Methods and compositions for treating amyotrophic lateral sclerosis

Abstract

The disclosure provides a method of treating at least one symptom of ALS in a subject who has received a first dose of a BSEP inhibitor, the method comprising administering to the subject a bile acid or a pharma- ceutically acceptable salt thereof and a phenylbutyric acid compound, monitoring the subject's response to the BSEP inhibitor, and administering a second dose of the BSEP inhibitor, wherein the second dose is less than the first dose.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 17 / 742,707 (filed May 12, 2022), the entire contents of which are incorporated herein by reference.

[0002] Technical Field This disclosure relates generally to compositions and methods for treating amyotrophic lateral sclerosis. [Background technology]

[0003] Amyotrophic lateral sclerosis (ALS) is the most common progressive motor neuron disease. ALS causes progressive degeneration of motor neurons, resulting in rapidly progressive muscle weakness and atrophy, eventually resulting in partial or total paralysis. The median survival from onset of symptoms is 2-3 years, with respiratory failure being the leading cause of death. At present, ALS treatment is centered on symptom management. Currently, only two FDA-approved drugs for ALS are available, riluzole and edaravone. Therefore, improved methods of treating ALS are needed.

[0004] The combined use of different drugs may cause adverse effects because the metabolism and / or excretion of each drug reduces or prevents the metabolism and / or excretion of the other drug, increasing the effective concentration of the drug compared to when it is administered alone. However, patients with neurodegenerative diseases, such as ALS patients, often require treatment with multiple drugs, and as a result, the potential toxicity of drug-drug interactions may have adverse consequences for these patients. Therefore, improved treatment methods that allow the administration of multiple drugs are desired. Summary of the Invention

[0005] The disclosure provides a method of treating at least one symptom of amyotrophic lateral sclerosis (ALS) in a subject, the method comprising: (a) administering to a subject who has received a first dose of an inhibitor of bile salt export pump (BSEP), a composition comprising about 1 g of taurursodiol (TURSO) and about 3 g of sodium phenylbutyrate; (b) determining, or having determined, a first level of serum transaminase and / or bilirubin in a first biological sample from the subject; and (c) administering to the subject a second dose of the inhibitor of BSEP, wherein the second dose is less than the first dose.

[0006] The specification also provides a method of administering TURSO and sodium phenylbutyrate to a subject exhibiting one or more symptoms of ALS, the method comprising: (a) administering to a subject who has received a first dose of an inhibitor of bile salt export pump (BSEP), a composition comprising about 1 gram of taurursodiol (TURSO) and about 3 grams of sodium phenylbutyrate; (b) determining, or having determined, a first level of serum transaminase and / or bilirubin in a first biological sample from the subject; and (c) administering to the subject a second dose of the inhibitor of BSEP, wherein the second dose is less than the first dose.

[0007] In some embodiments, the inhibitor of BSEP is cyclosporine. In some embodiments, the first dose of cyclosporine is about 0.5 to about 15 mg / kg / day.

[0008] In some embodiments, the method further comprises a step (d) of determining, or having determined, a second level of serum transaminase and / or bilirubin in a second biological sample from the subject.

[0009] In some aspects, the second level of serum transaminase and / or bilirubin is less than said first level, hi some aspects, the biological sample is plasma or serum.

[0010] In some embodiments, about 1 to about 2 g (inclusive) of TURSO is administered per day. In some embodiments, about 3 to about 6 g (inclusive) of sodium phenylbutyrate is administered per day. In some embodiments, about 1 g of TURSO is administered once per day. In some embodiments, about 1 g of TURSO is administered twice per day. In some embodiments, about 3 g of sodium phenylbutyrate is administered once per day. In some embodiments, about 3 g of sodium phenylbutyrate is administered twice per day.

[0011] In some embodiments, the composition is administered to the subject orally or through a feeding tube. In some embodiments, the subject has been diagnosed with ALS. In some embodiments, the subject is suspected of having ALS. In some embodiments, the subject is a human.

[0012] Unless otherwise defined, all scientific and technical 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 this invention, suitable methods and materials are described below.

[0013] Certain features of this disclosure are described in the context of separate embodiments for clarity, but it will be understood that they can also be provided in combination in a single embodiment. Conversely, various features of this disclosure are described in the context of a single embodiment for brevity, but they can also be provided separately or in any suitable subcombination. Any combination of the embodiments related to this disclosure is specifically embraced in this disclosure and disclosed in the specification just as if each and every combination were individually expressly disclosed. Furthermore, all subcombinations of the various embodiments and elements thereof are specifically embraced in this disclosure and disclosed in the specification just as if each and every subcombination were individually expressly disclosed herein.

[0014] All publications, patent applications, patents, and other references mentioned in this specification are incorporated by reference in their entirety. In case of conflict, this patent specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the invention will become apparent from the following detailed description and claims. [Brief description of the drawings]

[0015] [Figure 1] Figure 1 shows graphs and tables of the results of the primary and secondary endpoints. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Applicants have discovered that a combination of a bile acid (e.g., taurursodiol (TURSO)) and a phenylbutyric acid compound (e.g., sodium phenylbutyrate) can be used to treat one or more symptoms of ALS. Applicants have also surprisingly found that TURSO and its metabolites, ursodeoxycholic acid and glycoursodeoxycholic acid, are inhibitors of BSEP. Furthermore, it has been surprisingly found that phenylacetic acid, a metabolite of sodium phenylbutyrate, also inhibits BSEP. When an inhibitor of BSEP is administered simultaneously with a composition comprising a bile acid and a phenylbutyric acid compound, a drug-drug interaction can exacerbate the accumulation of conjugated bile salts in the liver, resulting in adverse events. Thus, the present disclosure provides a method of treating at least one symptom of ALS in a subject to which an inhibitor of BSEP is also administered.

[0017] The disclosure provides a method of treating at least one symptom of ALS in a subject, or administering TURSO and sodium phenylbutyrate to a subject exhibiting one or more symptoms of ALS, the method comprising: (a) administering to a subject who has received a first dose of a BSEP inhibitor, an effective amount of a composition comprising about 1 gram of TURSO and about 3 grams of sodium phenylbutyrate; (b) determining, or having determined, a first level of serum transaminase and / or bilirubin in a first biological sample from the subject; and (c) administering to the subject a second dose of the BSEP inhibitor, wherein the second dose is less than the first dose.

[0018] Where ranges are described, it is understood that, to the first decimal place of the unit of the lower limit unless otherwise clear from the context, each intervening value between the upper and lower limits of that range, and any other stated or intervening value in that stated range, is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the scope of this disclosure, provided that any limit in a stated range is specifically excluded. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

[0019] Certain ranges provided in this specification are preceded by the term "about." The term "about" is used in this specification to literally support the exact number that is followed by the term, as well as a number that is close to or approximately the number that is followed by the term. In determining whether a number is close to or approximately a specifically recited number, the unrecited near or approximate number may be a number that provides a substantially equivalent number to the specifically recited number in the context provided.

[0020] Unless otherwise defined, all technical terms, symbols, and other scientific or technical terms used in this specification are intended to have the meanings commonly understood by those of ordinary skill in the art to which this application pertains. In some cases, terms having commonly understood meanings are defined in this specification for clarity and / or ease of reference, and the inclusion of such definitions in this specification should not necessarily be construed as representing a substantial difference with respect to what is commonly understood in the art.

[0021] I. Amyotrophic lateral sclerosis (ALS) The terms "amyotrophic lateral sclerosis" and "ALS" are used interchangeably herein and include all of the classifications of ALS known in the art, including, but not limited to, classical ALS (e.g., ALS affecting both lower and upper motor neurons), primary lateral sclerosis (PLS, e.g., affecting only upper motor neurons), progressive bulbar palsy (PBP or bulbar onset, which is a version of ALS that usually begins with difficulty swallowing, chewing, and speaking), and progressive muscular atrophy (PMA, which usually affects only lower motor neurons). These terms include sporadic and familial (inherited) ALS, any rate of progression of ALS (e.g., rapid, non-indolent, or slowly progressing), and any stage of ALS (e.g., presymptomatic, symptomatic, and end-stage ALS).

[0022] The subject in the methods described herein may exhibit one or more symptoms associated with ALS and may have been diagnosed with ALS, hi some embodiments, the subject may be suspected of having and / or at risk of developing ALS.

[0023] The subject in the methods described herein may exhibit one or more symptoms associated with benign fasciculation syndrome (BFS) or cramp-fasciculation syndrome (CFS).

[0024] Some embodiments of the methods described herein can further include determining that a subject has or is at risk for developing ALS, diagnosing the subject as having or at risk for developing ALS, or selecting a subject as having or at risk for developing ALS. Similarly, some embodiments of the methods described herein can further include determining that a subject has or is at risk for developing benign fasciculation syndrome or fasciculation-fasciculation syndrome, diagnosing the subject as having or at risk for developing BFS or CFS, or selecting a subject as having or at risk for developing BFS or CFS.

[0025] In some embodiments of the methods described herein, the subject has exhibited one or more symptoms of ALS for about 24 months or less (e.g., about 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 month or less, or 1 week or less). In some embodiments, the subject has exhibited one or more symptoms of ALS for about 36 months or less (e.g., about 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, or 25 months or less).

[0026] The order and type of ALS symptoms a subject exhibits may depend on which motor neurons in the body are damaged first, and therefore which muscles in the body are damaged first. For example, bulbar-onset, limb-onset, or respiratory-onset ALS may exhibit similar or different symptoms. In general, ALS symptoms may include muscle weakness or atrophy (e.g., affecting the upper body, lower body, and / or speech), muscle fasciculations (twitching), painful muscle spasms, or stiffness of the affected muscles. Early symptoms of ALS may include arm or leg symptoms, difficulty speaking clearly or swallowing (e.g., bulbar-onset ALS). Other symptoms include loss of tongue mobility, difficulty breathing, shortness of breath or abnormal lung function, difficulty chewing, and / or difficulty walking (e.g., stumbling). The initial manifestation of ALS symptoms in a subject may be respiratory muscle weakness. Such subjects may have a very poor prognosis, with a median survival time of about 2 months from diagnosis in some cases. In some subjects, the time of onset of respiratory muscle weakness can be used as a prognostic factor.

[0027] ALS symptoms can also be classified by the location of the neuronal system that is degenerating, i.e., upper motor neuron or lower motor neuron. Lower motor neuron degeneration is manifested, for example, as a decline or weakness in one or more of the medulla oblongata, cervical spinal cord, thoracic spinal cord, and / or lumbosacral spinal cord regions. Upper motor neuron degeneration includes increased tendon reflexes, spasticity, pseudobulbar features, and worsening of reflexes (hyperreflexia), including Hoffman reflex, extensor plantar response, and hypersensitive gag reflex. Progression of neuronal degeneration or muscle weakness is a hallmark of the disease. Thus, some aspects of this disclosure provide methods of improving at least one symptom of lower motor neuron degeneration, at least one symptom of upper motor neuron degeneration, or at least one symptom each of lower motor neuron degeneration and upper motor neuron degeneration. In some aspects of the methods described herein, the onset of symptoms can be determined based on information from the subject and / or the subject's family. In some aspects, the median time from onset of symptoms to diagnosis is about 12 months.

[0028] In some cases, the subject has been diagnosed with ALS. For example, the subject may have been diagnosed with ALS within about 24 months (e.g., about 23, 22, 21, 20, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 month). For example, the subject may have been diagnosed with ALS within one week, or on the same day that the treatment of the disclosure is administered. The subject may have been diagnosed with ALS for more than about 24 months (e.g., about 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or 80 months). Methods for diagnosing ALS are known in the art. For example, the subject can be diagnosed based on medical history, family history, physical or neurological examination (e.g., signs of lower motor neuron or upper motor neuron degeneration). A subject can be confirmed or identified as having ALS, for example, by a medical professional. Multiple parties may be involved in the diagnostic process. For example, if a sample is obtained from a subject as part of a diagnosis, a first party can obtain the sample from the subject and a second party can test the sample. In some embodiments of human subjects described herein, the subject is diagnosed, selected, or referred by a medical professional (e.g., a general practitioner).

[0029] In some embodiments, the subject meets the El Escorial criteria for probable or definite ALS, i.e., the subject has: Electrophysiological evidence of other disease processes that may explain signs of lower motor neuron (LMN) and / or upper motor neuron (UMN) degeneration; and Neuroimaging evidence of other disease processes that may explain the observed clinical and electrophysiological signs; In addition to the lack of 1. Clinical, electrophysiological or neuropathological evidence of LMN degeneration; 2. Signs of UMN degeneration by clinical examination; and 3. Progressive spread of symptoms within one area or to other areas Shows.

[0030] Under the El Escorial criteria, signs of LMN and UMN degeneration are evaluated in four regions of the central nervous system, including the brainstem, cervical, thoracic and lumbosacral spinal cord. Subjects may be assessed into the following categories: A. Clinically definite ALS, which is defined on the basis of clinical evidence alone by the presence of UMN and LMN signs in three areas; B. Clinically probable ALS, which is defined based on clinical evidence alone, by UMN and LMN signs in at least two areas, with some UMN signs necessarily being more rostral (superior) than the LMN signs. C. Clinically probable ALS, supported by examination, defined as clinical signs of UMN and LMN dysfunction present in only one area, or UMN signs present in only one area and LMN signs as defined by EMG criteria present in at least two of the limbs, and other causes have been excluded by appropriate application of neuroimaging and clinical examination protocols. D. Clinically probable ALS, defined as clinical signs of UMN and LMN dysfunction present simultaneously in only one area or UMN signs present in two or more areas; or LMN signs present more rostral to UMN signs and a clinically probable diagnosis supported by testing.

[0031] In some embodiments, the subject is clinically definite (eg, based on El Escorial criteria) with ALS.

[0032] Subjects can be assessed and / or diagnosed using the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R). The ALSFRS-R is an ordinal rating scale (scored 0-4) used to determine a subject's assessment of their ability and independence in 12 functional activities associated with ALS. The ALSFRS-R score calculated at the time of diagnosis can be compared to scores over time to determine the rate of progression. Changes in ALSFRS-R scores can be correlated with changes in strength over time and can be related to quality of life measures and predicted survival. The ALSFRS-R demonstrates a mean linear slope and can be used as a prognostic indicator (see, e.g., Berry et al. Amyotroph Lateral Scler Frontotemporal Degener 15:1-8, 2014; Traynor et al., Neurology 63:1933-1935, 2004; Simon et al., Ann Neurol 76:643-657, 2014 and Moore et al. Amyotroph Lateral Scler Other Motor Neuron Disord 4:42, 2003).

[0033] In the ALSFRS-R, functions mediated by the cervical spinal cord, trunk, lumbosacral, and respiratory muscles are assessed with three items each. Each item is scored from 0 to 4, with 4 representing no disease involvement and 0 representing maximal involvement. A total is calculated by adding the scores of each item. The total score reflects the impact of ALS and is divided into the following representative categories: >40 (minimal-mild); 39-30 (mild-moderate); <30 (moderate-severe); <20 (advanced disease).

[0034] For example, a subject's ALSFRS-R score (e.g., baseline ALSFRS-R score) can be 40 or greater (e.g., at least 41, 42, 43, 44, 45, 46, 47, or 48), between 30 and 39 (inclusive) (e.g., 31, 32, 33, 34, 35, 36, 37, or 38), or less than 30 (e.g., 21, 22, 23, 24, 25, 26, 27, 28, or 29). In some embodiments of the methods described herein, the subject's ALSFRS-R score (e.g., baseline ALSFRS-R score) is 40 or less (e.g., 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 or less). In some embodiments, the subject's ALSFRS-R score (e.g., baseline ALSFRS-R score) is 20 or less (e.g., 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or less).

[0035] Since ALS is a progressive disease, generally all patients progress over time. However, the rate of progression varies widely between subjects, with some subjects dying or needing respiratory support within a few months, while others have a relatively long survival time. The subjects described herein may suffer from fast-progressing ALS or slow-progressing ALS. The rate of functional decline of subjects with ALS can be measured by the change in ALSFRS-R score per month. For example, the score may decrease by about 1.02 (±2.3) points per month.

[0036] One predictor of progression is the patient's historical disease progression rate (ΔFS), which is: ΔFS = (48-ALSFRS-R score at evaluation) / time from onset to evaluation (months) The ΔFS score represents the reduction in ALSFRS-R score at one month from the onset of symptoms and can be a significant predictor of progression and / or survival in subjects with ALS (see, e.g., Labra et al. J Neurol Neurosurg Psychiatry 87:628-632, 2016 and Kimura et al. Neurology 66:265-267, 2006). The subject's disease progression rate (ΔFS) may be about 0.50 or less (e.g., about 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, or 0.10 or less); about 0.50 to about 1.20 (inclusive) (e.g., about 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00, 1.05, 1.10, or 1.15); or about 1.20 or more (e.g., about 1.25, 1.30, 1.35, 1.40, 1.45, 1.50, 1.55, 1.60, 1.75, 1.80, 1.85, 1.90, 1.95, or 2.00 or more). In some embodiments of the methods described herein, the subject's ALS progression rate (ΔFS) may be about 0.50 or greater (e.g., about 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00, 1.05, 1.10, 1.15, 1.20, 1.25, 1.30, 1.35, 1.40, 1.45, 1.50, 1.55, 1.60, 1.75, 1.80, 1.85, 1.90, 1.95, or 2.00 or greater). However, it should be noted that the ΔFS score is a predictor of patient progression and, once assessed, may under- or over-estimate a patient's progression.

[0037] In some embodiments, the subject experiences an average decrease in ALSFRS-R score of about 0.8 to about 2 points (e.g., about 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9) per month from 3 to 12 months since the initial assessment. In some embodiments, the subject experiences an average decrease in ALSFRS-R score of more than about 1.2 points per month from 3 to 12 months since the initial assessment. The subject may experience an decrease in ALSFRS-R score of at least 3 points (e.g., at least 4, 6, 8, 10, 12, 14, 16, 20, 24, 28, or 32 points) from 3 to 12 months since the initial assessment. In some embodiments, the subject has experienced an average decline in the ALSFRS-R score of about 0.8 to about 2 points (e.g., about 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9 points) per month over the past 3 to 12 months. In some embodiments, the subject has experienced an average decline in the ALSFRS-R score of more than about 1.2 points (e.g., about 1.5, 1.8, 2.0, 2.5, or 3 points) per month over the past 3 to 12 months.

[0038] In some embodiments of the methods described herein, the presence or level of a marker in a sample obtained from a subject may be used to diagnose or prognose ALS, or to track disease activity and treatment response. Suitable samples include, for example, cells, tissues, or bodily fluids (e.g., blood, urine, or cerebrospinal fluid (CSF) samples). For example, the level of phosphorylated neurofilament heavy chain subunit (pNF-H) or neurofilament light chain (NfL) in CSF and / or blood can be used as a biomarker to diagnose or prognose ALS, or to track disease activity or treatment endpoints. pNF-H is a major component of the neuronal cytoskeleton and is released into the CSF and bloodstream upon neuronal injury. The level of pNF-H may correlate with the level of axonal loss and / or the burden of motor neuron dysfunction (see, for example, De Schaepdryver et al. Journal of Neurology, Neurosurgery & Psychiatry 89:367-373, 2018).

[0039] The concentration of pNF-H in the CSF and / or blood of ALS subjects may be significantly increased in the early stages of the disease. Higher levels of pNF-H in plasma, serum and / or CSF may be associated with faster ALS progression (e.g., faster ALSFRS-R decline) and / or shorter survival time. Plasma pNF-H concentrations may be higher in ALS subjects with bulbar onset than in ALS subjects with spinal onset. In some cases, an imbalance between the relative expression levels of neurofilament heavy chain subunits and neurofilament light chain subunits can be used to diagnose, prognose or track disease progression in ALS.

