Bile acids and phenylbutyrates with CYP P450 or transporter substrates for the treatment of amyotrophic lateral sclerosis

Abstract

Provided herein is a method for treating at least one symptom of ALS in a subject administered a first dose of a CYP or transporter substrate, the method comprising administering to the subject a composition comprising a bile acid or a pharma- ceutically acceptable salt thereof and a phenylbutyrate compound, monitoring the subject for response to the substrate, and administering a second dose of the substrate, 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. Application No. 17 / 684,845, filed March 2, 2022, which is incorporated by reference herein in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically as an XML file named 38709-0034WO1_SL_ST26.xml. The XML file was created on Feb. 21, 2023 and is 6,940 bytes in size. The contents of the XML file are incorporated herein by reference in their entirety.

[0003] Technical Field The present disclosure relates generally to compositions and methods for treating amyotrophic lateral sclerosis. [Background technology]

[0004] 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 that eventually leads to partial or total paralysis. Median survival from onset of symptoms is 2-3 years, with respiratory failure being the leading cause of death. Currently, ALS treatment is centered on symptom management. Only two FDA-approved medications for ALS are currently available: riluzole and edaravone. Thus, there is a need for improved methods of treating ALS.

[0005] 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

[0006] In one aspect, provided herein is a method of treating at least one symptom of amyotrophic lateral sclerosis (ALS) in a subject, the method comprising: (a) administering a composition comprising taurursodiol (TURSO) and sodium phenylbutyrate to a subject to which a first dose of a substrate of organic anion transporter 1 (OAT1) has been administered; (b) determining or having determined a first level of the substrate or a metabolite thereof in a first biological sample of the subject; and (c) administering a second dose of the substrate to the subject, wherein the second dose is less than the first dose. In some embodiments, the substrate of OAT1 is penicillin, a nonsteroidal anti-inflammatory drug (NSAID), an HIV protease inhibitor, or an antiviral drug. In some embodiments, the substrate of OAT1 is an NSAID selected from the group consisting of diclofenac, ketoprofen, and methotrexate. In some embodiments, the substrate of OAT1 is an antiviral drug selected from the group consisting of adefovir, cidofovir, and tenofovir. In some embodiments of the method of treating at least one symptom of ALS in a subject of the above aspects, the substrate of OAT1 has a narrow therapeutic index. In some embodiments, the method further comprises a step (d) of determining, or having determined, a second level of the substrate or a metabolite thereof in a second biological sample of the subject. In some embodiments, the second level of the substrate or a metabolite thereof is lower than the first level. In some embodiments of the method of treating at least one symptom of ALS in a subject of the above aspects, the biological sample is blood.

[0007] In another aspect, provided herein is a method of treating at least one symptom of ALS in a subject, the method comprising: (a) administering a composition comprising TURSO and sodium phenylbutyrate to a subject to which a first dose of a cytochrome P450 (CYP) substrate has been administered; (b) determining or having been determined a first level of the substrate or a metabolite thereof in a first biological sample of the subject; and (c) administering a second dose of the substrate to the subject, wherein the second dose is less than the first dose. In some embodiments, the CYP is CYP2B6. In some embodiments, the CYP substrate is bupropion or efavirenz. In some embodiments, the CYP is CYP2C8. In some embodiments, the CYP substrate is repaglinide, montelukast, pioglitazone, or rosiglitazone. In some embodiments, the CYP substrate has a narrow therapeutic index. In some embodiments of the method of treating at least one symptom of ALS in a subject of the above aspects, the method further comprises a step (d) of determining, or having determined, a second level of the substrate or a metabolite thereof in a second biological sample from the subject. In some embodiments, the second level of the substrate or a metabolite thereof is lower than the first level. In some embodiments, the biological sample is plasma or serum.

[0008] In a further aspect, provided herein is a method of treating at least one symptom of ALS in a subject, the method comprising: (a) administering to a subject administered a first dose of a narrow therapeutic index (NTI) medication, a composition comprising TURSO and sodium phenylbutyrate; (b) determining or having determined a first level of the NTI medication or a metabolite thereof in a first biological sample of the subject; and (c) administering to the subject a second dose of the NTI medication, wherein the second dose is less than the first dose. In some embodiments, the NTI medication is mexiletine, alfentanil, quinidine, cyclosporine, warfarin, or digoxin. In some embodiments of the method of treating at least one symptom of ALS in a subject of the above aspect, the method further comprises a step (d) of determining or having determined a second level of the NTI medication or a metabolite thereof in a second biological sample of the subject. In some embodiments, the second level of the NTI medication or a metabolite thereof is less than the first level.

[0009] In some embodiments of the methods disclosed herein, step (a) comprises administering about 1 g of TURSO and about 3 g of sodium phenylbutyrate to the subject once a day. In some embodiments, step (a) comprises administering about 1 g of TURSO and about 3 g of sodium phenylbutyrate to the subject twice a day. 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.

[0010] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.

[0011] It is understood that certain features of the present disclosure that are described in the context of separate embodiments for clarity of description may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure that are described in the context of a single embodiment for brevity of description may also be provided separately or in any suitable subcombination. All combinations of the embodiments related to the present disclosure are expressly embraced by the present disclosure and are disclosed herein as if each and every combination were individually and expressly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also expressly embraced by the present disclosure and are disclosed herein as if each and every such subcombination were individually and expressly disclosed herein.

[0012] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the present invention will become apparent from the following detailed description and claims. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 is a set of graphs showing the inhibition of CYP activity by positive time-dependent inhibitors. [Diagram 2] FIG. 2 shows individual plasma concentration (ng / mL) of tenofovir versus time data following oral administration of TDF alone (day 1, left panel) or AMX0035 and TDF (day 5, right panel) in male Sprague Dawley rats. [Diagram 3] FIG. 3 is a graph showing individual plasma concentration (ng / mL) of PB versus time data following oral administration of AMX0035 and TDF (day 5) in male Sprague Dawley rats. [Figure 4]FIG. 4 shows individual plasma concentration (ng / mL) of TUDCA versus time data in male Sprague Dawley rats following oral administration of TDF alone (day 1, left panel) or AMX0035 and TDF (day 5, right panel). [Diagram 5] FIG. 5 shows mean plasma concentrations (ng / mL) of tenofovir, PB, and TUDCA versus time data in male Sprague Dawley rats following oral administration of TDF alone (day 1, left panel) or AMX0035 and TDF (day 5, right panel). [Figure 6] FIG. 6 is a bar graph showing mean urinary recovery (ng) of tenofovir following oral dosing of TDF alone (day 1, left) or AMX0035 and TDF (day 5, right) in male Sprague Dawley rats. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Detailed Description Applicants have discovered that a combination of a bile acid (e.g., taurursodiol (TURSO)) and a phenylbutyrate compound (e.g., sodium phenylbutyrate) can be used to treat one or more symptoms of ALS, and have surprisingly found that the combination inhibits one or more cytochrome (CYP) P450 enzymes (e.g., CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A) and transporters (e.g., organic anion transporter 1 (OAT1)). For example, in some instances, the combination inhibits CYP2B6 or CYP2C8. When a substrate for a CYP P450 enzyme or transporter is co-administered with a composition comprising a bile acid and a phenylbutyrate compound, the level and / or effective dose of the substrate may be increased, which may result in increased toxic effects associated with the substrate. Thus, the present disclosure provides methods of treating at least one symptom of ALS in a subject to which a substrate for a CYP P450 enzyme (e.g., one with a narrow therapeutic index) or a substrate for a transporter (e.g., a substrate for OAT1) is also administered.

[0015] The disclosure provides a method of treating at least one symptom of ALS in a subject, the method comprising: (a) administering a composition (e.g., an effective amount of the composition) comprising TURSO and sodium phenylbutyrate to a subject who has been administered a first dose of a substrate for a cytochrome P450 enzyme, (b) determining, or having determined, a first level of the substrate in a first biological sample from the subject, and (c) administering a second dose of the substrate to the subject, where the second dose is less than the first dose. The composition of TURSO and sodium phenylbutyrate can comprise about 1 g of TURSO and about 3 g of sodium phenylbutyrate.

[0016] The disclosure also provides a method of treating at least one symptom of ALS in a subject, the method comprising: (a) administering a composition (e.g., an effective amount of the composition) comprising TURSO and sodium phenylbutyrate to a subject who has been administered a first dose of warfarin; (b) determining, or having determined, a first blood INR level of the subject; and (c) administering a second dose of warfarin to the subject, where the second dose is less than the first dose. The composition of TURSO and sodium phenylbutyrate can comprise about 1 g of TURSO and about 3 g of sodium phenylbutyrate.

[0017] Also provided is a method of treating at least one symptom of ALS in a subject, the method comprising: (a) administering a composition (e.g., an effective amount of the composition) comprising TURSO and sodium phenylbutyrate to a subject who has been administered a first dose of a substrate for organic anion transporter 1 (OAT1); (b) determining, or having determined, a first level of the substrate in a first biological sample from the subject; and (c) administering a second dose of the substrate to the subject, where the second dose is less than the first dose. The composition of TURSO and sodium phenylbutyrate can comprise about 1 g of TURSO and about 3 g of sodium phenylbutyrate.

[0018] When a range of values ​​is provided, it is understood that each value between the upper and lower limits of that range, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, and any other stated or intervening value within that stated range, is encompassed within the disclosure. The upper and lower limits of these narrower ranges may independently be included in the narrower ranges and are also encompassed within the disclosure, subject to any explicitly excluded limits in the stated range. When the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0019] Certain ranges are described herein with numerical values ​​preceded by the term "about". The term "about" is used herein to provide literal support for the exact number that follows the term and for a number that is close to or approximately the number that follows the term. When determining whether a number is close to or approximately a specifically stated number, the unstated number that is close or approximately may be a number that provides a substantial equivalent to the specifically stated number in the context in which it is stated.

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

[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 both 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 or may have been diagnosed with ALS, hi some embodiments, the subject may be suspected of 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 been showing 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), hi some embodiments, the subject has been showing 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 exhibited by a subject 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). A subject may have respiratory muscle weakness as an early manifestation of ALS symptoms. Such subjects may have a very poor prognosis, with a median survival time of about 2 months from diagnosis in some instances. 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 can include increased tendon reflexes, spasticity, pseudobulbar features, 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 the present disclosure provide methods for 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 examples, the subject has been diagnosed with ALS. For example, the subject may have been diagnosed with ALS for about 24 months or less (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 or less). For example, the subject may have been diagnosed with ALS for a week or less or on the same day that the treatment of the present 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 or more). 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 the 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 the signs of lower motor neuron (LMN) and / or upper motor neuron (UMN) degeneration; and In addition to the lack of neuroimaging evidence of other disease processes that might explain the observed clinical and electrophysiological signs. 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 region or UMN signs only present in one region and LMN signs 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 together in only one area or UMN signs only in two or more areas; or LMN signs more rostral to UMN signs, supported by testing, making the diagnosis clinically probable.

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

[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 can have an ALSFRS-R score (e.g., a baseline ALSFRS-R score) of 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 has an ALSFRS-R score (e.g., a baseline ALSFRS-R score) of 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 has an ALSFRS-R score (e.g., a baseline ALSFRS-R score) of 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 have 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 over a one-month period. For example, the score may decrease by about 1.02 (±2.3) points per month.

[0036] One predictor of patient progression is the patient's past rate of disease progression (ΔFS), which can be calculated as: ΔFS=(48-ALSFRS-R score at assessment) / time from onset to assessment (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 may have a disease progression rate (ΔFS) of 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, a subject may have an ALS disease progression rate (ΔFS) of about 0.50 or more (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 more). 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 has lost an average of about 0.8 to about 2 ALSFRS-R 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 over 3-12 months since the initial assessment. In some embodiments, the subject has lost an average of more than about 1.2 ALSFRS-R points per month over 3-12 months since the initial assessment. The subject may have lost an 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) over 3-12 months since the initial assessment. In some embodiments, the subject has lost an average of about 0.8 to about 2 ALSFRS-R 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 over the past 3-12 months. In some embodiments, the subject has lost an average of more than about 1.2 ALSFRS-R points (e.g., more than about 1.5, 1.8, 2.0, 2.5, or 3) per month over the past 3-12 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 for the diagnosis or prognosis of 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 for the diagnosis, prognosis, or to track disease activity or treatment outcome of ALS. 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 subjects with ALS 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, but are not limited to, 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. Neuropathol Appl Neurobiol 45(3):291-304, 2019). Commercially available pNF-H detection assays, such as those developed by EnCor Biotechnology, BioVendor, and Millipore-EMD, can also be used. Commercial NfL assay kits based on Simoa technology, such as those produced by Quanterix, 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] Subjects described herein may have a serum creatinine level of about 300 pg / mL or higher (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, 1900, 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, 6600, 6700, 6800, 6900, 7000, In some embodiments, the CSF or blood pNF-H level may be about 850, 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 may have a CSF or blood level of NfL of 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, serum NfL levels 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 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 of 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 biomarker for prognosing ALS (see, e.g., Atassi et al. Neurology 83(19):1719-1725, 2014). Akt phosphorylation can also be used as a biomarker for prognosing 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 having ALS or to determine that a subject is at risk of developing ALS.

[0044] Subjects may also be identified as having ALS 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). Subjects described herein may carry mutations 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., alanine to valine substitution 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 are a frequent cause of 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 acids and phenylbutyrate 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). Each subject's level of exposure to the administered compound (e.g., bile acids and phenylbutyrate compounds) can vary, i.e., there can be differences in the excretion level and pharmacokinetics of the compound in the subject being treated. Any of the factors described herein may affect the drug exposure to a subject. For example, a decrease in the clearance of a compound can result in increased drug exposure, while improved renal function can reduce actual drug exposure. The degree of drug exposure may correlate with the subject's response to the administered compound and the outcome of the 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 ~50, 30-40, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-100, 50-90, 50-80, 50-70, 50-60, 60-100, 60-90, 60-80, 60-70, 70-100, 70-90, 70-80, 80-100, 80-90 or 90-100 years of age). 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 2Having a mutation in an ALS-associated gene described herein or displaying a biomarker described herein may indicate that the 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 present disclosure provides a method of treating at least one symptom of ALS in a subject, comprising administering to the subject a bile acid or a pharma- ceutically acceptable salt thereof and a phenylbutyrate compound. In some embodiments, 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 to which the hydroxyl and carboxylic acid groups of the bile acid are attached along with 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 HCl, 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 of the present disclosure is a hydrophilic bile acid.Hydrophilic bile acids include, but are not limited to, TURSO, UDCA, chenodeoxycholic acid, cholic acid, hyodeoxycholic acid, lithocholic acid and glycoursodeoxycholic acid.Pharmaceutically acceptable salts or solvates of the bile acids disclosed herein are also contemplated. In some embodiments, bases commonly utilized to form pharma- ceutically acceptable salts of the bile acids of the present disclosure include hydroxides of alkali metals including sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals such as aluminum and zinc; 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 herein.

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

[0054] [ka]

[0055] The compound can be TURSO as shown in the following formula:

[0056] The bile acids described herein have the formula II (with carbons labeled to aid in understanding the positions at which substitutions may occur):

[0057] [ka]

[0058] The compound can be UDCA as shown in the following formula:

[0059] The bile acid derivatives of the present disclosure can be physiologically relevant bile acid derivatives. For example, combinations of substitution of hydrogen at the 3- or 7-position in the formula of TURSO or UDCA, 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 of the present disclosure include arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, cysteine, proline, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, and tryptophan, as well as β-alanine and γ-aminobutyric acid. One example of such a bile acid is represented by formula III:

[0061] [ka]

[0062] (In the formula, R is -H or C1-C4 alkyl; R1 is -CH2-SO3R3, CH2COOH or CH2CH2COOH and R2 is -H; or R1 is -COOH and R2 is -CH2-CH2-CONH2, -CH2-CONH2, -CH2-CH2-SCH3, CH2CH2CH2NH(C=NH)NH2, CH2(imidazolyl), CH2CH2CH2CH2NH2, CH2COOH, CH2CH2COOH, CH2OH, CH(OH)CH3, CH2SH, pyrrolidin-2-yl, CH3, 2-propyl, 2-butyl, 2-methylbutyl, CH2(phenyl), CH2(4-OH-phenyl), or -CH2-S-CH2-COOH; R3 is -H or a residue of an amino acid) 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 of the present disclosure has formula IV:

[0064] [ka]

[0065] (In the formula, R is -H or C1-C4 alkyl; R1 is -CH2-SO3R3 and R2 is -H; or R1 is -COOH and R2 is -CH2-CH2-CONH2, -CH2-CONH2, -CH2-CH2-SCH3 or -CH2-S-CH2-COOH; R3 is -H or a residue of a basic amino acid, or a 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 the mitochondrial membrane, 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, where long-term treatment (e.g., 1-2 years) is generally required to achieve complete dissolution. It is used for the treatment of cholestatic liver disease, including primary cirrhosis, pediatric familial intrahepatic cholestasis, and primary sclerosing cholangitis and cholestasis due to cystic fibrosis. TURSO is contraindicated in subjects with biliary tract infections, frequent biliary colic, or subjects with 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). Based on similar physicochemical characteristics, there are similarities in drug toxicity and interactions between TURSO and UDCA. 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 the solubility of UDCA 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 more proximally in the ileum. Ursodiol therapy is not associated with liver damage. Abnormalities in liver enzymes 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 when required by specific clinical circumstances. 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, thus counteracting the efficacy of ursodiol.

[0068] Phenylbutyrate Compounds Phenylbutyrate compounds are defined herein to include phenylbutyrate (low molecular weight aromatic carboxylic acid) as the free acid (4-phenylbutyrate (4-PBA), 4-phenylbutyric acid or phenylbutyric acid) and its pharma- ceutically acceptable salts, co-crystals, polymorphs, hydrates, solvates, conjugates, derivatives or prodrugs. Phenylbutyrate compounds described herein also include analogs of 4-PBA, including, but not limited to, glyceryl tri-(4-phenylbutyrate), phenylacetic acid (which is the active metabolite of PBA), 2-(4-methoxyphenoxy)acetic acid (2-POAA-OMe), 2-(4-nitrophenoxy)acetic acid (2-POAA-NO2) and 2-(2-naphthyloxy)acetic acid (2-NOAA) and pharma-ceutically acceptable salts thereof. Phenylbutyrate compounds also include physiologically relevant 4-PBA species, such as, but not limited to, those in which hydrogen is substituted for deuterium in the structure of 4-PBA. Other HDAC2 inhibitors are contemplated herein as alternatives to the phenylbutyrate compounds.

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

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

[0071] [ka]

[0072] has.

[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 embodiments, a combination of a bile acid (e.g., TURSO) or a pharma- ceutically acceptable salt thereof and a phenylbutyrate compound (e.g., sodium phenylbutyrate) has synergistic efficacy in treating one or more symptoms associated with ALS when dosed in a particular ratio (e.g., the ratios described herein). This combination can induce a mathematically synergistic increase in neuronal viability in a robust oxidative injury model (H2O2-mediated toxicity) by linear modeling, e.g., via simultaneous inhibition of endoplasmic reticulum stress and mitochondrial stress (see, e.g., U.S. Patent Nos. 9,872,865 and 10,251,896).

[0075] formulation The bile acid and phenylbutyrate compounds described herein can be formulated for use as or in pharmaceutical compositions.For example, the methods described herein can include administering an effective amount of a composition comprising TURSO and sodium phenylbutyrate.The term "effective amount" as used herein refers to the amount or concentration of one or more drugs over a period of time (including acute or chronic administration and regular or continuous administration) that is effective in administering one or more drugs to cause a desired effect or physiological outcome. The composition can include about 5 to about 15 w / w% (e.g., about 6% to about 14%, about 7% to about 13%, about 8% to about 12%, about 8% to about 11%, about 9% to about 10%, or about 9.7% w / w) 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, do not destroy the pharmacological activity thereof, and are non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the compound. As used herein, 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 pharmaceutical administration. Pharmaceutical compositions may contain conventional non-toxic pharmaceutically acceptable carriers, adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the compound to be formulated or its delivery form.

[0078] The compositions of the present disclosure 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 of the present disclosure 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 grade, available from JRS Pharma, Colonial Scientific, or Quadra).

[0079] The compositions of the present disclosure 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 composition of the present disclosure may contain about 22 to about 35 w / w% maltodextrin (e.g., about 22% to about 33%, about 24% to about 31%, about 25% to about 32%, about 26% to about 30% or about 28% to about 29% w / w, e.g., about 28.3% w / w maltodextrin). When solubilized in a solution, the maltodextrin can form a flexible spiral structure that allows for encapsulation of the active ingredient (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), thereby masking the taste of the active ingredient. 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 composition comprises about 28.3 w / w% pea maltodextrin. For example, pea maltodextrin available from Roquette (KLEPTOSE® LINECAPS) can be used.

