Taurursodiol and sodium phenylbutyrate combination in the treatment of amyotrophic lateral sclerosis

Abstract

Provided herein are methods and compositions for treating a neurodegenerative disease (e.g., ALS) that include administering to a subject a bile acid or a pharma- ceutically acceptable salt thereof and a phenylbutyric acid compound.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 17 / 746,458 (filed May 17, 2022), the entire contents of which are incorporated herein by reference.

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

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

[0004] The disclosure provides a method of treating at least one symptom of ALS in a subject, the method comprising: (a) administering to the subject one or more doses of a composition comprising about 1 g of taurursodiol (TURSO) and about 3 g of sodium phenylbutyrate; (b) administering to the subject (1) a C max is about 3 to about 425 μg / mL, and / or (2) the C of phenylacetic acid max is about 5 to about 50 μg / mL; and (c) further administering the composition to the subject. In some embodiments, the method includes determining that the C of sodium phenylbutyrate in the subject is about 5 to about 50 μg / mL. max In some embodiments, the method further comprises determining that the C of the sodium phenylbutyrate in the subject is about 90 to about 170 μg / mL. maxIn some embodiments, the method further comprises determining that the C of phenylacetic acid in the subject is about 110 to about 150 μg / mL. max In some embodiments, the method further comprises determining that C is about 10 to about 45 μg / mL. max is the steady-state C max It is.

[0005] The specification also provides a method of treating at least one symptom of ALS in a subject, the method comprising: (a) administering to the subject one or more doses of a composition comprising about 1 g of TURSO and about 3 g of sodium phenylbutyrate; (b) determining in the subject (1) an AUC 0-last is about 20 to about 550 μg × h / mL, and / or (2) the AUC of phenylacetic acid 0-last is about 20 to about 160 μg×h / mL, and (c) further administering the composition to the subject. 0-last is about 140 to about 300 μg×h / mL. 0-last is about 40 to about 80 μg×h / mL.

[0006] The specification also provides a method of treating at least one symptom of ALS in a subject, the method comprising: (a) administering to the subject one or more doses of a composition comprising about 1 g of TURSO and about 3 g of sodium phenylbutyrate; (b) determining in the subject (1) an AUC 0-∞ is about 25 to about 545 μg × h / mL, and / or the AUC 0-∞ is about 21 to about 155 μg×h / mL; and (c) further administering the composition to the subject. 0-∞ is about 140 to about 300 μg×h / mL. 0-∞is about 40 to about 80 μg×h / mL.

[0007] The disclosure also provides a method of administering TURSO and sodium phenylbutyrate to a subject exhibiting one or more symptoms of ALS, the method comprising: (a) administering to the subject one or more doses of a composition comprising about 1 g of taurursodiol (TURSO) and about 3 g of sodium phenylbutyrate; (b) administering to the subject (i) a C max is about 3 to about 425 μg / mL, or the C of phenylacetic acid max is about 5 to about 50 μg / mL; (ii) the AUC of sodium phenylbutyrate 0-last is about 20 to about 550 μg × h / mL, or the AUC 0-last is about 20 to about 160 μg × h / mL, or (iii) the AUC of sodium phenylbutyrate 0-∞ is about 25 to about 545 μg × h / mL, or the AUC 0-∞ is about 21 to about 155 μg×h / mL; and (c) further administering the composition to the subject.

[0008] In some embodiments, in the methods described herein, step (a) comprises administering the composition once a day or twice a day for about 1 day to about 40 weeks. In some embodiments, in the methods described herein, step (a) comprises administering the composition once a day or twice a day for about 10 weeks to about 26 weeks. In some embodiments, in the methods described herein, step (a) comprises administering the composition twice a day for about 9 weeks to about 21 weeks. In some embodiments, in the methods described herein, step (a) comprises administering the composition once a day for about 3 weeks, followed by administering the composition twice a day for about 9 weeks to about 21 weeks. In some embodiments, in the methods described herein, step (b) comprises drawing blood from the subject about 1 hour after the last administration of the composition. In some embodiments, in the methods described herein, step (b) comprises drawing blood from the subject about 4 hours after the last administration of the composition.

[0009] The present disclosure also provides a method of treating at least one symptom of ALS in a subject, or administering TURSO and sodium phenylbutyrate to a subject exhibiting one or more symptoms of ALS, the method comprising: (a) administering to the subject one or more times a composition comprising about 1 g of TURSO and about 3 g of sodium phenylbutyrate; (b) measuring in the subject a plasma concentration of one or more bile acids selected from TURSO, UDCA, or GUDCA; and (c) further administering to the subject the composition. In some embodiments, the plasma concentration is a steady-state plasma concentration. In some embodiments, step (a) comprises administering the composition once a day or twice a day for about 1 day to about 40 weeks. In some embodiments, step (a) comprises administering the composition once a day or twice a day for about 10 weeks to about 26 weeks. In some embodiments, step (a) comprises administering the composition twice a day for about 9 weeks to about 21 weeks. In some embodiments, step (a) comprises administering the composition once daily for about 3 weeks, followed by administering twice daily for about 9 weeks to about 21 weeks. In some embodiments, step (b) comprises measuring the plasma concentration about 1 hour after the last administration of the composition. In some embodiments, step (b) comprises measuring the plasma concentration about 4 hours after the last administration of the composition.

[0010] In some embodiments, step (b) comprises measuring the plasma concentration of TURSO in the subject about 1 hour after the last administration of the composition, wherein the plasma concentration of TURSO is about 20 to about 2570 ng / mL. In some embodiments, the plasma concentration of TURSO is about 20 to about 1045 ng / mL. In some embodiments, the plasma concentration of TURSO is about 88 to about 540 ng / mL. In some embodiments, step (b) comprises measuring the plasma concentration of TURSO in the subject about 4 hours after the last administration of the composition, wherein the plasma concentration of TURSO is about 20 to about 3250 ng / mL. In some embodiments, the steady-state plasma concentration of TURSO is about 20 to about 1125 ng / mL. In some embodiments, the steady-state plasma concentration of TURSO is about 155 to about 785 ng / mL.

[0011] In some embodiments, step (b) comprises measuring the plasma concentration of UDCA in the subject about 1 hour after the last administration of the composition, wherein the plasma concentration of UDCA is about 20 to about 6020 ng / mL. In some embodiments, the plasma concentration of UDCA is about 20 to about 1955 ng / mL. In some embodiments, the plasma concentration of UDCA is about 285 to about 1125 ng / mL. In some embodiments, step (b) comprises measuring the plasma concentration of UDCA in the subject about 4 hours after the last administration of the composition, wherein the plasma concentration of UDCA is about 20 to about 7340 ng / mL. In some embodiments, the plasma concentration of UDCA is about 20 to about 2550 ng / mL. In some embodiments, the plasma concentration of UDCA is about 305 to about 1395 ng / mL.

[0012] In some embodiments, step (b) comprises measuring the plasma concentration of GUDCA in the subject about 1 hour after the last administration of the composition, wherein the plasma concentration of GUDCA is about 20 to about 4600 ng / mL. In some embodiments, the plasma concentration of GUDCA is about 65 to about 2085 ng / mL. In some embodiments, the plasma concentration of GUDCA is about 340 to about 1635 ng / mL. In some embodiments, step (b) comprises measuring the plasma concentration of GUDCA in the subject about 4 hours after the last administration of the composition, wherein the plasma concentration of GUDCA is about 20 to about 5290 ng / mL. In some embodiments, the plasma concentration of GUDCA is about 320 to about 2315 ng / mL. In some embodiments, the plasma concentration of GUDCA is about 530 to about 1915 ng / mL.

[0013] In some embodiments, the method further comprises, prior to step (a), determining a baseline plasma concentration of a bile acid in the subject. In some embodiments, the method comprises determining a baseline plasma concentration of TURSO in the subject, wherein the baseline plasma concentration of TURSO is about 20 to about 577 ng / mL. In some embodiments, the baseline plasma concentration of TURSO is about 20 to about 125 ng / mL.

[0014] In some embodiments, the method includes determining a baseline plasma concentration of UDCA in a subject, wherein the baseline plasma concentration of UDCA is about 20 to about 5970 ng / mL. In some embodiments, the baseline plasma concentration of UDCA is about 20 to about 825 ng / mL. In some embodiments, the baseline plasma concentration of UDCA is about 20 to about 53 ng / mL.

[0015] In some embodiments, the method includes determining a baseline plasma concentration of GUDCA in a subject, wherein the baseline plasma concentration of GUDCA is about 20 to about 4540 ng / mL. In some embodiments, the baseline plasma concentration of GUDCA is about 20 to about 755 ng / mL. In some embodiments, the baseline plasma concentration of GUDCA is about 25 to about 180 ng / mL.

[0016] In some embodiments of the methods referred to herein, step (a) comprises administering the composition at least 2 hours after the subject ingests food, or at least 1 hour before the subject ingests food.

[0017] The present specification also provides a method of increasing the plasma concentration of a bile acid in a subject, the method comprising administering to the subject one or more times a composition comprising about 1 g of TURSO and about 3 g of sodium phenylbutyrate, the bile acid being selected from TURSO, UDCA, or GUDCA, and when the bile acid is TURSO, the plasma concentration is about 20 to about 3250 ng / mL, when the bile acid is UDCA, the plasma concentration is about 20 to about 7340 ng / mL, and when the bile acid is GUDCA, the plasma concentration is about 20 to about 5290 ng / mL. In some embodiments, the plasma concentration is a steady state plasma concentration. In some embodiments, the method comprises administering the composition once a day or twice a day for about 1 day to about 40 weeks. In some embodiments, the method comprises administering the composition once a day or twice a day for about 10 weeks to about 26 weeks. In some embodiments, the method includes administering the composition twice daily for about 9 weeks to about 21 weeks. In some embodiments, the method includes administering the composition once daily for about 3 weeks, followed by administering the composition twice daily for about 9 weeks to about 21 weeks. In some embodiments, the method includes measuring the plasma concentration of bile acid about 1 hour after the last administration of the composition. In some embodiments, the method includes measuring the plasma concentration of bile acid about 4 hours after the last administration of the composition.

[0018] In some embodiments of the methods referred to herein, the composition is administered orally. In some embodiments of the methods referred to herein, the composition is administered through a feeding tube. In some embodiments of the methods referred to herein, the composition is injected as a bolus. In some embodiments of the methods referred to herein, the composition is a powder.

[0019] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this invention, suitable methods and materials are described below.

[0020] Certain features of this disclosure are described in the context of separate embodiments for clarity, but it will be understood that they can also be provided in combination in a single embodiment. Conversely, various features of this disclosure are described in the context of a single embodiment for brevity, but they can also be provided separately or in any suitable subcombination. Any combination of the embodiments related to this disclosure is specifically embraced by this disclosure and disclosed in the specification just as if each and every combination were individually expressly disclosed. Furthermore, all subcombinations of the various embodiments and elements thereof are specifically embraced by this disclosure and disclosed in the specification just as if each and every subcombination were individually expressly disclosed herein.

[0021] All publications, patent applications, patents, and other references mentioned in this specification are incorporated by reference in their entirety. In case of conflict, this patent specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the invention will become apparent from the following detailed description and claims. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 is a flow chart of the test carried out in Example 1. [Diagram 2] FIG. 2 is a graph showing the geometric mean (× / ÷ geometric SD) plasma concentrations for PB following a single oral dose of AMX0035 in the fasted and fed state on a Log10 / linear scale. [Diagram 3] FIG. 3 is a graph showing the geometric mean (× / ÷ geometric SD) plasma concentrations for PAA following a single oral dose of AMX0035 in the fasted and fed state on a Log10 / linear scale. [Figure 4] FIG. 4 is a graph showing the geometric mean (× / ÷ geometric SD) plasma concentrations for TURSO following a single oral dose of AMX0035 in the fasted and fed state on a Log10 / linear scale. [Diagram 5]FIG. 5 is a graph showing the geometric mean (× / ÷ geometric SD) plasma concentrations for UDCA following a single oral dose of AMX0035 in the fasted and fed state on a Log10 / linear scale. [Figure 6] FIG. 6 is a graph showing the geometric mean (× / ÷ geometric SD) plasma concentrations for GUDCA following a single oral dose of AMX0035 in the fasted and fed state on a Log10 / linear scale. [Figure 7] Figure 7 shows the TUDCA box plots of visit groups pooled by gender. [Figure 8] Figure 8 shows the GUDCA box plots of visit groups pooled by gender. [Figure 9] Figure 9 shows UDCA box plots for visits pooled by age category. [Figure 10] Figure 10 shows the GUDCA box plots of visits pooled by age category. [Figure 11] Figure 11 shows the TUDCA boxplot of visits pooled by antibiotic use. [Figure 12] FIG. 12 shows UDCA box plots for visits pooled by antibiotic use. [Figure 13] FIG. 13 shows GUDCA boxplots of visits pooled by antibiotic use. [Figure 14] FIG. 14 shows TUDCA box plots of visits pooled by glomerular filtration rate classification. [Figure 15] FIG. 15 shows UDCA box plots for visits pooled by glomerular filtration rate classification. [Figure 16] FIG. 16 shows GUDCA box plots of visits pooled by glomerular filtration rate classification. [Figure 17] FIG. 17 shows the path visualization of changes in CC based on ANOVA results (week 12). [Figure 18] FIG. 18 shows the path visualization of changes in CC based on ANOVA results (week 24). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Detailed Description In one aspect, the disclosure provides a method of treating at least one symptom of ALS in a subject, or administering TURSO and sodium phenylbutyrate to a subject exhibiting one or more symptoms of ALS, the method comprising: (a) administering to the subject at least one dose of a composition comprising about 1 g of taurursodiol (TURSO) and about 3 g of sodium phenylbutyrate; (b) administering to the subject (1) a C of sodium phenylbutyrate; max is about 3 to about 425 μg / mL, and / or (2) the C of phenylacetic acid max is about 5 to about 50 μg / mL, and (c) further administering the composition to the subject. In another aspect, this specification provides a method of treating at least one symptom of ALS in a subject, or administering TURSO and sodium phenylbutyrate to a subject exhibiting one or more symptoms of ALS, the method comprising: (a) administering to the subject one or more doses of a composition comprising about 1 g of TURSO and about 3 g of sodium phenylbutyrate; (b) determining, in the subject, (1) an AUC of sodium phenylbutyrate of 0-last is about 20 to about 550 μg × h / mL, and / or (2) the AUC of phenylacetic acid 0-last is about 20 to about 160 μg×h / mL, and (c) further administering the composition to the subject. In another aspect, this specification provides a method of treating at least one symptom of ALS in a subject, or administering TURSO and sodium phenylbutyrate to a subject exhibiting one or more symptoms of ALS, the method comprising: (a) administering to the subject one or more doses of a composition comprising about 1 g of TURSO and about 3 g of sodium phenylbutyrate; (b) determining, in the subject, (1) an AUC of sodium phenylbutyrate of 0-∞ is about 25 to about 545 μg × h / mL, and / or the AUC 0-∞ is about 21 to about 155 μg×h / mL; and (c) further administering the composition to the subject.

