic acid, hyodeoxy cholic acid, deoxy cholic acid, 7-oxolithocholic acid, lithocholic acid, iododeoxycholic acid, iocholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, glycoursodeoxycholic acid, taurocholic acid, glycocholic acid, or an analog, derivative, or prodrug thereof.

[0067] In some embodiments, the bile acids of the present disclosure are hydrophilic bile acids. Hydrophilic bile acids include but are not limited to, TURSO, UDCA, chenodeoxycholic acid, cholic acid, hyodeoxycholic acid, lithocholic acid, and glycoursodeoxycholic acid. Pharmaceutically acceptable salts or solvates of any of the bile acids disclosed herein are also contemplated. In some embodiments, bases commonly employed to form pharmaceutically acceptable salts of the 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; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; tri ethylamine; mono-, bis-, or tris-(2-OH-(Cl-C6)-alkylamine), 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.

[0068] The terms “tauroursodeoxycholic acid” (TUDCA) and “taurursodiol” (TURSO) are used interchangeably herein.

[0069] The bile acid described herein can be TURSO, as shown in formula I (with labeled carbons to assist in understanding where substitutions may be made). In some embodiments, the TURSO is a hydrate, such as TURSO dihydrate.

[0070] I

[0071] The bile acid described herein can be UDCA as shown in formula II (with labeled carbons to assist in understanding where substitutions may be made). or a pharmaceutically acceptable salt thereof.

[0072] Derivatives of bile acids of the present disclosure can be physiologically related bile acid derivatives. For example, any combination of substitutions of hydrogen at position 3 or 7, a shift in the stereochemistry of the hydroxyl group at positions 3 or 7, in the formula of TURSO or UDCA are suitable for use in the present composition.

[0073] The “bile acid” can also be a bile acid conjugated with an amino acid. The amino acid in the conjugate can be, but are not limited to, taurine, glycine, glutamine, asparagine, methionine, or carbocysteine. Other amino acids that can be conjugated with a bile acid 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 P-alanine, and y-aminobutyric acid. One example of such a bile acid is a compound of formula III: wherein

[0074] R is -H or C1-C4 alkyl;

[0075] Ri is -CH2-SO3R3, CH2COOH, or CH2CH2COOH, and R2is -H; or Ri is -COOH and R2is -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; and

[0076] R3 is -H or the residue of an amino acid, or a pharmaceutically acceptable analog, derivative, prodrug thereof, or a mixture thereof. One example of the amino acid is a basic amino acid. Other examples of the amino acid 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 P-alanine, and y-aminobutyric acid.

[0077] Another example of a bile acid of the present disclosure is a compound of formula IV: wherein

[0078] R is -H or C1-C4 alkyl;

[0079] Ri is -CH2-SO3R3, and R2 is -H; or Ri is -COOH and R2is -CH2-CH2-CONH2, -CH2-CONH2, -CH2-CH2-SCH3, or - CH2-S-CH2-COOH; and

[0080] R3 is -H or the residue of a basic amino acid, or a pharmaceutically acceptable analog, derivative, prodrug thereof, or a mixture thereof. Examples of basic amino acids include lysine, histidine, and arginine.

[0081] In some embodiments, the bile acid is TURSO. TURSO is an ambiphilic bile acid and is the taurine conjugate form of UDCA. TURSO recovers mitochondrial bioenergetic deficits through incorporating into the mitochondrial membrane, reducing Bax translocation to the mitochondrial membrane, reducing mitochondrial permeability, and increasing the apoptotic threshold of the cell (Rodrigues et al. Biochemistry 42, 10: 3070-3080, 2003). It is used for the treatment of cholesterol gallstones, where long periods of treatment is generally required (e.g., 1 to 2 years) to obtain complete dissolution. It has been used for the treatment of cholestatic liver diseases including primary cirrhosis, pediatric familial intrahepatic cholestasis and primary sclerosing cholangitis and cholestasis due to cystic fibrosis. TURSO is contraindicated in subjects with biliary tract infections, frequent biliary colic, or in subjects who have trouble absorbing bile acids (e.g. ileal disease or resection). Drug interactions may include with substances that inhibit the absorption of bile acids, such as cholestyramine, and with drugs that increase the elimination of cholesterol in the bile (TURSO reduces biliary cholesterol content). Based on similar physicochemical characteristics, similarities in drug toxicity and interactions exist between TURSO and UDCA. The most common adverse reactions reported with the use of TURSO (>1%) are: abdominal discomfort, abdominal pain, diarrhea, nausea, pruritus, and rash. There are some cases of pruritus and a limited number of cases of elevated liver enzymes.

[0082] In some embodiments, the bile acid is UDCA. UDCA, or ursodiol, has been used for treating gallstones, and is produced and secreted endogenously by the liver as a taurine (TURSO) or glycine (GUDCA) conjugate. Taurine conjugation increases the solubility of UDCA by making it more hydrophilic. TURSO is taken up in the distal ileum under active transport and therefore likely has a slightly a longer dwell time within the intestine than UDCA which is taken up more proximally in the ileum. Ursodiol therapy has not been associated with liver damage. Abnormalities in liver enzymes have not been associated with Actigall® (Ursodiol USP capsules) therapy and, Actigall® has been shown to decrease liver enzyme levels in liver disease. However, subjects given Actigall® should have SGOT (AST) and SGPT (ALT) measured at the initiation of therapy and thereafter as indicated by the particular clinical circumstances. Previous studies have shown that bile acid sequestering agents such as cholestyramine and colestipol may interfere with the action of ursodiol by reducing its absorption. Aluminum-based antacids have been shown to adsorb bile acids in vitro and may be expected to interfere with ursodiol in the same manner as the bile acid sequestering agents. Estrogens, oral contraceptives, and clofibrate (and perhaps other lipid-lowering drugs) increase hepatic cholesterol secretion, and encourage cholesterol gallstone formation and hence may counteract the effectiveness of ursodiol.

[0083] Phenylbutyrate compounds

[0084] Phenylbutyrate compound is defined herein as encompassing phenylbutyrate (a low molecular weight aromatic carboxylic acid) as a free acid (4-phenylbutyrate (4-PBA), 4- phenylbutyric acid, or phenylbutyric acid), and pharmaceutically acceptable salts, co-crystals, polymorphs, hydrates, solvates, conjugates, derivatives or pro-drugs thereof. Phenylbutyrate compounds described herein also encompass analogs of 4-PBA, including but not limited to Glyceryl Tri-(4-phenylbutyrate), phenylacetic acid (which is the active metabolite of PBA), 2- (4-Methoxyphenoxy) acetic acid (2-P0AA-0Me), 2-(4-Nitrophenoxy) acetic acid (2-P0AA- NO2), and 2-(2 -Naphthyloxy) acetic acid (2-NOAA), and their pharmaceutically acceptable salts. Phenylbutyrate compounds also encompass physiologically related 4-PBA species, such as but not limited to any substitutions for Hydrogens with Deuterium in the structure of 4-PBA. Other HD AC2 inhibitors are contemplated herein as substitutes for phenylbutyrate compounds. Physiologically acceptable salts of phenylbutyrate, include, for example sodium, potassium, magnesium or calcium salts. Other example of salts include ammonium, zinc, or lithium salts, or salts of phenylbutyrate with an orgain amine, such as lysine or arginine.

[0085] In some embodiments of any of the methods described herein, the phenylbutyrate compound is sodium phenylbutyrate. Sodium phenylbutyrate has the following formula:

[0086] Phenylbutyrate is a pan-HDAC inhibitor and can ameliorate ER stress through upregulation of the master chaperone regulator DJ-1 and through 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). The large increase in chaperone production reduces activation of canonical ER stress pathways, folds misfolded proteins, and has been shown to increase survival in in vivo models including the G93A SOD1 mouse model of ALS (See e.g., Ryu, H et al. J Neurochem. 93: 1087-1098, 2005).

[0087] Formulation

[0088] Bile acids 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, refer to an amount or a concentration of one or more drugs administered for a period of time (including acute or chronic administration and periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome. The composition can include about 5% to about 15% w / w (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) of TURSO and about 15% to about 45% w / w (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) of sodium phenylbutyrate. In some embodiments, the composition includes about 9.7% w / w of TURSO and 29.2% w / w of sodium phenylbutyrate.

[0089] The sodium phenylbutyrate and TURSO can be present in the composition at a ratio by weight of between about 1 : 1 to about 4: 1 (e.g., about 2: 1 or about 3 : 1). In some embodiments, the ratio between sodium phenylbutyrate and TURSO is about 3: 1. The compositions described herein can include any pharmaceutically acceptable carrier, adjuvant, and / or vehicle. The term “pharmaceutically acceptable carrier or adjuvant” refers to a carrier or adjuvant that may be administered to a patient, together with a compound disclosed herein, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound. As used herein the language “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The pharmaceutical compositions may contain any conventional non-toxic pharmaceutically- acceptable carriers, adjuvants or vehicles. 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.

[0090] Compositions of the present disclosure can include about 8% to about 24% w / w of 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 of dextrates). Both anhydrous and hydrated dextrates are contemplated herein. The dextrates of the present disclosure can include a mixture of saccharides developed from controlled enzymatic hydrolysis of starch. Some embodiments of any of the compositions described herein include hydrated dextrates (e.g., NF grade, obtained from JRS Pharma, Colonial Scientific, or Quadra).

[0091] Compositions of the present disclosure can include about 1% to about 6% w / w of sugar alcohol (e.g., about 2% to about 5%, about 3% to about 4%, or about 3.9% w / w of sugar alcohol). Sugar alcohols can be derived from sugars and contain one hydroxyl group (-OH) attached to each carbon atom. Both disaccharides and monosaccharides can form sugar alcohols. Sugar alcohols can be natural or produced by hydrogenation of sugars. Exemplary sugar alcohols include but are not limited to, sorbitol, xylitol, and mannitol. In some embodiments, the composition comprises about 1% to about 6% w / w (e.g., about 2% to about 5%, about 3% to about 4%, or about 3.9% w / w) of sorbitol.

[0092] Compositions of the present disclosure can include about 22% to about 35% w / w of 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 of maltodextrin). Maltodextrin can form a flexible helix enabling the entrapment of the active ingredients (e.g., any of the phenylbutyrate compounds and bile acids described herein) when solubilized into solution, thereby masking the taste of the active ingredients. Maltodextrin produced from any suitable sources are contemplated herein, including but not limited to, pea, rice, tapioca, com, and potato. In some embodiments, the maltodextrin is pea maltodextrin. In some embodiments, the composition includes about 28.3% w / w of pea maltodextrin. For example, pea maltodextrin obtained from Roquette (KLEPTOSE® LINECAPS) can be used.

[0093] The compositions described herein can further include sugar substitutes (e.g. sucralose). For example, the compositions can include about 0.5% to about 5% w / w of 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 of sucralose). Other sugar substitutes contemplated herein include but are not limited to aspartame, neotame, acesulfame potassium, saccharin, and advantame.

