Bile acid and phenylbutyric acid with CYP P450 or transporter substrates for treatment of amyotrophic lateral sclerosis

Abstract

Provided herein are methods for treating at least one symptom of ALS in a subject having received a first dose of CYP or transporter substrate, the method comprises administering to the subject a composition comprising a bile acid or a pharmaceutically acceptable salt thereof and a phenylbutyric acid compound, monitoring the subject's reaction to the substrate, and administering a second dose of the substrate, wherein the second dose is less than the first dose.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Application No. 17 / 684,845, filed on March 2, 2022, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which is submitted in electronic form as an XML file named 38709-0034WO1_SL_ST26.xml. The XML file was created on February 21, 2023 and is 6,940 bytes in size. The information in the XML file is incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure generally relates to compositions and methods for treating amyotrophic lateral sclerosis. Background Art

[0006] 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, ultimately leading to partial or complete paralysis. The median survival from symptom onset is 2 to 3 years, and respiratory failure is the leading cause of death. ALS treatment currently centers on symptom management. There are currently only two FDA-approved ALS drugs, riluzole and edaravone. Therefore, improved therapies for the treatment of ALS are needed.

[0007] The simultaneous administration of different drugs may result in adverse reactions because the metabolism and / or excretion of each drug may reduce or interfere with the metabolism and / or excretion of one or more other drugs, thereby increasing the effective concentration of these drugs, compared to the effective concentration of these drugs when administered alone. However, patients with neurodegenerative diseases, such as ALS patients, often require treatment with multiple drugs, and the potential toxicity of drug-drug interactions has the disadvantage of having harmful consequences for these patients. Therefore, there is a need for improved treatment methods that allow the administration of multiple drugs.

[0008] Brief description

[0009] In one aspect, provided herein is a method for treating at least one symptom of amyotrophic lateral sclerosis (ALS) in a subject, the method comprising: (a) administering a composition comprising taurine diol (TURSO) and sodium phenylbutyrate to a subject who has received a first dose of a substrate for an organic anion transporter 1 (OAT1); (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 a second dose of the substrate to the subject, wherein the second dose is lower than the first dose. In some embodiments, the substrate for OAT1 is a penicillin, a nonsteroidal anti-inflammatory drug (NSAID), an HIV protease inhibitor, or an antiviral drug. In some embodiments, the substrate for OAT1 is an NSAID selected from the group consisting of diclofenac, ketoprofen, and methotrexate. In some embodiments, the substrate for OAT1 is an antiviral drug selected from the group consisting of adefovir, cidofovir, and tenofovir. In some embodiments of any of the methods for 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 step (d), determining or having determined a second level of the substrate or its metabolite in a second biological sample from the subject. In some embodiments, the second level of the substrate or its metabolite is lower than the first level. In some embodiments of any of the methods for treating at least one symptom of ALS in a subject of the above aspects, the biological sample is a blood sample.

[0010] In another aspect, provided herein is a method for 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 who has received a first dose of a substrate for cytochrome P450 (CYP); (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 a second dose of the substrate to the subject, wherein the second dose is lower than the first dose. In some embodiments, the CYP is CYP2B6. In some embodiments, the substrate for the CYP is bupropion or efavirenz. In some embodiments, the CYP is CYP2C8. In some embodiments, the substrate for the CYP is repaglinide, montelukast, pioglitazone, or rosiglitazone. In some embodiments, the substrate for the CYP has a narrow therapeutic index. In some embodiments of any of the methods for treating at least one symptom of ALS in a subject of the above aspects, the method further comprises a step (d), 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 metabolite thereof is lower than the first level. In some embodiments, the biological sample is a plasma or serum sample.

[0011] In another aspect, provided herein is a method for 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 who has received a first dose of a narrow therapeutic index (NTI) drug; (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 a second dose of the NTI drug to the subject, wherein the second dose is lower than the first dose. In some embodiments, the NTI drug is mexiletine, alfentanil, quinidine, cyclosporine, warfarin, or digoxin. In some embodiments of any of the methods for treating at least one symptom of ALS in a subject of the above aspects, the method further comprises step (d), determining or having determined a second level of the NTI drug or a metabolite thereof in a second biological sample from the subject. In some embodiments, the second level of the NTI drug or a metabolite thereof is lower than the first level.

[0012] In some embodiments of any 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 by a feeding tube. In some embodiments, the subject is diagnosed with ALS. In some embodiments, the subject is suspected of having ALS. In some embodiments, the subject is human.

[0013] 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 the 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.

[0014] It should be understood that certain features of the present disclosure, which are described in the context of separate embodiments for the sake of clarity, may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure, which are described in the context of a single embodiment for the sake of brevity, may also be provided separately or in any suitable sub-combination. All combinations of embodiments belonging to the present disclosure are specifically included by the present disclosure and are disclosed herein, just as each combination is individually and explicitly disclosed. In addition, all sub-combinations of various embodiments and elements thereof are also specifically included by the present disclosure and are disclosed herein, just as each such sub-combination is individually and explicitly disclosed herein.

[0015] All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail. 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 be apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a set of graphs showing the inhibition of CYP activity by positive time-dependent inhibitors.

[0017] Figure 2 Shown are individual tenofovir plasma concentration (ng / mL) versus time data following oral administration of TDF alone (Day 1, left graph) or AMX0035 and TDF (Day 5, right graph) to male Sprague Dawley rats.

[0018] Figure 3 is a graph showing individual PB plasma concentration (ng / mL) versus time data following oral administration of AMX0035 and TDF (Day 5) in male Sprague Dawley rats.

[0019] Figure 4 Shown are individual TUDCA plasma concentration (ng / mL) versus time data following oral administration of TDF alone (Day 1, left graph) or AMX0035 and TDF (Day 5, right graph) to male Sprague Dawley rats.

[0020] Figure 5Shown are mean tenofovir, PB, and TUDCA plasma concentrations (ng / mL) versus time data following oral administration of TDF alone (Day 1, left graph) or AMX0035 and TDF (Day 5, right graph) to male Sprague Dawley rats.

[0021] Figure 6 is a bar graph showing mean tenofovir urinary recovery (ng) data in male Sprague Dawley rats following oral administration of TDF alone (Day 1, left graph) or AMX0035 and TDF (Day 5, right graph). DETAILED DESCRIPTION OF THE INVENTION

[0023] Applicants have discovered that a combination of a bile acid (e.g., taurine diol (TURSO)) and a phenylbutyrate compound (e.g., sodium phenylbutyrate) can be used to treat one or more symptoms of ALS, and have surprisingly discovered 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 cases, the combination inhibits CYP2B6 or CYP2C8. When a substrate for a CYP P450 enzyme or transporter is administered simultaneously 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 an increase in any toxic effects associated with the substrate. Thus, the present disclosure provides methods of treating at least one symptom of ALS in a subject who is also receiving a substrate for a CYP P450 enzyme (eg, those with a narrow therapeutic index) or a substrate for a transporter (eg, a substrate for OAT1).

[0024] The present 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 a composition) comprising TURSO and sodium phenylbutyrate to a subject who has received a first dose of a substrate for a cytochrome P450 enzyme, (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 a second dose of the substrate to the subject, wherein the second dose is lower than the first dose. The composition of TURSO and sodium phenylbutyrate can include about 1 g of TURSO and about 3 g of sodium phenylbutyrate.

[0025] The present 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 received 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, wherein the second dose is lower than the first dose. The composition of TURSO and sodium phenylbutyrate may include about 1 g of TURSO and about 3 g of sodium phenylbutyrate.

[0026] 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 a composition) comprising TURSO and sodium phenylbutyrate to a subject who has received 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, wherein the second dose is lower than the first dose. The composition of TURSO and sodium phenylbutyrate can include about 1 g of TURSO and about 3 g of sodium phenylbutyrate.

[0027] Where a numerical range is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit (unless the context clearly dictates otherwise), between the upper and lower limits of the range, and any other stated value or intervening value within the stated range are included in the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also included in the disclosure, subject to any express exclusion limits in the stated range. If the stated range includes one or both limits, ranges excluding one or both of the included limits are also included in the disclosure.

[0028] Certain ranges are given 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 it as well as numbers that are close to or approximately the number that follows the term. In determining whether a number is close to or approximately a specifically recited number, a number that is close to or approximately an unrecited number may be a number that, in the context in which it is presented, provides a substantial equivalent to the specifically recited number.

[0029] Unless otherwise defined, all technical terms, symbols and other scientific terms or terminology used herein have the meanings commonly understood by those skilled in the art to which this application belongs. In some cases, for clarity and / or ease of reference, terms with commonly understood meanings are defined herein, and the inclusion of these definitions herein should not necessarily be interpreted as indicating a substantial difference from what is commonly understood in the art.

[0030] I. Amyotrophic lateral sclerosis (ALS)

[0031] The terms "amyotrophic lateral sclerosis" and "ALS" are used interchangeably herein and include all classifications of ALS known in the art, including but not limited to classic ALS (e.g., ALS that affects both lower motor neurons and upper motor neurons), primary lateral sclerosis (PLS, such as those that affect only upper motor neurons), progressive bulbar palsy (PBP or bulbar onset, a form 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 (hereditary) ALS, ALS of any rate of progression (e.g., rapid, non-slow, or slow progression), and ALS of any stage (e.g., before ALS onset, ALS morbidity, and ALS late stage).

[0032] The subject in the methods described herein may exhibit one or more symptoms associated with ALS, or have been diagnosed with ALS. In some embodiments, the subject may be suspected of having ALS and / or at risk of developing ALS.

[0033] The subject of the methods described herein may exhibit one or more symptoms associated with benign fasciculation syndrome (BFS) or cramps-fasciculation syndrome (CFS).

[0034] Some embodiments of any of the methods described herein may also include determining that a subject has or is at risk of developing ALS, diagnosing a subject with or at risk of developing ALS, or selecting a subject with or at risk of developing ALS. Similarly, some embodiments of any of the methods described herein may also include determining that a subject has or is at risk of developing benign fasciculation syndrome or spastic fasciculation syndrome, diagnosing a subject with or at risk of developing BFS or CFS, or selecting a subject with or at risk of developing BFS or CFS.

[0035] In some embodiments of any of the methods described herein, the subject has displayed 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 1 week or less). In some embodiments, the subject has displayed 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).

[0036] The order and type of ALS symptoms revealed 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, ALS with bulbar onset, limb onset, or respiratory onset may show similar or different symptoms. Typically, ALS symptoms may include muscle weakness or atrophy (e.g., affecting the upper body, lower body, and / or speech), fasciculations (twitches), cramps, or stiffness of affected muscles. Early symptoms of ALS may include symptoms in the arms or legs, slurred speech, or difficulty swallowing (e.g., in bulbar onset ALS). Other symptoms include loss of tongue mobility, respiratory difficulties, difficulty breathing, or abnormal lung function, difficulty chewing, and / or difficulty walking (e.g., causing stumbling). Subjects may have respiratory muscle weakness as the initial manifestation of ALS symptoms. The prognosis for these subjects may be poor, with a median survival time of about two months after diagnosis in some cases. In some subjects, the onset of respiratory muscle weakness can be used as a prognostic factor.

[0037] ALS symptoms can also be classified according to the position (i.e. upper motor neuron or lower motor neuron) of neuronal system degeneration. Lower motor neuron degeneration is manifested as weakness or emaciation in one or more regions of, for example, medulla oblongata, cervical segment, thoracic segment and / or lumbosacral segment region. Upper motor neuron degeneration can include increased tendon reflexes, increased spasms, increased pseudobulbar features, increased Hoffman reflexes, increased plantar extensor reactions and excessive reflexes (hyperreflexia) (including overactive pharyngeal reflexes). The progress of neuronal degeneration or myasthenia is the sign of the disease. Therefore, some embodiments 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 of each in lower motor neuron degeneration and upper motor neuron degeneration. In some embodiments of any method described herein, symptom onset can be determined based on information from experimenter and / or experimenter family members. In some embodiments, the median time from symptom onset to diagnosis is about 12 months.

[0038] In some cases, the subject is 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, 19, 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 1 week or less, or on the same day of applying the currently disclosed treatment. The subject may have been diagnosed with ALS for more than about 24 months (e.g., more than about 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76 or 80 months). Methods for diagnosing ALS are known in the art. For example, the subject may be diagnosed based on clinical history, family history, physical examination, or neurological examination (e.g., signs of lower motor neuron or upper motor neuron degeneration). The subject can be confirmed or identified (e.g., by a healthcare professional) as having ALS. 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 may obtain a sample from the subject and a second party may test the sample. In some embodiments of any of the human subjects described herein, the subject is diagnosed, selected, or referred by a physician (e.g., a general practitioner).

[0039] In some embodiments, the subject meets the El Escorial criteria for probable or definite ALS, i.e., the subject exhibits:

[0040] 1. Signs of lower motor neuron (LMN) degeneration as determined by clinical, electrophysiological or neuropathological examination;

[0041] 2. Signs of upper motor neuron (UMN) degeneration by clinical examination; and

[0042] 3. Gradual spread of signs in one area or another, with the absence of:

[0043] Electrophysiological evidence of other disease processes that may explain the signs of LMN and / or UMN degeneration; and

[0044] Neuroimaging evidence of other disease processes that may explain the observed clinical and electrophysiological signs.

[0045] According to the El Escorial criteria, signs of LMN and UMN degeneration are evaluated in four regions of the central nervous system, including the brainstem, cervical spinal cord, thoracic spinal cord, and lumbosacral spinal cord. The subject may be identified as one of the following categories:

[0046] A. Clinically defined ALS, defined solely by clinical evidence (presence of UMN and LMN signs in three areas).

[0047] B. Clinically probable ALS, defined based on clinical evidence alone (UMN and LMN signs in at least two areas, with some of the UMN signs having to be located rostral (superior) to the LMN signs).

[0048] C. Clinically probable ALS - laboratory supported, defined when clinical signs of UMN and LMN dysfunction are present in only one region, or when UMN signs are present in only one region and LMN signs defined by EMG criteria are present in at least two limbs, with appropriate application of neuroimaging and clinical laboratory protocols to exclude other causes.

[0049] D. Clinically potential ALS is defined when clinical signs of both UMN and LMN dysfunction are found in only one region, or UMN signs are found alone in two or more regions; or LMN signs are found rostral to UMN signs, and a clinically possible-laboratory supported diagnosis is present.

[0050] In some embodiments, the subject has clinically confirmed ALS (eg, based on El Escorial criteria).

[0051] The subject may be evaluated and / or diagnosed using the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R). The ALSFRS-R is an ordinal rating scale (score 0-4) that determines an assessment of the subject's ability and independence in 12 functional activities related to 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 the magnitude of change over time and can also be correlated with quality of life measures and predicted survival. The ALSFRS-R exhibits a linear mean slope and can be used as a prognostic indicator (see 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).

[0052] In the ALSFRS-R, the functions mediated by the cervical muscles, trunk muscles, lumbosacral muscles, and respiratory muscles are each assessed by 3 items. Each item is scored from 0 to 4, where 4 reflects no disease involvement and 0 reflects maximal disease involvement. The item scores are added together to give a total score. The total score reflects the impact of ALS, with the following exemplary classification:

[0053] >40 (mild to mild); 39-30 (mild to moderate); <30 (moderate to severe); <20 (advanced disease).

[0054] For example, the subject can have an ALSFRS-R score (e.g., a baseline ALSFRS-R score) of 40 or more (e.g., at least 41, 42, 43, 44, 45, 46, 47, or 48), 30 to 39 inclusive (e.g., 31, 32, 33, 34, 35, 36, 37, or 38), or 30 or less (e.g., 21, 22, 23, 24, 25, 26, 27, 28, or 29). In some embodiments of any 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).

[0055] Because ALS is a progressive disease, all patients will usually progress over time. However, since some subjects die or require respiratory support within a few months, while other subjects have a relatively long survival period, there is a large degree of inter-subject variability in the speed of progression. Subjects described herein may have rapidly progressive ALS or slowly progressive ALS. The rate of functional decline in subjects with ALS can be measured by the change in monthly ALSFRS-R scores. For example, the score can be reduced by about 1.02 (± 2.3) points per month.

[0056] One predictor of patient progression is the patient's previous disease progression rate (ΔFS), which can be calculated as: ΔFS = (48-ALSFRS-R score at evaluation) / duration from onset to evaluation (months). The ΔFS score represents the loss of ALSFRS-R scores per month since symptom onset and can serve as an important predictor of progression and / or survival in subjects with ALS (see Labra et al. J Neurol Neurosurg Psychiatry 87:628-632, 2016 and Kimura et al. Neurology 66:265-267, 2006). A 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); between about 0.50 and about 1.20 (including, for example, 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 any of the methods described herein, the subject can 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 may underestimate or overestimate a patient's progression if under evaluating.

[0057] In some embodiments, the subject has lost an average of about 0.8 to about 2 (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) ALSFRS-R scores per month over a period of 3-12 months since the initial evaluation. In some embodiments, the subject has lost an average of more than about 1.2 ALSFRS-R scores per month over a period of 3-12 months since the initial evaluation. The subject's ALSFRS-R score may have decreased by at least 3 points (e.g., at least 4, 6, 8, 10, 12, 14, 16, 20, 24, 28, or 32 points) over a period of 3-12 months since the initial evaluation. In some embodiments, the subject has lost an average of about 0.8 to about 2 (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) ALSFRS-R scores per month over the past 3-12 months. In some embodiments, the subject has lost an average of more than about 1.2 (e.g., more than about 1.5, 1.8, 2.0, 2.5, or 3) ALSFRS-R scores per month over the past 3-12 months.

[0058] In some embodiments of any method described herein, the presence or level of a marker in a sample obtained from a subject may be used for ALS diagnosis or prognosis, or for tracking disease activity and therapeutic response. Suitable samples include, for example, cells, tissues or body fluids (e.g., blood, urine or cerebrospinal fluid (CSF) samples). For example, the level of phosphorylated neurofilament heavy subunit (pNF-H) or neurofilament light chain (NfL) in CSF and / or blood can be used as a biomarker for ALS diagnosis, prognosis or tracking disease activity or therapeutic outcome. pNF-H is the main component of the neuronal cytoskeleton, which is released into CSF ​​and bloodstream as neuronal damage. The level of pNF-H may be associated with the level of axonal loss and / or the burden of motor neuron dysfunction (e.g., see De Schaepdryver et al. Journal of Neurology, Neurosurgery & Psychiatry 89: 367-373, 2018).

[0059] The concentration of pNF-H in the CSF and / or blood of subjects with ALS may be significantly increased in the early disease stage. Higher levels of pNF-H in plasma, serum and / or CSF may be associated with faster ALS progression (e.g., faster decline in ALSFRS-R) and / or shorter survival. Plasma pNF-H concentrations in ALS subjects with bulbar onset may be higher than plasma pNF-H concentrations in ALS subjects with spinal onset. In some cases, the imbalance between the relative expression levels of neurofilament heavy and light chain subunits can be used for ALS diagnosis, prognosis, or tracking of disease progression.

[0060] 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). Commercial pNF-H detection assays, such as those developed by EnCor Biotechnology, Biovandor, and Millipore EMD, can also be used. Commercially available NfL assay kits based on Simoa technology, such as those produced by Quanterix (see, e.g., Thouvenot et al. European Journal of Neurology 27:251-257,2020) can also be used. Factors affecting pNF-H and NfL levels or the relationship between the detection of pNF-H and NfL levels in serum or plasma and the course of the disease may be different from those in CSF. The levels of neurofilaments (e.g., pNF-H and / or NfL) in CSF and serum may be associated (see, e.g., Wilke et al. Clin Chem Lab Med 57(10):1556-1564,2019).

[0061] A subject described herein may have 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, 1 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, CSF pNF-H levels can be about 1000 to about 5000 pg / mL (eg, about 1500 to about 4000, or about 2000 to about 3000 pg / mL).

[0062] 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 can 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, CSF NfL levels can be between about 2000 and 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 8,000, about 2000 to about 6,000, 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 4,000 to about 20,000, about 4,000 to about 15,000, about 4,000 to about 10,000, about 4,000 to about 8,000, about 4,000 to about 6,000, about 6,000 to about 40,000, about 6,000 to about 35,000, about 6,000 to about 30,000, about 6,000 to about 25,000, about 6,000 to about 20,000, about 6,000 to about 15,000, about 6,000 to about 10,000, about 6,000 to about 8,000, about 8,000 to about 40,000, about 8 From about 10,000 to about 35,000, from about 8,000 to about 30,000, from about 8,000 to about 25,000, from about 8,000 to about 20,000, from about 8,000 to about 15,000, from about 8,000 to about 10,000, from about 10,000 to about 40,000, from about 10,000 to about 35,000, from about 10,000 to about 30,000, from about 10,000 to about 25,000, from about 10,000 to about 20,000, from about 10,000 to about 15,000, from about 15,000 to about 40,000, from about 15,000 to about 35,000, pg / mL).

[0063] Other biomarkers that can be used for ALS diagnosis, prognosis, and disease progression monitoring 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 ALS prognosis (see, e.g., Atassi et al. Neurology 83 (19): 1719-1725, 2014). Akt phosphorylation can also be used as a biomarker for ALS prognosis (see, e.g., WO 2012 / 160563). Urinary p75ECD and ketone body levels can be used as biomarkers for ALS diagnosis (see, e.g., Shepheard et al. Neurology 88: 1137-1143, 2017). Serum and urine creatinine levels can also be used as biomarkers. Other useful blood, CSF, neurophysiological, and neuroradiological biomarkers of ALS are described in Turner et al., Lancet Neurol 8:94-109, 2009. Any of the markers described herein can be used to diagnose a subject with ALS, or to determine that a subject is at risk for developing ALS.

[0064] Based on genetic analysis, the subject may also be identified as having ALS or having the risk of developing ALS. Genetic variation associated with ALS is 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. Exemplary 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. Additional 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 variations associated with ALS can affect the rate of ALS progression in a subject, the pharmacokinetics of a compound administered to a subject, and / or the efficacy of an administered compound to a subject.

[0065] The subject may have a mutation in a gene encoding copper-zinc superoxide dismutase (SOD1). The mutation causes the SOD1 protein to aggregate more easily, resulting in the deposition of cellular inclusions containing misfolded SOD1 aggregates (see, e.g., Andersen et al., Nature Reviews Neurology 7:603-615, 2011). More than 100 different mutations in SOD1 are associated with hereditary ALS, many of which result in single amino acid substitutions in the protein. In some embodiments, the SOD1 mutation is A4V (i.e., alanine at position 4 is substituted with valine). The SOD1 mutation is further described in, e.g., 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 common cause of ALS, and both loss of function of C9ORF72 and gain of toxic function of the repeat sequence are associated with ALS (see, e.g., Balendra and Isaacs, Nature Reviews Neurology 14:544-558, 2018). The methods described herein may include detecting SOD1 mutations and / or C9ORF72 mutations in a subject prior to administration of bile acid and butyrate compounds. Methods for screening 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.

[0066] Skilled practitioners will appreciate that certain factors can affect the bioavailability and metabolism of the compound administered to a subject, and adjustments can be made accordingly. These factors include, but are not limited to, liver function (e.g., levels of liver enzymes), renal function, and gallbladder function (e.g., levels of ion absorption and secretion, cholesterol transporters). Each subject has variability in exposure levels to the administered compound (e.g., bile acids and phenylbutyric acid compounds), differences in the levels of excretion, and differences in the pharmacokinetics of the compound in the treated subject. Any of the factors described herein may affect the drug exposure of the subject. For example, a reduction in the clearance of a compound can result in increased drug exposure, while improved renal function can reduce actual drug exposure. The extent of drug exposure may be related to the subject's response to the administered compound and the outcome of the treatment.

[0067] The subject can be, for example, older than about 18 years old (e.g., 18-100 years, 18-90 years, 18-80 years, 18-70 years, 18-60 years, 18-50 years, 18-40 years, 18-30 years, 18-25 years, 25-100 years, 25-90 years, 25-80 years, 25-70 years, 25-60 years, 25-50 years, 25-40 years, 25-30 years, 30-100 years, 30-90 years, 30-80 years, 30-70 years, 30- The subject may have a body weight of 18.5-30 kg / m2. The subject may have a body weight of 18.5-30 kg / m2. The subject may have a body weight of 18.5-30 kg / m2. The subject may have a body weight of 18.5-30 kg / m2. 2 between (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 28-30 kg / m 2 ) BMI. Mutations in any ALS-related genes described herein or the presence of any biomarkers described herein may indicate that the subject is at risk for developing ALS. For preventive and prophylaxis purposes, such subjects can be treated using the methods provided herein.

[0068] In some embodiments, the subject has one or more symptoms of benign fasciculation syndrome (BFS) or cramp-fasciculation syndrome (CFS). BFS and CFS are peripheral nerve hyperexcitability disorders that can cause fasciculation, cramps, pain, fatigue, muscle stiffness and paresthesia. Methods for identifying subjects with these disorders are known in the art, such as by clinical examination and electromyography.

[0069] II. Composition

[0070] The present disclosure provides a method of treating at least one symptom of ALS in a subject, the method comprising administering to the subject bile acid or a pharmaceutically acceptable salt thereof and a phenylbutyrate compound. In some embodiments, the method comprises administering to the subject a composition comprising TURSO and sodium phenylbutyrate.

[0071] Bile acid

[0072] As used herein, "bile acid" refers to a naturally occurring surfactant having a core derived from cholanic acid, which is typically substituted with a 3α-hydroxyl group at the C6, C7 or C12 position of the sterol core, and optionally also substituted with other hydroxyls. Bile acid derivatives (e.g., water-soluble bile acid derivatives) and bile acids conjugated to amines are also included in the term "bile acid". Bile acid derivatives include, but are not limited to, derivatives formed on carbon atoms, with hydroxyl and carboxylic acid groups of bile acids attached to other functional groups (including, but not limited to, halogens and amino groups). Soluble bile acids may include aqueous preparations of bile acids in the form of free acids combined with one of HCl, phosphoric acid, citric acid, acetic acid, ammonia or arginine. Suitable bile acids include, but are not limited to, taurodiol (TURSO), ursodeoxycholic acid (UDCA), chenodeoxycholic acid (also known as "chenodiol" or "chenic 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 an analog, derivative, or prodrug thereof.

[0073] 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 any bile acid disclosed herein are also contemplated. In some embodiments, bases commonly used to form pharmaceutically acceptable salts of bile acids of the present disclosure include hydroxides of alkali metals (including sodium, potassium and lithium); hydroxides of alkaline earth metals (e.g., calcium and magnesium); hydroxides of other metals (e.g., aluminum and zinc); ammonia, organic amines (e.g., unsubstituted or hydroxy-substituted mono-, di- or tri-alkylamines), dicyclohexylamine; tributylamine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, di- or tri-(2-OH-(C1-C6)-alkylamines), such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tris(2-hydroxyethyl)amine; N-methyl-D-glucamine; morpholine; thiomorpholine; piperidine; pyrrolidine; and amino acids (e.g., arginine, lysine), etc.