[0024] Yet another aspect of this disclosure includes a method of treating at least one symptom of ALS in a subject, or administering TURSO and sodium phenylbutyrate to a subject exhibiting one or more symptoms of ALS, the method comprising: (a) administering to the subject one or more times a composition comprising about 1 gram of TURSO and about 3 grams of sodium phenylbutyrate; (b) measuring in the subject a plasma concentration of one or more bile acids selected from TURSO, UDCA, or GUDCA; and (c) further administering to the subject the composition.

[0025] Additional aspects of this disclosure include a method of increasing a plasma concentration of a bile acid in a subject, the method comprising administering to the subject one or more times a composition comprising about 3 g of sodium phenylbutyrate and about 1 g of TURSO, wherein the bile acid is selected from TURSO, UDCA, and GUDCA, and wherein when the bile acid is TURSO, the plasma concentration is about 20 to about 3250 ng / mL, when the bile acid is UDCA, the plasma concentration is about 20 to about 7340 ng / mL, and when the bile acid is GUDCA, the plasma concentration is about 20 to about 5290 ng / mL.

[0026] Where ranges are described, it is understood that each intervening value between the upper and lower limits of that range, to one decimal place of the unit of the lower limit unless otherwise clear from the context, and any other stated or intervening value in that stated range, are encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the scope of this disclosure, provided that any limit in the stated range is specifically excluded. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure. It should be noted that throughout the specification, μg-h / mL, μg×h / mL, and μg.h / ml are used interchangeably. Similarly, ng-h / mL, ng×h / mL, and ng.h / ml are used interchangeably.

[0027] Certain ranges provided in this specification are preceded by the term "about." The term "about" is used in this specification to literally support the exact number that is followed by the term, as well as a number that is close to or approximately the number that is followed by the term. In determining whether a number is close to or approximately a specifically recited number, the unrecited near or approximate number may be a number that provides a substantially equivalent number to the specifically recited number in the context provided.

[0028] Unless otherwise defined, all technical terms, symbols, and other scientific or technical terms used in this specification are intended to have the meanings commonly understood by those of ordinary skill in the art to which this application pertains. In some cases, terms having commonly understood meanings are defined in this specification for clarity and / or ease of reference, and the inclusion of such definitions in this specification should not necessarily be construed as representing a substantial difference with respect to what is commonly understood in the art.

[0029] I. Amyotrophic lateral sclerosis (ALS) The terms "amyotrophic lateral sclerosis" and "ALS" are used interchangeably herein and include all of the classifications of ALS known in the art, including, but not limited to, classical ALS (e.g., ALS affecting both lower and upper motor neurons), primary lateral sclerosis (PLS, e.g., affecting only upper motor neurons), progressive bulbar palsy (PBP or bulbar onset, which is a version of ALS that usually begins with difficulty swallowing, chewing, and speaking), and progressive muscular atrophy (PMA, which usually affects only lower motor neurons). These terms include sporadic and familial (inherited) ALS, any rate of progression of ALS (e.g., rapid, non-indolent, or slowly progressing), and any stage of ALS (e.g., presymptomatic, symptomatic, and end-stage ALS).

[0030] The subject in the methods described herein may exhibit one or more symptoms associated with ALS and may have been diagnosed with ALS, hi some embodiments, the subject may be suspected of having and / or at risk of developing ALS.

[0031] The subject in the methods described herein may exhibit one or more symptoms associated with benign fasciculation syndrome (BFS) or cramp-fasciculation syndrome (CFS).

[0032] Some embodiments of the methods described herein can further include determining that a subject has or is at risk for developing ALS, diagnosing the subject as having or at risk for developing ALS, or selecting a subject as having or at risk for developing ALS. Similarly, some embodiments of the methods described herein can further include determining that a subject has or is at risk for developing benign fasciculation syndrome or fasciculation-fasciculation syndrome, diagnosing the subject as having or at risk for developing BFS or CFS, or selecting a subject as having or at risk for developing BFS or CFS.

[0033] In some embodiments of the methods described herein, the subject has exhibited one or more symptoms of ALS for about 24 months or less (e.g., about 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 month or less, or 1 week or less). In some embodiments, the subject has exhibited one or more symptoms of ALS for about 36 months or less (e.g., about 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, or 25 months or less).

[0034] The order and type of ALS symptoms a subject exhibits may depend on which motor neurons in the body are damaged first, and therefore which muscles in the body are damaged first. For example, bulbar-onset, limb-onset, or respiratory-onset ALS may exhibit similar or different symptoms. In general, ALS symptoms may include muscle weakness or atrophy (e.g., affecting the upper body, lower body, and / or speech), muscle fasciculations (twitching), painful muscle spasms, or stiffness of the affected muscles. Early symptoms of ALS may include arm or leg symptoms, difficulty speaking clearly or swallowing (e.g., bulbar-onset ALS). Other symptoms include loss of tongue mobility, difficulty breathing, shortness of breath or abnormal lung function, difficulty chewing, and / or difficulty walking (e.g., stumbling). The initial manifestation of ALS symptoms in a subject may be respiratory muscle weakness. Such subjects may have a very poor prognosis, with a median survival time of about 2 months from diagnosis in some cases. In some subjects, the time of onset of respiratory muscle weakness can be used as a prognostic factor.

[0035] ALS symptoms can also be classified by the location of the neuronal system that is degenerating, i.e., upper motor neuron or lower motor neuron. Lower motor neuron degeneration is manifested, for example, as a decline or weakness in one or more of the medulla oblongata, cervical spinal cord, thoracic spinal cord, and / or lumbosacral spinal cord regions. Upper motor neuron degeneration includes increased tendon reflexes, spasticity, pseudobulbar features, and worsening of reflexes (hyperreflexia), including Hoffman reflex, extensor plantar response, and hypersensitive gag reflex. Progression of neuronal degeneration or muscle weakness is a hallmark of the disease. Thus, some aspects of this disclosure provide methods of improving at least one symptom of lower motor neuron degeneration, at least one symptom of upper motor neuron degeneration, or at least one symptom each of lower motor neuron degeneration and upper motor neuron degeneration. In some aspects of the methods described herein, the onset of symptoms can be determined based on information from the subject and / or the subject's family. In some aspects, the median time from onset of symptoms to diagnosis is about 12 months.

[0036] In some cases, the subject has been diagnosed with ALS. For example, the subject may have been diagnosed with ALS within about 24 months (e.g., about 23, 22, 21, 20, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 month). For example, the subject may have been diagnosed with ALS within one week, or on the same day that the treatment of the disclosure is administered. The subject may have been diagnosed with ALS for more than about 24 months (e.g., about 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or 80 months). Methods for diagnosing ALS are known in the art. For example, the subject can be diagnosed based on medical history, family history, physical or neurological examination (e.g., signs of lower motor neuron or upper motor neuron degeneration). A subject can be confirmed or identified as having ALS, for example, by a medical professional. Multiple parties may be involved during diagnosis. For example, if a sample is obtained from a subject as part of the diagnosis, a first party may obtain the sample from the subject and a second party may test the sample. In some embodiments of human subjects described herein, the subject is diagnosed, selected, or referred by a medical professional (e.g., a general practitioner).

[0037] In some embodiments, the subject meets the El Escorial criteria for probable or definite ALS, i.e., the subject has: Electrophysiological evidence of other disease processes that may explain signs of lower motor neuron (LMN) and / or upper motor neuron (UMN) degeneration; and Neuroimaging evidence of other disease processes that may explain the observed clinical and electrophysiological signs; In addition to the lack of 1. Clinical, electrophysiological or neuropathological evidence of LMN degeneration; 2. Signs of UMN degeneration by clinical examination; and 3. Progressive spread of symptoms within one area or to other areas Shows.

[0038] Under the El Escorial criteria, signs of LMN and UMN degeneration are evaluated in four regions of the central nervous system, including the brainstem, cervical, thoracic and lumbosacral spinal cord. Subjects may be assessed into the following categories: A. Clinically definite ALS, which is defined on the basis of clinical evidence alone by the presence of UMN and LMN signs in three areas; B. Clinically probable ALS, which is defined based on clinical evidence alone, by UMN and LMN signs in at least two areas, with some UMN signs necessarily being more rostral (superior) than the LMN signs. C. Clinically probable ALS, supported by examination, defined as clinical signs of UMN and LMN dysfunction present in only one area, or UMN signs present in only one area and LMN signs as defined by EMG criteria present in at least two of the limbs, and other causes have been excluded by appropriate application of neuroimaging and clinical examination protocols. D. Clinically probable ALS, defined as clinical signs of UMN and LMN dysfunction present simultaneously in only one area or UMN signs present in two or more areas; or LMN signs present more rostral to UMN signs and a clinically probable diagnosis supported by testing.

[0039] In some embodiments, the subject is clinically definite (eg, based on El Escorial criteria) with ALS.

[0040] Subjects can be assessed and / or diagnosed using the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R). The ALSFRS-R is an ordinal rating scale (scored 0-4) used to determine a subject's assessment of their ability and independence in 12 functional activities associated with ALS. The ALSFRS-R score calculated at the time of diagnosis can be compared to scores over time to determine the rate of progression. Changes in ALSFRS-R scores can be correlated with changes in strength over time and can be related to quality of life measures and predicted survival. The ALSFRS-R demonstrates a mean linear slope and can be used as a prognostic indicator (see, e.g., Berry et al. Amyotroph Lateral Scler Frontotemporal Degener 15:1-8, 2014; Traynor et al., Neurology 63:1933-1935, 2004; Simon et al., Ann Neurol 76:643-657, 2014 and Moore et al. Amyotroph Lateral Scler Other Motor Neuron Disord 4:42, 2003).

[0041] In the ALSFRS-R, functions mediated by the cervical spinal cord, trunk, lumbosacral, and respiratory muscles are assessed with three items each. Each item is scored from 0 to 4, with 4 representing no disease involvement and 0 representing maximal involvement. A total is calculated by adding the scores of each item. The total score reflects the impact of ALS and is divided into the following representative categories: >40 (minimal to mild); 39 to 30 (mild to moderate); <30 (moderate to severe); <20 (advanced disease).

[0042] For example, a subject's ALSFRS-R score (e.g., baseline ALSFRS-R score) can be 40 or greater (e.g., at least 41, 42, 43, 44, 45, 46, 47, or 48), between 30 and 39 (inclusive) (e.g., 31, 32, 33, 34, 35, 36, 37, or 38), or less than 30 (e.g., 21, 22, 23, 24, 25, 26, 27, 28, or 29). In some embodiments of the methods described herein, the subject's ALSFRS-R score (e.g., baseline ALSFRS-R score) is 40 or less (e.g., 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 or less). In some embodiments, the subject's ALSFRS-R score (e.g., baseline ALSFRS-R score) is 20 or less (e.g., 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or less).

[0043] Since ALS is a progressive disease, generally all patients progress over time. However, the rate of progression varies widely between subjects, with some subjects dying or needing respiratory support within a few months, while others have a relatively long survival time. The subjects described herein may suffer from fast-progressing ALS or slow-progressing ALS. The rate of functional decline of subjects with ALS can be measured by the change in ALSFRS-R score per month. For example, the score may decrease by about 1.02 (±2.3) points per month.

[0044] One predictor of progression is the patient's historical disease progression rate (ΔFS), which is: ΔFS = (48-ALSFRS-R score at evaluation) / time from onset to evaluation (months) The ΔFS score represents the reduction in ALSFRS-R score at one month from the onset of symptoms and can be a significant predictor of progression and / or survival in subjects with ALS (see, e.g., Labra et al. J Neurol Neurosurg Psychiatry 87:628-632, 2016 and Kimura et al. Neurology 66:265-267, 2006). The subject's disease progression rate (ΔFS) may be about 0.50 or less (e.g., about 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, or 0.10 or less); about 0.50 to about 1.20 (inclusive) (e.g., about 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00, 1.05, 1.10, or 1.15); or about 1.20 or more (e.g., about 1.25, 1.30, 1.35, 1.40, 1.45, 1.50, 1.55, 1.60, 1.75, 1.80, 1.85, 1.90, 1.95, or 2.00 or more). In some embodiments of the methods described herein, the subject's ALS progression rate (ΔFS) may be about 0.50 or greater (e.g., about 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00, 1.05, 1.10, 1.15, 1.20, 1.25, 1.30, 1.35, 1.40, 1.45, 1.50, 1.55, 1.60, 1.75, 1.80, 1.85, 1.90, 1.95, or 2.00 or greater). However, it should be noted that the ΔFS score is a predictor of patient progression and, once assessed, may under- or over-estimate a patient's progression.

[0045] In some embodiments, the subject experiences an average decrease in ALSFRS-R score of about 0.8 to about 2 points (e.g., about 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9) per month from 3 to 12 months since the initial assessment. In some embodiments, the subject experiences an average decrease in ALSFRS-R score of more than about 1.2 points per month from 3 to 12 months since the initial assessment. The subject may experience an decrease in ALSFRS-R score of at least 3 points (e.g., at least 4, 6, 8, 10, 12, 14, 16, 20, 24, 28, or 32 points) from 3 to 12 months since the initial assessment. In some embodiments, the subject has experienced an average decline in the ALSFRS-R score of about 0.8 to about 2 points (e.g., about 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9 points) per month over the past 3 to 12 months. In some embodiments, the subject has experienced an average decline in the ALSFRS-R score of more than about 1.2 points (e.g., about 1.5, 1.8, 2.0, 2.5, or 3 points) per month over the past 3 to 12 months.

[0046] In some embodiments of the methods described herein, the presence or level of a marker in a sample obtained from a subject may be used for the diagnosis or prognosis of ALS, or to track disease activity and treatment response. Suitable samples include, for example, cells, tissues, or bodily fluids (e.g., blood, urine, or cerebrospinal fluid (CSF) samples). For example, the level of phosphorylated neurofilament heavy chain subunit (pNF-H) or neurofilament light chain (NfL) in CSF and / or blood can be used as a biomarker for the diagnosis or prognosis of ALS, or to track disease activity or treatment endpoints. pNF-H is a major component of the neuronal cytoskeleton and is released into the CSF and bloodstream upon neuronal injury. The level of pNF-H may correlate with the level of axonal loss and / or the burden of motor neuron dysfunction (see, for example, De Schaepdryver et al. Journal of Neurology, Neurosurgery & Psychiatry 89:367-373, 2018).

[0047] The concentration of pNF-H in the CSF and / or blood of ALS subjects may be significantly increased in the early stages of the disease. Higher levels of pNF-H in plasma, serum and / or CSF may be associated with faster ALS progression (e.g., faster ALSFRS-R decline) and / or shorter survival time. Plasma pNF-H concentrations may be higher in ALS subjects with bulbar onset than in ALS subjects with spinal onset. In some cases, an imbalance between the relative expression levels of neurofilament heavy chain subunits and the relative expression levels of neurofilament light chain subunits can be used to diagnose, prognose or track disease progression in ALS.