[0094] In some embodiments, the compositions include one or more flavorants. The compositions can include about 2% to about 15% w / w of flavorants (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). Flavorants can include substances that give another substance flavor, or alter the characteristics of a composition by affecting its taste. Flavorants can be used to mask unpleasant tastes without affecting physical and chemical stability, and can be selected based on the taste of the drug to be incorporated. Suitable flavorants include but are not limited to natural flavoring substances, artificial flavoring substances, and imitation flavors. Blends of flavorants can also be used. For example, the compositions described herein can include two or more (e.g., two, three, four, five or more) flavorants. Flavorants can be soluble and stable in water. Selection of suitable flavorants can be based on taste testing. For example, multiple different flavorants can be added to a composition separately, which are subjected to taste testing. Exemplary flavorants 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 include about 0.5% to about 1.5% w / w (e.g., about 1% w / w) of a mixed berry flavor powder and / or about 5% to about 7% w / w (e.g., about 6.3% w / w) of a masking flavor. Suitable masking flavors can be obtained from e.g., Firmenich.

[0095] The compositions described herein can further include silicon dioxide (or silica). Addition of silica to the composition can prevent or reduce agglomeration of the components of the composition. Silica can serve as an anti-caking agent, adsorbent, disintegrant, or glidant. In some embodiments, the compositions described herein include 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). Porous silica may have a higher H2O absorption capacity and / or a higher porosity as compared to fumed silica, at a relative humidity of about 20% or higher (e.g., about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or higher). The porous silica can have an H2O absorption capacity of about 5% to about 40% (e.g. about 20% to about 40%, or about 30% to about 40%) by weight at a relative humidity of about 50%. The porous silica can have a higher porosity at a relative humidity of about 20% or higher (e.g., about 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher) as compared to that of fumed silica. In some embodiments, the porous silica have an average particle size of about 2 pm to about 10 pm (e.g. about 3 pm to about 9 pm, about 4 pm to about 8 pm, about 5 pm to about 8 pm, or about 7.5 pm). In some embodiments, the porous silica have 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 have 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 include about 0.05% to about 2% w / w (e.g., any subranges of this range described herein) of Syloid® 63FP (WR Grace).

[0096] The compositions described herein can further include one or more buffering agents. For example, the compositions can include about 0.5% to about 5% w / w of buffering agents (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 of buffering agents). Buffering agents can include weak acid or base that maintain the acidity or pH of a composition near a chosen value after addition of another acid or base. Suitable buffering agents are known in the art. In some embodiments, the buffering agent in the composition provided herein is a phosphate, such as a sodium phosphate (e.g., sodium phosphate dibasic anhydrous). For example, the composition can include about 1% to about 4% w / w (e.g., about 2.7% w / w) of sodium phosphate dibasic.

[0097] The compositions can also include one or more lubricants. For example, the compositions can include about 0.05% to about 1% w / w of lubricants (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 lubricants). Exemplary lubricants include, but are not limited to sodium stearyl fumarate, magnesium stearate, stearic acid, metallic stearates, talc, waxes and glycerides with high melting temperatures, colloidal silica, polyethylene glycols, alkyl sulphates, glyceryl behenate, and hydrogenated oil. Additional lubricants are known in the art. In some embodiments, the composition includes about 0.05% to about 1% w / w (e.g., any of the subranges of this range described herein) of sodium stearyl fumarate. For example, the composition can include about 0.5% w / w of sodium stearyl fumarate.

[0098] In some embodiments, provided herein are compositions that include about 29.2% w / w of sodium phenylbutyrate, about 9.7% w / w of TURSO, about 15.6% w / w of dextrates, about 3.9% w / w of sorbitol, about 1.9% w / w of sucralose, about 28.3% w / w of maltodextrin, about 7.3% w / w of flavorants, about 0.9% w / w of silicon dioxide, about 2.7% w / w of sodium phosphate (e.g. sodium phosphate dibasic), and about 0.5% w / w of sodium stearyl fumerate.

[0099] In some embodiments, provided herein are compositions that include about 3000 mg of sodium phenylbutyrate, about 1000 mg of TURSO, about 1600 mg of dextrates, about 400 mg of sorbitol, about 200 mg of sucralose, about 97.2 mg of silicon dioxide, about 2916 mg of maltodextrin, about 746 mg of flavorants (e.g. about 102 mg of mixed berry flavor and about 644 mg of masking flavor), about 280 mg of sodium phosphate (e.g. sodium phosphate dibasic), and about 48.6 mg of sodium stearyl fumerate.

[0100] Additional suitable sweeteners or taste masking agents can also be included in the compositions, such as but not limited to, xylose, ribose, glucose, mannose, galactose, fructose, dextrose, sucrose, maltose, steviol glycosides, partially hydrolyzed starch, and corn syrup solid. Water soluble artificial sweeteners are contemplated herein, such as the soluble saccharin salts (e.g., sodium or calcium saccharin salts), cyclamate salts, acesulfam potassium (acesulfame K), and the free acid form of saccharin and aspartame based sweeteners such as L-aspartyl- phenylalanine methyl ester, Alitame® or Neotame®. The amount of sweetener or taste masking agents can vary with the desired amount of sweeteners or taste masking agents selected for a particular final composition.

[0101] Pharmaceutically acceptable binders in addition to those described above are also contemplated. Examples include cellulose derivatives including microcrystalline cellulose, low-substituted hydroxypropyl cellulose (e.g. LH 22, LH 21, LH 20, LH 32, LH 31, LH30); starches, including potato starch; croscarmellose sodium (i.e. cross-linked carboxymethylcellulose sodium salt; e.g. 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®).

[0102] Additional fillers, diluents or binders may be incorporated such as polyols, sucrose, sorbitol, mannitol, Erythritol®, Tagatose®, lactose (e.g., spray-dried lactose, a-lactose, P- lactose, Tabletose®, various grades of Pharmatose®, Microtose or Fast-Floc®), microcrystalline cellulose (e.g., various grades of Avicel®, such as Avicel® PH101, Avicel® PHI 02 or Avicel® PHI 05, Elcema® Pl 00, Emcocel®, Vivacel®, Ming Tai® and Solka- Floc®), hydroxypropylcellulose, L-hydroxypropylcellulose (low-substituted) (e.g. L-HPC- CH31, L-HPC-LH11, LH 22, LH 21, LH 20, LH 32, LH 31, LH30), dextrins, maltodextrins (e.g. Lodex® 5 and Lodex® 10), starches or modified starches (including potato starch, maize starch and rice starch), sodium chloride, sodium phosphate, calcium sulfate, and calcium carbonate.

[0103] Formulations containing the bile acids and phenylbutyrate compounds disclosed herein, e.g., TURSO and sodium phenylbutyrate, include those described in US20210177867, which is incorporated herein by reference in its entirety.

[0104] 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 CDERData Standards Manual, version number 004 (which is available at fda.give / cder / dsm / DRG / drg00301.html).

[0105] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (subcutaneous, intracutaneous, intravenous, intradermal, intramuscular, intra-articular, intraarterial, intrasynovial, intrastemal, intrathecal, intralesional and intracranial injection or infusion techniques), oral (e.g., inhalation or through a feeding tube), transdermal (topical), transmucosal, and rectal administration.

[0106] Pharmaceutical compositions can 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. Pharmaceutical compositions may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxy ethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions. Other commonly used surfactants such as Tweens or Spans and / or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

[0107] The compositions can be orally administered in any orally acceptable dosage form including, but not limited to, powders, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of powders for oral administration, the powders can be substantially dissolved in water prior to administration. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, may be added. For oral administration in a capsule form, useful diluents include lactose and dried com starch. When aqueous suspensions and / or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oily phase is combined with emulsifying and / or suspending agents. If desired, certain sweetening and / or flavoring and / or coloring agents may be added.

[0108] 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, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art.

[0109] In some embodiments, therapeutic compositions disclosed herein can be formulated for sale in the US, imported into the US, and / or exported from the US. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. In some embodiments, the invention provides kits that include the bile acid and phenylbutyrate compounds. The kit may also include instructions for the physician and / or patient, syringes, needles, box, bottles, vials, etc.

[0110] III. Methods of treatment

[0111] The present disclosure provides methods of treating one or more symptoms associated with Wolfram syndrome in a subject in need thereof. Also provided are methods of slowing, preventing, or reversing Wolfram syndrome disease progression in a subject in need thereof. Also provided are methods of increasing C-peptide response in a human subject having Wolfram syndrome. Also provided are methods of improving endocrinological, neurological, and / or ophthalmological function in a human subject having Wolfram syndrome. Also provided are methods of improving overall disease burden of a human subject having Wolfram syndrome. Also provided are methods of increasing survival time of a human subject having Wolfram syndrome. The provided methods include administering to the subject a combination of a bile acid or pharmaceutically acceptable salt there of (e.g. TURSO) and a phenylbutyrate compound (e.g., 4-phenylbutyrate or a pharmaceutically acceptable salt thereof, including, e.g., sodium phenylbutyrate).

[0112] Without being bound by theory, it is believed that the bile acid (e.g., TURSO) and the phenylbutyrate compound (e.g., sodium phenylbutyrate) are able to synergistically rescue ER stress and mitochondrial dysfunction across cell types affected by Wolfram syndrome, including e.g., pancreatic beta cells and neurons.

[0113] The bile acid or a pharmaceutically acceptable salt thereof and the phenylbutyrate compound can be administered separately or concurrently, including as a part of a regimen of treatment. The compounds can be administered daily (e.g. once a day, twice a day, or three times a day or more), weekly, monthly, or quarterly. The compounds can be administered over a period of weeks, months, or years. For example, the compounds can be administered over a period of at least or 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 or about 5 years, or more. The compounds can be administered once a day or twice a day for 60 days or less (e.g., 55 days, 50 days, 45 days, 40 days, 35 days, 30 days or less). Alternatively, the bile acid and phenylbutyrate compound can be administered once a day or twice a day for more than 60 days (e.g., more than 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 180, 200, 250, 300, 400, 500, 600 days).

[0114] In some embodiments, the bile acid is taursodiol (TURSO). TURSO can be administered at 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 to about 4.5, about 1.5 to about 4, about 1.5 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 at 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, about 1.8 to about 2.0 grams). In some embodiments, TURSO is administered at an amount of about 1 gram per day. For example, TURSO can be administered at an amount of about 1 gram once a day. In some embodiments, TURSO is administered at an amount of about 2 grams per day. For example, TURSO can be administered at an amount of about 1 gram twice a day.

[0115] In some embodiments, the phenylbutyrate compound is 4-phenylbutyrate or a pharmaceutically acceptable salt thereof, for example, sodium phenylbutyrate. Sodium phenylbutyrate can be administered at an amount of about 0.5 to about 10 grams 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 6.5, 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, about 8 to about 10 grams per day). In some embodiments, sodium phenylbutyrate is administered at 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 at an amount of about 3 grams per day. For example, sodium phenylbutyrate can be administered at an amount of about 3 grams once a day. In some embodiments, sodium phenylbutyrate is administered at an amount of about 6 grams per day. For example, sodium phenylbutyrate can be administered at an amount of about 3 grams twice a day. In some embodiments, the bile acid and phenylbutyrate compound are administered at a ratio by weight of about 2.5: 1 to about 3.5: 1 (e.g., about 3: 1).

[0116] The methods described herein can include administering about 1 gram of TURSO once a day and about 3 grams of sodium phenylbutyrate once a day, or about 1 gram of TURSO twice a day and about 3 grams of sodium phenylbutyrate twice a day. The methods can include administering about 1 gram of TURSO once a day and about 3 grams of sodium phenylbutyrate once a day 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 a day and about 3 grams of sodium phenylbutyrate twice a day for at least a day (e.g. at least 30, 40, 50, 60, 80, 100, 120, 150, 180, 250, 300, or 400 days). For example, the methods can include administering about 1 gram of TURSO once a day and about 3 grams of sodium phenylbutyrate once a day for about 14-21 days, followed by administering about 1 gram of TURSO twice a day and about 3 grams of sodium phenylbutyrate twice a day.