[0040] Methods for detecting pNF-H and NfL (e.g., in cerebrospinal fluid, plasma, or serum) are known in the art and include ELISA and Simoa assays (see, e.g., Shaw et al. Biochemical and Biophysical Research Communications 336:1268-1277, 2005; Ganesalingam et al. Amyotroph Lateral Scler Frontotemporal Degener 14(2):146-9, 2013; De Schaepdryver et al. Annals of Clinical and Translational Neurology 6(10): 1971-1979, 2019; Wilke et al. Clin Chem Lab Med 57(10):1556-1564, 2019; Poesen et al. Front Neurol 9:1167, 2018; Pawlitzki et al. Front. Neurol. 9:1037, 2018; Gille et al. al. Neuropathol Appl Neurobiol 45(3):291-304, 2019), but are not limited thereto. pNF-H detection assays developed and commercialized by EnCor Biotechnology, BioVendor and Millipore-EMD can also be used. Commercial NfL assay kits made by Quanterix and based on Simoa technology can also be used (see, e.g., Thouvenot et al. European Journal of Neurology 27:251-257, 2020). Factors in serum or plasma associated with disease course that affect pNF-H and NfL levels or their detection may differ from those in CSF. Levels of neurofilaments (e.g., pNF-H and / or NfL) in CSF and serum may be correlated (see, e.g., Wilke et al. Clin Chem Lab Med 57(10):1556-1564, 2019).

[0041] A subject described herein has a CSF or blood pNF-H level of about 300 pg / mL or greater (e.g., about 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, , 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 3000, 3200, 3500, 3800, or 4000 pg / mL or more). In some embodiments, the serum pNF-H level may be about 70 to about 1200 pg / mL (e.g., about 70 to about 1000, about 70 to about 800, about 80 to about 600, or about 90 to about 400 pg / mL). In some embodiments, the level of pNF-H in the CSF may be about 1000 to about 5000 pg / mL (eg, about 1500 to about 4000 or about 2000 to about 3000 pg / mL).

[0042] The subject's CSF or blood level of NfL may be about 50 pg / mL or more (e.g., about 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 pg / mL or more). In some embodiments, the NfL level in serum may be about 50 to about 300 pg / mL (e.g., about 50 to about 280, about 50 to about 250, about 50 to about 200, about 50 to about 150, about 50 to about 100, about 100 to about 300, about 100 to about 250, about 100 to about 200, about 100 to about 150, about 150 to about 300, about 150 to about 250, about 150 to about 200, about 200 to about 300, about 200 to about 250, or about 250 to about 300 pg / mL).In some embodiments, the NfL level in the CSF is about 2000 to about 40,000 pg / mL (e.g., about 2000 to about 35,000, about 2000 to about 30,000, about 2000 to about 25,000, about 2000 to about 20,000, about 2000 to about 15,000, about 2000 to about 10,000, about 2000 to about 8000, about 2000 to about 6000, about 2000 to about 4000, about 4000 to about 40,000, about 4000 to about 35,000, about 4000 to about 30,000, about 4000 to about 50,000, about 50 About 25,000, about 4000 to about 20,000, about 4000 to about 15,000, about 4000 to about 10,000, about 4000 to about 8000, about 4000 to about 6000, about 6000 to about 40,000, about 6000 to about 35,000, about 6000 to about 30,000, about 6000 to about 25,000, about 6000 to about 20,000, about 6000 to about 15,000, about 6000 to about 10,000, about 6000 to about 8000, about 8000 to about 40,000, about 8000 to about 35,000 , about 8000 to about 30,000, about 8000 to about 25,000, about 8000 to about 20,000, about 8000 to about 15,000, about 8000 to about 10,000, about 10,000 to about 40,000, about 10,000 to about 35,000, about 10,000 to about 30,000, about 10,000 to about 25,000, about 10,000 to about 20,000, about 10,000 to about 15,000, about 15,000 to about 40,000, about 15,000 to about 35,000, about 15,000 to about 30 ,000, about 15,000 to about 25,000, about 15,000 to about 20,000, about 20,000 to about 40,000, about 20,000 to about 35,000, about 20,000 to about 30,000, about 20,000 to about 25,000, about 25,000 to about 40,000, about 25,000 to about 35,000, about 25,000 to about 30,000, about 30,000 to about 40,000, about 30,000 to about 35,000, or about 35,000 to about 40,000 pg / mL).

[0043] Additional biomarkers useful for diagnosing, prognosing, and monitoring disease progression in ALS are contemplated herein, including, but not limited to, CSF levels of S100-β, cystatin C, and chitotriosidase (CHIT) (see, e.g., Chen et al. BMC Neurol 16:173, 2016). Serum levels of uric acid can be used as a prognostic biomarker for ALS (see, e.g., Atassi et al. Neurology 83(19):1719-1725, 2014). Akt phosphorylation can also be used as a prognostic biomarker for ALS (see, e.g., WO 2012 / 160563). Urinary levels of p75ECD and ketones can be used as biomarkers for diagnosing ALS (see, e.g., Shepheard et al. Neurology 88:1137-1143, 2017). Serum and urinary levels of creatinine can also be used as biomarkers. Other useful blood, CSF, neurophysiological and neuroradiological biomarkers for ALS are described, for example, in Turner et al. Lancet Neurol 8:94-109, 2009. Any of the markers described herein can be used to diagnose a subject as suffering from ALS or to determine that a subject is at risk of developing ALS.

[0044] A subject may also be identified as suffering from or at risk of developing ALS based on genetic analysis. Genetic variants associated with ALS are known in the art (see, for example, Taylor et al. Nature 539:197-206, 2016; Brown and Al-Chalabi N Engl J Med 377:162-72, 2017 and http: / / alsod.iop.kcl.ac.uk). A subject described herein may have a mutation in one or more genes associated with familial and / or sporadic ALS. Representative genes associated with ALS include, but are not limited to, ANG, TARDBP, VCP, VAPB, SQSTM1, DCTN1, FUS, UNC13A, ATXN2, HNRNPA1, CHCHD10, MOBP, C21ORF2, NEK1, TUBA4A, TBK1, MATR3, PFN1, UBQLN2, TAF15, OPTN, TDP-43 and DAO. Further descriptions of genes associated with ALS can be found in Therrien et al. Curr Neurol Neurosci Rep 16:59-71, 2016; Peters et al. J Clin Invest 125:2548, 2015 and Pottier et al. J Neurochem, 138:Suppl 1:32-53, 2016. Genetic variants associated with ALS may affect the rate of ALS progression in a subject, the pharmacokinetics of an administered compound in a subject, and / or the efficacy of an administered compound in a subject.

[0045] The subject may have a mutation in the gene encoding CuZn-superoxide dismutase (SOD1). The mutation makes the SOD1 protein more prone to aggregation, resulting in the accumulation of cellular inclusions containing misfolded SOD1 aggregates (see, e.g., Andersen et al., Nature Reviews Neurology 7:603-615, 2011). Over 100 different mutations in SOD1 have been associated with inherited ALS, many of which result in a single amino acid substitution in the protein. In some embodiments, the SOD1 mutation is A4V (i.e., substitution of alanine for valine at position 4). SOD1 mutations are further described, for example, in Rosen et al. Hum. Mol. Genet. 3, 981-987, 1994 and Rosen et al. Nature 362:59-62, 1993. In some embodiments, the subject has a mutation in the C9ORF72 gene. Repeat expansions in the C9ORF72 gene frequently cause ALS, and both loss of function of C9ORF72 and toxic gain of function of the repeat sequence have been implicated in ALS (see, e.g., Balendra and Isaacs, Nature Reviews Neurology 14:544-558, 2018). The methods described herein can include detecting SOD1 mutations and / or C9ORF72 mutations in a subject prior to administration of bile acid and phenylbutyric acid compounds. Methods for screening for mutations are well known in the art. Suitable methods include, but are not limited to, gene sequencing. See, e.g., Hou et al. Scientific Reports 6:32478, 2016 and Vajda et al. Neurology 88:1-9, 2017.

[0046] Those skilled in the art will appreciate that certain factors can affect the bioavailability and metabolism of a compound administered to a subject and adjustments can be made accordingly. These factors include, but are not limited to, liver function (e.g., liver enzyme levels), kidney function, and gallbladder function (e.g., ion absorption and secretion, cholesterol transport protein levels). There may be variability in the exposure level of each subject to the administered compound (e.g., bile acids and phenylbutyric acid compounds), and there may be differences in the excretion level and pharmacokinetics of the compound in treated subjects. Any of the factors described herein may affect the drug exposure to a subject. For example, a decrease in the clearance of a compound may result in increased drug exposure, while improved renal function may reduce actual drug exposure. The degree of drug exposure may correlate with the subject's response to the administered compound and the endpoint of treatment.

[0047] The subjects may be, for example, over about 18 years of age (e.g., 18-100, 18-90, 18-80, 18-70, 18-60, 18-50, 18-40, 18-30, 18-25, 25-100, 25-90, 25-80, 25-70, 25-60, 25-50, 25-40, 25-30, 30-100, 30-90, 30-80, 30-70, 30-60, 30-70, 30-80, 30-90, 30-60, 30-80, 30-90, 30-60, 30-100, 30-100, 30-90, 30-80, 30-70, 30-60, 30-10 ... The subject's BMI may be about 18.5 to 30 kg / m2. 2 (e.g. 18.5~28, 18.5~26, 18.5~24, 18.5~22, 18.5~20, 20~30, 20~28, 20~26, 20~24, 20~22, 22~30, 22~28, 22~26, 22~24, 24~30, 24~28, 24~26, 26~30, 26~28 or 28~30kg / m 2). Having a mutation in an ALS-associated gene described herein, or displaying a biomarker described herein, may indicate that a subject is at risk for developing ALS. Such subjects can be treated by the methods provided herein for prevention and prophylactic purposes.

[0048] In some embodiments, the subject has one or more symptoms of benign fasciculation syndrome (BFS) or fasciculation-fasciculation syndrome (CFS). BFS and CFS are disorders of peripheral nerve hyperexcitability that can cause fasciculations, fasciculations, pain, fatigue, muscle stiffness, and paresthesia. Methods for identifying subjects with these disorders, such as by clinical examination and electromyograms, are known in the art.

[0049] II. Composition The disclosure provides a method of treating at least one symptom of ALS in a subject, the method comprising administering a bile acid or a pharma- ceutically acceptable salt thereof and a phenylbutyrate compound, hi some aspects, the method comprises administering to the subject a composition comprising TURSO and sodium phenylbutyrate.

[0050] bile acids As used herein, "bile acid" refers to naturally occurring surfactants in which a nucleus derived from cholanic acid is substituted with a 3α-hydroxyl group, usually at the C6, C7 or C12 position of the sterol nucleus, and optionally with other hydroxyl groups. Bile acid derivatives (e.g., water-soluble bile acid derivatives) and bile acids conjugated with amines are also encompassed by the term "bile acid". Bile acid derivatives include, but are not limited to, derivatives formed at carbon atoms where the hydroxyl and carboxylic acid groups of the bile acid are bonded to other functional groups, including, but not limited to, halogens and amino groups. Soluble bile acids may include aqueous preparations of the free acid form of the bile acid in combination with hydrochloric acid, 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 "enoic acid"), cholic acid, hyodeoxycholic acid, deoxycholic acid, 7-oxolithocholic acid, lithocholic acid, iododeoxycholic acid, iocholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, glycoursodeoxycholic acid, taurocholic acid, glycocholic acid, or analogues, derivatives or prodrugs thereof.

[0051] In some embodiments, the bile acid disclosed herein is a hydrophilic bile acid. 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 the bile acids disclosed herein are also contemplated. In some aspects, bases commonly utilized to form pharma- ceutically acceptable salts of the bile acids disclosed herein 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; organic amines such as ammonia, unsubstituted or hydroxyl-substituted mono-, di-, or trialkylamines, dicyclohexylamine; tributylamine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-(C1-C6)-alkylamines such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; morpholine; thiomorpholine; piperidine; pyrrolidine; and amino acids such as arginine, lysine, and the like.

[0052] The terms "tauroursodeoxycholic acid" (TUDCA) and "taurursodiol" (TURSO) are used interchangeably in this specification.

[0053] The bile acids described herein have the formula I (with carbons numbered to aid in understanding the positions at which they may be substituted):

[0054] [ka]

[0055] The compound may be TURSO as shown below or a pharma- ceutically acceptable salt thereof.

[0056] The bile acids described herein have the formula II (with carbons numbered to aid in understanding the positions at which they may be substituted):

[0057] [ka]

[0058] The compound may be UDCA as shown in the following formula (I) or a pharma- ceutically acceptable salt thereof.

[0059] The bile acid derivatives disclosed herein may be physiologically relevant bile acid derivatives, for example, combinations of substitution of hydrogen at the 3- or 7-position in the TURSO or UDCA formula, i.e., changes in the stereochemistry of the hydroxyl group at the 3- or 7-position, are suitable for use in the compositions of the present invention.

[0060] A "bile acid" can also be a bile acid conjugated with an amino acid. The amino acid in the conjugate can be, but is not limited to, taurine, glycine, glutamine, asparagine, methionine, or carbocysteine. Other amino acids that can be conjugated with the bile acids disclosed herein include arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, cysteine, proline, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, and tryptophan, as well as β-alanine and γ-aminobutyric acid. One example of such a bile acid is represented by formula III:

[0061] [ka]

[0062] (In the formula, R is -H or C 1 ~C 4 is alkyl; R 1 Ha-CH 2 -SO 3 R 3 , C.H. 2 COOH or CH 2 CH 2 COOH, R 2 is -H; R1 is -COOH, R 2 Ha-CH 2 -CH 2 -CONH 2 , -CH 2 -CONH 2 , -CH 2 -CH 2 -SCH 3 , C.H. 2 CH 2 CH 2 NH(C=NH)NH 2 , C.H. 2 (Imidazolyl), CH 2 CH 2 CH 2 CH 2 NH 2 , C.H. 2 COOH, CH 2 CH 2 COOH, CH 2 OH, CH(OH)CH 3 , C.H. 2 SH, pyrrolidin-2-yl, CH 3 , 2-propyl, 2-butyl, 2-methylbutyl, CH 2 (phenyl), CH 2 (4-OH-phenyl) or -CH 2 -S-CH 2 -COOH; R 3 is -H or an amino acid residue) or a pharma- ceutically acceptable analog, derivative, prodrug, or mixture thereof. An example of an amino acid is a basic amino acid. Other examples of amino acids include glycine, glutamine, asparagine, methionine, carbocysteine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, cysteine, proline, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, and tryptophan, as well as β-alanine and γ-aminobutyric acid.

[0063] Another example of a bile acid disclosed herein has formula IV:

[0064] [ka]

[0065] (In the formula, R is -H or C 1 ~C 4 is alkyl; R 1 Ha-CH 2 -SO 3 R 3 So, R 2 is -H; R 1 is -COOH, R 2 Ha-CH 2 -CH 2 -CONH 2 , -CH 2 -CONH 2 , -CH 2 -CH 2 -SCH 3 or -CH 2 -S-CH 2 -COOH; R 3 is -H or a residue of a basic amino acid) or a pharma- ceutically acceptable analog, derivative, prodrug, or mixture thereof. Examples of basic amino acids include lysine, histidine, and arginine.

[0066] In some embodiments, the bile acid is TURSO. TURSO is an amphipathic bile acid and a taurine-conjugated form of UDCA. TURSO restores mitochondrial bioenergetic defects by incorporating into mitochondrial membranes, decreasing Bax translocation to the mitochondrial membrane, decreasing mitochondrial permeability, and increasing the apoptotic threshold of cells (Rodrigues et al. Biochemistry 42, 10: 3070-3080, 2003). It is used for the treatment of cholesterol gallstones, which generally require long periods of treatment (e.g., 1-2 years) to completely dissolve. It is used to treat cholestatic liver disease, including primary cirrhosis, pediatric familial intrahepatic cholestasis, and cholestasis due to primary sclerosing cholangitis and cystic fibrosis. TURSO is contraindicated in subjects who exhibit biliary tract infections, frequent biliary colic, or who have difficulty absorbing bile acids (e.g., ileal disease or ileal resection). Drug interactions may include those with substances that inhibit the absorption of bile acids, such as cholestyramine, and with drugs that increase the clearance of cholesterol in the bile (TURSO reduces bile cholesterol content). Similarities in drug toxicity and interactions exist between TURSO and UDCA due to similar physicochemical characteristics. The most common adverse reactions reported with the use of TURSO (1% or greater) are abdominal discomfort, abdominal pain, diarrhea, nausea, pruritus, and rash. There have been some cases of pruritus and a limited number of cases of liver enzyme elevations.

[0067] In some embodiments, the bile acid is UDCA. UDCA or ursodiol is used to treat gallstones and is endogenously produced and secreted by the liver as a taurine (TURSO) or glycine (GUDCA) conjugate. Taurine conjugation increases UDCA's solubility by making it hydrophilic. TURSO is taken up in the distal ileum during active transport and therefore likely has a slightly longer intestinal residence time than UDCA, which is taken up in the proximal ileum. Ursodiol therapy is not associated with liver damage. Liver enzyme abnormalities are not associated with Actigall® (Ursodiol USP Capsules) therapy, and Actigall® has been shown to reduce liver enzyme levels in liver disease. However, subjects receiving Actigall® should have SGOT (AST) and SGPT (ALT) measured at the beginning of therapy and thereafter as required by the specific clinical situation. Previous studies have shown that bile acid sequestrants 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 would be expected to interfere with ursodiol in a similar manner to the bile acid sequestrants. Estrogens, oral contraceptives, and clofibrate (and possibly other lipid-lowering drugs) increase hepatic cholesterol secretion and promote cholesterol gallstone formation, and thus may counteract the efficacy of ursodiol.

[0068] Phenylbutyric Acid Compounds Phenylbutyric acid compounds are defined herein to include phenylbutyric acid (low molecular weight aromatic carboxylic acid) as the free acid (4-phenylbutyric acid (4-PBA), 4-phenylbutyric acid or phenylbutyric acid) and its pharma- ceutically acceptable salts, co-crystals, polymorphs, hydrates, solvates, conjugates, derivatives or prodrugs. Phenylbutyric acid compounds described herein include tri-(4-phenylbutyric acid) glycerol, phenylacetic acid (which is the active metabolite of PBA), 2-(4-methoxyphenoxy)acetic acid (2-POAA-OMe), 2-(4-nitrophenoxy)acetic acid (2-POAA-NO 2) and 2-(2-naphthyloxy)acetic acid (2-NOAA), and pharma- ceutically acceptable salts thereof. Phenylbutyric acid compounds also include physiologically relevant species of 4-PBA, such as, but not limited to, substitutions of deuterium for hydrogen in the structure of 4-PBA. Other HDAC2 inhibitors are contemplated herein as alternatives to phenylbutyric acid compounds.

[0069] Physiologically acceptable salts of phenylbutyric acid include, for example, the sodium, potassium, magnesium or calcium salts. Other examples of salts include ammonium, zinc or lithium salts or salts of phenylbutyric acid with organic amines such as lysine or arginine.

[0070] In some embodiments of the methods described herein, the phenylbutyric acid compound is sodium phenylbutyrate. Sodium phenylbutyrate has the following formula:

[0071] [ka]

[0072] It is expressed as:

[0073] Phenylbutyrate is a pan-HDAC inhibitor that can ameliorate ER stress through upregulation of the master chaperone regulator DJ-1 and recruitment of other chaperone proteins (see, e.g., Zhou et al. J Biol Chem. 286: 14941-14951, 2011 and Suaud et al. JBC. 286:21239-21253, 2011). A large increase in chaperone production has been shown to reduce activation of the classical ER stress pathway, refold misfolded proteins, and extend 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).