[0048] Methods for detecting pNF-H and NfL (e.g., in cerebrospinal fluid, plasma, or serum) are known in the art and include ELISA and Simoa assays (see, e.g., Shaw et al. Biochemical and Biophysical Research Communications 336:1268-1277, 2005; Ganesalingam et al. Amyotroph Lateral Scler Frontotemporal Degener 14(2):146-9, 2013; De Schaepdryver et al. Annals of Clinical and Translational Neurology 6(10): 1971-1979, 2019; Wilke et al. Clin Chem Lab Med 57(10):1556-1564, 2019; Poesen et al. Front Neurol 9:1167, 2018; Pawlitzki et al. Front. Neurol. 9:1037, 2018; Gille et al. al. Neuropathol Appl Neurobiol 45(3):291-304, 2019), but are not limited thereto. pNF-H detection assays developed and commercialized by EnCor Biotechnology, BioVendor and Millipore-EMD can also be used. Commercial NfL assay kits made by Quanterix and based on Simoa technology can also be used (see, e.g., Thouvenot et al. European Journal of Neurology 27:251-257, 2020). Factors in serum or plasma associated with disease course that affect pNF-H and NfL levels or their detection may differ from those in CSF. Levels of neurofilaments (e.g., pNF-H and / or NfL) in CSF and serum may be correlated (see, e.g., Wilke et al. Clin Chem Lab Med 57(10):1556-1564, 2019).

[0049] A subject described herein has a CSF or blood pNF-H level of about 300 pg / mL or greater (e.g., about 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, , 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 3000, 3200, 3500, 3800, or 4000 pg / mL or more). In some embodiments, the serum pNF-H level may be about 70 to about 1200 pg / mL (e.g., about 70 to about 1000, about 70 to about 800, about 80 to about 600, or about 90 to about 400 pg / mL). In some embodiments, the level of pNF-H in the CSF may be about 1000 to about 5000 pg / mL (eg, about 1500 to about 4000 or about 2000 to about 3000 pg / mL).

[0050] The subject's CSF or blood level of NfL may be about 50 pg / mL or more (e.g., about 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 pg / mL or more). In some embodiments, the NfL level in serum may be about 50 to about 300 pg / mL (e.g., about 50 to about 280, about 50 to about 250, about 50 to about 200, about 50 to about 150, about 50 to about 100, about 100 to about 300, about 100 to about 250, about 100 to about 200, about 100 to about 150, about 150 to about 300, about 150 to about 250, about 150 to about 200, about 200 to about 300, about 200 to about 250, or about 250 to about 300 pg / mL).In some embodiments, the NfL level in the CSF is about 2000 to about 40,000 pg / mL (e.g., about 2000 to about 35,000, about 2000 to about 30,000, about 2000 to about 25,000, about 2000 to about 20,000, about 2000 to about 15,000, about 2000 to about 10,000, about 2000 to about 8000, about 2000 to about 6000, about 2000 to about 4000, about 4000 to about 40,000, about 4000 to about 35,000, about 4000 to about 30,000, about 4000 to about 50,000, about 50 About 25,000, about 4000 to about 20,000, about 4000 to about 15,000, about 4000 to about 10,000, about 4000 to about 8000, about 4000 to about 6000, about 6000 to about 40,000, about 6000 to about 35,000, about 6000 to about 30,000, about 6000 to about 25,000, about 6000 to about 20,000, about 6000 to about 15,000, about 6000 to about 10,000, about 6000 to about 8000, about 8000 to about 40,000, about 8000 to about 35,000 , about 8000 to about 30,000, about 8000 to about 25,000, about 8000 to about 20,000, about 8000 to about 15,000, about 8000 to about 10,000, about 10,000 to about 40,000, about 10,000 to about 35,000, about 10,000 to about 30,000, about 10,000 to about 25,000, about 10,000 to about 20,000, about 10,000 to about 15,000, about 15,000 to about 40,000, about 15,000 to about 35,000, about 15,000 to about 30 ,000, about 15,000 to about 25,000, about 15,000 to about 20,000, about 20,000 to about 40,000, about 20,000 to about 35,000, about 20,000 to about 30,000, about 20,000 to about 25,000, about 25,000 to about 40,000, about 25,000 to about 35,000, about 25,000 to about 30,000, about 30,000 to about 40,000, about 30,000 to about 35,000, or about 35,000 to about 40,000 pg / mL).

[0051] Additional biomarkers useful for diagnosing, prognosing, and monitoring disease progression in ALS are contemplated herein, including, but not limited to, CSF levels of S100-β, cystatin C, and chitotriosidase (CHIT) (see, e.g., Chen et al. BMC Neurol 16:173, 2016). Serum levels of uric acid can be used as a prognostic biomarker for ALS (see, e.g., Atassi et al. Neurology 83(19):1719-1725, 2014). Akt phosphorylation can also be used as a prognostic biomarker for ALS (see, e.g., WO 2012 / 160563). Urinary levels of p75ECD and ketones can be used as biomarkers for diagnosing ALS (see, e.g., Shepheard et al. Neurology 88:1137-1143, 2017). Serum and urinary levels of creatinine can also be used as biomarkers. Other useful blood, CSF, neurophysiological and neuroradiological biomarkers for ALS are described, for example, in Turner et al. Lancet Neurol 8:94-109, 2009. Any of the markers described herein can be used to diagnose a subject as suffering from ALS or to determine that a subject is at risk of developing ALS.

[0052] A subject may also be identified as suffering from or at risk of developing ALS based on genetic analysis. Genetic variants associated with ALS are known in the art (see, for example, Taylor et al. Nature 539:197-206, 2016; Brown and Al-Chalabi N Engl J Med 377:162-72, 2017 and http: / / alsod.iop.kcl.ac.uk). A subject described herein may have a mutation in one or more genes associated with familial and / or sporadic ALS. Representative genes associated with ALS include, but are not limited to, ANG, TARDBP, VCP, VAPB, SQSTM1, DCTN1, FUS, UNC13A, ATXN2, HNRNPA1, CHCHD10, MOBP, C21ORF2, NEK1, TUBA4A, TBK1, MATR3, PFN1, UBQLN2, TAF15, OPTN, TDP-43 and DAO. Further descriptions of genes associated with ALS can be found in Therrien et al. Curr Neurol Neurosci Rep 16:59-71, 2016; Peters et al. J Clin Invest 125:2548, 2015 and Pottier et al. J Neurochem, 138:Suppl 1:32-53, 2016. Genetic variants associated with ALS may affect the rate of ALS progression in a subject, the pharmacokinetics of an administered compound in a subject, and / or the efficacy of an administered compound in a subject.

[0053] The subject may have a mutation in the gene encoding CuZn-superoxide dismutase (SOD1). The mutation makes the SOD1 protein more prone to aggregation, resulting in the accumulation of cellular inclusions containing misfolded SOD1 aggregates (see, e.g., Andersen et al., Nature Reviews Neurology 7:603-615, 2011). Over 100 different mutations in SOD1 have been associated with inherited ALS, many of which result in a single amino acid substitution in the protein. In some embodiments, the SOD1 mutation is A4V (i.e., substitution of alanine for valine at position 4). SOD1 mutations are further described, for example, in Rosen et al. Hum. Mol. Genet. 3, 981-987, 1994 and Rosen et al. Nature 362:59-62, 1993. In some embodiments, the subject has a mutation in the C9ORF72 gene. Repeat expansions in the C9ORF72 gene frequently cause ALS, and both loss of function of C9ORF72 and toxic gain of function of the repeat sequence have been implicated in ALS (see, e.g., Balendra and Isaacs, Nature Reviews Neurology 14:544-558, 2018). The methods described herein can include detecting SOD1 mutations and / or C9ORF72 mutations in a subject prior to administration of bile acid and phenylbutyric acid compounds. Methods for screening for mutations are well known in the art. Suitable methods include, but are not limited to, gene sequencing. See, e.g., Hou et al. Scientific Reports 6:32478, 2016 and Vajda et al. Neurology 88:1-9, 2017.

[0054] Those skilled in the art will appreciate that certain factors can affect the bioavailability and metabolism of a compound administered to a subject and adjustments can be made accordingly. These factors include, but are not limited to, liver function (e.g., liver enzyme levels), kidney function, and gallbladder function (e.g., ion absorption and secretion, cholesterol transport protein levels). There may be variability in each subject's exposure level to the administered compound (e.g., bile acids and phenylbutyric acid compounds), and differences in the excretion level and pharmacokinetics of the compound in treated subjects. Any of the factors described herein may affect the drug exposure to a subject. For example, a decrease in the clearance of a compound may result in increased drug exposure, while improved renal function may reduce actual drug exposure. The degree of drug exposure may correlate with the subject's response to the administered compound and the endpoint of treatment.

[0055] The subjects may be, for example, over about 18 years of age (e.g., 18-100, 18-90, 18-80, 18-70, 18-60, 18-50, 18-40, 18-30, 18-25, 25-100, 25-90, 25-80, 25-70, 25-60, 25-50, 25-40, 25-30, 30-100, 30-90, 30-80, 30-70, 30-60, 30-70, 30-80, 30-90, 30-60, 30-80, 30-90, 30-60, 30-100, 30-100, 30-90, 30-80, 30-70, 30-60, 30-10 ... The subject's BMI may be about 18.5 to 30 kg / m2. 2 (e.g. 18.5~28, 18.5~26, 18.5~24, 18.5~22, 18.5~20, 20~30, 20~28, 20~26, 20~24, 20~22, 22~30, 22~28, 22~26, 22~24, 24~30, 24~28, 24~26, 26~30, 26~28 or 28~30kg / m 2). Having a mutation in an ALS-associated gene described herein, or displaying a biomarker described herein, may indicate that a subject is at risk for developing ALS. Such subjects can be treated by the methods provided herein for prevention and prophylactic purposes.

[0056] In some embodiments, the subject has one or more symptoms of benign fasciculation syndrome (BFS) or fasciculation-fasciculation syndrome (CFS). BFS and CFS are disorders of peripheral nerve hyperexcitability that can cause fasciculations, fasciculations, pain, fatigue, muscle stiffness, and paresthesia. Methods for identifying subjects with these disorders, such as by clinical examination and electromyograms, are known in the art.

[0057] II. Composition The disclosure provides a method of treating at least one symptom of ALS in a subject, the method comprising administering a bile acid or a pharma- ceutically acceptable salt thereof and a phenylbutyrate compound, hi some aspects, the method comprises administering to the subject a composition comprising TURSO and sodium phenylbutyrate.

[0058] bile acids As used herein, "bile acid" refers to naturally occurring surfactants in which a nucleus derived from cholanic acid is substituted with a 3α-hydroxyl group, usually at the C6, C7 or C12 position of the sterol nucleus, and optionally with other hydroxyl groups. Bile acid derivatives (e.g., water-soluble bile acid derivatives) and bile acids conjugated with amines are also encompassed by the term "bile acid". Bile acid derivatives include, but are not limited to, derivatives formed at carbon atoms where the hydroxyl and carboxylic acid groups of the bile acid are bonded to other functional groups, including, but not limited to, halogens and amino groups. Soluble bile acids may include aqueous preparations of the free acid form of the bile acid in combination with hydrochloric acid, phosphoric acid, citric acid, acetic acid, ammonia or arginine. Suitable bile acids include, but are not limited to, taurursodiol (TURSO), ursodeoxycholic acid (UDCA), chenodeoxycholic acid (also referred to as "chenodiol" or "enoic acid"), cholic acid, hyodeoxycholic acid, deoxycholic acid, 7-oxolithocholic acid, lithocholic acid, iododeoxycholic acid, iocholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, glycoursodeoxycholic acid, taurocholic acid, glycocholic acid, or analogues, derivatives or prodrugs thereof.

[0059] In some embodiments, the bile acid disclosed herein is a hydrophilic bile acid. Hydrophilic bile acids include, but are not limited to, TURSO, UDCA, chenodeoxycholic acid, cholic acid, hyodeoxycholic acid, lithocholic acid, and glycoursodeoxycholic acid. Pharmaceutically acceptable salts or solvates of the bile acids disclosed herein are also contemplated. In some aspects, bases commonly utilized to form pharma- ceutically acceptable salts of the bile acids disclosed herein include hydroxides of alkali metals including sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals such as aluminum and zinc; organic amines such as ammonia, unsubstituted or hydroxyl-substituted mono-, di-, or trialkylamines, dicyclohexylamine; tributylamine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-(C1-C6)-alkylamines such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; morpholine; thiomorpholine; piperidine; pyrrolidine; and amino acids such as arginine, lysine, and the like.

[0060] The terms "tauroursodeoxycholic acid" (TUDCA) and "taurursodiol" (TURSO) are used interchangeably in this specification.

[0061] The bile acids described herein have the formula I (with carbons numbered to aid in understanding the positions at which they may be substituted):

[0062] [ka]

[0063] The compound may be TURSO as shown below or a pharma- ceutically acceptable salt thereof.

[0064] The bile acids described herein have the formula II (with carbons numbered to aid in understanding the positions at which they may be substituted):

[0065] [ka]

[0066] The compound may be UDCA as shown in the following formula (I) or a pharma- ceutically acceptable salt thereof.

[0067] The bile acid derivatives disclosed herein may be physiologically relevant bile acid derivatives, for example, combinations of substitution of hydrogen at the 3- or 7-position in the TURSO or UDCA formula, i.e., changes in the stereochemistry of the hydroxyl group at the 3- or 7-position, are suitable for use in the compositions of the present invention.

[0068] A "bile acid" can also be a bile acid conjugated with an amino acid. The amino acid in the conjugate can be, but is not limited to, taurine, glycine, glutamine, asparagine, methionine, or carbocysteine. Other amino acids that can be conjugated with the bile acids disclosed herein include arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, cysteine, proline, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, and tryptophan, as well as β-alanine and γ-aminobutyric acid. One example of such a bile acid is represented by formula III:

[0069] [ka]

[0070] (In the formula, R is -H or C 1 ~C 4 is alkyl; R 1 Ha-CH 2 -SO 3 R 3 , C.H. 2 COOH or CH 2 CH 2 COOH, R 2 is -H; R1 is -COOH, R 2 Ha-CH 2 -CH 2 -CONH 2 , -CH 2 -CONH 2 , -CH 2 -CH 2 -SCH 3 , C.H. 2 CH 2 CH 2 NH(C=NH)NH 2 , C.H. 2 (Imidazolyl), CH 2 CH 2 CH 2 CH 2 NH 2 , C.H. 2 COOH, CH 2 CH 2 COOH, CH 2 OH, CH(OH)CH 3 , C.H. 2 SH, pyrrolidin-2-yl, CH 3 , 2-propyl, 2-butyl, 2-methylbutyl, CH 2 (phenyl), CH 2 (4-OH-phenyl) or -CH 2 -S-CH 2 -COOH; R 3 is -H or an amino acid residue) or a pharma- ceutically acceptable analog, derivative, prodrug, or mixture thereof. An example of an amino acid is a basic amino acid. Other examples of amino acids include glycine, glutamine, asparagine, methionine, carbocysteine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, cysteine, proline, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, and tryptophan, as well as β-alanine and γ-aminobutyric acid.