[0117] In some embodiments, the methods described herein include administering to a subject about 5 mg / kg to about 100 mg / kg of body weight of TURSO (e.g. about 10 to about 50, about 5 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 35, about 35 to about 40, about 40 to about 45, about 45 to about 50, about 50 to about 55, about 55 to about 60, about 60 to about 65, about 65 to about 70, about 70 to about 75, about 75 to about 80, about 80 to about 85, about 85 to about 90, about 90 to about 95, or about 95 to about 100 mg / kg).

[0118] In some embodiments, the methods described herein include administering to a subject about 10 mg / kg to about 400 mg / kg of body weight of sodium phenylbutyrate (e.g., about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 35, about 35 to about 40, about 40 to about 45, about 45 to about 50, about 50 to about 55, about 55 to about 60, about 60 to about 65, about 65 to about 70, about 70 to about 100, about 100 to about 150, about 150 to about 200, about 200 to about 300, or about 300 to about 400 mg / kg).

[0119] 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.

[0120] The compositions and methods described herein can be useful for treating or ameliorating at least one condition or symptom associated with Wolfram syndrome (e.g., any of the symptoms or clinical manifestations associated with Wolfram syndrome disclosed herein or known in the art). For example, administration of a combination of TURSO and 4- phenylbutyrate or a pharmaceutically acceptable salt thereof (e.g., sodium phenylbutyrate) can treat or ameliorate diabetes mellitus, diabetes insipidus, vision loss, hearing loss, neurogenic bladder or other conditions or symptoms associated with Wolfram syndrome. The compositions and methods described herein can be useful for improving pancreatic P-cell function, glycemic control, metabolic function, or visual acuity in a subject in need thereof (e.g., a subject having one of more symptoms of Wolfram syndrome, suspected as having, at risk for, or diagnosed with Wolfram syndrome). The compositions and methods are also useful for improving overall symptom burden in a subject having one or more symptoms of Wolfram syndrome. The compositions and methods disclosed herein can be used for prophylactically treating a subject at risk for developing Wolfram syndrome, as well as subjects who display one or more conditions including, e.g., diabetes insipidus, diabetes mellitus (e.g. juvenile-onset diabetes), optic nerve atrophy, progressive neurodegeneration, hearing loss, endocrine deficiencies and neurological and psychiatric conditions, cerebellar ataxia, autonomic dysfunction, dementia or intellectual disability, psychiatric disease, seizures, neurogenic bladder or bladder dyssynergia, bowel dysfunction, delayed / absent puberty, hypogonadism in males, non-autoimmune hypothyroidism, growth retardation, cardiomyopathy or structural congenital heart defects. In some embodiments, the subject has or is at risk for developing diabetes, for example, insulin-dependent diabetes or juvenile onset diabetes. In some embodiments, the subject has or is at risk for developing optic nerve atrophy or a hearing impairment.

[0121] The compositions and methods disclosed herein are useful for treating subjects with one or more mutations in the WFS1 gene, on one of both alleles. In some embodiments, the subjects has one or more functionally recessive mutations in the WFS1 gene. In some embodiments, the subject has functionally relevant recessive mutations on both alleles of the WFS1 gene. Some embodiments contemplate selecting a subject who has functionally relevant recessive mutations on one or both alleles of the WFS1 gene.

[0122] The compositions and methods disclosed herein are useful for treating subjects with a definitive diagnosis of Wolfram syndrome. For example, the subjects may have functionally relevant recessive mutations on both alleles of the WFS1 gene. In some embodiments, the methods of treating at least one symptom associated with Wolfram syndrome disclosed herein include selecting a subject who has a definitive diagnosis of Wolfram syndrome.

[0123] Mutations in the WFS1 gene are known in the art, including, for example, the C.1672OT, p.R558C mutation or the C.2654OT, p.P885L mutation. Additional mutations in the WFS1 gene include T699M, A716T, V779M, L829P, G831D (See, Hum Mol Genet. 2001 Oct 15; 10(22): 2501-2508.)

[0124] The compositions and methods disclosed herein are also useful for treating subjects with one or more mutations in the CDGSH iron sulfur domain protein 2 (CISD2) gene, on one of both alleles. Some embodiments contemplate selecting a subject who has functionally relevant recessive mutations on one or both alleles of the CISD2 gene.

[0125] The compositions and methods disclosed herein are useful for treating subjects with a stimulated C-peptide level > 0.2 ng / mL (e.g., > 0.5, 1, 1.5, 2, or 5 ng / mL). In some embodiments, the stimulated C-peptide level is determined by MMTT. In some embodiments, the methods of treating at least one symptom of Wolfram syndrome disclosed herein include selecting a subject who has a stimulated C-peptide level > 0.2 ng / mL (e.g., > 0.5, 1, 1.5, 2, or 5 ng / mL), for example, as determined by MMTT.

[0126] Subjects with insulin dependent diabetes mellitus, for example, as a clinical manifestation of Wolfram syndrome, can also be treated with the compositions and methods described herein. In some embodiments, the provided methods further include selecting a subject who has insulin dependent diabetes mellitus.

[0127] In some embodiments, subjects treated with the compositions and methods described herein have not received GLP-1 receptor agonists or GLP-1 agonists. In some embodiments, subjects treated with the compositions and methods described herein do not receive GLP-1 receptor agonists or GLP-1 agonists concurrently with the treatment disclosed herein. The disclosure also contemplates subjects that have received GLP-1 receptor agonists or GLP-1 agonists but is not receiving the same while being treated with the compositions and methods disclosed herein. In some embodiments, the subject is not receiving GLP-1 receptor agonists or GLP-1 agonists for the first week or more (e.g., first 4 weeks, 12 weeks, 32 weeks, or 48 weeks or more) while being treated with the compositions and methods disclosed herein.

[0128] Some embodiments of the provided methods include (a) selecting a subject that meet one or more of the following criteria: (1) has a definitive diagnosis of Wolfram syndrome (e.g., based on functionally relevant recessive mutations on both alleles of the WFS1 gene), (2) has a stimulated C-peptide level of > 0.2 ng / mL (e.g., > 0.5, 1, 1.5, 2, or 5 ng / mL) at baseline, (3) has insulin dependent diabetes mellitus due to Wolfram syndrome; and (b) administering a combination of a bile acid (e.g., TURSO) and a phenylbutyrate compound (e.g., sodium phenylbutyrate) to the subject.

[0129] In some embodiments, administration of the combination of the bile acid compound (e.g. TURSO) and the phenylbutyrate compound (e.g. sodium phenylbutyrate) results in improved treatment of one or more symptoms of Wolfram syndrome as compared to administration of each compound alone. For example, treatment with a combination of TURSO and sodium phenylbutyrate can lead to symptom reduction for the subjects described herein to a greater extent or at a faster rate than each compound administered alone.

[0130] Methods described in the present disclosure can include treatment of Wolfram syndrome per se, as well as treatment for one or more symptoms of Wolfram syndrome. “Treating” Wolfram syndrome does not require 100% abolition of the disease or disease symptoms in the subject. Any relief or reduction in the severity of symptoms or features of the disease is contemplated. “Treating” Wolfram syndrome also refers to a delay in onset of symptoms (e.g., in prophylaxis treatment) or delay in progression of symptoms or the loss of function associated with the disease. “Treating” Wolfram syndrome also refers to eliminating or reducing one or more side effects of a treatment (e.g. those caused by any of the therapeutic agents for treating Wolfram syndrome disclosed herein or known in the art). “Treating” Wolfram syndrome also refers to eliminating or reducing one or more direct or indirect effects of Wolfram syndrome disease progression. The subject may not exhibit signs of Wolfram syndrome but may be at risk for Wolfram syndrome. For instance, the subject may carry mutations in genes associated with Wolfram syndrome, have family history of having Wolfram syndrome. The subject may exhibit early signs of the disease or display symptoms of established or progressive disease. The disclosure contemplates any degree of delay in the onset of symptoms, alleviation of one or more symptoms of the disease, or delay in the progression of any one or more disease symptoms.

[0131] The treatment provided in the present disclosure can be initiated at any stage during disease progression. For example, treatment can be initiated prior to onset (e.g., for subjects at risk for developing Wolfram syndrome), at symptom onset or immediately following detection of symptoms associated with Wolfram syndrome, upon observation of any one or more symptoms that would lead a skilled practitioner to suspect that the subject may be developing Wolfram syndrome. Treatment can also be initiated at later stages. For example, treatment may be initiated at progressive stages of the disease.

[0132] Treatment methods can include a single administration, multiple administrations, and repeating administration as required for the prophylaxis or treatment of Wolfram syndrome, or at least one symptom associated with Wolfram syndrome. The duration of prophylaxis treatment can be a single dosage or the treatment may continue (e.g., multiple dosages), e.g., for years or indefinitely for 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 so as to prevent the disease from occurring or fulminating. In some embodiments treatment methods can include assessing a level of disease in the subject prior to treatment, during treatment, and / or after treatment. The treatment provided herein can be administered one or more times daily, or it can be administered weekly or monthly. In some embodiments, treatment can continue until a decrease in the level of disease in the subject is detected.

[0133] The terms “administer,” “administering,” or “administration” as used herein refers to administering drugs described herein to a subject using any art-known method, e.g., ingesting, injecting, implanting, absorbing, or inhaling, the drug, regardless of form. In some embodiments, one or more of the compounds disclosed herein can be administered to a subject by ingestion orally and / or topically (e.g., nasally). For example, the methods herein include administration of an effective amount of compound or compound composition to achieve the desired or stated effect. Specific dosage and treatment regimens for any particular subject will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the subject's disposition to the disease, condition or symptoms, and the judgment of the treating physician.

[0134] Outcome measurement

[0135] Following administration of the bile acid or a pharmaceutically acceptable salt thereof and the phenylbutyrate compound, the subject can be evaluated to detect, assess, or determine their level of disease (e.g., Wolfram syndrome or diabetes). For example, progression measurements can include measurements of diabetes mellitus severity (e.g., C-peptide response, HbAlc level, time in target glucose range, changes in exogenous insulin dose), visual acuity, and / or overall Wolfram syndrome symptom burden. In some embodiments, treatment can continue until a change (e.g., reduction) in the level of disease in the subject is detected. C-peptide

[0136] In some embodiments, the treatments described herein improve the subject’s beta cell function, which is correlated with diabetes mellitus severity. Beta cell function can be measured using methods known in the art. In some embodiments, beta cell function is assessed by monitoring C-peptide levels. C-peptide is the part of proinsulin which is cleaved prior to cosecretion with insulin from pancreatic beta cells. Produced in equimolar amounts to endogenous insulin, it is not a product of therapeutically administered exogenous insulin and can be used as a measure of insulin secretion and pancreatic beta cell function.