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

[0082] In some embodiments, the composition includes one or more flavoring agents. The composition can include about 2 to about 15 w / w% of the flavoring agent (e.g., about 3% to about 13%, about 3% to about 12%, about 4% to about 9%, about 5% to about 10%, or about 5% to about 8%, e.g., about 7.3% w / w). Flavoring agents can include substances that modify the characteristics of the composition by imparting a scent or affecting the taste of another substance. 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 to be incorporated. Suitable flavoring agents include, but are not limited to, natural flavoring agents, artificial flavoring agents, and simulants. Blends of flavoring agents can also be used. For example, the compositions described herein can include two or more (e.g., 2, 3, 4, 5 or more) flavoring agents. The flavoring agents can be soluble and stable in water. Selection of a suitable flavoring agent can be based on taste testing. For example, different flavoring agents can be added separately to the composition and subjected to taste testing. Exemplary flavoring agents include any fruit flavor powder (e.g., peach, strawberry, mango, orange, apple, grape, raspberry, cherry, or a mixture of various berries flavor powder). The compositions described herein can include about 0.5 to about 1.5 w / w% (e.g., about 1% w / w) of a mixed berry flavor powder and / or about 5 to about 7 w / w% (e.g., about 6.3% w / w) of a masking flavor. Suitable masking flavors 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% of porous silica (e.g., about 0.3% to about 1.5%, about 0.5% to about 1.2%, or about 0.8% to about 1%, e.g., 0.9% w / w). The porous silica may have a higher H2O absorption capacity and / or a higher porosity compared to 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). The porous silica can have an H2O absorption capacity of 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 can have a high porosity compared to the porosity 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 porous silica has an average particle size of about 2 μm to about 10 μm (e.g., about 3 μm to about 9 μm, about 4 μm to about 8 μm, about 5 μm to about 8 μm, or about 7.5 μm). In some embodiments, the porous silica has an average pore volume of about 0.1 cc / gm to about 2.0 cc / gm (e.g., about 0.1 cc / gm to about 1.5 cc / gm, about 0.1 cc / gm to about 1 cc / gm, about 0.2 cc / gm to about 0.8 cc / gm, about 0.3 cc / gm to about 0.6 cc / gm, or about 0.4 cc / gm). In some embodiments, the porous silica has a bulk density of about 50 g / L to about 700 g / L (e.g., about 100 g / L to about 600 g / L, about 200 g / L to about 600 g / L, about 400 g / L to about 600 g / L, about 500 g / L to about 600 g / L, about 540 g / L to about 580 g / L, or about 560 g / L). In some embodiments, the compositions described herein include about 0.05 to about 2 w / w% (eg, any range within this range) 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, such as sodium phosphate (e.g., anhydrous sodium dihydrogen phosphate). 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 dihydrogen 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. Water-soluble artificial sweeteners such as soluble saccharin salts (e.g., sodium or calcium saccharin salts), cyclamate salts, acesulfame potassium (acesulfame K), and the free acid form of saccharin and L-aspartyl-phenylalanine methyl ester, aspartame-based sweeteners such as Alitame® or Neotame® are contemplated herein. 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] In addition to those listed above, pharma- ceutically acceptable binders are also contemplated. Examples include cellulose derivatives, including microcrystalline cellulose, low-substituted hydroxypropyl cellulose (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 incorporated 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, corn 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 via 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 its 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 preparation for injection may also be a sterile solution or suspension for injection in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Acceptable vehicles and solvents that may be used include mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any bland fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injections, as are natural pharma- ceutical 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 or 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 commonly used in the manufacture of pharma- ceutically acceptable solid, liquid or other dosage forms, may also be used for formulation purposes.

[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 powders 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 and / or flavoring and / or coloring agents may be added.

[0095] Alternatively or additionally, the compositions may be administered by nasal aerosol or inhalation.Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as 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 together with instructions for administration. In some aspects, the present invention provides a kit comprising a bile acid and a phenylbutyrate compound. The kit can also include instructions for the physician and / or patient, syringes, needles, boxes, bottles, vials, etc.

[0097] III. Treatment method Applicants have discovered that a combination of a bile acid (e.g., TURSO) and a phenylbutyrate compound (e.g., sodium phenylbutyrate) can be used to treat one or more symptoms of ALS, and have surprisingly found that the combination inhibits one or more CYPs (e.g., CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A (CYP3A4)) and transporters (e.g., organic anion transporter 1 (OAT1)). For example, in some instances, the combination inhibits CYP2B6 and CYP2C8. When a CYP or transporter substrate is co-administered with a composition comprising a bile acid and a phenylbutyrate compound, the level and / or effective dose of the substrate may be increased, which may result in increased toxic effects associated with the substrate.

[0098] Thus, the present disclosure provides a method for treating at least one symptom of ALS in a subject to which a CYP substrate or a transporter substrate has been administered by adjusting the dose of the substrate, which may result in a plasma concentration of the substrate (or its metabolites) similar to, and may be as effective as, a dose of the substrate administered in the absence of bile acids and phenylbutyrate compounds.

[0099] Applicant discloses herein a method for treating at least one symptom of ALS in a subject administered a first dose of a CYP or transporter substrate, comprising administering to the subject a composition comprising TURSO and sodium phenylbutyrate, monitoring the subject for a response to the substrate, and administering a second dose of the substrate, where the second dose is less than the first dose. The composition can, for example, comprise 1 g of TURSO and 3 g of sodium phenylbutyrate. Monitoring the subject for a response to the substrate can include determining a level of the substrate or a metabolite thereof in a biological sample of the subject, determining the INR level in the subject's blood, or monitoring for known adverse events, overdose symptoms, or side effects associated with the substrate.

[0100] In one aspect, provided herein is a method of treating at least one symptom of ALS in a subject, the method comprising: (a) administering to a subject to whom a first dose of a substrate of a cytochrome P450 (CYP) has been administered a composition comprising TURSO and sodium phenylbutyrate; (b) determining, or having determined, a first level of the substrate or a metabolite thereof in a first biological sample of the subject; and (c) administering to the subject a second dose of the substrate, the second dose being less than the first dose. The composition can, for example, comprise 1 g of TURSO and 3 g of sodium phenylbutyrate. In some embodiments, the CYP is CYP2B6 or CYP2C8. In some embodiments, the substrate of the CYP is a drug with a narrow therapeutic index.

[0101] The disclosure also provides a method of treating at least one symptom of ALS in a subject, the method comprising: (a) administering to a subject who has been administered a first dose of warfarin a composition comprising TURSO and sodium phenylbutyrate; (b) determining, or having determined, a first blood INR level of the subject; and (c) administering to the subject a second dose of warfarin, wherein the second dose is less than the first dose. The composition can, for example, comprise 1 g of TURSO and 3 g of sodium phenylbutyrate.

[0102] Also provided is a method of treating at least one symptom of ALS in a subject, the method comprising: (a) administering to a subject to whom a first dose of a substrate for organic anion transporter 1 (OAT1) has been administered a composition comprising TURSO and sodium phenylbutyrate; (b) determining, or having determined, a first level of the substrate or a metabolite thereof in a first biological sample from the subject; and (c) administering to the subject a second dose of the substrate, the second dose being less than the first dose. The composition can, for example, comprise 1 g of TURSO and 3 g of sodium phenylbutyrate.

[0103] The disclosure further provides a method for treating at least one symptom of ALS in a subject administered a first dose of a narrow therapeutic index (NTI) drug, comprising administering to the subject a composition comprising TURSO and sodium phenylbutyrate, monitoring the subject for response to the NTI drug, and administering a second dose of the NTI drug, where the second dose is less than the first dose. The composition can comprise, for example, 1 g of TURSO and 3 g of sodium phenylbutyrate. In one aspect, a method for treating at least one symptom of ALS in a subject is provided herein, comprising: (a) administering to a subject administered a first dose of a narrow therapeutic index (NTI) drug a composition comprising TURSO and sodium phenylbutyrate; (b) determining or having determined a first level of the NTI drug or a metabolite thereof in a first biological sample of the subject; and (c) administering to the subject a second dose of the NTI drug, where the second dose is less than the first dose. A composition may contain, for example, 1 g of TURSO and 3 g of sodium phenylbutyrate.

[0104] In some embodiments of the methods described herein, the first and / or second dose of the substrate or NTI medicament can be a daily dose (once, twice, or three times daily), once every two days, three times a week, or weekly dose, or a dose based on some other standard. The second dose of the substrate or NTI medicament 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 dose. In some examples, the second dose involves administering the same or a similar amount per administration, but less frequently compared to the first dose.

[0105] In some embodiments of the methods described herein, the methods further comprise a step (d) of determining, or having determined, a second level of the substrate or a metabolite thereof, or the NTI drug or a metabolite thereof, in a second biological sample from the subject. In some embodiments, the second level is lower than the first level.

[0106] In some aspects of the methods described herein, a 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 phenylbutyrate. A 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 administration of a second dose of the substrate or NTI pharmaceutical.

[0107] In some embodiments of the methods described herein, the first and / or second biological sample can be serum, plasma, urine, or saliva.Methods for measuring the level of a substrate or its metabolite, or an NTI drug or its metabolite in a biological sample are known in the art.For example, immunoassays or liquid chromatography / tandem mass spectrometry (LC / MS) can be used.

[0108] Medicines with narrow therapeutic index Therapeutic index (TI) is the range of doses within which a drug is effective without producing unacceptable adverse events. A narrow therapeutic index (NTI) drug is one in which small differences in dose or blood concentration can cause significant therapeutic failure and / or adverse drug reactions that are life-threatening or result in permanent or significant disability or incapacity. Thus, the range of doses within which a drug can produce beneficial effects without causing serious and potentially fatal complications is very narrow.

[0109] In animal studies, the TI is the dose of a drug that is lethal to 50% of the population (LD 50 ) as the minimum effective dose in 50% of the population (ED 50 ) i.e. TI=LD 50 / ED 50 In clinical practice, the TI is the dose range at which a drug is expected to be effective in half of the participants in a clinical trial without causing unacceptable adverse effects. In general, a drug is considered to have a good safety profile if its TI is greater than a value of 10.

[0110] Many NTI drugs are known in the art. The FDA has established (a) the median lethal dose (LD 50 ) and median effective dose (ED 50 A pharmaceutical product is defined as having a narrow therapeutic index if (b) there is less than a two-fold difference between the minimal toxic concentration (MTC) and the minimal effective concentration (MEC) in the blood, and (c) the safe and effective use of the drug requires careful titration and patient monitoring.

[0111] In one aspect, provided herein is a method of treating at least one symptom of ALS in a subject, the method comprising: (a) administering to a subject who has been administered a first dose of a narrow therapeutic index (NTI) medication 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 the NTI medication or a metabolite thereof in a first biological sample of the subject; and (c) administering to the subject a second dose of the NTI medication, wherein the second dose is less than the first dose. The method can further comprise a step (d) of determining or having determined a second level of the NTI medication or a metabolite thereof in a second biological sample of the subject. Exemplary NTI medications include, but are not limited to, mexiletine, alfentanil, quinidine, cyclosporine, warfarin, and digoxin.

[0112] In some embodiments, the NTI medication is digoxin. Digoxin is a cardiac glycoside used in the treatment of mild to moderate heart failure and for ventricular rate control in chronic atrial fibrillation. The subject may be administered a first dose of digoxin of about 0.1 to about 0.6 mg / day (e.g., about 0.125 to about 0.5 mg / day, about 0.1 to 0.4 mg, or about 0.375 to 0.5 mg / day). The second dose of digoxin can be about 0.1 to about 0.475 mg / day (e.g., about 0.2, 0.25, 0.3, 0.35, 0.4, or 0.45 mg / day) less than the first dose.

[0113] Metabolites of digoxin are known in the art, representative metabolites include dihydrodigoxin and digoxigenin bisdigitoxoside.

[0114] In addition to or instead of determining the first and / or second level of the NTI drug or its metabolite in the biological sample of the subject, other methods of monitoring the subject's response to the first dose of the NTI drug are also contemplated herein.For example, known adverse events, side effects, or symptoms of overdose associated with NTI can be monitored.For example, symptoms of digoxin overdose include nausea, vomiting, visual changes, and arrhythmia.

[0115] Cytochrome P450 and Substrates Cytochrome P450 (CYP) is a superfamily of enzymes that contain heme as a cofactor and function as monooxygenases. CYP and CYP450 are used interchangeably herein. CYPs are membrane-associated proteins that are naturally located in the inner membrane of mitochondria or the endoplasmic reticulum of cells. These proteins are present in most tissues of the body and play important roles in the elimination of various compounds, the synthesis and degradation of hormones (e.g., estrogen and testosterone synthesis and metabolism), cholesterol synthesis, and vitamin D metabolism. CYPs also metabolize potentially toxic compounds, including drugs and products of endogenous metabolism such as bilirubin, primarily in the liver.

[0116] CYPs are the main enzymes involved in drug metabolism. Changes in CYP enzyme activity can affect the metabolism and elimination of various drugs, so effects on CYP activity are a major cause of adverse drug interactions. For example, if one drug inhibits the CYP-mediated metabolism of another drug, the second drug may accumulate to toxic levels in the body.

[0117] Cytochromes P450 are encoded by the CYP genes. In humans, CYP1 (CYP1A1, CYP1A2, CYP1B1, CYP1D1P), CYP2 (CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2R1, CYP2S1, CYP2U1, CYP2W1), CYP3 (CYP3A 4, CYP3A5, CYP3A7, CYP3A43, CYP3A51P, CYP3A52P, CYP3A54P, CYP3A137P), CYP4(CYP4A11, CYP4A22, CY P4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4V2, CYP4X1, CYP4Z1, CYP4F3A, CYP4F3B ), CYP5(CYP5A1), CYP7(CYP7A1, CYP7B1), CYP8(CYP8A1, CYP8B1), CYP11(CYP11A1, CYP11B1, CYP11B2) , CYP17(CYP17A1), CYP19(CYP19A1), CYP20(CYP20A1), CYP21(CYP21A2, CYP21A1P), CYP24(CYP24A1), There is a family of 18 cytochrome P450 genes, including CYP26 (CYP26A1, CYP26B1, CYP26C1), CYP27 (CYP27A1, CYP27B1, CYP27C1), CYP39 (CYP39A1), CYP46 (CYP46A1, CYP46A4P), and CYP51 (CYP51A1, CYP51P1, CYP51P2, CYP51P3).

[0118] The combination of the bile acid and the phenylbutyrate compound may inhibit one or more CYPs (e.g., CYPs described herein or known in the art). For example, the combination of TURSO and sodium phenylbutyrate may inhibit one or more of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, or CYP3A (e.g., CYP3A4). In some embodiments, the combination of TURSO and sodium phenylbutyrate inhibits CYP2B6 and / or CYP2C8. The combination of TURSO and sodium phenylbutyrate may be a strong or moderate inhibitor of CYP. According to the FDA definition, a strong CYP inhibitor is predicted to increase the AUC of a highly sensitive indicator substrate of CYP by 5-fold or more. A moderate CYP inhibitor is predicted to increase the AUC of a highly sensitive indicator substrate of CYP by 2-fold to 5-fold, inclusive. Examples of clinically relevant indicator substrates with high or intermediate sensitivity for representative CYPs are shown below.

[0119] [Table A]

[0120] An index substrate exhibits a predictable increase in exposure due to inhibition or induction of a given metabolic pathway and is commonly used in prospective clinical DDI studies. A highly sensitive index substrate is an index drug that demonstrates a 5-fold or greater increase in AUC with a potent index inhibitor of a given metabolic pathway in clinical DDI studies. An intermediately sensitive substrate is a drug that demonstrates a 2-fold or greater but less than 5-fold increase in AUC with a potent index inhibitor of a given metabolic pathway in clinical DDI studies. Representative potent index inhibitors of CYP are known in the art (see https: / / www.fda.gov / drugs / drug-interactions-labeling / drug-development-and-drug-interactions-table-substrates-inhibitors-and-inducers).

[0121] A composition comprising TURSO and sodium phenylbutyrate, when administered to a subject in combination with a substrate for CYP (e.g., CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, or CYP3A (e.g., CYP3A4)), may result in an increase in the level (e.g., plasma concentration) of the substrate in the subject. For example, a composition comprising TURSO and sodium phenylbutyrate, when administered to a subject in combination with a substrate for CYP2B6 or CYP2C8, may cause an increase in the level of the substrate in the subject. In some examples, a metabolite of TURSO or sodium phenylbutyrate is inhibitory to one or more CYPs. For example, phenylacetylacetate, a metabolite of phenylbutyrate, may be inhibitory to CYP1A2 and / or CYP2D6.

[0122] Substrates of CYPs can be drugs or other substances (e.g., xenobiotics) that are metabolized by CYPs. Substrates of CYPs can be pharmacologically active drugs that require metabolism to an inactive form for elimination from the subject's body or metabolically activated drugs (e.g., prodrugs) that require conversion to an active drug.

[0123] Many substrates of CYP are known in the art. Representative substrates of CYP1A2 include alosetron, caffeine, duloxetine, melatonin, ramelteon, tasimelteon, tizanidine, clozapine, pirfenidone, ramosetron and theophylline. Representative substrates of CYP2B6 include bupropion and efavirenz. Representative substrates of CYP2C8 include repaglinide, montelukast, pioglitazone and rosiglitazone. Representative substrates of CYP2C9 include celecoxib, glimepiride, phenytoin, warfarin, tolbutamide and warfarin. Representative substrates of CYP2C19 include S-mephenytoin, omeprazole, diazepam, lansoprazole, rabeprazole or voriconazole. Representative substrates of CYP2D6 include atomoxetine, desipramine, dextromethorphan, eliglustat, nebivolol, nortriptyline, perphenazine, tolterodine, R-venlafaxine, encainide, imipramine, metoprolol, propafenone, propranolol, tramadol, trimipramine, and S-venlafaxine. Representative substrates of CYP3A include alfentanil, avanafil, buspirone, conivaptan, cyclosporine, quinidine, darifenacin, darunavir, ebastine, everolimus, ibrutinib, lomitapide, lovastatin, midazolam, naloxegol, nisoldipine, saquinavir, simvastatin, sirolimus, tacrolimus, tipranavir, triazolam, vardenafil, budesonide, dasatinib, dronedarone, eletriptan, eplerenone, felodipine, indinavir, lurasidone, maraviroc, quetiapine, sildenafil, ticagrelor, tolvaptan, alprazolam, aprepitant, atorvastatin, colchicine, eliglustat, pimozide, rilpivirine, rivaroxaban or tadalafil. Representative substrates of CYP3A4 include alfentanil, cyclosporine, and quinidine.

[0124] Methods for identifying substrates of CYPs are known in the art, for example, cytochrome P450 reaction phenotyping can be used (see Zhang et al. Expert Opin Drug Metab Toxicol. 2007 Oct;3(5):667-87).

[0125] Provided herein is a method of treating at least one symptom of ALS in a subject, the method comprising: (a) administering to a subject to whom a first dose of a substrate of CYP has been administered a composition (e.g., an effective amount of the composition) comprising about TURSO and about sodium phenylbutyrate; (b) determining or having determined a first level of the substrate or a metabolite thereof in a first biological sample of the subject; and (c) administering to the subject a second dose of the substrate, the second dose being less than the first dose. For example, the composition can comprise about 1 g of TURSO and 3 g of sodium phenylbutyrate. The method can further comprise a step (d) of determining or having determined a second level of the substrate or a metabolite thereof in a second biological sample of the subject. In some embodiments, the second level of the substrate is less than the first level.

[0126] In some embodiments of the methods described herein, the CYP is CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, or CYP3A. The CYP substrate can be a CYP1A2 substrate, such as mexiletine. The CYP substrate can be a CYP3A4 substrate, such as alfentanil, cyclosporine, and quinidine. The CYP substrate can be a CYP2C9 substrate, such as warfarin. The CYP substrate can be a CYP2D6 substrate, such as mexiletine. In some embodiments, the CYP is CYP2B6 or CYP2C8. The CYP2B6 substrate can include bupropion or efavirenz. The CYP2C8 substrate can include repaglinide, montelukast, pioglitazone, or rosiglitazone.

[0127] CYP substrates can also be NTI drugs, for example, mexiletine, alfentanil, quinidine, cyclosporine, and warfarin are CYP substrates with narrow therapeutic indices.