[0071] Another example of a bile acid disclosed herein has formula IV:

[0072] [ka]

[0073] (In the formula, R is -H or C 1 ~C 4 is alkyl; R 1 Ha-CH 2 -SO 3 R 3 So, R 2 is -H; R 1 is -COOH, R 2 Ha-CH 2 -CH 2 -CONH 2 , -CH 2 -CONH 2 , -CH 2 -CH 2 -SCH 3 or -CH 2 -S-CH 2 -COOH; R 3 is -H or a residue of a basic amino acid) or a pharma- ceutically acceptable analog, derivative, prodrug, or mixture thereof. Examples of basic amino acids include lysine, histidine, and arginine.

[0074] In some embodiments, the bile acid is TURSO. TURSO is an amphipathic bile acid and a taurine-conjugated form of UDCA. TURSO restores mitochondrial bioenergetic defects by incorporating into mitochondrial membranes, decreasing Bax translocation to the mitochondrial membrane, decreasing mitochondrial permeability, and increasing the apoptotic threshold of cells (Rodrigues et al. Biochemistry 42, 10: 3070-3080, 2003). It is used for the treatment of cholesterol gallstones, which generally require long periods of treatment (e.g., 1-2 years) to completely dissolve. It is used to treat cholestatic liver disease, including primary cirrhosis, pediatric familial intrahepatic cholestasis, and cholestasis due to primary sclerosing cholangitis and cystic fibrosis. TURSO is contraindicated in subjects who exhibit biliary tract infections, frequent biliary colic, or who have difficulty absorbing bile acids (e.g., ileal disease or ileal resection). Drug interactions may include those with substances that inhibit the absorption of bile acids, such as cholestyramine, and with drugs that increase the clearance of cholesterol in the bile (TURSO reduces bile cholesterol content). Similarities in drug toxicity and interactions exist between TURSO and UDCA due to similar physicochemical characteristics. The most common adverse reactions reported with the use of TURSO (1% or greater) are abdominal discomfort, abdominal pain, diarrhea, nausea, pruritus, and rash. There have been some cases of pruritus and a limited number of cases of liver enzyme elevations.

[0075] In some embodiments, the bile acid is UDCA. UDCA or ursodiol is used to treat gallstones and is endogenously produced and secreted by the liver as a taurine (TURSO) or glycine (GUDCA) conjugate. Taurine conjugation increases UDCA's solubility by making it hydrophilic. TURSO is taken up in the distal ileum during active transport and therefore likely has a slightly longer intestinal residence time than UDCA, which is taken up in the proximal ileum. Ursodiol therapy is not associated with liver damage. Liver enzyme abnormalities are not associated with Actigall® (Ursodiol USP Capsules) therapy, and Actigall® has been shown to reduce liver enzyme levels in liver disease. However, subjects receiving Actigall® should have SGOT (AST) and SGPT (ALT) measured at the beginning of therapy and thereafter as required by the specific clinical situation. Previous studies have shown that bile acid sequestrants such as cholestyramine and colestipol may interfere with the action of ursodiol by reducing its absorption. Aluminum-based antacids have been shown to adsorb bile acids in vitro and would be expected to interfere with ursodiol in a similar manner to the bile acid sequestrants. Estrogens, oral contraceptives, and clofibrate (and possibly other lipid-lowering drugs) increase hepatic cholesterol secretion and promote cholesterol gallstone formation, and thus may counteract the efficacy of ursodiol.

[0076] Phenylbutyric Acid Compounds Phenylbutyric acid compounds are defined herein to include phenylbutyric acid (low molecular weight aromatic carboxylic acid) as the free acid (4-phenylbutyric acid (4-PBA), 4-phenylbutyric acid or phenylbutyric acid) and its pharma- ceutically acceptable salts, co-crystals, polymorphs, hydrates, solvates, conjugates, derivatives or prodrugs. Phenylbutyric acid compounds described herein include tri-(4-phenylbutyric acid) glycerol, phenylacetic acid (which is the active metabolite of PBA), 2-(4-methoxyphenoxy)acetic acid (2-POAA-OMe), 2-(4-nitrophenoxy)acetic acid (2-POAA-NO 2) and 2-(2-naphthyloxy)acetic acid (2-NOAA), and pharma- ceutically acceptable salts thereof. Phenylbutyric acid compounds also include physiologically relevant species of 4-PBA, such as, but not limited to, substitutions of deuterium for hydrogen in the structure of 4-PBA. Other HDAC2 inhibitors are contemplated herein as alternatives to phenylbutyric acid compounds.

[0077] Physiologically acceptable salts of phenylbutyric acid include, for example, the sodium, potassium, magnesium or calcium salts. Other examples of salts include ammonium, zinc or lithium salts or salts of phenylbutyric acid with organic amines such as lysine or arginine.

[0078] In some embodiments of the methods described herein, the phenylbutyric acid compound is sodium phenylbutyrate. Sodium phenylbutyrate has the following formula:

[0079] [ka]

[0080] It is expressed as:

[0081] Phenylbutyrate is a pan-HDAC inhibitor that can ameliorate ER stress through upregulation of the master chaperone regulator DJ-1 and recruitment of other chaperone proteins (see, e.g., Zhou et al. J Biol Chem. 286: 14941-14951, 2011 and Suaud et al. JBC. 286:21239-21253, 2011). A large increase in chaperone production has been shown to reduce activation of the classical ER stress pathway, refold misfolded proteins, and extend survival in in vivo models, including the G93A SOD1 mouse model of ALS (see, e.g., Ryu, H et al. J Neurochem. 93:1087-1098, 2005).

[0082] In some aspects, a combination of a bile acid (e.g., TURSO) or a pharma- ceutically acceptable salt thereof and a phenylbutyric acid compound (e.g., sodium phenylbutyrate) when dosed in a particular ratio (e.g., as described herein) is synergistically effective in treating one or more symptoms associated with ALS. This combination has been shown to be effective in treating one or more symptoms associated with ALS, e.g., through simultaneous inhibition of endoplasmic reticulum stress and mitochondrial stress, as demonstrated by a powerful oxidative injury model (HIL) by linear modeling. 2 O 2 In some cases, the inhibitors can induce a mathematically synergistic increase in neuronal survival in a number of different neuronal cell types (e.g., neuroprotection-mediated toxicity) (see, e.g., U.S. Pat. Nos. 9,872,865 and 10,251,896).

[0083] formulation The bile acid and phenylbutyric acid compounds described herein can be formulated as or for use in pharmaceutical compositions. For example, the methods described herein can include administering an effective amount of a composition comprising TURSO and sodium phenylbutyrate. The term "effective amount," as used herein, refers to an amount or concentration of one or more drugs effective for administration to cause a desired effect or physiological outcome over a period of time (including acute or chronic administration and regular or continuous administration). The composition can include about 5 to about 15 w / w% (e.g., about 6% to about 14%, about 7% to about 13%, about 8% to about 12%, about 8% to about 11%, about 9% to about 10%, or about 9.7% w / w) TURSO and about 15 to about 45 w / w% (e.g., about 20% to about 40%, about 25% to about 35%, about 28% to about 32%, or about 29% to about 30%, e.g., about 29.2% w / w) sodium phenylbutyrate. In some embodiments, the composition includes about 9.7 w / w% TURSO and 29.2 w / w% sodium phenylbutyrate.

[0084] The sodium phenylbutyrate and TURSO can be present in the composition in a weight ratio of about 1:1 to about 4:1 (e.g., about 2:1 or about 3:1). In some embodiments, the ratio of sodium phenylbutyrate to TURSO is about 3:1.

[0085] The compositions described herein may include pharmaceutically acceptable carriers, adjuvants and / or vehicles. The term "pharmaceutically acceptable carriers or adjuvants" refers to carriers or adjuvants that may be administered to a patient together with the compounds disclosed herein, that do not destroy the pharmacological activity thereof, and that are non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the compound. In this specification, the term "pharmaceutically acceptable carriers" includes saline, solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with the administration of a drug. A pharmaceutical composition may have a conventional non-toxic, pharmaceutically acceptable carrier, adjuvant or vehicle. In some cases, the pH of the formulation may be adjusted with a pharmaceutically acceptable acid, base or buffer to enhance the stability of the compound to be formulated or its delivery form.

[0086] The compositions disclosed herein can include about 8 to about 24% w / w dextrates (e.g., about 9% to about 23%, about 10% to about 22%, about 10% to about 20%, about 11% to about 21%, about 12% to about 20%, about 13% to about 19%, about 14% to about 18%, about 14% to about 17%, about 15% to about 16%, or about 15.6% w / w dextrates). Anhydrous and hydrated dextrates are contemplated herein. Dextrates disclosed herein can include a mixture of sugars resulting from the controlled enzymatic hydrolysis of starch. Some embodiments of the compositions described herein include hydrated dextrates (e.g., NF grades, available from JRS Pharma, Colonial Scientific, or Quadra).

[0087] The compositions disclosed herein can include about 1 to about 6 w / w% sugar alcohol (e.g., about 2% to about 5%, about 3% to about 4%, or about 3.9% w / w sugar alcohol). Sugar alcohols are derived from sugars and can contain one hydroxyl group (-OH) attached to each carbon atom. Disaccharides and monosaccharides can form sugar alcohols. Sugar alcohols can be natural or produced by hydrogenation of sugars. Exemplary sugar alcohols include, but are not limited to, sorbitol, xylitol, and mannitol. In some embodiments, the compositions include about 1 to about 6 w / w% (e.g., about 2% to about 5%, about 3% to about 4%, or about 3.9% w / w) sorbitol.

[0088] The compositions disclosed herein can include about 22 to about 35 w / w% maltodextrin (e.g., about 22% to about 33%, about 24% to about 31%, about 25% to about 32%, about 26% to about 30%, or about 28% to about 29% w / w, e.g., about 28.3% w / w maltodextrin). Maltodextrin dissolves in solution and forms a flexible helical structure that can entrap active ingredients (e.g., any of the phenylbutyric acid compounds and bile acids described herein), thereby masking the taste of the active ingredients. Maltodextrins produced from suitable sources, including, but not limited to, pea, rice, tapioca, corn, and potato, are contemplated herein. In some embodiments, the maltodextrin is pea maltodextrin. In some embodiments, the compositions include about 28.3 w / w% pea maltodextrin. For example, pea maltodextrin available from Roquette (KLEPTOSE® LINECAPS) can be used.

[0089] The compositions described herein can further include a sugar substitute (e.g., sucralose). For example, the compositions can include about 0.5 to about 5 w / w% sucralose (e.g., about 1% to about 4%, about 1% to about 3%, or about 1% to about 2%, e.g., about 1.9% w / w sucralose). Other sugar substitutes contemplated herein include, but are not limited to, aspartame, neotame, acesulfame potassium, saccharin, and advantame.

[0090] In some embodiments, the composition includes one or more flavoring agents. The composition may include about 2 to about 15 w / w% of the flavoring agent (e.g., about 3% to about 13%, about 3% to about 12%, about 4% to about 9%, about 5% to about 10%, or about 5% to about 8%, e.g., about 7.3% w / w). Flavoring agents may include substances that impart a scent to another substance or modify the characteristics of the composition by affecting its taste. Flavoring agents can be used to mask unpleasant tastes without affecting the physical and chemical stability and can be selected based on the taste of the drug involved. Suitable flavoring agents include, but are not limited to, natural flavoring agents, artificial flavoring agents, and simulants. Blends of flavoring agents can also be used. For example, the compositions described herein can include two or more (e.g., 2, 3, 4, 5, or more) flavoring agents. The flavoring agents may be soluble and stable in water. Selection of a suitable flavoring agent can be based on taste testing. For example, different flavoring agents can be added separately to the composition and taste tested. Exemplary flavoring agents include fruit flavor powders (e.g., peach, strawberry, mango, orange, apple, grape, raspberry, cherry, or a mixture of berries). The compositions described herein can include about 0.5 to about 1.5 w / w% (e.g., about 1% w / w) of a mixed berry flavor powder and / or about 5 to about 7 w / w% (e.g., about 6.3% w / w) of a masking flavor. Suitable masking flavors are available, for example, from Firmenich.

[0091] The compositions described herein may further comprise silicon dioxide (or silica). The addition of silica to the composition may prevent or reduce agglomeration of the components of the composition. Silica may function as an anti-caking agent, adsorbent, disintegrant, or glidant. In some embodiments, the compositions described herein comprise about 0.1 to about 2 w / w% porous silica (e.g., about 0.3% to about 1.5%, about 0.5% to about 1.2%, or about 0.8% to about 1%, e.g., 0.9% w / w). Porous silica has a higher H2O content than fumed silica at a relative humidity of about 20% or more (e.g., about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more). 2 The O absorption capacity and / or porosity of the porous silica may be high. 2 The O2 absorption capacity may be about 5 to about 40% by weight (e.g., about 20% to about 40% or about 30% to about 40%) at a relative humidity of about 50%. The porous silica may have a high porosity compared to that of fumed silica at a relative humidity of about 20% or more (e.g., about 30%, 40%, 50%, 60%, 70%, 80%, 90% or more). In some embodiments, the average particle size of the porous silica is about 2 μm to about 10 μm (e.g., about 3 μm to about 9 μm, about 4 μm to about 8 μm, about 5 μm to about 8 μm, or about 7.5 μm). In some embodiments, the average pore volume of the porous silica is about 0.1 cc / gm to about 2.0 cc / gm (e.g., about 0.1 cc / gm to about 1.5 cc / gm, about 0.1 cc / gm to about 1 cc / gm, about 0.2 cc / gm to about 0.8 cc / gm, about 0.3 cc / gm to about 0.6 cc / gm, or about 0.4 cc / gm). In some embodiments, the bulk density of the porous silica is about 50 g / L to about 700 g / L (e.g., about 100 g / L to about 600 g / L, about 200 g / L to about 600 g / L, about 400 g / L to about 600 g / L, about 500 g / L to about 600 g / L, about 540 g / L to about 580 g / L, or about 560 g / L). In some embodiments, the compositions described herein include about 0.05 to about 2 w / w % (eg, any range within this range) of Syloid® 63FP (WR Grace).

[0092] The compositions described herein may further comprise one or more buffering agents. For example, the compositions may comprise about 0.5 to about 5 w / w% of a buffering agent (e.g., about 1% to about 4% w / w, about 1.5% to about 3.5% w / w, or about 2% to about 3% w / w, e.g., about 2.7% w / w of a buffering agent). The buffering agent may comprise a weak acid or base that maintains the acidity or pH of the composition near a selected value after the addition of another acid or base. Suitable buffering agents are known in the art. In some embodiments, the buffering agent in the compositions provided herein is a phosphate salt, such as sodium phosphate (e.g., sodium dihydrogen phosphate anhydrous). For example, the composition may comprise about 2.7 w / w% of dibasic sodium phosphate.

[0093] The composition may also include one or more lubricants. For example, the composition may include about 0.05 to about 1 w / w% of a lubricant (e.g., about 0.1% to about 0.9%, about 0.2% to about 0.8%, about 0.3% to about 0.7%, or about 0.4% to about 0.6%, e.g., about 0.5% w / w of a lubricant). Representative lubricants include, but are not limited to, sodium stearyl fumarate, magnesium stearate, stearic acid, metal stearates, talc, high melting point waxes and glycerides, colloidal silica, polyethylene glycol, alkyl sulfates, glyceryl behenate, and hydrogenated oils. Additional lubricants are known in the art. In some embodiments, the composition includes about 0.05 to about 1 w / w% of sodium stearyl fumarate (e.g., within this range as described herein). For example, the composition may include about 0.5 w / w% of sodium stearyl fumarate.