[0137] The degradation rate of C-peptide in the body is slower than that of insulin (half-life of 20-30 min, compared with the half-life of insulin of just 3-5 min), which affords a more stable test window of fluctuating beta cell response. In healthy individuals the plasma concentration of C-peptide in the fasting state is 0.3-0.6 nmol / 1, with a postprandial increase to 1-3 nmol / 1. Half of all insulin secreted by the pancreas is metabolized in the liver by first-pass metabolism, whereas C-peptide has negligible hepatic clearance. C-peptide is cleared in the peripheral circulation at a constant rate, whereas insulin is cleared variably making direct measurement less consistent. In insulin-treated patients with diabetes, measurement of C-peptide also avoids the pitfail of cross-reaction of assay between exogenous and endogenous insulin.

[0138] Recent cross-sectional studies confirmed that C-peptide declines over time after diabetes mellitus onset in Wolfram syndrome. The decline can be related to age of onset with a younger age of onset (e.g., less than 10 years or less than 2 years) resulting in a more rapid C-peptide decline. Lower C-peptide values have been associated with poorer glycemic control and hence increased HbAlc values. C-peptide may also be a predictor of future outcomes independent of HbAlc levels.

[0139] C-peptide levels can be measured using methods known in the art, for example, including those described in Diabetes Ther. 2017 Jun; 8(3): 475-487. Common methods of C- peptide evaluation include, for example, Glucagon stimulation test (GST), Mixed meal tolerance test (MMTT), Oral glucose tolerance test (OGTT), Tolbutamide tolerance test (tCP), fasting C-peptide (fCP), Random non-fasting C-peptide (rCP), Urinary C-peptide creatinine ratio (UCPCR), Urinary C-peptide (UCP), 24 h urinary collection (24 h UCP).

[0140] The MMTT is a physiological stimulation test involving ingestion of a standardized liquid meal followed by timed measurements over the subsequent pre-determined time period. In the MMTT, a weight-based liquid meal, such as Sustacal or Boost, can be ingested over 5 min and timed samples for C-peptide determination can be taken 10 min prior to ingestion (t = -10), at baseline (t = 0), and at various timepoints after, for example, at 15, 30, 60, 90, 120, 180, or 240 minutes (and potentially at additional timepoints). Recent natural history studies suggest that during the first two years after diabetes onset, C-peptide reduces by 0.37 ng / mL per year; and after the first 2 years, C-peptide reduces by about 0.13 ng / mL per year, both as measured by a 30-minute MMTT test (120-min AUC not evaluated). In some embodiments, for non-diabetic individuals, C-peptide peaks approximately 30 minutes after a meal. In some embodiments, for individuals with Wolfram syndrome, C-peptide peaks more slowly than for non-diabetic individuals or individuals not living with Wolfram syndrome.

[0141] Various parameters related to C-peptide level changes in an MMTT test can be used to assess the effect of a treatment on glycemic control, metabolic function, or beta cell function, including, for example, the mean C-peptide, the Area Under the Curve (AUC) of C-peptide, the time to peak C-peptide level, the change from baseline between pre-dose to a specified timepoint of C-peptide (AC-peptide), and the AAC-peptide between timepoints.

[0142] The change in C-peptide (or AC-peptide) in an MMTT test can be determined according to the following formula:

[0143] Change in C-peptide (or AC-peptide) = (C-peptide at specific timepoint in MMTT [e.g., 90 minutes]) - (C-peptide at 0 minutes)

[0144] The change in AC-peptide (or AAC-peptide) between timepoint of interest and the baseline can be determined according to the following formula:

[0145] Change in AC-peptide (or AAC-peptide) = (AC-peptide over MMTT at timepoint of interest [e.g., 24 weeks]) - (AC-peptide over MMTT at baseline).

[0146] In some embodiments, administration of the compositions disclosed herein (e.g., a combination of TURSO and sodium phenylbutyrate) increases a human subject’s C-peptide levels as assessed using a MMTT. An increase in C-peptide level can be observed at various timepoints in an MMTT test compared to pre-treatment levels, e.g. 15 minutes, 30 minutes, 60 minutes, 90 minutes, or 120 minutes. For example, C-peptide levels (e.g., the peak C-peptide level) can be increased after about one day or more (e.g., about 1 week, 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, 44 weeks, or 48 weeks or more) of treatment with the combination of TURSO and sodium phenylbutyrate. C-peptide levels (e.g., the peak C-peptide level) can be increased by at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, or more relative to baseline (e.g., prior to administration of a combination of TURSO and sodium phenylbutyrate.)

[0147] The compositions and methods described herein are useful for reducing the time to peak C-peptide level in a subject in need thereof. In some embodiments, administration of a combination of TURSO and sodium phenylbutyrate shortens the time to peak C-peptide level, as compared to the time to peak C-peptide level pre-treatment. Time to peak C-peptide level can be assessed using methods known in the art, including, e.g., MMTT. A shortened time to peak C-peptide level can be observed, for example, after about 1 day, 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, 44 weeks, 48 weeks, 52 weeks or more of treatment with the compositions and methods disclosed herein. In some embodiments, the time to peak C-peptide level is reduced by about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, or more relative to baseline (e.g., prior to administration of a combination of TURSO and sodium phenylbutyrate.) In some embodiments, time to peak C-peptide level of the human subject is decreased by at least about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or more relative to baseline (e.g., prior to administration of a combination of TURSO and sodium phenylbutyrate.)

[0148] The compositions and methods described herein are useful for increasing the C-peptide AUC (as measured using an MMTT test) in a subject in need thereof. In some embodiments, administration of the compositions disclosed herein (e.g., a combination of TURSO and sodium phenylbutyrate) increases the C-peptide AUC. An increase in the C-peptide AUC can be observed at various timepoints in an MMTT test compared to pre-treatment levels, e.g., at 90 minutes, 120 minutes, and 240 minutes. Increases in the C-peptide AUC can be observed, for example, after about 1 day, 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, 44 weeks, 48 weeks, 52 weeks or more of treatment with the compositions and methods disclosed herein.

[0149] In some embodiments, the C-peptide AUC in a subject that has received a combination of TURSO and sodium phenylbutyrate (e.g., for about 1 to about 144 weeks or more, e.g., for about 12 weeks, 24 weeks, 36 weeks, or 48 weeks, or more) increases by about 15% or more (e.g., by about 18%, 20%, 22%, 25%, 28%, 30%, or more) as compared to pre-treatment levels, when assessed using MMTT. In some embodiments, C-peptide AUC in a subject that has received a combination of TURSO and sodium phenylbutyrate increases by about 1 min*ng / mL, 5 min*ng / mL, 10 min*ng / mL, 15 min*ng / mL, 20 min*ng / mL, 25 min*ng / mL, 30 min*ng / mL, 35 min*ng / mL, 40 min*ng / mL, 45 min*ng / mL, 50 min*ng / mL, 60 min*ng / mL, 70 min*ng / mL, 80 min*ng / mL, 90 min*ng / mL, 100 min*ng / mL, 110 min*ng / mL, 120 min*ng / mL, 130 min*ng / mL, 140 min*ng / mL, 150 min*ng / mL, 160 min*ng / mL, 170 min*ng / mL, 180 min*ng / mL, 190 min*ng / mL, 200 min*ng / mL, or more relative to baseline (e.g., prior to administration of a combination of TURSO and sodium phenylbutyrate.)

[0150] In some embodiments, administration of the compositions disclosed herein (e.g., a combination of TURSO and sodium phenylbutyrate) results in an increase in AC-peptide as assessed by MMTT. For example, administration of the combination for about one week or more (e.g., about 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, 44 weeks, 48 weeks, or more) results in an increase in AC-peptide compared to pre-administration. The increase can be observed at various timepoints in an MMTT test compared to pre-treatment levels, e.g., at 90 minutes, 120 minutes, and 240 minutes. In some embodiments, AC-peptide in a subject that has received a combination of TURSO and sodium phenylbutyrate increases by at least about 0.05 ng / mL, 0.06 ng / mL, 0.07 ng / mL, 0.08 ng / mL, 0.09 ng / mL, 0.1 ng / mL, 0.15 ng / mL, 0.2 ng / mL, 0.25 ng / mL, 0.3 ng / mL, 0.35 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.6 ng / mL, 0.7 ng / mL, 0.8 ng / mL, 0.9 ng / mL, 1.0 ng / mL, 1.5 ng / mL, 2.0 ng / mL, 2.5 ng / mL, 3.0 ng / mL, or more relative to baseline, as assessed at C- peptide peak or a timepoint of an MMTT.

[0151] Total daily insulin dose

[0152] In some embodiments, the compositions and methods disclosed herein lead to the reduction of the total daily insulin dose required by a subject. In some embodiments, the methods include assessing the total daily insulin dose by a subject. In some embodiments, the total daily insulin dose by a subject is decreased following administration of a combination of TURSO and sodium phenylbutyrate (e.g., for about 1 to about 96 weeks or more, e.g., for about 12 weeks, 24 weeks, 36 weeks, or 48 weeks or more). The total daily insulin dose can be patient-reported total daily insulin dose. In some embodiments, total daily insulin dose is relatively consistent week-to-week following administration of a combination of TURSO and sodium phenylbutyrate.

[0153] Continuous glucose monitoring

[0154] Continuous glucose monitoring can be useful to assess the subject’s degree of glycemic control and pancreatic function. The time that a subject’s glucose levels are within a target range (e.g., between about 70 mg / dL and about 180 mg / dL) can be used to evaluate the subject’s glycemic control. For subjects contemplated in this disclosure (e.g., those diagnosed with, suspected as having, or at risk for developing Wolfram syndrome), the time in target glucose range can become progressively difficult to maintain as disease progresses. The compositions and methods described herein can maintain or improve the amount of time that such subjects are within the target glucose range. In some embodiments, the compositions and methods disclosed herein improve the subject’s glycemic control, for example, by increasing the amount of time that the subject’s glucose levels are within the target range. In some embodiments, the target glucose range is between about 70 mg / dL and about 180 mg / dL. As described herein, administration of a combination of TURSO and sodium phenylbutyrate, for example, can increase the overall time in good glucose range (e.g., 70-180 mg / dL), and / or reduce the overall time above range (e.g., > 180 mg / dL).

[0155] In some embodiments, the methods disclosed herein further include tracking the time in good glucose range (70-180 mg / dL), time below range (<70 mg / dL), and / or time above range (>180 mg / dL). Glucose levels can be measured by methods known in the art. In some embodiments, glucose levels are monitored via a continuous glucose monitoring (CGM) device. In some embodiments, time in target glucose range is increased by at least about 1%, 1.5%, 2% , 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, or more relative to baseline (e.g., prior to administration of a combination of TURSO and sodium phenylbutyrate.)

[0156] HbAlc

[0157] Measurement of hemoglobin A1C (HbAlc) levels can also be useful for assessing diabetes mellitus progression. The HbAlc measures glycosylated hemoglobin and provides a sense of glycemic control of diabetes during the previous 2-3 months. HbAlc is inversely correlated to C-peptide, where improved metabolic function is associated with higher C- peptide and lower HbAlc.

[0158] For subjects contemplated in this disclosure (e.g., those diagnosed with, suspected as having, or at risk for developing Wolfram syndrome), HbAlc levels can become progressively difficult to maintain as disease progresses. The compositions and methods described herein can slow down the increase of HbAlc, maintain HbAlc levels, or reduce HbAlC levels in such subjects. In some embodiments, the HbAlc levels of a subject treated with the compositions and methods disclosed herein is reduced compared to pre-treatment HbAlc levels. For example, the HbAlc of a subject that has received a combination of TURSO and sodium phenylbutyrate (e.g., for about 1 to about 144 weeks or more, e.g., for about 12 weeks, about 24 weeks, about 36 weeks, about 48 weeks, or more) is reduced by about 0.05% or more (e.g., about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, or more) relative to baseline (e.g., prior to administration of a combination of TURSO and sodium phenylbutyrate.)