[0128] In some embodiments, the CYP substrate is mexiletine. Mexiletine can be used to treat ventricular arrhythmias, organ transplant rejection, myotonia, renal dysfunction, or hepatic dysfunction. Mexiletine can be metabolized by CYP2D6 and CYP1A2. The subject may be administered a first dose of about 100 to about 1200 mg / day (e.g., about 200 to about 1000 mg / day, about 250 to about 950 mg / day, about 300 to about 900 mg / day, about 300 mg / day, or about 900 mg / day) of mexiletine. The second dose of mexiletine can be about 10 to about 1150 mg / day (e.g., about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 900, 1000, or 1100 mg / day) less than the first dose.

[0129] In some embodiments, the CYP substrate is alfentanil. Alfentanil is a short-acting opioid anesthetic and analgesic derivative of fentanyl. Alfentanil can be used as an anesthetic during surgery, to supplement analgesia during surgical procedures, and as an analgesic for critically ill patients. Alfentanil can be metabolized by CYP3A4 and CYP3A5. The first dose of alfentanil may depend on the length of anesthesia. Exemplary first doses (e.g., induction or maintenance doses) of alfentanil are shown below:

[0130] [Table B]

[0131] In some embodiments, the CYP substrate is quinidine. Quinidine is a stereoisomer of quinine. Quinidine can be used to restore normal sinus rhythm, treat atrial fibrillation and flutter, and treat ventricular arrhythmias. Quinidine can be metabolized by CYP3A4. In the case of oral dosage forms, the first dose of quinidine can be about 200 to about 650 mg every 3 or 4 times a day, or about 300 to 660 mg every 8 to 12 hours, for example, when the subject is an adult. The second dose of quinidine can be about 10 mg to about 600 mg (e.g., about 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 350, 400, 450, 500, or 550 mg) less per administration than the first dose. The second dose of quinidine can also be administered less frequently (e.g., fewer times per day) than the first dose of quinidine. In the case of an infusion dosage form, the first dose of quinidine can be about 190 to about 380 mg injected intramuscularly every 2 to 4 hours or up to 0.25 mg / kg body weight / min in solution injected intravenously. The second dose of quinidine can be about 10 mg to about 350 mg (e.g., about 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, or 300 mg) less than the first dose per administration. If the subject is a child, the first dose of quinidine may be about 30 to about 40 mg / kg body weight / day for an oral dosage form.

[0132] In some embodiments, the CYP substrate is cyclosporine. Cyclosporine may be metabolized by CYP3A4 and CYP3A5. Cyclosporine can be used for the prevention of organ rejection in kidney, liver and heart allografts or for preventing bone marrow transplant rejection. Cyclosporine is used for the treatment of patients with severe active rheumatoid arthritis (RA) or severe refractory plaque psoriasis. Cyclosporine eye drops have been shown to increase tear production in patients suffering from keratoconjunctivitis sicca. In addition, cyclosporine has been approved for the treatment of steroid-dependent and steroid-resistant nephrotic syndrome resulting from glomerular disease, which may include minimal change nephrotic syndrome, focal segmental glomerulosclerosis or membranous glomerulonephritis. Cyclosporine is also commonly used for the treatment of various autoimmune and inflammatory conditions, such as atopic dermatitis, bullous disorders, ulcerative colitis, juvenile rheumatoid arthritis, uveitis, connective tissue diseases, and idiopathic thrombocytopenic purpura. The subject may be administered a first dose of cyclosporine of about 0.5 to about 15 mg / kg body weight / day (e.g., about 0.5 to about 5 mg / kg body weight / day, about 1 to about 4 mg / kg body weight / day, about 2.5 mg / kg body weight / day, or about 12 to about 15 mg / kg body weight / day). The second dose of cyclosporine can be about 0.1 to about 14 mg / kg body weight / day (e.g., about 0.5 to about 2.5 mg / kg body weight / day, about 1 to about 5 mg / kg body weight / day) less than the first dose.

[0133] In some embodiments, the CYP substrate is warfarin. Warfarin can be used as an anticoagulant to prevent blood clots (e.g., deep vein thrombosis and pulmonary embolism) and to prevent stroke in subjects with atrial fibrillation, valvular heart disease, or artificial heart valves. Warfarin can be metabolized by CYP2C9, CYP1A2, and CYP3A. The subject may be administered a first dose of warfarin of about 0.5 to about 12 mg / day (e.g., about 2 to about 6 mg / day, about 5 to about 8 mg / day, or about 7 to about 10 mg / day). The second dose of warfarin can be less than the first dose of warfarin and can be about 0.5 to about 9.5 mg / day (e.g., about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, or 9 mg / day).

[0134] Metabolites of the CYP substrates described herein are known in the art. For example, metabolites of mexiletine include, but are not limited to, p-hydroxymexiletine, hydroxy-methylmexiletine, N-hydroxy-mexiletine, and N-methylmexiletine. Representative metabolites of alfentanil include noralfentanil and N-phenylpropionamide. Representative metabolites of quinidine include 3-hydroxyquinidine, 2'-oxoquinidinone, O-desmethylquinidine, and quinidine-N-oxide. Representative metabolites of cyclosporine include M1, M9, and M4N. Further examples of cyclosporine metabolites can be found in Yatscoff et al. Clin Biochem, Vol 24, pp.23-35, 1991. Representative metabolites of R-warfarin include hydroxywarfarin, 10-hydroxywarfarin, and diastereoisomeric alcohols. S-warfarin can be metabolized to 7-hydroxywarfarin.

[0135] In addition to or instead of determining the first and / or second levels of the substrate or its metabolites in the subject's biological sample, other methods of monitoring the subject's response to a first dose of the substrate are also contemplated herein. For example, known adverse events, side effects, or overdose symptoms associated with the substrate can be monitored. Symptoms of mexiletine overdose can include nausea, hypotension, sinus bradycardia, paresthesia, seizures, bundle branch block, AV heart block, asystole, ventricular tachycardia including ventricular fibrillation, cardiovascular collapse or coma. Symptoms of alfentanil overdose can include characteristic contractures of skeletal muscles, cardiac and respiratory depression, and constriction of the pupils. Symptoms of quinidine overdose can include irregular heartbeat, diarrhea, vomiting, headache, tinnitus or loss of hearing, visual changes (blurred vision or photosensitivity), or confusion. Symptoms of cyclosporine overdose can include hepatotoxicity and nephrotoxicity. Symptoms of warfarin overdose primarily involve bleeding (e.g., blood in the stool or urine, hematuria, excessive menstrual bleeding, stool, petechiae, excessive bruising or persistent oozing from superficial wounds, unexplained decrease in hemoglobin) that is a manifestation of excessive anticoagulation.

[0136] When CYP substrate is warfarin, subject's response can also be monitored by prothrombin time-international normalized ratio (PT-INR). PT-INR measures the time it takes for subject's blood to form a clot, and can be used to determine whether the appropriate dose of warfarin has been administered. Methods for monitoring subject's INR level are known in the art. Typical INR targets are 2.0-3.5 (e.g. 2.0-3.3, 2.0-3.0, 2.0-2.8, 2.0-2.6, 2.0-2.4, 2.0-2.2, 2.2-3.5, 2.2-3.3, 2.2-3.0, 2.2-2.8, 2.2-2.6, 2.2-2.4, 2.4-3.5, 2.4-3.3, 2.4-3.0, 2.4-2.8, 2.4-2.6, 2.6-3.5, 2.6-3.3, 2.6-3.0, 2.6-2.8, 2.8-3.5, 2.8-3.3, 2.8-3.0, 3.0-3.5, 3.0-3.3, or 3.3-3.5). The INR can be monitored once daily, twice weekly, once weekly, once every two weeks, once every three weeks, or once every four weeks.

[0137] The warfarin dose can be adjusted so that the PT-INR blood test is in the target range. The INR can be monitored more frequently if the dose is changed, if the subject starts or stops another medication, or if the subject's medical condition changes. It can be monitored less frequently if the dose is stable. A typical monitoring frequency for stable dosing is approximately every 4 to 6 weeks (e.g., every 4, 5, or 6 weeks).

[0138] In one aspect, provided herein is a method of treating at least one symptom of ALS in a subject, the method comprising: (a) administering to a subject who has been administered a first dose of warfarin a composition (e.g., an effective amount of the composition) comprising TURSO and sodium phenylbutyrate; (b) determining, or having determined, a first blood INR level of the subject; and (c) administering to the subject a second dose of warfarin, where the second dose is less than the first dose. For example, the composition can comprise 1 g of TURSO and 3 g of sodium phenylbutyrate.

[0139] The first dose of warfarin can be about 0.5 to about 12 mg / day (e.g., about 2 to about 6 mg / day, about 5 to about 8 mg / day, or about 7 to about 10 mg / day). The second dose of warfarin can be less than the first dose of warfarin and can be about 0.5 to about 9.5 mg / day (e.g., about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, or 9 mg / day).

[0140] In some embodiments, step (b) determines or includes having determined the subject's first blood INR level once daily. The subject's first blood INR level can be determined at any suitable frequency, e.g., once every other day, twice a week, once a week, once every two weeks, once every three weeks, or once every four weeks. In some embodiments, the first blood INR level is about 3.0 or greater (e.g., about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, or about 10.0). In some embodiments, the first blood INR level is about 4.0 or greater (e.g., about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, or about 10.0). In some embodiments of the methods described herein, the method further comprises a step (d) of determining, or having determined, a second blood INR level of the subject. In some embodiments, the second blood INR level is lower than the first blood INR level.

[0141] Transporter Transporters are membrane proteins involved in the uptake or excretion of drugs and are present in various tissues such as lymphocytes, intestine, liver, kidney, placenta and the central nervous system. Transporters can significantly affect the pharmacokinetics of endogenous compounds (e.g., ions, vitamins or amino acids) and exogenous compounds. Coadministration of drugs or nutrients can affect transporter activity, which can cause changes in the pharmacokinetics of drugs and, in some cases, result in reduced efficacy or increased toxicity (e.g., drug-drug or drug-nutrient interactions). Representative transporters include P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), organic anion transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3), organic anion transporters 1 and 3 (OAT1 and OAT3), organic cation transporters 1 and 2 (OCT1 and OCT2), and multidrug and toxin extrusion proteins 1 and 2K (MATE1 and MATE2K).

[0142] 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 or bidirectional movement across cell membranes. Uptake transporters at the blood-brain barrier (BBB) ​​are responsible for bringing solutes from the circulation into endothelial cells (i.e., apical / luminal membranes) and then through the basolateral membrane to the brain. Representative uptake transporters include H+ / ditripeptide transporters, organic anion transporting polypeptides (OATPs, e.g., OATP1B1 or OATP1B3), organic anion transporters (e.g., OAT1 or OAT3), and organic cation transporters (OCT) 1 and OCT2.

[0143] Efflux transporters pump compounds across the cell apical membrane (i.e., the blood side) back into the blood and on the basolateral side pump compounds out of the cell to the brain. Exemplary efflux transporters include P-glycoprotein (P-gp, ABCB1), multidrug resistance associated protein (MRP) 1 and MRP2, and breast cancer resistance protein (BCRP, ABCG2).

[0144] Applicants have discovered that a combination of a bile acid or a pharma- ceutically acceptable salt thereof and a phenylbutyrate compound can inhibit one or more transporters, e.g., transporters described herein or known. For example, a combination of TURSO and sodium phenylbutyrate can inhibit one or more transporters, e.g., OAT1. Accordingly, Applicants disclose herein a method for treating at least one symptom of ALS in a subject administered a first dose of a substrate of a transporter, comprising administering to the subject a composition comprising about 1 g of TURSO and about 3 g of sodium phenylbutyrate, monitoring the subject for a response to the substrate, and administering a second dose of the substrate, wherein the second dose is less than the first dose. Monitoring the subject for a response to the substrate can include determining, or having determined, a level of the substrate or a metabolite thereof in a biological sample of the subject, or monitoring for a known adverse event, overdose symptom, or side effect associated with the substrate.

[0145] Substrates of transporters are known in the art. Representative substrates of P-gp include digoxin, fexofenadine, loperamide, quinidine, talinolol and vinblastine. Representative substrates of BCRP include 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), coumestrol, daidzein, dantrolene, estrone-3-sulfate, genistein, prazosin and sulfasalazine. Representative substrates of OATP1B1 or OATP1B3 include cholecystokinin octapeptide (CCK-8), estradiol-17β-glucuronide, estrone-3-sulfate, pitavastatin, pravastatin, telmisartan and rosuvastatin. Representative substrates of OAT1 include penicillin, nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., diclofenac, ketoprofen, or methotrexate), HIV protease inhibitors, and antivirals (e.g., adefovir, cidofovir, and tenofovir). Representative substrates of OAT3 include benzylpenicillin, estrone-3-sulfate, methotrexate, and pravastatin. Representative substrates of MATE1 or MATE-2K include metformin, 1-methyl-4-phenylpyridinium (MPP+), and tetraethylammonium (TEA). Representative substrates of OCT2 include metformin, 1-methyl-4-phenylpyridinium (MPP+), and tetraethylammonium.

[0146] In one aspect, provided herein is a method of treating at least one symptom of ALS in a subject, the method comprising: (a) administering to a subject to whom a first dose of a substrate for organic anion transporter 1 (OAT1) has been administered a composition (e.g., an effective amount of the composition) comprising TURSO and sodium phenylbutyrate; (b) determining or having determined a first level of the substrate in a first biological sample of the subject; and (c) administering to the subject a second dose of the substrate, the second dose being less than the first dose. For example, the composition can comprise about 1 g of TURSO and about 3 g of sodium phenylbutyrate. The method can further comprise a step (d) of determining or having determined a second level of the substrate in a second biological sample of the subject. In some embodiments, the second level of the substrate is less than the first level.

[0147] Representative substrates of OAT1 include penicillin, nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g. diclofenac, ketoprofen or methotrexate), HIV protease inhibitors and antivirals (e.g. adefovir, cidofovir and tenofovir).

[0148] 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 phenylbutyrate compound. The bile acid or a pharma- ceutically acceptable salt thereof and a phenylbutyrate 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 a period of 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 at least or about 5 years or more. The compound can be administered once or twice a day for 60 days or less (e.g., 55 days, 50 days, 45 days, 40 days, 35 days, 30 days or less). Alternatively, the bile acid and phenylbutyrate compounds can be administered once or twice daily for more than 60 days (e.g., more than 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 180, 200, 250, 300, 400, 500, 600 days).

[0149] 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 is 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, TURSO is administered at about 1 to about 2 g, inclusive (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). In some embodiments, TURSO is administered at about 1 g per day. In some embodiments, TURSO is administered at about 2 g per day. For example, TURSO can be administered at about 1 g twice a day.

[0150] 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, inclusive (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). 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 per day. In some embodiments, the bile acid and the phenylbutyrate compound are administered in a weight ratio of about 2.5:1 to about 3.5:1 (e.g., about 3:1).

[0151] The methods described herein can include administering about 1 g of TURSO once a day and about 3 g of sodium phenylbutyrate once a day or about 1 g of TURSO twice a day and about 3 g of sodium phenylbutyrate twice a day. The methods can include administering about 1 g of TURSO once a day and about 3 g of sodium phenylbutyrate once a 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 g of TURSO twice a day and about 3 g of sodium phenylbutyrate twice a day or at least one day (e.g., at least 30, 40, 50, 60, 80, 100, 120, 150, 180, 250, 300, or 400 days). For example, the method can include administering about 1 g of TURSO once per day and about 3 g of sodium phenylbutyrate once per day for 14 to 21 days, followed by administering about 1 g of TURSO twice per day and about 3 g of sodium phenylbutyrate twice per day.

[0152] In some embodiments, the methods described herein provide for 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 10 mg / kg to about 36 mg / kg. mg / 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 14mg / kg, about 10mg / kg to about 12mg / kg, about 12mg / kg to about 50mg / kg, about 12mg / kg to about 48mg / kg, about 12mg / kg to about 46mg / kg, about 12mg / kg to about 44mg / kg, about 12mg / kg to about 42mg / kg, about 12mg / kg ~40mg / kg, 12mg / kg~38mg / kg, 12mg / kg~36mg / kg, 12mg / kg~34mg / kg, 12mg / kg~32mg / kg, 12mg / kg~30mg / kg, 12mg / kg~28mg / kg, 12mg / kg~28mg / 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,about 14 mg / kg to about 22 mg / kg, about 14 mg / kg to about 20 mg / kg, about 14 mg / kg to about 18 mg / kg, about 14 mg / kg to about 16 mg / kg, about 16 mg / kg to about 50 mg / kg, about 16 mg / kg to about 48 mg / kg, about 16 mg / kg to about 46 mg / kg, about 16 mg / kg to about 44 mg / kg, about 16 mg / kg to about 42 mg / kg, about 16 mg / kg to about 40 mg / kg, about 16 mg / kg to about 38 mg / kg, about 16 mg / kg to about 36 mg / kg, about 16 mg / kg to about 34 mg / kg, about 16 mg / kg to about 32 mg / kg, about 16 mg / kg to about 30 mg / kg, about 16 mg / kg to about 28 mg / kg, about 16 mg / kg to about 26 mg / kg, about 16 mg / kg to about 24 mg / kg, about 16 mg / kg to about 22 mg / kg, about 16 mg / kg to about 20 mg / kg, about 16 mg / kg to about 18 mg / kg, about 18 mg / kg to about 50 mg / kg, about 18 mg / kg to about 48 mg / kg, about 18 mg / kg to about 46 mg / kg, about 18 mg / kg to about 44 mg / kg, about 18 mg / kg to about 42 mg / kg, about 18 mg / kg to about 40 mg / kg, about 18 mg / kg to about 38 mg / kg, about 18 mg / kg to about 36 mg / kg, about 18 mg / kg to about 34 mg / kg, about 18 mg / kg to about 32 mg / kg, about 18 mg / kg to about 30 mg / kg, about 18 mg / kg to about 28 mg / kg, about 18 mg / kg to about 26 mg / kg, about 18 mg / kg to about 24 mg / kg, about 18 mg / kg to about 22 mg / kg, about 18 mg / kg to about 20 mg / kg, about 20 mg / kg to about 50 mg / kg, about 20 mg / kg to about 48 mg / kg, about 20 mg / kg to about 46 mg / kg, about 20 mg / kg to about 44 mg / kg, about 20 mg / kg to about 42 mg / kg, about 20 mg / kg to about 40 mg / kg, about 20 mg / kg to about 38 mg / kg, about 20 mg / kg to about 36 mg / kg, about 20 mg / kg to about 34 mg / kg, about 20 mg / kg to about 32 mg / kg, about 20 mg / kg to about 30 mg / kg, about 20 mg / kg to about 28 mg / kg, about 20 mg / kg to about 26 mg / kg, about 20 mg / kg to about 24 mg / kg, about 20 mg / kg to about 22 mg / kg, about 22 mg / kg to about 50 mg / kg, about 22 mg / kg to about 48 mg / kg, about 22 mg / kg to about 46 mg / kg,About 22 mg / kg to about 44 mg / kg, about 22 mg / kg to about 42 mg / kg, about 22 mg / kg to about 40 mg / kg, about 22 mg / kg to about 38 mg / kg, about 22 mg / kg to about 36 mg / kg, about 22 mg / kg to about 34 mg / kg, about 22 mg / kg to about 32 mg / kg, about 22 mg / kg to about 30 mg / kg, about 22 mg / kg to about 28 mg / kg, about 22 mg / kg to about 26 mg / kg, about 22 mg / kg to about 24 mg / kg, about 24 mg / kg to about 50 mg / kg, about 24 mg / kg to about 48 mg / kg, about 24 mg / kg to about 46 mg / kg, about 24 mg / kg to about 44 mg / kg, about 24 mg / kg to about 42 mg / kg, about 24 mg / kg to about 40 mg / kg, about 24 mg / kg to about 38 mg / kg, about 24 mg / kg to about 36 mg / kg, about 24 mg / kg to about 34 mg / kg, about 24 mg / kg to about 32 mg / kg, about 24 mg / kg to about 30 mg / kg, about 24 mg / kg to about 28 mg / kg, about 24 mg / kg to about 26 mg / kg, about 26 mg / kg to about 50 mg / kg, about 26 mg / kg to about 48 mg / kg, about 26 mg / kg to about 46 mg / kg, about 26 mg / kg to about 44 mg / kg, about 26 mg / kg to about 42 mg / kg, about 26 mg / kg to about 40 mg / kg, about 26 mg / kg to about 38 mg / kg, about 26 mg / kg to about 36 mg / kg, about 26 mg / kg to about 34 mg / kg, about 26 mg / kg to about 32 mg / kg, about 26 mg / kg to about 30 mg / kg, about 26 mg / kg to about 28 mg / kg, about 28 mg / kg to about 50 mg / kg, about 28 mg / kg to about 48 mg / kg, about 28 mg / kg to about 46 mg / kg, about 28 mg / kg to about 44 mg / kg, about 28 mg / kg to about 42 mg / kg, about 28 mg / kg to about 40 mg / kg, about 28 mg / kg to about 38 mg / kg, about 28 mg / kg to about 36 mg / kg, about 28 mg / kg to about 34 mg / kg, about 28 mg / kg to about 32 mg / kg, about 28 mg / kg to about 30 mg / kg, about 30 mg / kg to about 50 mg / kg, about 30 mg / kg to about 48 mg / kg, about 30 mg / kg to about 46 mg / kg, about 30 mg / kg to about 44 mg / kg, about 30 mg / kg to about 42 mg / kg, about 30 mg / kg to about 40 mg / kg, about 30 mg / kg to about 38 mg / kg, 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 34mg / kg to approximately 36mg / kg, approximately 36mg / kg to approximately 50mg / kg, approximately 36mg / kg to approximately 48mg / kg, approximately 36mg / kg to approximately 46mg / kg, approximately 36mg / kg to approximately 44mg / kg, approximately 36mg / kg to approximately 42mg / 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.