[0074] In some aspects, a combination of a bile acid (e.g., TURSO) or a pharma- ceutically acceptable salt thereof and a phenylbutyric acid compound (e.g., sodium phenylbutyrate) when dosed in a particular ratio (e.g., as described herein) is synergistically effective in treating one or more symptoms associated with ALS. This combination has been shown to be effective in treating one or more symptoms associated with ALS, e.g., through simultaneous inhibition of endoplasmic reticulum stress and mitochondrial stress, as demonstrated by a powerful oxidative injury model (HIL) by linear modeling. 2 O 2 In some cases, the inhibitors can induce a mathematically synergistic increase in neuronal survival in a number of different neuronal cell types (e.g., neuroprotection-mediated toxicity) (see, e.g., U.S. Pat. Nos. 9,872,865 and 10,251,896).

[0075] formulation The bile acid and phenylbutyric acid compounds described herein can be formulated as or for use in pharmaceutical compositions. For example, the methods described herein can include administering an effective amount of a composition comprising TURSO and sodium phenylbutyrate. The term "effective amount," as used herein, refers to an amount or concentration of one or more drugs effective for administration to cause a desired effect or physiological outcome over a period of time (including acute or chronic administration and regular or continuous administration). 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) 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) sodium phenylbutyrate. In some embodiments, the composition includes about 9.7 w / w% TURSO and 29.2 w / w% sodium phenylbutyrate.

[0076] The sodium phenylbutyrate and TURSO can be present in the composition in a weight ratio of about 1:1 to about 4:1 (e.g., about 2:1 or about 3:1). In some embodiments, the ratio of sodium phenylbutyrate to TURSO is about 3:1.

[0077] The compositions described herein may include pharmaceutically acceptable carriers, adjuvants and / or vehicles. The term "pharmaceutically acceptable carriers or adjuvants" refers to carriers or adjuvants that may be administered to a patient together with the compounds disclosed herein, that do not destroy the pharmacological activity thereof, and that are non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the compound. In this specification, the term "pharmaceutically acceptable carriers" includes saline, solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with the administration of a drug. A pharmaceutical composition may have a conventional non-toxic, pharmaceutically acceptable carrier, adjuvant or vehicle. In some cases, the pH of the formulation may be adjusted with a pharmaceutically acceptable acid, base or buffer to enhance the stability of the compound to be formulated or its delivery form.

[0078] The compositions disclosed herein can include about 8 to about 24% w / w dextrates (e.g., about 9% to about 23%, about 10% to about 22%, about 10% to about 20%, about 11% to about 21%, about 12% to about 20%, about 13% to about 19%, about 14% to about 18%, about 14% to about 17%, about 15% to about 16%, or about 15.6% w / w dextrates). Anhydrous and hydrated dextrates are contemplated herein. Dextrates disclosed herein can include a mixture of sugars resulting from the controlled enzymatic hydrolysis of starch. Some embodiments of the compositions described herein include hydrated dextrates (e.g., NF grades, available from JRS Pharma, Colonial Scientific, or Quadra).

[0079] The compositions disclosed herein can include about 1 to about 6 w / w% sugar alcohol (e.g., about 2% to about 5%, about 3% to about 4%, or about 3.9% w / w sugar alcohol). Sugar alcohols are derived from sugars and can contain one hydroxyl group (-OH) attached to each carbon atom. 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 compositions include 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) sorbitol.

[0080] The compositions disclosed herein can include about 22 to about 35 w / w% maltodextrin (e.g., about 22% to about 33%, about 24% to about 31%, about 25% to about 32%, about 26% to about 30%, or about 28% to about 29% w / w, e.g., about 28.3% w / w maltodextrin). Maltodextrin dissolves in solution and forms a flexible helical structure that can entrap active ingredients (e.g., any of the phenylbutyric acid compounds and bile acids described herein), thereby masking the taste of the active ingredients. Maltodextrins produced from suitable sources, including, but not limited to, pea, rice, tapioca, corn, and potato, are contemplated herein. In some embodiments, the maltodextrin is pea maltodextrin. In some embodiments, the compositions include about 28.3 w / w% pea maltodextrin. For example, pea maltodextrin available from Roquette (KLEPTOSE® LINECAPS) can be used.

[0081] The composition described in the specification can further include a sugar substitute (e.g., sucralose). For example, the composition can include from about 0.5 to about 5 w / w% sucralose (e.g., from about 1% to about 4%, from about 1% to about 3% or from about 1% to about 2%, e.g., about 1.9% w / w sucralose). Other sugar substitutes contemplated in this specification include, but are not limited to, aspartame, neotame, acesulfame potassium, saccharin, and advantame.

[0082] In some embodiments, the composition includes one or more flavoring agents. The composition can include from about 2 to about 15 w / w% flavoring agent (e.g., from about 3% to about 13%, from about 3% to about 12%, from about 4% to about 9%, from about 5% to about 10% or from about 5% to about 8%, e.g., about 7.3% w / w). Flavoring agents can include substances that change the characteristics of the composition by imparting a scent to another substance or affecting the taste. Flavoring agents can be used to mask unpleasant tastes without affecting physical and chemical stability and can be selected based on the taste of the drug contained. Suitable flavoring agents include, but are not limited to, natural flavoring substances, artificial flavoring substances, and imitation flavors. Blends of flavoring agents can also be used. For example, the composition described in the specification can include two or more (e.g., 2, 3, 4, 5 or more) flavoring agents. The flavoring agent may be water-soluble and stable. The selection of a suitable flavoring agent can be based on taste tests. For example, a plurality of different flavoring agents can be added separately to the composition and taste tests thereof can be performed. Representative flavoring agents include fruit flavor powders (e.g., flavor powders of peach, strawberry, mango, orange, apple, grape, raspberry, cherry or a mixture of various berries). The composition described in the specification can include from about 0.5 to about 1.5 w / w% (e.g., about 1% w / w) of mixed berry flavor powder and / or from about 5 to about 7 w / w% (e.g., about 6.3% w / w) of masking flavor. Suitable masking flavors are available, for example, from Firmenich.

[0083] The compositions described herein may further comprise silicon dioxide (or silica). The addition of silica to the composition may prevent or reduce agglomeration of the components of the composition. Silica may function as an anti-caking agent, adsorbent, disintegrant, or glidant. In some embodiments, the compositions described herein comprise about 0.1 to about 2 w / w% porous silica (e.g., about 0.3% to about 1.5%, about 0.5% to about 1.2%, or about 0.8% to about 1%, e.g., 0.9% w / w). The porous silica has a higher H2O content than fumed silica at a relative humidity of about 20% or more (e.g., about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more). 2 The O absorption capacity and / or porosity of the porous silica may be high. 2 The O2 absorption capacity may be about 5 to about 40% by weight (e.g., about 20% to about 40% or about 30% to about 40%) at a relative humidity of about 50%. The porous silica may have a high porosity compared to that of fumed silica at a relative humidity of about 20% or more (e.g., about 30%, 40%, 50%, 60%, 70%, 80%, 90% or more). In some embodiments, the average particle size of the porous silica is about 2 μm to about 10 μm (e.g., about 3 μm to about 9 μm, about 4 μm to about 8 μm, about 5 μm to about 8 μm, or about 7.5 μm). In some embodiments, the average pore volume of the porous silica is 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 bulk density of the porous silica is 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 % (eg, any range within this range) of Syloid® 63FP (WR Grace).

[0084] The compositions described herein may further comprise one or more buffering agents. For example, the compositions may comprise about 0.5 to about 5 w / w% of a buffering agent (e.g., about 1% to about 4% w / w, about 1.5% to about 3.5% w / w, or about 2% to about 3% w / w, e.g., about 2.7% w / w of a buffering agent). The buffering agent may comprise a weak acid or base that maintains the acidity or pH of the composition near a selected value after the addition of another acid or base. Suitable buffering agents are known in the art. In some embodiments, the buffering agent in the compositions provided herein is a phosphate salt, such as sodium phosphate (e.g., sodium dihydrogen phosphate anhydrous). For example, the composition may comprise about 2.7 w / w% of dibasic sodium phosphate.

[0085] The composition may also include one or more lubricants. For example, the composition may include about 0.05 to about 1 w / w% of a lubricant (e.g., about 0.1% to about 0.9%, about 0.2% to about 0.8%, about 0.3% to about 0.7%, or about 0.4% to about 0.6%, e.g., about 0.5% w / w of a lubricant). Representative lubricants include, but are not limited to, sodium stearyl fumarate, magnesium stearate, stearic acid, metal stearates, talc, high melting point waxes and glycerides, colloidal silica, polyethylene glycol, alkyl sulfates, glyceryl behenate, and hydrogenated oils. Additional lubricants are known in the art. In some embodiments, the composition includes about 0.05 to about 1 w / w% of sodium stearyl fumarate (e.g., within this range as described herein). For example, the composition may include about 0.5 w / w% of sodium stearyl fumarate.

[0086] In some embodiments, the composition comprises about 29.2 w / w% sodium phenylbutyrate, about 9.7 w / w% TURSO, about 15.6 w / w% dextrates, about 3.9 w / w% sorbitol, about 1.9 w / w% sucralose, about 28.3 w / w% maltodextrin, about 7.3 w / w% flavorings, about 0.9 w / w% silicon dioxide, about 2.7 w / w% sodium phosphate (e.g., sodium hydrogen phosphate), and about 0.5 w / w% sodium stearyl fumarate.

[0087] The composition may include about 3000 mg sodium phenylbutyrate, about 1000 mg TURSO, about 1600 mg dextrates, about 400 mg sorbitol, about 200 mg sucralose, about 97.2 mg silicon dioxide, about 2916 mg maltodextrin, about 746 mg flavorings (e.g., about 102 mg assorted berry flavor and about 644 mg masking flavor), about 280 mg sodium phosphate (e.g., sodium hydrogen phosphate), and about 48.6 mg sodium stearyl fumarate.

[0088] Additional suitable sweeteners or taste masking agents may also be included in the composition, such as, but not limited to, xylose, ribose, glucose, mannose, galactose, fructose, dextrose, sucrose, maltose, steviol glycosides, partial starch hydrolysates, and corn syrup solids. Contemplated herein are soluble saccharin salts (e.g., sodium or calcium saccharin salts), cyclamate salts, acesulfame potassium (acesulfame K), and water-soluble artificial sweeteners such as the free acid form of saccharin and L-aspartyl-phenylalanine methyl ester, aspartame-based sweeteners such as Alitame® or Neotame®. The amount of sweetener or taste masking agent may be varied to select the desired amount of sweetener or taste masking agent for a particular final composition.

[0089] Pharmaceutically acceptable binders are contemplated in addition to those previously described, examples of which include cellulose derivatives including microcrystalline cellulose, low substituted hydroxypropylcellulose (e.g., LH22, LH21, LH20, LH32, LH31, LH30), starches including potato starch, croscarmellose sodium (i.e., cross-linked carboxymethylcellulose sodium salt; e.g., Ac-Di-Sol®), alginic acid or alginates, insoluble polyvinylpyrrolidones (e.g., Polyvidon® CL, Polyvidon® CL-M, Kollidon® CL, Polyplasdone® XL, Polyplasdone® XL-10), and sodium carboxymethyl starch (e.g., Primogel® and Explotab®).

[0090] Polyols, sucrose, sorbitol, mannitol, Erythritol®, Tagatose®, lactose (e.g. spray dried lactose, α-lactose, β-lactose, Tabletose®, various grades of Pharmatose®, Microtose or Fast-Floc®), microcrystalline cellulose (e.g. various grades of Avicel® such as Avicel® PH101, Avicel® PH102 or Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Further excipients, diluents or binders may be included such as cellulose acetate, cellulose esters such as cellulose acetate esters, cellulose esters such as cellulose acetate Tai® and Solka-Floc®, hydroxypropylcellulose, L-hydroxypropylcellulose (low substituted) (e.g. L-HPC-CH31, L-HPC-LH11, LH22, LH21, LH20, LH32, LH31, LH30), dextrins, maltodextrins (e.g. Lodex® 5 and Lodex® 10), starch or modified starches (including potato starch, maize starch and rice starch), sodium chloride, sodium phosphate, calcium sulfate and calcium carbonate.

[0091] The compositions described herein can be formulated or adapted for administration to a subject by any route, e.g., a route approved by the Food and Drug Administration (FDA). Exemplary methods are described in FDA's CDER Data Standards Manual, version number 004 (available at fda.give / cder / dsm / DRG / drg00301.html).

[0092] Pharmaceutical compositions are typically formulated to be compatible with the intended route of administration, which include parenteral (subcutaneous, intradermal, intravenous, intradermal, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial infusion or injection techniques), oral (e.g., via inhalation or feeding tube), transdermal (topical), transmucosal and rectal administration.

[0093] The pharmaceutical composition may be in the form of a solution or powder for inhalation and / or nasal administration. In some embodiments, the pharmaceutical composition is formulated as a powder-in-a-sachet. Suitable powders may include those that are substantially soluble in water. The pharmaceutical composition may be formulated using suitable dispersing or wetting agents (such as, for example, Tween® 80) and suspending agents according to techniques known in the art. The sterile injection preparation may also be a sterile injection solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as, for example, a 1,3-butanediol solution. Acceptable vehicles and solvents that may be used include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, fixed oils are conventionally employed as solvents or suspending media. For this purpose, non-irritating fixed oils may be employed, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the manufacture of injections, as are natural pharma-ceutically acceptable oils such as olive oil or castor oil, especially in polyoxyethylated form. These oil solutions or suspensions may also contain long chain alcohol diluents or dispersants, carboxymethylcellulose, or similar dispersing agents commonly used in formulating pharma- ceutically acceptable dosage forms, such as emulsions and / or suspensions. Other commonly used surfactants, such as Tween® or Span®, and / or other similar emulsifiers or bioavailability enhancers, that are pharma- ceutically acceptable and commonly used in the manufacture of solid, liquid, or other dosage forms, may also be used for the formulation.

[0094] The composition 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 powder can be substantially dissolved in water before administration. In the case of tablets for oral use, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate may be added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions and / or emulsions are orally administered, the active ingredient may be suspended or dissolved in a combined oily phase using emulsifying and / or suspending agents. If desired, certain sweetening, flavoring, and / or coloring agents may be added.

[0095] Alternatively or additionally, the compositions may be administered by nasal aerosol or inhalation.Such compositions may be prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as saline solutions utilizing benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art.

[0096] In some aspects, the therapeutic compositions disclosed herein can be formulated for sale in the United States, imported into the United States, and / or exported from the United States. The pharmaceutical compositions can be included in a container, pack, or dispenser device along with instructions for administration. In some aspects, the present invention provides kits that include a bile acid and a phenylbutyric acid compound. The kits can also include instructions for the physician and / or patient, syringes, needles, boxes, bottles, vials, and the like.

[0097] III. Treatment method Applicant has discovered that a combination of a bile acid (e.g., TURSO) and a phenylbutyric acid compound (e.g., sodium phenylbutyrate) can be used to treat one or more symptoms of ALS. Applicant has discovered that TURSO and its metabolites, ursodeoxycholic acid and glycoursodeoxycholic acid, are inhibitors of BSEP. Surprisingly, phenylacetic acid, a metabolite of sodium phenylbutyrate, has also been found to inhibit BSEP. Thus, when a BSEP inhibitor is administered simultaneously with a composition comprising a bile acid and a phenylbutyric acid compound, a drug-drug interaction may result in an adverse event due to an exacerbation of the accumulation of conjugated bile salts in the liver. When a BSEP inhibitor is administered simultaneously with a composition comprising, for example, TURSO and sodium phenylbutyrate to a subject in need of both treatments, signs of a drug-drug interaction may be monitored and the dose of the BSEP inhibitor may be adjusted accordingly. For example, when a BSEP inhibitor is administered simultaneously with a composition comprising TURSO and sodium phenylbutyrate, serum transaminase and bilirubin levels may be elevated, indicating liver toxicity. Therefore, adverse effects associated with drug-drug interactions can be prevented or mitigated by monitoring serum transaminases and bilirubin and adjusting the dosing of BSEP inhibitors.

[0098] Therefore, this disclosure provides a method for treating at least one symptom of ALS in a subject to which a BSEP inhibitor is administered by adjusting the administration of the BSEP inhibitor.Such adjustment may result in a plasma concentration of the BSEP inhibitor similar to that obtained when the BSEP inhibitor is administered without the bile acid and phenylbutyric acid compound, and may have the same degree of efficacy.

[0099] Applicant discloses herein a method of treating at least one symptom of ALS in a subject who has received a first dose of a BSEP inhibitor, or a method of administering TURSO and sodium phenylbutyrate to a subject who exhibits at least one symptom of ALS and has received a first dose of a BSEP inhibitor, the method comprising administering to the subject a composition comprising about 1 g of TURSO and about 3 g of sodium phenylbutyrate, monitoring the subject's response to the BSEP inhibitor, and administering a second dose of the BSEP inhibitor, wherein the second dose is less than the first dose. Monitoring the subject's response to the BSEP inhibitor can include determining a first level of serum transaminase and / or bilirubin in a biological sample from the subject, monitoring for known adverse events and / or overdose symptoms or side effects associated with inhibition of BSEP, or elevated serum transaminase or bilirubin levels.

[0100] In some embodiments of the methods described herein, the first and / or second administration of the BSEP inhibitor can be daily (once, twice or three times a day), once every other day, three times a week or once a week, or on other basis. The second administration of the BSEP inhibitor can be about 1% to about 95% (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%) less than the first administration. In some cases, the second administration is administered in the same or similar amount as the first administration, but less frequently. In some cases, administration of the BSEP inhibitor is discontinued, i.e., a second administration is not administered.

[0101] In some aspects of the methods described herein, the methods include determining, or having determined, a second level of serum transaminase and / or bilirubin in a second biological sample from the subject, hi some aspects, the second level is lower than the first level.

[0102] In some embodiments of the methods described herein, the first biological sample can be obtained from the subject about 1 hour to about 72 hours (e.g., about 2, 4, 6, 8, 10, 16, 24, 32, 48, or 56 hours) after administration of a composition comprising TURSO and phenylbutyric acid. The second biological sample can be obtained from the subject about 1 hour to about 72 hours (e.g., about 2, 4, 6, 8, 10, 16, 24, 32, 48, or 56 hours) after a second administration of a BSEP inhibitor.

[0103] In some aspects of the methods described herein, the first and / or second biological sample may be a serum, plasma, urine or saliva sample. Methods for measuring serum transaminase and / or bilirubin levels in a biological sample are known in the art.

[0104] BSEP inhibitors Transporters can be divided into (i) efflux transporters belonging to the ATP-binding cassette (ABC) family and (ii) uptake transporters belonging to the solute carrier (SLC) family, which mediate influx across or bidirectional movement within the cell membrane. Efflux transporters return compounds to the blood as they pass through the apical (i.e., blood side) side of the cell membrane and also deliver compounds from outside the cell to the basolateral side of the brain.

[0105] Bile formation is one of the important functions of the mammalian liver. It involves hepatocyte-mediated secretion of bile acids and other bile-tropic substances from the plasma of the sinusoids into the bile canaliculi. This results in bile acids or bile salts being concentrated in the bile by more than 500 times compared to plasma. This concentration is dependent on ATP and is mainly driven by the bile canalicular bile salt export pump (BSPE). BSEP belongs to family B of the ATP-binding cassette (ABC) superfamily of transporters and is classified as ABCB11.