[0074] The terms "taurodeoxycholic acid" (TUDCA) and "taurine diol" (TURSO) are used interchangeably herein.

[0075] The bile acid described herein may be a TURSO as shown in Formula I (with labeled carbons to help understand where substitutions may be made).

[0076]

[0077] or a pharmaceutically acceptable salt thereof.

[0078] The bile acid described herein may be UDCA, as shown in Formula II (with labeled carbons to help understand where substitutions may be made).

[0079]

[0080] or a pharmaceutically acceptable salt thereof.

[0081] The bile acid derivatives disclosed herein may be physiologically relevant bile acid derivatives. For example, any combination of substitution of hydrogen at position 3 or 7, shift in stereochemistry of the hydroxyl group at position 3 or 7 in the formula of TURSO or UDCA is suitable for the present composition.

[0082] "Bile acid" may also be a bile acid conjugated to an amino acid. The amino acid in the conjugate may be, but is not limited to, taurine, glycine, glutamine, asparagine, methionine, or carboxymethylcysteine. Other amino acids that may be conjugated to the bile acids of the present disclosure include arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, cysteine, proline, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, and tryptophan, as well as β-alanine and γ-aminobutyric acid. An example of such a bile acid is a compound of formula III:

[0083]

[0084] in:

[0085] R is -H or C1-C4 alkyl;

[0086] R1 is -CH2-SO3R3, CH2COOH or CH2CH2COOH, and R2 is -H;

[0087] 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; and

[0088] R3 is -H or an amino acid residue, or a pharmaceutically acceptable analog, derivative, prodrug or mixture thereof. An example of an amino acid is a basic amino acid. Other examples of amino acids include glycine, glutamine, asparagine, methionine, carboxymethylcysteine, 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.

[0089] Another example of a bile acid of the present disclosure is a compound of formula IV:

[0090]

[0091] in:

[0092] R is -H or C1-C4 alkyl;

[0093] R1 is -CH2-SO3R3, and R2 is -H;

[0094] or R1 is -COOH and R2 is -CH2-CH2-CONH2, -CH2-CONH2, -CH2-CH2-SCH3 or -CH2-S-CH2-COOH; and

[0095] R3 is -H or a basic amino acid residue, or a pharmaceutically acceptable analog, derivative, prodrug or mixture thereof. Examples of basic amino acids include lysine, histidine and arginine.

[0096] In some embodiments, bile acid is TURSO. TURSO is an amphipathic bile acid, which is a taurine conjugate form of UDCA. TURSO restores mitochondrial bioenergetic defects by incorporating into mitochondrial membranes, reducing Bax translocation to mitochondrial membranes, reducing mitochondrial permeability and increasing the apoptosis threshold of cells (Rodrigues et al. Biochemistry 42, 10: 3070-3080 2003). It is used to treat cholesterol gallstones, which usually require long-term treatment (e.g., 1 to 2 years) to completely dissolve. It has been used to treat cholestatic liver diseases, including primary cirrhosis, familial intrahepatic cholestasis in children, primary sclerosing cholangitis, and cholestasis caused by cystic fibrosis. TURSO is contraindicated in subjects with biliary infections, frequent biliary colic, or subjects with difficulty absorbing bile acids (e.g., ileal disease or resection). Drug interactions may include interactions with substances that inhibit bile acid absorption (e.g., cholestyramine), and with drugs that increase cholesterol elimination in bile (TURSO reduces the cholesterol content of bile). Based on similar physicochemical characteristics, similarities in drug toxicity and interactions exist between TURSO and UDCA. The most common adverse reactions (≥1%) reported with TURSO were: abdominal discomfort, abdominal pain, diarrhea, nausea, pruritus, and rash. Pruritus was reported in some cases, and elevated liver enzymes were reported in a few cases.

[0097] In some embodiments, the bile acid is UDCA. UDCA or ursodiol has been 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 more hydrophilic. TURSO is absorbed in the distal ileum under active transport and therefore may have a slightly longer residence time in the intestine than UDCA absorbed in the proximal ileum. Ursodiol treatment is not associated with liver damage. Liver enzyme abnormalities and (Ursodiol USP capsules) therapy is irrelevant, It has been shown to reduce liver enzyme levels in liver disease. Subjects should have SGOT (AST) and SGPT (ALT) measured at the start of treatment and thereafter based on specific clinical circumstances. Previous studies have suggested that bile acid sequestrants (e.g., cholestyramine and colestipol) may interfere with the action of ursodeoxycholic acid by reducing its absorption. Aluminum-based antacids have been shown to adsorb bile acids in vitro and might be expected to interfere with ursodeoxycholic acid in the same manner as bile acid sequestrants. Estrogens, oral contraceptives, and clofibrate (and perhaps other lipid-lowering drugs) increase hepatic cholesterol secretion and promote cholesterol gallstone formation and therefore might counteract the effectiveness of ursodeoxycholic acid.

[0098] Phenylbutyrate compounds

[0099] Phenylbutyric acid compounds are defined herein as comprising phenylbutyric acid (a low molecular weight aromatic carboxylic acid) as a free acid (4-phenylbutyric acid (4-PBA), 4-phenylbutyric acid (4-phenylbutyric acid) or phenylbutyric acid (phenylbutyric acid)) and pharmaceutically acceptable salts, co-crystals, polymorphs, hydrates, solvates, conjugates, derivatives or prodrugs thereof. Phenylbutyric acid compounds described herein also include analogs of 4-PBA, including but not limited to triglycerol-(4-phenylbutyric acid), phenylacetic acid (which is an 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 pharmaceutically acceptable salts thereof. Phenylbutyric acid compounds also include physiologically relevant 4-PBA species, such as, but not limited to, any substitutions in which deuterium replaces hydrogen in the 4-PBA structure. Other HDAC2 inhibitors are contemplated herein as alternatives to phenylbutyric acid compounds.

[0100] Physiologically acceptable salts of phenylbutyric acid include, for example, sodium, potassium, magnesium or calcium salts. Examples of other salts include ammonium, zinc or lithium salts, or salts of phenylbutyric acid with an organic amine such as lysine or arginine.

[0101] In some embodiments of any of the methods described herein, the phenylbutyric acid compound is sodium phenylbutyrate. Sodium phenylbutyrate has the following structural formula:

[0102]

[0103] Phenylbutyric acid is a pan-HDAC inhibitor that can improve ER stress by upregulating the master chaperone regulator DJ-1 and by recruiting other chaperone proteins (see Zhou et al. Biol Chem. 286: 14941-14951, 2011 and Suaud et al. JBC. 286: 21239-21253, 2011). A large increase in the production of chaperone proteins reduces the activation of the classical ER stress pathway, folds misfolded proteins, and has been shown to increase survival in in vivo models (including the G93A SOD1 mouse model of ALS) (see, for example, Ryu, H et al. J Neurochem. 93: 1087-1098, 2005).

[0104] In some embodiments, when administered at a specific ratio (e.g., any ratio described herein), the combination of a bile acid (e.g., TURSO) or a pharmaceutically acceptable salt thereof and a phenylbutyric acid compound (e.g., sodium phenylbutyrate) has a synergistic effect in treating one or more symptoms associated with ALS. This combination can, for example, induce a mathematically synergistic increase in neuronal viability in a model of strong oxidative damage (H2O2-mediated toxicity) using linear modeling by simultaneously inhibiting endoplasmic reticulum stress and mitochondrial stress (see, e.g., U.S. Pat. No. 9,872,865 and U.S. Pat. No. 10,251,896).

[0105] preparation

[0106] Bile acids and phenylbutyric acid compounds described herein can be formulated into pharmaceutical compositions for use or for use in pharmaceutical compositions. For example, the methods described herein can include administering an effective amount of a composition comprising TURSO and sodium phenylbutyrate. As used herein, the term "effective amount" refers to the amount or concentration of one or more drugs over a period of time (including acute or chronic administration and periodic or continuous administration), which is effective within its application range to cause a desired effect or physiological result. The composition may include about 5% to about 15% w / w (e.g., about 6% to about 14%, about 7% to about 13%, about 8% to about 12%, about 8% to about 11%, about 9% to about 10%, or about 9.7% w / w) of TURSO and about 15% to about 45% w / w (e.g., about 20% to about 40%, about 25% to about 35%, about 28% to about 32%, or about 29% to about 30%, such as about 29.2% w / w) of sodium phenylbutyrate. In some embodiments, the composition comprises about 9.7% w / w TURSO and 29.2% w / w sodium phenylbutyrate.

[0107] Sodium phenylbutyrate and TURSO may be present in the composition in a weight ratio of about 1:1 to about 4:1, such as about 2:1 or about 3:1. In some embodiments, the ratio between sodium phenylbutyrate and TURSO is about 3:1.

[0108] The compositions described herein may include any pharmaceutically acceptable carrier, adjuvant and / or excipient. The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that may be administered to a patient together with the compounds disclosed herein, and when administered at a dose sufficient to deliver a therapeutic amount of the compound, the carrier or adjuvant does not destroy the pharmacological activity of the compound and is non-toxic. As used herein, the language "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, etc., compatible with drug administration. The pharmaceutical composition may contain any conventional non-toxic pharmaceutically acceptable carrier, adjuvant or excipient. In some cases, the pH of the preparation may be adjusted using a pharmaceutically acceptable acid, base or buffer to enhance the stability of the formulated compound or its delivery form.

[0109] The compositions of the present disclosure may 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). Both anhydrous and hydrated dextrates are contemplated herein. The dextrates of the present disclosure may include a mixture of saccharides produced by controlled enzymatic hydrolysis of starch. Some embodiments of any of the compositions described herein include a hydrated dextrates (e.g., NF grade, available from JRS Pharma, Colonial Scientific, or Quadra).

[0110] The compositions of the present disclosure may include about 1% to about 6% w / w of sugar alcohol (e.g., about 2% to about 5%, about 3% to about 4%, or about 3.9% w / w of sugar alcohol). Sugar alcohols can be derived from sugars and contain a hydroxyl group (-OH) attached to each carbon atom. Both disaccharides and monosaccharides can form sugar alcohols. Sugar alcohols can be natural or produced by hydrogenation of sugars. Exemplary sugar alcohols include, but are not limited to, sorbitol, xylitol, and mannitol. In some embodiments, the composition comprises about 1% to about 6% w / w (e.g., about 2% to about 5%, about 3% to about 4%, or about 3.9% w / w) of sorbitol.

[0111] The compositions of the present disclosure may include about 22% to about 35% w / w maltodextrin (e.g., about 22% to about 33%, about 24% to about 31%, about 25% to about 32%, about 26% to about 30%, or about 28% to about 29% w / w, such as about 28.3% w / w maltodextrin). When dissolved in a solution, maltodextrin can form a flexible spiral that enables the embedding of active ingredients (e.g., any phenylbutyric acid compound and bile acid described herein), thereby masking the taste of the active ingredients. Maltodextrin produced from any suitable source is contemplated herein, including but not limited to peas, rice, cassava, corn, and potatoes. In some embodiments, the maltodextrin is pea maltodextrin. In some embodiments, the composition comprises about 28.3% w / w pea maltodextrin. For example, maltodextrin obtained from Roquette ( LINECAPS) pea maltodextrin.

[0112] The compositions described herein may also include a sugar substitute (e.g., sucralose). For example, the composition may 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.

[0113] In some embodiments, the composition includes one or more flavoring agents. The composition may include about 2% to about 15% w / w of 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%, such as about 7.3% w / w). Flavoring agent may include a substance that gives another substance an odor, or a substance that changes the characteristics of the composition by affecting its taste. Flavoring agent may be used to cover up unpleasant tastes without affecting physical and chemical stability, and may be selected based on the taste of the drug to be incorporated. Suitable flavoring agents include, but are not limited to, natural flavoring substances, artificial flavoring substances, and imitation spices. A mixture of flavoring agents may also be used. For example, the composition described herein may include two or more (e.g., two, three, four, five or more) flavoring agents. Flavoring agent is soluble and stable in water. Suitable flavoring agent may be selected according to taste testing. For example, a variety of different flavoring agents may be added to the composition, and the composition may be taste tested. Exemplary flavoring agents include any fruit flavor powder (e.g., peach, strawberry, mango, orange, apple, grape, raspberry, cherry, or mixed berry flavor powder). The compositions described herein can include about 0.5% to about 1.5% w / w (e.g., about 1% w / w) of mixed berry flavor powder and / or about 5% to about 7% w / w (e.g., about 6.3% w / w) of a masking flavor. Suitable masking flavors can be obtained, for example, from Firmenich.

[0114] The compositions described herein may also include silicon dioxide (silicon dioxide or silica). Adding silicon dioxide to the composition can prevent or reduce agglomeration of the components of the composition. Silicon dioxide can act as an anti-caking agent, adsorbent, disintegrant, or glidant. In some embodiments, the compositions described herein include about 0.1% to about 2% w / w porous silicon dioxide (e.g., about 0.3% to about 1.5%, about 0.5% to about 1.2%, or about 0.8% to about 1%, such as 0.9% w / w). Porous silicon dioxide may have a higher H2O absorption capacity and / or a higher porosity than fumed silica at a relative humidity of about 20% or more (e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more). At a relative humidity of about 50%, the porous silica can have a H2O absorption capacity of about 5% to about 40% (e.g., about 20% to about 40%, or about 30% to about 40%) by weight. Compared with fumed silica, the porous silica can have a higher porosity 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 from about 0.05% to about 2% w / w (e.g., any sub-range of the ranges described herein) of 63FP (WR-Grace).

[0115] Compositions described herein may also include one or more buffers. For example, the composition may include about 0.5% to about 5% w / w of a buffer (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, such as a buffer of about 2.7% w / w). Buffers may include a weak acid or a weak base that maintains the acidity or pH of the composition near a selected value after adding another acid or base. Suitable buffers are known in the art. In some embodiments, the buffer in the composition provided herein is a phosphate, such as sodium phosphate (e.g., anhydrous disodium hydrogen phosphate). For example, the composition may include about 2.7% w / w of disodium hydrogen phosphate.

[0116] The composition can also include one or more lubricants. For example, the composition can include a lubricant of about 0.05% to about 1% w / w (for example, 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%, such as a lubricant of about 0.5% w / w). Exemplary lubricants include but are not limited to sodium stearyl fumarate, magnesium stearate, stearic acid, stearic acid metal salts, talc, waxes and glycerides with high melting points, colloidal silicon dioxide, polyethylene glycol, alkyl sulfates, behenic acid glyceride and hydrogenated oil. Other lubricants are known in the art. In some embodiments, the composition includes sodium stearyl fumarate of about 0.05% to about 1% w / w (for example, any sub-range of the scope described herein). For example, the composition can include sodium stearyl fumarate of about 0.5% w / w.

[0117] In some embodiments, the composition includes about 29.2% w / w sodium phenylbutyrate, about 9.7% w / w TURSO, about 15.6% w / w dextrates, about 3.9% w / w sorbitol, about 1.9% w / w sucralose, about 28.3% w / w maltodextrin, about 7.3% w / w flavoring, about 0.9% w / w silicon dioxide, about 2.7% w / w sodium phosphate (e.g., disodium hydrogen phosphate), and about 0.5% w / w sodium stearyl fumarate.

[0118] The composition can include about 3000 mg of sodium phenylbutyrate, about 1000 mg of TURSO, about 1600 mg of dextrates, about 400 mg of sorbitol, about 200 mg of sucralose, about 97.2 mg of silicon dioxide, about 2916 mg of maltodextrin, about 746 mg of flavoring (e.g., about 102 mg of mixed berry flavor and about 644 mg of masking flavor), about 280 mg of sodium phosphate (e.g., disodium hydrogen phosphate), and about 48.6 mg of sodium stearyl fumarate.

[0119] Other 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, glucose, sucrose, maltose, steviol glycosides, partially hydrolyzed starch, and corn syrup solids. Water-soluble artificial sweeteners are contemplated herein, such as soluble saccharin salts (e.g., saccharin sodium or saccharin calcium), sodium cyclamate, acesulfame potassium (acesulfame K), and saccharin and aspartame-based sweeteners (e.g., L-aspartame-phenylalanine methyl ester, or The amount of sweetener or taste-masking agent may vary depending on the desired amount of sweetener or taste-masking agent selected for the particular final composition.

[0120] In addition to the above, pharmaceutically acceptable binders are also contemplated. Examples include cellulose derivatives, including microcrystalline cellulose, low-substituted hydroxypropyl cellulose (e.g., LH 22, LH 21, LH 20, LH 32, LH 31, LH30); starch, including potato starch; cross-linked carboxymethyl cellulose sodium (i.e., cross-linked carboxymethyl cellulose sodium salt; e.g., Ac-Di- ); alginic acid or alginate; insoluble polyvinyl pyrrolidone (e.g. CL, CL-M, CL, XL, XL-10); and sodium carboxymethyl starch (e.g. Primogel and ).

[0122] Other fillers, diluents or binders may be added, such as polyols, sucrose, sorbitol, mannitol, Lactose (such as spray-dried lactose, α-lactose, β-lactose, Different levels Microtose or Fast- ), microcrystalline cellulose (e.g. different grades of For example PH101, PH102 or PH105, P100, Ming and Solka- ), hydroxypropyl cellulose, L-hydroxypropyl cellulose (low-substituted) (e.g., L-HPC-CH31, L-HPC-LH11, LH 22, LH 21, LH 20, LH 32, LH 31, LH30), dextrin, maltodextrin (e.g. 5 and 10), starch or modified starch (including potato starch, corn starch and rice starch), sodium chloride, sodium phosphate, calcium sulfate and calcium carbonate.

[0123] The compositions described herein can be formulated to be administered to a subject or suitable for administration to a subject by any route, such as any route approved by the Food and Drug Administration (FDA). Exemplary methods are described in the FDA's CDER Data Standards Manual (Release No. 004) (available at fda.give / cder / dsm / DRG / drg00301.html).

[0124] Pharmaceutical compositions are typically formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral (subcutaneous, intradermal, intravenous, intradermal, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques), oral (e.g., inhalation or by feeding tube), transdermal (topical), transmucosal, and rectal administration.

[0125] 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 into a powder-filled capsule. Suitable powders may include powders that are substantially soluble in water. The pharmaceutical composition may be formulated using suitable dispersants or wetting agents (e.g., Tween 80) and suspending agents according to techniques known in the art. Sterile injectable preparations may also be sterile injectable solutions or suspensions in nontoxic parenteral acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable excipients and solvents that may be used are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are routinely used as solvents or suspension media. For this purpose, any mild fixed oil may be used, including synthetic monoglycerides or diglycerides. Fatty acids (e.g., oleic acid and its glyceride derivatives) are useful in the preparation of injections, as are natural pharmaceutically acceptable oils (e.g., olive oil or castor oil), especially their polyoxyethylated forms. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents, which are commonly used in the preparation of pharmaceutically acceptable dosage forms (e.g., emulsions and or suspensions). Other commonly used surfactants, such as Tweens or Spans and / or other similar emulsifiers or bioavailability enhancers, which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid or other dosage forms, may also be used for formulation purposes.

[0126] The composition can be orally administered in any oral acceptable dosage form, including but not limited to powders, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. For powders for oral administration, the powder can be substantially dissolved in water before administration. For tablets for oral use, conventional 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 administered orally, the active ingredient may be suspended or dissolved in an oil phase and combined with an emulsifier and / or suspending agent. If desired, certain sweeteners and / or flavorings and / or coloring agents may be added.

[0127] Alternatively or additionally, the compositions may be administered by nasal aerosol or inhalation. These compositions are formulated according to techniques well known in the art of pharmaceutical formulation and are formulated as solutions in normal saline, using benzyl alcohol or other suitable preservatives to enhance bioavailability, absorption enhancers, fluorocarbons and / or other solubilizing or dispersing agents known in the art.

[0128] In some embodiments, the therapeutic compositions disclosed herein can be formulated for sale in the United States, imported into the United States, and / or exported from the United States. The pharmaceutical composition can be included in a container, packaging bag, or dispenser with instructions for administration. In some embodiments, the present invention provides a kit including bile acid and phenylbutyric acid compounds. The kit also includes instructions for use for doctors and / or patients, syringes, needles, boxes, bottles, vials, etc.

[0129] III. Treatment Methods

[0130] 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 surprisingly discovered 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 cases, the combination inhibits CYP2B6 and CYP2C8. When a substrate for a CYP 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 an increase in any toxic effects associated with the substrate.

[0131] Thus, the present disclosure provides methods for treating at least one symptom of ALS in a subject receiving a CYP substrate or transporter substrate by adjusting the dosage of the substrate. Such adjustment may provide a plasma concentration of the substrate (or its metabolite) similar to the dosage of the administered substrate in the absence of bile acid and phenylbutyrate compounds, and may be as effective as the dosage of the administered substrate.

[0132] Applicants disclose herein a method for treating at least one symptom of ALS in a subject who has received a first dose of a substrate for a CYP or transporter, the method comprising administering to the subject a composition comprising TURSO and sodium phenylbutyrate, monitoring the subject's response to the substrate, and administering a second dose of the substrate, wherein the second dose is less than the first dose. The composition may include, for example, 1 g of TURSO and 3 g of sodium phenylbutyrate. Monitoring the subject's response to the substrate may include determining the level of the substrate or its metabolite in a biological sample from the subject, determining the subject's blood INR level, or monitoring known adverse events, overdose symptoms, or side effects associated with the substrate.

[0133] In one aspect, provided herein is a method for treating at least one symptom of ALS in a subject, comprising: (a) administering a composition comprising TURSO and sodium phenylbutyrate to a subject who has received a first dose of a substrate for cytochrome P450 (CYP), (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 a second dose of the substrate to the subject, wherein the second dose is lower than the first dose. The composition may include, for example, 1 g of TURSO and 3 g of sodium phenylbutyrate. In some embodiments, the CYP is CYP2B6 or CYP2C8. In some embodiments, the substrate for the CYP is a drug with a narrow therapeutic index.

[0134] The present disclosure also provides a method of treating at least one symptom of ALS in a subject, comprising: (a) administering a composition comprising TURSO and sodium phenylbutyrate to a subject who has received 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, wherein the second dose is lower than the first dose. The composition may include, for example, 1 g of TURSO and 3 g of sodium phenylbutyrate.

[0135] Also provided is a method of treating at least one symptom of ALS in a subject, comprising: (a) administering a composition comprising TURSO and sodium phenylbutyrate to a subject who has received a first dose of an organic anion transporter 1 (OAT1) substrate; (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 a second dose of the substrate to the subject, wherein the second dose is lower than the first dose. The composition may include, for example, 1 g of TURSO and 3 g of sodium phenylbutyrate.

[0136] The present disclosure also provides a method for treating at least one symptom of ALS in a subject who has received a first dose of a narrow therapeutic index (NTI) drug, the method comprising administering a composition comprising TURSO and sodium phenylbutyrate to the subject, monitoring the subject's response to the NTI drug, and administering a second dose of the NTI drug, wherein the second dose is less than the first dose. The composition may include, for example, 1 g of TURSO and 3 g of sodium phenylbutyrate. In one aspect, provided herein is a method for treating at least one symptom of ALS in a subject, comprising: (a) administering a composition comprising TURSO and sodium phenylbutyrate to a subject who has received a first dose of a narrow therapeutic index (NTI) drug; (b) determining or having determined a first level of the NTI drug or its metabolite in a first biological sample from the subject, and (c) administering a second dose of the NTI drug to the subject, wherein the second dose is lower than the first dose. The composition may include, for example, 1 g of TURSO and 3 g of sodium phenylbutyrate.

[0137] In some embodiments of any method described herein, the first and / or second dose of the substrate or NTI drug can be a dose of daily (once, twice or three times a day), once every two days, three times a week or once a week, or a dose based on some other basis. The second dose of the substrate or NTI drug can be less than the first dose by 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%). In some cases, the second dose involves the same or similar amount of each administration, but the frequency is low compared to the first dose.

[0138] In some embodiments of any of the methods described herein, the method further comprises step (d), determining or having determined a second level of the substrate or its metabolite or the NTI drug or its metabolite in a second biological sample from the subject. In some embodiments, the second level is lower than the first level.

[0139] In some embodiments of any of the methods described herein, a first biological sample can be obtained from the subject at 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 at 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 a substrate or NTI drug.

[0140] In some embodiments of any of the methods described herein, the first and / or second biological sample can be a serum, plasma, urine or saliva sample. 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, an immunoassay or liquid chromatography / tandem mass spectrometry (LC / MS) can be used.

[0141] Narrow therapeutic index drugs

[0142] The therapeutic index (TI) is the dose range within which a drug is effective without unacceptable adverse events. Narrow therapeutic index (NTI) drugs are those for which small differences in dose or blood concentration may result in serious treatment failure and / or life-threatening adverse drug reactions, or may cause persistent or severe disability or incapacity. Therefore, only within a very small dose range does the drug produce a beneficial effect without causing serious and potentially fatal complications.

[0143] In animal studies, the TI can be calculated as the lethal dose of the drug that kills 50% of the population (LD 50 ) divided by the minimum effective dose (ED 50 ), that is, TI=LD 50 / ED 50 In clinical practice, the TI is the dose range within which a drug appears to be effective in half of the participants in a clinical trial without unacceptable adverse effects. It is generally considered that a drug has a good safety profile if its TI is over 10.

[0144] Many NTI drugs are known in the art. 50 FDA defines a drug as having a narrow therapeutic index when (a) the difference between the minimum toxic concentration (MTC) and the median effective dose (ED50) is less than two-fold, or (b) the difference between the minimum toxic concentration (MTC) and the minimum effective concentration (MEC) in the blood is less than two-fold, and (c) the safe and effective use of the drug requires careful titration and patient monitoring.

[0145] In one aspect, provided herein is a method for treating at least one symptom of ALS in a subject, comprising: (a) administering an effective amount of a composition comprising about 1 g of TURSO and about 3 g of sodium phenylbutyrate to a subject who has received a first dose of a narrow therapeutic index (NTI) drug; (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 a second dose of the NTI drug to the subject, wherein the second dose is lower than the first dose. The method may further comprise step (d), determining or having determined a second level of the NTI drug or a metabolite thereof in a second biological sample from the subject. Exemplary NTI drugs include, but are not limited to, mexiletine, alfentanil, quinidine, cyclosporine, warfarin, and digoxin.

[0146] In some embodiments, the NTI drug is digoxin. Digoxin is a cardiac glycoside used for the treatment of mild to moderate heart failure and for ventricular reaction rate control in chronic atrial fibrillation. The subject may have received 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 0.1-0.4 mg or about 0.375-0.5 mg / day). The second dose of digoxin can be less than the first dose by 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).

[0147] Metabolites of digoxin are known in the art. Exemplary metabolites include dihydrodigoxin and digoxigenin bisdigitoxoside.