[0153] In some embodiments, the methods described herein provide for 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 200 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 260 mg / kg, from about 10 mg / kg to about 200 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 200 mg / kg, from about 10 mg / kg to about 260 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 260 mg / kg, from about 10 mg / kg to about 200 mg / kg, from about 10 mg / kg to about 260 mg / kg, from about 10 mg / kg to about 260 mg / mg / 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 / kg kg, 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 320mg / 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.

[0154] 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.

[0155] The methods described herein can be used to treat or ameliorate at least one symptom of ALS in a subject, slow ALS disease progression, extend survival time of a subject with 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 skills. 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.

[0156] 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.

[0157] As disclosed herein, the methods can be used to treat a subject who has been diagnosed with ALS, is at risk for developing ALS, or is suspected of having ALS. The subject may have been diagnosed with ALS, for example, for 24 months or less (e.g., within a range therein). For example, the subject may have been diagnosed with ALS for one week or less, or on the same day that the treatment of the present disclosure is administered. The subject may have one or more symptoms of ALS for 24 months or less (e.g., any range within this range); may have an ALS disease progression rate (ΔFS) of about 0.50 or more (e.g., any range within this range); may have an ALSFRS-R score of 40 or less (e.g., any range within this range); may have lost an average of about 0.8 to about 2 points (e.g., any range within this range) on the ALSFRS-R per month over the past 3 to 12 months; and / or a serum albumin concentration 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, 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 of the subject in the methods described herein can 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 can 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 may have a CSF or blood level of NfL of 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 can be about 50 to about 300 pg / mL (e.g., a range within this range). In some embodiments, the subject in the methods described herein can have a CSF NfL level of about 2000 to about 40,000 pg / mL (eg, any range within this range).

[0158] 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 also refers to delaying the onset of symptoms (e.g., prevention) or delaying the progression of symptoms or loss of function associated with the disease. "Treating" ALS also refers 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 also refers to eliminating or reducing one or more direct or indirect effects of ALS disease 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 carry a mutation in a gene associated with ALS, may have a family history of having 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. The present disclosure contemplates any degree of delay in onset of symptoms, alleviation of one or more of the disease, or slowing of progression of one or more disease symptoms (e.g., improvement as measured by the ALSFRS-R or maintenance of an ALSFRS-R rating, signaling slower disease progression). Relief or reduction in severity of symptoms or features of benign fasciculation syndrome and fasciculation-fasciculation syndrome is also contemplated herein.

[0159] The treatment provided in the present disclosure can be initiated during the progression of the disease. For example, treatment can be initiated pre-symptomatically (e.g., muscle weakness, muscle fasciculations and / or muscle spasms), at the onset of symptoms, or immediately after detection of ALS symptoms, based on the observation of one or more symptoms that lead a person skilled in the art to suspect that the subject may be suffering from ALS (e.g., a subject at risk of developing ALS). Treatment can also be initiated at a later stage. For example, treatment may 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 initiation of treatment, the subject may experience muscle tension 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).

[0160] 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. The duration of preventative treatment can be a single dose or treatment can be continued indefinitely (e.g., multiple doses), for example, for years or over the lifespan of the subject. For example, a subject at risk for ALS can be treated 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., relief of one or more symptoms of ALS, improvement as measured by ALSFRS-R or 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 less than 60 administrations (e.g., less than 50, 45, 40, 35, 30, 25, 20, 15, or 10 administrations).

[0161] 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 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., nasal) 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, general 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.

[0162] After administration of the bile acid or a pharma- ceutically acceptable salt thereof and the phenylbutyrate 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.

[0163] Once a patient's condition has improved (e.g., a change (e.g., a decrease) in the level of disease in the subject), a maintenance dose of a compound, composition, or combination of the 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.

[0164] IV. Symptoms and Outcome Measures The present disclosure further provides methods for assessing ALS symptoms, monitoring ALS progression, and assessing a subject's response to treatment methods, 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.

[0165] The subject's respiratory function can be measured, for example, by 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 (FEV1). Muscle strength can be evaluated, for example, by handheld dynamometry (HHD), grip dynamometry, manual muscle testing (MMT), electrical impedance myography (EIM), maximum voluntary isometric contraction test (MVICT), motor unit number estimation (MUNE), accurate limb isometric muscle strength test (ATLIS) or combinations 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 pAktdAkt ratios can also be used to assess a subject's disease progression and response to treatment (see, for example, WO 2012 / 160563).

[0166] 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 in urine samples can be analyzed. 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), may also be utilized.

[0167] Muscle strength The subject's muscle strength can be assessed using methods known in the art. Quantitative measures 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).

[0168] 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 subject's lower limb, upper limb strength, and the subject's grip strength. In some embodiments, the ATLIS test detects changes in muscle strength before changes in function are observed.

[0169] 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.

[0170] 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 phenylbutyrate compound.

[0171] 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 of the present disclosure can result in a rate of decline in the subject's ATLIS total score of about 3.50 PPN / month 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 / month or less). The methods of the present disclosure 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 / month (e.g., at least about 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 PPN / month) compared to untreated control subjects. The average rate of decline in the subject's upper extremity ATLIS score can be reduced by at least about 0.50 PPN / month (e.g., at least about 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, or 0.90 PPN / month) compared to a control 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 / month (e.g., at least about 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, or 0.60 PPN / month) compared to a control subject not receiving a treatment described herein. In some embodiments, the improvement or maintenance of the subject's muscle strength can begin to 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 normal strength expected based on age, sex, weight, and height.

[0172] Pulmonary function ALS is a progressive neurodegenerative disease that eventually leads to respiratory insufficiency and death. Pulmonary function tests such as vital capacity (VC), maximum mid-expiratory flow rate (MMERF), forced vital capacity (FVC), resting vital capacity (SVC) and forced expiratory volume in 1 second (FEV1) can be used to monitor ALS progression and / or a subject's response to treatment. On average, 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 over one month. In some embodiments, pulmonary function test measures are related to survival time (see, for example, 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 standing and supine positions may also provide an earlier indicator of ventilatory muscle decline.

[0173] The methods provided herein may improve or maintain respiratory muscle and / or lung function in a subject, or may slow the deterioration of respiratory muscle and / or lung function in a subject. The subject's respiratory muscle and / or lung function may be assessed by any suitable method described herein or known in the art. In some embodiments, the human subject's respiratory muscle function is assessed based on the subject's SVC. In some embodiments of the methods of improving, maintaining, or slowing the 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 / month 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 / month or less). In some embodiments, the treatment reduces the average rate of decline in the subject's SVC by at least about 0.5 PPN / month (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 / month) compared to a control subject not receiving the treatment. In some embodiments, the improvement or maintenance of the subject's pulmonary function may begin to 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 becomes less progressive than would be expected within 60 days of the first administration.

[0174] 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 control 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 embodiments, the adverse event is a serious adverse event, such as, but not limited to, a respiratory adverse event, a fall, or a laceration.

[0175] In some aspects, administration of a combination of a bile acid and a phenylbutyrate compound can result in fewer adverse events (e.g., adverse events disclosed herein) or less severe adverse events compared to administration of either the bile acid or the phenylbutyrate compound alone.

[0176] The average survival time of ALS patients may vary. Median survival times 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 of subjects with ALS by at least one 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 embodiments, may delay the onset of ventilator dependency or tracheotomy by at least one 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).

[0177] 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 untreated control subjects. 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 embodiments, 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).

[0178] 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 shown the onset of ALS symptoms 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 shown the onset of ALS symptoms for more than about 24 months (e.g., more than 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 the onset of ALS symptoms 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 the onset of ALS symptoms 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).

[0179] In some embodiments, the 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 outcome measures 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 outcome measures described herein) than male subjects when treated at similar stages of disease progression. The pharmacokinetics of bile acids and phenylbutyrate compounds may be the same or different in female and male subjects.

[0180] V. Further Therapeutic Agents The methods described herein may further comprise administering to the subject one or more additional therapeutic agents, for example in an amount effective to treat or achieve regulation of at least one symptom of ALS. Any known ALS therapeutic agent known in the art may be used as the additional therapeutic agent. Representative therapeutic agents include riluzole (C8H5F3N2OS, e.g., sold under the names Rilutek® and Tiglutik®), edaravone (e.g., sold under the names Radicava® and Radicut®), dextromethorphan, anticholinergic medications, and psychiatric medications (e.g., antidepressants, antipsychotics, anxiolytics / hypnotics, mood stabilizers, and stimulants).

[0181] 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 (Tobyace®), flavoxate (Urispas®), glycopyrrolate, homatropine hydrobromide, hyoscyamine, serotonin ... These include scopolamine (Levsinex™), ipratropium (Atrovent®), orphenadrine, oxybutynin (Ditropan™ XL), propantheline (Pro-vansine®), 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.

[0182] The additional therapeutic agent can be administered during a period prior to administration of the first dose of a composition comprising a bile acid or a pharma- ceutically acceptable salt thereof (e.g., TURSO) and a phenylbutyrate compound (e.g., sodium phenylbutyrate) and / or during a period after administration of the final dose of the composition. 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 composition of the present disclosure. The absorption, metabolism, and / or excretion of the additional therapeutic agent may be affected by the bile acid or a pharma- ceutically acceptable salt thereof and / or the phenylbutyrate compound. For example, co-administration of sodium phenylbutyrate with riluzole or edaravone may 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 phenylbutyrate compound can improve riluzole tolerance by a subject compared to administering riluzole alone.

[0183] The combination of a bile acid or a pharma- ceutically acceptable salt thereof, a phenylbutyrate 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 phenylbutyrate 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 phenylbutyrate 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 used when the additional therapeutic agent is administered alone. Additionally or alternatively, the methods of the present disclosure may reduce the number of doses required of the other therapeutic agent (e.g., other ALS therapeutic agent) to achieve the same pharmacological effect.

[0184] The bile acid or its pharma- ceutically acceptable salt and the phenylbutyrate compound can be administered immediately after a meal (e.g., within 2 hours after a meal) or under fasting conditions. The subject may have 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 the phenylbutyrate 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 the phenylbutyrate 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 nutrient and caloric content 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., 1.5 hours, 1 hour, or 0.5 hours) before or after administering the bile acid or pharma- ceutically acceptable salt thereof and / or phenylbutyrate compound. EXAMPLES

[0185] Further aspects are disclosed in further detail in the following examples, which are provided by way of illustration and are in no way intended to limit the scope of the disclosure or the claims.

[0186] Example 1 Evaluation of the potential for induction of cytochrome P450 enzymes in human hepatocytes by sodium phenylbutyrate and tauroursodeoxycholic acid The cytotoxicity and induction potential of CYP mRNA (CYP1A2, CYP2B6, and CYP3A4) by the combination of sodium phenylbutyrate (PB) and tauroursodeoxycholic acid (TUDCA) was evaluated in human hepatocytes.

[0187] cytotoxicity The cytotoxicity of sodium phenylbutyrate (PB) and tauroursodeoxycholic acid (TUDCA) in combination was evaluated using human hepatocytes. Cryopreserved human hepatocytes ready for seeding and induction were thawed and isolated in Human Hepatocyte Thawing Medium. Cells were suspended in Human Hepatocyte Plating Medium, counted (cell viability was assessed by trypan blue exclusion), and plated (day 0) at 750,000 cells / mL (150,000 cells / well in 48-well plates) in BioCoat™ collagen-coated 48-well plates (Corning Life Sciences, catalog#354505, Tewksbury, MA) and incubated at 37°C in a 95% air / 5% CO2 incubator. After attachment (4 hours), the medium was replaced with fresh Hepatocyte Medium for overnight cell recovery. Hepatocytes were then treated with hepatocyte medium enriched with five concentrations of PB / TUDCA (PB / TUDCA, 14.8 / 1.62, 148 / 16.2, 826 / 108, 1480 / 1080 and 7400 / 1600 μM). A positive control (100 μM chlorpromazine) was treated in parallel. Vehicle controls were treated with hepatocyte medium containing the same content of solvent (1% MeOH instead of PB / TUDCA and 0.1% DMSO instead of chlorpromazine). Hepatocyte incubation was performed at 37° C. in a 95% air / 5% CO2 incubator for 3 days (72 hours), with daily replacement of hepatocyte medium containing PB / TUDCA, positive control or vehicle. Cell viability was measured by analyzing the intracellular conversion of the tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] to a formazan product by a dehydrogenase that is active only in viable cells. The absorbance of the formazan, which is proportional to the number of viable cells, was measured spectrophotometrically using the CellTiter96® AQueous One Solution Cell Proliferation Assay (MTS).Briefly, wells were rinsed with DPBS, then hepatocyte medium (200 μL) and CellTiter96® AQueous One Solution Cell Proliferation Assay reagent (40 μL) were added to each well, and cells were incubated for 1 h at 37° C. in a 95% air / 5% CO2 incubator. The absorbance of formazan in each well was measured at 492 nm using a FLUOstar® OPTIMA microplate reader (BMG Lab Technologies, Durham, NC, USA).

[0188] The cytotoxicity of PB / TUDCA and the positive control (100 μM chlorpromazine) in human hepatocytes (expressed as cell viability, ie, as a percentage of MTS absorbance relative to vehicle control) is summarized in Table 1.

[0189] [Table 1]

[0190] Cytotoxicity of PB and TUDCA combinations at five concentrations (PB / TUDCA, 14.8 / 1.62, 148 / 16.2, 826 / 108, 1480 / 1080 and 7400 / 1600 μM) was evaluated using one human hepatocyte donor. At 14.8 / 1.62 μM and 148 / 16.2 μM, PB / TUDCA was not cytotoxic, whereas at 826 / 108 μM, 1480 / 1080 μM and 7400 / 1600 μM, it was cytotoxic to the donor. As the cytotoxicity results for 826 / 108 μM PB / TUDCA were close to the baseline, this test concentration combination was included in the subsequent CYP induction study (using a different donor).

[0191] CYP induction Cryopreserved human hepatocytes ready for seeding and induction were thawed and isolated in Human Hepatocyte Thawing Medium. Cells were suspended in Human Hepatocyte Plating Medium, counted (cell viability was assessed by trypan blue exclusion), and plated (day 0) at 750,000 cells / mL (150,000 cells / well in 48-well plates) in BioCoat™ collagen-coated 48-well plates (Corning Life Sciences, catalog# 354505) and incubated at 37°C in a 95% air / 5% CO2 incubator. After attachment (4 hours), the medium was replaced with fresh hepatocyte medium for overnight cell recovery. Hepatocytes were then treated with hepatocyte medium enriched with three concentrations of PB / TUDCA (14.8 / 1.62, 148 / 16.2, and 826 / 108 μM based on cytotoxicity test results).

[0192] Positive controls were treated in parallel with hepatocyte medium fortified with known inducers of each CYP of interest, i.e., 50 μM omeprazole (OME) for CYP1A2, 1,000 μM phenobarbital for CYP2B6, or 50 μM rifampicin (RIF) for CYP3A4. Negative controls were treated with 10 μM flumazenil, and vehicle controls were treated with hepatocyte medium. All experiments were performed in triplicate. Hepatocyte incubation was performed at 37° C. in a 95% air / 5% CO2 incubator for 3 days (72 h) with daily changes of hepatocyte medium containing TA, positive or negative inducer, or vehicle. Experimental conditions for CYP induction and sample treatment are summarized in Table 2.

[0193] [Table 2]

[0194] Cell viability assay After CYP induction treatment, cells were used for cell viability assays. Cell viability (expressed as a percentage of MTS absorbance relative to vehicle control) was measured as described herein.

[0195] qPCR-mRNA assay After induction treatment, CYP mRNA expression was measured by qPCR. Total RNA was isolated from treated cells using RNeasy® Mini Kit (Qiagen, Valencia, CA, USA) and treated with RNase-free DNase (Qiagen) according to the manufacturer's protocol. RNA concentration was determined in a Qubit® fluorometer using Qubit RNA HS Assay Kit (Invitrogen). cDNA was synthesized from up to 1 μg of total RNA recovered from cells using QuantiTect® RT Kit (Qiagen). Analysis of CYP gene expression by qPCR (Table 3) was performed using a LightCycler® 480II system (Roche Diagnostics Corporation, Indianapolis, IN, USA).

[0196] [Table 3]

[0197] Relative mRNA was calculated as the fold increase (2 -ΔΔCt The percentage of mRNA fold increase relative to the positive control was calculated using the following formula: Induction rate (%) relative to positive control = 100 × (mRNA TA -mRNA ビヒクル ) / (mRNA 陽性対照 -mRNA ビヒクル ) was used to calculate.

[0198] Induction of CYP1A2, CYP2B6 and CYP3A4 mRNA by three concentrations of PB and TUDCA (14.8 / 1.62, 148 / 16.2 and 826 / 108 μM PB / TUDCA) was evaluated using three human hepatocyte donors. In donor 1, PB and TUDCA did not increase CYP1A2 and CYP3A4 mRNA at any of the three concentrations tested (less than 2-fold over vehicle control and less than 20% of positive control). PB and TUDCA did not increase CYP2B6 mRNA at 14.8 / 1.62 and 148 / 16.2 μM, but a 5.56-fold induction was observed at 826 / 108 μM.

[0199] In donor 2, PB and TUDCA did not increase CYP1A2 or CYP3A4 mRNA at any of the three concentrations tested. PB and TUDCA did not increase CYP2B6 mRNA at 14.8 / 1.62 μM, but 3.18- and 6.82-fold induction was observed at 148 / 16.2 μM and 826 / 108 μM, respectively.

[0200] In donor 3, PB and TUDCA did not increase CYP1A2 or CYP3A4 mRNA at any of the three concentrations tested. PB and TUDCA did not increase CYP2B6 mRNA at 14.8 / 1.62 and 148 / 16.2 μM, but a 6.66-fold induction was observed at 826 / 108 μM.

[0201] Induction of CYP1A2, CYP2B6 and CYP3A4 mRNA in human hepatocytes by PB / TUDCA, positive control and negative control are summarized in Tables 4 to 6. Cell viability results after CYP induction treatment are summarized in Table 7.

[0202] [Table 4]

[0203] [Table 5]

[0204] [Table 6]

[0205] [Table 7]

[0206] In conclusion, PB and TUDCA are unlikely to be inducers of CYP1A2 or CYP3A4 at concentrations between 14.8 / 1.62 μM and 826 / 108 μM. Induction of CYP2B6 by PB and TUDCA was observed primarily at 826 / 108 μM.

[0207] Example 2 Inhibition of Cytochrome P450 Enzymes in Human Liver Microsomes by Sodium Phenylbutyrate and Tauroursodeoxycholic Acid The inhibition of cytochrome P450 (CYP) enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A) in human liver microsomes (HLM) by sodium phenylbutyrate (PB) and tauroursodeoxycholic acid (TUDCA) was evaluated. Eight concentrations of PB and TUDCA were administered in the presence of MgCl2 (5 mM), CYP-specific probe substrates (K m The CYP enzyme activities were determined by measuring the formation of each CYP probe metabolite by LC-MS / MS.