[0094] In some embodiments, the composition comprises about 29.2 w / w% sodium phenylbutyrate, about 9.7 w / w% TURSO, about 15.6 w / w% dextrates, about 3.9 w / w% sorbitol, about 1.9 w / w% sucralose, about 28.3 w / w% maltodextrin, about 7.3 w / w% flavorings, about 0.9 w / w% silicon dioxide, about 2.7 w / w% sodium phosphate (e.g., sodium hydrogen phosphate), and about 0.5 w / w% sodium stearyl fumarate.

[0095] The composition may include about 3000 mg sodium phenylbutyrate, about 1000 mg TURSO, about 1600 mg dextrates, about 400 mg sorbitol, about 200 mg sucralose, about 97.2 mg silicon dioxide, about 2916 mg maltodextrin, about 746 mg flavorings (e.g., about 102 mg assorted berry flavor and about 644 mg masking flavor), about 280 mg sodium phosphate (e.g., sodium hydrogen phosphate), and about 48.6 mg sodium stearyl fumarate.

[0096] Additional suitable sweeteners or taste masking agents may also be included in the composition, such as, but not limited to, xylose, ribose, glucose, mannose, galactose, fructose, dextrose, sucrose, maltose, steviol glycosides, partial starch hydrolysates, and corn syrup solids. Contemplated herein are soluble saccharin salts (e.g., sodium or calcium saccharin salts), cyclamate salts, acesulfame potassium (acesulfame K), and water-soluble artificial sweeteners such as the free acid form of saccharin and L-aspartyl-phenylalanine methyl ester, aspartame-based sweeteners such as Alitame® or Neotame®. The amount of sweetener or taste masking agent may be varied to select the desired amount of sweetener or taste masking agent for a particular final composition.

[0097] Pharmaceutically acceptable binders are contemplated in addition to those previously described, examples of which include cellulose derivatives including microcrystalline cellulose, low substituted hydroxypropylcellulose (e.g., LH22, LH21, LH20, LH32, LH31, LH30), starches including potato starch, croscarmellose sodium (i.e., cross-linked carboxymethylcellulose sodium salt; e.g., Ac-Di-Sol®), alginic acid or alginates, insoluble polyvinylpyrrolidones (e.g., Polyvidon® CL, Polyvidon® CL-M, Kollidon® CL, Polyplasdone® XL, Polyplasdone® XL-10), and sodium carboxymethyl starch (e.g., Primogel® and Explotab®).

[0098] Polyols, sucrose, sorbitol, mannitol, Erythritol®, Tagatose®, lactose (e.g. spray dried lactose, α-lactose, β-lactose, Tabletose®, various grades of Pharmatose®, Microtose or Fast-Floc®), microcrystalline cellulose (e.g. various grades of Avicel® such as Avicel® PH101, Avicel® PH102 or Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Further excipients, diluents or binders may be included such as cellulose acetate, cellulose esters such as cellulose acetate esters, cellulose esters such as cellulose acetate Tai® and Solka-Floc®, hydroxypropylcellulose, L-hydroxypropylcellulose (low substituted) (e.g. L-HPC-CH31, L-HPC-LH11, LH22, LH21, LH20, LH32, LH31, LH30), dextrins, maltodextrins (e.g. Lodex® 5 and Lodex® 10), starch or modified starches (including potato starch, maize starch and rice starch), sodium chloride, sodium phosphate, calcium sulfate and calcium carbonate.

[0099] The compositions described herein can be formulated or adapted for administration to a subject by any route, e.g., a route approved by the Food and Drug Administration (FDA). Exemplary methods are described in FDA's CDER Data Standards Manual, version number 004 (available at fda.give / cder / dsm / DRG / drg00301.html).

[0100] Pharmaceutical compositions are typically formulated to be compatible with the intended route of administration, which include parenteral (subcutaneous, intradermal, intravenous, intradermal, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial infusion or injection techniques), oral (e.g., via inhalation or feeding tube), transdermal (topical), transmucosal and rectal administration.

[0101] The pharmaceutical composition may be in the form of a solution or powder for inhalation and / or nasal administration. In some embodiments, the pharmaceutical composition is formulated as a powder-in-a-sachet. Suitable powders may include those that are substantially soluble in water. The pharmaceutical composition may be formulated using suitable dispersing or wetting agents (such as, for example, Tween® 80) and suspending agents according to techniques known in the art. The sterile injection preparation may also be a sterile injection solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as, for example, a 1,3-butanediol solution. Acceptable vehicles and solvents that may be used include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, fixed oils are conventionally employed as solvents or suspending media. For this purpose, non-irritating fixed oils may be employed, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the manufacture of injections, as are natural pharma-ceutically acceptable oils such as olive oil or castor oil, especially in polyoxyethylated form. These oil solutions or suspensions may also contain long chain alcohol diluents or dispersants, carboxymethylcellulose, or similar dispersing agents commonly used in formulating pharma- ceutically acceptable dosage forms, such as emulsions and / or suspensions. Other commonly used surfactants, such as Tween® or Span®, and / or other similar emulsifiers or bioavailability enhancers, that are pharma- ceutically acceptable and commonly used in the manufacture of solid, liquid, or other dosage forms, may also be used for the formulation.

[0102] The composition can be orally administered in any orally acceptable dosage form, including, but not limited to, powders, capsules, tablets, emulsions, and aqueous suspensions, dispersions, and solutions. In the case of powders for oral administration, the powder can be substantially dissolved in water before administration. In the case of tablets for oral use, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate may be added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions and / or emulsions are orally administered, the active ingredient may be suspended or dissolved in a combined oily phase using emulsifying and / or suspending agents. If desired, certain sweetening, flavoring, and / or coloring agents may be added.

[0103] Alternatively or additionally, the compositions may be administered by nasal aerosol or inhalation.Such compositions may be prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as saline solutions utilizing benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art.

[0104] In some aspects, the therapeutic compositions disclosed herein can be formulated for sale in the United States, imported into the United States, and / or exported from the United States. The pharmaceutical compositions can be included in a container, pack, or dispenser device along with instructions for administration. In some aspects, the present invention provides kits that include a bile acid and a phenylbutyric acid compound. The kits can also include instructions for the physician and / or patient, syringes, needles, boxes, bottles, vials, and the like.

[0105] III. Treatment Methods / Pharmacokinetics In one aspect, the disclosure provides a method of treating at least one symptom of ALS in a subject, or administering TURSO and sodium phenylbutyrate to a subject exhibiting at least one symptom of ALS, the method comprising: (a) administering to the subject one or more doses of a composition comprising about 1 gram of taurursodiol (TURSO) and about 3 grams of sodium phenylbutyrate; (b) administering to the subject a C of sodium phenylbutyrate in the subject. max , AUC 0-last , and / or AUC 0-∞ and / or the C of the metabolite phenylacetic acid. max , AUC 0-last , and / or AUC 0-∞ determining that the level of f is constant; and (c) further administering the composition to the subject.

[0106] This method is the C max is about 3 to about 425 μg / mL (e.g., about 10 to about 425, about 20 to about 425, about 30 to about 425, about 40 to about 425, about 50 to about 425, about 60 to about 425, about 70 to about 425, about 80 to about 425, about 100 to about 425, about 150 to about 425, about 200 to about 425, about 300 to about 425, about 90 to about 170, or about 110 to about 150 μg / mL).

[0107] Sodium phenylbutyrate is rapidly excreted by metabolism (β-oxidation in the liver and kidney) to the primary metabolite phenylacetic acid (PAA). Therefore, this method allows for the determination of the C of phenylacetic acid of interest. max In some embodiments of the methods described herein, the C may be determined to be about 5 to about 50 μg / mL (e.g., about 9 to about 45, about 9 to about 40, or about 15 to about 35 μg / mL). max is the steady-state C max (e.g. steady-state average C max ).

[0108] This method uses the AUC 0-lastis about 20 to about 550 μg×h / mL (e.g., about 40 to about 500, about 60 to about 450, about 80 to about 400, about 100 to about 350, or about 140 to about 300 μg×h / mL). 0-last In some embodiments of the methods described herein, the AUC 0-last is the steady-state AUC 0-last (e.g. steady-state average AUC 0-last ).

[0109] In some examples, the method includes: (1) determining an AUC of sodium phenylbutyrate in a subject; 0-∞ is about 25 to about 545 μg × h / mL, and / or (2) the AUC of phenylacetic acid 0-∞ is about 21 to about 155 μg×h / mL. For example, the method can include determining an AUC 0-∞ The method can include determining that the AUC of phenylacetic acid in the subject is about 40 to about 500, about 60 to about 450, about 80 to about 400, about 100 to about 350, or about 140 to about 300 μg×h / mL. 0-∞ In some embodiments of the methods described herein, the AUC is about 30 to about 150, about 40 to about 120, or about 40 to about 80 μg×h / mL. 0-∞ is the steady-state AUC 0-∞ (e.g. steady-state average AUC 0-∞ ).

[0110] In some embodiments of the methods described herein, step (a) can include administering the composition once a day or twice a day for about 1 day to about 40 weeks (e.g., about 3 days to about 38 weeks, about 1 week to about 34 weeks, about 3 weeks to about 32 weeks, about 4 weeks to about 32 weeks, about 6 weeks to about 32 weeks, about 8 weeks to about 32 weeks, about 10 weeks to about 32 weeks, about 10 weeks to about 26 weeks, or about 12 weeks to about 24 weeks). For example, the composition can be administered twice a day for about 9 weeks to about 21 weeks. In some examples, the composition is administered once a day for about 3 weeks, followed by administration twice a day for about 9 weeks to about 21 weeks.

[0111] Step (b) of the methods described herein involves drawing blood from the subject about 30 minutes to about 8 hours (eg, about 1, 2, 3, 4, 5, 6, or 7 hours) after the last administration of the composition.

[0112] In another aspect, the disclosure provides a method of treating at least one symptom of ALS in a subject, or administering TURSO and sodium phenylbutyrate to a subject exhibiting at least one symptom of ALS, the method comprising: (a) administering to the subject one or more times a composition comprising about 1 g of TURSO and about 3 g of sodium phenylbutyrate; (b) determining a plasma concentration of one or more bile acids in the subject; and (c) further administering the composition to the subject. For example, the method can include determining a plasma concentration of TURSO, UDCA, GUDCA, cholic acid (CA), CDCA (chenodeoxycholic acid), DCA (deoxycholic acid), glycocholic acid (GCA), glycodeoxycholic acid (GDCA), taurocholic acid (TCA), taurochenodeoxycholic acid (TCDCA), or taurochenodeoxycholic acid (TDCA). In some embodiments, the plasma concentration is a steady-state plasma concentration. In some embodiments, step (b) can include determining the plasma concentration about 30 minutes to about 8 hours (e.g., about 1, 2, 3, 4, 5, 6, or 7 hours) after the last administration of the composition.

[0113] In some embodiments of the above aspects, the method includes determining the plasma concentration of TURSO in the subject about 1 hour after the last administration of the composition, where the plasma concentration of TURSO is about 20 to about 2570 ng / mL (e.g., about 20 to about 2000, about 20 to about 1800, about 20 to about 1500, about 20 to about 1100, about 20 to about 1045, about 40 to about 800, about 60 to about 700, or about 88 to about 540 ng / mL). The plasma concentration of TURSO may also be determined about 4 hours after the last administration of the composition, in which case the plasma concentration is about 20 to about 3250 ng / mL (e.g., about 20 to about 2800, about 20 to about 2500, about 20 to about 2000, about 20 to about 1800, about 20 to about 1500, about 20 to about 1200, about 20 to about 1125, about 50 to about 1000, about 80 to about 900, about 100 to about 800, or about 155 to about 785 ng / mL).

[0114] In some embodiments of the above aspects, the method includes determining a plasma concentration of UDCA in the subject about 1 hour after the last administration of the composition, where the plasma concentration of UDCA is about 20 to about 6020 ng / mL (e.g., about 20 to about 5500, about 20 to about 5000, about 20 to about 4500, about 20 to about 4000, about 20 to about 3500, about 20 to about 3000, about 20 to about 2500, about 20 to about 2000, about 20 to about 1955, about 50 to about 1800, about 80 to about 1500, or about 285 to about 1125 ng / mL). The plasma concentration of UDCA may also be determined about 4 hours after the last administration of the composition, in which case the plasma concentration is about 20 to about 7340 ng / mL (e.g., about 20 to about 7000, about 20 to about 6000, about 20 to about 5000, about 20 to about 4000, about 20 to about 3000, about 20 to about 2550, about 50 to about 2000, about 100 to about 1500, or about 305 to about 1395 ng / mL).

[0115] In some embodiments of the above aspects, the method includes determining the plasma concentration of GUDCA in the subject about 1 hour after the last administration of the composition, where the plasma concentration of GUDCA is about 20 to about 4600 ng / mL (e.g., about 20 to about 4000, about 20 to about 3500, about 20 to about 3000, about 40 to about 2500, about 65 to about 2085, or about 340 to about 1635 ng / mL). The plasma concentration of GUDCA can also be determined about 4 hours after the last administration of the composition, where the plasma concentration is about 20 to about 5290 ng / mL (e.g., about 40 to about 4500, about 80 to about 4000, about 150 to about 3500, about 320 to about 2315 ng / mL, or about 530 to about 1915 ng / mL).

[0116] In some embodiments of the above aspects, the method further comprises, prior to step (a), determining a baseline plasma concentration of a bile acid in the subject. For example, the method can comprise determining a baseline plasma concentration of TURSO in the subject, where the baseline plasma concentration of TURSO is about 20 to about 577 ng / mL (e.g., about 20 to about 400, about 20 to about 300, about 20 to about 200, or about 20 to about 125 ng / mL). The method can comprise determining a baseline plasma concentration of UDCA in the subject, where the baseline plasma concentration of UDCA is about 20 to about 5970 ng / mL (e.g., about 20 to about 5000, about 20 to about 4000, about 20 to about 3000, about 20 to about 2000, about 20 to about 1000, about 20 to about 825, about 20 to about 500, or about 20 to about 52 ng / mL). The method can include determining a baseline plasma concentration of GUDCA in the subject, where the baseline plasma concentration of GUDCA is about 20 to about 4540 ng / mL (e.g., about 20 to about 4000, about 20 to about 3000, about 20 to about 2000, about 20 to about 755, or about 25 to about 180 ng / mL).

[0117] In some embodiments of the methods described herein, the subject fasts for a period of time before the composition is administered. For example, step (a) of the method can include administering the composition 2 hours or more after the subject ingests food, or 1 hour or more before the subject ingests food. In some embodiments, the subject ingests food within 2 hours (e.g., within 1 hour or within 30 minutes) of the composition being administered.