[0148] In addition to or in lieu of determining the first and / or second levels of the NTI drug or its metabolites in a biological sample from the subject, other methods of monitoring the subject's response to the first dose of the NTI drug are contemplated herein. For example, known adverse events, side effects, or overdose symptoms associated with the NTI can be monitored. For example, symptoms of digoxin overdose include nausea, vomiting, vision changes, and cardiac arrhythmias.

[0149] Cytochrome P450 and substrates

[0150] Cytochrome P450 (CYP) is a superfamily enzyme containing heme as a cofactor, and works as a monooxygenase. CYP and CYP450 are used interchangeably herein. CYP is the main membrane-associated protein located in the inner membrane of mitochondria or the endoplasmic reticulum of cells. These proteins are present in most tissues of health, and play an important role in the removal, hormone synthesis and decomposition (synthesis and metabolism of estrogen and testosterone), cholesterol synthesis and vitamin D metabolism of various compounds. CYP also plays the function of metabolizing potential toxic compounds, which include drugs mainly in the liver and the product of endogenous metabolism, such as bilirubin.

[0151] CYPs are the main enzymes involved in drug metabolism. Effects on CYP activity are a major source of adverse drug interactions because changes in CYP enzyme activity may affect the metabolism and elimination of various drugs. For example, if one drug inhibits the CYP-mediated metabolism of another drug, the second drug may accumulate in the body to toxic levels.

[0152] Cytochrome P450 is encoded by CYP genes. Humans have 18 cytochrome P450 gene families, including CYP1 (CYP1A1, CYP1A2, CYP1B1, CYP1D1P), CYP2 (CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2R1, CYP2S1, CYP2 U1, CYP2W1), CYP3 (CYP3A4, CYP3A5, CYP3A7, CYP3A43, CYP3A51P, CYP3A52P, CYP3A54P, CYP3A137P), CYP4(CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4V2, CYP4 X1, CYP4Z1, CYP4F3A, CYP4F3B), CYP5(CYP5A1), CYP7(CYP7A1, CYP7B1), CYP8(CYP8A1, CYP8B1), CYP 11(CYP11A1, CYP11B1, CYP11B2), CYP17(CYP17A1), CYP19(CYP19A1), CYP20(CYP20A1), CYP21(CYP21 A2, CYP21A1P), CYP24 (CYP24A1), CYP26 (CYP26A1, CYP26B1, CYP26C1), CYP27 (CYP27A1, CYP27B1, CYP 27C1), CYP39 (CYP39A1), CYP46 (CYP46A1, CYP46A4P) and CYP51 (CYP51A1, CYP51P1, CYP51P2, CYP51P3).

[0153] The combination of bile acid and phenylbutyric acid compound may inhibit one or more CYP (e.g., any CYP 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 inhibitor of CYP or a moderate inhibitor of CYP. According to the FDA definition, a strong CYP inhibitor is expected to increase the AUC of a sensitive index substrate of a CYP by 5 times or more. A moderate CYP inhibitor is expected to increase the AUC of a sensitive index substrate of a CYP by 2 to 5 times (including 2 and 5 times).

[0154] Examples of clinically sensitive or moderately sensitive index substrates for exemplary CYPs are shown below.

[0155]

[0156] Exponential substrates predictably show increased exposure due to inhibition or induction of a given metabolic pathway, and exponential substrates are commonly used in prospective clinical DDI studies. A sensitive exponential substrate is an index drug that demonstrates an increase of greater than or equal to 5 times the AUC of a strong exponential inhibitor of a given metabolic pathway in a clinical DDI study. A moderately sensitive exponential substrate is a drug that demonstrates an increase of greater than or equal to 2 to less than 5 times the AUC of a strong exponential inhibitor of a given metabolic pathway in a clinical DDI study. Exemplary strong exponential 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).

[0157] When administered to a subject simultaneously with a CYP substrate (e.g., CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, or CYP3A (e.g., CYP3A4)), a composition comprising TURSO and sodium phenylbutyrate may result in an increase in the level (e.g., plasma concentration) of the substrate in the subject. For example, when administered to a subject simultaneously with a substrate for CYP2B6 or CYP2C8, a composition comprising TURSO and sodium phenylbutyrate may result in an increase in the level of the substrate in the subject. In some cases, a metabolite of TURSO or sodium phenylbutyrate has an inhibitory effect on one of the multiple CYPs. For example, phenylacetoacetate, a metabolite of phenylbutyric acid, may have an inhibitory effect on CYP1A2 and / or CYP2D6.

[0158] A substrate for a CYP can be a drug or other substance (e.g., a xenobiotic) that is metabolized by a CYP. A substrate for a CYP can be a pharmacologically active drug that needs to be metabolized to an inactive form to be eliminated from the subject's body, or a metabolically activated drug (e.g., a prodrug) that needs to be converted into an active drug.

[0159] Many substrates of CYP are known in the art. Exemplary substrates of CYP1A2 include alosetron, caffeine, duloxetine, melatonin, ramelteon, tasimelteon, tizanidine, clozapine, pirfenidone, ramosetron and theophylline. Exemplary substrates of CYP2B6 include bupropion and efavirenz. Exemplary substrates of CYP2C8 include repaglinide, montelukast, pioglitazone and rosiglitazone. Exemplary substrates of CYP2C9 include celecoxib, glimepiride, phenytoin, warfarin, tolbutamide and warfarin. Exemplary substrates of CYP2C19 include S-mephenytoin, omeprazole, diazepam, lansoprazole, rabeprazole or voriconazole. Exemplary substrates of CYP2D6 include atomoxetine, desipramine, dextromethorphan, eliglukast, nebivolol, nortriptyline, perphenazine, tolterodine, R-venlafaxine, encainide, imipramine, metoprolol, propafenone, propranolol, tramadol, trimipramine, and S-venlafaxine. Exemplary 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, iplerenone, felodipine, indinavir, lurasidone, maraviroc, quetiapine, sildenafil, ticagrelor, tolvaptan, alprazolam, aprepitant, atorvastatin, colchicine, eliglokast, pimozide, rilpivirine, rivaroxaban, or tadalafil. Exemplary substrates of CYP3A4 include alfentanil, cyclosporine, and quinidine.

[0160] Methods for identifying CYP substrates are known in the art. For example, cytochrome P450 reaction-typing can be used (see Zhang et al. Expert Opin Drug Metab Toxicol. 2007 Oct; 3(5): 667-87).

[0161] Provided herein is a method for treating at least one symptom of ALS in a subject, the method comprising (a) administering a composition (e.g., an effective amount of a composition) comprising about TURSO and about sodium phenylbutyrate to a subject who has received a first dose of a CYP substrate, (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 a second dose of the substrate to the subject, wherein the second dose is lower than the first dose. For example, the composition may include about 1 g of TURSO and 3 g of sodium phenylbutyrate. The method may also include step (d), 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 is lower than the first level.

[0162] In some embodiments of any of the methods described herein, CYP is CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 or CYP3A. The substrate of CYP can be a substrate of CYP1A2, such as mexiletine. The substrate of CYP can be a substrate of CYP3A4, such as alfentanil, cyclosporine and quinidine. The substrate of CYP can be a substrate of CYP2C9, such as warfarin. The substrate of CYP can be a substrate of CYP2D6, such as mexiletine. In some embodiments, CYP is CYP2B6 or CYP2C8. The substrate of CYP2B6 can include bupropion or efavirenz. The substrate of CYP2C8 can include repaglinide, montelukast, pioglitazone or rosiglitazone.

[0163] CYP substrates can also be NTI drugs. For example, mexiletine, alfentanil, quinidine, cyclosporine, warfarin are CYP substrates with narrow therapeutic index.

[0164] In some embodiments, the substrate of CYP is mexiletine. Mexiletine can be used to treat ventricular arrhythmias, organ transplant rejection, myotonia, renal impairment or liver impairment. Mexiletine can be metabolized by CYP2D6 and CYP1A2. The subject may have received a first dose of mexiletine 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 or about 900 mg / day). The second dose of mexiletine can be less than the first dose by 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).

[0165] In some embodiments, the substrate of CYP is alfentanil. Alfentanil is a short-acting opioid anesthetic and analgesic derivative of fentanyl. Alfentanil can be used as an anesthetic during surgery, for supplemental analgesia during surgery, and can also be used 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. An exemplary first dose of alfentanil (e.g., an induction or maintenance dose) is shown below.

[0166]

[0167] In some embodiments, the substrate of CYP 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. For oral dosage forms, the first dose of quinidine may be about 200 to about 650 mg 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 less than the first dose by about 10 mg to about 600 mg per administration (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 per administration). The frequency of the second dose of quinidine can also be lower than the quinidine of the first dose (for example, less number of times per day). For injection dosage forms, the first dose of quinidine may be injected into muscle about 190 to about 380 mg every 2 to 4 hours, or up to 0.25 mg / kg body weight per minute in the solution injected into the vein. Each time it is used, the second dose of quinidine can be less than the first dose by about 10 mg to about 350 mg (for example, less about 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280 or 300 mg). If the subject is a child, for oral dosage forms, the first dose of quinidine may be about 30 to about 40 mg / kg body weight per day.

[0168] In some embodiments, the substrate of CYP is cyclosporine. Cyclosporine can be metabolized by CYP3A4 and CYP3A5. Cyclosporine can be used to prevent organ rejection in allogeneic kidney, liver and heart transplants, or to prevent bone marrow transplant rejection. Cyclosporine is used to treat patients with severe active rheumatoid arthritis (RA) or severe, refractory, plaque psoriasis. The ophthalmic solution of cyclosporine shows that it can increase the tear secretion of patients with keratoconjunctivitis sicca. In addition, cyclosporine is approved for the treatment of steroid-dependent and steroid-resistant nephrotic syndrome caused by glomerular diseases, which may include minimal change nephropathy, focal and segmental glomerulosclerosis or membranous glomerulonephritis. Cyclosporine is also commonly used to treat various autoimmune and inflammatory diseases, such as atopic dermatitis, blistering diseases, ulcerative colitis, juvenile rheumatoid arthritis, uveitis, connective tissue diseases and idiopathic thrombocytopenic purpura. The subject may have received a first dose of cyclosporine of about 0.5 to about 15 mg / kg / day body weight (e.g., about 0.5 to about 5 mg / kg / day, about 1 to about 4 mg / kg / day, about 2.5 mg / kg / day, or about 12 to about 15 mg / kg / day). The second dose of cyclosporine may be about 0.1 to about 14 mg / kg / day less than the first dose (e.g., about 0.5 to about 2.5 mg / kg / day, about 1 to about 5 mg / kg / day).

[0169] In some embodiments, the substrate of CYP is warfarin.Warfarin can be used as an anticoagulant to prevent thrombus (e.g., deep vein thrombosis and pulmonary embolism), and can be used to prevent a stroke in a subject with atrial fibrillation, valvular heart disease or an artificial heart valve.Warfarin can be metabolized by CYP2C9, CYP1A2 and CYP3A.The subject may have received a first dose of warfarin of about 0.5 to about 12 mg / day (e.g., about 2 to about 6 mg / day, about 6 to about 8 mg or about 7 to about 10 mg / day).The second dose of warfarin can be less than the first dose of warfarin by 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).

[0170] Metabolites of CYP substrates described herein are known in the art. For example, metabolites of mexiletine include, but are not limited to, para-hydroxymexiletine, hydroxymethylmexiletine, N-hydroxymexiletine, and N-methylmexiletine. Exemplary metabolites of alfentanil include noralfentanil and N-phenylpropionamide. Exemplary metabolites of quinidine include 3-hydroxyquinidine, 2'-oxoquinidinone, O-desmethylquinidine, and quinidine-N-oxide. Exemplary metabolites of cyclosporine include M1, M9, and M4N. Other examples of cyclosporine metabolites can be found in Yatscoff et al. Clin Biochem, Vol 24, pp. 23-35, 1991. Exemplary metabolites of R-warfarin include hydroxywarfarin, 10-hydroxywarfarin, and diastereoisomeric alcohols. S-warfarin can be metabolized to 7-hydroxywarfarin.

[0171] In addition to or in lieu of determining the first and / or second levels of a substrate or its metabolites in a biological sample from a subject, other methods of monitoring the subject's response to the substrate of the first dose are contemplated herein. For example, known adverse events, side effects, or overdose symptoms associated with the substrate may be monitored. Symptoms of mexiletine overdose may include nausea, hypotension, sinus bradycardia, paresthesia, seizures, bundle branch block, AV heart block, cardiac arrest, ventricular tachyarrhythmias, including ventricular fibrillation, cardiovascular failure, or coma. Symptoms of alfentanil overdose may include characteristic stiffness of skeletal muscle, cardiac and respiratory depression, and miosis. Symptoms of quinidine overdose include irregular heartbeat, diarrhea, vomiting, headache, tinnitus or hearing loss, visual changes (blurred vision or photosensitivity), or confusion. Symptoms of cyclosporine overdose may include hepatotoxicity and nephrotoxicity. Symptoms of warfarin overdose primarily involve bleeding (eg, blood in stool or urine, hematuria, menorrhagia, melena, ecchymoses, excessive bruising or persistent oozing from superficial injury, unexplained decrease in hemoglobin), which is a manifestation of excessive anticoagulation.

[0172] If the substrate of CYP is warfarin, the reaction of the subject can also be monitored by prothrombin time-international normalized ratio (PT-INR). PT-INR measures the time required for the subject's blood to form a clot and can be used to determine whether an appropriate dose of warfarin has been administered. Methods for monitoring the INR level of a subject are known in the art. A typical INR target range is 2.0 to 3.5 (e.g., 2.0 to 3.3, 2.0 to 3.0, 2.0 to 2.8, 2.0 to 2.6, 2.0 to 2.4, 2.0 to 2.2, 2.2 to 3.5, 2.2 to 3.3, 2.2 to 3.0, 2.2 to 2.8, 2.2 to 2.6, 2.2 to 2.4, 2.4 to 3.5, 2.4 to 3.3, 2.4 to 3.0, 2.4 to 2.8, 2.4 to 2.6, 2.6 to 3.5, 2.6 to 3.3, 2.6 to 3.0, 2.6 to 2.8, 2.8 to 3.5, 2.8 to 3.3, 2.8 to 3.0, 3.0 to 3.5, 3.0 to 3.3, or 3.3 to 3.5). The INR can be monitored daily, twice a week, weekly, every two weeks, every three weeks, or every four weeks.

[0173] The warfarin dosage can be adjusted so that the PT-INR blood test reaches the target range. When the subject starts or stops another drug, or when the subject's medical condition changes, the INR can be monitored more frequently when the dosage changes. When the dosage is stable, the monitoring frequency can be reduced. The typical monitoring frequency of stable administration is about once every four weeks or six weeks (e.g., every four weeks, every five weeks, or every six weeks).

[0174] In one aspect, provided herein 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 a composition) comprising TURSO and sodium phenylbutyrate to a subject who has received 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, wherein the second dose is lower than the first dose. For example, the composition may include 1 g of TURSO and 3 g of sodium phenylbutyrate.

[0175] 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 about 0.5 to about 9.5 mg / day less than the first dose of warfarin (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).

[0176] In some embodiments, step (b) includes measuring once a day or has measured the first blood INR level of experimenter.Can measure the first blood INR level of experimenter with any suitable frequency, for example, 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 higher (for example, 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 higher (for example, 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 any method described herein, the method further comprises step (d), 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.

[0177] Transporter

[0178] Transporters are membrane proteins involved in drug uptake or efflux, and are present in various tissues such as lymphocytes, intestines, liver, kidneys, placenta and central nervous system. Transporters can have a significant impact on the pharmacokinetics of endogenous compounds (such as ions, vitamins or amino acids) and exogenous compounds. Co-administered drugs or nutrients can affect transporter activity, which may lead to changes in the pharmacokinetics of the drug, and therefore may lead to reduced efficacy or increased toxicity (e.g., drug-drug or drug-nutrient interactions). Exemplary 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 efflux proteins 1 and 2K (MATE1 and MATE2K).

[0179] Transporters can be divided into (i) efflux transporters belonging to the ATP binding component (ABC) family and (ii) uptake transporters belonging to the solute carrier (SLC) family, which mediate influx or bidirectional movement of 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 across the basolateral membrane into the brain. Exemplary uptake transporters include H+ / ditripeptide transporters, organic anion transporting polypeptides (OATPs, such as OATP1B1 or OATP1B3), organic anion transporters (e.g., OAT1 or OAT3), and organic cation transporters (OCT) 1 and OCT2.

[0180] Efflux transporters pump compounds back into the blood as they cross the apical cell membrane (i.e., the blood side), and also pump compounds out of the cell into the brain on the basolateral side. Exemplary efflux transporters include P-glycoprotein (P-gp, ABCB1), multidrug resistance-associated proteins (MRP) 1 and MRP2, and breast cancer resistance protein (BCRP, ABCG2).

[0181] Applicants have discovered that the combination of bile acids or pharmaceutically acceptable salts thereof with phenylbutyric acid compounds can inhibit one or more transporters (e.g., any transporter described herein or known in the art). For example, the combination of TURSO and sodium phenylbutyrate can inhibit one or more transporters (e.g., OAT1). Therefore, applicants disclose herein a method for treating at least one symptom of ALS in a subject who has received a first dose of a substrate for a transporter, the method comprising administering to the subject a composition comprising about 1 g of TURSO and about 3 g of sodium phenylbutyrate, monitoring the subject's response to the substrate, and administering a second dose of the substrate, wherein the second dose is less than the first dose. Monitoring the subject's response to the substrate may include determining or having determined the level of the substrate or its metabolite in a biological sample from the subject, or monitoring known adverse events, symptoms of overdose, or side effects associated with the substrate.

[0182] Substrates of transport proteins are known in the art. Exemplary substrates of P-gp include digoxin, fexofenadine, loperamide, quinidine, talinolol, and vinblastine. Exemplary substrates of BCRP include 2-amino-1-methyl-6-phenylimidazole [4,5-b] pyridine (PhIP), coumestrol, daidzein, dantrolene, estrone-3-sulfate, genistein, prazosin, and sulfasalazine. Exemplary substrates of OATP1B1 or OATP1B3 include cholecystokinin octapeptide (CCK-8), estradiol-17β-glucuronide, estrone-3-sulfate, pitavastatin, pravastatin, telmisartan, and rosuvastatin. Exemplary substrates of OAT1 include penicillin, nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., diclofenac, ketoprofen or methotrexate), HIV protease inhibitors and antiviral drugs (e.g., adefovir, cidofovir and tenofovir). Exemplary substrates of OAT3 include benzylpenicillin, estrone-3-sulfate, methotrexate and pravastatin. Exemplary substrates of MATE1 or MATE-2K include metformin, 1-methyl-4-phenylpyridinium (MPP+) and tetraethylammonium (TEA). Exemplary substrates of OCT2 include metformin, 1-methyl-4-phenylpyridinium (MPP+) and tetraethylammonium.

[0183] In one aspect, provided herein is a method for treating at least one symptom of ALS in a subject, the method comprising: (a) administering a composition (e.g., an effective amount of a composition) comprising TURSO and sodium phenylbutyrate to a subject who has received a first dose of a substrate for an 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, wherein the second dose is lower than the first dose. For example, the composition may comprise about 1 g of TURSO and about 3 g of sodium phenylbutyrate. The method may also comprise step (d), determining or having determined a second level of the substrate in a second biological sample from the subject. In some embodiments, the second level of the substrate is lower than the first level.

[0184] Exemplary substrates of OAT1 include penicillins, nonsteroidal anti-inflammatory drugs (NSAIDs) (eg, diclofenac, ketoprofen, or methotrexate), HIV protease inhibitors, and antiviral drugs (eg, adefovir, cidofovir, and tenofovir).

[0185] Administration of TURSO and sodium phenylbutyrate

[0186] The methods described herein include administering bile acid or a pharmaceutically acceptable salt thereof and a phenylbutyric acid compound to a subject. Bile acid or a pharmaceutically acceptable salt thereof and a phenylbutyric acid compound can be administered alone or simultaneously, including administration as part of a treatment regimen. The compound can be administered daily (e.g., once a day, twice a day, or three times a day or more), weekly, monthly, or quarterly. The compound can be administered over a period of weeks, months, or years. For example, the compound can be administered over a period of at least or about 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, or at least or about 5 years or longer. The compound can be administered once a day or twice a day for 60 days or less (e.g., 55 days, 50 days, 45 days, 40 days, 35 days, 30 days, or less). Alternatively, the bile acid and phenylbutyrate compounds can be administered once a day or twice a day for more than 60 days (e.g., more than 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 180, 200, 250, 300, 400, 500, 600 days).

[0187] 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 can be administered in an amount of about 0.5 to about 5 g per day (e.g., about 0.5 to about 4.5, about 0.5 to about 4, about 0.5 to about 3.5, about 0.5 to about 3, about 0.5 to about 2.5, about 0.5 to about 2, about 0.5 to about 1.5, about 0.5 to about 1, about 1 to about 5, about 1 to about 4.5, about 1 to about 4, about 1 to about 3.5, about 1 to about 3, about 1 to about 2.5, about 1 to about 2, about 1 to about 1.5, about 1.5 to about 5, about 1.5 to about 4.5, about 1.5 to about 4, about 1.5 to about 3.5, about 1 .5 to about 3, about 1.5 to about 2.5, about 1.5 to about 2, about 2 to about 5, about 2 to about 4.5, about 2 to about 4, about 2 to about 3.5, about 2 to about 3, about 2 to about 2.5, about 2.5 to about 5, about 2.5 to about 4.5, about 2.5 to about 4, about 2.5 to about 3.5, about 2.5 to about 3, about 3 to about 5, about 3 to about 4.5, about 3 to about 4, about 3 to about 3.5, about 3.5 to about 5, about 3.5 to about 4.5, about 3.5 to about 4, about 4 to about 5, about 4 to about 4.5, or about 4.5 to about 5 g) is administered. In some embodiments, TURSO is administered in an amount of about 1 to about 2 g per day, including values ​​therebetween (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 1.2 g, about 1.2 to about 2.0 g, about 1.2 to about 1.8 g, about 1.2 to 1.6 g, about 1.2 to about 1.4 g, about 1.4 to about 2.0 g, about 1.4 to 1.8 g, about 1.4 to 1.6 g, about 1.6 to 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 in an amount of about 1 g per day. In some embodiments, TURSO is administered in an amount of about 2 g per day. For example, TURSO can be administered twice a day in an amount of about 1 g.

[0188] Sodium phenylbutyrate can be taken in an amount of about 0.5 to about 10 g per day (e.g., about 1 to about 10, about 1 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 1 to about 2, about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, about 2.5 to about 9.5, about 2.5 to about 8.5, about 2.5 to about 7.5, about 2.5 to about 6.5, about 2.5 to about 4.5, about 2.5 to about 6.5, about 2.5 to about 8.5, about 2.5 to about 6.5, about 2.5 to about 4.5, about 2.5 to about 6 ...10. 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) is administered. In some embodiments, sodium phenylbutyrate is administered in an amount of about 3 to about 6 g per day, including values ​​therebetween (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 in an amount of about 3 g per day. In some embodiments, sodium phenylbutyrate is administered in an amount of about 6 g per day. For example, sodium phenylbutyrate can be administered twice daily in an amount of about 3 g. 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 (eg, about 3:1).

[0189] The methods described herein may include administering about 1 g of TURSO once a day and about 3 g of sodium phenylbutyrate once a day, or administering about 1 g of TURSO twice a day and about 3 g of sodium phenylbutyrate twice a day. The methods may 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 a 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 TURSO once daily and about 3 g sodium phenylbutyrate once daily for 14-21 days, followed by administering about 1 g TURSO twice daily and about 3 g sodium phenylbutyrate twice daily.

[0190] In some embodiments, the methods described herein comprise administering to a subject about 10 mg / kg to about 50 mg / kg per day (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, about 10 mg / kg to about 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 14 mg / kg, about 10 mg / kg to about 12 mg / kg, about 12 mg / kg to about 50 mg / kg, about 12 mg / kg to about 48 mg / kg, about 12 mg / kg to about 46 mg / kg, about 12 mg / kg to about 44 mg / kg, about 12 mg / kg to about 42 mg / kg, about 12 mg / kg to about 40 mg / kg, about 12 mg / kg to about g / kg to about 38mg / kg, about 12mg / kg to about 36mg / kg, about 12mg / kg to about 34mg / kg, about 12mg / kg to about 32mg / kg, about 12mg / kg to about 30mg / kg, about 12mg / kg to about 28mg / kg, about 12mg / kg to about 26mg / kg, about 12mg / kg to about 24mg / kg, about 12mg / kg to about 22mg / kg, about 12mg / kg to about 20mg / kg, about 12mg / kg to about 18mg / kg, about 12mg / kg to about 16mg / kg, about 12mg / kg to about 14mg / kg, about 14mg / kg to about 50mg / kg, about 1 4mg / kg to about 48mg / kg, about 14mg / kg to about 46mg / kg, about 14mg / kg to about 44mg / kg, about 14mg / kg to about 42mg / kg, about 14mg / kg to about 40mg / kg, about 14mg / kg to about 38mg / kg, about 14mg / 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 14mg / kg to about 22mg / 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 g / 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 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 g / kg to about 40mg / kg, about 20mg / kg to about 38mg / kg, about 20mg / kg to about 36mg / kg, about 20mg / kg to about 34mg / kg, about 20mg / kg to about 32mg / kg, about 20mg / kg to about 30mg / kg, about 20mg / kg to about 28mg / kg, about 20mg / kg to about 26mg / kg, about 20mg / kg to about 24mg / kg, about 20mg / kg to about 22mg / kg, about 22mg / kg to about 50mg / kg, about 22mg / kg to about 48mg / kg, about 22mg / kg to about 46mg / kg, about 22mg / kg to about 44mg / 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 g / 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 about 42mg / kg, about 26mg / kg to about 40mg / kg, about 26mg / kg to about 38mg / kg, about 26mg / kg to about 36mg / kg, about 26mg / kg to about 34mg / kg, about 26mg / kg to about 32mg / kg, about 26mg / kg to about 30mg / kg, about 26mg / kg to about 28mg / kg, about 28mg / kg to about 50mg / kg, about 28mg / kg to about 48mg / kg, about 28mg / kg to about 46mg / kg, about 28mg / kg to about 44mg / kg, about 28mg / kg to about 42mg / kg, about 28mg / kg to about 40mg / kg, about 28m g / kg to about 38mg / kg, about 28mg / kg to about 36mg / kg, about 28mg / kg to about 34mg / kg, about 28mg / kg to about 32mg / kg, about 28mg / kg to about 30mg / kg, about 30mg / kg to about 50mg / kg, about 30mg / kg to about 48mg / kg, about 30mg / kg to about 46mg / kg, about 30mg / kg to about 44mg / kg, about 30mg / kg to about 42mg / kg, about 30mg / kg to about 40mg / kg, about 30mg / kg to about 38mg / kg, about 30mg / kg to about 36mg / kg, about 30mg / kg to about 34mg / kg,about 30 mg / kg to about 32 mg / kg, about 32 mg / kg to about 50 mg / kg, about 32 mg / kg to about 48 mg / kg, about 32 mg / kg to about 46 mg / kg, about 32 mg / kg to about 44 mg / kg, about 32 mg / kg to about 42 mg / kg, about 32 mg / kg to about 40 mg / kg, about 32 mg / kg to about 38 mg / kg, about 32 mg / kg to about 36 mg / kg, about 32 mg / kg to about 34 mg / kg, about 34 mg / kg to about 50 mg / kg, about 34 mg / kg to about 4 8mg / kg, about 34mg / kg to about 46mg / kg, about 34mg / kg to about 44mg / kg, about 34mg / kg to about 42mg / kg, about 34mg / kg to about 40mg / kg, about 34mg / kg to about 38mg / kg, about 34mg / kg to about 36mg / kg, about 36mg / kg to about 50mg / kg, about 36mg / kg to about 48mg / kg, about 36mg / kg to about 46mg / kg, about 36mg / kg to about 44mg / kg, about 36mg / kg to about 42mg / kg, about 36m g / kg to about 40mg / kg, about 36mg / kg to about 38mg / kg, about 38mg / kg to about 50mg / kg, about 38mg / kg to about 48mg / kg, about 38mg / kg to about 46mg / kg, about 38mg / kg to about 44mg / kg, about 38mg / kg to about 42mg / kg, about 38mg / kg to about 40mg / kg, about 40mg / kg to about 50mg / kg, about 40mg / kg to about 48mg / kg, about 40mg / kg to about 46mg / kg, about 40mg / kg to about 44mg / kg kg, 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 per kg body weight.