[0208] Eight concentrations of PB (0-7400 μM, 50 × C) were used for each test article. max,u = 7400 μM, 0.1 × I gut = 6444 μM) and TUDCA (0 to 1600 μM, 50 × C max,u= 81 μM, 0.1 × I gut = 1600 μM) was incubated with pooled HLM (0.25 mg protein / mL) in phosphate buffer (100 mM, pH 7.4) containing MgCl2 (5 mM), NADPH (1 mM) and CYP probe substrate (PB and TUDCA co-incubation). The total organic solvent content in the final incubation was less than 1% (DMSO ≤ 0.1%, other organic solvents ≤ 1%). The reaction mixture without NADPH was equilibrated at 37 °C in a shaking water bath for 5 min. The reaction was initiated by the addition of NADPH followed by incubation at 37 °C for 10–30 min depending on the CYP isoform. The reaction was terminated by the addition of 2 volumes of ice-cold acetonitrile (ACN) containing an internal standard (IS, stable isotope-labeled CYP probe metabolite). Negative (vehicle) controls were performed without TA. Positive controls were performed in parallel using known CYP inhibitors. After removal of proteins by centrifugation (1,640 g) for 10 min at 4° C., the supernatant was transferred to an HPLC autosampler plate. The formation of CYP probe metabolites was determined by LC-MS / MS. The experimental conditions for CYP reactions and sample analysis are summarized in Table 8.

[0209] The percentage of control enzyme activity was calculated using the following formula: Percentage of control enzyme activity (%) = 100 x (enzyme activity in the presence of TA / enzyme activity in the absence of TA) was used to calculate.

[0210] [Table 8]

[0211] Enzyme activity was expressed as the peak area ratio between the probe metabolite and IS measured by LC-MS / MS. IC was calculated by fitting the experimental data (ratio of control enzyme activity to log [inhibitor concentration]) to a sigmoid model using GraphPad Prism (Version 5.0 or higher, GraphPad Software, San Diego, CA, USA) followed by nonlinear regression analysis.50 The values ​​were estimated.

[0212] The inhibition of CYP activity in HLM by PB and TUDCA is summarized in Table 9. The inhibition of CYP activity by positive inhibitors is summarized in Table 10.

[0213] [Table 9]

[0214] [Table 10]

[0215] Combinations of PB (up to 274 μM) and TUDCA (up to 59.3 μM) showed little to no inhibition (less than 20%) of any of the seven major CYPs tested. Combinations of PB (822 μM) and TUDCA (178 μM) showed 7% inhibition of CYP1A2, 12% inhibition of CYP2C8, 15% inhibition of CYP2C19, 1.5% inhibition of CYP2D6, 7-17% inhibition of CYP3A, 37% inhibition of CYP2B6, and 31% inhibition of CYP2C9. The combination of PB (2467 μM) and TUDCA (533 μM) showed 18% inhibition of CYP1A2, 8% inhibition of CYP2D6, 74% inhibition of CYP2B6, 26% inhibition of CYP2C8, 50% inhibition of CYP2C9, 24% inhibition of CYP2C19, and 27-37% inhibition of CYP3A. The combination of PB (7400 μM) and TUDCA (1600 μM), the highest concentration tested in this study, showed >50% inhibition of all seven major CYPs tested.

[0216] Example 3 Cytochrome P450 Response Phenotyping of Sodium Phenylbutyrate and Tauroursodeoxycholic Acid Using Human Liver Microsomes and Small Molecule Inhibitors The aim of this study was to perform cytochrome P450 (CYP) response phenotyping of sodium phenylbutyrate (PB) and tauroursodeoxycholic acid (TUDCA) using human liver microsomes (HLM) and small molecule inhibitors.

[0217] CYP reaction phenotypes of PB and TUDCA were assessed using pooled human liver microsomes (HLM, 0.5 mg protein / mL) and CYP selective inhibitors. The amounts of PB and TUDCA remaining after incubation (0, 5, 10, 20, 30, and 60 min) were measured by LC-MS / MS.

[0218] After 60 min of incubation with HLM, the percentages of PB and TUDCA remaining were greater than 95% in the absence and presence of NADPH, with or without a 15 min preincubation. These results suggest that PB and TUDCA are not metabolized by major CYPs in HLM.

[0219] method PB and TUDCA at one concentration each (5 μM in the final incubation) were incubated with HLM (0.5 mg protein / mL) in phosphate buffer (100 mM, pH 7.4) containing MgCl2 (5 mM) and NADPH (1 mM) in the absence and presence of CYP selective inhibitors (Table 11). For reversible inhibition, the reaction mixture without NADPH was equilibrated at 37°C for 5 min in a shaking water bath and the reaction was initiated by the addition of NADPH (except for the control without NADPH) and subsequently incubated at 37°C. For irreversible inhibition, the inhibitors were preincubated with HLM in the presence of NADPH for 15 min at 37°C and the reaction was initiated by the addition of TA. Aliquots of the incubated solution were sampled at 0, 5, 10, 20, 30 and 60 min. The reaction was terminated by the addition of ice-cold acetonitrile (ACN) containing 0.1% formic acid. After removal of proteins by centrifugation at 1,640 g (3,000 rpm) for 10 min at 4° C., the supernatant was transferred to an HPLC autosampler plate. The remaining percentage of TA (expressed as peak area ratio of test article to IS) was determined by LC-MS / MS. CYP enzyme activity of HLM was confirmed in parallel by determining the formation of CYP probe metabolites using standard curves by LC-MS / MS. The experimental conditions for CYP reaction phenotyping and sample analysis are summarized in Tables 12 and 13.

[0220] 4. Data Analysis The remaining percentage of TA was calculated using the following formula: TA survival rate (%) = 100 x A t / A0 (In the formula, A t and A0 are the peak area ratios of TA and IS at time t and time 0, respectively. was used to calculate.

[0221] result The percent survival of PB using pooled HLM (0.5 mg protein / mL) in the presence and absence of CYP selective inhibitors is summarized in Tables 14 and 15. The percent survival of TUDCA using pooled HLM (0.5 mg protein / mL) in the presence and absence of CYP selective inhibitors is summarized in Tables 16 and 17. CYP enzyme activity of HLM was confirmed in parallel by determining the formation of CYP probe metabolites using LC-MS / MS, and the results are summarized in Tables 18 and 19.

[0222] [Table 11]

[0223] [Table 12]

[0224] [Table 13]

[0225] [Table 14]

[0226] [Table 15]

[0227] [Table 16]

[0228] [Table 17]

[0229] [Table 18]

[0230] [Table 19]

[0231] Example 4 Cytochrome P450 reaction phenotype analysis of sodium phenylbutyrate and tauroursodeoxycholic acid using human recombinant CYP enzymes Cytochrome P450 (CYP) reaction phenotype analysis of sodium phenylbutyrate (PB) and tauroursodeoxycholic acid (TUDCA) was evaluated using human recombinant CYP enzymes (hrCYP). PB and TUDCA (5 μM each) were incubated with hrCYP (20 pmol CYP / mL) in phosphate buffer (100 mM, pH 7.4) containing MgCl2 (5 mM) and NADPH (1 mM). The amount of PB and TUDCA remaining after incubation (0, 5, 10, 20, 30, and 60 min) was measured by LC-MS / MS.

[0232] PA and TUDCA at one concentration each (5 μM in final incubation) were incubated with hrCYP (20 pmol CYP / mL) or CYP control (negative control without CYP enzyme, 0.1 mg protein / mL) in phosphate buffer (100 mM, pH 7.4) containing MgCl2 (5 mM) and NADPH (1 mM). The incubation mixture without NADPH was equilibrated at 37°C for 5 min in a shaking water bath. The reaction was initiated by the addition of NADPH and subsequently incubated at 37°C. Aliquots (100 μL) of the incubation solution were sampled at 0, 5, 10, 20, 30 and 60 min. The reaction was terminated by the addition of ice-cold acetonitrile (ACN) containing 0.1% formic acid. After removal of proteins by centrifugation at 1,640 g (3,000 rpm) for 10 min at 4°C, the supernatant was transferred to an HPLC autosampler plate and stored at -20°C until analysis. Test article (TA) retention (expressed as peak area ratio of TA to IS) was determined by LC-MS / MS. hrCYP activity was confirmed in parallel by determining the formation of CYP probe metabolites after 20 min incubation with CYP probe substrates using standard curves by LC-MS / MS. The experimental conditions for CYP reaction phenotyping and sample analysis are summarized in Table 20.

[0233] [Table 20]

[0234] The remaining percentage of TA was calculated using the following formula: TA survival rate (%) = 100 × A t / A0 (In the formula, A t where A is the peak area ratio of TA to internal standard (IS) at time t, and A0 is the average peak area ratio of TA to IS at time 0. Values ​​of the remaining percentages of PB and TUDCA in hrCYP (20 pmol CYP / mL) are summarized in Tables 21 and 22, respectively. hrCYP activity was confirmed in parallel by determining the formation of CYP probe metabolites using LC-MS / MS, and the results are summarized in Table 23.

[0235] [Table 21]

[0236] [Table 22]

[0237] [Table 23]

[0238] After 60 min of incubation with hrCYPs, >90% of PB and TUDCA remained (<10% lost) in the presence of hrCYP1A2, hrCYP2B6, hrCYP2C8, hrCYP2C9, hrCYP2C19, hrCYP2D6, and hrCYP3A4. These results suggest that PB and TUDCA are not metabolized by the major hepatic CYPs.

[0239] Example 5. Evaluation of the potential for time-dependent inhibition of cytochrome P450 enzymes in human liver microsomes by sodium phenylbutyrate and tauroursodeoxycholic acid. The potential for time-dependent inhibition (TDI) of cytochrome P450 (CYP) enzymes in human liver microsomes (HLM) by sodium phenylbutyrate (PB) and tauroursodeoxycholic acid (TUDCA) was evaluated. Eight concentrations of PB and TUDCA were preincubated with pooled HLM (0.25 mg protein / mL) for 30 min in the presence and absence of NADPH (co-incubation with PB and TUDCA), followed by CYP enzyme activity assays for CYP probe substrates and quantification of the formation of each CYP probe metabolite by LC-MS / MS.

[0240] The results showed that the difference in CYP inhibition by PB and TUDCA was less than 20% for all seven major hepatic CYPs tested in this study at all concentrations of PB and TUDCA after 30 min preincubation in the absence and presence of NADPH, indicating that the IC 50 This suggests that the shift is less than 1.5 (the threshold for TDI), but the ICs for PB and TUDCA 50 and IC 50 Shift values ​​could not be calculated due to the simultaneous incubation of PB and TUDCA. The results suggest that TDI of CYPs by PB and TUDCA is unlikely to occur.

[0241] CYP TDI was assessed by 30 min preincubation of TA with HLM in the presence and absence of NADPH, followed by CYP enzyme activity assay. CYP reactions were performed in an incubation volume of 200 μL. Briefly, eight concentrations of PB (0–7400 μM, 50 × C) were added per test article. max,u = 7400 μM, 0.1 × I gut = 6444 μM) and TUDCA (0 to 1600 μM, 50 × C max,u = 81 μM, 0.1 × I gutHLM (0.25 mg protein / mL) in phosphate buffer (100 mM, pH 7.4) containing MgCl2 (5 mM) in the presence (irreversible incubation conditions) and absence (reversible incubation conditions) of NADPH (1 mM) was preincubated for 30 min at 37 °C (PB and TUDCA co-incubation). The total organic solvent content in the final incubation was less than 1% (DMSO ≦ 0.1%, other organic solvents ≦ 1%). CYP reactions were initiated by adding the CYP probe substrate with (if NADPH was not added in the pre-incubation step) or without (if NADPH was added in the pre-incubation step) the addition of NADPH (1 mM). The reaction mixture was incubated at 37 °C for 10 to 30 min depending on the CYP isoform (Table 24). Reactions were terminated with ice-cold acetonitrile (ACN) containing an internal standard (IS, stable isotope-labeled CYP probe metabolite). Negative (vehicle) controls were performed using incubation mixtures that did not contain TA. Positive controls were run in parallel using known CYP time-dependent inhibitors. After removal of protein by centrifugation at 1,640 g (3,000 rpm) for 10 min at 4° C., the supernatant was transferred to an HPLC autosampler plate. The formation of CYP probe metabolites was determined by LC-MS / MS. The experimental conditions for CYP reactions and sample analysis are summarized in Table 24.

[0242] The percentage of control enzyme activity was calculated using the following formula: Percentage of control enzyme activity (%) = 100 x (enzyme activity in the presence of TA / enzyme activity in the absence of TA) was used to calculate.

[0243] Enzyme activity was expressed as the peak area ratio between CYP probe metabolite and IS measured by LC-MS / MS. IC was calculated by fitting the experimental data (ratio of control enzyme activity to log [inhibitor concentration]) to a sigmoid model using GraphPad Prism (Version 5.0 or higher, GraphPad Software, San Diego, CA, USA) followed by nonlinear regression analysis. 50 The values ​​were estimated.

[0244] [Table 24]

[0245] IC during reversible incubation conditions (30 min preincubation without NADPH) or irreversible incubation conditions (30 min preincubation with NADPH) 50 The shift is an indication of possible TDI, and the threshold for a positive result is IC 50 The shift is >1.5.

[0246] The potential for CYP TDI in HLM by PB and TUDCA is summarized in Table 25. Inhibition of CYP activity by positive time-dependent inhibitors is summarized in Tables 26-27 and Figure 1.

[0247] [Table 25]

[0248] [Table 26]

[0249] [Table 27]

[0250] Example 6 Evaluation of drug-drug interactions by transporters The objective of this study was to evaluate the ability of AMX0035 as a substrate and inhibitor (IC) of the following transporters: P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), organic anion transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3), organic anion transporters 1 and 3 (OAT1 and OAT3), organic cation transporters 1 and 2 (OCT1 and OCT2), and multidrug and toxin extrusion proteins 1 and 2K (MATE1 and MATE2K). 50 The objective of this study was to evaluate the efficacy and safety of AMX0035 in treating chronic bronchitis. AMX0035 is a combination of TUDCA and PB. In this study, both TUDCA and PB were present simultaneously in all dosing solutions.

[0251] The inhibitory potency of test articles was determined (IC) using HEK cell lines transfected with each of the uptake transporters of interest (OATP1B1, OATP1B3, OAT1, OAT3, OCT2, MATE1 or MATE2K). 50 Estimated IC for OAT1 50 The estimated IC for OCT2 ranged from 0.0741 to 0.222 μM for TUDCA and 6.76 to 20.3 μM for PB. 50 The estimated IC for OAT3 was greater than 162 μM for TUDCA and 14785 μM for PB. 50 The estimated IC for OATP1B1 ranged from 54.0 to 162 μM for TUDCA and from 4928 to 14785 μM for PB. 50 The estimated IC for OATP1B3 ranged from 12.9 to 38.7 μM for TUDCA and from 1208 to 3625 μM for PB. 50 The estimated IC for MATE1 ranged from 38.7 to 116 μM for TUDCA and 3625 to 10875 μM for PB. 50 The estimated IC for MATE2K was 270–810 μM for TUDCA and 24642–73927 μM for PB. 50 was greater than 810 μM for TUDCA and 73,927 μM for PB.

[0252] AMX0035 was evaluated as an inhibitor of P-gp in MDR1-MDCK and C2BBe1 cells. Transport of the P-gp probe substrate digoxin (10 μM) across cell monolayers was used as an indicator of P-gp activity. Assays were performed bidirectionally from apical to basolateral (AP to BL) and basolateral to apical (BL to AP). In MDR1-MDCK cells, the estimated IC 50 The estimated IC values ​​for TUDCA and PB ranged from 889 to 2668 μM and 7161 to 21482 μM, respectively. In C2BBe1 cells, the estimated IC values ​​for P-gp were 50 was 222-667 μM for TUDCA and 1790-5371 μM for PB.

[0253] AMX0035 was evaluated as an inhibitor of BCRP in BCRP-MDCK and C2BBe1 cells. Transport of the BCRP probe substrate cladribine (10 μM) across cell monolayers was used as an indicator of BCRP activity. The assay was performed bidirectionally. In BCRP-MDCK cells, the estimated IC 50 The estimated IC values ​​for TUDCA and PB were 296–889 μM and 2387–7161 μM, respectively. In C2BBe1 cells, the estimated IC values ​​for P-gp were 50 was 222-667 μM for TUDCA and 1790-5371 μM for PB.

[0254] AMX0035 was evaluated as a substrate of P-gp and BCRP in both directions in C2BBe1 cells. The efflux ratios of TUDCA were 0.506, 0.305, 0.230, and 0.302 at 81.0, 16.2, 1.62, and 0.810 μM, respectively. The efflux ratios of PB were 0.740, 0.620, 0.940, and 1.65 at 1479, 148, 73.9, and 14.8 μM, respectively.

[0255] In summary, in vitro, TUDCA and PB are not substrates for either P-gp or BCRP. The estimated IC of TUDCA for each transporter was 50The estimated IC of PB for each transporter was as follows: 0.0741-0.222 μM (OAT1), >162 μM (OCT2), 54.0-162 μM (OAT3), 12.9-38.7 μM (OATP1B1), 38.7-116 μM (OATP1B3), 270-810 μM (MATE1), >810 μM (MATE2K), 889-2668 μM (P-gp in MDR1-MDCK cells), 222-667 μM (P-gp in C2BBe1 cells), 296-889 μM (BCRP in BCRPMDCK cells) and 222-667 μM (BCRP in C2BBe1 cells). 50 are as follows: 6.76-20.3 μM (OAT1), >14785 μM (OCT2), 4928-14785 μM (OAT3), 1208-3625 μM (OATP1B1), 3625-10875 μM (OATP1B3), 24642-73927 μM (MATE1), >73927 μM (MATE2K), 7161-21482 μM (P-gp in MDR1-MDCK cells), 1790-5371 μM (P-gp in C2BBe1 cells), 2387-7161 μM (BCRP in BCRPMDCK cells) and 1790-5371 μM (BCRP in C2BBe1 cells).

[0256] introduction The relevant concentrations of TUDCA and PB are listed in Table 28. The test concentrations of TUDCA and PB selected based on analytical sensitivity and clinical relevance in this example are summarized in Table 29.

[0257] For substrate and inhibitor potential evaluation of efflux transporters such as P-gp and BCRP, the preferred in vitro method was a cell-based bidirectional permeability assay. In this example, MDCK and C2BBe1 cells transfected with human P-gp and BCRP were used. For substrate and inhibitor potential evaluation of uptake transporters such as OATP1B1 and OATP1B3, cell lines with overexpression of the target genes were used.

[0258] [Table 28]

[0259] [Table 29]

[0260] method Chemical Stability Evaluation The chemical stability of TUDCA and PB was tested in HBSSg_7.4 and HBSS_8.5. Test articles were prepared in DMSO stocks (0.5 mM) and aliquots of the DMSO stocks were added to each matrix. The nominal concentration of each component was 0.5 μM in each matrix. Aliquots were transferred to separate vials (one vial per time point) and the vials were incubated at 37°C, 5% CO2 in a humidified incubator. At the designated time points, an equal volume of acetonitrile containing an internal standard was added to each vial and the mixtures were placed at 4°C until the end of the assay. The peak areas of the test article and IS for each sample were determined by LC-MS / MS and the peak area ratios (analyte:IS) were calculated.

[0261] Solubility evaluation All solubility samples were prepared in silanized glass tubes. An aliquot of powder of each component was added to each matrix in an amber vial (Table 30). After addition of buffer, the samples were incubated overnight (1020-1080 min) in a shaking water bath at 37 °C. After incubation, the mixtures were centrifuged at 20,800 g for 15 min. The supernatants were appropriately diluted and the concentrations of each component were determined by LC-MS / MS.

[0262] [Table 30]

[0263] Non-specific binding assessment Since the assay plates used for efflux and uptake transporters were made of different materials, nonspecific binding assessment of TUDCA and PB was performed using the corresponding cell-free plates.

[0264] (i) Efflux transporter format Rat-tail collagen-coated transwell plates (12 wells) were used and all incubations were performed in a humidified incubator at 37°C and 5% CO2. Aliquots of TUDCA and PB DMSO stocks were added to HBSSg_7.4. Triplicate wells were used for the assay (Table 31). Samples were removed from the receiver and donor compartments at preselected time points and mixed with an equal volume of acetonitrile containing IS. Concentrations of TUDCA and PB were determined by LC-MS / MS.

[0265] (ii) Uptake transporter format Poly-D-lysine coated plates (24 wells) were used and all incubations were performed in a humidified incubator at 37°C and 5% CO2. TUDCA and PB dosing solutions were prepared in HBSSg_7.4 and HBSSg_8.5. Two treatments (3 wells each) were included, in the first treatment, wells were pre-incubated with HBSSg_7.4_BSA or HBSSg_8.5_BSA for 5 min; in the second treatment, no pre-incubation was performed (Table 32). After pre-incubation, the solution was aspirated and dosing solution (500 μL) was added to each well. The plate was placed in a humidified incubator at 37°C and 5% CO2 for 5 min. After incubation, aliquots (200 μL) were removed from each chamber and combined with an equal volume of acetonitrile containing IS and stored at 4°C until analysis. Concentrations of TUDCA and PB were determined by LC-MS / MS.