[0118] In yet another aspect, the present disclosure provides a method of increasing the plasma concentration of a bile acid (e.g., any of the bile acids described herein) in a subject, the method comprising administering to the subject one or more doses of a composition comprising about 3 g of sodium phenylbutyrate and about 1 g of TURSO. The composition can be administered according to a suitable dosing regimen disclosed herein. In some embodiments, the bile acid is selected from TURSO, UDCA, and GUDCA. For example, the bile acid can be TURSO, and the plasma concentration of TURSO after administration of the composition is about 20 to about 3250 ng / mL (e.g., any range within this range). The bile acid can be UDCA, and the plasma concentration of UDCA after administration of the composition is about 20 to about 7340 ng / mL (e.g., any range within this range). The bile acid can also be GUDCA, and the plasma concentration of GUDCA after administration of the composition is about 20 to about 5290 ng / mL (e.g., any range within this range).

[0119] This specification relates to administering a composition comprising about 1 g of taurursodiol (TURSO) and about 3 g of sodium phenylbutyrate to a subject (e.g., a healthy subject) who has fasted for 24 hours, and measuring one or more pharmacokinetic parameters (e.g., T max , C max and AUC 0-last Also provided are methods for measuring the T of sodium phenylbutyrate in a subject. max In some embodiments, the C of the subject sodium phenylbutyrate is about 0.25 hours to about 0.50 hours. max In some embodiments, the AUC of sodium phenylbutyrate in a subject is about 64.4 μg / mL to about 260 μg / mL. (0-last)In some embodiments, the AUC of sodium phenylbutyrate in a subject is about 73.5 μg×h / mL to about 423 μg×h / mL. (0-∞) In some embodiments, the TURSO of a subject is about 74.8 μg×h / mL to about 425 μg×h / mL. max In some embodiments, the C of the subject TURSO is about 1.50 hours to about 10.00 hours. max In some embodiments, the AUC of the subject's TURSO is about 0.219 μg / mL to about 1.74 μg / mL. (0-last) In some embodiments, the AUC of the subject's TURSO is about 1.18 μg×h / mL to about 11.6 μg×h / mL. (0-∞) In the embodiments described herein, T max is the steady-state T max (e.g. steady-state average T max In the embodiment described herein, C max is the steady-state C max (e.g. steady-state average C max In the embodiments described herein, AUC (0-last) is the steady-state AUC (0-last) (e.g. steady-state average AUC (0-last) In the embodiments described herein, AUC (0-∞) is the steady-state AUC (0-∞) (e.g. steady-state average AUC (0-∞) ) may be the case.

[0120] This disclosure relates to administering to a subject (e.g., a healthy subject) a composition comprising about 1 g of taurursodiol (TURSO) and about 3 g of sodium phenylbutyrate, and measuring one or more pharmacokinetic parameters (e.g., T) of one or more metabolites of sodium phenylbutyrate and / or TURSO. max , C max and AUC 0-lastAlso provided are methods for measuring the T of phenylacetic acid in a subject. Specifically, phenylacetic acid is believed to be a metabolic product of sodium phenylbutyrate. Furthermore, under physiological conditions, enterohepatic recirculation leads to active deconjugation of TURSO to UDCA by the gut microbiota and reconjugation of UDCA with glycine or taurine in the liver (GUDCA and TURSO, respectively). In some embodiments, the T of phenylacetic acid in a subject is measured. max In some embodiments, the C of the phenylacetic acid of interest is about 1.50 hours to about 3.50 hours. max In some embodiments, the AUC of phenylacetic acid in a subject is about 13.3 μg / mL to about 42.3 μg / mL. (0-last) In some embodiments, the AUC of phenylacetic acid in a subject is about 43.8 μg×h / mL to about 141 μg×h / mL. (0-∞) In some embodiments, the T of UDCA in a subject is about 45.2 μg×h / mL to about 142 μg×h / mL. max In some embodiments, the C of the UDCA of interest is about 0.25 hours to about 20.00 hours. max In some embodiments, the AUC of UDCA in a subject is about 195 ng / mL to about 1380 ng / mL. (0-last) In some embodiments, the AUC of UDCA in a subject is about 1970 μg×h / mL to about 14900 μg×h / mL. (0-∞) In some embodiments, the T of the GUDCA of the subject max In some embodiments, the C of the GUDCA of interest is about 6.00 hours to about 20.00 hours. max In some embodiments, the AUC of GUDCA in a subject is about 143 ng / mL to about 1420 ng / mL. (0-last) In the embodiment described herein, T max is the steady-state T max (e.g. steady-state average T max In the embodiment described herein, C max is the steady-state C max (e.g. steady-state average C max In the embodiments described herein, AUC (0-last) is the steady-state AUC (0-last)(e.g. steady-state average AUC (0-last) In the embodiments described herein, AUC (0-∞) is the steady-state AUC (0-∞) (e.g. steady-state average AUC (0-∞) ) may be the case.

[0121] This specification relates to administering a composition containing about 1 g of taurursodiol (TURSO) and about 3 g of sodium phenylbutyrate to a subject (e.g., a healthy subject) who has consumed a high-fat meal, and measuring one or more pharmacokinetic parameters (e.g., T max , C max and AUC 0-last Also provided are methods for measuring the T of sodium phenylbutyrate in a subject. max In some embodiments, the C of the subject sodium phenylbutyrate is about 0.25 hours to about 1.50 hours. max In some embodiments, the AUC of sodium phenylbutyrate in a subject is about 12.9 μg / mL to about 70.5 μg / mL. (0-last) In some embodiments, the AUC of sodium phenylbutyrate in a subject is about 34.6×h / mL to about 198 μg×h / mL. (0-∞) In some embodiments, the TURSO of a subject is about 76.2 μg×h / mL to about 200 μg×h / mL. max In some embodiments, the C of the subject TURSO is about 4.50 hours to about 10.00 hours. max In some embodiments, the AUC of the subject's TURSO is about 0.435 μg / mL to about 2.16 μg / mL. (0-last) In some embodiments, the AUC of the subject's TURSO is about 2.45 μg×h / mL to about 22.4 μg×h / mL. (0-∞) In the embodiments described herein, T max is the steady-state T max (e.g. steady-state average T max In the embodiment described herein, C max is the steady-state C max (e.g. steady-state average C max In the embodiments described herein, AUC (0-last)is the steady-state AUC (0-last) (e.g. steady-state average AUC (0-last) In the embodiments described herein, AUC (0-∞) is the steady-state AUC (0-∞) (e.g. steady-state average AUC (0-∞) ) may be the case.

[0122] This disclosure relates to administering a composition comprising about 1 g of taurursodiol (TURSO) and about 3 g of sodium phenylbutyrate to a subject (e.g., a healthy subject) who has consumed a high-fat meal, and measuring one or more pharmacokinetic parameters (e.g., T) of one or more metabolites of sodium phenylbutyrate and / or TURSO. max , C max and AUC 0-last Also provided are methods for measuring the T of phenylacetic acid in a subject. max In some embodiments, the C of the subject phenylacetic acid is about 2.00 hours to about 4.50 hours. max In some embodiments, the AUC of phenylacetic acid in a subject is about 8.39 μg / mL to about 36.5 μg / mL. (0-last) In some embodiments, the AUC of phenylacetic acid in a subject is about 30.6 μg×h / mL to about 128 μg×h / mL. (0-∞) In some embodiments, the T of UDCA in a subject is about 31.3 μg×h / mL to about 130 μg×h / mL. max In some embodiments, the C of the UDCA of interest is about 6.00 hours to about 24.00 hours. max In some embodiments, the AUC of UDCA in a subject is about 181 ng / mL to about 6250 ng / mL. (0-last) In some embodiments, the AUC of UDCA in a subject is about 1590 μg×h / mL to about 33400 μg×h / mL. (0-∞) In some embodiments, the T of the GUDCA of the subject is about 8580 μg×h / mL. max In some embodiments, the C of the GUDCA of interest is from about 0.50 hours to about 24.00 hours. max In some embodiments, the AUC of GUDCA in a subject is about 114 ng / mL to about 2430 ng / mL. (0-last)In the embodiment described herein, T max is the steady-state T max (e.g. steady-state average T max In the embodiment described herein, C max is the steady-state C max (e.g. steady-state average C max In the embodiments described herein, AUC (0-last) is the steady-state AUC (0-last) (e.g. steady-state average AUC (0-last) In the embodiments described herein, AUC (0-∞) is the steady-state AUC (0-∞) (e.g. steady-state average AUC (0-∞) ) may be the case. EXAMPLES

[0123] The present invention will be described in detail with specific examples. The following examples are provided for illustrative purposes and are not intended to limit the present invention in any way. Those skilled in the art will readily recognize various non-critical parameters that can be changed or modified to produce essentially the same results. It is understood that certain features of the present invention that are described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the present invention that are described in the context of a single embodiment for brevity may also be provided separately or in any suitable subcombination.

[0124] Example 1 Pharmacokinetic study The pharmacokinetics of AMX0035 are based on plasma concentration data from two studies: a single-dose PK study in healthy volunteers and sparse sampling from patients in clinical efficacy and safety studies.

[0125] Protocol 1. Protocol AMX3500: Evaluation of the safety, tolerability, efficacy and activity of AMX0035, a fixed combination of phenylbutyrate (PB) and tauroursodeoxycholic acid (TUDCA), for the treatment of amyotrophic lateral sclerosis (ALS) A 28-week, multicenter, randomized, double-blind, placebo-controlled Phase II study to investigate the safety, tolerability, efficacy, pharmacokinetics, and bioactivity of AMX0035 Population: 132 men or women aged 18-80 years with sporadic or familial ALS, lung capacity greater than 60% predicted, and onset of ALS symptoms within the past 18 months. Treatment: Subjects will be randomly assigned in a 2:1 ratio to receive oral (or feeding tube) AMX0035 (approximately 88 subjects) or placebo (approximately 44 subjects). Treatment will be as one sachet per day for the first 3 weeks, then increased to one sachet twice daily if tolerated. Sachets in the active treatment group contain 1g TUDCA and 3g PB. Pharmacokinetic Sampling: Blood samples for analysis of plasma concentrations of PB, phenylacetate (PAA), TUDCA, UDCA, and GUDCA will be drawn at the baseline visit (pre-dose) and at treatment visits at weeks 12 and 24. Sampling times will be randomized to occur either 1 or 4 hours post-dose at the week 12 visit and the remaining time at the week 24 visit. Treatment period: 24 weeks 2. Protocol A35-002: A Phase I study of single-dose oral AMX0035 under fasted and fed conditions in healthy volunteers to evaluate plasma pharmacokinetics A Phase I, open-label, two-period, crossover, single-dose study of AMX0035 in healthy adult volunteers to investigate the pharmacokinetics of PB, TUDCA, and major metabolites following a single oral dose of AMX0035 with and without food Population: 40-65 years old, body mass index 18.5-32 kg / m 2 Fourteen healthy male or female volunteers Treatment: Subjects will receive a single dose of one sachet of AMX0035 (1g TUDCA and 3g PB) under fasted (overnight and 4 hours after dosing) and fed (standard high-fat breakfast 30 minutes prior to dosing) conditions with a minimum of 4 days washout between treatments. The order of dosing with and without food will be determined randomly. Pharmacokinetic Sampling: Blood samples for analysis of plasma concentrations of PB, PAA, TUDCA, UDCA, and GUDCA will be collected pre-dose and at 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 10, 12, 16, 20, and 24 hours after a single dose of AMX0035 on both study days. In addition, serial plasma samples will be obtained the day before the first dose of AMX0035 to characterize endogenous concentrations of TUDCA and metabolites.

[0126] Pharmacokinetic data characteristics Protocol AMX3500: Sparse sampling in 87 subjects with ALS. Approximately two samples will be collected for each subject, at 1 hour or 4 hours after administration, after 12 and 24 weeks of treatment. Protocol A35-002: Rich sampling in 14 healthy adults. For each subject, approximately 21 samples will be collected over a 24-hour period, once after a high-fat meal and once under fasting conditions.

[0127] result The primary metabolite of sodium phenylbutyrate (PB) is phenylacetate (PAA). PAA occurs endogenously as a metabolite of phenylalanine. Population PK analyses show that PAA is eliminated in a nonlinear (saturable) manner.

[0128] Population PK analysis showed an inverse correlation between PAA exposure and body weight, with a possible additive effect of fasting on low body weight on increased PAA exposure.

[0129] TURSO, UDCA (ursodiol), and GUDCA (glycoursodeoxycholic acid) are naturally occurring hydrophilic bile acids that represent a minor portion of the total bile acid pool in humans. All three undergo extensive enterohepatic circulation. Oral administration of TURSO results in increased plasma levels of all three.

[0130] Pharmacokinetic parameters following a single dose of ALBRIOZA® under fasting conditions in healthy subjects are shown in Table 1 for phenylbutyrate and the primary metabolite PAA and in Table 2 for ursodoxycortaurine and its metabolites.

[0131] [Table 1]

[0132] [Table 2]

[0133] The steady-state plasma concentrations of ursodoxycortaurine, UDCA, and GUDCA following twice-daily administration of ALBRIOZA® in ALS patients are shown in Table 3. Oral administration of TURSO results in increased plasma levels of all three.

[0134] [Table 3]

[0135] absorption Following a single oral dose of AMX0035 in healthy subjects under fasting conditions (as described in Example 2), sodium phenylbutyrate was rapidly absorbed, with a mean C max The estimated steady-state mean C of sodium phenylbutyrate in ALS patients based on population PK max is 131 μg / mL (range 3.8 to 423).

[0136] Under physiological conditions, bile acids in the intestine are absorbed primarily in the distal part (ileum) by both passive diffusion (unconjugated bile acids) and active uptake by the apical sodium-dependent transporter (ASBT) (conjugated bile acids). Specific transporters in enterocytes ensure that bile acids are directed to the liver via the portal vein, where they undergo extensive enterohepatic recirculation. Bile acids can be recycled between hepatocytes in the liver and enterocytes in the intestine 4 to 12 times per day. After a single oral dose of AMX0035 in healthy subjects under fasting conditions, TURSO showed a mean C max reached 871 ng / mL (range 219-1740). The plasma concentration profile of TURSO in most subjects had 2-3 peaks consistent with storage and release of bile acids during meals / snacks.

[0137] Dietary effects Administration of a single dose of 3 g sodium phenylbutyrate and 1 g TURSO to healthy volunteers with a high-fat, high-calorie meal (approximately 800-1000 calories: 500-600, 250, and 150 calories from fat, carbohydrate, and protein, respectively) decreased the rate and extent of absorption of sodium phenylbutyrate (Cmax and AUC decreased by 75% and 55%, respectively). The high-fat, high-calorie meal did not affect the Cmax of TURSO, but exposure (AUC) increased by 46%. In phase II safety and efficacy studies, patients were advised to take the drug before meals.

[0138] distribution When coadministered in vitro, plasma protein binding of sodium phenylbutyrate and ursodoxycortaurine is 82% and 98%, respectively.

[0139] Under physiological conditions, less than 10% of the total bile acid pool reaches the systemic circulation, as the liver removes most from the hepatic circulation for reuse. Serum concentrations of bile acids reflect the balance between intestinal uptake and hepatic extraction, and hepatic extraction of conjugated bile acids is known to be more efficient than that of unconjugated forms.