[0191] In some embodiments, the methods described herein comprise administering to a subject about 10 mg / kg to about 400 mg / kg per day (e.g., about 10 mg / kg to about 380 mg / kg, about 10 mg / kg to about 360 mg / kg, about 10 mg / kg to about 340 mg / kg, about 10 mg / kg to about 320 mg / kg, about 10 mg / kg to about 300 mg / kg, about 10 mg / kg to about 280 mg / kg, about 10 mg / kg to about 260 mg / kg, about 10 mg / kg to about 240 mg / kg, about 10 mg / kg to about 220 mg / kg, about 10 mg / kg to about 200 mg / kg, about 10 mg / kg to about 180 mg / kg, about 10 mg / kg to about 160 mg / kg, about 10 mg / kg to about 140 mg / kg, about 10 mg / kg to about 120 mg / kg, about 10 mg / kg to about 100 mg / kg, about 10 mg / kg to about 80 mg / kg, about 10 mg / kg to about 60 mg / 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 320 mg / kg / kg, about 20 mg / kg to about 300 mg / kg, about 20 mg / kg to about 280 mg / kg, about 20 mg / kg to about 260 mg / kg, about 20 mg / kg to about 240 mg / kg, about 20 mg / kg to about 220 mg / kg, about 20 mg / kg to about 200 mg / kg, about 20 mg / kg to about 180 mg / kg, about 20 mg / kg to about 160 mg / kg, about 20 mg / kg to about 140 mg / kg, about 20 mg / kg to about 120 mg / kg, about 20 mg / kg to about 100 mg / kg, about 20 mg / kg to about 80 mg / kg, about 20 mg / kg to about 60 mg / kg g, about 20 mg / kg to about 40 mg / kg, about 40 mg / kg to about 400 mg / kg, about 40 mg / kg to about 380 mg / kg, about 40 mg / kg to about 360 mg / kg, about 40 mg / kg to about 340 mg / kg, about 40 mg / kg to about 320 mg / kg, about 40 mg / kg to about 300 mg / kg, about 40 mg / kg to about 280 mg / kg, about 40 mg / kg to about 260 mg / kg, about 40 mg / kg to about 240 mg / kg, about 40 mg / kg to about 220 mg / kg, about 40 mg / kg to about 200 mg / kg, about 40 mg / kg to about 180 mg / 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 g / kg to about 260mg / kg, about 60mg / kg to about 240mg / kg, about 60mg / kg to about 220mg / kg, about 60mg / kg to about 200mg / kg, about 60mg / kg to about 180mg / kg, about 60mg / kg to about 160mg / kg, about 60mg / kg to about 140mg / kg, about 60mg / kg to about 120mg / kg, about 60mg / kg to about 100mg / kg, about 60mg / kg to about 80mg / kg, about 80mg / kg to about 400mg / kg, about 80mg / kg to about 380mg / kg, about 80mg / kg to about 360mg / kg, about 80mg / 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 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 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 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 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 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,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 per body weight.

[0192] In some embodiments, TURSO is administered in an amount of 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 in an amount of 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.

[0193] The methods described herein can be used to treat or improve at least one symptom of ALS in a subject, slow down ALS disease progression, increase the survival time of a subject with one or more ALS symptoms, prevent or reduce at least one adverse event (e.g., severe adverse events) associated with ALS or its treatment, and reduce the decline of muscle strength, respiratory muscles / lung function, and / or fine motor skills, maintain or improve muscle strength, respiratory muscles / lung function, and / or fine motor skills. The methods can also be used for preventive treatment of subjects at risk of developing ALS (e.g., subjects with a family history of ALS) or subjects suspected of developing ALS (e.g., subjects showing at least one ALS symptom, upper motor neuron degeneration symptom, and / or lower motor neuron degeneration symptom, but the symptoms at the time were not sufficient to support a comprehensive diagnosis of ALS). The methods are useful for improving the symptoms of at least one lower motor neuron degeneration or upper motor neuron degeneration.

[0194] The methods disclosed herein are also useful for preventing or reducing constipation (eg, constipation associated with ALS), and ameliorating at least one symptom of benign fasciculation syndrome or spastic fasciculation syndrome.

[0195] As disclosed herein, the methods can be used to treat a subject diagnosed with ALS, at risk of developing ALS, or suspected of having ALS. The subject may, for example, have been diagnosed with ALS for 24 months or less (e.g., any subrange within the ranges described herein). For example, the subject may have been diagnosed with ALS for 1 week or less, or on the same day of administering a currently disclosed treatment. The subject may have displayed one or more ALS symptoms for 24 months or less (e.g., any subrange within the range described herein), an ALS disease progression rate (ΔFS) of about 0.50 or more in the past 3-12 months (e.g., any subrange within the range described herein), an ALSFRS-R score of 40 or less in the past 3-12 months (e.g., any subrange within the range described herein), an average monthly loss of about 0.8 to about 2 ALSFRS-R points in the past 3-12 months (e.g., any subrange within the range described herein), and the subject has a chromosomal marker selected from SOD1, C9ORF72, ANG, TARDBP, VCP, VAPB, SQSTM1, DCTN1, FUS, UNC13A, ATXN2, HNRNPA1, CHCHD10, MOBP, C21ORF2, NEK1, TUBA4A, TBK1, MATR3, PFN1, UBQLN2, TAF15, , OPTN and TDP-43, and / or having a level 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, 1800, 185 ... 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 CSF pNF-H level of the subject 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 serum NfL level of the subject in the methods described herein can be about 50 to about 300 pg / mL (e.g., any subrange within the range described herein). In some embodiments, a subject in the methods described herein may have a CSF NfL level of about 2000 to about 40,000 pg / mL (eg, any subrange within the ranges described herein).

[0198] The methods described in the present disclosure may include treatment of ALS itself, as well as treatment of one or more symptoms of ALS. "Treating" ALS does not require 100% elimination of the disease or disease symptoms in the subject. Any alleviation or reduction in the severity of the symptoms or characteristics of the disease is contemplated. "Treating" ALS also refers to a delay in the onset of symptoms (e.g., in preventive treatment) or a delay in 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., those caused by any therapeutic agent disclosed herein or known in the art for treating ALS). "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. The subject may not show signs of ALS, but may be at risk of ALS. For example, the subject may carry a genetic mutation associated with ALS, have a family history of ALS, or have elevated biomarker levels suggesting a risk of developing ALS. The subject may show early signs of disease or show symptoms of a determined or progressive disease. The present disclosure contemplates any degree of delay in symptom onset, alleviation of one or more symptoms of a disease, or delay in progression of any one or more disease symptoms (e.g., any improvement as measured by the ALSFRS-R, or maintenance of the ALSFRS-R score (signaling delayed disease progression)). Also contemplated herein is any alleviation or reduction in severity of a symptom or feature of benign fasciculation syndrome or spasticity-fasciculation syndrome.

[0199] Treatment provided in the present disclosure can be started at any stage during the progression of the disease. For example, treatment can be started before the onset of disease (e.g., for subjects with a risk of developing ALS), at the onset of symptoms, or immediately after the detection of ALS symptoms, when any one or more symptoms (e.g., muscle weakness, fasciculations, and / or muscle spasms) that would cause a skilled practitioner to suspect that the subject may be developing ALS are observed. Treatment can also be started at a later stage. For example, treatment can be started at the progression stage of the disease, for example, when muscle weakness and atrophy spread to different parts of the body and the subject already has increased movement problems. At or before the start of treatment, the subject may suffer from muscle tightness and stiffness (spasm), excessive reflexes (hyperreflexia), muscle weakness and atrophy, muscle cramps, and / or short-term twitches (fasciculations) visible under the skin, difficulty swallowing (dysphagia), difficulty speaking or organizing speech (dysarthria).

[0200] The treatment method may include a single administration, multiple administrations, and repeated administrations required for preventing or treating ALS or at least one ALS symptom. The duration of the preventive treatment may be a single dose, or the treatment may continue (e.g., multiple doses) for example, for several years or indefinitely in the lifetime of the subject. For example, a subject at risk of ALS may be treated with the methods provided herein for several days, weeks, months, or even years to prevent the occurrence or outbreak of the disease. In some embodiments, the treatment method may include assessing the disease level in the subject before, during, and / or after treatment. The treatment provided herein may be administered once or more per day, or may also be administered weekly or monthly. In some embodiments, treatment may continue until a decrease in the disease level in the subject is detected. The methods provided herein may, in some embodiments, begin to show efficacy (e.g., reduction in one or more symptoms of ALS, improvement as measured by ALSFRS-R, or maintenance of ALSFRS-R score) 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).

[0201] The terms "administer", "administering" or "administration" as used herein refer to administering a drug described herein to a subject using any method known in the art (e.g., ingestion, injection, implantation, absorption or inhalation of the drug, regardless of the form). In some embodiments, one or more compounds disclosed herein can be administered to a subject by oral and / or topical (e.g., intranasal) ingestion. For example, the methods herein include the administration of an effective amount of a compound or a combination of compounds to achieve the desired or stated effect. The specific dosage and treatment regimen for any particular subject will depend on a variety of factors, including the activity of the specific compound used, age, weight, general health, sex, diet, time of administration, excretion rate, drug combination, severity and course of the disease, condition or symptoms, subject's treatment of the disease, condition or symptoms, and the judgment of the attending physician.

[0202] After administration of bile acid or pharmaceutically acceptable salt thereof and phenylbutyrate compound, the subject can be evaluated to detect, assess or determine their level of ALS disease. In some embodiments, treatment can continue until a change (e.g., decrease) in the level of disease in the subject is detected.

[0203] After the patient's condition improves (e.g., a change (e.g., a decrease) in the level of disease in the subject), a maintenance dose of a compound, composition, or combination of the present disclosure may be administered, if necessary. Subsequently, the dose or frequency of administration, or both, may be reduced, as a function of symptoms, to a level that maintains the improved condition. However, the patient may require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.

[0204] IV. Symptom and outcome measures

[0205] The present disclosure also provides methods for evaluating ALS symptoms, monitoring ALS progression, and evaluating the response of a subject to a treatment method. Non-limiting examples include physical evaluation by a physician, weight analysis, electrocardiogram (ECG) analysis, ALS Functional Rating Scale (ALSFRS or ALSFRS-R) score analysis, respiratory function analysis, muscle strength analysis, cognitive / behavioral function analysis, quality of life analysis, and speech analysis.

[0206] The respiratory function of the subject can be measured by, for example, vital capacity (including forced vital capacity and slow 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 by, for example, handheld dynamometer (HHD), hand grip strength dynamometry, manual muscle strength test (MMT), electrical resistance myography (EIM), maximum voluntary isometric contraction test (MVICT), motor unit number estimation (MUNE), accurate limb isometric strength test (ATLIS) or a combination thereof. Cognitive / behavioral function can be evaluated by, for example, ALS Depression Inventory (ADI-12) questionnaire, Beck Depression Inventory (Beck Depression Inventory) (BDI) questionnaire and Hospital Anxiety and Depression Scale (Hospital Anxiety Depression Scale) (HADS) questionnaire. Quality of life can be evaluated by, for example, ALS assessment questionnaire (ALSAQ-40). Akt levels, Akt phosphorylation and / or the pAktdAkt ratio can also be used to assess a subject's disease progression and response to treatment (see, eg, WO 2012 / 160563).

[0207] The level of biomarkers in the CSF or blood sample of the subject is a useful indicator of the ALS progression of the subject and the responsiveness to the treatment methods provided herein. For example, but not limited to the biomarkers of phosphorylated neurofilament heavy chain (pNF-H), neurofilament medium chain, neurofilament light chain (NFL), S100-β, cystatin C, chitotriosidase, CRP, TDP-43, uric acid and some microRNA can be used for analysis for this purpose. Urinalysis can also be used to assess the response of the subject to treatment. The level of biomarkers (such as but not limited to p75ECD and ketone bodies) in the urine sample can be analyzed. The level of creatinine can be measured in urine and blood samples. In some embodiments, the method provided herein causes the ketone body level in the urine sample of the subject to increase or decrease. Medical imaging may also be utilized, including but not limited to MRI and PET imaging of markers such as translocation protein (TSPO).

[0208] Muscle strength

[0209] The muscle strength of the subject can be evaluated using known methods in the art. Quantitative strength measurement generally shows linear, predictable strength loss in ALS patients. Tufts quantitative neuromuscular examination (TQNE) can be used to provide quantitative measurement using fixed strain gauges. TQNE measures the isometric strength of 20 muscle groups and produces interval strength data in strong and weak muscles (see, for example, Andres et al., Neurology 36:937–941, 1986). Handheld dynamometer (HHD) tests the isometric strength of specific muscles of arms and legs, and produces interval level data (see, for example, Shefne JM, Neurotherapeutics 14:154–160, 2017).

[0210] The accurate test of limb isometric muscle strength (ATLIS) can be used to measure strong and weak muscle groups using fixed wireless load cells (e.g., Andres et al., Muscle Nerve 56 (4): 710-715, 2017). The strength of 12 muscle groups was evaluated in the ATLIS test, which reflects the strength of the subject's lower limbs, upper limbs, and the grip strength of the subject. In some embodiments, the ATLIS test detects changes in muscle strength before any functional changes are observed.

[0211] As evaluated by any suitable method described herein, the methods provided herein may improve, maintain or slow down the decline of subject's muscle strength (e.g., lower limb strength, upper limb strength or grip strength). The method may result in the improvement of subject's upper limb strength more significantly than other muscle groups. For example, the impact on muscle strength can be reflected in one or more muscle groups selected from quadriceps, biceps, hamstrings, triceps and tibialis anterior.

[0212] Muscle strength can be assessed by HHD, handgrip dynamometer, MMT, EIM, MVICT, MUNE, ATLIS, or a combination thereof before, during, and / or after administration of bile acid or a pharmaceutically acceptable salt thereof and a phenylbutyrate compound.

[0213] In some embodiments, muscle strength is assessed by ATLIS.Total ATLIS score and upper and lower limb ATLIS score can be assessed.The disclosed method can cause the total ATLIS score decline rate of the experimenter to be about 3.50PPN / month or lower (e.g., about 3.45, 3.40, 3.35, 3.30, 3.25, 3.20, 3.15, 3.10, 3.05, 3.00PPN / month or lower).Compared with the control subjects not receiving administration, the disclosed method can also cause the average decline rate of the total ATLIS score of the experimenter to be at least about 0.2PPN / month (e.g., at least about 0.25, 0.30, 0.35, 0.40, 0.45 or 0.50PPN / month). Compared with the control subjects who did not receive the administration described herein, the average rate of decline of the upper limb ATLIS score of the subject can be at least about 0.50PPN / month (e.g., at least about 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85 or 0.90PPN / month). Compared with the control subjects who did not receive the administration described herein, the average rate of decline of the lower limb ATLIS score of the subject can be at least about 0.20PPN / month (e.g., at least about 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55 or 0.60PPN / month). In some embodiments, the improvement or maintenance of the muscle strength of the subject may begin to occur less than 60 days (e.g., less than 55, 50, 45, 40, 30, 25 or 20 days) after the initial administration. PPN represents the percentage of normal strength based on the prediction of age, sex weight and height.

[0214] Lung function

[0215] ALS is a progressive neurodegenerative disease that eventually leads to respiratory failure and death. Pulmonary function tests such as vital capacity (VC), maximum mid-expiratory flow (MMEF), forced vital capacity (FVC), slow vital capacity (SVC) and forced expiratory volume in 1 second (FEV1) can be used to monitor ALS progression and / or the subject's response to treatment. On average, the rate of decline in respiratory function in ALS patients measured by vital capacity (VC) can be about 2.24% of the predicted value per month (± 6.9). In some embodiments, measurements from pulmonary function tests are associated with survival (see, for example, Moufavi et al. Iran J Neurol 13 (3): 131–137, 2014). Other measurements, such as maximum inspiratory and expiratory pressures, arterial blood gas measurements and overnight oxygen saturation, may provide early evidence of dysfunction. Comparison of upright and supine vital capacity may also provide early indications of ventilatory muscle weakness.

[0216] Methods provided herein may improve or maintain the respiratory muscles and / or lung function of a subject, or slow down the decline of the respiratory muscles and / or lung function of a subject. The respiratory muscles and / or lung function of a subject can be evaluated by any suitable method as described herein or other known in the art. In some embodiments, the respiratory muscle function of a human subject is assessed based on the SVC of the subject. In some embodiments of any method for improving, maintaining or slowing down the decline of respiratory muscle function in a human subject as described herein, the average rate of decline of the SVC of the subject resulting in the treatment is about 3.50PPN / month or lower (e.g., about 3.45, 3.40, 3.35, 3.30, 3.25, 3.20, 3.15, 3.10, 3.05 or 3.00PPN / month or lower). In some embodiments, the treatment reduces the average rate of decline of 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 who did not receive treatment. In some embodiments, the improvement or maintenance of the subject's pulmonary function may begin to occur less than 60 days (e.g., less than 55, 50, 45, 40, 30, 25, or 20 days) after the initial administration. In some embodiments, less than 60 days after the initial administration, the subject's pulmonary function progresses less than expected.

[0217] Adverse Events

[0218] Subjects treated with any of the methods provided herein may experience fewer adverse events (e.g., any adverse events disclosed herein), or experience one or more adverse events to a lesser extent than untreated control subjects. Exemplary adverse events include gastrointestinal-related adverse events (e.g., abdominal pain, gastritis, nausea and vomiting, constipation, rectal bleeding, peptic ulcer disease, and pancreatitis); hematological adverse events (e.g., aplastic anemia and ecchymosis); cardiovascular adverse events (e.g., arrhythmias and edema); renal adverse events (e.g., renal tubular acidosis); psychiatric adverse events (e.g., depression); skin adverse events (e.g., rash); and other adverse events (e.g., syncope and weight gain). In some embodiments, the methods provided herein do not result in constipation, neck pain, headache, falling, dry mouth, muscle weakness, falls, tearing, and elevated alanine aminotransferase (ALT), or result in mild symptoms of constipation, neck pain, headache, falling, dry mouth, muscle weakness, falls, tearing, and elevated alanine aminotransferase (ALT). 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.

[0219] In some embodiments, administration of a combination of a bile acid and a phenylbutyrate compound can result in fewer adverse events (eg, any adverse events disclosed herein) or less severe adverse events compared to administration of either a bile acid or a phenylbutyrate compound alone.

[0220] The average survival time of ALS patients may be different. The median survival time can be about 30 to about 32 months after symptom onset, or about 14 to about 20 months after diagnosis. The survival time of patients with bulbar onset ALS can be from about 6 months to 84 months after symptom onset, with a median of about 27 months. The methods provided herein may improve 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) in some embodiments. The methods provided herein may, in some embodiments, delay the onset of ventilator dependence 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).

[0221] The methods provided herein may reduce the rate of disease progression, wherein the average ALSFRS-R score loss per month in a subject is reduced by at least about 0.2 (e.g., at least about 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, or 1.5) compared to a control subject not receiving treatment. The methods provided herein may slow the progression of one or more categories of ALSFRS scale assessment, including: speech, drooling, swallowing, handwriting, cutting food and handling utensils, dressing and hygiene, turning over in bed and adjusting bedding, walking, climbing stairs, dyspnea, orthopnea, respiratory insufficiency. In some embodiments, the methods provided herein improve or slow the decline of a subject's fine motor function as assessed by one or more categories of the ALSFRS-R scale (e.g., handwriting, cutting food and handling utensils, or dressing and hygiene).

[0222] In some embodiments, the methods provided herein are more effective in treating subjects aged about 18 to about 50 years (e.g., about 18 to about 45 years, about 18 to about 40 years, about 18 to about 35 years, about 18 to about 30 years, about 18 to about 25 years, or about 18 to about 22 years) compared to subjects aged 50 years or older (e.g., 55, 60, 65, 70, 75, or 80 years or older). In some embodiments, the methods provided herein are more effective in treating subjects diagnosed with ALS and / or exhibiting 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 diagnosed with ALS and / or exhibiting 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 embodiments, the methods provided herein are more effective in treating subjects diagnosed with ALS and / or exhibiting ALS symptom onset 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 diagnosed with ALS and / or exhibiting 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).

[0223] In some embodiments, the responsiveness to the treatment methods provided herein is gender-dependent. Compared with male subjects, the methods provided herein can be more effective or less effective in treating female subjects. For example, when treated at similar stages of disease progression, female subjects may show improvement earlier or later than male subjects (e.g., measured by ALSFRS-R or any other outcome measurement described herein). When treated at similar stages of disease progression, female subjects may show greater or less improvement (e.g., measured by ALSFRS-R or any other outcome measurement described herein) than male subjects in some embodiments. The pharmacokinetics of bile acids and phenylbutyric acid compounds may be the same or different in female and male subjects.

[0224] V. Additional Therapeutic Agents

[0225] The methods described herein may include administering to the subject one or more additional therapeutic agents, e.g., in an amount effective for treating or achieving modulation of at least one symptom of ALS. Any known ALS therapeutic agent known in the art may be used as an additional therapeutic agent. Exemplary therapeutic agents include riluzole (C8H5F3N2OS, e.g., sold under the trade name and Edaravone (sold under the trade name and sold), dextromethorphan, anticholinergics, and psychotropics (e.g., antidepressants, antipsychotics, anxiolytics / hypnotics, mood stabilizers, and stimulants).

[0226] A combination of dextromethorphan and quinidine can be used to treat pseudobulbar emotions (inappropriate laughing or crying). Anticholinergics and antidepressants can be used to treat excessive salivation. Exemplary anticholinergic drugs include glycopyrrolate, scopolamine, atropine (Atropen), belladonna alkaloids, benztropine mesylate (Cogentin), clidinium, cyclopentolate (Cyclogyl), darifenacin (Enablex), dicylomine, fesoterodine (Toviaz), flavoxate (Urispas), glycopyrrolate, homatropine hydrobromide, hyoscyamine (Levsinex), ipratropium (Atrovent), orthophenadrine, oxybutynin (Ditropan XL), propantheline (Pro-banthine), scopolamine, methscopolamine, solifenacin (VESIcare), tiotropium (Spiriva), tolterodine (Detrol), trihexyphenidyl, trospium chloride, and diphenhydramine (Benadryl). Exemplary 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.

[0227] One or more additional therapeutic agents may be administered for a period of time before administering an initial dose of a composition comprising bile acid or a pharmaceutically acceptable salt thereof (e.g., TURSO) and a phenylbutyric acid compound (e.g., sodium phenylbutyrate), and / or for a period of time after administering a final dose of the composition. In some embodiments, the subject in the methods described herein has previously been treated with one or more additional therapeutic agents (e.g., any additional therapeutic agent described herein, such as riluzole and edaravone). In some embodiments, before administering the composition of the present disclosure, the subject has been administered a stable dose of one or more therapeutic agents (e.g., riluzole and / or edaravone) for at least 30 days (e.g., at least 40 days, 50 days, 60 days, 90 days, or 120 days). The absorption, metabolism, and / or excretion of one or more additional therapeutic agents may be affected by bile acid or a pharmaceutically acceptable salt thereof and / or a phenylbutyric acid compound. For example, the co-administration of sodium phenylbutyrate with riluzole or edaravone may increase the subject's exposure to riluzole or edaravone. Compared with the administration of riluzole alone, co-administration of riluzole with bile acid or a pharmaceutically acceptable salt thereof and a phenylbutyric acid compound can improve the tolerability of riluzole in a subject.

[0228] The combination of bile acid or a pharmaceutically acceptable salt thereof, a phenylbutyrate compound and one or more additional therapeutic agents can have a synergistic effect in treating ALS. When administered in combination with bile acid or a pharmaceutically acceptable salt thereof and a phenylbutyrate compound, a smaller dose of the additional therapeutic agent may be required to obtain the same pharmacological effect. In some embodiments, the amount of one or more additional therapeutic agents administered in combination with bile acid or a pharmaceutically acceptable salt thereof and a phenylbutyrate compound can 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 dose used when one or more additional therapeutic agents are administered alone. Additionally or alternatively, the methods of the present disclosure can reduce the frequency of administration of other therapeutic agents (e.g., other ALS therapeutic agents) required to obtain the same pharmacological effect.

[0229] Bile acid or its pharmaceutically acceptable salt and phenylbutyrate compound can be administered shortly after a meal (e.g., within two hours after a meal) or in a fasting state. The subject may have consumed food (e.g., solid food or liquid food) less than 2 hours before the administration of bile acid or its pharmaceutically acceptable salt and / or phenylbutyrate compound; or will consume food within less than 2 hours after taking one or both compounds. Food may affect the rate and extent of absorption of bile acid or its pharmaceutically acceptable salt and / or phenylbutyrate compound. For example, food can change the bioavailability of the compound by delaying gastric emptying, stimulating bile flow, changing gastrointestinal pH, increasing visceral blood flow, changing the intraluminal metabolism of the substance, or physically or chemically interacting with the dosage form or substance. The nutritional and caloric content of the meal, the volume of the meal, and the temperature of the meal can cause physiological changes in the gastrointestinal tract, thereby affecting drug transit time, luminal dissolution, drug permeability, and systemic availability. In general, meals with high total calorie and fat content are more likely to affect GI physiology, thereby having a greater impact on the bioavailability of the drug. The methods provided herein can also include administering a variety of foods to the subject, for example, less than 2 hours (e.g., less than 1.5 hours, 1 hour, or 0.5 hours) before or after administration of the bile acid or pharmaceutically acceptable salt thereof and / or phenylbutyrate compound. DETAILED DESCRIPTION

[0230] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not intended to limit the scope of the disclosure or the claims in any way.