[0159] Visual Acuity

[0160] Wolfram syndrome results in progressive optic nerve atrophy leading to loss of both visual acuity and color vision, eventually leading to blindness. In some aspects, the compositions and methods disclosed herein are useful for improving visual acuity in a subject in need thereof. In some embodiments, the methods disclosed herein include determining the visual acuity for one or both eyes of the subject. Methods of determining visual acuity are known in the art. For example, the best- corrected visual acuity (BCVA) can be measured using the LogMAR scale by sight tests using the Snellen chart. To conduct the LogMAR test, values are taken for each eye after correction, and can range from 0, which represents perfect vision (values of -0.1 and -0.2 are also possible representing better than perfect vision), to +2 which represents near blindness. Increases in LogMAR represent deterioration. Some studies have found that visual acuity decline over time in Wolfram syndrome patients with a mean slope of about 0.059 LogMAR / year. Some patients have been found to exhibit more rapid decline in visual acuity, e.g., at a rate of about 0.16 LogMAR / year.

[0161] In some embodiments, compositions and methods disclosed herein (e.g., a combination of TURSO and sodium phenylbutyrate, e.g., at about 1 gram of TURSO and 3 grams of sodium phenylbutyrate once a day or twice a day) results in improved or stable visual acuity (e.g., BCVA) in a subject in need thereof. Improvement or stabilization of visual acuity can be observed after treatment for about 1 day to about 144 weeks or more (e.g., for about 1 week, 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, 44 weeks, or 48 weeks or more. Improvement in visual acuity can include improved visual perception in one or both eyes for a subject who has limited vision (e.g., blind or legally blind, for example, as a condition associated with Wolfram syndrome). In some embodiments, administration of a combination of TURSO and sodium phenylbutyrate (e.g., for about 1 day, 4 weeks, 12 weeks, 24 weeks, 36 weeks, or 48 weeks or more) results in a mean change (e.g., decrease) from baseline in the BCVA of the subject by at least about 0.01 units (e.g., 0.02, 0.03, 0.04, 0.05, or more units) on the LogMAR scale for one or both eyes (e.g., for the best eye).

[0162] Overall symptom burden

[0163] In addition, measures of overall symptom burden can be used to assess treatment outcomes. For example, Patient-reported global impression of change (PGI-C) and Clinician- reported global impression of change (CGLC), both of which use a 7-point scale to rate the change in symptoms associated with Wolfram syndrome can be useful for assessing benefit from treatment. The 7-point scale include the following: 1 - very much improved; 2 - much improved, 3 - minimally improved; 4 - no change; 5 - minimally worse; 6 - much worse; 7 - very much worse. Responders to the treatment disclosed herein can include those with a 1, 2, 3, or 4 rating in the PGI-C or CGLC 7-point scale.

[0164] In some embodiments, treatment with the compositions and methods disclosed herein improves the symptom burden as reported by patients or physicians. In some embodiments, symptom burden can be assessed using the PGI-C or CGI-C scale. For example, treatment with a combination of TURSO and sodium phenylbutyrate can result in improvement on the PGI-C or CGI-C scale.

[0165] Upon improvement of a patient's condition (e.g., a change (e.g., decrease) in the level of disease in the subject), a maintenance dose of a compound, composition or combination of this disclosure may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.

[0166] The methods described herein can further include administering to the subject one or more additional therapeutic agents, e.g. in amounts effective for treating or achieving a modulation of at least one symptom of Wolfram syndrome. Any Wolfram syndrome therapeutic agents known in the art can be used as an additional therapeutic agent. Exemplary therapeutic agents include valproic acid, glucagon-like peptide (GLP)-l receptor agonists, dantrolene sodium, and ER Ca2+ stabilizer, Sigma-1 Receptor agonists (e.g., pridopidine), and Ibudilast. The compositions and methods disclosed herein can also be used in combine with one or more additional therapies for treating at least one symptom of Wolfram syndrome. The additional therapies include gene therapies that transfer wild type WFS1 alleles, for example, via AAV vectors, or that correct the pathogenic WFS1 variants; introduction of the Mesencephalic astrocyte-derived neurotrophic factor (MANF) using gene therapy techniques; and transplantation of iPSC-derived tissues.

[0167] The bile acid or a pharmaceutically acceptable salt thereof and the phenylbutyrate compound can be administered shortly after a meal (e.g., within two hours of a meal) or under fasting conditions. The subject may have consumed food items (e.g., solid foods or liquid foods) less than 2 hours before administration of a bile acid or a pharmaceutically acceptable salt thereof and / or a phenylbutyrate compound; or will consume food items less than 2 hours after administration of one or both of the compounds. Food items may affect the rate and extent of absorption of the bile acid or a pharmaceutically acceptable salt thereof and / or the phenylbutyrate compound. For instance, food can change the bioavailability of the compounds by delaying gastric emptying, stimulating bile flow, changing gastrointestinal pH, increasing splanchnic blood flow, changing luminal metabolism of the substance, or physically or chemically interacting with a dosage form or the substance. The nutrient and caloric contents of the meal, the meal volume, and the meal temperature can cause physiological changes in the GI tract in a way that affects drug transit time, luminal dissolution, drug permeability, and systemic availability. In general, meals that are high in total calories and fat content are more likely to affect the GI physiology and thereby result in a larger effect on the bioavailability of a drug. The methods provided herein can further include administering to the subject a plurality of food items, for example, less than 2 hours (e.g., less than 1.5 hour, 1 hour, or 0.5 hour) before or after administering the bile acid or a pharmaceutically acceptable salt thereof, and / or the phenylbutyrate compound.

[0168] EXAMPLES

[0169] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.

[0170] Example 1. A Phase II Study of Safety and Efficacy of AMX0035 in Adult Patients with Wolfram Syndrome

[0171] This Example describes the Phase II study design, which is a single-centre, open-label study where up to 12 participants are treated with AMX0035 for up to 144 weeks. The overall study design is shown in FIG. 1.

[0172] The 152-week study consists of a Screening period of up to 4 weeks, a 48-week Open- Label Treatment Period, a 96-week Extension Phase, and a Safety Follow-up visit occurring 4 weeks after the End of Treatment visit. Upon completion of Screening and baseline procedures, eligible participants receive standard of care and AMX0035 at predefined doses.

[0173] Eligible participants were enrolled into the Open-Label Treatment Period of the study on Day 1 and received their first dose of study drug. During the first three weeks of dosing, participants took one sachet of AMX0035 daily, and if tolerated, increase to one sachet twice daily (morning and evening). Participants returned to the study site every approximately twelve weeks for study procedures and assessments as well as blood collection during the Open-Label Treatment Period. During the Treatment Extension Phase, participants come into the clinic every 24 weeks for study procedures and assessments as well as blood collection.

[0174] Study Objectives:

[0175] Primary Objectives • To evaluate the effect of AMX0035 on residual beta cell functions by monitoring C-peptide levels during a 0-240 minutes mixed-meal tolerance test (MMTT)

[0176] • To assess the safety and tolerability of AMX0035 administered orally for up to 144 weeks in adult patients with diabetes mellitus due to Wolfram syndrome

[0177] Secondary Objectives

[0178] • Estimate the treatment effect size of AMX0035 on best-corrected visual acuity for both eyes measured on the LogMAR scale by sight tests in clinic using Snellen chart

[0179] • To evaluate the effect of AMX0035 by tracking changes in total daily insulin dose (with percentage basal versus bolus)

[0180] • To evaluate the effect of AMX0035 by tracking the time in good glucose range (70-180 mg / dL), time below range (54-69 mg / dL), time above range (181-250 mg / dL) measured by continuous glucose monitoring (CGM)

[0181] • To evaluate the effect of AMX0035 by tracking the reduction of 14b Ale

[0182] Exploratory Objectives

[0183] To evaluate the effects of AMX0035 on:

[0184] • Wolfram Unified Rating Scale (WURS)

[0185] • Scale for the assessment and rating of ataxia (SARA)

[0186] • Visual function using Visual Functioning Questionnaire - 25 (VFQ-25)

[0187] • Diabetic measurements

[0188] • Blood biomarker (panel) levels of neurodegeneration and neuroinflammation

[0189] • Changes in retinal pathologies, based on optical coherence tomography (OCT), including OCT-angiography

[0190] • Global Impression Scales, patient reported global impression of change (PGIC) and clinician reported global impression of change (CGIC)

[0191] • Most bothersome symptom (MBS)

[0192] Table 1: Study endpoints.

[0193]

[0194]

[0195] Study Population:

[0196] Inclusion Criteria (To be eligible for entry into the study, participants must have met all of the following criteria): 1. Provides a signed informed consent form (ICF) and has the mental capability to understand the ICF. If participant is unable to sign the ICF, the ICF must be signed by a representative in accordance with local regulatory requirements

[0197] 2. The participant has a definitive diagnosis of Wolfram syndrome, as determined by the following: a. Documented functionally relevant recessive mutations on both alleles of the WFS1 gene based on historical test results (if available) or from a qualified laboratory at Screening.

[0198] 3. A stimulated C-peptide level of >0.2 ng / mL during the Screening Visit

[0199] 4. Insulin dependent diabetes mellitus due to Wolfram syndrome

[0200] 5. At least 17 years of age at the time of written informed consent

[0201] 6. Participant must be willing to wear a CGM device for the duration of the study (e.g., 144 weeks / EOT or until the Safety Follow-up visit)

[0202] 7. Women of child-bearing potential (e.g., not post-menopausal for at least one year or surgically sterile) must agree to use adequate birth control* for the duration of the study and 6 months after last dose of study drug. Women must not be planning to become pregnant for the duration of the study and 6 months after the last dose of study drug

[0203] 8. Men must agree to practice contraception* for the duration of the study and for at least 6 months after the last dose of study drug. Men must not plan to father a child or provide sperm for donation for the duration of the study and 6 months after the last dose of study drug

[0204] * Acceptable birth control methods for use in this study are: a. Hormonal methods, such as birth control pills, patches, injections, vaginal ring, or implants b. Barrier methods (such as a condom or diaphragm) used with a spermicide (a foam, cream, or gel that kills sperm) c. Intrauterine device (IUD) d. Abstinence (no heterosexual sex) e. Unique partner who is surgically sterile (men) or not of childbearing potential (female) Exclusion Criteria

[0205] 1. Clinically significant non- Wolfram related central nervous system (CNS) involvement which is judged by the Investigator to likely interfere with the accurate administration and interpretation of protocol assessments

[0206] 2. Clinically significant unstable medical condition (other than Wolfram syndrome) that would pose a risk to the participant if they were to participate in the study, according to Investigator judgment

[0207] 3. Clinically significant, in the opinion of the Investigator, infection or inflammation at the time of Screening or admission. If infection and inflammation has been cured, participants can be rescreened

[0208] 4. Acute gastrointestinal symptoms (e.g., nausea, vomiting, diarrhea) at the time of Screening or admission

[0209] 5. Presence of pathologies that can alter the enterohepatic circulation of bile acids (e.g., ileal resection and stoma, regional ileitis)