[0266] [Table 31]

[0267] [Table 32]

[0268] Uptake format transporter-associated HEK cell culture Human embryonic kidney (HEK293) cells transfected with uptake transporter genes (OATP1B1, OATP1B3, OAT1, OAT3, OCT2, MATE1 or MATE2K) or blank vector control genes (VC) were used to evaluate the substrate and inhibitory potential of test articles against the corresponding transporters. Cells were maintained in DMEM supplemented with PEST, 10% FBS, 1% NEAA and 1 mM sodium pyruvate in a humidified incubator at 37°C and 5% CO2. The medium was changed three times weekly and cell growth was observed under a light microscope. After the cells reached confluence, they were harvested by trypsinization and the harvested cells were seeded in plates for uptake studies. The plates were placed in a humidified incubator at 37°C and 5% CO2. One day after seeding, MATE and vector control transfected cells were incubated with medium containing 10 mM sodium butyrate for approximately 16-20 hours and then used.

[0269] Prior to the assay (same day), each cell line was probed with a transporter-specific fluorescent marker compound (quality control [QC] compound) to confirm functionality of the transfected transporter (Table 33).

[0270] [Table 33]

[0271] Validation of HEK cells for uptake format transporter studies QC dosing solutions were prepared by diluting aliquots of DMSO stocks of QC compounds into the appropriate buffer based on the transporter; the final concentration of each QC compound is shown in Table 33. The medium was gently aspirated without disturbing the cells. QC dosing solution was added to each well and the cells were incubated for a preselected period in a humidified incubator at 37°C, 5% CO2. The reaction was stopped by washing the cells twice with ice-cold HBSSg_7.4 buffer. After the second rinse, the cells were lysed using 150 μL of RIPA buffer and incubated at 4°C for 10 min. An aliquot of the supernatant (100 μL) was collected into an opaque white fluorescence reader plate and the fluorescence of each compound was read using the optimal excitation and emission wavelengths.

[0272] Additional wells (n=3) from the same batch of cells were used for protein determination. The medium was gently aspirated without disturbing the cells, and the wells were washed twice with HBSSg buffer. After the second rinse, the cells were lysed with 150 μL of RIPA buffer for 15 min at 4° C. on a shaker. Aliquots of cell lysate (12.5 μL) were collected in microcentrifuge tubes and stored at −20° C. until protein concentration analysis.

[0273] Format Transporter Related Cell Culture C2BBe1 and MDCK cells were obtained from the American Type Culture Collection (Manassas, VA, USA). MDR1-MDCK cells were obtained from the National Institute of Health (Bethesda, MD, USA). BCRP-MDCK cells were generated by Absorption Systems LLC (Exton, PA, USA). Cells were maintained in DMEM medium containing 10% FBS, 1% NEAA, 4 mM L-glutamine, 1 mM sodium pyruvate, PEST and appropriate selection reagents in a humidified incubator at 37°C and 5% CO2 (Table 34). The medium was changed three times weekly, and cell growth was observed under a light microscope. After the cells reached 80-90% confluence, they were harvested by trypsinization and seeded (60,000 cells / cm) in Costar transwell plates. 2 ) to grow cell monolayers for permeation studies. Fresh medium (1.5 mL) was added to each well and cell suspension (0.5 mL) was added to each insert. Plates were placed in a humidified incubator at 37 °C and 5% CO2, and medium was changed every other day until use. Prior to use of BCRP-MDCK cells, cell monolayers were supplemented with medium containing sodium butyrate (2.5 mM) overnight (approximately 16-20 h).

[0274] [Table 34]

[0275] Validation of cells for efflux transporter-related assays Prior to the assay, permeation of QC compounds was measured in each batch of cell monolayers. QC compounds were prepared in HBSSg_7.4. Cell monolayers used for QC testing were washed twice with HBSSg_7.4. For apical to basolateral (AP to BL) transport, 0.5 mL of dosing solution containing QC compounds was added to the insert and 1.5 mL of HBSSg_7.4 was added to the well. For basolateral to apical (BL to AP) transport, 1.5 mL of dosing solution was added to the well and 0.5 mL of HBSSg_7.4 was added to the insert. Cells were incubated for 120 min in a humidified incubator. Samples were taken from the receiver compartment at 120 min and the concentration of each QC compound was determined by LC-MS / MS.

[0276] Tolerance assessment in efflux transporter-related cells The effect of TUDCA and PB on cell monolayer integrity was evaluated in C2BBe1 and MDCK cells (Table 35). MDCK was used as a representative cell line for MDR1-MDCK and BCRPMDCK cells. Triplicate wells of cell monolayers of each cell line were used for each treatment. Dosing solutions were prepared in HBSSg_7.4 containing LY. Dosing solutions and plain HBSSg_7.4 were placed on the BL and AP sides, respectively, in a humidified incubator at 37°C and 5% CO2 for 150 minutes. After incubation, an aliquot (100 μL) from the AP side was withdrawn and the concentration of LY was determined by fluorescence at excitation and emission wavelengths of 485 nm and 540 nm, respectively, using a BMG microplate reader.

[0277] The concentrations of TUDCA and PB in the dosing solutions were determined by LC-MS / MS.

[0278] [Table 35]

[0279] Tolerance assessment in uptake format transporter transfected cells TUDCA and PB were prepared in the appropriate assay buffer at the indicated concentrations (Table 36). The medium was aspirated. Vector control transfected cells (Group 1) received a 30-minute preincubation with the test article dosing solution, and vector control transfected cells (Group 2) received no preincubation. For the MATE-related tolerability assay, cells were preincubated with plain HBSSg_8.5 for 20 minutes. After preincubation, the preincubation solution was aspirated and the appropriate buffer or test article-containing dosing solution (500 μL) was added to the corresponding wells. The cells were placed in a humidified incubator at 37° C., 5% CO2. After 20 minutes, the buffer or dosing solution was removed from the wells, the cells were rinsed twice with HBSSg_7.4, and 500 μL of medium was added. Afterwards, 100 μL of CellTiter96® AQueous ONE Solution Cell Proliferation Assay reagent was added to each well, and the cells were then returned to a humidified incubator at 37°C and 5% CO2. After 1 hour of incubation, the absorbance of each well was measured at 492 nm using a FLUOstar® spectrophotometer (BMG Labtech, Ortenberg, Germany). The concentrations of TUDCA and PB in the dosing solutions were determined by LC-MS / MS. Vector control transfected HEK cells were used as a representative cell line in the tolerability evaluation. Puromycin-resistant vector control cells (Puro-VC) were for MATE-related cells, and G418-resistant vector control (Neo-VC) were for all other transporter-transfected cells.

[0280] The CellTiter96 AQueous Non-Radioactive Cell Proliferation Assay is a homogeneous colorimetric method for determining the number of viable cells in cytotoxicity assays. The CellTiter96 AQueous Assay consists of a solution of a tetrazolium compound (MTS; 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) and an electron coupling reagent (phenazine methosulfate). MTS is bioreduced by cells to a formazan product that is soluble in tissue culture medium. The amount of formazan product, measured by absorbance at 492 nm, is directly proportional to the number of viable cells in culture.

[0281] Uptake format transporter inhibitor evaluation TUDCA and PB were prepared in appropriate matrix at preselected concentrations (Table 37). The medium was gently aspirated; the inhibition assay was started as follows: 1) Incubation with 500 μL of probe substrate in the absence and presence of TUDCA and PB or known inhibitors at 37° C., 5% CO2 for the desired time; 2) At the end of the incubation period, the solution was gently aspirated; cells were rinsed twice with ice-cold HBSSg_7.4 buffer (1000 μL each time); 3) cells were dissolved in 400 μL of acetonitrile:water (3:1, v / v) containing IS; 4) lysate was collected for analysis of probe substrate concentration (Table 38). The dosing solution concentrations of TUDCA and PB in inhibitor evaluation were also determined by LC-MS / MS.

[0282] [Table 36]

[0283] [Table 37]

[0284] [Table 38]

[0285] P-gp and BCRP IC 50 evaluation This evaluation was performed in both MDR1-MDCK and C2BBe1 cells (for P-gp) and BCRPMDCK and C2BBe1 cells (for BCRP). TUDCA and PB were prepared in the appropriate matrix at preselected concentrations (Table 39). Bidirectional transport of digoxin was measured in the absence and presence of TUDCA and PB, valspodar (for digoxin only) and Ko143 (for cladribine only). Digoxin and cladribine were used as probe substrates for P-gp and BCRP, respectively (Table 40). All incubations were performed in a humidified incubator at 37°C and 5% CO2. Probe substrates, known inhibitors, as well as TUDCA and PB were prepared in HBSSg_7.4. All probe substrate-containing solutions also contained 200 μM LY.

[0286] A 30-min preincubation with TUDCA and PB or a known inhibitor was performed on both sides preloaded with cells. After 30 min, the preincubation solution was aspirated. An aliquot of fresh dosing solution with or without TUDCA and PB or a known inhibitor (0.55 mL from AP to BL, 1.55 mL from BL to AP) was added to the donor compartment, and HBSSg_7.4 buffer with or without TUDCA and PB or a known inhibitor (1.5 mL from AP to BL, 0.5 mL from BL to AP) was added to the receiver compartment. Receiver samples (300 μL) were taken at preselected time points. For receiver samples, one portion (200 μL) was used for analysis of TUDCA and PB, and the remaining sample (100 μL) was collected for LY measurement at the end of the permeation assay. Donor samples (50 μL) were taken at preselected time points.

[0287] After completion of the permeation assay, cell monolayer integrity was examined by LY assessment. The concentration of LY was measured at 438 nm (excitation) and 540 nm (emission). The concentrations of probe substrate in the collected dosing solution, donor and receiver samples, as well as TUDCA and PB in the dosing solution were determined by LC-MS / MS.

[0288] [Table 39]

[0289] [Table 40]

[0290] P-gp and BCRP substrate evaluation P-gp and BCRP substrate evaluation was performed in C2BBe1 cell monolayers. TUDCA and PB were prepared in the appropriate matrix at preselected concentrations (Table 41). All incubations were performed in a humidified incubator at 37°C, 5% CO2. LY was co-dosed with each dosing solution at a nominal concentration of 200 μM. Bidirectional permeability assays were performed with TUDCA and PB in the absence of P-gp or BCRP inhibitors (Table 42). Digoxin (P-gp substrate) or Cladribine (BCRP substrate) were flowed in parallel (Table 43).

[0291] The cell medium was aspirated and the cell monolayer was rinsed once with HBSSg_7.4. For TUDCA and PB groups, fresh aliquots of dosing solution (0.55 mL for AP to BL, 1.55 mL for BL to AP) were added to the donor compartment, and inhibitor-free HBSSg_7.4 buffer (1.5 mL for AP to BL, 0.5 mL for BL to AP) was added to the receiver compartment. For receiver samples, aliquots (300 μL) were taken at preselected time points. For receiver samples, one portion (200 μL) was used for TUDCA and PB analysis, and the remaining sample (100 μL) was collected for LY measurement at the end of the permeation assay. For donor samples, aliquots (50 μL) were taken at preselected time points without replenishment.

[0292] The concentrations of TUDCA, PB, digoxin, and cladribine in the dosing solutions, receiver, and donor samples were determined by LC-MS / MS. The concentration of LY was determined by fluorescence at excitation and emission wavelengths of 485 nm and 540 nm, respectively, using a BMG microplate reader.

[0293] [Table 41]

[0294] [Table 42]

[0295] [Table 43]

[0296] 4. Data Analysis Uptake transporter related analysis The influx rates of TUDCA and PB into transporter and vector control transfected cells were determined. Negative values ​​of percent inhibition are reported as 0 and maximum inhibition is reported as 100%. IC 50Values ​​were calculated using GraphPad Prism (version 5.0).

[0297]

number

[0298] IR: average influx rate (amount of substrate normalized to average protein content and incubation time, pmol / mg protein / min) CS: substrate concentration in cell lysate in μM VS: Substrate assessment cell lysate volume in mL CP: protein concentration in cell lysate in mg / mL VP: Cell lysate volume for protein content determination in mL Vesicle protein: Protein content of vesicles (50 μg) VC: vector control transfected cells TF: Transporter-transfected cells TA: TUDCA and PB or known inhibitors ATP: In the presence of ATP AMP: In the presence of AMP X: logarithm of the nominal concentration of TUDCA and PB Y: Percentage of transporter remaining active at a given concentration YL: lowest response (using the highest concentrations of TUDCA and PB) YH: highest response (vehicle control)

[0299] Efflux transporter-related analysis

[0300]

number

[0301] dC R / dt: slope of cumulative concentration in the receiver compartment versus time (μM / s); C R C : cumulative receiver concentration (μM); C R 60 : Receiver concentration at 60 min (μM); V R : volume of receiver compartment (mL); V D : volume of donor compartment (mL); A: Cell membrane surface area (1.13cm 2 ); C D0 : donor concentration at 0 min (μM); C D5 : donor concentration at 5 min (μM); C DN : nominal concentration of dosing solution (μM); C R final : cumulative receiver concentration at the end of incubation (μM); C D final : donor concentration at the end of incubation (μM); TA: Test article or known inhibitor

[0302] For withdrawal and replenishment on the receiver side, the gradient was constructed after calculating the cumulative concentration. The cumulative concentration at each time point, C R C is the measured concentration at that time (C m ) and was equal to 3 / 15 (3 / 5 in the BL to AP direction) of the measured concentration at that point because 300 μL of the 1.5 mL total sample (or 300 μL of 0.5 mL in the BL to AP direction) was removed and replenished.

[0303] Regarding the permeation test, C R C 60=C m 60 C R C 120=C m 120+3 / 15(or 3 / 5)×C m 60 It was.

[0304] Using equations (7-A) and (7-B), the P of TUDCA and PB app Using formula (7-C), the P of digoxin and cladribine were determined. app Using equations (7-D) and (7-E), the P of LY was determined. app Using equation (7-E), the P of the control compound in the batch QC of the cell monolayer was determined. app It was decided.

[0305] The origin was not included in the fit because no receiver sample was taken at 0 min. The rationale for equation (10) is as follows: an emission ratio of 1 indicates zero emission. Therefore, the residual emission was determined after subtracting a value of 1 from the calculated emission ratio. The minimum value of the inhibition rate was "0%" and the maximum value was "100%."

[0306] result chemical stability TUDCA and PB appeared to be stable in the test matrix for the duration of the study (Table 44). In subsequent solubility evaluations, TUDCA and PB were incubated overnight.

[0307] [Table 44]

[0308] Solubility evaluation The acceptance criterion for the solubility assessment is that the measured concentration in the matrix must be between 80 and 120% of the nominal value in at least two out of three replicates.

[0309] In the solubility evaluation, the measured concentrations of TUDCA and PB were greater than 80% of the nominal concentration in at least two replicates in both matrices (Table 45). These results indicate that TUDCA and PB are soluble up to the target concentrations, i.e., 8005 μM in HBSSg_7.4 and 810 μM in HBSSg_8.5 for TUDCA, and 64447 μM in HBSSg_7.4 and 73927 μM in HBSSg_8.5 for PB.

[0310] [Table 45]

[0311] Non-specific binding assessment The acceptance criteria for the dosing solution preparations was that the measured concentrations of the dosing solutions must be between 80-120% of the nominal value in at least two of the three replicates. The measured dosing concentrations of TUDCA and PB in all three replicates were between 80-120% of the nominal value for each treatment, indicating that the dosing solution preparations were acceptable.

[0312] For the efflux transporter format, recoveries of TUDCA and PB were greater than 80% in all three replicates, indicating that the test articles had little nonspecific binding to the experimental device. No further procedures were used in the efflux transporter substrate evaluation.

[0313] For the uptake transporter format, the recoveries of TUDCA and PB were greater than 90% in both matrices without BSA preincubation. These results indicate that TUDCA and PB have negligible nonspecific binding to the experimental devices. No BSA preincubation was performed in the uptake transporter evaluation.

[0314] Tolerance assessment in efflux transporter-related cells Efflux transporter-related tolerability assessment The concentrations of TUDCA and PB in the dosing solutions were analyzed. The measured concentrations of the test articles in all three replicates were within 80-120% of the nominal values ​​for each concentration, indicating that the dosing solution preparations were acceptable.

[0315] In trial 1, LY's P app 0.8 × 10 in all monolayers of C2BBe1 in the presence of TUDCA and PB. -6 In trial 2, the P of LY was greater than 1.0 cm / s (Table 46), indicating that cell monolayer integrity was affected by the presence of the test article. app 0.8 × 10 in monolayers in the presence of 3202 μM and 25778 μM TUDCA and PB, respectively. -6 cm / s, whereas the other two concentrations were 0.8 × 10 -6 The mean mean airflow velocity was less than 1.0 cm / s (Table 46).

[0316] In the following assays, the highest concentrations of TUDCA and PB did not exceed 2001 μM and 16112 μM, respectively, in C2BBe1 cells.

[0317] [Table 46]

[0318] For MDCK cells, LY P app was 0.8 × 10 in all monolayers in the presence of TUDCA and PB. -6 cm / s (Table 47), indicating that the cell monolayer was not affected by the presence of the test article.

[0319] In the following assays, the highest concentrations of TUDCA and PB did not exceed 8005 μM and 64447 μM, respectively, in MDR1- or BCRP-transfected MDCK cells.

[0320] [Table 47]

[0321] Tolerance assessment in uptake transporter-related cells Concentrations of TUDCA and PB were analyzed in the uptake transporter-related tolerability assessment. Measured concentrations in at least two of three replicates were within 80-120% of the nominal values ​​in the pH 7.4 and pH 8.5 dosing solutions, indicating that the dosing solution preparations were acceptable.

[0322] In cells preincubated with TUDCA and PB for 30 min, the formazan absorbance was greater than 75% of the control value in all groups (Table 48). These results indicate that TUDCA and PB at the concentrations tested had no adverse effect on the metabolic activity of cells. In subsequent assays, the highest concentrations of TUDCA and PB did not exceed 348 and 32625 μM, respectively, in these cells.

[0323] In cells that were not preincubated with TUDCA and PB, the formazan absorbance in the presence of TUDCA and PB was higher than 75% of the control value in all groups, indicating that TUDCA and PB did not affect the viability of HEK cells at the concentrations tested (Tables 49 and 50).

[0324] In subsequent assays, the highest concentrations of TUDCA did not exceed 348 μM in HBSSg_7.4 and 810 μM in HBSSg_8.5 in these cells, and 32625 μM in HBSSg_7.4 and 73927 μM in HBSSg_8.5 in PB.

[0325] [Table 48]

[0326] [Table 49]

[0327] [Table 50]

[0328] Uptake transporter inhibitor evaluation (i) OAT1 OAT1 IC 50 The measured concentrations of TUDCA and PB in the dosing solutions evaluated were within 80-120% of the nominal values ​​for all three replicates at all concentrations, indicating that the test article dosing solution preparations were acceptable. Detailed annotations for Solutions 1 through 8 are provided in Table 28. The PAH dosing solution preparations were also acceptable.

[0329] In the absence of TUDCA and PB or probenecid, the ratio of the influx rates of PAH was 385 (OAT1 to vector control) (Table 51). The presence of probenecid caused an 86.5% inhibition of PAH uptake in OAT1 transfected cells (Table 52). These results indicate normal functioning of the test system.

[0330] In the presence of TUDCA and PB, the mean flux rate of PAHs through OAT1 ranged from 4.90 to 411 pmol / mg protein / min (Table 51). AMX0035 (combination of TUDCA and PB) showed concentration-dependent inhibition of OAT1-mediated uptake of PAHs.

[0331] OAT1 IC 50 In the evaluation, the simultaneous presence of TUDCA and PB in the dosing solution resulted in a significant difference in the IC 50 It is not possible to determine the inhibition of OAT1. The inhibition of OAT1 was 44.8% in the presence of solution 8 (0.0741 μM TUDCA and 6.76 μM PB) and 65.2% in the presence of solution 7 (0.222 μM TUDCA and 20.3 μM PB) (Table 52). Thus, the estimated IC 50 was 0.0741-0.222 μM for TUDCA and 6.76-20.3 μM for PB.

[0332] Currently, the FDA has the following criteria for the in vitro inhibitor evaluation of test substances against OAT1: unbound [I]1 / IC 50 If [I]1 is less than 0.1, in vivo drug-drug interaction studies are not necessary. The unbound [I]1 of TUDCA and PB is 1.62 and 148 μM, respectively (Table 28). The ratio is greater than 0.1 for TUDCA (1.62 / [0.0741-2.22]) and PB (148 / [6.76-20.3]). Since the ratio is greater than 0.1, in vivo drug-drug interaction studies with OAT1 substrates are recommended for AMX0035.