[0106] Inhibition of BSEP reduces the secretion of bile salts, decreasing bile flow and causing cholestasis. Cholestasis is a disease of the liver. People with cholestasis experience abdominal pain, abdominal distension, fatty stools, nausea, pale stools, loss of appetite or fatigue, dark urine, failure to thrive, itching or yellowing of the skin and eyes, abnormal bilirubin levels, and / or abnormal liver enzyme levels.

[0107] There are many drugs known to be BSEP inhibitors. Such drugs can cause acquired cholestasis in susceptible individuals, but this is rapidly resolved when administration is discontinued. Representative inhibitors of BSEP include, but are not limited to, cyclosporine, glibenclamide, rifamycin, bosentan, troglitazone, fluvastatin, and ketoconazole.

[0108] As previously described, applicants have discovered that TURSO and its metabolites, ursodeoxycholic acid and glycoursodeoxycholic acid, are inhibitors of BSEP. It has also been surprisingly discovered that phenylacetic acid, a metabolite of sodium phenylbutyrate, inhibits BSEP. Coadministration of compositions containing bile acids and phenylbutyric acid compounds with BSEP inhibitors may result in drug-drug interactions and adverse events, such as exacerbated accumulation of conjugated bile salts in the liver, resulting in elevated serum transaminase and bilirubin levels and toxic effects.

[0109] Thus, in some aspects, there is provided herein a method for treating at least one symptom of ALS in a subject, comprising: (a) administering to a subject who has received a first dose of a BSEP inhibitor an effective amount of a composition comprising about 1 g of TURSO and about 3 g of sodium phenylbutyrate; (b) determining or having determined a first level of serum transaminase and / or bilirubin in a first biological sample of the subject; and (c) administering to the subject a second dose of the BSEP inhibitor, wherein the second dose is less than the first dose. The method may further comprise a step (d) of determining or having determined a second level of serum transaminase and / or bilirubin in a second biological sample of the subject.

[0110] In some embodiments, the BSEP inhibitor is cyclosporine. Cyclosporine (also called cyclosporine A) can be used to prevent rejection in allogeneic kidney, liver and heart transplants, or to prevent bone marrow transplant rejection. Cyclosporine is used to treat patients with severe active rheumatoid arthritis (RA) or severe refractory plaque psoriasis. Cyclosporine eye drops have been shown to increase tear production in patients with keratoconjunctivitis sicca. In addition, cyclosporine is approved for the treatment of steroid-dependent and steroid-resistant nephrotic syndrome due to glomerular diseases, including minimal change nephropathy, focal and segmental glomerulosclerosis, or membranous glomerulonephritis. Cyclosporine is also commonly used to treat various autoimmune and inflammatory diseases, such as atopic dermatitis, blistering diseases, ulcerative colitis, juvenile rheumatoid arthritis, uveitis, connective tissue diseases, and idiopathic thrombocytopenic purpura. The subject may receive a first dose of cyclosporine at about 0.5 to about 15 mg / kg body weight / day (e.g., about 0.5 to about 5 mg / kg / day, about 1 to about 4 mg / kg / day, about 2.5 mg / kg / day, or about 12 to about 15 mg / kg / day). The second dose of cyclosporine may be about 0.1 to about 14 mg / kg / day (e.g., about 0.5 to about 2.5 mg / kg / day, about 1 to about 5 mg / kg / day) less than the first dose.

[0111] In addition to or instead of determining the first and / or second levels of the BSEP inhibitor in the subject's biological sample, other methods of monitoring the subject's response to the first administration of the BSEP inhibitor are also contemplated herein. For example, known adverse events, side effects, or symptoms of overdose associated with BSEP inhibitors can be monitored. For example, in some embodiments, the subject is monitored for cholestasis. As previously described, symptoms of cholestasis include abdominal pain, abdominal distension, fatty stools, nausea, pale stools, anorexia or fatigue, dark urine, growth retardation, pruritus, or yellowing of the skin and eyes. Symptoms of cyclosporine overdose include hepatotoxicity and nephrotoxicity.

[0112] Measurement of liver function Drug metabolism can affect liver function. Liver function tests check the levels of certain enzymes and proteins in the blood. Levels higher or lower than normal may indicate a liver problem. For example, levels of one or more of alanine transaminase, aspartate transaminase, alkaline phosphatase, albumin and total protein, bilirubin, gamma-glutamyltransferase, L-lactate dehydrogenase, and prothrombin time (to measure blood clotting factors) may be checked.

[0113] Serum transaminases Serum transaminases include alanine transaminase (ALT) and aspartate transaminase (AST). Serum transaminases (also called aminotransferases) are enzymes that catalyze the interconversion of amino acids and oxoacids by transfer of an amino group. ALT and AST are the most reliable markers of hepatocellular injury or necrosis. Their levels can be elevated in various liver diseases.

[0114] Normal ranges for ALT and AST vary by gender, age, and laboratory. Individuals with elevated serum transaminase levels can be classified as "mild" (less than 5 times normal), "moderate" (5-10 times normal), or "marked" (more than 10 times normal). Thus, some embodiments of the methods described herein include determining a first level of ALT or AST in a subject's sample. In some instances, the first level of ALT or AST can be about 5 times, about 5-10 times higher than the normal range for ALT or AST, or can be more than 5 times the normal range.

[0115] Methods for measuring serum transaminase levels are widely known in the art. Usually, ALT and AST levels are measured from the patient's blood, usually together with other liver enzymes and compounds in the blood. For example, immuno / enzyme immunoassay or liquid chromatography / tandem mass spectrometry (LC / MS) can be used.

[0116] Bilirubin Bilirubin (BR) is a yellowish-orange compound produced in the normal catabolic pathway of heme breakdown in the vertebrate liver. Elevated bilirubin levels are a sign of liver disease. There are three types of bilirubin: unconjugated bilirubin, conjugated bilirubin, and total bilirubin (the sum of unconjugated and conjugated bilirubin). Unconjugated (also called "indirect") bilirubin is bilirubin that is produced by the breakdown of red blood cells. It travels through the bloodstream to the liver. Conjugated (also called "direct") bilirubin is bilirubin that reaches the liver and is chemically altered. It travels to the intestine and is excreted in urine and stool. Bilirubin tests can determine the total amount of unconjugated bilirubin, conjugated bilirubin, and bilirubin. For example, in adults over 18 years of age, a normal total bilirubin level is up to 1.2 milligrams per deciliter (mg / dl) of blood. In people under the age of 18, the normal level is about 1 mg / dL. The normal level for conjugated (direct) bilirubin is less than 0.3 mg / dL.

[0117] Some embodiments of the methods described herein include determining, e.g., a first level of bilirubin in a sample from a subject. The first level of bilirubin can be about X higher than the normal range for bilirubin. In some embodiments, the subject's total bilirubin level can be elevated above 1.2 mg / dl (e.g., 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 mg / dl or more). In some embodiments, the subject's total bilirubin level can be elevated above 0.3 mg / dl (e.g., 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mg / dl or more).

[0118] Methods for measuring bilirubin levels are well known in the art. Typically, bilirubin levels are measured from a patient's blood, typically along with other liver enzymes and compounds in the blood, as well as ALT and AST. Bilirubin blood tests measure the amount of total bilirubin present in the blood. Bilirubin can also be tested in urine. Urinary bilirubin tests measure the presence of bilirubin in the urine. As with ALT and AST, bilirubin levels can be measured using immuno / enzyme immunoassays or liquid chromatography / tandem mass spectrometry (LC / MS).

[0119] Administration of TURSO and sodium phenylbutyrate The methods described herein include administering to a subject a bile acid or a pharma- ceutically acceptable salt thereof and a phenylbutyric acid compound. The bile acid or a pharma- ceutically acceptable salt thereof and a phenylbutyric acid compound can be administered separately or simultaneously, for example, as part of a treatment regimen. The compound can be administered daily (e.g., once a day, twice a day, three times a day or more), weekly, monthly, or quarterly. The compound can be administered for weeks, months, or years. For example, the compound can be administered for 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 5 years, or more. The compound can be administered once a day or twice a day for up to 60 days (e.g., up to 55 days, 50 days, 45 days, 40 days, 35 days, 30 days). Alternatively, the bile acid and phenylbutyric acid compounds can be administered once daily or twice daily for more than 60 days (e.g., 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 180, 200, 250, 300, 400, 500, 600 days).

[0120] In some embodiments, the methods provided herein include administering an effective amount of a composition comprising about 1 g of TURSO and about 3 g of sodium phenylbutyrate. TURSO may be administered in an amount of about 0.5 to about 5 g (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 about 2.5 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 g). In some embodiments, about 1 to about 2 g (e.g., about 1 to about 1.8 g, about 1 to about 1.6 g, about 1 to about 1.4 g, about 1 to about 1.2 g, about 1.2 to about 2.0 g, about 1.2 to about 1.8 g, about 1.2 to about 1.6 g, about 1.2 to about 1.4 g, about 1.4 to about 2.0 g, about 1.4 to about 1.8 g, about 1.4 to about 1.6 g, about 1.6 to about 2.0 g, about 1.6 to about 1.8 g, about 1.8 to about 2.0 g) (inclusive) of TURSO is administered per day. In some embodiments, about 1 g of TURSO is administered per day. In some embodiments, about 2 g of TURSO is administered per day. For example, about 1 g of TURSO can be administered twice a day.

[0121] Sodium phenylbutyrate is taken at a daily dose of about 0.5 to about 10 g (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 g). In some embodiments, sodium phenylbutyrate is administered at about 3 to about 6 g (e.g., about 3 to about 5.5 g, about 3 to about 5.0 g, about 3 to about 4.5 g, about 3 to about 4.0 g, about 3 to about 3.5 g, about 3.5 to about 6 g, about 3.5 to about 5.5 g, about 3.5 to about 5.0 g, about 3.5 to about 4.5 g, about 3.5 to about 4.0 g, about 4.0 to about 6 g, about 4.0 to about 5.5 g, about 4.0 to about 5.0 g, about 4.0 to about 4.5 g, about 4.5 to about 6 g, about 4.5 to about 5.5 g, about 4.5 to about 5.0 g, about 5.0 to about 6 g, about 5.0 to about 5.5 g, or about 5.5 to about 6.0 g) (inclusive). In some embodiments, sodium phenylbutyrate is administered at about 3 g per day. In some embodiments, sodium phenylbutyrate is administered at about 6 g per day. For example, sodium phenylbutyrate can be administered at about 3 g twice daily. In some embodiments, the bile acid and the phenylbutyric acid compound are administered at a weight ratio of about 2.5:1 to about 3.5:1 (e.g., about 3:1).

[0122] The methods described herein can include administering about 1 gram of TURSO once per day and about 3 grams of sodium phenylbutyrate once per day, or about 1 gram of TURSO twice per day and about 3 grams of sodium phenylbutyrate twice per day. The methods can include administering about 1 gram of TURSO once per day and about 3 grams of sodium phenylbutyrate once per day for at least 14 days (e.g., at least 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 per day and about 3 grams of sodium phenylbutyrate twice per day or at least every day (e.g., at least 30, 40, 50, 60, 80, 100, 120, 150, 180, 250, 300, or 400 days). For example, the method can include administering about 1 gram of TURSO once per day and about 3 grams of sodium phenylbutyrate once per day for 14 to 21 days, followed by administering about 1 gram of TURSO twice per day and about 3 grams of sodium phenylbutyrate twice per day.

[0123] In some embodiments, the methods described herein involve administering to a subject a daily dose of about 10 mg / kg to about 50 mg / kg of body weight (e.g., about 10 mg / kg to about 48 mg / kg, about 10 mg / kg to about 46 mg / kg, about 10 mg / kg to about 44 mg / kg, about 10 mg / kg to about 42 mg / kg, about 10 mg / kg to about 40 mg / kg, about 10 mg / kg to about 38 mg / kg, about 10 mg / kg to about 36 mg / kg, about 10 mg / kg to about 34 mg / kg, about 10 mg / kg to about 32 mg / kg, about 10 mg / kg to about 30 mg / kg, about 10 mg / kg to about 28 mg / kg, or about 28 ... g / kg, about 10 mg / kg to about 26 mg / kg, about 10 mg / kg to about 24 mg / kg, about 10 mg / kg to about 22 mg / kg, about 10 mg / kg to about 20 mg / kg, about 10 mg / kg to about 18 mg / kg, about 10 mg / kg to about 16 mg / kg, about 10 mg / kg to about 1 4mg / kg, approximately 10mg / kg to approximately 12mg / kg, approximately 12mg / kg to approximately 50mg / kg, approximately 12mg / kg to approximately 48mg / kg, approximately 12mg / kg to approximately 46mg / kg, approximately 12mg / kg to approximately 44mg / kg, approximately 12mg / kg to approximately 42mg / kg, approximately 12mg / kg to About 40mg / kg, about 12mg / kg to about 38mg / kg, about 12mg / kg to about 36mg / kg, about 12mg / kg to about 34mg / kg, about 12mg / kg to about 32mg / kg, about 12mg / kg to about 30mg / kg, about 12mg / kg to about 28mg / kg, about 12mg / kg kg~about 26mg / kg, about 12mg / kg~about 24mg / kg, about 12mg / kg~about 22mg / kg, about 12mg / kg~about 20mg / kg, about 12mg / kg~about 18mg / kg, about 12mg / kg~about 16mg / kg, about 12mg / kg~about 14mg / kg, about 14m g / kg ~ approx. 50 mg / kg, approx. 14 mg / kg ~ approx. 48 mg / kg, approx. 14 mg / kg ~ approx. 46 mg / kg, approx. 14 mg / kg ~ approx. 44 mg / kg, approx. 14 mg / kg ~ approx. 42 mg / kg, approx. 4mg / kg to about 36mg / kg, about 14mg / kg to about 34mg / kg, about 14mg / kg to about 32mg / kg, about 14mg / kg to about 30mg / kg, about 14mg / kg to about 28mg / kg, about 14mg / kg to about 26mg / kg, about 14mg / kg to about 24mg / kg,from about 14 mg / kg to about 22 mg / kg, from about 14 mg / kg to about 20 mg / kg, from about 14 mg / kg to about 18 mg / kg, from about 14 mg / kg to about 16 mg / kg, from about 16 mg / kg to about 50 mg / kg, from about 16 mg / kg to about 48 mg / kg, from about 16 mg / kg to about 46 mg / kg, from about 16 mg / kg to about 44 mg / kg, from about 16 mg / kg to about 42 mg / kg, from about 16 mg / kg to about 40 mg / kg, from about 16 mg / kg to about 38 mg / kg, from about 16 mg / kg to about 36 mg / kg, from about 16 mg / kg to about 34 mg / kg, from about 16 mg / kg to about 32 mg / kg, from about 16 mg / kg to about 30 mg / kg, from about 16 mg / kg to about 28 mg / kg, from about 16 mg / kg to about 26 mg / kg, from about 16 mg / kg to about 24 mg / kg, from about 16 mg / kg to about 22 mg / kg, from about 16 mg / kg to about 20 mg / kg, from about 16 mg / kg to about 18 mg / kg, from about 18 mg / kg to about 50 mg / kg, from about 18 mg / kg to about 48 mg / kg, from about 18 mg / kg to about 46 mg / kg, from about 18 mg / kg to about 44 mg / kg, from about 18 mg / kg to about 42 mg / kg, from about 18 mg / kg to about 40 mg / kg, from about 18 mg / kg to about 38 mg / kg, from about 18 mg / kg to about 36 mg / kg, from about 18 mg / kg to about 34 mg / kg, from about 18 mg / kg to about 32 mg / kg, from about 18 mg / kg to about 30 mg / kg, from about 18 mg / kg to about 28 mg / kg, from about 18 mg / kg to about 26 mg / kg, from about 18 mg / kg to about 24 mg / kg, from about 18 mg / kg to about 22 mg / kg, from about 18 mg / kg to about 20 mg / kg, from about 20 mg / kg to about 50 mg / kg, from about 20 mg / kg to about 48 mg / kg, from about 20 mg / kg to about 46 mg / kg, from about 20 mg / kg to about 44 mg / kg, from about 20 mg / kg to about 42 mg / kg, from about 20 mg / kg to about 40 mg / kg, from about 20 mg / kg to about 38 mg / kg, from about 20 mg / kg to about 36 mg / kg, from about 20 mg / kg to about 34 mg / kg, from about 20 mg / kg to about 32 mg / kg, from about 20 mg / kg to about 30 mg / kg, from about 20 mg / kg to about 28 mg / kg, from about 20 mg / kg to about 26 mg / kg, from about 20 mg / kg to about 24 mg / kg, from about 20 mg / kg to about 22 mg / kg, from about 22 mg / kg to about 50 mg / kg, from about 22 mg / kg to about 48 mg / kg, from about 22 mg / kg to about 46 mg / kg,from about 22 mg / kg to about 44 mg / kg, from about 22 mg / kg to about 42 mg / kg, from about 22 mg / kg to about 40 mg / kg, from about 22 mg / kg to about 38 mg / kg, from about 22 mg / kg to about 36 mg / kg, from about 22 mg / kg to about 34 mg / kg, from about 22 mg / kg to about 32 mg / kg, from about 22 mg / kg to about 30 mg / kg, from about 22 mg / kg to about 28 mg / kg, from about 22 mg / kg to about 26 mg / kg, from about 22 mg / kg to about 24 mg / kg, from about 24 mg / kg to about 50 mg / kg, from about 24 mg / kg to about 48 mg / kg, from about 24 mg / kg to about 46 mg / kg, from about 24 mg / kg to about 44 mg / kg, from about 24 mg / kg to about 42 mg / kg, from about 24 mg / kg to about 40 mg / kg, from about 24 mg / kg to about 38 mg / kg, from about 24 mg / kg to about 36 mg / kg, from about 24 mg / kg to about 34 mg / kg, from about 24 mg / kg to about 32 mg / kg, from about 24 mg / kg to about 30 mg / kg, from about 24 mg / kg to about 28 mg / kg, from about 24 mg / kg to about 26 mg / kg, from about 26 mg / kg to about 50 mg / kg, from about 26 mg / kg to about 48 mg / kg, from about 26 mg / kg to about 46 mg / kg, from about 26 mg / kg to about 44 mg / kg, from about 26 mg / kg to about 42 mg / kg, from about 26 mg / kg to about 40 mg / kg, from about 26 mg / kg to about 38 mg / kg, from about 26 mg / kg to about 36 mg / kg, from about 26 mg / kg to about 34 mg / kg, from about 26 mg / kg to about 32 mg / kg, from about 26 mg / kg to about 30 mg / kg, from about 26 mg / kg to about 28 mg / kg, from about 28 mg / kg to about 50 mg / kg, from about 28 mg / kg to about 48 mg / kg, from about 28 mg / kg to about 46 mg / kg, from about 28 mg / kg to about 44 mg / kg, from about 28 mg / kg to about 42 mg / kg, from about 28 mg / kg to about 40 mg / kg, from about 28 mg / kg to about 38 mg / kg, from about 28 mg / kg to about 36 mg / kg, from about 28 mg / kg to about 34 mg / kg, from about 28 mg / kg to about 32 mg / kg, from about 28 mg / kg to about 30 mg / kg, from about 30 mg / kg to about 50 mg / kg, from about 30 mg / kg to about 48 mg / kg, from about 30 mg / kg to about 46 mg / kg, from about 30 mg / kg to about 44 mg / kg, from about 30 mg / kg to about 42 mg / kg, from about 30 mg / kg to about 40 mg / kg, from about 30 mg / kg to about 38 mg / kg, from about 30 mg / kg to about 36 mg / kgApproximately 30mg / kg to approximately 34mg / kg, approximately 30mg / kg to approximately 32mg / kg, approximately 32mg / kg to approximately 50mg / kg, approximately 32mg / kg to approximately 48mg / kg, approximately 32mg / kg to approximately 46mg / kg, approximately 32mg / kg to approximately 44mg / kg, Approximately 32mg / kg to approximately 42mg / kg, approximately 32mg / kg to approximately 40mg / kg, approximately 32mg / kg to approximately 38mg / kg, approximately 32mg / kg to approximately 36mg / kg, approximately 32mg / kg to approximately 34mg / kg, approximately 34mg / kg to approximately 50mg / kg, Approximately 34mg / kg to approximately 48mg / kg, approximately 34mg / kg to approximately 46mg / kg, approximately 34mg / kg to approximately 44mg / kg, approximately 34mg / kg to approximately 42mg / kg, approximately 34mg / kg to approximately 40mg / kg, approximately 34mg / kg to approximately 38mg / kg, Approximately 34 mg / kg to approximately 36 mg / kg, approximately 36 mg / kg to approximately 50 mg / kg, approximately 36 mg / kg to approximately 48 mg / kg, approximately 36 mg / kg to approximately 46 mg / kg, approximately 36 mg / kg to approximately 44 mg / kg, approximately 36 mg / kg to approximately 42 mg / kg, Approximately 36mg / kg to approximately 40mg / kg, approximately 36mg / kg to approximately 38mg / kg, approximately 38mg / kg to approximately 50mg / kg, approximately 38mg / kg to approximately 48mg / kg, approximately 38mg / kg to approximately 46mg / kg, approximately 38mg / kg to approximately 44mg / kg, Approximately 38mg / kg to approximately 42mg / kg, approximately 38mg / kg to approximately 40mg / kg, approximately 40mg / kg to approximately 50mg / kg, approximately 40mg / kg to approximately 48mg / kg, approximately 40mg / kg to approximately 46mg / kg, approximately 40mg / kg to approximately 44mg / kg, The method includes administering about 40 mg / kg to about 42 mg / kg, about 42 mg / kg to about 50 mg / kg, about 42 mg / kg to about 48 mg / kg, about 42 mg / kg to about 46 mg / kg, about 42 mg / kg to about 44 mg / kg, about 44 mg / kg to about 50 mg / kg, about 44 mg / kg to about 48 mg / kg, about 44 mg / kg to about 46 mg / kg, about 46 mg / kg to about 50 mg / kg, about 46 mg / kg to about 48 mg / kg, or about 46 mg / kg to about 50 mg / kg of TURSO.