[0210] 6. Presence of unstable psychiatric disease, cognitive impairment, dementia or substance abuse that would impair the ability of the participant to provide informed consent and follow instructions, according to Investigator judgment

[0211] 7. Any major surgery within 4 weeks of Screening

[0212] 8. Unable to comply with the protocol, (e.g., has a clinically relevant medical condition making implementation of the protocol difficult, unstable social situation, known clinically significant psychiatric / behavioral instability, is unable to travel to site as required for study evaluations, or is otherwise unlikely to complete the study), as determined by the Investigator

[0213] 9. History of known allergy to phenylbutyrate (PB) or bile salts

[0214] 10. Abnormal liver function defined as aspartate transaminase (AST) and / or alanine transaminase (ALT) > 3 times the upper limit of the normal (ULN)

[0215] 11. Renal insufficiency as defined by estimated glomerular filtration rate (eGFR) < 60 mL / min / 1.73 m2

[0216] 12. Anemia with hemoglobin (Hgb) concentration < 10.0 g / dL at screening 13. Pregnant women or women currently breastfeeding

[0217] 14. Current biliary disease which may lead to biliary obstruction or impedes biliary flow including active cholecystitis, primary biliary cirrhosis, sclerosing cholangitis, gallbladder cancer, gallbladder polyps, gangrene of the gallbladder, abscess of the gallbladder

[0218] 15. Any History of heart failure per New York Heart Association (NYHA)

[0219] 16. History of or family history of breast and / or ovarian cancer

[0220] 17. Participant under severe salt restriction where the added salt intake due to treatment would put the patient at risk, in the Investigator’s judgment

[0221] 18. Received treatment with any investigational drug or device within the 30 days prior to Screening / study entry

[0222] 19. Received blood product transfusions within 90 days prior to Screening 0. Previous treatment with gene or cellular therapy 1. Evidence of organ dysfunction or any clinically significant deviation from normal in physical examination, vital signs, or clinical laboratory determinations beyond what is consistent with the target population in the opinion of the PI. 2. Clinically significant abnormality on 12-lead ECG prior to study treatment administration, confirmed by repeat. 3. Any history of clinically significant suicidal ideation and / or behavior within 1 year of Screening as determined by the Investigator. 4. Anything that, in the opinion of the Investigator, precludes the participant's full compliance with or completion of the study 5. Currently or previously treated within the last 30 days prior to Screening or planned exposure to any prohibited medications listed below.

[0223] Except for the study drug, any investigational therapy being used or evaluated for the treatment of Wolfram syndrome is prohibited beginning 30 days (or 5 half-lives, whichever is longer) prior to the first dose (or screening, in case same visit) and throughout the trial. Use of any gene or cellular therapy (such as NurOwn, Q-Cells, T regulatory therapy) prior to this trial excludes participants from enrollment. These are also prohibited during the trial. Unless approved by the Investigator in consultation with the Sponsor, participants should not receive the following medications (not a comprehensive list) described in Table 2.

[0224] Table 2. List of Prohibited Medication.

[0225] Study Treatment:

[0226] All participants receive oral AMX0035 treatment. For the first 3 weeks of dosing, participants took 1 sachet daily, and if tolerated, increased to 1 sachet twice daily (morning and evening).

[0227] AMX0035 is supplied by the Sponsor to the site pharmacy as a carton box containing single use sachets. Each AMX0035 sachet contains active ingredients (3 g PB and 1 g taurursodiol [TURSO]) and excipients in a powder formulation. Study drug is mixed with ~1 cup of water and taken orally. Duration of Study and Treatment:

[0228] Treatment lasts up to 144 weeks. The planned overall study duration is up to 152 weeks for participants who complete the study.

[0229] Statistical methods:

[0230] 12 participants enrolled in the open- label treatment period, and AAC-peptide at each visit, change of Week 24 AC-peptide from baseline, and change of Week 24 C-peptide AUCo- 4hrs from baseline were reviewed. No formal statistical hypothesis are tested in this signal finding study. The primary and secondary efficacy endpoints are summarized descriptively.

[0231] The sample size is estimated based on enrollment feasibility with a 15% drop out rate, and the level of precision the study provides around the estimated mean change from baseline in AC-peptide at Week 24 (AAC-peptide).

[0232] Assuming a mean AAC-peptide (baseline to Week 24) of 0.12 ng / mL and a standard deviation of 0.18 ng / mL is observed, the corresponding 95% confidence interval (CI) is (0.006, 0.234). Hence, 12 participants gives enough precision such that the anticipated lower confidence limit is greater than 0 (where AAC-peptide=0 ng / mL suggests no treatment effect, and AAC-peptide>0 ng / mL suggests an improvement).

[0233] The safety and efficacy data is summarized descriptively. A detailed statistical analysis plan (SAP) is written and finalized in advance of database lock. The all-treated population consists of all participants dosed. Both the efficacy and safety analyses are based on the alltreated population.

[0234] Descriptive summaries (n, mean, SD, 95% CI of mean, median, standard error, minimum and maximum) on efficacy endpoints are provided on the all-treated population. The evaluation of safety is performed on the all-treated population. AEs occurring after the start of study drug dosing at baseline are summarized descriptively. All AEs are coded according to system organ class (SOC) and preferred term (PT) using MedDRA. Any AEs of special interest such as hypoglycemic events are separately summarized.

[0235] Laboratory parameters (e.g., chemistry / hematology panel, fasting glucose, urinalysis) change from baseline are summarized by visit. Descriptive statistics denoting the changes from baseline to the Week 144 / End of Treatment (EOT) visits with respect to key laboratory parameters are also provided. Frequencies of high and low laboratory values outside the normal range are displayed, as are shift tables comparing each treatment visit and baseline visit by time point and treatment group.

[0236] Individual Stopping Criteria:

[0237] All AEs, safety laboratory results and use of concomitant medications are monitored closely by the Investigator throughout the study. If any clinically significant laboratory or clinical abnormality occurs, the participants are monitored closely until resolution or clinically stable.

[0238] Management of dose limiting treatment-emergent adverse events (TEAE), intolerable to the participant and possibly related to study drug in the opinion of the Investigator, may be managed by stepwise dose reduction(s) to a lower dose of 1 sachet of study drug per day. If this first level dose reduction does not result in improvement within 7 to 14 days, the dose may be reduced to 1 sachet of study drug once every 2 days. The Investigator may decide at any time to interrupt treatment.

[0239] If a participant demonstrates treatment-emergent signs of neurotoxicity including, but not limited to, vomiting, nausea, headache, dizziness, somnolence, dysgeusia hypoacusis, disorientation, confusion, memory loss, neuropathy, possibly related to study drug in the opinion of the Investigator, the Investigator should consider a dose reduction or interruption. Any dosage adjustment, including the reason for and dates of adjustment, are documented in the source documentation and the electronic case report form (eCRF) Any dose modifications may be discussed with the Medical Monitor.

[0240] The new regimen, reduced dose or interruption, may be maintained for as long as necessary until the event improves. The Investigator may then choose to resume the higher dosage or maintain the participant at a reduced dosage. Any dose interruptions should be discussed with the Medical Monitor.

[0241] Recurrence of the dose-limiting TEAE upon re-introduction of the study drug will result in stopping treatment permanently.

[0242] The following AEs trigger temporary dose interruption:

[0243] • Persistent diarrhea: Persistence of several (>5) loose, watery stools for more than 3 days after the start of the treatment associated with the need of rehydration therapy.

[0244] • Treatment emergent increase in serum creatinine or liver enzymes according to the following guidance: o Confirmed increase > 50% from baseline in serum creatinine o ALT or AST > 8 x ULN o ALT or AST > 5 x ULN for more than 2 weeks o ALT or AST > 3 x ULN and (serum total bilirubin > 2 x ULN or international normalized ratio > 1.5) o ALT or AST > 3 x ULN with the appearance of fatigue, nausea, vomiting, right upper quadrant pain or tenderness, fever, rash, and / or eosinophilia (>5%)

[0245] • Treatment emergent Grade 3 AE per NCI Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 unless otherwise specified.

[0246] STUDY ASSESSMENTS AND PROCEDURES

[0247] Wolfram Syndrome Clinical Features A detailed assessment of Wolfram syndrome signs and symptoms present at Screening were collected and included the following:

[0248] • Neurological examination

[0249] • Ophthalmic examination

[0250] • Physical examination

[0251] Neurological Examination

[0252] A brief standard neurological examination was performed. The neurological exam served as the baseline for clinical assessment. Symptoms identified during the Screening period were not recorded as AEs; however, new symptoms or current symptoms that change in severity or frequency after the first day of study drug will be recorded as AEs. The neurological examination assesses:

[0253] • Mental Status - assessment of orientation, speech, and memory

[0254] • Cranial nerves - assessment of cranial nerves II-XII.

[0255] • Motor system - brief assessment of tone and strength, tremors

[0256] • Sensory system - brief assessment of light touch and temperature sensation

[0257] • Reflexes - assessment of deep tendon reflexes and plantar responses (Babinski sign)

[0258] • Coordination - assessment of upper and lower extremities, including assessment for tremor

[0259] • Gait - assessment of tandem gait (if clinically indicated and safe)

[0260] • Station - assessment of posture and stability as defined by the following:

[0261] If clinically indicated and safe, postural instability may be assessed by determining the impairment of postural reflexes on neurological examination (i.e., retropulsion with or without unaided recovery after a backward pull) in the absence of any other medical cause to explain this impairment (e.g., primary sensory deficit, vestibular dysfunction, pontine infarction, cerebellar syndrome, prominent upper or lower motor neuron signs).

[0262] DIAGNOSTIC TOOLS AND RATING SCALES

[0263] The diagnostic tools and rating scales include the following:

[0264] • 0-240 minutes MMTT (Primary Efficacy Endpoint)

[0265] The MMTT tests measure residual P-cell functions (Buss 1982). The night before the MMTT, the participant receives an evening dose of Lantus insulin and fast from midnight until the test at 8:00 AM. The mixed meal consists of 6 mL / kg (maximum 360 mL) of Boost Original (Societe des Produits Nestle S.A., Vevey, Switzerland).

[0266] The following instructions should be followed:

[0267] • Prior to the MMTT, participants should not take short-acting insulin, short-acting GLP- 1 receptor agonists, metformin, and SGLT inhibitors

[0268] • The timeframe for consumption of Boost Original is within 5 minutes

[0269] • Blood for glucose and C-peptide measurement is be drawn at Times -10, 0, 15, 30, 60, 90, 120, 180, and 240 minutes with ±5 minutes sample collection windows

[0270] • If a participant’s fasting glucose exceeds 11.1 mmol / L, the test is not be performed, but fasting glucose and C-peptide are obtained

[0271] • Participants using continuous subcutaneous insulin infusion (CSII) for their diabetes management should receive appropriate insulin adjustment leading up to the MMTT test

[0272] • Appropriate clinical safety measure should be put in place to ensure safety of the participants during the 0-240 minutes MMTT. o The PI and study staff is responsible for appropriate clinical monitoring over the entire study day duration, this includes safety monitoring (e.g. ketone monitoring) during the MMTT. In the opinion of the PI, appropriate clinical safety measure should be put in place to ensure safety of the participants during the 0-240 minutes MMTT.