[0231] Example 1 - Evaluation of the Inducing Potential of Sodium Phenylbutyrate and Tauroursodeoxycholic Acid on Cytochrome P450 Enzymes in Human Hepatocytes

[0232] The cytotoxicity and induction potential of CYP mRNA (CYP1A2, CYP2B6 and CYP3A4) by a combination of sodium phenylbutyrate (PB) and tauroursodeoxycholic acid (TUDCA) were evaluated in human hepatocytes.

[0233] Cytotoxicity

[0234] The cytotoxicity of the combination of sodium phenylbutyrate (PB) and tauroursodeoxycholic acid (TUDCA) was evaluated using human hepatocytes. Plateable and inducible cryopreserved human hepatocytes were thawed and isolated in human hepatocyte thawing medium. Cells were suspended in human hepatocyte plating medium, counted (cell viability assessed by trypan blue exclusion), and plated (day 0) at 750,000 cells / mL (150,000 cells / well in a 48-well plate) onto BioCoat plates. TMCollagen-coated 48-well plates (Corning Life Sciences, Cat. No. 354505, Tewksbury, MA) were placed on the plates and incubated in a 95% air / 5% CO2 incubator at 37°C. After attachment (4 hours), the culture medium was replaced with fresh hepatocyte culture medium for overnight cell recovery. Hepatocytes were then treated with hepatocyte culture medium reinforced with PB / TUDCA at five concentrations (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. The vehicle control was treated with a hepatocyte culture medium containing the same amount of solvent (1% MeOH for PB / TUDCA and 0.1% DMSO for chlorpromazine). Hepatocytes were incubated in a 95% air / 5% CO2 incubator at 37°C for three days (72 hours), with daily changes of hepatocyte culture medium containing PB / TUDCA, positive control, or vehicle. Cell viability was determined by analyzing the intracellular conversion of tetrazolium compounds [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] to formazan products by dehydrogenases (active only in living cells). CellTiter The AQueous One Solution Cell Proliferation Assay (MTS) measures the absorbance of formazan spectrophotometrically, which is proportional to the number of viable cells. Briefly, wells were rinsed with DPBS, and then hepatocyte culture medium (200 μL) and CellTiter AQueous-solution cell proliferation assay reagent (40 μL) was added and the cells were incubated at 37°C in a 95% air / 5% CO2 incubator for 1 hour. The absorbance of formazan in each well was measured at 492 nm using an OPTIMA microplate reader (BMG Lab Technologies, Durham, NC, USA).

[0235] Table 1 summarizes the cytotoxicity of PB / TUDCA and the positive control (100 μM chlorpromazine) in human hepatocytes (expressed as percentage of cell viability, MTS absorbance relative to vehicle control).

[0236] [Table 1] Cell viability after treatment with PB / TUDCA or positive control

[0237]

[0238] aAn MTS absorbance <75% of the vehicle control was considered a positive cytotoxicity result.

[0239] QC acceptance criteria: MTS absorbance of the excipient control ≤ 50% relative to the positive control.

[0240] The cytotoxicity of the combination of PB and TUDCA at five concentrations (PB / TUDCA, 14.8 / 1.62, 148 / 16.2, 826 / 108, 1480 / 1080 and 7400 / 1600 μM) was evaluated using a human hepatocyte donor. The results showed that PB / TUDCA was not cytotoxic at 14.8 / 1.62 μM and 148 / 16.2 μM, but showed cytotoxicity to the donor at 826 / 108 μM, 1480 / 1080 μM and 7400 / 1600 μM. Since the cytotoxicity results were critical at 826 / 108 μM PB / TUDCA, this combination of test concentrations was included in subsequent CYP induction tests (using different donors).

[0241] CYP induction

[0242] Thaw and isolate plateable and inducible cryopreserved human hepatocytes in human hepatocyte thawing medium. Resuspend cells in human hepatocyte plating medium, count (assess cell viability by trypan blue exclusion), and plate (day 0) at 750,000 cells / mL (150,000 cells / well in a 48-well plate) onto BioCoat TM The cells were plated on a collagen-coated 48-well plate (Corning Life Sciences, catalog #354505) and incubated in a 95% air / 5% CO2 incubator at 37°C. After attachment (4 hours), the medium was replaced with fresh hepatocyte medium for overnight cell recovery. The hepatocytes were then treated with PB / TUDCA-fortified hepatocyte medium at three concentrations (14.8 / 1.62, 148 / 16.2, and 826 / 108 μM, based on cytotoxicity test results).

[0243] Positive controls were treated in parallel with hepatocyte culture medium fortified with known inducers of each CYP of interest: 50 μM omeprazole (OME) for CYP1A2, 1000 μM phenobarbital for CYP2B6, and 50 μM rifampicin (RIF) for CYP3A4. Negative controls were treated with 10 μM flumazenil, and vehicle controls were treated with hepatocyte culture medium. All experiments were performed in triplicate. Hepatocyte incubation was performed in a 95% air / 5% CO2 incubator at 37°C for three days (72 hours), and hepatocyte culture medium containing TA, positive or negative inducers, or vehicles was replaced every day. The experimental conditions for CYP induction and sample treatment are summarized in Table 2.

[0244] [Table 2] CYP induction conditions

[0245]

[0246] Cell viability assay

[0247] After CYP induction treatment, the cells were used for cell viability assay. Cell viability (expressed as percentage of MTS absorbance relative to vehicle control) was measured as described herein.

[0248] qPCR mRNA assay

[0249] CYP mRNA expression was measured by qPCR after induction treatment. Total RNA was isolated from treated cells using the QIAQUICK mini kit (Qiagen, Valencia, CA, USA) and treated with RNase-free DNase (Qiagen) according to the manufacturer's protocol. RNA was expressed as 500 ng / mL using the Qubit RNA HS Assay Kit (Invitrogen). The concentration of RNA was measured using a fluorometer. cDNA was synthesized from up to 1 μg of total RNA collected from cells using the RT kit (Qiagen). Analysis of CYP gene expression was performed by qPCR (Table 3) using the 480II system (Roche Diagnostics Corporation, Indianapolis, IN, USA).

[0250] [Table 3]

[0251]

[0252]

[0253] Relative mRNA is expressed as the normalized mRNA level (2 -ΔΔCt ) was calculated as the fold increase. The percentage of mRNA fold increase relative to the positive control was calculated using the following equation:

[0254] Induction % relative to positive control = 100 × (mRNA TA -mRNA 赋形剂 ) / (mRNA 阳性对照 -mRNA 赋形剂 )

[0255] The induction of CYP1A2, CYP2B6 and CYP3A4 mRNA by PB and TUDCA at three concentrations (PB / TUDCA at 14.8 / 1.62, 148 / 16.2 and 826 / 108 μM) was evaluated using three human hepatocyte donors. In donor 1, PB and TUDCA did not increase the mRNA of CYP1A2 and CYP3A4 at any of the three tested concentrations (less than 2-fold compared to the vehicle control, less than 20% of the positive control). PB and TUDCA did not increase the mRNA of CYP2B6 at 14.8 / 1.62 and 148 / 16.2 μM, but a 5.56-fold induction was observed at 826 / 108 μM.

[0256] In donor 2, PB and TUDCA did not increase the mRNA of CYP1A2 and CYP3A4 at any of the three concentrations tested. PB and TUDCA did not increase the mRNA of CYP2B6 at 14.8 / 1.62 μM, but 3.18- and 6.82-fold inductions were observed at 148 / 16.2 μM and 826 / 108 μM, respectively.

[0257] In donor 3, PB and TUDCA did not increase the mRNA of CYP1A2 and CYP3A4 at any of the three concentrations tested. PB and TUDCA did not increase the mRNA of CYP2B6 at 14.8 / 1.62 μM and 148 / 16.2 μM, but a 6.66-fold induction was observed at 826 / 108 μM.

[0258] The induction of CYP1A2, CYP2B6 and CYP3A4 mRNA in human hepatocytes by PB / TUDCA, positive controls and negative controls are summarized in Tables 4 to 6. The cell viability results after CYP induction treatment are summarized in Table 7.

[0259] [Table 4] Induction of CYP1A2 mRNA

[0260]

[0261] a Fold increase is the normalized mRNA level from TA, positive inducer, or negative inducer treated cells relative to vehicle treated cells (2 -ΔΔCt ) calculated.

[0262] b The percentage of mRNA fold increase relative to cells treated with positive inducers. Negative values ​​were considered zero. [Table 5] Induction of CYP2B6 mRNA

[0263] [Table 6] Induction of CYP3A4 mRNA

[0264] [Table 7] Cell viability after CYP induction treatment

[0265]

[0266] In summary, PB and TUDCA are unlikely to be inducers of CYP1A2 or CYP3A4 in the concentration range of 14.8 / 1.62 μM to 826 / 108 μM. Induction of CYP2B6 by PB and TUDCA (mainly at 826 / 108 μM) was observed.

[0267] Example 2 - Inhibition of Cytochrome P450 Enzymes in Human Liver Microsomes by Sodium Phenylbutyrate and Tauroursodeoxycholic Acid

[0268] The inhibition of cytochrome P450 (CYP) enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A) in human liver microsomes (HLM) was evaluated by sodium phenylbutyrate (PB) and tauroursodeoxycholic acid (TUDCA). Eight concentrations of PB and TUDCA were added to mixed HLM (0.25 mg protein / mL) in a solution containing MgCl2 (5 mM), CYP-specific probe substrate (at about K m ) and NADPH (1mM) in phosphate buffer (100mM, pH 7.4). After a period of incubation, the reaction was terminated by adding an ice-cold protein precipitation solvent (acetonitrile containing an internal standard) and the sample was centrifuged. The CYP enzyme activity was determined by measuring the formation of each CYP probe metabolite by LC-MS / MS.

[0269] For each test product, PB (0-7400 μM, 50×C 最大,游离的 =7400μM,0.1×I 肠道 =6444 μM) and TUDCA (0-1600 μM, 50×C 最大,游离的 =81μM,0.1×I 肠道

[0270] =1600μM) were incubated with mixed HLM (0.25mg protein / mL) in phosphate buffer (100mM, pH 7.4) containing MgCl2 (5mM), NADPH (1mM) and a separate CYP probe substrate (co-incubation of PB and TUDCA). 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 balanced in a shaking water bath at 37°C for 5 minutes. The reaction was initiated by adding NADPH and then incubated at 37°C for 10-30 minutes depending on the individual CYP isoform. The reaction was terminated by adding two volumes of ice-cold acetonitrile (ACN) containing an internal standard (IS, a stable isotope-labeled CYP probe metabolite). Negative (excipient) controls were performed without TA. Positive controls were performed in parallel using known CYP inhibitors. After removal of proteins by centrifugation at 1640 g for 10 min at 4°C, the supernatant was transferred to an HPLC autosampler plate. The formation of individual CYP probe metabolites was determined by LC-MS / MS. The experimental conditions for CYP reactions and sample analysis are summarized in Table 8.

[0271] Calculate the percentage of control enzyme activity using the following equation:

[0272] Control enzyme activity % = 100 × (enzyme activity in the presence of TA / enzyme activity in the absence of TA)

[0273] [Table 8] CYP reactions and sample analysis

[0274]

[0275] Enzyme activity was expressed as the peak area ratio of the probe metabolite to the IS, measured by LC-MS / MS. IC50 values ​​were estimated by fitting the experimental data (percentage of enzyme activity of the control vs. log[inhibitor concentration]) to a sigmoidal model followed by nonlinear regression analysis using GraphPad Prism (version 5.0 or higher, GraphPad Software, San Diego, CA, USA).

[0276] 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.

[0277] [Table 9] Inhibition of CYP activity in HLM by PB and TUDCA

[0278]

[0279] aPercentage of control enzyme activity = 100 x (enzyme activity in the presence of TA / enzyme activity in the absence of TA). Enzyme activity was calculated from the peak area ratio of CYP probe metabolites to IS by LC-MS / MS. SD: standard deviation.

[0280] [Table 10] Inhibition of CYP activity in HLM by positive inhibitors

[0281]

[0282] The combination of PB (up to 274 μM) and TUDCA (up to 59.3 μM) showed little or no inhibition (<20%) on any of the seven major CYPs tested. The combination 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 greater than 50% inhibition of all seven major CYPs tested.

[0283] Example 3 - Phenotyping of Cytochrome P450 Responses to Sodium Phenylbutyrate and Tauroursodeoxycholic Acid Using Human Liver Microsomes and Chemical Inhibitors

[0284] The aim of this study was to characterize the cytochrome P450 (CYP) response phenotype using human liver microsomes (HLM) and the chemical inhibitors sodium p-phenylbutyrate (PB) and tauroursodeoxycholic acid (TUDCA).

[0285] The CYP response phenotype of PB and TUDCA was evaluated using pooled human liver microsomes (HLM, 0.5 mg protein / mL) and CYP selective inhibitors. The amount of PB and TUDCA remaining after incubation for a period of time (0, 5, 10, 20, 30 and 60 minutes) was measured by LC-MS / MS.

[0286] After incubation with HLM for 60 min, the residual percentages of PB and TUDCA were 95% or higher in the absence and presence of NADPH, regardless of preincubation for 15 min. These results indicate that neither PB nor TUDCA is metabolized by the major CYPs in HLM.

[0287] method

[0288] In the absence and presence of a single CYP selective inhibitor, PB and TUDCA were co-incubated with HLM (0.5 mg protein / mL) at a concentration (5 μM in the final incubation) in a phosphate buffer (100 mM, pH 7.4) containing MgCl2 (5 mM) and NADPH (1 mM) (Table 11). For reversible inhibition, the reaction mixture (without NADPH) was balanced in a shaking water bath at 37°C for 5 minutes, and the reaction was initiated by adding NADPH (except for the control without NADPH), and then incubated at 37°C. For irreversible inhibition, the inhibitor was pre-incubated with HLM in the presence of NADPH at 37°C for 15 minutes, and the reaction was initiated by adding TA. Aliquots of the incubated solution were sampled at 0, 5, 10, 20, 30 and 60 minutes. The reaction was terminated by adding ice-cold acetonitrile (ACN) containing 0.1% formic acid. After protein removal by centrifugation at 1640 g (3,000 rpm) for 10 min at 4 °C, the supernatant was transferred to an HPLC autosampler plate. The residual TA (expressed as the peak area ratio of the test sample to the IS) was determined by LC-MS / MS. The CYP enzyme activity of HLM was verified in parallel using a standard curve by determining the formation of individual CYP probe metabolites using LC-MS / MS. The experimental conditions for CYP reaction phenotype identification and sample analysis are summarized in Tables 12 and 13.

[0289] Data analysis

[0290] Calculate the residual percentage of TA using the following equation:

[0291] Residual TA % = 100 × A t / A0

[0292] Wherein, At and A0 are the peak area ratios of TA to IS at time t and time 0, respectively.

[0293] result

[0294] The residual percentage of PB containing mixed HLM (0.5 mg protein / mL) in the presence and absence of CYP selective inhibitors is summarized in Tables 14 and 15. The residual percentage of TUDCA containing mixed HLM (0.5 mg protein / mL) in the presence and absence of CYP selective inhibitors is summarized in Tables 16 and 17. The CYP enzyme activity of HLM was verified in parallel by determining the formation of CYP probe metabolites using LC-MS / MS, and the results are summarized in Tables 18 and 19.

[0295] Table 11: CYP Selective Chemical Inhibitors

[0296] CYP Chemical inhibitors supplier Item# CYP1A2 Furaphylline MilliporeSigma F124 CYP2B6 Thio-TEPA MilliporeSigma T6069 CYP2C8 Montelukast Bosche Scientific M2936 CYP2C9 Sulfadiazine MilliporeSigma S0758 CYP2C19 (+)-N-3-Benzyl Nivacol (N3B) MilliporeSigma B8686 CYP2D6 Quinidine MilliporeSigma Q3625 CYP3A Ketoconazole MilliporeSigma K1003

[0297] Table 12: Incubation conditions for CYP reactions using HLM

[0298]

[0299] Table 13: CYP responses and sample analysis using HLM

[0300]

[0301] a The residual % (n=3) was calculated by LC-MS / MS from the peak area ratio of TA and IS. SD: standard deviation.

[0302] Table 14: Residual % of PB in HLM (irreversible incubation conditions)

[0303]

[0304] Table 15: Residual % of PB in HLM (reversible incubation conditions)

[0305]

[0306] a The residual % (n=3) was calculated by LC-MS / MS from the peak area ratio of TA to IS.

[0307] SD: standard deviation.

[0308] Table 16: Residual % of TUDCA in HLM (irreversible incubation conditions)

[0309]

[0310] a Residual % (n=3) was calculated by LC-MS / MS from the peak area ratio of TA to IS.

[0311] SD: standard deviation.

[0312] Table 17: Residual % of TUDCA in HLM (reversible incubation conditions)

[0313]

[0314] a The residual % (n=3) was calculated by LC-MS / MS from the peak area ratio of TA to IS.

[0315] SD: standard deviation.

[0316] Table 18: Formation of CYP probe metabolites in HLM

[0317]

[0318] a The concentrations of CYP probe metabolites (mean, n=2) were measured by LC-MS / MS using a standard curve.

[0319] b The formation rate of CYP probe metabolites is expressed in pmol / min / mg protein. When no probe metabolite could be detected, the formation rate was reported as zero.

[0320] c Formation rate ratio = [formation rate (+NADPH-抑制剂) -Formation rate (-NADPH-抑制剂) ] / [Formation rate (+NADPH+抑制剂) -Formation rate (-NADPH-抑制剂) ].

[0321] QC acceptance criteria: formation rate ratio ≥ 2.

[0322] SD: standard deviation.

[0323] Table 19: Formation of CYP probe metabolites in HLM (continued)

[0324]

[0325] Example 4 - Phenotypic characterization of cytochrome P450 responses to sodium phenylbutyrate and tauroursodeoxycholic acid using human recombinant Cyp enzyme

[0326] The cytochrome P450 (CYP) reaction phenotype identification of sodium phenylbutyrate (PB) and tauroursodeoxycholic acid (TUDCA) was evaluated using human recombinant CYP enzymes (hrCYP). PB and TUDCA (5 μM each) were co-incubated with individual hrCYP (20 pmol CYP / mL) in a phosphate buffer (100 mM, pH 7.4) containing MgCl2 (5 mM) and NADPH (1 mM). The amount of PB and TUDCA remaining after incubation for a period of time (0, 5, 10, 20, 30 and 60 minutes) was measured by LC-MS / MS.

[0327] PA and TUDCA were co-incubated at a concentration (5 μM in the final incubation) with individual 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 balanced in a shaking water bath at 37°C for 5 minutes. The reaction was initiated by adding NADPH and then incubated at 37°C. Aliquots (100 μL) of the incubation solution were sampled at 0, 5, 10, 20, 30 and 60 minutes. The reaction was terminated by adding ice-cold acetonitrile (ACN) containing 0.1% formic acid. After removing the protein by centrifugation at 1640g (3,000 rpm) for 10 minutes at 4°C, the supernatant was transferred to an HPLC autosampler plate and stored at -20°C until analysis. The residual test article (TA) was determined by LC-MS / MS (expressed as the peak area ratio of TA to IS). After incubation with the individual CYP probe substrates for 20 minutes, the formation of CYP probe metabolites was determined by LC-MS / MS, and the hrCYP activity was verified in parallel using the standard curve. The experimental conditions for CYP reaction phenotype identification and sample analysis are summarized in Table 20.

[0328] [Table 20] CYP reactions and sample analysis using hrCYP

[0329]

[0330] Calculate the residual percentage of TA using the following equation:

[0331] Residual TA % = 100 × A t / A0

[0332] Among them, A tis 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. The residual percentages of PB and TUDCA in hrCYP (20 pmol CYP / mL) are summarized in Tables 21 and 22, respectively. The hrCYP activity was verified in parallel by determining the formation of CYP probe metabolites using LC-MS / MS, and the results are summarized in Table 23.

[0333] [Table 21] Residual % of PB in hrCYP

[0334]

[0335] a The residual % of TA was calculated from the peak area ratio of TA to IS measured by LC-MS / MS.

[0336] SD: standard deviation.

[0337] [Table 22] Residual % of TUDCA in hrCYP

[0338]

[0339] [Table 23] Formation of CYP probe metabolites in hrCYP

[0340]

[0341] a The concentrations of CYP probe metabolites were measured by LC-MS / MS (mean, n=2).

[0342] b The formation rates of CYP probe metabolites were normalized and expressed as pmol metabolite / min / mg Supersome protein. When no probe metabolite could be detected, the formation rate was reported as zero.

[0343] c Formation rate ratio = formation rate (hrCYP) / Formation rate (阴性对照) . QC acceptance criteria: formation rate ratio ≥2.

[0344] After 60 minutes of incubation with hrCYP, more than 90% of PB and TUDCA remained (<10% disappeared) in the presence of hrCYP1A2, hrCYP2B6, hrCYP2C8, hrCYP2C9, hrCYP2C19, hrCYP2D6, and hrCYP3A4. The results indicate that neither PB nor TUDCA is metabolized by the major liver CYPs.

[0345] Example 5 - Evaluation of the Time-Dependent Inhibitory Potential of Sodium Phenylbutyrate and Tauroursodeoxycholic Acid on Cytochrome P450 Enzymes in Human Liver Microsomes

[0346] Sodium phenylbutyrate (PB) and tauroursodeoxycholic acid (TUDCA) were evaluated for their potential to induce time-dependent inhibition (TDI) of cytochrome P450 (CYP) enzymes in human liver microsomes (HLM). Eight concentrations of PB and TUDCA were preincubated (co-incubated with PB and TUDCA) with mixed HLM (0.25 mg protein / mL) in the presence and absence of NADPH for 30 min, followed by CYP enzyme activity assays with individual CYP probe substrates and quantification by measuring the formation of each CYP probe metabolite by LC-MS / MS.

[0347] The results showed that the differences in CYP inhibition of all seven major hepatic CYPs tested in this study by PB and TUDCA at all concentrations were less than 20% in the absence and presence of NADPH preincubation for 30 minutes. This suggests that the IC50 shift will be less than 1.5 (the threshold for TDI), although the individual IC50 and IC50 shift values ​​for PB and TUDCA could not be calculated due to the co-incubation of PB and TUDCA. The results suggest that TDI of PB and TUDCA for CYPs is unlikely.

[0348] CYP TDI was evaluated by preincubating TA with HLM in the presence and absence of NADPH for 30 min followed by CYP enzyme activity assay. CYP reactions were performed in an incubation volume of 200 μL. Briefly, eight concentrations of each test article in PB (0-7400 μM, 50×C) were incubated in the presence (irreversible incubation conditions) and absence (reversible incubation conditions) of NADPH (1 mM) in phosphate buffer (100 mM, pH 7.4) containing MgCl2 (5 mM). 最大，未结合的 =7400μM, 0.1×I 肠道 =6444 μM) and TUDCA (0-1600 μM, 50×C 最大，未结合的 =81μM, 0.1×I 肠道=1600 μM) was pre-incubated (co-incubation of PB and TUDCA) with HLM (0.25 mg protein / mL) at 37°C for 30 minutes. The total organic solvent content in the final incubation was less than 1% (DMSO ≤ 0.1%, other organic solvents ≤ 1%). The CYP reaction was initiated by adding a separate CYP probe substrate with added (when NADPH was not added in the pre-incubation step) or without added (when NADPH was added in the pre-incubation step) NADPH (1 mM). Depending on the individual CYP isoform, the reaction mixture was incubated at 37°C for 10-30 minutes (Table 24). The reaction was terminated using ice-cold acetonitrile (ACN) containing an internal standard (IS, a stable isotope-labeled CYP probe metabolite). A negative (excipient) control was performed using an incubation mixture without TA. A positive control was performed in parallel using a known CYP time-dependent inhibitor. After protein removal by centrifugation at 1640 g (3,000 rpm) for 10 min at 4°C, the supernatant was transferred to an HPLC autosampler plate. The formation of individual CYP probe metabolites was determined by LC-MS / MS. The experimental conditions for CYP reactions and sample analysis are summarized in Table 24.

[0349] Calculate the percentage of control enzyme activity using the following equation:

[0350] % of control enzyme activity = 100 x (enzyme activity in the presence of TA / enzyme activity in the absence of TA).

[0351] Enzyme activity was expressed as the peak area ratio of the CYP probe metabolite to the IS, measured by LC-MS / MS. IC50 values ​​were estimated by fitting the experimental data (percentage of enzyme activity of the control vs. log[inhibitor concentration]) to a sigmoidal model followed by nonlinear regression analysis using GraphPadPrism (version 5.0 or higher, GraphPad Software, San Diego, CA, USA).

[0352] [Table 24] CYP reactions and sample analysis

[0353]

[0354] IC50 shifts between reversible (without 30 min preincubation with NADPH) or irreversible (with 30 min preincubation with NADPH) incubation conditions are indicators of TDI potential: the threshold for a positive result is an IC50 shift > 1.5.

[0355] The potential of PB and TUDCA for CYP TDI in HLM is summarized in Table 25. Tables 26-27 and Figure 1 The inhibition of CYP activity by positive time-dependent inhibitors is summarized in.

[0356] [Table 25] Potential of PB and TUDCA for CYP TDI in HLM

[0357]

[0358] a Percentage of control enzyme activity = 100 x (enzyme activity in the presence of TA / enzyme activity in the absence of TA). Enzyme activity was calculated from the peak area ratio of CYP probe metabolites to IS by LC-MS / MS.

[0359] b Although individual IC50 and IC50 shift values ​​for PB and TUDCA could not be calculated due to the co-incubation of PB and TUDCA, the difference in CYP inhibition by PB and TUDCA was less than 20% at the highest concentration (7400 and 1600 μM for PB and TUDCA, respectively) after 30 min of preincubation in the absence and presence of NADPH, suggesting that the IC50 shift may be less than 1.5 and the conclusion may be that TDI is unlikely. SD: standard deviation.

[0360] [Table 26] IC50 shift of positive time-dependent inhibitors on CYP-TDI in HLM

[0361] a Percentage of control enzyme activity = 100 x (enzyme activity in the presence of inhibitor / enzyme activity in the absence of inhibitor). Enzyme activity was calculated from the peak area ratio of CYP probe metabolites to IS by LC-MS / MS.