[0124] In some embodiments, the methods described herein involve administering to a subject a daily dose of from about 10 mg / kg to about 400 mg / kg of body weight (e.g., from about 10 mg / kg to about 380 mg / kg, from about 10 mg / kg to about 360 mg / kg, from about 10 mg / kg to about 340 mg / kg, from about 10 mg / kg to about 320 mg / kg, from about 10 mg / kg to about 300 mg / kg, from about 10 mg / kg to about 280 mg / kg, from about 10 mg / kg to about 260 mg / kg, from about 10 mg / kg to about 240 mg / kg, from about 10 mg / kg to about 220 mg / kg, from about 10 mg / kg to about 20 ... g / kg~about 180mg / kg, about 10mg / kg~about 160mg / kg, about 10mg / kg~about 140mg / kg, about 10mg / kg~about 120mg / kg, about 10mg / kg~about 100mg / kg, about 10mg / kg~about 80mg / kg, about 10mg / kg~about 60mg / k g, about 10 mg / kg to about 40 mg / kg, about 10 mg / kg to about 20 mg / kg, about 20 mg / kg to about 400 mg / kg, about 20 mg / kg to about 380 mg / kg, about 20 mg / kg to about 360 mg / kg, about 20 mg / kg to about 340 mg / kg, about 20 mg / kg to about 3 20mg / kg, about 20mg / kg to about 300mg / kg, about 20mg / kg to about 280mg / kg, about 20mg / kg to about 260mg / kg, about 20mg / kg to about 240mg / kg, about 20mg / kg to about 220mg / kg, about 20mg / kg to about 200mg / kg, about 2 0mg / kg to about 180mg / kg, about 20mg / kg to about 160mg / kg, about 20mg / kg to about 140mg / kg, about 20mg / kg to about 120mg / kg, about 20mg / kg to about 100mg / kg, about 20mg / kg to about 80mg / kg, about 20mg / kg to about 60mg / kg, about 20mg / kg to about 40mg / kg, about 40mg / kg to about 400mg / kg, about 40mg / kg to about 380mg / kg, about 40mg / kg to about 360mg / kg, about 40mg / kg to about 340mg / kg, about 40mg / kg to about 320mg / kg, about 40mg / kg ~300mg / kg, 40mg / kg~280mg / kg, 40mg / kg~260mg / kg, 40mg / kg~240mg / kg, 40mg / kg~220mg / kg, 40mg / kg~200mg / kg, 40mg / kg~180mg / kg,about 40 mg / kg to about 160 mg / kg, about 40 mg / kg to about 140 mg / kg, about 40 mg / kg to about 120 mg / kg, about 40 mg / kg to about 100 mg / kg, about 40 mg / kg to about 80 mg / kg, about 40 mg / kg to about 60 mg / kg, about 60 mg / kg to about 400 mg / kg, about 60 mg / kg to about 380 mg / kg, about 60 mg / kg to about 360 mg / kg, about 60 mg / kg to about 340 mg / kg, about 60 mg / kg to about 320 mg / kg, about 60 mg / kg to about 300 mg / kg, about 60 mg / kg to about 280 mg / kg, about 60 mg / kg to about 260 mg / kg, about 60 mg / kg to about 240 mg / kg, about 60 mg / kg to about 220 mg / kg, about 60 mg / kg to about 200 mg / kg, about 60 mg / kg to about 180 mg / kg, about 60 mg / kg to about 160 mg / kg, about 60 mg / kg to about 140 mg / kg, about 60 mg / kg to about 120 mg / kg, about 60 mg / kg to about 100 mg / kg, about 60 mg / kg to about 80 mg / kg, about 80 mg / kg to about 400 mg / kg, about 80 mg / kg to about 380 mg / kg, about 80 mg / kg to about 360 mg / kg, about 80 mg / kg to about 340 mg / kg, about 80 mg / kg to about 320 mg / kg, about 80 mg / kg to about 300 mg / kg, about 80 mg / kg to about 280 mg / kg, about 80 mg / kg to about 260 mg / kg, about 80 mg / kg to about 240 mg / kg, about 80 mg / kg to about 220 mg / kg, about 80 mg / kg to about 200 mg / kg, about 80 mg / kg to about 180 mg / kg, about 80 mg / kg to about 160 mg / kg, about 80 mg / kg to about 140 mg / kg, about 80 mg / kg to about 120 mg / kg, about 80 mg / kg to about 100 mg / kg, about 100 mg / kg to about 400 mg / kg, about 100 mg / kg to about 380 mg / kg, about 100 mg / kg to about 360 mg / kg, about 100 mg / kg to about 340 mg / kg, about 100 mg / kg to about 320 mg / kg, about 100 mg / kg to about 300 mg / kg, about 100 mg / kg to about 280 mg / kg, about 100 mg / kg to about 260 mg / kg, about 100 mg / kg to about 240 mg / kg, about 100 mg / kg to about 220 mg / kg, about 100 mg / kg to about 200 mg / kg, about 100 mg / kg to about 180 mg / kg, about 100 mg / kg to about 160 mg / kg,About 100 mg / kg to about 140 mg / kg, about 100 mg / kg to about 120 mg / kg, about 120 mg / kg to about 400 mg / kg, about 120 mg / kg to about 380 mg / kg, about 120 mg / kg to about 360 mg / kg, about 120 mg / kg to about 340 mg / kg, about 120 mg / kg to about 320 mg / kg, about 120 mg / kg to about 300 mg / kg, about 120 mg / kg to about 280 mg / kg, about 120 mg / kg to about 260 mg / kg, about 120 mg / kg to about 240 mg / kg, about 120 mg / kg to about 220 mg / kg, about 120 mg / kg to about 200 mg / kg, about 120 mg / kg to about 180 mg / kg, about 120 mg / kg to about 160 mg / kg, about 120 mg / kg to about 140 mg / kg, about 140 mg / kg to about 400 mg / kg, about 140 mg / kg to about 380 mg / kg, about 140 mg / kg to about 360 mg / kg, about 140 mg / kg to about 340 mg / kg, about 140 mg / kg to about 320 mg / kg, about 140 mg / kg to about 300 mg / kg, about 140 mg / kg to about 280 mg / kg, about 140 mg / kg to about 260 mg / kg, about 140 mg / kg to about 240 mg / kg, about 140 mg / kg to about 220 mg / kg, about 140 mg / kg to about 200 mg / kg, about 140 mg / kg to about 180 mg / kg, about 140 mg / kg to about 160 mg / kg, about 160 mg / kg to about 400 mg / kg, about 160 mg / kg to about 380 mg / kg, about 160 mg / kg to about 360 mg / kg, about 160 mg / kg to about 340 mg / kg, about 160 mg / kg to about 320 mg / kg, about 160 mg / kg to about 300 mg / kg, about 160 mg / kg to about 280 mg / kg, about 160 mg / kg to about 260 mg / kg, about 160 mg / kg to about 240 mg / kg, about 160 mg / kg to about 220 mg / kg, about 160 mg / kg to about 200 mg / kg, about 160 mg / kg to about 180 mg / kg, about 180 mg / kg to about 400 mg / kg, about 180 mg / kg to about 380 mg / kg, about 180 mg / kg to about 360 mg / kg, about 180 mg / kg to about 340 mg / kg, about 180 mg / kg to about 320 mg / kg, about 180 mg / kg to about 300 mg / kg, about 180 mg / kg to about 280 mg / kg, about 180 mg / kg to about 260 mg / kg, about 180 mg / kg to about 240 mg / kg,About 180 mg / kg to about 220 mg / kg, about 180 mg / kg to about 200 mg / kg, about 200 mg / kg to about 400 mg / kg, about 200 mg / kg to about 380 mg / kg, about 200 mg / kg to about 360 mg / kg, about 200 mg / kg to about 340 mg / kg, about 200 mg / kg to about 320 mg / kg, about 200 mg / kg to about 300 mg / kg, about 200 mg / kg to about 280 mg / kg, about 200 mg / kg to about 260 mg / kg, about 200 mg / kg to about 240 mg / kg, about 200 mg / kg to about 220 mg / kg, about 220 mg / kg to about 400 mg / kg, about 220 mg / kg to about 380 mg / kg, about 220 mg / kg to about 360 mg / kg, about 220 mg / kg to about 340 mg / kg, about 220 mg / kg to about 320 mg / kg, about 220 mg / kg to about 300 mg / kg, about 220 mg / kg to about 280 mg / kg, about 220 mg / kg to about 260 mg / kg, about 220 mg / kg to about 240 mg / kg, about 240 mg / kg to about 400 mg / kg, about 240 mg / kg to about 380 mg / kg, about 240 mg / kg to about 360 mg / kg, about 240 mg / kg to about 340 mg / kg, about 240 mg / kg to about 320 mg / kg, about 240 mg / kg to about 300 mg / kg, about 240 mg / kg to about 280 mg / kg, about 240 mg / kg to about 260 mg / kg, about 260 mg / kg to about 400 mg / kg, about 260 mg / kg to about 380 mg / kg, about 260 mg / kg to about 360 mg / kg, about 260 mg / kg to about 340 mg / kg, about 260 mg / kg to about 320 mg / kg, about 260 mg / kg to about 300 mg / kg, about 260 mg / kg to about 280 mg / kg, about 280 mg / kg to about 400 mg / kg, about 280 mg / kg to about 380 mg / kg, about 280 mg / kg to about 360 mg / kg, about 280 mg / kg to about 340 mg / kg, about 280 mg / kg to about 320 mg / kg, about 280 mg / kg to about 300 mg / kg, about 300 mg / kg to about 400 mg / kg, about 300 mg / kg to about 380 mg / kg, about 300 mg / kg to about 360 mg / kg, about 300 mg / kg to about 340 mg / kg, about 300 mg / kg to about 320 mg / kg, about 320 mg / kg to about 400 mg / kg, about 320 mg / kg to about 380 mg / kg, about 320 mg / kg to about 360 mg / kg,The method includes administering about 320 mg / kg to about 340 mg / kg, about 340 mg / kg to about 400 mg / kg, about 340 mg / kg to about 380 mg / kg, about 340 mg / kg to about 360 mg / kg, about 360 mg / kg to about 400 mg / kg, about 360 mg / kg to about 380 mg / kg, or about 380 mg / kg to about 400 mg / kg of sodium phenylbutyrate.

[0125] In some embodiments, TURSO is administered at about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, or about 70 mg / kg of body weight per day. In some embodiments, sodium phenylbutyrate is administered at about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 120 mg / kg, about 140 mg / kg, about 160 mg / kg, about 180 mg / kg, about 200 mg / kg, about 220 mg / kg, about 240 mg / kg, about 260 mg / kg, about 280 mg / kg, about 300 mg / kg, about 320 mg / kg, about 340 mg / kg, about 360 mg / kg, about 380 mg / kg, or about 400 mg / kg of body weight per day.

[0126] The methods described herein can be used to treat or ameliorate at least one symptom of ALS in a subject, slow the progression of ALS, extend the survival of a subject exhibiting one or more symptoms of ALS, prevent or reduce at least one adverse event (e.g., a serious adverse event) associated with ALS or its treatment, and reduce, maintain or improve deterioration of muscle strength, respiratory muscle / pulmonary function and / or fine motor function. The methods can also be used to prophylactically treat a subject at risk of developing ALS (e.g., a subject with a family history of ALS) or suspected of developing ALS (e.g., a subject with at least one symptom of ALS, a symptom of upper motor neuron degeneration and / or a symptom of lower motor neuron degeneration, but not yet sufficiently symptomatic to be fully diagnosed with ALS at the time). The methods are useful for ameliorating at least one symptom of lower motor neuron degeneration or upper motor neuron degeneration.

[0127] The methods disclosed herein are also useful for preventing or alleviating constipation (e.g., constipation associated with ALS) and ameliorating at least one symptom of benign fasciculation syndrome or fasciculations-fasciculations syndrome.

[0128] As disclosed herein, the methods can be used to treat a subject who has been diagnosed with ALS, who is at risk for developing ALS, or who is suspected of having ALS. For example, the subject may have been diagnosed with ALS for 24 months or less (e.g., within a range therein). For example, the subject may have been diagnosed with ALS within one week, or may have been diagnosed with ALS on the same day that the treatment of the disclosure is administered. The subject may have exhibited one or more symptoms of ALS for 24 months or less (e.g., within a range), the rate of progression of ALS (ΔFS) may be about 0.50 or more (e.g., within a range), the ALSFRS-R score may be 40 or less (e.g., within a range), the ALSFRS-R score may have decreased by an average of about 0.8 to about 2 points (e.g., within a range) per month for the past 3 to 12 months, and the subject may have an ALS-related symptom that is associated with a disease ... The patient may have a mutation in one or more genes and / or a CSF or blood level of pNF-H of about 300 pg / mL or more (e.g., about 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 3000, 3200, 3500, 3800 or 4000 pg / mL or more). In some aspects, the pNF-H level in the serum of a subject in the methods described herein may be about 70 to about 1200 pg / mL (e.g., about 70 to about 1000, about 70 to about 800, about 80 to about 600, or about 90 to about 400 pg / mL).In some embodiments, the subject's CSF pNF-H level in the methods described herein may be about 1000 to about 5000 pg / mL (e.g., about 1500 to about 4000 or about 2000 to about 3000 pg / mL). The subject's CSF or blood NfL level may be about 50 pg / mL or more (e.g., about 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 pg / mL or more). In some embodiments, the subject's serum NfL level in the methods described herein may be about 50 to about 300 pg / mL (e.g., a range within this range). In some embodiments, a subject in the methods described herein may have a level of NfL in the CSF of about 2000 to about 40,000 pg / mL (eg, any range within this range).

[0129] The methods described in this disclosure can include treatment of ALS itself as well as treatment of one or more symptoms of ALS. "Treating" ALS does not require 100% disappearance of the disease or disease symptoms in the subject. Alleviation or reduction in the severity of the symptoms or characteristics of the disease is contemplated. "Treating" ALS can also refer to delaying the onset of symptoms (e.g., prevention), delaying the progression of symptoms, or delaying the loss of function associated with the disease. "Treating" ALS can also refer to eliminating or reducing one or more side effects of treatment (e.g., caused by an ALS therapeutic agent disclosed herein or known in the art). "Treating" ALS can also refer to eliminating or reducing one or more direct or indirect effects of ALS progression, such as an increase in the number of falls, lacerations, or GI problems. A subject may be at risk for ALS rather than exhibiting symptoms of ALS. For example, a subject may have a mutation in a gene associated with ALS, may have a family history of ALS, or may have elevated levels of a biomarker indicative of a risk of developing ALS. The subject may exhibit early signs of disease or symptoms of established or progressive disease. This disclosure contemplates any degree of delay in onset of symptoms, alleviation of one or more of the disease symptoms, or slowing of progression of one or more of the disease symptoms (e.g., improvement as measured by the ALSFRS-R or maintenance of an ALSFRS-R rating, which signals a slowing of disease progression). This specification also contemplates alleviation or reduction in the severity of symptoms or characteristics of benign fasciculation syndrome and fasciculation-fasciculation syndrome.

[0130] The treatments provided in this disclosure can be initiated at an advanced stage of the disease. For example, treatment can be initiated before the onset of ALS symptoms (e.g., in subjects at risk of developing ALS), at the onset, or shortly after onset, when a skilled physician observes one or more symptoms that lead the subject to suspect that the subject is developing ALS (e.g., muscle weakness, muscle fasciculations, and / or muscle spasms). Treatment can also be initiated at a later stage. For example, treatment can be initiated at an advanced stage of the disease, for example, when muscle weakness and muscle atrophy spread to different parts of the body and the subject has more serious problems with mobility. At or before the onset of treatment, the subject may experience muscle tightness and stiffness (spasticity), worsening of reflexes (hyperreflexia), muscle weakness and muscle atrophy, muscle spasms, and / or sudden twitching of muscles seen under the skin (fasciculations), difficulty swallowing (dysphagia), difficulty speaking or forming words (dysarthria).

[0131] Treatment methods can include single doses, multiple doses, and repeated doses as needed to prevent or treat ALS or at least one symptom of ALS. There can be a single dose for the duration of preventative treatment, and treatment can continue (e.g., multiple doses), for example, for years or for the life of the subject. For example, a subject at risk for ALS can continue treatment for days, weeks, months, or even years using the methods provided herein to prevent the disease from developing or occurring. In some embodiments, treatment methods can include assessing the level of disease in the subject before, during, and / or after treatment. The treatments provided herein can be performed one or more times daily, or can be performed weekly or monthly. In some embodiments, treatment can be continued until a reduction in the level of disease in the subject is detected. The methods provided herein, in some embodiments, may begin to show efficacy (e.g., alleviation of one or more symptoms of ALS, improvement as measured by the ALSFRS-R, or maintenance of an ALSFRS-R rating) in less than 60 days (e.g., less than 50, 45, 40, 35, 30, 25, 20, 15, or 10 days) after the first administration, or after fewer than 60 administrations (e.g., less than 50, 45, 40, 35, 30, 25, 20, 15, or 10 administrations).