[0333] [Table 51]

[0334] [Table 52]

[0335] (ii) OCT2 OCT2 IC 50 The measured concentrations of TUDCA and PB in the dosing solutions evaluated were within 80-120% of the nominal values ​​for all three replicates at all concentrations, indicating that the test article dosing solution preparations were acceptable. Detailed annotations for Solutions 1 through 8 are provided in Table 37. The MPP+ dosing solution preparations were also acceptable.

[0336] In the absence of TUDCA and PB or imipramine, the ratio of the influx rates of MPP+ was 48.9 (OCT2 to vector control) (Table 53). The presence of imipramine caused a 97.9% inhibition of MPP+ uptake in OCT2 transfected cells (Table 54). These results indicate normal functioning of the test system.

[0337] In the presence of TUDCA and PB, the mean influx rate of MPP+ through OCT2 was 680-991 pmol / mg protein / min (Table 53). AMX0035 (a combination of TUDCA and PB) did not inhibit OCT2-mediated uptake of MPP+ at the concentrations tested. The inhibition rate was less than 50%, so the estimated IC 50 was greater than 162 μM for TUDCA and 14785 μM for PB.

[0338] [Table 53]

[0339] [Table 54]

[0340] The criteria used to evaluate the in vitro inhibition of OCT2 are the same as for OAT1. The unbound [I]1 of TUDCA and PB are 1.62 and 148 μM, respectively (Table 28). The ratios are less than 0.1 for TUDCA (1.62 / [>162]) and PB (148 / [>14785]). Since the ratios are less than 0.1, in vivo drug-drug interaction studies with OCT2 substrates are not recommended for AMX0035.

[0341] (iii) OAT3 OAT3 IC 50 The measured concentrations of TUDCA and PB in the dosing solutions evaluated were within 80-120% of the nominal values ​​for all three replicates at all concentrations, indicating that the test article dosing solution preparations were acceptable. Detailed annotations for Solutions 1 through 8 are provided in Table 37. The furosemide dosing solution preparation was also acceptable.

[0342] In the absence of TUDCA and PB or probenecid, the flux rate ratio of furosemide was 20.1 (OAT3 to vector control) (Table 55). The presence of probenecid caused a 99.1% inhibition of furosemide uptake in OAT3 transfected cells (Table 56). These results indicate normal functioning of the test system.

[0343] In the presence of TUDCA and PB, the mean flux of furosemide through OAT3 ranged from 11.3 to 62.0 pmol / mg protein / min (Table 55). AMX0035 (combination of TUDCA and PB) showed greater than 50% inhibition (80.3%) of OAT3-mediated uptake of furosemide at the highest concentration tested (Table 56).

[0344] [Table 55]

[0345] The inhibition of OAT3 was 46.8% in the presence of solution 2 (54.0 μM TUDCA and 4928 μM PB) and 80.3% in the presence of solution 1 (162 μM TUDCA and 14785 μM PB) (Table 56). Thus, the estimated IC 50 was 5.40-162 μM for TUDCA and 4928-14785 μM for PB.

[0346] [Table 56]

[0347] The criteria used to evaluate the in vitro inhibition of OAT3 are the same as for OAT1. The unbound [I]1 of TUDCA and PB are 1.62 and 148 μM, respectively (Table 28). The ratios are less than 0.1 for TUDCA (1.62 / [54.0-162]) and PB (148 / [4928-14785]). Since the ratios are less than 0.1, in vivo drug-drug interaction studies with OAT3 substrates are not recommended for AMX0035.

[0348] (iv) OATP1B1 and OATP1B3 OATP1B1 and OATP1B3 IC 50 The measured concentrations of TUDCA and PB in the dosing solutions evaluated were within 80-120% of the nominal values ​​for at least two of the three replicates at all concentrations, indicating that the test article dosing solution preparations were acceptable. Detailed annotations for Solutions 1 through 8 are provided in Table 37. The atorvastatin dosing solution preparation was also acceptable.

[0349] In the absence of TUDCA and PB or rifamycin SV (RSV), the influx rate ratios of atorvastatin were 15.1 (OATP1B1 to vector control) and 8.23 ​​(OATP1B3 to vector control) (Table 57). The presence of RSV caused 96.7% and 98.1% inhibition of atorvastatin uptake in OATP1B1 and OATP1B3 transfected cells, respectively (Table 58). These results indicate normal functioning of the test system.

[0350] In the presence of TUDCA and PB, the mean influx rates of atorvastatin ranged from 0.660 to 6.26 pmol / mg protein / min through OATP1B1 and 0.813 to 2.93 pmol / mg protein / min through OATP1B3 (Table 57).

[0351] [Table 57]

[0352] [Table 58]

[0353] The inhibition of OATP1B1 was 38.9% in the presence of solution 4 (12.9 μM TUDCA and 1208 μM PB) and 65.1% in the presence of solution 3 (38.7 μM TUDCA and 3625 μM PB) (Table 58). Thus, the estimated IC50 The estimated IC values ​​for TUDCA and PB ranged from 12.9 to 38.7 μM and 1208 to 3625 μM, respectively. Similarly, the estimated IC values ​​for OATP1B3 were 50 was 38.7-116 μM for TUDCA and 3625-10875 μM for PB.

[0354] Currently, the FDA has recommended that oral administration of 5-aminoethyl 1,4-diphenylmethane (1,2,3,4-tetrahydrofuran) be administered at the maximum plasma unbound inhibitor concentration at the entrance to the liver (unbound I in,max ) is used. Non-bonded type I in,max and IC 50 If the ratio is greater than 0.1, the test article has the ability to inhibit OATP1B1 / 3. in,max are 3.48 and 326 μM, respectively (Table 28).

[0355] For OATP1B1, the ratio for TUDCA is [3.48 / (12.9-38.7)] and for PB is [326 / (1208-3625)]. Based on the current data, IC 50 Since can not be estimated more precisely than the range of concentrations, it is uncertain whether the ratio exceeds 0.1.

[0356] For OATP1B3, the ratios are less than 0.1 for TUDCA [3.48 / (38.7-116)] and PB [326 / (3625-10875)]. Because the ratios are less than 0.1, in vivo drug-drug interaction studies with OATP1B3 substrates are not recommended for AMX0035.

[0357] (v) MATE1 and MATE2K MATE1 and MATE2K ICs 50 The measured concentrations of TUDCA and PB in the dosing solutions evaluated were within 80-120% of the nominal values ​​for at least two of the three replicates at all concentrations, indicating that the test article dosing solution preparations were acceptable. Detailed annotations for Solutions 1 through 8 are provided in Table 37. The metformin dosing solution preparation was also acceptable.

[0358] In the absence of TUDCA and PB or positive inhibitors, the metformin influx rate ratios were 43.4 (MATE1 vs. vector control, Table 59) and 2.70 (MATE2K vs. vector control, Table 60). The presence of cimetidine or pyrimethamine caused 99.2% and 85.3% inhibition of metformin uptake in MATE1 and MATE2K transfected cells, respectively (Table 61). These results indicate normal functioning of the test system.

[0359] In the presence of TUDCA and PB, the mean flux rates of metformin ranged from 189 to 435 pmol / mg protein / min through MATE1 (Table 59) and 36.7 to 52.0 pmol / mg protein / min through MATE2K (Table 60).

[0360] [Table 59]

[0361] The inhibition of MATE1 was 37.5% in the presence of solution 2 (270 μM TUDCA and 24642 ​​μM PB) and 68.9% in the presence of solution 1 (810 μM TUDCA and 73927 μM PB) (Table 61). Thus, the estimated IC 50 The estimated IC values ​​for TUDCA and PB were 270–810 μM and 24642–73927 μM, respectively. 50 was greater than 810 μM for TUDCA and 73,927 μM for PB.

[0362] [Table 60]

[0363] [Table 61]

[0364] Currently, the FDA has the following criteria for the in vitro inhibitor evaluation of test substances against MATE1 and MATE2K: unbound [I]1 / IC 50 If the ratio is greater than 0.02, the test article has the ability to inhibit MATE1 / 2K. The projected unbound Cmax of TUDCA and PB are 1.62 μM and 148 μM, respectively (Table 28).

[0365] For MATE1, the ratios are less than 0.02 for TUDCA [1.62 / (270-810)] and PB [148 / (24642-73927)]. Similarly, for MATE2K, these ratios are less than 0.02. Since the ratios are less than 0.02, in vivo drug-drug interaction studies with MATE1 or MATE2K substrates are not required for AMX0035.

[0366] P-gp and BCRP IC 50 evaluation (i) P-gp IC 50 evaluation P-gp IC in MDR1-MDCK cells 50 The concentrations of TUDCA and PB in the dosing solutions for evaluation were determined. The measured concentrations of each component in at least two of the three replicates were in each case 80-120% of the nominal value, indicating that the test article dosing solution preparations were acceptable. Detailed annotations for Solutions 1 through 8 are provided in Table 39. Digoxin preparations were also acceptable in all cases except in the presence of Solution 2.

[0367] In MDR1-MDCK cells, in the absence of TUDCA and PB or valspodar, the efflux ratio of digoxin was 304, and the addition of valspodar reduced it to 1.23 (Table 62), which corresponds to 99.9% inhibition (Table 63). These results indicate normal function of P-gp in the test system.

[0368] In the presence of TUDCA and PB, the efflux ratios for digoxin ranged from 60.4 to 270 (Table 62). Inhibition of P-gp was 80.4% (>50%) in the presence of solution 2 (2668 μM TUDCA and 21482 μM PB), but was variable at lower concentrations (Table 63). Such variability was due to cell-to-cell variability, rather than P-gp inhibition by TUDCA and PB. Thus, the estimated IC 50 was 889-2668 μM for TUDCA and 7161-21482 μM for PB.

[0369] Currently, for orally administered drugs, the FDA requires that the ratio of [I] of the test article to its calculated IC 50 The ratio of IC to IC was used to evaluate the in vitro P-gp inhibitor potency. 50 If the [I]2 for is less than 10, in vivo drug-drug interaction studies with P-gp substrates are not necessary.

[0370] The estimated [I]2 for TUDCA and PB are 8005 and 64447 μM (Table 28). The projected ratios are less than 10 for TUDCA [8005 / (889-2668)] and less than 10 for PB [64447 / (7161-21482)] in MDR1-MDCK cells.

[0371] [Table 62]

[0372] [Table 63]

[0373] P-gp IC in C2BBe1 cells 50The concentrations of TUDCA and PB in the dosing solutions for evaluation were determined. The measured concentrations of each component in at least two of the three replicates were in each case 80-120% of the nominal value, indicating that the test article dosing solution preparations were acceptable. Detailed annotations for solutions 1 through 8 are provided in Table 39. The digoxin preparation was also acceptable.

[0374] In C2BBe1 cells, in the absence of TUDCA and PB or valspodar, the efflux ratio of digoxin was 20.5, and the addition of valspodar reduced it to 1.14 (Table 64), which corresponds to 99.3% inhibition (Table 65). These results indicate normal function of P-gp in the test system.

[0375] In the presence of TUDCA and PB, the efflux ratio of digoxin ranged from 2.80 to 18.9 (Table 64). Inhibition of P-gp was 69.2% (>50%) in the presence of solution 2 (667 μM TUDCA and 5371 μM PB) and 40.0% in the presence of solution 3 (222 μM TUDCA and 1790 μM PB) (Table 65). Thus, the estimated IC 50 was 222-667 μM for TUDCA and 1790-5371 μM for PB.

[0376] Currently, for orally administered drugs, the FDA requires that the ratio of [I] of the test article to its calculated IC 50 The ratio of IC to IC was used to evaluate the in vitro P-gp inhibitor potency. 50 If the [I]2 for is less than 10, in vivo drug-drug interaction studies with P-gp substrates are not necessary.

[0377] The estimated [I]2 for TUDCA and PB are 8005 and 64447 μM (Table 28). The projected ratios are >10 for TUDCA [8005 / (222-667)] and >10 for PB [64447 / (1790-5371)] in C2BBE1 cells.

[0378] Based on the current results, in vivo drug-drug interactions of AMX0035 with P-gp substrates may be necessary.

[0379] (ii) BCRP IC 50 evaluation BCRP IC in BCRP-MDCK cells 50 The concentrations of TUDCA and PB in the dosing solutions for evaluation were determined. The measured concentrations of each component in at least two of the three replicates were in each case 80-120% of the nominal value, indicating that the test article dosing solution preparations were acceptable. Detailed annotations for solutions 1 through 8 are provided in Table 39. The cladribine preparation was also acceptable.

[0380] In the absence of TUDCA and PB or Ko143, the efflux ratio of cladribine was 223, and the addition of Ko143 reduced it to 0.922 (Table 66), which corresponds to 100% inhibition (Table 67). These results indicate normal function of BCRP in the test system.

[0381] In the presence of TUDCA and PB, the efflux ratio of cladribine was 4.94-264 (Table 66). Inhibition of BCRP was 28.2% in the presence of solution 4 (296 μM TUDCA and 2387 μM PB) and 90.7% in the presence of solution 3 (889 μM TUDCA and 7161 μM PB) (Table 67). Thus, the estimated IC 50 was 296-889 μM for TUDCA and 2387-7161 μM for PB.

[0382] Currently, for orally administered drugs, the FDA requires that the ratio of [I] of the test article to its calculated IC 50 The ratio of IC to IC was used to evaluate the in vitro BCRP inhibitor potency. 50 If the [I]2 for BCRP is less than 10, in vivo drug-drug interaction studies with BCRP substrates are not necessary.

[0383] The estimated [I]2 for TUDCA and PB are 8005 and 64447 μM (Table 28). The projected ratios are [8005 / (296-889)] for TUDCA and [64447 / (2387-7161)] for PB in BCRP-MDCK cells. Based on the current data, the IC 50 Because the range of concentrations cannot be estimated with precision, it is not certain whether the ratio is less than 10. Definitive in vitro-in vivo extrapolation is not available for BCRP inhibition using BCRP-MDCK cells.

[0384] [Table 64]

[0385] [Table 65]

[0386] [Table 66]

[0387] [Table 67]

[0388] BCRP IC in C2BBe1 cells 50 The concentrations of TUDCA and PB in the dosing solutions for evaluation were determined. The measured concentrations of each component in at least two of the three replicates were in each case 80-120% of the nominal value, indicating that the test article dosing solution preparations were acceptable. Detailed annotations for solutions 1 through 8 are provided in Table 39. The cladribine preparation was also acceptable.

[0389] In the absence of TUDCA and PB or Ko143, the efflux ratio of cladribine was 27.6, and the addition of Ko143 reduced it to 1.73 (Table 68), which corresponds to 97.2% inhibition (Table 69). These results indicate normal function of BCRP in the test system.

[0390] In the presence of TUDCA and PB, the efflux ratio of cladribine ranged from 3.20 to 25.5 (Table 68). Inhibition of BCRP was 46.1% in the presence of solution 3 (222 μM TUDCA and 1790 μM PB) and 78.0% in the presence of solution 2 (667 μM TUDCA and 5371 μM PB) (Table 69). Thus, the estimated IC 50 is 222-667 μM for TUDCA and 1790-5371 μM for PB.

[0391] Currently, for orally administered drugs, the FDA requires that the ratio of [I] of the test article to its calculated IC 50 The ratio of IC to IC was used to evaluate the in vitro BCRP inhibitor potency. 50 If the [I]2 for BCRP is less than 10, in vivo drug-drug interaction studies with BCRP substrates are not necessary.

[0392] The estimated [I]2 for TUDCA and PB are 8005 and 64447 μM (Table 28). The projected ratios are [8005 / (222-667)] for TUDCA and [64447 / (1790-5371)] for PB in C2BBe1 cells. The ratios are greater than 10 for both TUDCA and PB.

[0393] In vivo drug-drug interactions of AMX0035 with BCRP substrates may be necessary based on current data.

[0394] P-gp and BCRP substrate evaluation Concentrations of TUDCA and PB in dosing solutions for P-gp and BCRP substrate evaluation in C2BBe1 cells were determined. The measured concentrations of each component in at least two of three replicates were in each case 80-120% of the nominal value, indicating that the test article dosing solution preparations were acceptable. Detailed annotations of solutions 1 to 4 are given in Table 41. Bidirectional permeation of solutions 1, 3, and 4 was performed with one batch of C2BBe1 cells, and bidirectional permeation of solution 2 was performed with another batch. Preparations of digoxin and cladribine were also acceptable.

[0395] When bidirectional permeation of the probe substrate was performed using solutions 1, 3, and 4, the efflux ratios of digoxin and cladribine were 41.6 and 27.0, respectively; when bidirectional permeation of the probe substrate was performed using solution 2, the efflux ratios of digoxin and cladribine were 38.2 and 19.7, respectively (Table 70). These results indicate normal function of P-gp and BCRP in the test system.

[0396] [Table 68]

[0397] [Table 69]

[0398] [Table 70]

[0399] Currently, the procedure for determining the ability of a test article to be a P-gp and BCRP substrate is as follows: if the efflux ratio (determined using Equation 9) is greater than or equal to 2.00, the results indicate a potential substrate for the transporters. The bidirectional permeation of the test article is further determined in the presence of transporter-specific inhibitors, and if inhibition is greater than 50%, the test article is a substrate for the transporters.

[0400] The efflux ratios of TUDCA and PB were less than 2.00 at all concentrations tested (Tables 71 and 72). TUDCA and PB are not P-gp or BCR substrates since the results did not meet the criteria.

[0401] [Table 71]

[0402] [Table 72]

[0403] In summary, in vitro, TUDCA and PB are not substrates for either P-gp or BCRP. The estimated IC 50 The estimated IC of PB for each transporter was as follows: 0.0741-0.222 μM (OAT1), >162 μM (OCT2), 54.0-162 μM (OAT3), 12.9-38.7 μM (OATP1B1), 38.7-116 μM (OATP1B3), 270-810 μM (MATE1), >810 μM (MATE2K), 889-2668 μM (P-gp in MDR1-MDCK cells), 222-667 μM (P-gp in C2BBe1 cells), 296-889 μM (BCRP in BCRP-MDCK cells) and 222-667 μM (BCRP in C2BBe1 cells). 50 are as follows: 6.76-20.3 μM (OAT1), >14785 μM (OCT2), 4928-14785 μM (OAT3), 1208-3625 μM (OATP1B1), 3625-10875 μM (OATP1B3), 24642-73927 μM (MATE1), >73927 μM (MATE2K), 7161-21482 μM (P-gp in MDR1-MDCK cells), 1790-5371 μM (P-gp in C2BBe1 cells), 2387-7161 μM (BCRP in BCRP-MDCK cells) and 1790-5371 μM (BCRP in C2BBe1 cells).

[0404] Example 7 Drug-Drug Interaction Study of Tenofovir Disoproxil Fumarate and AMX0035 The potential for drug-drug interactions between tenofovir disoproxil fumarate (TDF) and AMX0035 was evaluated. TDF is a prodrug that is rapidly converted to the active moiety, tenofovir, in plasma. Tenofovir is a known substrate of the renal organic anion transporter OAT1 (SLC22A6), and this study was designed to determine whether coadministration of AMX0035 alters the plasma or urinary concentrations of tenofovir in rats.

[0405] The study consisted of one group of three male CD® (Sprague-Dawley) IGS rats. To achieve steady-state concentrations of tenofovir prior to administration of AMX0035, rats received 25 mg / kg / dose of TDF by PO gavage in a dose volume of 5 mL / kg once daily on days 1-4. On day 5, rats received 840 mg / kg / dose of AMX0035 by PO gavage in a dose volume of 9.94 mL / kg, followed by 25 mg / kg / dose of TDF by PO gavage in a dose volume of 5 mL / kg. The dose of tenofovir was determined by the T max The study was administered 1.5 hours after AMX0035, which was designed to approximate the

[0406] Clinical observations were recorded approximately 1 hour post-dose on days 1-4 and twice on day 5, once after AMX0035 and once after TDF. Body weight measurements were recorded prior to randomization and daily thereafter until study termination. Plasma samples were collected from all animals at 0.25, 0.5, 1, 2, 4, 8, and 24 hours post-dose on days 1 and 5 (day 5 collection was performed after TDF administration) for analysis of systemic exposure to tenofovir, TDF, PB, and TUDCA. Urine samples were collected from all animals at 0-4, 4-8, and 8-24 hours post-dose on days 1 and 5 (day 5 collection was performed after TDF administration) for calculation of urinary recovery of tenofovir, TDF, PB, and TUDCA.