[0273] • Diabetic Measurements

[0274] The primary responsibility for diabetes management remains with the treating or referring diabetes care provider, but the Investigator study team will provide close additional support through interaction by phone as needed. Diabetes management is monitored by the Investigator study staff with phone calls between study visits as needed.

[0275] Diabetic measurements include:

[0276] • fasting glucose, fasting proinsulin, AUC C-peptide / AUC-glucose, delta proinsulin

[0277] The diabetic measurements allow for the following:

[0278] • continuous glucose monitoring (CGM) collected through the participants personal CGM device. The study endocrinologist is responsible for extracting data from the personal CGM devices. Time in range based on a set timeframe prior to each visit (standardized period of 2 weeks) is assessed. Baseline is data collected from the current device used prior to first dose.

[0279] • tracking changes in total daily insulin dose. Baseline is data collected from the current device used prior to first dose. For participants self-injecting insulin, self-reported data from medical records is used.

[0280] • tracking the reduction of Hb Al c

[0281] • WURS

[0282] The WURS is a clinical scale to measure disease severity and progression in Wolfram syndrome. The scale consists of three (3) domains:

[0283] • Domain A = Physical - Physician Rated

[0284] • Domain B = Physical - Parent Rated

[0285] • Domain C = Behavioral - Parent Rated.

[0286] The Physical domains are rated 0 - 4, with zero (0) corresponding to the absence of symptoms, and four (4) to the presence of symptoms with the greatest severity. The behavioral domain is rated 0 - 3, with zero (0) corresponding to a normal behavior, and three (3) indicating the presence of a disorder of greater severity.

[0287] • SARA

[0288] The Scale for the Rating and Assessment of Ataxia is an 8-item performance-based scale, yielding a total score 0 (no ataxia) to 40 (most severe ataxia). The scores are based on participant performance of: 1) gait, 2) stance, 3) sitting, 4) speech disturbance, 5) finger chase, 6) nose-finger test, 7) fast alternating hand movements, 8) heel-shin slide.

[0289] • VFQ-25

[0290] The 25-Item National Eye Institute Visual Functioning Questionnaire (VFQ-25) is designed to measure vision-related functioning and the influence of vision-related problems. The VFQ-25 represents 11 vision-related constructs and contains up to 39 items, plus an additional singleitem general health rating question. Scoring involves raw scores being converted to a 100-point scale with higher scores associated with worse performance. CGI-C

[0291] The CGI-C rates improvement by 7 categories: very much improved, much improved, minimally improved, no change, minimally worse, much worse, very much worse. These assessments are administered to the participant by the Site (Study PI).

[0292] • PGI-C

[0293] Participants evaluate the change in their Wolfram syndrome-related symptoms since initiation of study drug by choosing one of seven responses. The PGI-C is a 7-point response scale. The participant is asked by the Investigator or qualified designee to rate their change in status using the following 7-point scale:

[0294] 1 = Very much improved, 2 = Much improved, 3 = Minimally improved, 4 = No change,

[0295] 5 = Minimally worse, 6 = Much worse, 7 = Very much worse.

[0296] The responses of "Very much improved," "Much improved," "Minimally improved" and "No change" on the PGI-C are used to define responders.

[0297] • MBS

[0298] The patient-identified Most Bothersome Symptom (MBS) is identified at Screening, where participants describe the MBS they associate with Wolfram syndrome. At follow-up visits, participants are asked to rate the overall change in that symptom since study initiation, using a 7 -item Likert-type scale ranging from “very much improved” to “very much worse”:

[0299] 1 = Very much improved, 2 = Much improved, 3 = Minimally improved, 4 = No change,

[0300] 5 = Minimally worse, 6 = Much worse, 7 = Very much worse.

[0301] It is administered to the participant by a clinician.

[0302] • OCT

[0303] Optical Coherence Tomography (OCT) is a non-invasive imaging test of the eye. OCT uses light waves to take cross-section pictures of the participants retina for the diagnosis and study of eye disorders. The OCT measurements study also includes angiography and testing for visual acuity. A detailed OCT manual is included as separate document. C-SSRS

[0304] The C-SSRS is a systematically administered instrument developed to track suicidal AEs across a treatment study. The instrument is designed to assess suicidal behavior and ideation, track and assess all suicidal events, as well as the lethality of attempts. Additional features assessed include frequency, duration, controllability, reason for ideation, and deterrents. The C-SSRS is considered a low-burden instrument as it takes less than 5 minutes to administer. It is administered to the participant by the Investigator or qualified designee.

[0305] Any participant noted to have suicidal ideation with plan within the prior month, either via answering "yes" to Questions 4 or 5 to the suicidal ideation portion of the C-SSRS or via clinical interview, is evaluated immediately by the Investigator. The Medical Monitor and Sponsor are also informed. Appropriate steps are taken to protect the participant, including but not limited to possible discontinuation (decided by either the Investigator or Medical Monitor) from the study and referral for appropriate psychiatric care. Any such participant at Screening or on Day 1 was also excluded from the study.

[0306] There are two types of C-SSRS questionnaires administered over the course of this study:

[0307] • C-SSRS - Baseline

[0308] • C-SSRS - Since Last Visit

[0309] The C-SSRS - Baseline questionnaire should cover all suicidal behavior from the participants birth to the baseline visit. The C-SSRS - Since Last Visit questionnaire should cover all suicidal behavior from the time of the most recent C-SSRS questionnaire administration to current day.

[0310] Adverse events

[0311] Summary tables showing the number of participants and percent within each category are generated for each of the following types of AEs:

[0312] • All TEAEs;

[0313] • Serious AEs;

[0314] • Deaths;

[0315] • AEs leading to treatment interruption or treatment discontinuation and / or withdrawal;

[0316] Fatal AEs; • AEs by maximum severity level;

[0317] • Treatment related AEs. Example 2. Results from the Phase II Study of Safety and Efficacy of AMX0035 in Adult Patients with Wolfram Syndrome

[0318] This Example describes results of the Phase II study obtained through Week 48. Table 3 shows the baseline demographic characteristics for the twelve patients enrolled in the Phase II Study of Safety and Efficacy of AMX0035 in Adult Patients with Wolfram Syndrome described in Example 1.

[0319] Table 3. Characteristics of Study Participants. At screening, the median time from Wolfram syndrome diagnosis for the participants was about 5 years (with a range of about 0.4 years to about 15 years). An interim analysis was conducted when eight of the twelve participants completed 24 weeks of treatment. One of the twelve patients in the Intent to Treat (ITT) group was later found to not meet the inclusion / exclusion criteria upon genetic review, as the patient was found to have a pathogenic autosomal recessive mutation on just one of the two alleles and a variant of uncertain significance on the other. Additionally, the participant was within the normal range for C- peptide, glycemic measures, and vision throughout, suggesting lack of typical Wolfram Syndrome phenotype. This participant also discontinued insulin and was switched to oral antidiabetic medication. Therefore, data for the remaining eleven patients with genetically confirmed Wolfram syndrome, or the “Per Protocol” group, was analyzed separately for each analysis, including upon completion of 48 weeks of treatment.

[0320] As noted in Example 1, the effect of AMX0035 on residual beta cell function was assessed by monitoring C-peptide levels during a 240-minute mixed-meal tolerance test (MMTT). A 240-minute MMTT was expected to be a more comprehensive measure and allowed for the evaluation of earlier timepoints, such as the 90-minute MMTT, which has been found to be a highly sensitive and specific measure of peak insulin secretion. The night before each MMTT, each participant received an evening dose of Lantus insulin and were instructed to fast from midnight until the test at 8:00 AM the next day. The mixed meal consisted of 6 mL / kg (maximum 360 mL) of Boost Original (Societe des Produits Nestle S.A., Vevey, Switzerland). Prior to the MMTT, participants were instructed to not take short-acting insulin, short-acting GLP-1 receptor agonists, metformin, and SGLT inhibitors. They were also instructed to consume Boost Original within 5 minutes.

[0321] Blood was drawn for C-peptide measurement at the following time points: 10 minutes prior to consuming Boost Original (-10 minutes), when Boost Original was consumed (0 minutes), 15 minutes after Boost Original was consumed, 30 minutes after Boost Original was consumed, 60 minutes after Boost Original was consumed, 90 minutes after Boost Original was consumed, 120 minutes after Boost Original was consumed, 180 minutes after Boost Original was consumed, and 240 minutes after Boost Original was consumed, with ±5 minutes sample collection windows. As described below, a partial reversal in C-peptide phenotype was observed following treatment for 12, 24, 36, or 48 weeks compared to baseline.

[0322] Mean C-peptide and C-peptide AUC'.

[0323] Mean C-peptide levels decrease with natural Wolfram syndrome disease progression; however, surprisingly, with AMX0035 treatment, an improved C-peptide response to the MMTT was observed. Analyses following AMX0035 treatment for 12, 24, 36, or 48 weeks demonstrated that mean C-peptide levels were increased at various time points during the 240- minute MMTT, in particular, 60, 90, and 120 minutes post-consumption of Boost Original. As shown in FIG. 2A for the Per Protocol group and in FIG. 2B for the ITT group, higher and earlier C-peptide peaks were observed compared to baseline.

[0324] Similarly, with normal disease progression, it was expected that C-peptide AUC would decrease compared to baseline. However, increased C-peptide AUC was observed following AMX0035 treatment. FIG. 3 shows C-peptide AUC change from baseline at the 120-minute timepoint of the MMTT. The 120-minute timepoint was chosen to capture the C-peptide peak, as more time is needed to reach the C-peptide peak for individuals with Wolfram syndrome. As shown in FIG. 3, the Per Protocol group and ITT group exhibited a sustained increase in C-peptide AUC relative to baseline, with the Per Protocol group’s C-peptide AUC change from baseline exceeding that of the ITT group. The change from baseline to Week 48 for C-peptide AUC at 120 minutes was determined to be +34.5 min*ng / mL [standard error (SE): 13.0] for the Per Protocol group (N=10) and +14.2 min*ng / mL [SE 23.4] in the Intent to Treat group (N=l l).

[0325] A C-Peptide:

[0326] Change from baseline in AC-peptide, which provides insight into changes in beta cell responsiveness overtime, was also assessed. Positive values indicate increased responsiveness, while negative values indicate decreased responsiveness.

[0327] AC-peptide was expected to decrease over time in Wolfram syndrome. However, following AMX0035 treatment, the Per Protocol group exhibited an increase in AC-peptide compared to baseline. FIG. 4A shows AC-peptide mean change from baseline at the MMTT peak, and FIG. 4B shows AC-peptide mean change from baseline at the 120-minute MMTT timepoint. These results thus demonstrate an improvement in average beta cell responsiveness in the Per Protocol group at Weeks 12, 24, 36 and 48 compared to that at screening.

[0328] Time to Peak C-Peptide:

[0329] The time to peak C-peptide was expected to increase from baseline with natural Wolfram syndrome disease progression; however, shorter time to peak C-peptide was observed in the majority of participants in this study, suggesting more rapid beta cell response to glucose challenge. The interim analysis showed that, under the MMTT test, the time to peak C-peptide was shortened as early as 12 weeks of treatment, and this effect was sustained for most participants at 24, 36, and 48 weeks on treatment, as shown in FIG. 5. The shorter time to peak C-peptide suggests stable or improved pancreatic function, as the pancreas was able to respond more quickly to a glucose challenge via the MMTT than at screening.