[0132] The terms "administer", "administering" or "administration" as used herein refer to administering a drug described herein to a subject using methods known in the art, e.g., ingesting, injecting, implanting, absorbing or inhaling the drug, regardless of its form. In some embodiments, one or more of the compounds disclosed herein can be administered to a subject by oral and / or topical (e.g., intranasal) ingestion. For example, the methods herein include administration of an effective amount of a compound or composition of a compound to achieve a desired or described effect. The specific dosage and treatment regimen for a particular subject will depend on a variety of factors, including the activity of the specific compound employed, age, weight, health, sex, diet, time of administration, excretion rate, drug combination, severity and course of the disease, condition or symptom, predisposition of the subject to the disease, condition or symptom, and the judgment of the treating physician.

[0133] After administration of the bile acid or a pharma- ceutically acceptable salt thereof and the phenylbutyric acid compound, the subject may be evaluated to detect, assess, or determine the level of ALS disease. In some embodiments, treatment can be continued until a change (e.g., a decrease) in the level of disease in the subject is detected.

[0134] After a patient's condition improves (e.g., there is a change (e.g., a decrease) in the level of disease in the subject), a maintenance dose of a compound, composition, or combination of the disclosure may be administered, if necessary. Thereafter, the dosage or frequency of administration, or both, may be reduced, depending on the symptoms, to a level at which the improvement in the condition is maintained. However, patients may require intermittent treatment in the long term for recurrence of disease symptoms.

[0135] IV. Symptom and Outcome Measurements The present disclosure further provides methods for assessing ALS symptoms, monitoring ALS progression, and assessing a subject's response to treatment, including, but not limited to, physical assessment by a physician, weight, electrocardiogram (ECG), ALS Functional Rating Scale (ALSFRS or ALSFRS-R) score, respiratory function, muscle strength, cognitive / behavioral function, quality of life, and voice analysis.

[0136] Respiratory function of a subject may be measured, for example, by measuring vital capacity (including forced vital capacity and resting vital capacity), maximum mid-expiratory flow rate (MMERF), forced vital capacity (FVC), and forced expiratory volume in 1 second (FEV 1 ). Muscle strength can be measured, for example, by handheld dynamometry (HHD), grip dynamometry, manual muscle testing (MMT), electrical impedance myography (EIM), maximal voluntary isometric contraction test (MVICT), motor unit number estimation (MUNE), accurate limb isometric muscle strength test (ATLIS), or a combination thereof. Cognitive / behavioral function can be evaluated, for example, by the ALS Depression Scale (ADI-12), Beck Depression Scale (BDI) and Hospital Anxiety and Depression Scale (HADS) questionnaires. Quality of life can be evaluated, for example, by the ALS Assessment Questionnaire (ALSAQ-40). Akt levels, Akt phosphorylation and / or the ratio of pAkt to dAkt can also be used to assess the progression of disease and response to treatment in subjects (see, for example, WO 2012 / 160563).

[0137] The level of biomarkers in a subject's CSF or blood sample is a useful indicator of the subject's ALS progression and responsiveness to the treatment methods provided herein. Biomarkers such as, but not limited to, phosphorylated neurofilament heavy chain (pNF-H), neurofilament medium chain, neurofilament light chain (NFL), S100-β, cystatin C, chitotriosidase, CRP, TDP-43, uric acid, and certain microRNAs can be analyzed for this purpose. Urine tests can also be used to assess the subject's response to treatment. The levels of biomarkers, such as, but not limited to, p75ECD and ketones, can be analyzed in urine samples. Creatinine levels can be measured in urine and blood samples. In some embodiments, the methods provided herein result in an increase or decrease in ketone levels in a subject's urine sample. Medical imaging, including but not limited to, MRI and PET imaging of markers such as translocator protein (TSPO), can also be utilized.

[0138] Muscle strength The subject's muscle strength can be assessed using methods known in the art. Quantitative measurements of force generally demonstrate a linear and predictable loss of force in ALS patients. The Tufts Quantitative Neuromuscular Test (TQNE) can be used to perform quantitative measurements using fixed strain gauges. The TQNE measures isometric strength of 20 muscle groups and produces interval strength data for both strong and weak muscles (see, e.g., Andres et al., Neurology 36:937-941, 1986). Handheld dynamometry (HHD) tests isometric strength of specific muscles in the arms and legs and produces interval level data (see, e.g., Shefne JM, Neurotherapeutics 14:154-160, 2017).

[0139] The Accurate Limb Isometric Strength Test (ATLIS) can be used to measure both strong and weak muscle groups using fixed wireless load cells (see, e.g., Andres et al., Muscle Nerve 56(4):710-715, 2017). The ATLIS test assesses the strength of 12 muscle groups, which reflects the power of a subject's lower limbs, upper limbs, and the subject's grip strength. In some embodiments, the ATLIS test detects changes in muscle strength before changes in function are observed.

[0140] The methods provided herein may improve, maintain, or slow the decline of a subject's muscle strength (e.g., lower limb strength, upper limb strength, or grip strength) as assessed by a suitable method described herein. The methods may improve the subject's upper limb strength to a greater extent than other muscle groups. For example, the effect on muscle strength may be reflected in one or more muscle groups selected from the quadriceps, biceps, femoral flexors, triceps, and tibialis anterior.

[0141] Muscle strength can be assessed by HHD, grip strength dynamometry, MMT, EIM, MVICT, MUNE, ATLIS, or a combination thereof, before, during, and / or after administration of the bile acid or a pharma- ceutically acceptable salt thereof and the phenylbutyric acid compound.

[0142] In some embodiments, muscle strength is assessed with the ATLIS. ATLIS total score and upper and lower limb ATLIS scores can be assessed. The methods disclosed herein can result in a rate of decline in the subject's ATLIS total score of about 3.50 PPN or less (e.g., about 3.45, 3.40, 3.35, 3.30, 3.25, 3.20, 3.15, 3.10, 3.05, 3.00 PPN or less) per month. The methods disclosed herein can also result in a reduction in the average rate of decline in the subject's ATLIS total score of at least about 0.2 PPN (e.g., at least about 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 PPN) per month compared to untreated subjects. The average rate of decline in the subject's upper extremity ATLIS score can be reduced by at least about 0.50 PPN (e.g., at least about 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, or 0.90 PPN) per month compared to a subject not receiving a treatment described herein. The average rate of decline in the subject's lower extremity ATLIS score can be reduced by at least about 0.20 PPN (e.g., at least about 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, or 0.60 PPN) per month compared to a subject not receiving a treatment described herein. In some embodiments, the improvement or maintenance of the subject's muscle strength can occur in less than 60 days (e.g., less than 55, 50, 45, 40, 30, 25, or 20 days) from the first treatment. PPN represents a percentage of expected normal strength based on age, sex, weight, and height.

[0143] Pulmonary function ALS is a progressive neurodegenerative disease that ultimately leads to respiratory failure and death. Vital capacity (VC), maximum mid-expiratory flow rate (MMERF), forced vital capacity (FVC), resting vital capacity (SVC) and forced expiratory volume in 1 second (FEV 1Pulmonary function tests such as pulmonary function test (PFRT) can be used to monitor ALS progression and / or a subject's response to treatment. The rate of decline in respiratory function in ALS patients, as measured by vital capacity (VC), may be about 2.24% (±6.9) of predicted value per month, on average. In some embodiments, pulmonary function test measures are related to survival time (see, e.g., Moufavi et al. Iran J Neurol 13(3): 131-137, 2014). Additional measures such as maximum inspiratory and expiratory pressures, arterial blood gas measurements, and overnight oxygen saturation measurements may provide earlier evidence of functional impairment. Comparison of vital capacity in the upright and supine positions may also provide an earlier indicator of ventilatory muscle decline.

[0144] The methods provided herein may improve or maintain respiratory muscle and / or lung function or delay deterioration of respiratory muscle and / or lung function of a subject. The respiratory muscle and / or lung function of a subject can be assessed by any suitable method described herein or known in the art. In some embodiments, the function of the human subject's respiratory muscle is assessed based on the subject's SVC. In some embodiments of the methods of improving, maintaining, or delaying deterioration of respiratory muscle function in a human subject described herein, the treatment results in an average rate of decline in the subject's SVC of about 3.50 PPN or less (e.g., about 3.45, 3.40, 3.35, 3.30, 3.25, 3.20, 3.15, 3.10, 3.05, or 3.00 PPN or less) per month. In some embodiments, the treatment reduces the average rate of decline in the subject's SVC by at least about 0.5 PPN (e.g., at least about 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1.00 PPN) per month compared to subjects not receiving the treatment. In some embodiments, the improvement or maintenance of the subject's pulmonary function can occur in less than 60 days (e.g., less than 55, 50, 45, 40, 30, 25, or 20 days) from the first administration. In some embodiments, the subject's pulmonary function progresses more slowly than expected within 60 days of the first administration.

[0145] Adverse events Subjects treated with the methods provided herein may exhibit fewer adverse events (e.g., adverse events disclosed herein) or may exhibit one or more of the adverse events to a lesser extent than untreated subjects. Exemplary adverse events include gastrointestinal-related adverse events (e.g., abdominal pain, gastritis, nausea, vomiting, constipation, rectal bleeding, peptic ulcer, pancreatitis); hematological adverse events (e.g., aplastic anemia and ecchymosis); cardiovascular adverse events (e.g., arrhythmia and edema); renal adverse events (e.g., renal tubular acidosis); psychiatric adverse events (e.g., depression); skin adverse events (e.g., rash); and miscellaneous adverse events (e.g., syncope and weight gain). In some embodiments, the methods provided herein do not result in constipation, neck pain, headache, falls, dry mouth, muscle weakness, falls, lacerations, and alanine aminotransferase (ALT) increases, or result in minimal symptoms thereof. In some aspects, the adverse event is a serious adverse event, such as, but not limited to, a respiratory adverse event, a fall, or a laceration.

[0146] In some aspects, administration of a combination of a bile acid and a phenylbutyric acid compound can result in fewer adverse events (e.g., the adverse events disclosed herein) or less severe adverse events compared to administration of a bile acid or a phenylbutyric acid compound alone.

[0147] The average survival time of ALS patients may vary. The median survival time may be about 30 to about 32 months from onset of symptoms, or about 14 to about 20 months from diagnosis. Subjects with bulbar onset ALS may have a survival time of about 6 months to about 84 months from onset of symptoms, with a median of about 27 months. The methods provided herein, in some embodiments, may extend the survival time of a subject with ALS by at least 1 month (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 28, 32, 36, 40, 50, 60, 70, 80, or 90 months). The methods provided herein, in some aspects, may delay the onset of ventilator dependency or tracheotomy by at least 1 month (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 28, 32, 36, 40, 50, 60, 70, 80, or 90 months).

[0148] The methods provided herein may reduce the rate of disease progression, where the subject's average ALSFRS-R score reduction over one month is at least about 0.2 points (e.g., at least about 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, or 1.5) compared to a subject not receiving the treatment. The methods provided herein may slow the progression of one or more categories assessed on the ALSFRS scale, including speech, salivation, swallowing, writing, feeding activities, dressing and care activities, activities in bed, ambulation, stair climbing, dyspnea, orthopnea, and respiratory failure. In some aspects, the methods provided herein improve or slow the deterioration of a subject's fine motor function as assessed in one or more categories of the ALSFRS-R scale (e.g., writing, feeding, or dressing and self-care).

[0149] In some embodiments, the methods provided herein are more effective in treating subjects who are about 18 to about 50 years of age (e.g., about 18 to about 45, about 18 to about 40, about 18 to about 35, about 18 to about 30, about 18 to about 25, or about 18 to about 22 years of age) compared to subjects who are 50 years of age or older (e.g., 55, 60, 65, 70, 75, or 80 years of age or older). In some embodiments, the methods provided herein are more effective in treating subjects who have been diagnosed with ALS and / or have exhibited symptoms of ALS for less than about 24 months (e.g., less than about 22, 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, or 1 month) compared to subjects who have been diagnosed with ALS and / or have exhibited symptoms of ALS for more than about 24 months (e.g., about 26, 28, 30, 32, 34, 36, 40, 45, 50, 55, or 60 months). In some aspects, the methods provided herein are more effective in treating subjects who have been diagnosed with ALS and / or have exhibited symptoms of ALS for more than about 24 months (e.g., more than about 26, 28, 30, 32, 34, 36, 40, 45, 50, 55, or 60 months) compared to subjects who have been diagnosed with ALS and / or have exhibited symptoms of ALS for less than about 24 months (e.g., less than about 22, 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, or 1 months).

[0150] In some embodiments, responsiveness to the treatment methods provided herein is gender-dependent. The methods provided herein may be more or less effective in treating female subjects compared to male subjects. For example, female subjects may show improvement (e.g., as measured by the ALSFRS-R or other endpoints described herein) sooner or later than male subjects when treated at similar stages of disease progression. Female subjects may, in some embodiments, show greater or less improvement (e.g., as measured by the ALSFRS-R or other endpoints described herein) than male subjects when treated at similar stages of disease progression. The pharmacokinetics of bile acids and phenylbutyric acid compounds may be the same or different in female and male subjects.

[0151] V. Further Therapeutic Agents The methods described herein can further include administering to the subject one or more additional therapeutic agents, e.g., in an amount effective to treat or achieve modulation of at least one symptom of ALS. Any known ALS therapeutic agent known in the art can be used as the additional therapeutic agent. Exemplary therapeutic agents include riluzole (C 8 H 5 F 3 N 2 OS (e.g. sold under the names Rilutek® and Tiglutik®), edaravone (e.g. sold under the names Radicava® and Radicut®), dextromethorphan, anticholinergic medicines and psychiatric medicines (e.g. antidepressants, antipsychotics, anxiolytics / hypnotics, mood stabilizers and stimulants).

[0152] Neudexta® is a combination of dextromethorphan and quinidine and can be used to treat emotional dysregulation (inappropriate laughing or crying). Anticholinergic medications and antidepressants can be used to treat excessive salivation. Representative anticholinergic medications include glycopyrrolate, scopolamine, atropine (Atropen®), belladonna alkaloids, benztropine mesylate (Cogentin®), clidinium, cyclopentolate (Cyclogyl®), darifenacin (Enablex®), dicyclomine, fesoterodine (Tobie®), flavoxate (Urispas®), glycopyrrolate, homatropine hydrobromide, and hyoscyamine. (Levsinex®), ipratropium (Atrovent®), orphenadrine, oxybutynin (Ditropan® XL), propantheline (Pro-Bansine®), scopolamine, methscopolamine, solifenacin (Vesicare®), tiotropium (Spiriva®), tolterodine (Detrol®), trihexyphenidyl, trospium, and diphenhydramine (Benadryl®). Representative antidepressants include selective serotonin inhibitors, serotonin-norepinephrine reuptake inhibitors, serotonin modulators and stimulants, serotonin antagonists and reuptake inhibitors, norepinephrine reuptake inhibitors, norepinephrine-dopamine reuptake inhibitors, tricyclic antidepressants, tetracyclic antidepressants, monoamine oxidase inhibitors, and NMDA receptor antagonists.

[0153] The additional therapeutic agent can be administered before the first administration and / or after the last administration of the composition comprising the bile acid or a pharma- ceutically acceptable salt thereof (e.g., TURSO) and the phenylbutyric acid compound (e.g., sodium phenylbutyrate). In some embodiments, the subject in the methods described herein has been previously treated with one or more additional therapeutic agents (e.g., an additional therapeutic agent described herein, such as riluzole or edaravone). In some embodiments, the subject has been administered a stable dose of a therapeutic agent (e.g., riluzole and / or edaravone) for at least 30 days (e.g., at least 40, 50, 60, 90, or 120 days) prior to administration of the compositions disclosed herein. The absorption, metabolism, and / or excretion of the additional therapeutic agent can be affected by the bile acid or a pharma- ceutically acceptable salt thereof and / or the phenylbutyric acid compound. For example, co-administration of sodium phenylbutyrate with riluzole or edaravone can increase the subject's exposure to riluzole or edaravone. Co-administration of riluzole with a bile acid or a pharma- ceutically acceptable salt thereof and a phenylbutyric acid compound can improve riluzole tolerance by a subject compared to administration of riluzole alone.

[0154] The combination of a bile acid or a pharma- ceutically acceptable salt thereof, a phenylbutyric acid compound, and one or more additional therapeutic agents may have a synergistic effect in the treatment of ALS. When administered in combination with a bile acid or a pharma- ceutically acceptable salt thereof and a phenylbutyric acid compound, a smaller dose of the additional therapeutic agent may be required to achieve the same pharmacological effect. In some embodiments, the amount of the additional therapeutic agent administered in combination with a bile acid or a pharma- ceutically acceptable salt thereof and a phenylbutyric acid compound may be reduced by at least about 10% (e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or 55%) compared to the dosage of the additional therapeutic agent administered alone. Additionally or alternatively, the methods disclosed herein may reduce the number of doses required of the other therapeutic agent (e.g., another ALS therapeutic agent) to achieve the same pharmacological effect.

[0155] The bile acid or its pharma- ceutically acceptable salt and phenylbutyric acid compound can be administered immediately after a meal (e.g., within 2 hours after a meal) or under fasting conditions. The subject may have consumed food (e.g., solid or liquid food) within 2 hours before administration of the bile acid or its pharma- ceutically acceptable salt and / or phenylbutyric acid compound; or consume food within 2 hours after administration of one or both of the compounds. Food may affect the rate and extent of absorption of the bile acid or its pharma- ceutically acceptable salt and / or phenylbutyric acid compound. For example, food may alter the bioavailability of a compound by delaying gastric emptying, stimulating bile flow, altering gastrointestinal pH, increasing splanchnic blood flow, altering the intraluminal metabolism of a substance, or physically or chemically interacting with a dosage form or substance. The nutrients and calories of the meal, the amount of the meal, and the temperature of the meal may cause physiological changes in the GI tract in ways that affect drug transit time, intraluminal dissolution, drug permeability, and systemic availability. In general, meals that are high in total calories and fat content are more likely to affect GI physiology, thereby resulting in a greater impact on drug bioavailability. The methods provided herein can further include feeding the subject multiple foods, e.g., within 2 hours (e.g., within 1.5 hours, 1 hour, or 0.5 hours) before or after administering the bile acid or a pharma- ceutically acceptable salt thereof and / or phenylbutyric acid compound. EXAMPLES

[0156] Additional aspects are disclosed in further detail in the following examples, which are provided for illustrative purposes and are not intended to limit the scope of this disclosure or the claims.

[0157] Example 1 Evaluation of the Safety, Tolerability, Efficacy, and Activity of AMX0035, a Fixed Combination of Phenylbutyrate (PB) and Taurursodiol (TURSO), for the Treatment of ALS The safety, tolerability, efficacy, pharmacokinetics, and bioactivity of AMX0035 (a combination of sodium phenylbutyrate 3 g and TURSO 1 g) were evaluated in a multicenter, randomized, double-blind, placebo-controlled, 24-week study. Participants were randomized 2:1 to receive AMX0035 (once daily for 3 weeks, then twice daily) or placebo.