[0140] metabolism Sodium phenylbutyrate is rapidly cleared by metabolism (β-oxidation in the liver and kidney) to the potentially pharmacologically active primary metabolite phenylacetate (PAA). Plasma concentrations of the metabolite PAA in healthy subjects were evident from 0.25 hours onwards in all 13 dosed subjects, with mean C max The estimated steady-state mean C in ALS patients based on population PK analysis reached 26.44 μg / mL (range 13.3 to 42.3). max was 24.1 μg / mL (range 9.08 to 46.3).

[0141] Phenylacetate is rapidly conjugated with glutamine in the liver and kidney via acetylation to form phenylacetylglutamine, which is excreted by the kidney.

[0142] Under physiological conditions, the enterohepatic recirculation results in active deconjugation of TURSO to UDCA by the gut microbiota and reconjugation of UDCA with glycine or taurine (GUDCA and ursodoxycortaurine, respectively) in the liver; approximately 95% of bile acids in the gut are (re)absorbed into the enterohepatic circulation. All metabolites resulting from TURSO detected in human hepatocytes in vitro were also detected in rat and minipig hepatocytes.

[0143] Disappearance Phenylacetate exhibits nonlinear pharmacokinetics characterized by saturable metabolism. By 6 hours after dosing, phenylbutyrate and phenylacetate had cleared from the systemic circulation, with estimated terminal half-lives of 0.46 and 0.81 hours, respectively. Based on this rapid elimination of PB and PA, there was no accumulation in plasma after once or twice daily dosing in patients. Other minor metabolites of phenylbutyrate were identified. The majority (approximately 80-100%) of administered sodium phenylbutyrate is excreted in the urine within 24 hours as the conjugation product, phenylacetylglutamine.

[0144] Terminal half-lives of bile acids could not be reliably determined in most subjects, primarily due to insufficient sampling periods during the terminal elimination phase (see also Tables 6 and 7). Comparisons across studies (single-dose pharmacokinetic studies and sparsely sampled data from Phase II safety and efficacy studies) indicate that there appears to be little accumulation of ursodoxycortaurine after twice-daily dosing, but there was substantial accumulation of UDCA and GUDCA.

[0145] Bile acids that are not (re)absorbed in the intestine (5% under physiological conditions) are further modified by bacteria before excretion mainly in the stool. Bile acids found in the stool are unconjugated and consist mainly of hydrophobic secondary bile acids, lithocholic and deoxycholic acids, as well as other complex mixtures. Administration of ursodoxycortaurine is known to increase UDCA levels in the stool.

[0146] Special Populations and Conditions ·Geriatric medicine Model-based analyses showed no significant differences in the pharmacokinetics of sodium phenylbutyrate or its metabolite phenylacetate between ALS patients aged 65-76 years and those <65 years (data included: 18 and 39 patients, respectively). There were no differences in steady-state plasma concentrations of ursodoxycortaurine or its major derivatives UDCA and GUDCA between ALS patients aged 65-76 years and those <65 years (data included: 24-27 and 78-82 patients, respectively).

[0147] ·sex Following a single oral dose of ALBRIOZA® to healthy volunteers, differences in exposure to sodium phenylbutyrate and phenylacetate were observed between men (N=8) and women (N=6) and were consistent between fed and fasted conditions, with approximately 35% and 31% higher exposure levels in women compared to men, respectively. However, due to moderate variability between subjects, this difference was not determined to be statistically significant. Similarly, gender was not identified as a significant covariate influencing sodium phenylbutyrate or phenylacetate PK parameters in the across-study pharmacometric analysis in healthy subjects and ALS patients (26 females and 56 males contributing data).

[0148] Following a single oral dose of ALBRIOZA® to healthy volunteers, differences in exposure to ursodoxycortaurine and UDCA were observed between men (N=8) and women (N=6) and were consistent between the two dietary conditions: approximately 55% higher exposure was seen in women for ursodoxycortaurine and approximately 29% lower exposure was seen in women for UDCA when compared to men. This effect was statistically significant only for ursodoxycortaurine AUC(0-last). No such effect was seen for GUDCA. There was no consistent discernible effect of gender on steady-state plasma concentrations of ursodoxycortaurine, UDCA, and GUDCA in ALS patients (34 women; 75 men).

[0149] Liver dysfunction Sodium phenylbutyrate and ursodoxcortaurine are metabolized in the liver and kidney, so increased plasma levels would be expected with liver dysfunction. Ursodoxcortaurine and derivatives such as UDCA and GUDCA are bile salts that are recycled by enterohepatic recirculation and stored in the gallbladder. Lithocholic acid, one of the products of bile acid metabolism in the intestine, is known to have strong toxicity. Although not observed in the Phase 2 safety and efficacy study, increased serum levels of lithocholic acid with TURSO administration have been reported, and liver dysfunction would be expected to contribute to potential harm.

[0150] ·Kidney dysfunction The Phase II safety and efficacy study included 32 ALS patients with mild renal impairment (estimated glomerular filtration rate (eGFR) 60-90 mL / min). Model-based analysis showed no significant correlation in the pharmacokinetics of sodium phenylbutyrate or its metabolite phenylacetate in healthy subjects and ALS patients with normal renal function and mild renal insufficiency (eGFR >60 mL / min). There were no discernible differences in steady-state plasma concentrations of ursodoxycortaurine or its major metabolites UDCA and GUDCA between ALS patients with mild renal insufficiency (eGFR = 71-76 >60 mL / min and <90 mL / min). However, these data are inconclusive because i) a limited number of patients (N=8) had an eGFR <75 and ii) eGFR overestimates renal function in ALS patients.

[0151] The main end product, phenylacetylglutamine, is excreted by the kidney, and in addition, phenylbutyrate and its major metabolite, phenylacetate, are metabolized by the kidney and liver. Therefore, increased plasma levels of phenylbutyrate and metabolites are expected with renal dysfunction. Moreover, due to the high protein binding of ALBRIOZA® (approximately 99%), renal insufficiency may alter the distribution and efficacy of TURSO.

[0152] Example 2 Phase 1 Study of Single-Dose Oral AMX0035 Under Fasted and Fed Conditions in Healthy Volunteers the purpose The primary objective of the study was to determine the plasma levels and PK parameters of PB sodium, TURSO and major metabolites following a single oral dose of AMX0035 in a population of men and women over 40 years of age.

[0153] Secondary objectives of the study were to evaluate the effect of a standardized high-fat breakfast on the extent and rate of absorption of PB, TURSO, and active metabolites in healthy subjects; to evaluate the effect of gender on the extent and rate of absorption of PB, TURSO, and active metabolites in healthy subjects; and to quantify the association between plasma concentrations of PB, TURSO, and active metabolites and QT / corrected QT interval (QTc) prolongation and proarrhythmic potential of a single dose of AMX0035 in healthy subjects.

[0154] Overall study design and planning This was a Phase I, open-label, two-sequence, two-period, crossover, single-dose study of AMX0035 in healthy subjects. A total of 14 healthy subjects were enrolled, with 12 or more subjects completing. A predefined gender mix was selected. Subjects received two single oral doses of AMX0035 under fasting conditions after a washout of 4 or more days or after a standardized high-fat meal in a balanced, two-period, crossover design.

[0155] Treatment period 1 was preceded by 24-hour matched sampling at several time points to ascertain endogenous levels of TURSO in each subject.

[0156] Each study period followed the same study design (Figure 1). Subjects were screened for inclusion in the study up to 28 days prior to dosing. Subjects were admitted to the clinical unit on study day -2 in Period 1 and on day -1 in Period 2 and were dosed on the morning of Day 1. On the evening before Period 1, subjects in the fasted / fed or fed / fasted sequence fasted for a minimum of 10 hours prior to dosing.

[0157] Subjects did not leave the clinical unit until discharge on day 2, according to the study scheme. The minimum washout period was 4 days. A follow-up call was made 7 days after the last dose to ensure the subjects' continuing health status.

[0158] Each subject received the following treatment (Table 4):

[0159] [Table 4]

[0160] Test Design AMX0035 is a fixed-dose combination of two small molecules, PB and TURSO, designed to prevent neuronal cell death and neurotoxic inflammation through simultaneous inhibition of ER stress and mitochondrial cell stress. PB and TURSO act with unique modes of action on two independent cellular pathways.

[0161] This study was designed to determine plasma levels and PK parameters of AMX0035 PB, TURSO, and major metabolites, and to evaluate the effect of food and gender on the extent and rate of absorption.AMX0035 formulation is predicted to simultaneously target both ER stress and mitochondrial bioenergetics pathways.

[0162] Selection of study population Subjects were selected from volunteers recruited by Quotient Sciences and screened for study inclusion within 28 days prior to dosing.

[0163] Inclusion criteria To be eligible for study enrollment, subjects had to meet all of the following criteria: 1. Healthy men or women aged 40 to 65 years. 2. Body mass index is 18.5 kg / m 2 More than 32.0kg / m 2 below. 3. Women of non-childbearing potential due to surgery (bilateral tubal ligation, hysterectomy ± oophorectomy, bilateral oophorectomy, bilateral salpingectomy) or menopause (menopause confirmed by absence of menses for at least 12 months and follicle-stimulating hormone level <40 IU / L at screening). 4. All subjects agree to use one acceptable method of contraception during study participation and for 30 days after the last dose. Acceptable methods of contraception include: a. Targeted sterilization (vasectomy with documented azoospermia); b. Use of a non-hormonal intrauterine device (IUD) in the female partner of the study subject, with a failure rate of less than 1% per year, that was inserted by a qualified physician at least 1 month prior to study drug administration and remained in place for at least 30 days after the last dose of study drug; c. Barrier methods such as male condoms or caps, diaphragms or sponges with spermicide; d. Men who were infertile (at least 3 months after vasectomy), truly celibate with respect to heterosexual intercourse, or who lived an exclusively homosexual lifestyle were not required to use two methods of contraception, but were required to maintain this lifestyle during the study and for 30 days after their last dose. 5. Negative for hepatitis B surface antigen (HBsAg), hepatitis C antibody (HCV Ab), or human immunodeficiency virus (HIV) 1 and 2 antibodies at screening. 6. Subjects provide informed consent prior to participating in this study.

[0164] In addition to the above criteria, subjects also agreed to the following restrictions: -No alcohol consumption for 48 hours before administration and until 24 hours after discharge. No foods or beverages containing caffeine or xanthine products (e.g., coffee, tea, cola drinks, energy drinks, and chocolate) from 24 hours before administration of the drug until 24 hours after discharge. -No consumption of grapefruit, grapefruit juice, Seville oranges, Seville orange marmalade, any food or beverage containing Seville orange juice, or any other products containing grapefruit or Seville oranges, from 7 days prior to hospitalization until 24 hours after discharge. -No unaccustomed strenuous exercise from 72 hours before dosing until 24 hours after release from the study. Subjects were advised not to donate blood or plasma for at least 3 months after their last drug dose. Subjects were following all contraceptive measures appropriate to their individual circumstances.

[0165] Exclusion criteria Subjects were excluded from the study if one or more of the following applied to them: 1. Subjects with an ongoing clinically significant disease or disorder including, for example, cardiovascular disease; hypertension; cancer or neoplasm; diabetes; hepatic disease, endocrine disease, metabolic disease, respiratory disease, renal disease, biliary disease or gastrointestinal disease including cholecystectomy (excluding appendectomy), hematological disease, or Axis I or II psychiatric disorder. 2. Clinically significant laboratory abnormalities at screening or admission in the opinion of the investigator. 3. Clinically significant infection or inflammation at screening or admission, in the opinion of the investigator. 4. Acute gastrointestinal symptoms (e.g., nausea, vomiting, diarrhea) at screening or admission or clinical diagnosis of irritable bowel syndrome (IBS) according to the ROME criteria. 5. Subject had a mean QT interval (QTcF) corrected by Fridericia's formula of >450 ms from electrocardiograms (ECGs) at screening and first hospitalization or had subsequent clinically significant QTcF prolongation. 6. Current or former illicit use of class A drugs such as opiates, cocaine, ecstasy, LSD, and amphetamines (class B). Subjects who were permitted to use cannabis on a special basis were not excluded unless they had negative drug abuse screens at screening and admission and had not used the drug for at least 3 months. 7. Consumption of more than 14 units of alcohol per week or an unwillingness to not consume alcohol during the study. Note: 1 unit = 8 g of ethanol (250 mL of beer or approximately 10 oz, 1 glass of wine [100 mL or approximately 3 oz], 1 measure of spirits [30 mL or approximately 1 oz]). 8. Use of any medication (over-the-counter [OTC]) within 14 days of hospitalization or within 5 elimination half-lives (whichever is longer). 9. Use of prescribed centrally acting or psychotropic medications within 28 days of admission. 10. Use of medications with enzyme-inducing properties, such as St. John's wort, within 3 weeks prior to first dose of investigational product. 11. Continuing medical needs during the study. 12. Use of an investigational drug within 3 months prior to the start of this study, or scheduled to receive an investigational drug during this study. 13. Participation in another clinical trial within 30 days prior to screening. 14. History of plasma / blood donation within the past 2 months. 15. History of severe allergic or multiple adverse drug reactions, including to penicillins and cephalosporins. 16.Any condition that impairs the ability to provide consent or communicate with the investigator as required for the completion of this study. 17. Unwillingness to comply with all lifestyle considerations and restrictions as dictated by the protocol. 18. Previous exposure to AMX0035 in this study. 19. Subject has consumed grapefruit, grapefruit juice, Seville oranges, Seville orange marmalade, Seville orange juice, or other products containing grapefruit or Seville oranges during the 7 days prior to hospitalization.

[0166] Removal of a subject from a study Subjects were allowed to discontinue the study if they wished. All reasonable efforts were made by Quotient Sciences to complete the final evaluation / discharge procedures. Quotient Sciences notified the sponsor of the subject's withdrawal from the study.

[0167] The date of early withdrawal was defined as the date of the decision to withdraw the subject from the study. The date of subject completion was defined as the date of the last procedure or last contact (i.e., phone call) made for that subject.

[0168] If a subject requested to leave the clinical unit earlier than the scheduled discharge time, for example due to unforeseen personal circumstances, but intended to return to the unit to complete the study, this was recorded as the subject's voluntary discharge and protocol deviation. Subjects had to complete the planned evaluation / discharge procedures prior to discharge from the clinical unit and returned for the next study / evaluation as scheduled.

[0169] Subjects were withdrawn from study medication for the following reasons: - Including but not limited to: A clinically significant QTc(F) of more than 500 milliseconds or an increase in QTc(F) of more than 60 milliseconds from baseline (confirmed after repeat ECG) b. Baseline was considered to be the closest QTcF of the three pre-dose ECGs for each period. C. Alanine aminotransferase (ALT) concentration more than 3 times the upper limit of the normal range and total bilirubin more than 2 times the upper limit of the normal range Experiencing serious or severe AEs, including Termination of a trial by a sponsor, regulatory authority, or IRB At the request of the subject (withdrawal of consent) -Serious comorbidities or the need for prohibited medications Subjects of non-compliance Investigator discretion

[0170] For purposes of withdrawal criteria, baseline was considered to be the most recent pretreatment assessment.