[0330] The C-peptide results discussed above demonstrate that treatment with AMX0035 led to improvement in average beta cell responsiveness in the participants.

[0331] HbAlc: HbAlc is expected to stay stable if blood glucose is well controlled; however, it may get more difficult for levels to remain stable as the disease progresses. Participants’ HbAlc levels were assessed at baseline and after 12, 24, 36 and 48 weeks of treatment. Following AMX0035 treatment, the average HbAlc after treatment for 12 weeks was reduced as compared to that from the baseline, and this reduction was sustained after treatment for 24, 36, and 48 weeks (FIG. 6A). In the full 48-week analysis, the ITT group showed an HblAc mean change from baseline of about -0.25%, and the per protocol group showed an HblAc mean change from baseline of about -0.40%.

[0332] Contrary to the natural history of the disease, AMX0035 led to an improvement in glycemic control across all weeks shown compared to screening. The improved glycemic control was despite consistent insulin use (FIG. 6B). These results suggest that AMX0035 led to improved glycemic control and metabolic function.

[0333] Overall Time in Target Glucose Range:

[0334] As Wolfram syndrome progresses, the time in target glucose range becomes more challenging to control over time. Therefore, changes in the degree of glycemic control were also assessed through monitoring the time in target glucose range via continuous glucose monitoring. All participants had continuous glucose monitoring in the study, allowing for a rigorous measurement of the time in target glucose range. Specifically, the change from baseline to Week 24, Week 36, and Week 48 in the overall time in target glucose range was assessed, which is defined as the percentage of time glucose values are between 70 and 180 mg / dL. Consistent with HbAlc findings, most participants demonstrated increased time in target glucose range from screening to the 48-week timepoint, with the majority meeting goal times in range as defined by recommendations from an international consensus report endorsed by the American Diabetes Association and American Association of Clinical Endocrinologists. As shown in FIG. 7, at Week 48, the absolute time in target glucose range improved on average by about 7.9% in the ITT group and about 9.6% in the Per Protocol group. Overall, the increased time in glucose range was sustained across Weeks 12, 24, 36, and 48. These results suggest that AMX0035 was able to improve pancreatic P-cell function and glycemic control in study participants living with Wolfram syndrome.

[0335] Best Corrected Visual Acuity (BCVA):

[0336] The effects of AMX0035 on best-corrected visual acuity were evaluated in participants that reached Week 24 and 48. Visual acuity was measured for both eyes after correction, using the Snellen chart. LogMAR values range from 0 (perfect vision) to +2 (near blindness). Natural disease progression is expected to result in decreased Best-Corrected Visual Acuity (BCVA); however, treatment with AMX0035 led to improvement in BCVA. As shown in FIG. 8, at Week 24 and Week 48, visual acuity improved compared to screening. Seven of the twelve patients experienced improvement in visual acuity in their best eye. Of the remaining participants, two were stable in their best eye, one was stable in one eye, and two worsened from baseline to Week 24 but stabilized from 24 to 48 weeks. These results are encouraging in view of a recent 10-year analysis of 38 individuals with Wolfram syndrome in which visual acuity decline over time in all patients with a mean slope of 0.059 LogMAR / year.

[0337] Exploratory endpoints

[0338] Wolfram symptom burden was also assessed on a more global, holistic scale. For example, the patient- and clinician-reported global impression of change was used to assess symptom burden; both use a 7-point scale to evaluate the change in Wolfram Syndrome related symptoms since initiating drug. Participants demonstrated improvement under the Patient- Reported Global Impression of Change (PGI-C) and Clinician-Reported Global Impression of Change (CGI-C) scales. The responses of “Very much improved,” “Much improved,” “Minimally improved,” and “No change” on the PGI-C and CGI-C are used to define responders. Given the progressive nature of Wolfram syndrome, even no change in symptom burden is considered a difference from the normal course of the disease. All participants showed disease stability or improvement, as shown in FIG. 9A and FIG. 9B. In on-study qualitative interviews, 9 of 11 interviewed participants reported improvements in at least one Wolfram Syndrome-related symptom, with nearly all nothing these changes were meaningful.

[0339] Moreover, participants reported improvements from baseline in their most bothersome symptom (MBS). As shown in Table 3, at screening, vision impairment was the most frequently reported MBS; other participants listed headaches, diabetes mellitus, and overheating as their MBS. FIG. 10 shows patient descriptions of their MBS relative to baseline following AMX0035 treatment for 24, 36, or 48 weeks. Only one participant reported their MBS as being minimally worse than baseline. The remaining participants reported their MBS as being much improved, minimally improved, or as having no change.

[0340] Participant disease burden was also assessed using the Assessment and Rating of Ataxia (SARA) and Wolfram Unified Rating Scale (WURS) measurements. For both SARA and WURS, lower values are indicative of improvement in participant physical ability (SARA) or disease progression and severity (WURS). Most participants demonstrated stable or decreased SARA scores at Week 48 (Table 4). Similarly, most participants demonstrated stable or decreased WURS scores at Week 48 (Table 5). Table 4. SARA scores per participant at screening and at Week 48.

[0341] Table 5. WURS scores per participant at screening and at Week 48. Furthermore, AMX0035 was found to be well-tolerated by study participants. There were no serious, severe (Grade > 3) or fatal (Grade 5) Treatment Emergent Adverse Events (TEAEs). See summary in Table 6. Most TEAEs did not lead to treatment reduction or interruption, and none led to total treatment discontinuation. Diarrhea was the most common adverse event.

[0342] Table 6. TEAEs observed through Week 48. In summary, AMX0035 demonstrated improvement or stabilization across measures in different organ systems, including pancreatic beta cell function, glycemic control, vision, and overall disease burden. From the perspective of diabetic measures, based on the natural history of the Wolfram syndrome, progressively declining pancreatic function and challenging glycemic control are expected. However, treatment with AMX0035 resulted in partial reversal of diabetic phenotypes such as decreased HbAlc levels and increased time in target glucose range (70-180 mg / dL). Stabilization / improvement in visual acuity and perceived symptom burden were also observed with AMX0035. These results were sustained in the majority of participants following 48 weeks of treatment.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of slowing Wolfram syndrome disease progression in a human subject, the method comprising administering to the human subject a pharmaceutically effective amount of a combination of taurursodiol (TURSO) and sodium phenylbutyrate, to thereby slow Wolfram syndrome progression in the human subject.

2. A method of increasing C-peptide response in a human subject having Wolfram syndrome, the method comprising administering to the human subject a pharmaceutically effective amount of a combination of TURSO and sodium phenylbutyrate, to thereby increase C-peptide response in the human subject.

3. A method of improving endocrinological, neurological, and / or ophthalmological function in a human subject having Wolfram syndrome, the method comprising administering to the human subject a pharmaceutically effective amount of a combination of TURSO and sodium phenylbutyrate, to thereby improve endocrinological, neurological, and / or ophthalmological function in the human subject.

4. A method of improving overall disease burden of a human subject having Wolfram syndrome, the method comprising administering to the subject a combination of TURSO and sodium phenylbutyrate, to thereby improve overall disease burden of the human subject.

5. A method of increasing survival time of a human subject having Wolfram syndrome, the method comprising administering to the human subject a pharmaceutically effective amount of a combination of TURSO and sodium phenylbutyrate, to thereby increase survival time of the human subject.

6. The method of any one of the above claims, wherein the human subject has or is at risk for developing diabetes.

7. The method of claim 6, wherein the diabetes is insulin-dependent diabetes.

8. The method of claim 6, wherein the diabetes is juvenile onset diabetes.

9. The method of any one of the above claims, wherein the human subject has or is at risk for developing optic nerve atrophy.

10. The method of any one of the above claims, wherein the human subject has or is at risk for developing a hearing impairment.

11. The method of any one of the above claims, wherein the human subject has one or more mutations in the WFS1 gene.

12. The method of claim 11, wherein the human subject has the c.1672OT, p.R558C mutation in the WFS1 gene.

13. The method of claim 11, wherein the human subject has the C.2654OT, p.P885L mutation in the WFS1 gene.

14. The method of any one of the above claims, wherein the human subject has one or more mutations in the CISD2 gene.

15. The method of any one of the above claims, wherein the TURSO and the sodium phenylbutyrate are administered once a day or twice a day.

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

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

18. The method of any one of the above claims, wherein the TURSO is administered at an amount of about 0.5 g to about 5 g per day.

19. The method of any one of the above claims, wherein the sodium phenylbutyrate is administered at an amount of about 0.5 g to about 10 g per day.

20. The method of any one of the above claims, comprising administering to the human subject about 1 g of TURSO and about 3 g of sodium phenylbutyrate once a day or twice a day.

21. The method of any one of the above claims, comprising administering to the human subject about 1 g of TURSO once a day and about 3 g of sodium phenylbutyrate once a day for about 14 days or more, followed by administering to the human subject about 1 g of TURSO twice a day and 3 g of sodium phenylbutyrate twice a day.

22. The method of any one of the above claims, wherein the TURSO and the sodium phenylbutyrate are administered orally.

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

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

25. The method of claim 20, wherein the one or more additional therapeutic agents is valproic acid, glucagon-like peptide (GLP)-l receptor agonists, dantrolene sodium, or ER Ca2+ stabilizers.

26. The method of any one of the above claims, wherein peak C-peptide levels of the human subject are increased after a meal relative to baseline.

27. The method of claim 26, wherein the peak C-peptide levels are increased by at least about 5% relative to baseline.

28. The method of claim 27, wherein the peak C-peptide levels are measured with a mixed meal tolerance test (MMTT).

29. The method of claim 28, wherein the MMTT is used to calculate C-peptide area under the curve (AUC), AC -peptide, and / or time to peak C-peptide of the human subject.

30. The method of claim 29, wherein the C-peptide AUC is increased relative to baseline.

31. The method of claim 30, wherein the C-peptide AUC is increased by at least about 10 min*ng / mL relative to baseline when measured at or around peak C-peptide.

32. The method of claim 29, wherein AC-peptide is increased relative to baseline.

33. The method of claim 32, wherein the AC-peptide is increased by at least about 0.08 ng / mL relative to baseline when measured at or around the C-peptide peak.

34. The method of any one of the above claims, wherein time to peak C-peptide of the human subject is shortened relative to baseline.

35. The method of claim 34, wherein the time to peak C-peptide of the human subject is shortened by at least about 10% relative to baseline.

36. The method of any one of the above claims, wherein hemoglobin Ale (HbAlc) levels of the human subject are decreased relative to baseline.

37. The method of claim 36, wherein the HbAlc levels of the human subject are decreased by at least about 0.1% relative to baseline.

38. The method of any one of the above claims, wherein time in target glucose range of the human subject is increased relative to baseline.

39. The method of claim 38, wherein the time in target glucose range of the human subject is increased by at least about 2% relative to baseline.

40. The method of any one of the above claims, wherein the human subject exhibits improved or stabilized best-corrected visual acuity (BCVA).

41. The method of claim 40, wherein BCVA is measured on the LogMAR scale.

42. The method of claim 41, wherein a LogMAR scale reading of the human subject decreases by at least about 0.01 units in one or both eyes.

43. The method of claim 4, wherein overall disease burden is assessed using patient reported global impression of change (PGI-C).

44. The method of claim 4, wherein overall disease burden is assessed using clinician reported global impression of change (CGLC).

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