[0362] b IC50 shift = IC50 (-NADPH) / IC50 (+NADPH) QC of positive control: IC50 shift ≥ 2. SD: standard deviation; ND: not determined (n=2)

[0363] [Table 27] IC50 shift of positive time-dependent inhibitors on CYP-TDI in HLM (continued)

[0364]

[0365] Example 6 - Transporter Drug-Drug Interaction Assessment

[0366] The purpose of this study was to evaluate the substrate and inhibitor potential (IC50) of AMX0035 against the following transporters: P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), organic anion transporting polypeptide 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 efflux proteins 1 and 2K (MATE1 and MATE2K). AMX0035 is a combination of TUDCA and PB. In this study, TUDCA and PB were present simultaneously in all dosing solutions.

[0367] The inhibitory potential (IC50 evaluation) of the test articles was determined using HEK cell lines transfected with each uptake transporter of interest (OATP1B1, OATP1B3, OAT1, OAT3, OCT2, MATE1, or MATE2K). The estimated IC50 for OAT1 will be between 0.0741 and 0.222 μM TUDCA, with 6.76 and 20.3 μM PB, respectively. The estimated IC50 for OCT2 will be greater than 162 μM TUDCA and greater than 14785 μM PB. The estimated IC50 for OAT3 will be between 54.0 and 162 μM TUDCA, with 4928 and 14785 μM PB, respectively. The estimated IC50 for OATP1B1 will be between 12.9 and 38.7 μM TUDCA, with 1208 and 3625 μM PB, respectively. The estimated IC50 for OATP1B3 will be between 38.7 and 116 μM TUDCA, with 3625 and 10875 μM PB, respectively. The estimated IC50 for MATE1 will be between 270 and 810 μM TUDCA, with 24642 ​​and 73927 μM PB, respectively. The estimated IC50 for TUDCA and PB for MATE2K will be greater than 810 μM and greater than 73927 μM, respectively.

[0368] Evaluation of AMX0035 as a P-gp inhibitor was performed in MDR1-MDCK and C2BBel cells. Transport of the P-gp probe substrate digoxin (10 μM) across the cell monolayer was used as an indicator of P-gp activity. The assay was performed using a bidirectional approach: apical to basolateral (AP to BL) and basolateral to apical (BL to AP). In MDR1-MDCK cells, the estimated IC50 for P-gp ranged from 889 to 2668 μM TUDCA, with 7161 and 21482 μM PB, respectively. In C2BBel cells, the estimated IC50 for P-gp ranged from 222 and 667 μM TUDCA, with 1790 and 5371 μM PB, respectively.

[0369] Evaluation of AMX0035 as a BCRP inhibitor was performed in BCRP-MDCK and C2BBe1 cells. Transport of the BCRP probe substrate cladribine (10 μM) across the cell monolayer was used as an indicator of BCRP activity. The assay was performed using a bidirectional approach. In BCRP-MDCK cells, the estimated IC50 for BCRP was between 296 and 889 μM TUDCA, with 2387 and 7161 μM PB, respectively. In C2BBe1 cells, the estimated IC50 for P-gp would be between 222 and 667 μM TUDCA, with 1790 and 5371 μM PB, respectively.

[0370] AMX0035 was evaluated as a substrate for P-gp and BCRP using a bidirectional approach in C2BBe1 cells. The efflux ratios of TUDCA at 81.0, 16.2, 1.62, and 0.810 μM were 0.506, 0.305, 0.230, and 0.302, respectively. The efflux ratios of PB at 1479, 148, 73.9, and 14.8 μM were 0.740, 0.620, 0.940, and 1.65, respectively.

[0371] In conclusion, neither TUDCA nor PB is a substrate for P-gp or BCRP in vitro. The estimated IC50 of TUDCA for each transporter is as follows: 0.0741-0.222 μM (OAT1), greater than 162 μM (OCT2), 54.0-162 μM (OAT3), 12.9-38.7 μM (OATP1B1), 38.7-116 μM (OATP1B3), 270-810 μM (MATE1), greater than 810 μM (MATE2K), 889-2668 μM (P-gp in MDR1-MDCK cells), 222-667 μM (P-gp in C2BBel cells), 296-889 μM (BCRP BCRP in MDCK cells), and 222-667 μM (BCRP in C2BBel cells). The estimated IC50 of PB for each transporter was as follows: 6.76-20.3 μM (OAT1), greater than 14785 μM (OCT2), 4928-14785 μM (OAT3), 1208-3625 μM (OATP1B1), 3625-10875 μM (OATP1B3), 24642-73927 μM (MATE1), greater than 73927 μM (MATE2K), 7161-21482 μM (P-gp in MDR1-MDCK cells), 1790-5371 μM (P-gp in C2BBel cells), 2387-7161 μM (BCRP in MDCK cells), and 1790-5371 μM (BCRP in C2BBel cells).

[0372] Introduction

[0373] The relevant concentrations of TUDCA and PB are listed in Table 28. The tested concentrations of TUDCA and PB selected based on analytical sensitivity in the current example and clinical relevance are summarized in Table 29.

[0374] For the evaluation of substrate and inhibitor potential of efflux transporters (e.g., P-gp and BCRP), the preferred in vitro method is a cell-based bidirectional permeability assay. In the current example, both MDCK and C2BBel cells transfected with human P-gp and BCRP were used. For the evaluation of substrate and inhibitor potential of uptake transporters (e.g., OATP1B1 and OATP1B3), cell lines with target gene overexpression were used.

[0375] [Table 28] Relevant concentrations of TUDCA and PB for in vitro transporter assessment

[0376]

[0377] [Table 29] TUDCA and PB concentrations used in this example

[0378]

[0379] a Used in MDR1-MDCK and BCRP-MDCK cells.

[0380] b Used in C2BBe1 cells.

[0381] method

[0382] Chemical stability assessment

[0383] The chemical stability of TUDCA and PB was tested in HBSSg_7.4 and HBSS_8.5. The test samples were prepared in DMSO stock solution (0.5mM) and aliquots of the DMSO stock solution were added to each matrix. The nominal concentration of each component in each matrix was 0.5μM. The aliquots were transferred to separate vials (one vial per time point) and the vials were incubated in a humidified incubator at 37°C and 5% CO2. At pre-confirmed time points, an equal volume of acetonitrile with internal standard was added to each vial, and the mixture was placed at 4°C until the end of the assay. The peak areas of the test sample and IS for each sample were determined by LC-MS / MS, and the ratio of the peak areas (analyte: IS) was calculated.

[0384] Solubility assessment

[0385] All solubility samples were prepared in silanized glass tubes. Powder aliquots of each component were added to each matrix in an amber vial (Table 30). After adding the buffer, the samples were incubated overnight (1020-1080 minutes) in a 37°C shaking water bath. After incubation, the mixture was centrifuged at 20800 g for 15 minutes. The supernatant was appropriately diluted and the concentration of each component was determined by LC-MS / MS.

[0386] [Table 30] Conditions for evaluating the solubility of TUDCA and PB

[0387] Components HBSSg_7.4 HBSSg_8.5 TUDCA (nominal concentration, μM) 8005 810 PB (nominal concentration, μM) 64447 73927

[0388] Nonspecific binding assessment

[0389] Since the assay plates for efflux and uptake transporters were made of different materials, nonspecific binding assessments of TUDCA and PB were performed using the corresponding cell-free plates.

[0390] (i) Efflux transporter form

[0391] Transwell plates (12 wells) coated with rat tail collagen were used, and all incubations were carried out in a humid incubator at 37°C, 5% CO2. Aliquots of TUDCA and PB DMSO stock solutions were added to HBSSg-7.4. Triple wells were used for determination (Table 31). Samples were taken out from the receptor and donor compartments at preselected time points and mixed with an equal volume of acetonitrile containing IS. The concentrations of TUDCA and PB were measured by LC-MS / MS.

[0392] (ii) Uptake transporter form

[0393] Poly-D-lysine coated plates (24 wells) were used, and all incubations were performed in a humid incubator at 37°C and 5% CO2. TUDCA and PB dosing solutions were prepared in HBSSg_7.4 and HBSSg_8.5. Two treatments (three wells each) included: in the first treatment, the wells were pre-incubated with HBSSg_7.4_BSA or HBSSg_8.5_BSA for 5 minutes; in the second treatment, no pre-incubation was performed (Table 32). After pre-incubation, the solution was aspirated and the dosing solution (500 μL) was added to each well. The plate was placed in a humid incubator at 37°C and 5% CO2 for 5 minutes. After incubation, an aliquot (200 μL) was taken out from each chamber, combined with an equal volume of acetonitrile containing IS, and stored at 4°C until analysis. The concentrations of TUDCA and PB were determined by LC-MS / MS.

[0394] [Table 31] Nonspecific binding evaluation conditions (efflux transporter form)

[0395]

[0396] [Table 32] Nonspecific binding evaluation conditions (uptake transporter form)

[0397]

[0398] HEK cell culture related to uptake form transporter

[0399] Human embryonic kidney (HEK293) cells transfected with individual uptake transporter genes (OATP1B1, OATP1B3, OAT1, OAT3, OCT2, MATE1, or MATE2K) or a blank vector control gene (VC) were used to evaluate the inhibitory potential of substrates and test articles on 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, 5% CO2. The medium was changed three times a week, and cell growth was observed by light microscopy. When cells were confluent, cells were harvested by trypsinization, and the harvested cells were plated on plates for uptake studies. The plates were placed in a humidified incubator at 37°C, 5% CO2. One day after plating, MATE-transfected and vector control-transfected cells were incubated with medium containing 10 mM sodium butyrate for approximately 16-20 hours before use.

[0400] Prior to the assay (same day), each cell line was checked with a transporter-specific fluorescent marker compound (quality control [QC] compound) to confirm functionality of the transfected transporter (Table 33).

[0401] [Table 33] Culture conditions of HEK cells transfected with uptake form transporter

[0402]

[0403] a ASP was prepared in HBSSg_8.5; cells were pre-incubated with HBSSg_8.5 for 20 min prior to QC assay.

[0404] Demonstration of HEK cells for uptake form transporter studies

[0405] QC dosing solution is prepared by diluting aliquots of DMSO stock solution of QC compound in appropriate buffer based on transporter; The final concentration of each QC compound is shown in Table 33. Without disturbing the cells, the culture medium is gently aspirated. QC dosing solution is added to each well, and the cells are incubated for a pre-selected period of time in a humid incubator at 37°C and 5% CO2. The reaction is terminated by washing the cells 2 times with ice-cold HBSSg_7.4 buffer. After the second rinse, the cells are lysed with 150 μL RIPA buffer and incubated at 4°C for 10 minutes. Aliquots (100 μL) of the supernatant are collected into an opaque white fluorescent ELISA plate, and the fluorescence of each compound is read at the optimal excitation and emission wavelengths.

[0406] Other wells (n=3) from the same batch of cells were used for protein determination. Without disturbing the cells, the medium was gently aspirated and the wells were washed twice with HBSSg buffer. After the second rinse, the cells were lysed with 150 μL of RIPA buffer at 4°C on a shaker for 15 minutes. Aliquots (12.5 μL) of the cell lysate were collected in microcentrifuge tubes and stored at -20°C until protein concentration analysis.

[0407] Design of cell culture related transporters

[0408] C2BBe1 and MDCK cells are obtained from the American Type Culture Collection (Manassas, VA, USA). MDR1-MDCK cells are obtained from the National Institutes of Health (Bethesda, MD, USA). BCRP-MDCK cells are produced by Absorption Systems LLC (Exton, PA, USA). The cells are maintained in a humid incubator at 37°C 5% CO2 in DMEM culture medium containing 10% FBS, 1% NEAA, 4mM L-glutamine, 1mM sodium pyruvate, PEST and appropriately selected reagents (Table 34). The culture medium is replaced 3 times per week, and cell growth is observed by optical microscopy. When the cells reach 80-90% confluence, the cells are harvested by trypsinization and inoculated into Costar Transwell plates (60000 cells / cm 2 ) to generate a cell monolayer for permeability studies. Fresh culture medium (1.5 mL) was added to each well, and cell suspension (0.5 mL) was added to each insert well. The plate was placed in a humidified incubator at 37°C 5% CO2, and the culture medium was changed every other day until use. Prior to using BCRP-MDCK cells, the cell monolayer was supplemented with culture medium containing sodium butyrate (2.5 mM) overnight (approximately 16-20 hours).

[0409] [Table 34] Cell culture conditions related to efflux transporters

[0410]

[0411] Validation of cells for efflux transporter-related assays

[0412] Before measuring, the permeability of QC compound is measured in each batch of cell monolayer. The QC compound is prepared in HBSSg-7.4. The cell monolayer for QC test is washed twice with HBSSg-7.4. For the top to the basolateral (AP to BL) transport, 0.5mL of the dosing solution containing QC compound is added to the insertion hole, and 1.5mL HBSSg-7.4 is added to the hole. For the basolateral to the top (BL to AP) transport, 1.5mL dosing solution is added to the hole, and 0.5mL HBSSg-7.4 is added to the insertion hole. The cells are incubated in a humid incubator for 120 minutes. Samples are taken from the receptor compartment at 120 minutes, and the concentration of each QC compound is measured by LC-MS / MS.

[0413] Tolerance assessment of efflux transporter-associated cells

[0414] The effects of TUDCA and PB on cell monolayer integrity were evaluated in C2BBe1 and MDCK cells (Table 35). MDCK was used as a representative cell line for MDR1-MDCK and BCRPMDCK cells. The cell monolayer of each cell line was treated with triplicate wells for each treatment. The dosing solution was prepared in HBSSg_7.4 containing LY. The dosing solution and simple HBSSg_7.4 were placed on the BL and AP sides, respectively, and placed in a humid incubator at 37°C, 5% CO2 for 150 minutes. After incubation, aliquots (100 μL) on the AP side were collected, and the concentration of LY was determined by fluorescence using a BMG microplate reader, with excitation and emission wavelengths of 485nm and 540nm, respectively.

[0415] The concentrations of TUDCA and PB in the dosing solution were determined by LC-MS / MS.

[0416] [Table 35] Tolerance evaluation conditions (efflux transporter)

[0417]

[0418] a The concentrations of TUDCA and PB were 8005 and 64447 μM, 4002 and 32223 μM, 3202 and 25778 μM, 2001 and 16112 μM, and 800 and 6445 μM, respectively.

[0419] b The concentration of LY was 200 μM.

[0420] Tolerance assessment of cells transfected with uptake form transporters

[0421] TUDCA and PB were prepared at predetermined concentrations in the appropriate assay buffer (Table 36). The culture medium was aspirated. The vector control transfected cells (Group 1) were preincubated with the test article dosing solution for 30 minutes, while the vector control transfected cells (Group 2) were not preincubated. For tolerance assays associated with MATE, the cells were preincubated with simple HBSSg_8.5 for 20 minutes. After preincubation, the preincubation solution was aspirated and the appropriate buffer or dosing solution containing the test article (500 μL) was added to the corresponding wells. The cells were placed in a humidified incubator at 37°C and 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 culture medium was added. Subsequently, 100 μL of CellTiter was added to each well. AQueous ONE solution cell proliferation assay reagent was then added and the cells were returned to a 37°C, 5% CO2 humidified incubator. After one hour of incubation, the cells were assayed using The absorbance of each well was measured at 492 nm by a spectrophotometer (BMGLabtech, Ortenberg, Germany). The concentrations of TUDCA and PB in the dosing solution were determined by LC-MS / MS. HEK cells transfected with vector controls were used as representative cell lines in the tolerance assessment. Puromycin-resistant vector control cells (Puro-VC) were used for MATE-related cells, and G418-resistant vector controls (Neo-VC) were used for all other transporter-transfected cells.

[0422] The CellTiter 96AQueous non-radioactive cell proliferation assay is a homogenous colorimetric method for determining the number of viable cells in cytotoxicity assays. The CellTiter 96AQueous 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 (as measured by absorbance at 492 nm) is proportional to the number of viable cells in culture.

[0423] Uptake form transporter inhibitor assessment

[0424] TUDCA and PB were prepared in appropriate matrices at preselected concentrations (Table 37). The culture medium was gently aspirated; and the inhibition assay was started as follows: 1) in the absence and presence of TUDCA and PB or known inhibitors, incubated with 500 μL probe substrate for the required time at 37°C, 5% CO2; 2) at the end of the incubation period, the solution was gently aspirated; the cells were rinsed 2 times with ice-cold HBSSg-7.4 buffer (1000 μL per rinse); 3) the cells were lysed in 400 μL acetonitrile: water (3:1, v / v) containing IS; and 4) lysates were collected to analyze the probe substrate concentration (Table 38). The dosing solution concentrations of TUDCA and PB in the inhibitor assessment were also determined by LC-MS / MS.

[0425] [Table 36] Tolerance measurement conditions (uptake form transporter)

[0426]

[0427] [Table 37] Concentrations of TUDCA and PB in IC50 evaluation of uptake transporters

[0428]

[0429] a Prepare dosing solution in HBSSg_7.4.

[0430] b Dosing solutions were used for OAT1, OAT3, and OCT2 IC50 assessments.

[0431] c Dosing solutions were used for OATP1B1 and OATP1B3 IC50 assessments.

[0432] d Dosing solutions were prepared in HBSSg_8.5 for MATE1 and MATE2K IC50 assessments.

[0433] [Table 38] Evaluation conditions of uptake form transporter inhibitors (IC50)

[0434]

[0435] a Duplicate wells of each cell line were used for each treatment.

[0436] b No preincubation was performed unless otherwise stated.

[0437] c Prior to the assay, cells were pre-incubated with the test article, RSV or simple HBSSg-7.4 for 30 minutes.

[0438] d The dosing solution was prepared using HBSSg_7.4. e Prior to inhibition assessment, cells were pre-incubated with HBSSg_8.5 for 20 min.

[0439] f The dosing solution was prepared using HBSSg_8.5.

[0440] P-gp and BCRP IC50 assessment

[0441] Evaluate in both MDR1-MDCK and C2BBe1 cells (for P-gp) and BCRPMDCK and C2BBe1 cells (for BCRP). TUDCA and PB (Table 39) were prepared in a suitable matrix at a preselected concentration. The bidirectional transport of digoxin was measured in the absence and presence of TUDCA and PB, valspodar (containing only digoxin) and Ko143 (containing only cladribine). Digoxin and cladribine were used as probe substrates for P-gp and BCRP, respectively (Table 40). All incubations were carried out in a humid incubator at 37°C 5% CO2. Probe substrates, known inhibitors, and TUDCA and PB were prepared in HBSSg-7.4. All solutions containing probe substrates also contained 200 μM LY.

[0442] Pre-incubate for 30 minutes on both sides with TUDCA and PB or known inhibitors to preload cells. After 30 minutes, aspirate the pre-incubation solution. An aliquot of a fresh dosing solution with or without TUDCA and PB or known inhibitors (0.55mL in AP to BL direction, 1.55mL in BL to AP direction) is added to the donor compartment, and HBSSg-7.4 buffer with or without TUDCA and PB or known inhibitors (1.5mL in AP to BL direction, 0.5mL in BL to AP direction) is added to the receptor compartment. Collect receptor samples (300 μL) at pre-selected time points. For receptor samples, a portion (200 μL) is used for analysis of TUDCA and PB, and the remaining samples (100 μL) are used for LY measurement at the end of permeability determination. Collect donor samples (50 μL) at pre-selected time points.

[0443] After the permeability assay was completed, the cell monolayer integrity was checked with LY assessment. The concentration of LY was measured at 438 nm (excitation) and 540 nm (emission). The concentration of probe substrate in the collected dosing solution, donor sample and acceptor sample, as well as the concentration of TUDCA and PB in the dosing solution were determined by LC-MS / MS.

[0444] [Table 39] Concentrations of TUDCA and PB in P-gp and BCRP IC50 evaluation

[0445] a Prepare dosing solution in HBSSg_7.4.

[0446] b The dosing solution was used in MDR1-MDCK and BCRP-MDCK cells.

[0447] c The dosing solution was used in C2BBe1 cells.

[0448] [Table 40] P-gp and BCRP IC50 evaluation conditions

[0449]

[0450] a The concentrations of TUDCA and PB are listed in Table 39.

[0451] b The concentration of the probe substrate for digoxin was 10 μM, and the concentration of the probe substrate for cladribine was 10 μM.

[0452] c Penspodar (1 μM) was used for P-gp inhibition; Ko143 (0.5 μM) was used for BCRP inhibition.

[0453] P-gp and BCRP substrate assessment

[0454] P-gp and BCRP substrate assessments were performed in C2BBe1 cell monolayers. TUDCA and PB were prepared at preselected concentrations in appropriate substrates (Table 41). All incubations were performed in a humidified incubator at 37°C 5% CO2. LY was co-administered with each dosing solution at a nominal concentration of 200 μM. In the absence of P-gp or BCRP inhibitors, bidirectional permeability assays were performed with TUDCA and PB (Table 42). Digoxin (P-gp substrate) or cladribine (BCRP substrate) were run in parallel (Table 43).

[0455] Aspirate the cell culture medium and rinse the cell monolayer once with HBSSg_7.4. For the TUDCA and PB groups, aliquots of fresh dosing solution (0.55 mL from AP to BL, 1.55 mL from BL to AP) were added to the donor compartment, and HBSSg_7.4 buffer without inhibitors (1.5 mL from AP to BL, 0.5 mL from BL to AP) was added to the receptor compartment. For receptor samples, aliquots (300 μL) were collected at pre-selected time points. For receptor samples, a portion (200 μL) was used for analysis of TUDCA and PB, and the remaining samples (100 μL) were used for LY measurement at the end of the permeability assay. For donor samples, aliquots (50 μL) were collected at pre-selected time points without replacement.

[0456] The concentrations of TUDCA and PB, digoxin and cladribine in the dosing solution, acceptor and donor samples were determined by LC-MS / MS. The concentration of LY was determined by fluorescence using a BMG microplate reader with excitation and emission wavelengths of 485 nm and 540 nm, respectively.

[0457] [Table 41] Concentrations of TUDCA and PB in P-gp and BCRP substrate assessment

[0458]

[0459] a Prepare dosing solution in HBSSg_7.4

[0460] [Table 42] Efflux transporter substrate evaluation conditions 1

[0461]

[0462] [Table 43] Efflux transporter substrate evaluation condition 2

[0463]

[0464] a The concentrations of digoxin and cladribine were both 10 μM

[0465] Data analysis

[0466] Uptake transporter-related analysis

[0467] The influx rates of TUDCA and PB were determined in transporter-transfected and vector control-transfected cells. Negative values ​​of percent inhibition were reported as 0 and maximal inhibition was reported as 100%. IC50 values ​​were calculated using GraphPad Prism (version 5.0).

[0468]

[0469] Net Inflow Rate (NIR) = IR (TF) -IR (VC) (3)

[0470]

[0471] IR: average influx rate (substrate amount normalized to average protein content and incubation time, pmol / mg protein / min)

[0472] CS: Substrate concentration in cell lysate (μM)

[0473] VS: Substrate evaluation cell lysate volume (mL)

[0474] CP: protein concentration in cell lysate (mg / mL)

[0475] VP: Volume of cell lysate for protein content determination (mL)

[0476] Vesicle protein: protein content of vesicles (50 μg)

[0477] VC: vector control transfected cells

[0478] TF: transporter-transfected cells

[0479] TA: TUDCA and PB or known inhibitors

[0480] ATP: In the presence of ATP

[0481] AMP: In the presence of AMP

[0482] X: Logarithm of the nominal concentrations of TUDCA and PB

[0483] Y: The remaining percentage of activity of the transporter at a given concentration

[0484] YL: lowest response (highest concentration of TUDCA and PB)

[0485] YH: Highest response (vehicle control)

[0486] Efflux transporter-related analysis

[0487]

[0488] Corrected efflux ratio = efflux ratio - 1 (10)

[0489]

[0490] dC R / dt: slope of cumulative concentration versus time in the receptor compartment (μM / s);

[0491] C R C : cumulative receptor concentration (μM);

[0492] C R 60 : receptor concentration at 60 min (μM);

[0493] V R : volume of the receptor compartment (mL);

[0494] V D : Volume of donor compartment (mL);

[0495] A: Cell membrane surface area (1.13 cm 2 );

[0496] C D0 : Donor concentration at 0 min (μM);

[0497] C D5 : Donor concentration at 5 min (μM);

[0498] C DN : Nominal concentration of dosing solution (μM);

[0499] C R 最终 : Cumulative receptor concentration at the end of incubation (μM);

[0500] C D 最终 : Donor concentration at the end of incubation (μM);

[0501] TA: Test article or known inhibitor

[0502] Due to the removal and replacement of the receptor side, the cumulative concentration was calculated before slope construction. The cumulative concentration C at each time point R C Equal to the measured concentration at that time point (C m ) and 3 / 15 (3 / 5 for BL to AP direction) of the concentration measured at the previous time point, because 300 μL was removed from the total sample of 1.5 mL (or 300 μL from 0.5 mL for BL to AP direction) and replaced.

[0503] For the permeability test,

[0504] C R C 60=C m 60

[0505] C R C 120 = Cm 120+3 / 15(or 3 / 5)×C m 60

[0506] Equations (7-A) and (7-B) are used to determine the P of TUDCA and PB. app Equation (7-C) was used to determine the P values ​​of digoxin and cladribine. app Equations (7-D) and (7-E) are used to determine the P of LY. app Equation (7-E) was used to determine the P of the control compound in the batch QC of the cell monolayer. app .

[0507] Since no receptor samples were collected at 0 min, the origin was not included in the fit. The rationale for equation (10) is as follows: An efflux ratio of 1 indicates zero efflux.

[0508] Therefore, a value of 1 was subtracted from the calculated efflux ratio before determining the residual efflux. The minimum value for the inhibition percentage is "0%" and the maximum value is "100%".

[0509] result

[0510] Chemical stability

[0511] During the test period, TUDCA and PB appeared to be stable in the test matrix (Table 44). In subsequent solubility assessments, TUDCA and PB were incubated overnight.

[0512] [Table 44] Chemical stability of TUDCA and PB

[0513]

[0514] a ~1020 to 1080 minutes.

[0515] Solubility assessment

[0516] The acceptance criterion for the solubility assessment was that the concentration measured in the matrix had to be within 80–120% of the nominal value in at least two of the three replicates.

[0517] In the solubility evaluation, the measured TUDCA and PB concentrations were greater than 80% of the nominal concentrations in at least two replicates of the two matrices (Table 45). The results showed that TUDCA and PB were soluble to the target concentrations: 8005 μM in HBSSg_7.4 and 810 μM in HBSSg_8.5 for TUDCA; 64447 μM in HBSSg_7.4 and 73927 μM in HBSSg_8.5 for PB.

[0518] [Table 45] Solubility measurement of TUDCA and PB

[0519]

[0520] Nonspecific binding assessment

[0521] The acceptance criteria for the dosing solution formulation was that the measured concentration of the dosing solution must be within 80-120% of the nominal value in at least two of the three replicates. The dosing concentrations of TUDCA and PB measured in all three replicates were within 80-120% of the nominal value for each treatment, indicating that the formulation of the dosing solution was acceptable.

[0522] For the efflux transporter forms, recoveries of TUDCA and PB were greater than 80% in all three replicates, indicating little nonspecific binding of the test article to the experimental setup. No additional steps were used in the efflux transporter substrate assessment.