[0158] Primary endpoint Safety and tolerability, defined as the proportion of subjects who continue on study drug until planned discontinuation Rate of decline (slope of decline) of the ALS Functional Rating Scale (ALSFRS-R)

[0159] Secondary endpoints -Evaluate the effect of AMX0035 on the rate of decline in isometric strength as measured by the Accurate Limb Isometric Strength Test (ATLIS) -Evaluate the effect of AMX0035 on disease progression as measured by decline in resting vital capacity (SVC) -To evaluate the effect of AMX0035 on survival, length of hospital stay and tracheotomy Assessment of the effect of AMX0035 on biomarkers including phosphorylated axonal neurofilament H subunit (pNF-H) levels and translocator protein 18 kDa (TSPO) uptake Evaluation of concentration-response models of TUDCA and phenylbutyrate at steady state following twice daily administration of 4 g AMX0035

[0160] Selection Criteria 1. Male or female aged 18-80 2. Sporadic or familial ALS diagnosed as definite cases as defined by the revised El Escorial criteria of the World Federation of Neurology 3. Less than 18 months since the onset of ALS symptoms 4. Ability to provide informed consent and comply with study procedures 5. Geographical access to the facility 6. Resting vital capacity (SVC) at the time of screening visit is greater than 60% of the value predicted for sex, height, and age. 7. Subjects must be either riluzole-naive or on a stable dose of riluzole for at least 30 days prior to the screening visit. Riluzole-naive subjects are eligible to participate in the study. 8. Females of childbearing potential (e.g., not postmenopausal for at least 1 year and not surgically sterile) must agree to practice adequate birth control for the duration of the study and for 3 months after the last dose of study drug. a. Women must not plan to become pregnant during the study and for 3 months after the last dose of study drug. 9. Men must agree to use contraception for the duration of the study and for 3 months after the last dose of study drug. a. Men must not plan to father a child or donate sperm during the study period or for 3 months after the last dose of study drug.

[0161] Exclusion criteria 1. Presence of tracheotomy 2. Exposure to PB, TUDCA, or UDCA within 3 months prior to the screening visit or plans to use these medications during the course of the study 3. History of allergy to PB or bile salts 4. Abnormal liver function defined as AST and / or ALT greater than 3 times the upper limit of normal 5.eGFR<60mL / min / 1.73m 2 Renal failure as defined by 6.Uncontrolled arterial hypertension (SBP>160mmHg or DBP>100mmHg) at the time of screening visit 7. Pregnant or breastfeeding women 8. History of cholecystectomy 9. Biliary tract disease that impedes biliary flow, including active cholecystitis, primary biliary cirrhosis, sclerosing cholangitis, gallbladder cancer, gallbladder polyps, gallbladder gangrene, and gallbladder abscess 10. History of Class III / IV heart failure (NYHA) 11. Severe pancreatic or intestinal disorders that may alter the enterohepatic circulation and absorption of TUDCA, including biliary tract infection, pancreatitis, and ileal resection. 12. The presence of unstable psychiatric illness, cognitive impairment, dementia, or substance abuse that, in the opinion of the institution's responsible physician, would impair the subject's ability to provide informed consent. 13. Patients with cancer, except for: basal cell carcinoma or successfully treated squamous cell carcinoma of the skin; cervical intraepithelial carcinoma; prostatic intraepithelial carcinoma; or other malignancies that have been treated and have had no evidence of recurrence for at least 3 years. 14.A clinically significant unstable medical condition (other than ALS) that would pose a risk to the subject if the subject were to participate in the study. 15. Active participation in an ALS clinical trial evaluating a small molecule within 30 days of the screening visit. 16. Exposure at any time to investigational cell and gene therapies in subjects with ALS (off-label use or investigational drugs) 17. Exposure to an investigational monoclonal antibody for ALS (off-label use or investigational drug) within 90 days of screening. If already exposed to a monoclonal antibody during an investigational ALS trial, a 90-day washout period is required prior to screening. 18. Implantation of Diaphragmatic Pacing System (DPS) 19. Any matter that, in the opinion of the Investigator, may expose the subject to increased risk or prevent the subject from fully complying with or completing the study. 20. Exposure to any of the following prohibited drugs: Prohibited medications for all subjects include HDAC inhibitors including valproate, vorinostat (Zolinza), romidepsin, thidamide, panobinostat, lithium, butyrate, and suramin; probenecid; bile acid sequestrants including cholestyramine and cholestyramine light, questran and questran light, Welchol, colestid and flavored colestid, and Prevalite.

[0162] result At baseline, the placebo and AMX0035 arms were comparable and did not differ statistically significantly with respect to demographics (gender, age, race and ethnicity, weight, height and body mass index) and disease characteristics (time since onset of ALS diagnosis, time since onset of ALS symptoms, riluzole use, duration of edaravone use, duration of riluzole use, family history of ALS, site of ALS onset, predicted SVC%, ALSFRS-R subscale and total scores, and ATLIS subscale and total scores). However, more patients in the placebo arm (50%) were receiving edaravone at or before study entry compared with the AMX0035 arm (25.8%) (p=0.0078).

[0163] Primary endpoint Treatment with AMX0035 slowed the rate of decline in ALSFRS-R scores, a measure of function in daily activities. The estimated mean slopes of ALSFRS-R total scores in the partially modified all-inclusive (mITT) population were -1.24 and -1.66 points / month for active and placebo, respectively (difference = 0.42 points / month; 95% confidence interval [CI], 0.03 to 0.81; P = .03) (Figure 1). After 24 weeks, there was an estimated absolute difference of 2.32 points (on a 48-point scale) between the treatment and placebo groups in least-squares mean ALSFRS-R total scores.

[0164] Secondary endpoints As shown in Figure 1, the mean rate of decline in ATLIS total score was -3.03 PPN / month vs. -3.54 PPN / month for active vs. placebo, respectively (difference = 0.51 PPN / month; 95% CI, -0.12 to 1.14). Between-group differences (active minus placebo) in the mean rate of decline in upper and lower limb ATLIS scores were 0.77 PPN / month (95% CI, 0.03 to 1.52) and 0.38 PPN / month (95% CI, -0.40 to 1.16), respectively. The mean rate of decline in SVC was -3.10 PPN / month vs. -4.03 PPN / month for active vs. placebo, respectively (difference = 0.93 PPN / month; 95% CI, -0.10 to 1.95).

[0165] The cumulative hazard ratio for death, tracheotomy, or hospitalization in the active treatment group compared with the placebo group was 0.53 (95% CI, 0.27 to 1.05) (Figure 1).

[0166] Open-label study extension At the end of the 24-week, parallel-group phase of the study, participants were eligible to enroll in an open-label extension (OLE) study in which participants and investigators remained blinded to the original treatment arm.

[0167] Overall survival was analyzed for all subjects randomly assigned initially to the trial (intention-to-treat analysis) and patients assigned to AMX0035 were compared with patients assigned to placebo. Participants initially randomly assigned to AMX0035 received AMX0035 for a median of 4.8 months longer than participants assigned to placebo. The risk of death was 36% lower in participants initially randomly assigned to AMX0035 compared with participants assigned to placebo (HR 0.64; 95% CI 0.42 to 0.94; P = 0.048). Median survival was 23.5 months (95% CI 19.5 to 26.2 months) in the group previously randomly assigned to AMX0035 and 18.7 months (95% CI 13.5 to 24.4 months) in the group previously randomly assigned to placebo.

[0168] Examples 2 to 7 Evaluation of human drug transporter interactions of six test products The purpose of Examples 2 to 7 was to evaluate the interaction of six test articles, which are substrates and / or inhibitors of the bile salt efflux pump (BSEP), an efflux transporter, with human drug transporters.

[0169] The criteria used to determine whether a test article is a substrate for an efflux transporter were an efflux ratio of greater than 2 and greater than 50% inhibition by the inhibitor. The criteria used to determine whether a test article is an inhibitor of an efflux transporter were greater than 25% inhibition of the probe substrate net efflux activity; inhibition should be concentration-dependent by the test article. If inhibited, the 50% inhibitory concentration (IC 50 ) values ​​were estimated based on the dose used for each test article. Drug-drug interaction (DDI) potentials for each test article were calculated using the appropriate R value formula from the FDA guidance (Food and Drug Administration, 2020).

[0170] Materials and Methods The following materials and methods were used for Examples 2-7.

[0171] Vesicle uptake procedure Membrane vesicles prepared from baculovirus-infected insect cells (Sf9) expressing the BSEP protein were obtained from Genomembrane (Thermo Fisher Scientific, Waltham, Massachusetts).

[0172] BSEP-transfected Sf9 membrane vesicles were thawed and placed on wet ice prior to use. Assay uptake buffer and membrane vesicles were diluted with the appropriate substrate ( 3H-taurocholic acid, TCA), inhibitor (cyclosporine A) or test article solution, and the mixture was added to the appropriate wells of a 96-well filter plate (50 μg / well). The plate and adenosine triphosphate (ATP) and adenosine monophosphate (AMP) solutions were pre-incubated at 37° C. for 5 minutes. The reaction was initiated by adding ATP to the wells; AMP was added to the negative control wells to a final concentration of 5 mM. The plate was incubated at 37° C. for 30 minutes. The reaction was terminated by the addition of cold wash buffer, and the plate was kept on wet ice until filtration. Cold blocking buffer was added to the filter plate and positive pressure was applied. The filter plate was then washed with ice-cold wash buffer, and the incubation medium was transferred to the pre-treated filter plate and gentle positive pressure was applied. The filter plate was washed four times with cold wash buffer, ethanol was added to the filter plate, and the samples were eluted into a deep-well 96-well plate at ambient temperature for 10 minutes. Vesicles were extracted from the filter plates for LC-MS quantification. Where applicable, samples were stored at -20°C until analysis.

[0173] Evaluation of test products as substrates for BSEP Adenosine triphosphate (ATP)-dependent uptake of test articles (at concentrations shown in Table 1) by the transporters was performed according to the vesicular uptake procedure in the presence of vehicle or inhibitors.

[0174] [Table 1]

[0175] Uptake of the probe substrate in the presence or absence of inhibitors served as a control. The information on the probe substrates and selective inhibitors is summarized in Table 2.

[0176] [Table 2]

[0177] Evaluation of test articles as BSEP inhibitors Transporter uptake of radiolabeled probe substrates was performed according to the vesicular uptake procedure in the presence of a) vehicle, b) selective inhibitors, and c) test articles (at concentrations shown in Table 3). Probe substrate and selective inhibitor information is summarized in Table 2.

[0178] [Table 3]

[0179] Sample identification Samples were uniquely identified by tube / well number or plate location, cross-referenced with study number, transporter, incubation time, test article / substrate / inhibitor concentration and donor / receiver, if applicable.

[0180] calculation Efflux transporters (membrane vesicle assay: vesicle uptake) Uptake activation rate

[0181]

number

[0182] During the ceremony, amount ves Amount of substrate or test article in vesicles in each well (pmol) Time Incubation time (min) Protein Total protein in each well (mg)

[0183] ATP-dependent uptake activity ATP-dependent uptake activity = uptake ATP -Import AMP

[0184] Signal-to-Noise Ratio (SNR)

[0185]

number

[0186] During the ceremony, Import ATP Uptake in the presence of ATP (pmol / min / mg) Import AMP Uptake in the presence of AMP (pmol / min / mg)

[0187] Inhibition of uptake

[0188]

number

[0189] During the ceremony, Import +inh ATP-dependent uptake of substrate or test article in the presence of inhibitor or ATP-dependent uptake of substrate in the presence of test article (pmol / min / mg) Import -inh ATP-dependent uptake of substrate or test article alone (pmol / min / mg)

[0190] 4. Data Analysis Statistical analysis was limited to descriptive statistics such as means, standard deviations, relative standard deviations and regression analyses, where appropriate.

[0191] Data Acceptance Criteria The membrane vesicle efflux system was considered fully functional if the ATP-dependent uptake fold over vehicle or vector control was 4 or greater and selective inhibitors demonstrated greater than 50% inhibition of probe substrate uptake.

[0192] Test articles were identified as substrates if there was greater than 2-fold ATP-dependent uptake relative to vehicle control and greater than 50% inhibition by selective inhibitors. Test articles were identified as inhibitors if there was greater than 25% inhibition of probe substrate uptake.

[0193] Example 2 Evaluation of sodium phenylbutyrate as a substrate and / or inhibitor of BSEP Experiments were performed to assess whether sodium phenylbutyrate is a substrate and / or inhibitor of BSEP. Sodium phenylbutyrate at 1 μM and 10 μM was not detected in the uptake samples, indicating that sodium phenylbutyrate is not a substrate for BSEP. Sodium phenylbutyrate (25 μM and 250 μM) was not an inhibitor of BSEP.

[0194] Solubility, stability, and nonspecific binding of sodium phenylbutyrate. solubility The test article, sodium phenylbutyrate, was soluble up to 50,000 μM in methanol and 250 μM in assay buffer, with a final methanol content of 0.5% (v:v) for all concentrations tested.

[0195] stability The stability of 25 μM and 250 μM sodium phenylbutyrate was determined before and after freezing once in glass vials and after freezing / thawing aliquots three times in assay plates. Test articles were tested in transport buffer alone or in transport buffer containing 0.002% PS-80 or 1% HSA and recovery was assessed. Data are shown in Table 4. In transport buffer alone, recovery of sodium phenylbutyrate after a single freeze or after three freeze-thaw cycles was 98.9% or greater. In buffer containing 0.002% PS-80, recovery of sodium phenylbutyrate was 91.6% or greater after a single freeze and 95.3% or greater after three freeze-thaw cycles. In transport buffer with 1% HSA, recovery of sodium phenylbutyrate was 90.5% or greater after a single freeze and 94.8% or greater after three freeze-thaw cycles.

[0196] [Table 4]

[0197] non-specific binding Nonspecific binding of sodium phenylbutyrate to the transwell assay plate in the absence of cells was also evaluated. Sodium phenylbutyrate solutions (25 μM and 250 μM) were placed in the apical (250 μL) or basolateral (700 μL) chambers (donor chambers) of the transwell plate in triplicate wells, and the corresponding blank buffer was placed in the opposite receiver chamber and incubated at 37° C. for 2 h. Data are shown in Table 5. With transport buffer alone, recovery was ≥107% in the apical to basolateral direction and ≥82.8% in the basolateral to apical direction. With transport buffer containing 0.002% PS-80, recovery was ≥110% in the apical to basolateral direction and ≥86.9% in the basolateral to apical direction. With transport buffer containing 1% HSA, recovery was ≥107% in the apical to basolateral direction and ≥92.8% in the basolateral to apical direction. Stability and nonspecific binding results were similar, with acceptable recoveries from all buffer systems tested. Other test articles showed best recoveries in a transport buffer containing 0.002% PS-80; therefore, this buffer was also used in the sodium phenylbutyrate assay conditions.

[0198] [Table 5]

[0199] Evaluation of sodium phenylbutyrate as a substrate and inhibitor of BSEP (using membrane vesicles) ATP-dependent uptake of sodium phenylbutyrate (1 μM and 10 μM) into BSEP-transfected Sf9 membrane vesicles was tested alone or with the BSEP inhibitor cyclosporine A (20 μM) and the data are shown in Table 6. Sodium phenylbutyrate at 1 μM and 10 μM was not detected in the experimental samples, indicating that no uptake occurred. Sodium phenylbutyrate was not a substrate for BSEP.

[0200] [Table 6]

[0201] BSEP substrate 3 The ATP-dependent uptake of H-taurocholic acid (TCA) into BSEP vesicles, alone or with cyclosporine A (20 μM) or sodium phenylbutyrate (25 μM and 250 μM), is shown in Table 7. 3 The mean ATP-dependent uptake activity for 3H-taurocholate was 25.0 pmol / min / mg protein with a signal-to-noise ratio of 41.9. 3 H-TCA uptake was reduced to 2.85% in the presence of the BSEP inhibitor cyclosporine A. Sodium phenylbutyrate 3 It slightly inhibited H-TCA uptake, with 77.2% activity remaining at 25 μM, but the inhibition was not concentration-dependent and did not meet the criteria for identification as an inhibitor.

[0202] [Table 7]

[0203] Example 3 Evaluation of phenylacetic acid as a substrate and / or inhibitor of BSEP Experiments were performed to assess whether phenylacetic acid is a substrate and / or inhibitor of BSEP. The adenosine triphosphate (ATP)-dependent BSEP uptake activity for 1 μM and 10 μM phenylacetic acid was more than twice as high as adenosine monophosphate (AMP) uptake but was not inhibited by cyclosporine A, suggesting that phenylacetic acid is not a substrate for BSEP. Phenylacetic acid (750 μM and 7500 μM) potently inhibited probe substrate uptake by BSEP at 7500 μM, with an IC 50 was estimated to be less than 7500 μM.

[0204] Solubility, stability, and nonspecific binding of phenylacetic acid. solubility The test article, phenylacetic acid, was soluble up to 1500 mM in methanol and 7500 μM in assay buffer, with a final methanol content of 0.5% (v:v) at all concentrations tested.

[0205] stability The stability of phenylacetic acid at 250 μM and 2500 μM was determined before and after freezing once in glass vials and after three freeze / thaw cycles of aliquots in assay plates. Test articles were tested in transport buffer alone or in transport buffer containing 0.002% PS-80 or 1% HSA and recovery was assessed. Data are shown in Table 8. In transport buffer alone, recovery of phenylacetic acid after a single freeze or after three freeze-thaw cycles was 90.8% or greater. In buffer containing 0.002% PS-80, recovery of phenylacetic acid was 96.1% and 54.7% at 250 μM and 2500 μM, respectively, after a single freeze and 77.9% and 57.7% after three freeze-thaw cycles. Recovery was acceptable in this buffer at concentrations up to 250 μM. In transport buffer containing 1% HSA, recovery of phenylacetic acid was greater than 99.5% after a single freeze and greater than 26.6% after three freeze-thaw cycles.

[0206] [Table 8]

[0207] non-specific binding There was no nonspecific binding of phenylacetic acid to the transwell assay plate in the absence of cells. However, recovery of the test article (1 μM and 10 μM) was determined in the presence of cells in a Caco-2 efflux assay and was acceptable. Based on the stability results, a transporter assay for phenylacetic acid was performed in transport buffer containing 0.002% PS-80.

[0208] Evaluation of phenylacetic acid as a substrate and inhibitor of BSEP (using membrane vesicles) ATP-dependent uptake of phenylacetic acid (1 μM and 10 μM) into BSEP-transfected Sf9 membrane vesicles was tested alone or with the BSEP inhibitor cyclosporine A (20 μM) and the data are shown in Table 9. ATP-dependent uptake of phenylacetic acid at 1 μM and 10 μM was observed to be 24.2 and 814 pmol / min / mg protein, respectively, with signal-to-noise ratios of 9.95 and 12.6, respectively. However, activity in the presence of inhibitor was 75.6% and 94.9% of control at 1 μM and 10 μM, respectively. Inhibition was not concentration-dependent; therefore, phenylacetic acid is unlikely to be a substrate for BSEP.

[0209] [Table 9]

[0210] BSEP substrate 3 The ATP-dependent uptake of H-TCA into BSEP vesicles, alone or with cyclosporine A (20 μM) or phenylacetic acid (750 μM and 7500 μM), is shown in Table 10. 3 The average ATP-dependent uptake activity for H-TCA was 4.69 pmol / min / mg protein with a signal-to-noise ratio of 9.97. 3 H-TCA uptake was reduced to 3.49% in the presence of the BSEP inhibitor cyclosporine A. 3 It inhibited H-TCA uptake, with 99.6% activity remaining at 750 μM and 0.00% activity remaining at 7500 μM, and the IC 50 was estimated to be less than 7500 μM. Therefore, phenylacetic acid was identified as an inhibitor of BSEP.

[0211] [Table 10]

[0212] Example 4 Evaluation of phenylacetyl-L-glutamine as a substrate and / or inhibitor of BSEP Experiments were performed to evaluate whether phenylacetyl-L-glutamine is a substrate and / or inhibitor of BSEP. No ATP-dependent BSEP uptake activity was observed for 1 μM and 10 μM phenylacetyl-L-glutamine, suggesting that phenylacetyl-L-glutamine is not a substrate for BSEP. Phenylacetyl-L-glutamine (50 μM and 500 μM) was not an inhibitor of BSEP.