[0407] Results showed that all rats survived until scheduled sacrifice on day 6. No test article-related changes in body weight occurred, and no test article-related clinical observations were noted. Tenofovir was rapidly formed through systemic absorption of TDF. The prodrug (TDF) was not detected in plasma at any time point post-dose, either on days 1 or 5. The mean peak (C max ) and total (AUC last ) Plasma exposure was increased by 1.24- and 2.17-fold, respectively, in the presence of AMX0035. The Cs of TUDCA (298 ng / mL) and PB (10,200 ng / mL) detected in this study were max Plasma levels, converted into total drug concentrations, were approximately 1.1 times higher than IC reported for OAT1 inhibition in previous in vitro transporter studies. 50 These results suggest that AMX0035 significantly reduces tenofovir exposure (AUC last ) is consistent with resulting in a moderate increase in

[0408] introduction The objective of this study was to evaluate whether AMX0035 is an inhibitor of the OAT1 transporter in rats. To do this, the study evaluated the drug-drug interaction between AMX0035 and tenofovir disoproxil fumarate (TDF), a known substrate of the organic anion transporter OAT1. TDF was administered by PO gavage on 4 consecutive days to achieve steady-state concentrations before AMX0035 was administered by PO gavage on day 5. Following AMX0035 administration on day 5, TDF was administered 1.5 hours after AMX0035 administration.

[0409] method Experimental design The objective of this study was to evaluate the drug-drug interaction between TDF and AMX0035 after oral administration. TDF was administered once daily for 5 days. AMX0035 was administered once on day 5, followed by TDF administration 1.5 hours after AMX0035 administration.

[0410] Pharmacokinetic evaluation Pharmacokinetic analysis was performed on the individual plasma concentration versus time data for tenofovir, PB, and TUDCA using Phoenix WinNonlin noncompartmental analysis (linear trapezoidal rule for AUC calculation). Nominal dose values ​​and nominal sampling times were used for calculations. All concentrations reported as BLQ (<50 ng / mL) were set equal to 0. For calculation of AUC on study days 1 and 5, plasma levels at time 0 for tenofovir, PB, and TUDCA were set equal to 0. PK parameters were not assessed for PB and TDF on day 1 and TDF on day 5 because the plasma concentration values ​​were BLQ. Pharmacokinetic analysis included C max , T max , AUC last , T last and AUC 0-24 The decision included:

[0411] Individual and mean plasma concentration versus time data for tenofovir, TDF, PB, and TUDCA are presented with standard deviations (SD) reported to three significant figures and percent coefficient of variation (CV%) reported to one decimal place. Pharmacokinetic parameters were presented as individual and mean values. max and T last are reported to two significant figures, all other values ​​are reported to three significant figures. Mean PK parameters for tenofovir, PB, and TUDCA were max and T last Except for , the values ​​are shown to three significant digits, and CV% is shown to one decimal place. max and T last The average values ​​are shown to two significant figures.

[0412] Total urinary recovery data for tenofovir, TDF and PB were calculated for the 24-hour collection period on days 1 and 5. Recovery data were not calculated because urinary concentrations were at BLQ (<20 ng / mL) for the PB analysis in all animals on day 1, the TUDCA analysis in all animals on days 1 and 5, and the TDF analysis in two animals on day 1 and one animal on day 5. Urinary recovery data reported included the amount recovered (ng) and percent recovery (%) for TDF only. Individual and mean urinary recovery data for tenofovir, TDF and PB are presented with SDs reported to three significant figures and CVs reported to one decimal place.

[0413] Individual and mean plasma concentration versus time data for tenofovir, TDF, PB, and TUDCA are presented in Tables 73-76 and illustrated in Figures 2-5. Individual animal and mean PK parameters for tenofovir, PB, and TUDCA in male Sprague Dawley rats are presented in Tables 77-78. Individual and mean total urinary recovery data for tenofovir, TDF, and PB are presented in Tables 79-80 and illustrated in Figure 6.

[0414] Pharmacokinetic Abbreviations: AUC: Area under the concentration-time curve AUC 0-24 : Area under the curve from time of dosing to time of last measurable concentration AUC last : Area under the curve from time of dosing to time of last measurable concentration BLQ: below limit of quantification C max : Maximum observed concentration CV%: Coefficient of variation percent N: Number of observations NA: Not applicable SD: standard deviation T last : Time of last time point with quantifiable concentration T max : Time when Cmax occurred

[0415] [Table 73]

[0416] [Table 74]

[0417] [Table 75]

[0418] [Table 76]

[0419] [Table 77]

[0420] [Table 78]

[0421] [Table 70]

[0422] [Table 80]

[0423] TDF was rapidly converted to the active moiety, tenofovir, so quantifiable plasma TDF concentrations were not observed in any animals on any sampling days following single oral doses of 25 mg / kg / dose of TDF on day 1 and 840 mg / kg / dose of AMX0035 + 25 mg / kg / dose of TDF on day 5.

[0424] Quantifiable tenofovir concentrations were observed in all animals by the first collection time at 0.25 hours post-dose. Tenofovir peak time (Tmax) occurred 0.25 hours post-dose on days 1 and 5. The mean Cmax of tenofovir on day 1 was 63.3 ng / mL, and the compound was rapidly cleared from the plasma compartment, with the compound being undetectable at 1 hour on day 1 and 2 hours on day 5 (BLQ). However, in the presence of AMX0035, the mean Cmax and AUClast of tenofovir were increased by 1.24 and 2.17-fold, respectively. The mean plasma concentrations of tenofovir were higher in the presence of AMX0035 at each measurable time point (1.24, 1.67, and 1.5-fold at 0.25, 0.5, and 1 hour, respectively).

[0425] TUDCA is an endogenous bile acid and was therefore quantifiable on day 1 in the absence of AMX0035. Mean endogenous levels of TUDCA measured on day 1 ranged from 130 to 222 ng / mL over the 24-hour time course. Following dosing with AMX0035 on day 5, the Tmax of TUDCA occurred 1 to 2 hours after dosing. Mean plasma concentrations of TUDCA on day 5 ranged from 154 ng / mL to 277 ng / mL, and were overall 30 to 50% higher than day 1 at each time point, reflecting dosing with AMX0035. Plasma levels of TUDCA remained fairly constant throughout the time course.

[0426] Quantifiable plasma PB concentrations were observed 0.25 hours after AMX0035 dosing on day 5 and remained quantifiable for an overall period of 4 hours. PB was rapidly absorbed, with the Tmax of PB occurring 0.25-0.5 hours after dosing. Similar to tenofovir, PB was rapidly eliminated, with concentrations undetectable after the 4 hour time point.

[0427] Previous studies have shown that TUDCA and PB inhibit human OAT1 in vitro using IC 50These values ​​ranged from 74 to 222 nM (35 to 111 ng / mL) for TUDCA and 6.76 to 20.3 μM (1103 to 3313 ng / mL) for PB. The peak plasma Cmax values ​​observed in this study for TUDCA (298 ng / mL) and PB (10,200) were approximately equal to the IC values ​​of OAT1 inhibition. 50 beyond the scope of.

[0428] Three of the four analytes were detected in the urine. As expected, TUDCA was not detected in any of the urine samples because bile acids are cleared by the liver. Samples were collected over three time points (0-4, 4-8, and 8-24 hours) on days 1 and 5, but for the 4-8 hour time point on day 1, no urine was produced by any animal. In addition, one of the three animals did not produce urine at the 0-4 hour time point. Thus, four of the nine samples on day 1 had no samples available for measurement. This is problematic given the rapid clearance of tenofovir and the need to compare data from days 1 and 5. Concentrations at these early time points are likely to be high, significantly affecting the total amount recovered in urine.

[0429] conclusion Tenofovir was rapidly formed due to systemic absorption of TDF. The prodrug (TDF) was not detected in plasma at any time point after dosing on either day 1 or day 5. The mean peak (C max ) and total (AUC last ) Plasma exposure was increased by 1.24- and 2.17-fold, respectively, in the presence of AMX0035. The Cs of TUDCA (298 ng / mL) and PB (10,200 ng / mL) detected in this study were max Plasma levels, in terms of total drug concentration, were approximately 1.1 times higher than the IC reported for OAT1 inhibition in previous in vitro transporter studies (see Example 6). 50 These results are consistent with AMX0035 producing a modest increase in tenofovir exposure (AUClast) mediated by an inhibitory effect on OAT1.

[0430] Further Aspects Aspect 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 been administered a first dose of a cytochrome P450 (CYP) substrate an effective amount of 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 the substrate or a metabolite thereof in a first biological sample from the subject; and (c) administering to the subject a second dose of the substrate, wherein the second dose is less than the first dose; The method includes: Aspect 2: The method of aspect 1, wherein the CYP is CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 or CYP3A. Embodiment 3: The method of embodiment 1, wherein the CYP is CYP1A2. Aspect 4: The method of aspect 3, wherein the substrate is alosetron, caffeine, duloxetine, melatonin, ramelteon, tasimelteon, tizanidine, clozapine, pirfenidone, ramosetron or theophylline. Embodiment 5: The method of embodiment 1, wherein the CYP is CYP2B6. Embodiment 6: The method of embodiment 5, wherein the substrate is bupropion or efavirenz. Embodiment 7: The method of embodiment 1, wherein the CYP is CYP2C8. Aspect 8: The method of aspect 7, wherein the substrate is repaglinide, montelukast, pioglitazone or rosiglitazone. Embodiment 9: The method according to embodiment 1, wherein the CYP is CYP2C9. Aspect 10: The method of aspect 9, wherein the substrate is celecoxib, glimepiride, phenytoin, tolbutamide or warfarin.

[0431] Embodiment 11: The method of embodiment 1, wherein the CYP is CYP2C19. Aspect 12: The method of aspect 11, wherein the substrate is S-mephenytoin, omeprazole, diazepam, lansoprazole, rabeprazole, or voriconazole. Embodiment 13: The method of embodiment 1, wherein the CYP is 2D6. Aspect 14: The method of aspect 13, wherein the substrate is atomoxetine, desipramine, dextromethorphan, eliglustat, nebivolol, nortriptyline, perphenazine, tolterodine, R-venlafaxine, encainide, imipramine, metoprolol, propafenone, propranolol, tramadol, trimipramine or S-venlafaxine. Embodiment 15: The method of embodiment 1, wherein the CYP is CYP3A. Aspect 16: The method of aspect 15, wherein the substrate is alfentanil, avanafil, buspirone, conivaptan, cyclosporine, quinidine, darifenacin, darunavir, ebastine, everolimus, ibrutinib, lomitapide, lovastatin, midazolam, naloxegol, nisoldipine, saquinavir, simvastatin, sirolimus, tacrolimus, tipranavir, triazolam, vardenafil, budesonide, dasatinib, dronedarone, eletriptan, eplerenone, felodipine, indinavir, lurasidone, maraviroc, quetiapine, sildenafil, ticagrelor, tolvaptan, alprazolam, aprepitant, atorvastatin, colchicine, eliglustat, pimozide, rilpivirine, rivaroxaban, or tadalafil. Embodiment 17: The method according to embodiment 15, wherein the CYP is CYP3A4. Embodiment 18: The method of embodiment 16, wherein the substrate is alfentanil. Embodiment 19: The method according to embodiment 16, wherein the substrate is cyclosporine. Aspect 20: The method of aspect 19, wherein the first dose of cyclosporine is about 0.5 to about 15 mg / kg / day.

[0432] Embodiment 21: The method of embodiment 16, wherein the substrate is quinidine. Aspect 22: The method of aspect 1, wherein the CYP substrate is mexiletine, alfentanil, quinidine, cyclosporine or warfarin. Aspect 23: The method of aspect 1, wherein the substrate of CYP has a narrow therapeutic index. Embodiment 24: The method according to any one of embodiments 1 to 23, further comprising a step (d) of determining, or having determined, a second level of the substrate or a metabolite thereof in a second biological sample of the subject. Embodiment 25 The method of embodiment 24, wherein the second level of the substrate or metabolite thereof is lower than the first level. Embodiment 26: The method according to any one of embodiments 1 to 25, wherein the biological sample is plasma or serum. Embodiment 27: A method of treating at least one symptom of ALS in a subject, comprising: (a) administering to a subject who has been administered a first dose of warfarin 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 blood international normalized ratio (INR) level of the subject; and (c) administering to the subject a second dose of warfarin, wherein the second dose is less than the first dose; The method includes: Aspect 28: The method of aspect 27, wherein the first dose of warfarin is about 0.5 to 12 mg / day. Embodiment 29. The method of embodiment 27 or 28, wherein step (b) determines, or comprises having determined, the subject's first blood INR level once daily. Embodiment 30. The method of embodiment 27 or 28, wherein step (b) determines, or comprises having determined, the subject's first blood INR level once every 1 to 4 weeks.

[0433] Embodiment 31: The method of any one of embodiments 27 to 30, wherein the first blood INR level is greater than or equal to about 3.0. Embodiment 32: The method of embodiment 31, wherein the first blood INR level is greater than or equal to about 4.0. Embodiment 33: The method according to any one of embodiments 27 to 32, further comprising a step (d) of determining, or having determined, a second blood INR level of the subject. Embodiment 34: The method of embodiment 33, wherein the second blood INR level is lower than the first blood INR level. Embodiment 35: A method of treating at least one symptom of ALS in a subject, comprising: (a) administering to a subject who has been administered a first dose of a narrow therapeutic index (NTI) medication 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 an NTI drug or a metabolite thereof in a first biological sample from the subject; and (c) administering to the subject a second dose of the NTI medication, wherein the second dose is less than the first dose; The method includes: Aspect 36: The method of aspect 35, wherein the NTI medication is mexiletine, alfentanil, quinidine, cyclosporine, warfarin, or digoxin. Embodiment 37: The method of embodiment 35 or 36, further comprising a step (d) of determining, or having determined, a second level of the NTI drug or a metabolite thereof in a second biological sample from the subject. Aspect 38: The method of aspect 37, wherein the second level of the NTI drug or metabolite thereof is lower than the first level. Embodiment 39: A method of treating at least one symptom of ALS in a subject, comprising: (a) administering to a subject who has been administered a first dose of a substrate for organic anion transporter 1 (OAT1), 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 the substrate or a metabolite thereof in a first biological sample from the subject; and (c) administering to the subject a second dose of the substrate, wherein the second dose is less than the first dose; The method includes: Embodiment 40 The method according to embodiment 39, wherein the substrate of OAT1 is a penicillin, a nonsteroidal anti-inflammatory drug (NSAID), an HIV protease inhibitor or an antiviral drug.

[0434] Aspect 41: The method of aspect 40, wherein the NSAID is diclofenac, ketoprofen or methotrexate. Aspect 42: The method of aspect 40, wherein the antiviral agent is adefovir, cidofovir or tenofovir. Embodiment 43: The method according to any one of embodiments 39 to 42, further comprising a step (d) of determining, or having determined, a second level of the substrate or a metabolite thereof in a second biological sample of the subject. Embodiment 44 The method of embodiment 43, wherein the second level of the substrate or metabolite thereof is lower than the first level. Embodiment 45: A method according to any one of embodiments 35 to 44, wherein the biological sample is blood. Aspect 46: The method of any one of aspects 1 to 45, wherein TURSO is administered from about 1 to about 2 g / day, inclusive. Aspect 47: The method of any one of aspects 1 to 46, wherein the sodium phenylbutyrate is administered from about 3 to about 6 g / day, inclusive. Embodiment 48: The method according to any one of embodiments 1 to 47, wherein TURSO is administered once a day, at about 1 g. Embodiment 49: The method according to any one of embodiments 1 to 47, wherein TURSO is administered about 1 g twice a day. Aspect 50: The method of any one of aspects 1 to 49, wherein the sodium phenylbutyrate is administered in a dose of about 3 g once a day.

[0435] Aspect 51: The method of any one of aspects 1 to 49, wherein the sodium phenylbutyrate is administered about 3 g twice daily. Embodiment 52: The method according to any one of embodiments 1 to 51, wherein the composition is administered to the subject orally or through a feeding tube. Embodiment 53: The method according to any one of embodiments 1 to 52, wherein the subject has been diagnosed with ALS. Embodiment 54: The method according to any one of embodiments 1 to 52, wherein the subject is suspected of having ALS. Embodiment 55: The method according to any one of embodiments 1 to 54, wherein the subject is a human.

[0436] Other Aspects Although the invention has been described in conjunction with its detailed description, it is to be understood that this description is intended to illustrate, and not to limit, the scope of the invention, which is defined by the claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A method for treating at least one symptom of amyotrophic lateral sclerosis (ALS) in a subject, comprising: (a) administering to a subject who has been administered a first dose of a substrate for organic anion transporter 1 (OAT1), a composition comprising taurursodiol (TURSO) and sodium phenylbutyrate; (b) determining, or having determined, a first level of the substrate or a metabolite thereof in a first biological sample from the subject; and (c) administering to the subject a second dose of the substrate, wherein the second dose is less than the first dose; The method includes:

2. 2. The method of claim 1, wherein the substrate of OAT1 is penicillin, a nonsteroidal anti-inflammatory drug (NSAID), an HIV protease inhibitor, or an antiviral drug.

3. 3. The method of claim 2, wherein the substrate of OAT1 is an NSAID selected from the group consisting of diclofenac, ketoprofen and methotrexate.

4. 3. The method of claim 2, wherein the substrate of OAT1 is an antiviral drug selected from the group consisting of adefovir, cidofovir and tenofovir.

5. The method according to any one of claims 1 to 4, wherein said substrate of OAT1 has a narrow therapeutic index.

6. 6. The method of any one of claims 1 to 5, further comprising a step (d) of determining or having determined a second level of said substrate or a metabolite thereof in a second biological sample of said subject.

7. The method of claim 6 , wherein the second level of the substrate or its metabolite is lower than the first level.

8. The method according to any one of claims 1 to 7, wherein the biological sample is blood.

9. 1. A method for treating at least one symptom of ALS in a subject, comprising: (a) administering to a subject who has been administered a first dose of a cytochrome P450 (CYP) substrate a composition comprising TURSO and sodium phenylbutyrate; (b) determining, or having determined, a first level of the substrate or a metabolite thereof in a first biological sample from the subject; and (c) administering to the subject a second dose of the substrate, wherein the second dose is less than the first dose; The method includes:

10. 10. The method of claim 9, wherein the CYP is CYP2B6.

11. 11. The method of claim 10, wherein the CYP substrate is bupropion or efavirenz.

12. 10. The method of claim 9, wherein the CYP is CYP2C8.

13. 13. The method of claim 12, wherein the CYP substrate is repaglinide, montelukast, pioglitazone or rosiglitazone.

14. The method according to any one of claims 9 to 13, wherein the CYP substrate has a narrow therapeutic index.

15. 15. The method of any one of claims 9 to 14, further comprising a step (d) of determining or having determined a second level of said substrate or a metabolite thereof in a second biological sample of said subject.

16. 16. The method of claim 15, wherein the second level of the substrate or metabolite thereof is less than the first level.

17. The method according to any one of claims 9 to 16, wherein the biological sample is plasma or serum.

18. 1. A method for treating at least one symptom of ALS in a subject, comprising: (a) administering to a subject who has been administered a first dose of a narrow therapeutic index (NTI) medication a composition comprising TURSO and sodium phenylbutyrate; (b) determining, or having determined, a first level of the NTI drug or a metabolite thereof in a first biological sample from the subject; and (c) administering to the subject a second dose of the NTI medicament, wherein the second dose is less than the first dose; The method includes:

19. 19. The method of claim 18, wherein the NTI medication is mexiletine, alfentanil, quinidine, cyclosporine, warfarin, or digoxin.

20. 20. The method of claim 18 or 19, further comprising a step (d) of determining, or having determined, a second level of the NTI drug, or a metabolite thereof, in a second biological sample from the subject.

21. 21. The method of claim 20, wherein the second level of the NTI drug or a metabolite thereof is less than the first level.

22. 22. The method of any one of claims 1 to 21, wherein step (a) comprises administering to the subject about 1 g of TURSO and about 3 g of sodium phenylbutyrate once daily.

23. 22. The method of any one of claims 1 to 21, wherein step (a) comprises administering to the subject about 1 g of TURSO and about 3 g of sodium phenylbutyrate twice daily.

24. The method of any one of claims 1 to 23, wherein the composition is administered to the subject orally or through a feeding tube.

25. The method of any one of claims 1 to 24, wherein the subject has been diagnosed with ALS.

26. The method of any one of claims 1 to 24, wherein the subject is suspected of having ALS.

27. The method of any one of claims 1 to 26, wherein the subject is a human.

Images