[0523] For the uptake transporter format, recoveries of TUDCA and PB were greater than 90% in both matrices without BSA preincubation. The results suggest that nonspecific binding of TUDCA and PB to the experimental setup was minimal. No BSA preincubation was performed in the uptake transporter evaluation. Tolerance assessment of efflux transporter-associated cells

[0524] The concentrations of TUDCA and PB in the dosing solutions for efflux transporter-related tolerance assessment were analyzed. The measured concentrations of the test articles in all three replicates were within the range of 80-120% of the nominal value for each concentration, indicating that the preparation of the dosing solutions was acceptable.

[0525] In run 1, in the presence of TUDCA and PB, the P of LY in all monolayers of C2BBe1 app The P values ​​of LY in the monolayer were greater than 0.8×10-6 cm / s (Table 46), indicating that the integrity of the cell monolayer was affected by the presence of the test article. In Run 2, three low concentrations of TUDCA and PB were tested. In the presence of TUDCA and PB (3202 μM and 25778 μM, respectively), the P values ​​of LY in the monolayer were 2.34×10-6 cm / s and 1.22×10-6 cm / s, respectively. app Greater than 0.8×10-6cm / s, but less than 0.8×10-6cm / s at the other two concentrations (Table 46).

[0526] In the following assays, the highest concentrations of TUDCA and PB in C2BBe1 cells did not exceed 2001 μM and 16112 μM, respectively.

[0527] [Table 46] Effects of TUDCA and PB on the integrity of C2BBe1 cell monolayer

[0528]

[0529] a Run 1. b Run 2

[0530] c Acceptance criteria are LY P app ≤0.8×10 -6 cm / s.

[0531] d The measured concentration of LY was greater than 5 μM (the upper limit of quantification). A value of 5 was used to estimate the P value of LY. app . Actual LY P app Greater than 1.2×10 -6 cm / s.

[0532] For MDCK cells, the P of LY in all monolayers was app Less than 0.8×10-6 cm / s (Table 47), indicating that the cell monolayer was not affected by the presence of the test article.

[0533] 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.

[0534] [Table 47] Effects of TUDCA and PB on the integrity of MDCK cell monolayer

[0535]

[0536] a Acceptance criteria are LY P app ≤0.8×10 -6 cm / s.

[0537] b The measured concentration of LY was less than 0.313 μM (lower limit of quantification). The value of 0.313 was used to estimate the P value of LY. app .

[0538] Actual LY P app Less than 0.1×10 -6 cm / s.

[0539] Assessment of tolerance of uptake transporter-related cells

[0540] The concentrations of TUDCA and PB were analyzed for uptake transporter-related tolerance assessment. In both pH 7.4 and pH 8.5 dosing solutions, the concentrations measured in at least two of the three replicates were within the range of 80-120% of the nominal values, indicating that the preparation of the dosing solutions was acceptable.

[0541] In the cells pre-incubated with TUDCA and PB for 30 minutes, the formazan absorbance in all groups was greater than 75% of the control value (Table 48). The results show that the TUDCA and PB of the test concentrations have no adverse effect on the metabolic activity of the cells. In subsequent assays, the highest concentrations of TUDCA and PB in these cells were no more than 348 and 32625 μM, respectively.

[0542] In cells not pre-incubated 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 tested concentrations (Tables 49 and 50).

[0543] In subsequent assays, the highest concentration of TUDCA in these cells did not exceed 348 μM in HBSSg_7.4 and 810 μM in HBSSg_8.5; the highest concentration of PB in HBSSg_7.4 did not exceed 32625 μM and 73927 μM in HBSSg_8.5.

[0544] [Table 48] Effects of TUDCA and PB on the viability of VC-transfected HEK cells (pre-incubated with test article)

[0545]

[0546] a Cells are pre-incubated with test article solution.

[0547] b The percentage was obtained by dividing the individual absorbance of each well in the presence of the test article by the mean absorbance value in the absence of the test article.

[0548] ND: Not determined.

[0549] [Table 49] Effects of TUDCA and PB on the viability of VC-transfected HEK cells (without pre-incubation with the test article)

[0550]

[0551] a Cells are pre-incubated with test article solution.

[0552] b The percentage was obtained by dividing the individual absorbance of each well in the presence of the test article by the mean absorbance value in the absence of the test article.

[0553] ND: Not determined.

[0554] [Table 50] Effects of TUDCA and PB on the viability of VC-transfected HEK cells (without pre-incubation with the test article, pH 8.50)

[0555]

[0556] Uptake transporter inhibitor evaluation (i) OAT1

[0557] At all concentrations, in all three replicates, the measured concentrations of TUDCA and PB in the dosing solutions in the OAT1 IC50 assessment were within 80-120% of the nominal values, indicating that the preparation of the test article dosing solutions was acceptable. Detailed annotations for solutions 1 to 8 are described in Table 28. The preparation of the PAH dosing solutions was also acceptable.

[0558] In the absence of TUDCA and PB or probenecid, the PAH influx rate ratio was 385 (OAT1 compared to vehicle control) (Table 51). The presence of probenecid caused an 86.5% inhibition of PAH uptake in OAT1 transfected cells (Table 52). These results indicate that the test system is functioning properly.

[0559] The mean influx rate of PAH through OAT1 in the presence of TUDCA and PB ranged from 4.90 to 411 pmol / mg protein / min (Table 51). AMX0035 (a combination of TUDCA and PB) showed concentration-dependent inhibition of OAT1-mediated uptake of PAH.

[0560] Since both TUDCA and PB were present in the dosing solutions for the OAT1 IC50 evaluation, the IC50 for either component could not be determined. In the presence of solution 8 (0.0741 μM TUDCA and 6.76 μM PB), OAT1 was inhibited by 44.8%, and in the presence of solution 7 (0.222 μM TUDCA and 20.3 μM PB), OAT1 was inhibited by 65.2% (Table 52). Therefore, the estimated IC50 for OAT1 would be between 0.0741 and 0.222 μM TUDCA, with 6.76 and 20.3 μM PB, respectively.

[0561] Currently, the FDA has the following ratio criteria for the in vitro evaluation of test articles as inhibitors of OAT1: If the unbound [I]1 / IC50 is less than 0.1, then no in vivo drug-drug interaction studies are required. The unbound [I]1 for TUDCA and PB are 1.62 and 148 μM, respectively (Table 28). For both TUDCA (1.62 / [0.0741-2.22]) and PB (148 / [6.76-20.3]), the ratios will be greater than 0.1. Since the ratios are greater than 0.1, in vivo drug-drug interaction studies with OAT1 substrates are recommended for AMX0035.

[0562] [Table 51] Uptake of PAH in the absence and presence of TUDCA and PB or probenecid (OAT1 IC50 evaluation)

[0563]

[0564] [Table 52] Net influx rate of PAH and percentage inhibition of OAT1 by AMX0035 and probenecid

[0565] NA: Not applicable (ii) OCT2

[0566] At all concentrations, in all three replicates, the measured concentrations of TUDCA and PB in the dosing solutions in the OCT2 IC50 assessment were within 80-120% of the nominal values, indicating that the preparation of the test article dosing solutions was acceptable. Detailed annotations for solutions 1 to 8 are described in Table 37. The preparation of the MPP+ dosing solution was also acceptable.

[0567] In the absence of TUDCA and PB or imipramine, the influx rate ratio of MPP+ was 48.9 (OCT2 compared 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 that the test system is functioning properly.

[0568] In the presence of TUDCA and PB, the average influx rate of MPP+ through OCT2 was 680 to 991 pmol / mg protein / minute (Table 53). AMX0035 (a combination of TUDCA and PB) did not inhibit the uptake of MPP+ mediated by OCT2 in the tested concentration range. Since the inhibition percentage was less than 50%, the estimated IC50 for OCT2 would be greater than 162 μM and greater than 14785 μM, respectively.

[0569] [Table 53] Uptake of MPP+ in the absence and presence of TUDCA and PB or imipramine (OCT2 IC50 assessment)

[0570]

[0571] [Table 54] Net influx rate of MPP+ and percentage inhibition of OCT2 by AMX0035 and imipramine

[0572]

[0573] a Negative inhibition percentages indicate no inhibition.

[0574] The criteria used to evaluate in vitro inhibition of OCT2 are the same as those for OAT1. The unbound [I]1 for TUDCA and PB are 1.62 and 148 μM, respectively (Table 28). For both TUDCA (1.62 / [>162]) and PB (148 / [>14785]), the ratios will be less than 0.1. Since the ratios are less than 0.1, in vivo drug-drug interaction studies with OCT2 substrates are not recommended for AMX0035.

[0575] (iii) OAT3

[0576] At all concentrations, in all three replicates, the measured concentrations of TUDCA and PB in the dosing solutions in the OAT3 IC50 assessment were within 80-120% of the nominal values, indicating that the preparation of the test article dosing solutions was acceptable. Detailed annotations for solutions 1 to 8 are described in Table 37. The preparation of furosemide was also acceptable.

[0577] In the absence of TUDCA and PB or probenecid, the ratio of the influx rate of furosemide was 20.1 (OAT3 compared 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 that the test system is functioning properly.

[0578] In the presence of TUDCA and PB, the mean influx rate of furosemide through OAT3 ranged from 11.3 to 62.0 pmol / mg protein / min (Table 55). At the highest concentration tested, AMX0035 (a combination of TUDCA and PB) showed greater than 50% inhibition (80.3%) of OAT3-mediated furosemide uptake (Table 56).

[0579] [Table 55] Uptake of furosemide in the absence and presence of TUDCA and PB or probenecid (OAT3 IC50 evaluation)

[0580]

[0581] In the presence of solution 2 (54.0 μM TUDCA and 4928 μM PB), the inhibition of OAT3 was 46.8%; in the presence of solution 1 (162 μM TUDCA and 14785 μM PB), the inhibition of OAT3 was 80.3% (Table 56). Therefore, the estimated IC50 for OAT3 will be between 5.40 and 162 μM TUDCA, with 4928 and 14785 μM PB, respectively.

[0582] [Table 56] Net influx rate of furosemide and percentage inhibition of OAT3 by AMX0035 and probenecid

[0583]

[0584] The criteria used to evaluate in vitro inhibition of OAT3 are the same as those for OAT1. The unbound [I]1 of TUDCA and PB were 1.62 and 148 μM, respectively (Table 28). The ratio of TUDCA (1.62 / [54.0-162]) and PB (148 / [4928-14785]) will be less than 0.1. Since this ratio is less than 0.1, in vivo drug-drug interaction studies with OAT3 substrates are not recommended for AMX0035.

[0585] (iv) OATP1B1 and OATP1B3

[0586] The measured concentrations of TUDCA and PB in the dosing solutions in the OATP1B1 and OATP1B3 IC50 assessments were within the range of 80-120% of the nominal values ​​in at least two of the three replicates at all concentrations, indicating that the preparation of the test article dosing solutions was acceptable. Detailed annotations for Solution 1 to Solution 8 are described in Table 37. The preparation of atorvastatin was also acceptable.

[0587] In the absence of TUDCA and PB or rifamycin SV (RSV), the influx rate ratios of atorvastatin were 15.1 (OATP1B1 compared to vector control) and 8.23 ​​(OATP1B3 compared 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 that the test system is functioning properly.

[0588] In the presence of TUDCA and PB, the mean influx rates of atorvastatin through OATP1B1 ranged from 0.660 to 6.26 pmol / mg protein / min, and the mean influx rates through OATP1B1 ranged from 0.813 to 2.93 pmol / mg protein / min (Table 57).

[0589] [Table 57] Uptake of atorvastatin in the absence and presence of TUDCA and PB or rifamycin SV (OATP1B1 and OATP1B3 IC50 evaluation)

[0590]

[0591] [Table 58] Net influx rate of atorvastatin and percentage inhibition of AMX0035 and RSV on OATP1B1 and OATP1B3

[0592]

[0593] In the presence of solution 4 (12.9 μM TUDCA and 1208 μM PB), the inhibition rate of OATP1B1 was 38.9%, and in the presence of solution 3 (38.7 μM TUDCA and 3625 μM PB), the inhibition rate of OATP1B1 was 65.1% (Table 58). Therefore, the estimated IC50 for OATP1B1 will be between 12.9 and 38.7 μM TUDCA, with 1208 and 3625 μM PB, respectively. Similarly, the estimated IC50 for OATP1B3 will be between 38.7 and 116 μM TUDCA, with 3625 and 10875 μM PB, respectively.

[0594] Currently, the FDA uses the maximum unbound plasma inhibitor concentration (UNBPI) at the liver entrance for oral administration. 入口，最大 ). If the unbound I 入口，最大 If the ratio to IC50 is greater than 0.1, the test article may inhibit OATP1B1 / 3. 入口，最大 3.48 and 326 μM respectively (Table 28).

[0595] For OATP1B1, the ratio for TUDCA is [3.48 / (12.9-38.7)]; for PB, the ratio for TUDCA is [326 / (1208-3625)]. Based on the current data, it is unclear whether this ratio is greater than 0.1, as the IC50 cannot be estimated more accurately than a range of concentrations.

[0596] For OATP1B3, the ratios for TUDCA [3.48 / (38.7-116)] and PB [326 / (3625-10875)] would be less than 0.1. Since the ratios are less than 0.1, in vivo drug-drug interaction studies with OATP1B3 substrates are not recommended for AMX0035.

[0597] (v)MATE1 and MATE2K

[0598] In at least two of the three replicates at all concentrations, the measured concentrations of TUDCA and PB in the dosing solutions were within the range of 80-120% of the nominal values ​​in the MATE1 and MATE2K IC50 assessments, indicating that the preparation of the test article dosing solutions was acceptable. Detailed annotations for solutions 1 to 8 are described in Table 37. The preparation of the metformin dosing solution was also acceptable.

[0599] In the absence of TUDCA and PB or positive inhibitors, the influx rate ratios of metformin were 43.4 (MATE1 compared to vector control, Table 59) and 2.70 (MATE2K compared to 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 that the test system is functioning properly.

[0600] In the presence of TUDCA and PB, the average influx rates of metformin through MATE1 ranged from 189 to 435 pmol / mg protein / min (Table 59) and the average influx rates through MATE2K ranged from 36.7 to 52.0 pmol / mg protein / min (Table 60).

[0601] [Table 59] Uptake of metformin in the absence and presence of TUDCA and PB or cimetidine (MATE1 IC50 evaluation)

[0602]

[0603] In the presence of solution 2 (270 μM TUDCA and 24642 ​​μM PB), the inhibition of MATE1 was 37.5%, and in the presence of solution 1 (810 μM TUDCA and 73927 μM PB), the inhibition of MATE1 was 68.9% (Table 61). Therefore, the estimated IC50 for MATE1 will be between 270 and 810 μM TUDCA, with 24642 ​​and 73927 μM PB, respectively. Similarly, the estimated IC50s of TUDCA and PB for MATE2K will be greater than 810 μM TUDCA and greater than 73927 μM, respectively.

[0604] [Table 60] Uptake of metformin in the absence and presence of TUDCA and PB or cimetidine (MATE2KIC50 assessment)

[0605]

[0606] [Table 61] Net influx rate of metformin, inhibition percentage and IC50 of TUDCA and PB on MATE1 and MATE2K

[0607]

[0608] Currently, the FDA has the following ratio criteria for the evaluation of test articles as in vitro inhibitors of MATE1 and MATE2K: If the ratio of unbound [I]1 / IC50 is greater than 0.02, the test article has the potential to inhibit MATE1 / 2K. 最大 1.62 μM and 148 μM respectively (Table 28).

[0609] For MATE1, the ratios of TUDCA [1.62 / (270-810)] and PB [148 / (24642-73927)] to their IC50s will be less than 0.02. Similarly, for MATE2K, the ratios will be 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.

[0610] P-gp and BCRP IC50 assessment (i) P-gp and IC 50 Evaluate

[0611] The concentrations of TUDCA and PB in the dosing solutions for P-gp IC50 evaluation in MDR1-MDCK cells were determined. The measured concentrations of each component were within 80-120% of the nominal value of each component in at least two of the three replicates, indicating that the preparation of the test article dosing solutions was acceptable. Detailed annotations for solutions 1 to 8 are described in Table 39. The preparation of digoxin was acceptable in all cases except in the presence of solution 2.

[0612] In MDR1-MDCK cells, in the absence of TUDCA and PB or valspodar, the efflux ratio of digoxin was 304, which was reduced to 1.23 by the addition of valspodar (Table 62), equivalent to 99.9% inhibition (Table 63). These results indicate that P-gp is functioning properly in the test system.

[0613] In the presence of TUDCA and PB, the efflux ratio of digoxin was 60.4-270 (Table 62). In the presence of solution 2 (2668 μM TUDCA and 21482 μM PB), the inhibition rate of P-gp was 80.4% (greater than 50%), but it was variable at low concentrations (Table 63). This variability was due to cell-to-cell variability rather than TUDCA and PB inhibition of P-gp. Therefore, the estimated IC50 for P-gp was between 889 and 2668 μM TUDCA, with 7161 and 21482 μM PB, respectively.

[0614] Currently, for orally administered drugs, FDA uses the following ratio to assess the potential of a P-gp inhibitor in vitro: the [I]2 of the test article divided by its calculated IC 50 Correspondingly, if [I]2 is equal to IC 50 If the ratio is less than 10, in vivo drug-drug interaction studies with P-gp substrates are not necessary.

[0615] The estimated [I]2 of TUDCA and PB were 8005 and 64447 μM (Table 28). In MDR1-MDCK cells, the predicted ratio of TUDCA was less than 10 [8005 /

[0616] (889~2668)], and the predicted ratio of PB was less than 10 [64447 / (7161~21482)].

[0617] [Table 62] Permeability and recovery of digoxin in the absence and presence of TUDCA and PB or valspodar in MDR1-MDCK cells

[0618]

[0619] a Value P app It is calculated using formula (7-C).

[0620] b LY's P app Greater than 0.8×10 -6 cm / s, which indicates that the integrity of the cell monolayer is damaged. app are excluded from the calculation of the average.

[0621] NA: Not applicable.

[0622] [Table 63] Corrected efflux ratio of digoxin and inhibition by TUDCA, PB and valspodar (MDR1-MDCK cells)

[0623] deal with Corrected efflux ratio Inhibition percentage 10 μM Digoxin 303 NA 10 μM Digoxin + Solution 1 NA NA 10 μM Digoxin + Solution 2 59.4 80.4 10 μM Digoxin + Solution 3 206 32.0 10 μM Digoxin + Solution 4 180 40.6 10 μM Digoxin + Solution 5 257 15.1 10 μM Digoxin + Solution 6 252 16.7 10 μM Digoxin + Solution 7 269 11.0 10 μM Digoxin + Solution 8 181 40.2 10μM digoxin + 1μM pentaspodar 0.226 99.9

[0624] NA: Not applicable.

[0625] The concentrations of TUDCA and PB in the dosing solutions for P-gp IC50 evaluation in C2BBe1 cells were determined. The measured concentrations of each component were within the range of 80-120% of the nominal value of each component in at least two of the three replicates, indicating that the preparation of the test article dosing solutions was acceptable. Detailed annotations for solutions 1 to 8 are described in Table 39. The preparation of digoxin was also acceptable.

[0626] In C2BBe1 cells, in the absence of TUDCA and PB or valspodar, the efflux ratio of digoxin was 20.5, which was reduced to 1.14 by the addition of valspodar (Table 64), equivalent to 99.3% inhibition (Table 65). These results indicate that P-gp is functioning properly in the test system.

[0627] In the presence of TUDCA and PB, the efflux ratio of digoxin was 2.80-18.9 (Table 64). The inhibition rate of P-gp was 69.2% (greater than 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). Therefore, the estimated IC50 for P-gp will be between 222 and 667 μM TUDCA, with 1790 and 5371 μM PB, respectively.

[0628] Currently, for orally administered drugs, FDA uses the following ratio to assess the potential of a P-gp inhibitor in vitro: the [I]2 of the test article divided by its calculated IC 50 Correspondingly, if [I]2 is equal to IC 50 If the ratio is less than 10, in vivo drug-drug interaction studies with P-gp substrates are not required.

[0629] The estimated [I]2 of TUDCA and PB were 8005 and 64447 μM, respectively (Table 28). In C2BBE1 cells, the predicted ratio of TUDCA was less than 10 [8005 / (222-667)], and the predicted ratio of PB was greater than 10 [64447 / (1790-5371)].

[0630] Based on the current results, in vivo drug-drug interactions of AMX0035 with P-gp substrates may need to be investigated.

[0631] (ii) BCRP IC 50 Evaluate

[0632] The concentrations of TUDCA and PB in the dosing solutions for BCRP IC50 evaluation in BCRP-MDCK cells were determined. The measured concentrations of each component were within 80-120% of the nominal value of each component in at least two of the three replicates, indicating that the preparation of the test article dosing solutions was acceptable. Detailed annotations for solutions 1 to 8 are described in Table 39. The preparation of cladribine was also acceptable.

[0633] In the absence of TUDCA and PB or Ko143, the efflux ratio of cladribine was 223, which was reduced to 0.922 (Table 66) by the addition of Ko143, equivalent to 100% inhibition (Table 67). These results indicate that BCRP is functioning properly in the test system.

[0634] In the presence of TUDCA and PB, the efflux ratio of cladribine was 4.94-264 (Table 66). In the presence of solution 4 (296 μM TUDCA and 2387 μM PB), the inhibition rate of BCRP was 28.2%, and in the presence of solution 3 (889 μM TUDCA and 7161 μM PB), the inhibition rate of BCRP was 90.7% (Table 67). Therefore, the estimated IC50 for BCRP will be between 296 and 889 μM TUDCA, with 2387 and 7161 μM PB, respectively.

[0635] Currently, for orally administered drugs, FDA uses the following ratio to assess the potential of a BCRP inhibitor in vitro: the [I]2 of the test article divided by its calculated IC 50 Correspondingly, if [I]2 is equal to IC 50 If the ratio is less than 10, in vivo drug-drug interaction studies with BCRP substrates are not necessary.

[0636] The estimated [I]2 for TUDCA and PB were 8005 and 64447 μM, respectively (Table 28). In BCRP-MDCK cells, the predicted ratio for TUDCA was [8005 / (296-889)] and for PB was [64447 / (2387-7161)]. Based on the current data, it is unclear whether this ratio is less than 10, as the IC50 cannot be estimated more accurately than a range of concentrations. No clear in vitro-in vivo extrapolation can be made for the inhibition of BCRP using BCRP-MDCK cells.

[0637] [Table 64] Permeability and recovery of digoxin in the absence and presence of TUDCA and PB or valspodar in C2BBe1 cells

[0638]

[0639] a P app The value is calculated using equation (7-C).

[0640] b LY's P app Greater than 0.8×10 -6 cm / s, which indicates that the integrity of the cell monolayer is damaged. app are excluded from the calculation of the average.

[0641] NA: Not applicable.

[0642] [Table 65] Corrected efflux ratio of digoxin and inhibition of TUDCA, PB and valspodar (C2BBE1 cells)

[0643] deal with Corrected efflux ratio Inhibition percentage 10 μM Digoxin 19.5 0 10 μM Digoxin + Solution 1 1.80 90.8 10 μM Digoxin + Solution 2 6.01 69.2 10 μM Digoxin + Solution 3 11.7 40.0 10 μM Digoxin + Solution 4 12.1 38.2 10 μM Digoxin + Solution 5 17.9 8.27 10 μM Digoxin + Solution 6 17.7 9.11 10 μM Digoxin + Solution 7 13.3 31.8 10 μM Digoxin + Solution 8 17.3 11.4 10μM digoxin + 1μM pentaspodar 0.144 99.3

[0644] [Table 66] Permeability and recovery of cladribine in the absence and presence of TUDCA and PB or Ko143 in BCRP-MDCK cells (BCRP-MDCK cells)

[0645]

[0646] a P app The value is calculated using equation (7-C).

[0647] b LY's P app Greater than 0.8×10 -6 cm / s, which indicates that the integrity of the cell monolayer is damaged. app are excluded from the calculation of the average.

[0648] NA: Not applicable.

[0649] [Table 67] Corrected efflux ratio of cladribine and inhibition of TUDCA, PB and Ko143 (BCRP-MDCK cells)

[0650] deal with Corrected efflux ratio Inhibition percentage 10 μM Cladribine 222 NA 10 μM Cladribine + Solution 1 NA NA 10 μM Cladribine + Solution 2 3.94 98.2 10 μM Cladribine + Solution 3 20.7 90.7 10 μM Cladribine + Solution 4 159 28.2 10 μM Cladribine + Solution 5 148 33.2 10 μM Cladribine + Solution 6 263 -18.8 10 μM Cladribine + Solution 7 235 -5.81 10 μM Cladribine + Solution 8 198 10.8 10μM Cladribine + 0.5μM Ko143 -0.0782 100

[0651] NA: Not applicable.

[0652] The concentrations of TUDCA and PB in the dosing solutions for BCRP IC50 evaluation in C2BBe1 cells were determined. The measured concentrations of each component were within 80-120% of the nominal value of each component in at least two of the three replicates, indicating that the preparation of the test article dosing solutions was acceptable. Detailed annotations for solutions 1 to 8 are described in Table 39. The preparation of cladribine was also acceptable.

[0653] In the absence of TUDCA and PB or Ko143, the efflux ratio of cladribine was 27.6, which was reduced to 1.73 by addition of Ko143 (Table 68), corresponding to 97.2% inhibition (Table 69). These results indicate that BCRP is functioning properly in the test system.

[0654] In the presence of TUDCA and PB, the efflux ratio of cladribine was 3.20-25.5 (Table 68). In the presence of solution 3 (222 μM TUDCA and 1790 μM PB), the inhibition rate of BCRP was 46.1%, and in the presence of solution 2 (667 μM TUDCA and 5371 μM PB), the inhibition rate of BCRP was 78.0% (Table 69). Therefore, it is estimated that the IC50 for BCRP will be between 222 and 667 μM TUDCA, with 1790 and 5371 μM PB, respectively.

[0655] Currently, for orally administered drugs, FDA uses the following ratio to assess the potential of a BCRP inhibitor in vitro: the [I]2 of the test article divided by its calculated IC 50 Correspondingly, if [I]2 is equal to IC 50 If the ratio is less than 10, in vivo drug-drug interaction studies with BCRP substrates are not necessary.

[0656] The estimated [I]2 of TUDCA and PB were 8005 and 64447 μM, respectively (Table 28). In C2BBe1 cells, the predicted ratio of TUDCA was 10 [8005 / (222-667)] and the predicted ratio of PB was [64447 / (1790-5371)]. For both TUDCA and PB, the ratios were greater than 10.

[0657] Based on the current data, in vivo drug-drug interactions of AMX0035 with BCRP substrates may need to be investigated.