[0213] Solubility, stability and non-specific binding of phenylacetyl-L-glutamine solubility The test article, phenylacetyl-L-glutamine, was soluble in DMSO up to 100,000 μM. Stock solutions were further diluted from DMSO stocks with methanol.

[0214] Phenylacetyl-L-glutamine was soluble in the assay buffer up to 500 μM, and the final organic content was 0.5% (v:v) at all concentrations tested.

[0215] stability The stability of 50 μM and 500 μM phenylacetyl-L-glutamine was determined before and after freezing once in glass vials and after three freeze / thaw cycles of aliquots in assay plates. Test articles were tested in transport buffer alone or in transport buffer containing 0.002% PS-80 or 1% HSA and recovery was assessed. Data are shown in Table 11. In transport buffer alone, recovery of phenylacetyl-L-glutamine was 95.0% or greater after a single freeze or after three freeze-thaw cycles. In buffer containing 0.002% PS-80, recovery of phenylacetyl-L-glutamine was 96.3% or greater after a single freeze and 98.8% or greater after three freeze-thaw cycles. In transport buffer containing 1% HSA, recovery of phenylacetyl-L-glutamine was 96.9% or greater after a single freeze and 106% or greater after three freeze-thaw cycles.

[0216] [Table 11]

[0217] non-specific binding Nonspecific binding of phenylacetyl-L-glutamine to the transwell assay plate in the absence of cells was also evaluated. Phenylacetyl-L-glutamine solutions (50 μM and 500 μM) were placed in the apical (250 μL) or basolateral (700 μL) chambers (donor chambers) of the transwell plate in triplicate wells, and the corresponding blank buffer was placed in the receiver chambers and incubated at 37° C. for 2 hours. Data are shown in Table 12. With transport buffer alone, recovery was ≥107% in the apical to basolateral direction and ≥103% in the basolateral to apical direction. With transport buffer containing 0.002% PS-80, recovery was ≥105% in the apical to basolateral direction and ≥98.6% in the basolateral to apical direction. With transport buffer containing 1% HSA, recovery was ≥106% in the apical to basolateral direction and ≥99.4% in the basolateral to apical direction.

[0218] [Table 12]

[0219] Stability and nonspecific binding results were similar, with acceptable recoveries from all buffer systems tested. Other test articles showed best recoveries in a transport buffer containing 0.002% PS-80; therefore, this buffer was also used in the phenylacetyl-L-glutamine assay conditions.

[0220] Evaluation of phenylacetyl-L-glutamine as a substrate and inhibitor of BSEP (using membrane vesicles) ATP-dependent uptake of phenylacetyl-L-glutamine (1 μM and 10 μM) into BSEP-transfected Sf9 membrane vesicles was tested alone or with the BSEP inhibitor cyclosporine A (20 μM), and the data are shown in Table 13. No ATP-dependent uptake activity was observed for 1 μM and 10 μM phenylacetyl-L-glutamine. These data indicated that phenylacetyl-L-glutamine is not a substrate for BSEP.

[0221] [Table 13]

[0222] BSEP substrate 3 The ATP-dependent uptake of H-TCA into BSEP vesicles, alone or with cyclosporine A (20 μM) or phenylacetyl-L-glutamine (50 μM and 500 μM), is shown in Table 14. 3 The average ATP-dependent uptake activity for H-TCA was 15.4 pmol / min / mg protein with a signal-to-noise ratio of 25.5. 3 H-TCA uptake was reduced to 2.09% in the presence of the BSEP inhibitor cyclosporine A. 3 It did not inhibit H-TCA uptake, and 86.4% activity remained at 500 μM.

[0223] [Table 14]

[0224] Example 5 Evaluation of tauroursodeoxycholic acid as a substrate and / or inhibitor of BSEP Experiments were performed to assess whether tauroursodeoxycholic acid is a substrate and / or inhibitor of BSEP. ATP-dependent BSEP uptake activity for 1 μM and 10 μM tauroursodeoxycholic acid was more than twice as high as AMP uptake and was inhibited by cyclosporine A, identifying tauroursodeoxycholic acid as a substrate for BSEP. Tauroursodeoxycholic acid (5 μM and 50 μM) potently inhibited probe substrate uptake by BSEP, with IC 50 was estimated to be less than 5 μM.

[0225] Solubility, stability, and nonspecific binding of tauroursodeoxycholic acid. solubility The test article, tauroursodeoxycholic acid, was soluble up to 10,000 μM in methanol and 50 μM in assay buffer, with a final methanol content of 0.5% (v:v) for all concentrations tested.

[0226] stability The stability of 1 μM and 10 μM tauroursodeoxycholic acid was determined before and after freezing once in glass vials and after three freeze / thaw cycles of aliquots in assay plates. Test articles were tested in transport buffer alone or in transport buffer containing 0.002% PS-80 or 1% HSA and recovery was assessed. Data are shown in Table 15. In transport buffer alone, recovery of tauroursodeoxycholic acid was 77.7% or greater after a single freeze or after three freeze-thaw cycles. In buffer containing 0.002% PS-80, recovery of tauroursodeoxycholic acid was 108% or greater after a single freeze and 106% or greater after three freeze-thaw cycles. In transport buffer containing 1% HSA, recovery of tauroursodeoxycholic acid was 102% or greater after a single freeze and 82.7% or greater after three freeze-thaw cycles.

[0227] [Table 15]

[0228] non-specific binding Nonspecific binding of tauroursodeoxycholic acid to the transwell assay plate in the absence of cells was also evaluated. Tauroursodeoxycholic acid solutions (1 μM and 10 μM) were placed in the apical (250 μL) or basolateral (700 μL) chambers (donor chambers) of the transwell plate in triplicate wells, and the corresponding blank buffer was placed in the receiver chamber and incubated at 37° C. for 2 hours. Data are shown in Table 16. With transport buffer alone, recovery was ≥112% in the apical to basolateral direction and ≥74.4% in the basolateral to apical direction, suggesting some adhesion to plastic in the absence of cells when excipient-free buffer was used. With transport buffer containing 0.002% PS-80, recovery was ≥136% in the apical to basolateral direction and ≥95.3% in the basolateral to apical direction. In transport buffer containing 1% HSA, recovery was ≧124% in the apical to basolateral direction and ≧69.3% in the basolateral to apical direction.

[0229] Based on the stability and nonspecific binding results, the transporter assay was performed in transport buffer containing 0.002% PS-80 to optimize recovery of tauroursodeoxycholic acid under the assay conditions.

[0230] [Table 16]

[0231] Evaluation of tauroursodeoxycholic acid as a substrate and inhibitor of BSEP (using membrane vesicles) ATP-dependent uptake of tauroursodeoxycholic acid (1 μM and 10 μM) into BSEP-transfected Sf9 membrane vesicles was tested alone or with the BSEP inhibitor cyclosporine A (20 μM), and the data are shown in Table 17. The average ATP-dependent uptake activity for 1 μM and 10 μM tauroursodeoxycholic acid was 14.4 and 22.4 pmol / min / mg protein, respectively, with signal-to-noise ratios of 3.94-fold and 10.6-fold relative to the AMP control. Cyclosporine A treatment resulted in less than 12.6% residual BSEP activity. These data indicated that tauroursodeoxycholic acid is actively transported and is a substrate for BSEP.

[0232] [Table 17]

[0233] BSEP substrate 3 The ATP-dependent uptake of H-TCA into BSEP vesicles, alone or with cyclosporine A (20 μM) or tauroursodeoxycholic acid (5 μM and 50 μM), is shown in Table 18. 3 The average ATP-dependent uptake activity for H-TCA was 14.5 pmol / min / mg protein with a signal-to-noise ratio of 28.3. 3 H-TCA uptake was reduced to 4.88% in the presence of the BSEP inhibitor cyclosporine A. Tauroursodeoxycholic acid 3 Inhibits H-TCA uptake with 5.14% activity remaining at 50 μM; estimated IC 50 is less than 5 μM.

[0234] [Table 18]

[0235] Example 6 Evaluation of ursodeoxycholic acid as a substrate and / or inhibitor of BSEP Experiments were performed to assess whether ursodeoxycholic acid is a substrate and / or inhibitor of BSEP. ATP-dependent BSEP uptake with 1 μM and 10 μM ursodeoxycholic acid was less than twice that in the AMP sample, indicating that the test article is not a substrate for BSEP. Ursodeoxycholic acid (50 μM and 500 μM) inhibited probe substrate uptake by BSEP, with IC 50 was estimated to be less than 500 μM.

[0236] Solubility, stability, and nonspecific binding of ursodeoxycholic acid. solubility The test article, ursodeoxycholic acid, was soluble up to 100,000 μM in methanol and 500 μM in assay buffer, with a final methanol content of 0.5% (v:v) for all concentrations tested.

[0237] stability The stability of ursodeoxycholic acid at 1 μM and 10 μM was determined before and after freezing once in glass vials and after three freeze / thaw cycles of aliquots in assay plates. Test articles were tested in transport buffer alone or in transport buffer containing 0.002% PS-80 or 1% HSA and recovery was assessed. Data are shown in Table 19. In transport buffer alone, recovery of ursodeoxycholic acid was 85.8% or greater after a single freeze or after three freeze-thaw cycles. In buffer containing 0.002% PS-80, recovery of ursodeoxycholic acid was 110% after a single freeze and 98.4% or greater after three freeze-thaw cycles. In transport buffer containing 1% HSA, recovery of ursodeoxycholic acid was 95.0% or greater after a single freeze and 89.8% or greater after three freeze-thaw cycles.

[0238] [Table 19]

[0239] non-specific binding Nonspecific binding of ursodeoxycholic acid to the Transwell assay plate in the absence of cells was also evaluated. Ursodeoxycholic acid solutions (1 μM and 10 μM) were placed in the apical (250 μL) or basolateral (700 μL) chambers (donor chambers) of the Transwell plate in triplicate wells, and the corresponding blank buffer was placed in the receiver chambers and incubated at 37° C. for 2 hours. Data are shown in Table 20. With transport buffer alone, recovery was ≥118% in the apical to basolateral direction and ≥84.8% in the basolateral to apical direction. With transport buffer containing 0.002% PS-80, recovery was ≥104% in the apical to basolateral direction and ≥96.9% in the basolateral to apical direction. With transport buffer containing 1% HSA, recovery was ≥107% in the apical to basolateral direction, but the test article was undetectable in the apical chamber in the basolateral to apical direction.

[0240] [Table 20]

[0241] Based on the stability and nonspecific binding results, the transporter assay was performed in transport buffer containing 0.002% PS-80 to optimize recovery of ursodeoxycholic acid under assay conditions.

[0242] Evaluation of ursodeoxycholic acid as a substrate and inhibitor of BSEP (using membrane vesicles) ATP-dependent uptake of ursodeoxycholic acid (1 μM and 10 μM) into BSEP-transfected Sf9 membrane vesicles was tested alone or with the BSEP inhibitor cyclosporine A (20 μM) and the data are shown in Table 21. The average ATP-dependent uptake activity for ursodeoxycholic acid was undetectable at 1 μM. The ATP-dependent uptake activity at 10 μM was 0.792 pmol / min / mg protein in one experiment, with a signal-to-noise ratio of only 1.26-fold relative to the AMP control. These data indicated that ursodeoxycholic acid is not a substrate for BSEP.

[0243] [Table 21]

[0244] BSEP substrate 3 The ATP-dependent uptake of H-TCA into BSEP vesicles, alone or with cyclosporine A (20 μM) or ursodeoxycholic acid (50 μM and 500 μM), is shown in Table 22. 3 The average ATP-dependent uptake activity for H-TCA was 9.43 pmol / min / mg protein with a signal-to-noise ratio of 21.0. 3 H-TCA uptake was reduced to 4.39% in the presence of the BSEP inhibitor cyclosporine A. Ursodeoxycholic acid 3 It inhibited H-TCA uptake, with 64.0% activity remaining at 50 μM and 4.50% remaining at 500 μM, and an IC 50 was estimated to be less than 500 μM.

[0245] [Table 22]

[0246] Example 7 Evaluation of glycoursodeoxycholic acid as a substrate and / or inhibitor of BSEP Experiments were performed to assess whether glycoursodeoxycholic acid is a substrate and / or inhibitor of BSEP. ATP-dependent BSEP uptake activity for 1 μM and 10 μM glycoursodeoxycholic acid was more than twice as high as AMP uptake and was inhibited by cyclosporine A, identifying glycoursodeoxycholic acid as a substrate for BSEP. Glycoursodeoxycholic acid (10 μM and 100 μM) potently inhibited probe substrate uptake by BSEP, with IC 50 was estimated to be less than 10 μM.

[0247] Solubility, stability, and nonspecific binding of glycoursodeoxycholic acid. solubility The test article, glycoursodeoxycholic acid, was soluble up to 50,000 μM in methanol and 100 μM in assay buffer, with a final methanol content of 0.5% (v:v) for all concentrations tested.

[0248] stability The stability of 1 μM and 10 μM glycoursodeoxycholic acid was determined before and after a single freeze in glass vials and after three freeze / thaw cycles of aliquots in assay plates. Test articles were tested in transport buffer alone or in transport buffer containing 0.002% PS-80 or 1% HSA and recovery was assessed. Data are shown in Table 23. In transport buffer alone, recovery of glycoursodeoxycholic acid was 84.1% or greater after a single freeze or three freeze-thaw cycles. In buffer containing 0.002% PS-80, recovery of glycoursodeoxycholic acid was 99.8% or greater after a single freeze and 107% or greater after three freeze-thaw cycles. In transport buffer containing 1% HSA, the dosing solution was approximately twice the expected concentration; thus, recovery of glycoursodeoxycholic acid was greater than 94.8% after a single freeze and greater than 98.4% after three freeze-thaw cycles.

[0249] [Table 23]

[0250] non-specific binding Nonspecific binding of glycoursodeoxycholic acid to the transwell assay plate in the absence of cells was also evaluated. Glycoursodeoxycholic acid solutions (1 μM and 10 μM) were placed in the apical (250 μL) or basolateral (700 μL) chambers (donor chambers) of the transwell plate in triplicate wells, and the corresponding blank buffer was placed in the receiver chambers and incubated at 37° C. for 2 hours. Data are shown in Table 24. With transport buffer alone, recovery was ≥138% in the apical to basolateral direction and ≥85.3% in the basolateral to apical direction. With transport buffer containing 0.002% PS-80, recovery was ≥81.7% in the apical to basolateral direction and ≥99.1% in the basolateral to apical direction. With transport buffer containing 1% HSA, recovery was ≥140% in the apical to basolateral direction and ≥68.4% in the basolateral to apical direction.

[0251] [Table 24]

[0252] Based on the stability and nonspecific binding results, the transporter assay was performed in transport buffer containing 0.002% PS-80 to optimize recovery of glycoursodeoxycholic acid under the assay conditions.

[0253] Evaluation of glycoursodeoxycholic acid as a substrate and inhibitor of BSEP (using membrane vesicles) ATP-dependent uptake of glycoursodeoxycholic acid (1 μM and 10 μM) into BSEP-transfected Sf9 membrane vesicles was tested alone or with the BSEP inhibitor cyclosporine A (20 μM) and the data are shown in Table 25. The average ATP-dependent uptake activities for 1 μM and 10 μM glycoursodeoxycholic acid were 11.2 and 14.2 pmol / min / mg protein, respectively. Signal-to-noise ratios could not be determined because AMP incubation was below the limit of quantification. No residual BSEP activity was observed after treatment with cyclosporine A. These data indicated that glycoursodeoxycholic acid is actively transported and is a substrate for BSEP.

[0254] [Table 25]

[0255] BSEP substrate 3 The ATP-dependent uptake of H-TCA into BSEP vesicles, alone or with cyclosporine A (20 μM) or glycoursodeoxycholic acid (10 μM and 100 μM), is shown in Table 26. 3 The mean ATP-dependent uptake activity for H-TCA was 17.7 pmol / min / mg protein with a signal-to-noise ratio of 19.2. 3 H-TCA uptake was reduced to 3.64% in the presence of the BSEP inhibitor cyclosporine A. Glycoursodeoxycholic acid 3 Inhibits H-TCA uptake with 10.4% activity remaining at 100 μM; estimated IC 50 is less than 10 μM.

[0256] [Table 26]

[0257] conclusion As these examples show, sodium phenylbutyrate was not identified as a substrate for the BSEP transporter. Sodium phenylbutyrate (25 μM and 250 μM) did not inhibit the BSEP transporter. Phenylacetic acid (1 μM and 10 μM) was not identified as a substrate for the BSEP transporter. Phenylacetic acid (750 μM and 7500 μM) inhibited BSEP, indicating a possible drug-drug interaction. Phenylacetyl-L-glutamine was neither a substrate (1 μM and 10 μM) nor an inhibitor (50 μM and 500 μM) of the BSEP transporter. Tauroursodeoxycholic acid (1 μM and 10 μM) was a substrate for the BSEP transporter. Tauroursodeoxycholic acid (5 μM and 50 μM) inhibited BSEP. The BSEP inhibitory interaction indicated a possible drug-drug interaction. Ursodeoxycholic acid (1 μM and 10 μM) was not a substrate for the BSEP transporter. Ursodeoxycholic acid (50 μM and 500 μM) inhibited the uptake of the BSEP probe substrate. Glycoursodeoxycholic acid (1 μM and 10 μM) was a substrate for the BSEP transporter. Glycoursodeoxycholic acid (10 μM and 100 μM) inhibited the BSEP transporter, indicating a possible drug-drug interaction.

Claims

1. 1. A method of treating at least one symptom of amyotrophic lateral sclerosis (ALS) in a subject, comprising: (a) administering to a subject who has received a first dose of an inhibitor of the bile salt export pump (BSEP), a composition comprising about 1 g of taurursodiol (TURSO) and about 3 g of sodium phenylbutyrate; (b) determining or having determined a first level of serum transaminase and / or bilirubin in a first biological sample from the subject; and (c) administering to the subject a second dose of the inhibitor of BSEP, wherein the second dose is less than the first dose; A method comprising:

2. 2. The method of claim 1, wherein the inhibitor of BSEP is cyclosporine.

3. 3. The method of claim 2, wherein the first dose of cyclosporine is from about 0.5 to about 15 mg / kg / day.

4. The method of any one of claims 1 to 3, further comprising a step (d) of determining or having determined a second level of serum transaminase and / or bilirubin in a second biological sample from the subject.

5. 5. The method of claim 4, wherein the second level of serum transaminase and / or bilirubin is lower than the first level.

6. The method according to any one of claims 1 to 5, wherein the biological sample is plasma or serum.

7. 7. The method of any one of claims 1 to 6, wherein the TURSO is administered in an amount of from about 1 to about 2 g, inclusive, per day.

8. 8. The method of any one of claims 1 to 7, wherein the sodium phenylbutyrate is administered in an amount of from about 3 to about 6 g, inclusive, per day.

9. The method of any one of claims 1 to 8, wherein the TURSO is administered at about 1 g once per day.

10. 9. The method of any one of claims 1 to 8, wherein the TURSO is administered at about 1 g twice daily.

11. 11. The method of any one of claims 1 to 10, wherein the sodium phenylbutyrate is administered in a dose of about 3 g once a day.

12. 11. The method of any one of claims 1 to 10, wherein the sodium phenylbutyrate is administered in an amount of about 3 g twice daily.

13. The method of any one of claims 1 to 12, wherein the composition is administered to the subject orally or through a feeding tube.

14. The method of any one of claims 1 to 13, wherein the subject has been diagnosed with ALS.

15. The method of any one of claims 1 to 13, wherein the subject is suspected of suffering from ALS.

16. The method of any one of claims 1 to 15, wherein the subject is a human.

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