[0658] P-gp and BCRP substrate assessment

[0659] The concentrations of TUDCA and PB in the dosing solutions for P-gp and BCRP substrate evaluation in C2BBel cells were determined. In at least two of the three replicates, the measured concentrations of each component were within 80-120% of the nominal value of each component, indicating that the preparation of the test article dosing solutions was acceptable. Detailed annotations for solutions 1 to 4 are described in Table 41. Bidirectional permeability of solutions 1, 3, and 4 was run with one batch of C2BBel cells, and bidirectional permeability of solution 2 was run with another batch of cells. The preparation of digoxin and cladribine was also acceptable.

[0660] When the bidirectional permeability of the probe substrate was performed with solutions 1, 3, and 4, the efflux ratios of digoxin and cladribine were 41.6 and 27.0, respectively; when the bidirectional permeability of the probe substrate was performed with solution 2, the efflux ratios of digoxin and cladribine were 38.2 and 19.7, respectively (Table 70). These results indicate that P-gp and BCRP functioned normally in the test system.

[0661] [Table 68] Permeability and recovery of cladribine in the absence and presence of TUDCA and PB or Ko143 in C2BBe1 cells (BCRP IC50 evaluation)

[0662]

[0663] a P app The value is calculated using equation (7-C).

[0664] b LY's P app Greater than 0.8×10 -6 cm / s, which indicates that the integrity of the cell monolayer is damaged. app are excluded from the calculation of the average.

[0665] NA: Not applicable.

[0666] [Table 69] Corrected efflux ratio of cladribine and inhibition of TUDCA, PB and Ko143

[0667] (C2BBe1 cells)

[0668] deal with Corrected efflux ratio Inhibition percentage 10 μM Cladribine 26.6 0 10 μM Cladribine + Solution 1 2.20 91.7 10 μM Cladribine + Solution 2 5.87 78.0 10 μM Cladribine + Solution 3 14.3 46.1 10 μM Cladribine + Solution 4 14.8 44.2 10 μM Cladribine + Solution 5 23.2 12.8 10 μM Cladribine + Solution 6 21.8 18.1 10 μM Cladribine + Solution 7 24.2 9.09 10 μM Cladribine + Solution 8 24.5 7.81 10μM Cladribine + 0.5μM Ko143 0.733 97.2

[0669] [Table 70] Permeability and recovery of digoxin and cladribine (P-gp and BCRP substrate evaluation)

[0670]

[0671] a For use with Sol_1, Sol_3, and Sol_4.

[0672] b For use with Sol_2.

[0673] c P app The value is calculated using equation (7-C).

[0674] Currently, the method for determining the P-gp and BCRP substrate potential of a test article is as follows: If the efflux ratio (determined using Equation 9) is greater than or equal to 2.00, the result indicates a possible substrate for the transporter. In the presence of a transporter-specific inhibitor, the bidirectional permeability of the test article will be further determined, and if the inhibition is greater than 50%, the test article is a substrate for the transporter.

[0675] At all tested concentrations, the efflux ratios of TUDCA and PB were less than 2.00 (Table 71 and Table 72). As the results did not meet the criteria, neither TUDCA nor PB were P-gp or BCRP substrates.

[0676] [Table 71] Permeability and recovery of TUDCA (P-gp and BCRP substrate evaluation)

[0677]

[0678] a P app The value is calculated using equation (7-A).

[0679] [Table 72] Permeability and recovery of PB (P-gp and BCRP substrate evaluation)

[0680]

[0681] a P app The value is calculated using equation (7-A).

[0682] b P app The value is calculated using equation (7-B).

[0683] In conclusion, neither TUDCA nor PB is a substrate for P-gp or BCRP in vitro. The estimated IC50 of TUDCA for each transporter is as follows: 0.0741-0.222 μM (OAT1), greater than 162 μM (OCT2), 54.0-162 μM (OAT3), 12.9-38.7 μM (OATP1B1), 38.7-116 μM (OATP1B3), 270-810 μM (MATE1), greater than 810 μM (MATE2K), 889-2668 μM (P-gp in MDR1-MDCK cells), 222-667 μM (P-gp in C2BBel cells), 296-889 μM (BCRP in BCRP-MDCK cells), and 222-667 μM (BCRP in C2BBel cells). The estimated IC50 of PB for each transporter was as follows: 6.76-20.3 μM (OAT1), greater than 14785 μM (OCT2), 4928-14785 μM (OAT3), 1208-3625 μM (OATP1B1), 3625-10875 μM (OATP1B3), 24642-73927 μM (MATE1), greater than 73927 μM (MATE2K), 7161-21482 μM (P-gp in MDR1-MDCK cells), 1790-5371 μM (P-gp in C2BBel cells), 2387-7161 μM (BCRP in BCRP-MDCK cells), and 1790-5371 μM (BCRP in C2BBel cells).

[0684] Example 7 - Drug-drug interaction study of Tenofovir Disoproxil Fumarate and AMX0035

[0685] The potential drug-drug interaction 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 organic anion transporter OAT1 (SLC22A6) in the kidney, and this study was designed to determine whether coadministration of AMX0035 would alter tenofovir concentrations in plasma or urine in rats.

[0686] This study involved a group of three male (Sprague Dawley) IGS rats. On days 1 to 4, rats were administered 25 mg / kg / dose of TDF once daily by PO gavage at a dose volume of 5 mL / kg to achieve steady-state concentrations of tenofovir before administration of AMX0035. On day 5, rats were administered 840 mg / kg / dose of AMX0035 by PO gavage at a dose of 9.94 mL / kg, followed by 25 mg / kg / dose of TDF by PO gavage at a dose of 5 mL / kg. 1.5 hours after administration of AMX0035 (designed to be close to the T of AMX0035) max ) The dose of tenofovir administered.

[0687] Clinical observations were recorded approximately 1 hour after dosing on days 1-4, twice on day 5, once after AMX0035 administration, and once after TDF administration. Body weight measurements were recorded before randomization and then daily until the end of the study. Plasma samples were collected from all animals at 0.25, 0.5, 1, 2, 4, 8, and 24 hours after dosing on days 1 and 5 (collection on day 5 was 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 after dosing on days 1 and 5 (collection on day 5 was after TDF administration) for calculation of urinary recovery of tenofovir, TDF, PB, and TUDCA.

[0688] Results showed that all rats survived until the scheduled sacrifice time on day 6. No test article-related weight changes or clinical observations were observed. Tenofovir was rapidly formed after systemic absorption of TDF. No prodrug (TDF) was detected in plasma at any time point after administration on day 1 or day 5. In the presence of AMX0035, the mean peak value (C 最大 ) and total (AUC 最后 ) plasma exposure increased by 1.24-fold and 2.17-fold, respectively. The C levels of TUDCA (298 ng / mL) and PB (10200 ng / mL) detected in this study 最大 Plasma levels, both in terms of total drug concentration, exceeded the IC for OAT1 inhibition reported in previous in vitro transporter studies. 50 These results are consistent with the fact that AMX0035 can modestly increase tenofovir exposure (AUC 最后 ), this increase was mediated by inhibition of OAT1.

[0689] Introduction

[0690] The aim of this study was to evaluate whether AMX0035 is an inhibitor of the OAT1 transporter in rats. To this end, this study evaluated the drug-drug interaction of tenofovir disoproxil fumarate (TDF), a known substrate of the organic anion transporter OAT1, with AMX0035. TDF was administered by PO gavage for four consecutive days to achieve steady-state concentrations before AMX0035 was administered by PO gavage on day 5. After AMX0035 administration on day 5, TDF was administered 1.5 hours after AMX0035 administration.

[0691] method

[0692] Experimental design

[0693] The purpose of this study was to evaluate the drug-drug interaction of TDF and AMX0035 after oral administration. TDF was administered once daily for five days. AMX0035 was administered once on day 5, followed by TDF administered 1.5 hours after AMX0035 administration.

[0694] Pharmacokinetic evaluation

[0695] Pharmacokinetic analysis was performed on individual plasma concentration versus time data for tenofovir, PB, and TUDCA using Phoenix WinNonlin non-compartmental analysis (linear trapezoidal rule for AUC calculations). Nominal dose values ​​and nominal sampling times were used for calculations. Any concentration reported as BLQ (<50 ng / mL) was set to zero. For the calculation of AUC on study days 1 and 5, the plasma levels of tenofovir, PB, and TUDCA at time zero were set to zero. The PK parameters for PB and TDF on day 1 and TDF on day 5 were not evaluated because the plasma concentration values ​​were BLQ. Pharmacokinetic analysis included C 最大 , T 最大 , AUC 最后 , T 最后 and AUC 0-24 Determination of.

[0696] Individual and mean tenofovir, TDF, PB, and TUDCA plasma concentration versus time data are presented as standard deviation (SD) reported to three significant figures, and the coefficient of variation percentage (CV%) is reported to one decimal place. Pharmacokinetic parameters are presented as individual values ​​and mean values. 最大 and T 最后 are reported to two significant figures, while all other values ​​are reported to three significant figures. 最大 and T 最后In addition, the mean PK parameters of tenofovir, PB, and TUDCA are presented with three significant figures, and the CV% is presented with one decimal place. 最大 and T 最后 Values ​​are presented to two significant figures.

[0697] Total urine recovery data for tenofovir, TDF, and PB were calculated for the 24-hour collection period on Days 1 and 5. Urine PB concentrations were analyzed for all animals on Day 1, TUDCA was analyzed for all animals on Days 1 and 5, two animals were analyzed for TDF on Day 1, and one animal was BLQ (<20 ng / mL) on Day 5, so recovery data were not calculated. Urine recovery data reported include the amount recovered (ng) and the percentage recovery (%) of TDF alone. Individual and mean urine recovery data for tenofovir, TDF, and PB are presented as SD, reported to three significant figures, and CV% is reported to one decimal place.

[0698] Individual and mean tenofovir, TDF, PB, and TUDCA plasma concentration versus time data are shown in Tables 73-76. Figure 2-5 Tables 77-78 list individual animal and mean tenofovir, PB, and TUDCA PK parameters in male Sprague Dawley rats. Individual and mean tenofovir, TDF, and PB total urine recovery data are shown in Tables 79-80. Figure 6 In the description.

[0699] Pharmacokinetic Abbreviation:

[0700] AUC: Area under the concentration-time curve

[0701] AUC 0-24 : Area under the curve (AUC) from the time of administration to the time of the last measurable concentration 最后 : Area under the curve from the time of administration to the time of the last measurable concentration BLQ: Below the limit of quantification

[0702] C 最大 : Maximum observed concentration

[0703] CV%: coefficient of variation percentage

[0704] N: number of observations

[0705] NA: Not applicable

[0706] SD: Standard deviation

[0707] T 最后 : The time of the last point with a quantifiable concentration

[0708] T 最大 :C最大 Table 73: Individual and mean tenofovir, TDF, PB, and TUDCA plasma concentrations (ng / mL) versus time data following oral administration of TDF alone (Day 1) or AMX0035 and TDF (Day 5) in male Sprague Dawley rats

[0709]

[0710] [Table 74] Individual and mean tenofovir, TDF, PB, and TUDCA plasma concentrations (ng / mL) versus time data following oral administration of TDF alone (Day 1) or AMX0035 and TDF (Day 5) in male Sprague Dawley rats

[0711]

[0712] [Table 75] Individual and mean tenofovir, TDF, PB, and TUDCA plasma concentrations (ng / mL) versus time data following oral administration of TDF alone (Day 1) or AMX0035 and TDF (Day 5) in male Sprague Dawley rats

[0713]

[0714] [Table 76] Individual and mean tenofovir, TDF, PB, and TUDCA plasma concentrations (ng / mL) versus time data following oral administration of TDF alone (Day 1) or AMX0035 and TDF (Day 5) in male Sprague Dawley rats

[0715]

[0716] Note: BLQ = below limit of quantitation (<50 ng / mL)

[0717] [Table 77] Individual and mean PK parameters of tenofovir, PB, and TUDCA in male Sprague Dawley rats following oral administration of TDF alone (Day 1) or AMX0035 and TDF (Day 5)

[0718]

[0719] [Table 78] Individual and mean PK parameters of tenofovir, PB, and TUDCA in male Sprague Dawley rats following oral administration of TDF alone (Day 1) or AMX0035 and TDF (Day 5)

[0720]

[0721] Note: Because the concentration values ​​were BLQ, the PK parameters of PB were not evaluated on day 1, and the PK parameters of TDF were not evaluated on days 1 and 5.

[0722] [Table 79] Individual and mean total urine recovery data of tenofovir, TDF, and PB in male Sprague Dawley rats following oral administration of TDF alone (Day 1) or AMX0035 and TDF (Day 5)

[0723]

[0724] [Table 80] Individual and mean total urine recovery data of tenofovir, TDF, and PB in male Sprague Dawley rats following oral administration of TDF alone (Day 1) or AMX0035 and TDF (Day 5)

[0725]

[0726] Note: Urine concentrations for PB analysis in all animals on day 1, TUDCA analysis in all animals on days 1 and 5, TDF analysis in 2 animals on day 1, and TDF analysis in 1 animal on day 5 were BLQ (20 ng / mL), so no recovery data were calculated.

[0727] Following single oral administration of 25 mg / kg / dose TDF on day 1 and 840 mg / kg / dose AMX0035 + 25 mg / kg / dose TDF on day 5, no quantifiable TDF plasma concentrations were observed in any animal on either sampling day due to the rapid conversion of TDF to the active ingredient tenofovir.

[0728] At the time of the first collection at 0.25 hours post-dose, quantifiable tenofovir concentrations were observed in all animals. The time of individual peaks of tenofovir (T 最大 ) occurred 0.25 hours after dosing on days 1 and 5. The mean C of tenofovir on day 1 最大 The compound was rapidly cleared from the plasma compartment and no compound was detected after 1 hour on day 1 and 2 hours on day 5 (BLQ). However, in the presence of AMX0035, the mean C of tenofovir was 最大 and AUC 最后 In the presence of AMX0035, mean plasma concentrations of tenofovir were higher at every measurable time point (1.24-fold, 1.67-fold, and 1.5-fold at the 0.25, 0.5, and 1 hour time points, respectively).

[0729] TUDCA is an endogenous bile acid and therefore quantifiable on day 1 in the absence of any dose of AMX0035. The mean endogenous levels of TUDCA measured on day 1 ranged from 130 to 222 ng / mL over a 24-hour time course. After dosing with AMX0035 on day 5, the individual T values ​​of TUDCA were 最大 The mean plasma concentrations of TUDCA on Day 5 ranged from 154 ng / mL to 277 ng / mL, with each time point generally 30-50% higher than Day 1, reflecting the dose of AMX0035. The levels of TUDCA in plasma remained fairly constant over the entire time course.

[0730] Quantifiable PB plasma concentrations were observed on day 5 at 0.25 hours after AMX0035 administration and generally remained quantifiable over 4 hours. PB was rapidly absorbed, with individual T values ​​of PB being 最大 This occurred between 0.25 and 0.5 hours after dosing. Similar to tenofovir, PB was rapidly cleared, with undetectable concentrations after the 4-hour time point.

[0731] Previous studies have provided IC50 values ​​for the in vitro inhibition of human OAT1 by TUDCA and PB. These values ​​were 74-222 nM (35-111 ng / mL) for TUDCA and 6.76-20.3 μM (1103-3313 ng / mL) for PB. The peaks (C 最大 ) plasma values ​​all exceeded the IC50 range for OAT1 inhibition.

[0732] Three of the four analytes were detected in the urine. As expected, since bile acids are cleared through the liver, TUDCA was not detected in any urine samples. Although samples were collected at three time points on Days 1 and 5 (0-4, 4-8, and 8-24 hours), no urine was produced by any animal at the 4-8 hour time point on Day 1. In addition, one of the three animals did not produce urine at the 0-4 hour time point. Therefore, four of the nine potential samples on Day 1 did not have sample volume available for measurement. This poses a problem due to the rapid clearance of tenofovir and the need to compare data from Days 1 and 5. Concentrations in samples from these early time points would likely be high and would significantly impact the total amount recovered in the urine.

[0733] in conclusion

[0734] Tenofovir is rapidly formed following systemic absorption of TDF. No prodrug (TDF) was detected in plasma at any time point after dosing on Day 1 or Day 5. The mean peak (C 最大 ) and total (AUC 最后 ) plasma exposure increased by 1.24-fold and 2.17-fold, respectively. The C levels of TUDCA (298 ng / mL) and PB (10200 ng / mL) detected in this study 最大 The plasma levels, in terms of total drug concentration, exceeded the IC50 values ​​for OAT1 inhibition reported in previous in vitro transporter studies (see Example 6). These results are consistent with the modest increase in tenofovir exposure (AUC 最后 ), this increase was mediated by inhibition of OAT1.

[0735] Additional embodiments

[0736] Embodiment 1: A method of treating at least one symptom of amyotrophic lateral sclerosis (ALS) in a subject, the method comprising:

[0737] (a) administering to a subject who has received a first dose of a cytochrome P450 (CYP) substrate an effective amount of a composition comprising about 1 g of taurine diol (TURSO) and about 3 g of sodium phenylbutyrate,

[0738] (b) determining or having determined a first level of the substrate or a metabolite thereof in a first biological sample from the subject, and

[0739] (c) administering to the subject a second dose of the substrate, wherein the second dose is lower than the first dose.

[0740] Embodiment 2: The method of embodiment 1, wherein the CYP is CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 or CYP3A.

[0741] Embodiment 3: The method of Embodiment 1, wherein the CYP is CYP1A2.

[0742] Embodiment 4: The method of Embodiment 3, wherein the substrate is alosetron, caffeine, duloxetine, melatonin, ramelteon, tasimelteon, tizanidine, clozapine, pirfenidone, ramosetron, or theophylline.

[0743] Embodiment 5: The method of Embodiment 1, wherein the CYP is CYP2B6.

[0744] Embodiment 6: The method of Embodiment 5, wherein the substrate is bupropion or efavirenz.

[0745] Embodiment 7: The method of Embodiment 1, wherein the CYP is CYP2C8.

[0746] Embodiment 8: The method of Embodiment 7, wherein the substrate is repaglinide, montelukast, pioglitazone or rosiglitazone.

[0747] Embodiment 9: The method of Embodiment 1, wherein the CYP is CYP2C9.

[0748] Embodiment 10: The method of Embodiment 9, wherein the substrate is celecoxib, glimepiride, phenytoin, tolbutamide or warfarin.

[0749] Embodiment 11: The method of Embodiment 1, wherein the CYP is CYP2C19.

[0750] Embodiment 12: The method of Embodiment 11, wherein the substrate is S-mephenytoin, omeprazole, diazepam, lansoprazole, rabeprazole or voriconazole.

[0751] Embodiment 13: The method of Embodiment 1, wherein the CYP is 2D6.

[0752] Embodiment 14: The method of embodiment 13, wherein the substrate is atomoxetine, desipramine, dextromethorphan, eliglukast (e), nebivolol, nortriptyline, perphenazine, tolterodine, R-venlafaxine, encainide, imipramine, metoprolol, propafenone, propranolol, tramadol, trimipramine or S-venlafaxine.

[0753] Embodiment 15: The method of Embodiment 1, wherein the CYP is CYP3A.

[0754] Embodiment 16: The method of embodiment 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, iplerenone, felodipine, indinavir, lurasidone, maraviroc, quetiapine, sildenafil, ticagrelor, tolvaptan, alprazolam, aprepitant, atorvastatin, colchicine, eliglokast, pimozide, rilpivirine, rivaroxaban or tadalafil.

[0755] Embodiment 17: The method of Embodiment 15, wherein the CYP is CYP3A4.

[0756] Embodiment 18: The method of Embodiment 16, wherein the substrate is alfentanil.

[0757] Embodiment 19: The method of Embodiment 16, wherein the substrate is cyclosporine.

[0758] Embodiment 20: The method of Embodiment 19, wherein the first dose of cyclosporine is about 0.5 to about 15 mg / kg / day.

[0759] Embodiment 21: The method of Embodiment 16, wherein the substrate is quinidine.

[0760] Embodiment 22: The method of Embodiment 1, wherein the substrate of CYP is mexiletine, alfentanil, quinidine, cyclosporine or warfarin.

[0761] Embodiment 23: The method of Embodiment 1, wherein the substrate of the CYP has a narrow therapeutic index.

[0762] Embodiment 24: The method of any one of Embodiments 1-23, further comprising step (d), determining or having determined a second level of the substrate or its metabolite in a second biological sample from the subject.

[0763] Embodiment 25: The method of Embodiment 24, wherein the second level of the substrate or metabolite thereof is lower than the first level.

[0764] Embodiment 26: The method of any one of Embodiments 1-25, wherein the biological sample is a plasma or serum sample.

[0765] Embodiment 27: A method of treating at least one symptom of ALS in a subject, the method comprising:

[0766] (a) administering to a subject who has received a first dose of warfarin an effective amount of a composition comprising about 1 g of TURSO and about 3 g of sodium phenylbutyrate;

[0767] (b) determining or having determined a first blood international normalized ratio (INR) level of the subject; and

[0768] (c) administering to the subject a second dose of warfarin, wherein the second dose is lower than the first dose.

[0769] Embodiment 28: The method of Embodiment 27, wherein the first dose of warfarin is about 0.5 to about 12 mg / day.

[0770] Embodiment 29: The method of Embodiment 27 or 28, wherein step (b) comprises determining or has determined the subject's first blood INR level once a day.

[0771] Embodiment 30: The method of Embodiment 27 or 28, wherein step (b) comprises determining or has determined the subject's first blood INR level once every 1-4 weeks.

[0772] Embodiment 31: The method of any one of Embodiments 27-30, wherein the first blood INR level is about 3.0 or higher.

[0773] Embodiment 32: The method of Embodiment 31, wherein the first blood INR level is about 4.0 or higher.

[0774] Embodiment 33: The method of any one of Embodiments 27-32 further comprises step (d), determining or having determined a second blood INR level of the subject.

[0775] Embodiment 34: The method of Embodiment 33, wherein the second blood INR level is lower than the first blood INR level.

[0776] Embodiment 35: A method of treating at least one symptom of ALS in a subject, the method comprising:

[0777] (a) administering to a subject who has received a first dose of a narrow therapeutic index (NTI) drug an effective amount of a composition comprising about 1 g of TURSO and about 3 g of sodium phenylbutyrate;

[0778] (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

[0779] (c) administering to the subject a second dose of the NTI drug, wherein the second dose is lower than the first dose.

[0780] Embodiment 36: The method of Embodiment 35, wherein the NTI drug is mexiletine, alfentanil, quinidine, cyclosporine, warfarin, or digoxin.

[0781] Embodiment 37: The method of Embodiment 35 or 36 further comprises step (d), determining or having determined a second level of the NTI drug or its metabolite in a second biological sample from the subject.

[0782] Embodiment 38: The method of Embodiment 37, wherein the second level of the NTI drug or its metabolite is lower than the first level.

[0783] Embodiment 39: A method of treating at least one symptom of ALS in a subject, the method comprising:

[0784] (a) administering to a subject who has received 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,

[0785] (b) determining or having determined a first level of the substrate or a metabolite thereof in a first biological sample from the subject; and

[0786] (c) administering to the subject a second dose of the substrate, wherein the second dose is lower than the first dose.

[0787] Embodiment 40: The method of Embodiment 39, wherein the substrate of OAT1 is penicillin, a nonsteroidal anti-inflammatory drug (NSAID), an HIV protease inhibitor, or an antiviral drug.

[0788] Embodiment 41: The method of Embodiment 40, wherein the NSAID is diclofenac, ketoprofen, or methotrexate.

[0789] Embodiment 42: The method of Embodiment 40, wherein the antiviral drug is adefovir, cidofovir or tenofovir.

[0790] Embodiment 43: The method of any one of Embodiments 39-42 further comprises step (d), determining or having determined a second level of the substrate or its metabolite in a second biological sample from the subject.

[0791] Embodiment 44: The method of Embodiment 43, wherein the second level of the substrate or metabolite thereof is lower than the first level.

[0792] Embodiment 45: The method of any one of Embodiments 35-44, wherein the biological sample is a blood sample.

[0793] Embodiment 46: The method of any of Embodiments 1-45, wherein TURSO is administered in an amount of about 1 to about 2 g per day, including 1 g or 2 g per day.

[0794] Embodiment 47: The method of any one of Embodiments 1-46, wherein sodium phenylbutyrate is administered in an amount of about 3 to about 6 g per day, inclusive.

[0795] Embodiment 48: The method of any one of Embodiments 1-47, wherein TURSO is administered in an amount of about 1 g once daily.

[0796] Embodiment 49: The method of any of Embodiments 1-47, wherein TURSO is administered in an amount of about 1 g twice daily.

[0797] Embodiment 50: The method of any one of Embodiments 1-49, wherein sodium phenylbutyrate is administered in an amount of about 3 g once a day.

[0798] Embodiment 51: The method of any of Embodiments 1-49, wherein sodium phenylbutyrate is administered in an amount of about 3 g twice a day.

[0799] Embodiment 52: The method of any of Embodiments 1-51, wherein the composition is administered to the subject orally or via a feeding tube.

[0800] Embodiment 53: The method of any one of Embodiments 1-52, wherein the subject is diagnosed with ALS.

[0801] Embodiment 54: The method of any one of Embodiments 1-52, wherein the subject is suspected of having ALS.

[0802] Embodiment 55: The method of any one of Embodiments 1-54, wherein the subject is human.

[0803] Other Implementations

[0804] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims

1. A method of treating at least one symptom of amyotrophic lateral sclerosis (ALS) in a subject, the method comprising: include: (a) administering a composition comprising taurine diol (TURSO) and sodium phenylbutyrate to a subject who has received a first dose of a substrate for organic anion transporter 1 (OAT1); (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 lower than the first dose.

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

4. 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 of any one of claims 1-4, wherein the substrate of OAT1 has a narrow therapeutic index.

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

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

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

9. A method of treating at least one symptom of ALS in a subject, the method include: (a) administering a composition comprising TURSO and sodium phenylbutyrate to a subject receiving a first dose of a substrate for cytochrome P450 (CYP); (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 lower than the first dose.

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

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

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

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

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

15. The method of any one of claims 9 to 14, further comprising 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.

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

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

18. A method of treating at least one symptom of ALS in a subject, the method include: (a) administering a composition comprising TURSO and sodium phenylbutyrate to a subject who has received a first dose of a narrow therapeutic index (NTI) drug; (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 drug, wherein the second dose is lower than the first dose.

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

20. The method of claim 18 or 19, further comprising 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. The method of claim 20, wherein the second level of the NTI drug or its metabolite is lower than the first level.

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

23. The method of any one of claims 1-21, 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 1-23, wherein the composition is administered to the subject orally or via a feeding tube. E 25. The method of any one of E 1-24, wherein the subject is diagnosed with ALS. E 26. The method of any one of E 1-24, wherein the subject is suspected of having ALS. E 27. The method of any one of E 1-26, wherein the subject is a human.

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