[0171] For subjects who withdrew due to an IMP-related AE, every effort was made to ensure that subjects completed follow-up procedures. Subjects who withdrew or discontinued the study early due to an IMP-related AE or study termination were considered to have completed the study and were not replaced.

[0172] Early termination for any of the above reasons should be distinguished from a withdrawal of consent by the subject to participate in further activities.

[0173] Subjects withdrawing for other reasons may be replaced at the discretion of the investigator and sponsor.

[0174] Administered Investigational Medicinal Product (IMP) Subject mixed and consumed the drink thoroughly under supervision. It was normal for a small amount of powder to remain undissolved. Subject consumed within 60 minutes of adding the powder to the water. Subject was allowed a small amount of water (1 oz) to wash down the bitter taste of the AMX0035.

[0175] The following information was recorded in the clinical database: Date and time of administration Confirmation that all doses were administered

[0176] Identification of investigational drug AMX0035 was supplied by the sponsor to local pharmacies in carton boxes containing 40 single-use sachets. The active pharmaceutical ingredients were TURSO and PB (AMX0035), supplied by the sponsor in sachets as white powders. Each sachet contained 1 g of TURSO and 3 g of PB.

[0177] Qualified pharmacy personnel prepared the study medication by opening one sachet of AMX0035, placing the powder in a container, and adding approximately 240 mL of room temperature water. The solution was stirred until the powder was mostly dissolved.

[0178] Utilization accountability records were maintained throughout the study. Unused IMPs were destroyed at the request of the sponsor.

[0179] How subjects are assigned to treatment groups At the time of admission (period 1), enrolled subjects were randomly assigned to one of the two sequences in a 1:1 ratio, such that seven subjects were randomly assigned to sequence 1 and seven subjects were randomly assigned to sequence 2. Sequence 1: AMX0035 was administered in the fasted state in Period 1 and in the fed state in Period 2 Sequence 2: AMX0035 was administered in the fed state in Period 1 and in the fasted state in Period 2

[0180] Treatment allocation lists were generated prior to dosing using a random allocation schedule.

[0181] The randomization schedule and treatment allocation list were generated according to Quotient Sciences standard operating procedures (SOPs). The treatment allocation list provided the dosing team with information about which treatment would be administered to each subject in each period and was kept on file at the investigator's site.

[0182] Dose Selection for the Study PB has been evaluated in subjects with neurodegenerative diseases in a dose-escalation study and was found to be generally safe and well tolerated at doses significantly higher than those evaluated in this study. Specifically, the most common AEs included; falls or other accidental injuries, dizziness, diarrhea, edema, dry mouth, headache, nausea, and rash. Apart from the occurrence of headache in subjects, these AEs occurred in a higher proportion compared to the placebo cohort and are expected side effects from PB. No clinically significant changes in laboratory values, ECGs, or vital signs were observed, and no deaths, unexpected or related serious AEs occurred. Importantly, these studies evaluated daily doses of PB of 9-21 g and 12-18 g, whereas this study used 6 g daily. (Cudkowicz ME, Andres PL, Macdonald SA, et al. Phase 2 study of sodium phenylbutyrate in ALS. Amyotroph Lateral Scler. 2009; 10 (2): 99-106;Hogarth P, Lovrecic L, Krainc D. Sodium phenylbutyrate in Huntington's disease: A dose-finding study. Mov Disord. 2007; 22 (13): 1962-1964).

[0183] Choice and timing of administration for each subject AMX0035 was taken orally as a single dose on the morning of Day 1 in Periods 1 and 2. Any deviations from the intended regimen were recorded in the eCRF. All drug administration occurred at the study site under the supervision of appropriately trained staff.

[0184] Meals were required to be administered by a clinical staff member on Day 1. Meals were served at nominal times. Meal start and end times were recorded in the source workbook.

[0185] Regarding feeding Subjects were provided with a light meal and then abstained from all food and drink (except water) until the next morning when a high-fat breakfast was provided. Breakfast was consumed over a maximum of 25 minutes and dosing occurred 30 minutes after breakfast began. Subjects were encouraged to consume their meal over a 25-minute period. For this study, subjects were considered compliant if they consumed a standard breakfast with >80% high fat.

[0186] Fasting Administration Subjects in the 24-hour baseline and fasting periods continued to fast until 4 hours post-dose and were provided with a standardized meal to be consumed from 4.00 to 5.00 hours post-dose. Subjects in the fed condition fasted overnight, consumed a high-fat standardized breakfast 30 minutes prior to dosing, and then continued to fast from 4.00 to 5.00 hours post-dose and consumed a standardized meal.

[0187] No water was permitted one hour before or after drug administration, except for a small (1 oz) rinse for taste (except for administration of study medication as indicated), at which time water was available ad libitum. Subjects were encouraged to hydrate regularly during their hospital stay, including fasting periods, but no formal baseline amounts were required.

[0188] Previous and concomitant therapy No medications were allowed from 14 days or 5 half-lives (whichever was longer) prior to IMP administration until a follow-up call 7 days after administration and as deemed necessary by the investigator to treat AEs. Medications used were recorded in the source workbook.

[0189] Adherence to treatment During all clinical phases of the study, subjects were observed by study staff to ensure compliance with all study procedures, including drug administration.

[0190] Subject mixed and consumed the beverage thoroughly under supervision. It was normal for a small amount of powder to remain undissolved. Subject consumed within 60 minutes of adding the powder to the water. Subject was able to use a small amount of water (1 oz) to wash down the bitter taste of the AMX0035.

[0191] The date and time each subject was dosed was recorded at the subject's source, as well as confirmation that all doses were administered. Any non-compliance required evaluation by the investigator and sponsor to determine whether the subject could continue in the study.

[0192] Pharmacokinetic and safety variables The evaluation schedule for PK and safety procedures is shown in Tables 5 to 7.

[0193] When multiple procedures were scheduled at the same time point, the order of procedures was as follows:

[0194] [ka]

[0195] Vital signs were measured before ECG if both measurements were scheduled at the same time. Other assessments, e.g., physical examination, were performed within the required time. ECG and blood samples should be measured / collected within 10 minutes.

[0196] All safety evaluations were timed relative to the start of dosing.

[0197] [Table 5-1]

[0198] [Table 5-2]

[0199] [Table 6]

[0200] [Table 7]

[0201] Pharmacokinetic measurements Sample collection Venous blood samples were drawn via an indwelling cannula or by venipuncture according to the time schedules presented in Tables 5, 6 and 7.

[0202] Approximately 4 mL of blood samples were collected into 4 mL BD Vacutainer Plastic K2EDTA tubes with Lavender Hemogard Closure and gently inverted approximately 8 times. Tubes were then immediately stored on ice and centrifuged at 1500 g for 10 minutes at 2-8°C and processed within 60 minutes of blood collection. The resulting plasma was divided equally into two aliquots and transferred to appropriately labeled 2 mL primary and backup polypropylene cryovials. Samples were stored at or below -20°C until transport to WWCT for analysis of AMX0035.

[0203] Analysis method Plasma concentrations of PB sodium, PAA, TURSO, ursodeoxycholic acid (UDCA), and glycoursodeoxycholic acid (GUDCA) were determined at Worldwide Clinical using validated analytical methods. The lower limits of quantification were 1.6 μg / mL for PB sodium, 0.8 μg / mL for PAA, 0.02 μg / mL for TURSO, and 20 ng / mL for UDCA and GUDCA.

[0204] Safety measurements Adverse events An AE was an untoward medical occurrence in a subject administered a medicinal product that did not necessarily have a causal relationship to this treatment. An AE could be any adverse or unintended sign (including abnormal clinical laboratory findings), symptom, or disease temporally related to the administration of an IMP, whether or not considered related to the IMP. Pre-existing conditions that worsened during the study were reported as AEs. An adverse drug reaction (ADR) is an AE with at least a reasonable possibility of a causal relationship (probably related or related) to the IMP.

[0205] Clinically significant abnormalities in clinical laboratory parameters, vital signs, or ECG could be reported as AEs according to the judgment of the PI, taking into account relevant clinical signs and symptoms and pre-dose values.

[0206] All AEs were recorded from the time of submission of consent until 30 days after the last dose of study drug. During each study visit, subjects were questioned and / or surveyed by the investigator or his / her designee for evidence of AEs. Each AE was recorded in the subject's source workbook, describing the date and time of onset, description of the AE, duration, severity (yes / no), severity (mild [grade 1], moderate [grade 2], severe [grade 3], very severe or fatal [grade 4]), action taken, outcome, and the investigator's opinion on the relationship of the event to the study treatment. A diagnosis and final opinion on the relationship of the event to the study treatment (not related, probably related, or related) was provided by the investigator at the end of the study.

[0207] Subjects who withdrew from the study due to an AE were followed until the outcome was determined and a written report was provided by the investigator.

[0208] Serious Adverse Events A serious adverse event was an untoward medical occurrence or effect at any dose that resulted in death, was fatal, required or prolonged hospitalization, resulted in persistent or significant disability / incapacity, was a congenital anomaly / birth defect, or was considered a significant medical event as recognized by the PI.

[0209] A suspected unexpected serious adverse reaction (SUSAR) was an unintended response to an IMP related to any dose, i.e., serious, inconsistent with applicable product information, for which there is at least a reasonable likelihood of a causal relationship between the IMP and the AE. All SUSARs were subject to expedited reporting.

[0210] Clinical Laboratory Parameters The following clinical laboratory evaluations were performed at the time points specified in Tables 2, 3, and 4.

[0211] Blood tests: Blood samples were collected in K2EDTA coated tubes (4 mL) with Lavender Hemogard Closure. The following analyses were performed: hemoglobin, hematocrit, red blood cells, red blood cell distribution width, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, mean platelet volume, white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, and basophils.

[0212] Clinical Chemistry: Blood samples were collected in serum separator tubes (7.5 mL) containing a clot activator and serum gel separator. The following analyses were performed: sodium, potassium, chloride, blood urea nitrogen, uric acid, creatinine, total bilirubin, direct bilirubin, alkaline phosphatase, aspartate aminotransferase, ALT, total protein, albumin, lactate dehydrogenase, calcium, phosphate, and glucose.

[0213] Coagulation: Blood samples were collected in sodium citrate tubes (2.7 mL). The following analyses were performed: prothrombin time (PT), prothrombin international normalized ratio, and activated partial thromboplastin time (aPTT).

[0214] Urinalysis: The following analyses were performed on urine samples (>20 mL) using urine test strips: pH, specific gravity, glucose, ketones, nitrites, leukocyte esterase, protein, urobilinogen, blood, and bilirubin.

[0215] Drug Screening: At screening and admission, urine samples (>20 mL) were tested for drugs, including drugs of abuse.

[0216] Viral testing: Clinical chemistry blood samples were used to screen for HBsAg, HCV Ab, and HIV (1 and 2).

[0217] Alcohol testing: These were administered at screening and upon admission to each study.

[0218] If clinical laboratory findings were outside the normal range and the investigator considered the results to be potentially clinically significant, resampling was requested, except in the case of a positive viral test result. If the abnormal findings were clinically significant, appropriate action was taken, e.g. not enrolling the subject in the study or withdrawing the subject from the study. Subjects were referred to their general practitioner or other appropriate health care provider (e.g. genitourinary clinic) for further care. The same was true if HBsAg, HCV Ab or HIV test results were positive and the investigator ensured that adequate counselling was available if requested.

[0219] Abnormal findings at follow-up also required reexamination if the investigator considered the results to be potentially clinically significant.

[0220] Vital signs Vital signs (systolic and diastolic blood pressure [BP], heart rate, and oral temperature) were measured by an automatic recorder using the arm opposite to the one used for blood sampling after the subjects were in a supine position for a minimum of 5 minutes, according to the time schedules presented in Tables 2, 3, and 4. BP and pulse measurements were performed after the subjects had rested for 5 minutes. Pre-dose vital signs were measured within 2 hours prior to dosing. Post-dose vital sign measurements were taken ±15 minutes from the nominal post-dose time point. Vital signs measurements at discharge were taken ±1 hour from the nominal time point. At the time of the return visit, vital signs were measured ±2 hours from the nominal return visit.

[0221] electro-cardiogram Digital 12-lead ECGs were recorded after subjects were in supine position for a minimum of 15 min as detailed in Tables 2, 3, and 4. Three ECGs were taken 1–2 min apart.

[0222] ECGs were assessed for QT, QTcF, PR, QRS, RR and HR intervals and data were collected in a clinical database. ECGs were read by Quotient's principal investigator. -Pre-dose ECG measurement was performed within 2 hours prior to dosing. Post-dose ECG measurements were taken ±15 minutes from the nominal post-dose time point. ECG measurements at discharge were taken ±1 hour from the nominal time point. Return-visit ECG measurements were taken ±2 hours from the nominal return visit time.

[0223] Physical Examination Subjects underwent a complete physical examination, including a neurological examination, as detailed in Tables 2, 3, and 4. Any changes were recorded. Height was measured once and weight was measured according to the assessment schedule.

[0224] pregnancy Male subjects agreed to inform the PI if their partner became pregnant during the study. Any pregnancies were followed and the status of the mother and child was reported to the sponsor after delivery, when possible.

[0225] Measurement Appropriateness The nature and timing of the safety evaluations, as detailed in the final protocol, were deemed appropriate to evaluate the safety of the active compound, taking into account the nature of the compound and its route of administration.

[0226] The timing of blood sample and urine collection intervals was considered adequate to evaluate the PK profiles of the active drug and metabolites.

[0227] Data Quality Assurance All study data recorded in the source workbook were transcribed into a validated database (InForm v5.0). Quality control, data validation and query resolution were performed according to Quotient Sciences SOPs. All source documentation generated during the study was available to the PI for review and to sponsor representatives, regulatory authorities and appropriate IRBs for source data validation.

[0228] Statistical methods and sample size determination Analysis population The safety population included all subjects who received any dose of IMP.

[0229] The safety analysis population was defined based on treatment and included all relevant data from subjects included in the safety population who received that treatment. For purposes of subject disposition, demographics, and baseline, the safety analysis population was defined based on sequence (i.e., all subjects randomized to that sequence who received at least one treatment).

[0230] The PK population included all subjects who received at least one dose of IMP, had no missing samples or invalid post-dose analytical results at critical time points (e.g., near Cmax), had no relevant protocol deviations that could affect the study objectives regarding PK endpoints (e.g., subjects were considered compliant if they consumed >80% of a high-fat standardized breakfast), and had no relevant AEs such as vomiting that suggested the full dose was not absorbed in a particular subject.

[0231] The PK analysis set was a subset of the PK population, defined based on treatment and included all relevant data from subjects in the treated PK population. Individual subject profiles (i.e., periods) were excluded from the PK analysis set when deemed appropriate, such as when subjects in the affected study period did not meet the above criteria or when there were other new study questions relevant to the PK analysis or interpretation.

[0232] If additional subjects needed to be excluded from the statistical analysis, if necessary, the PK analysis subpopulation was determined at the same time as the PK population and was based on the PK analysis population (see "Analyzed Populations" section).

[0233] The safety-PK / analysis population included all subjects included in both the safety and PK / analysis populations.

[0234] Pharmacokinetic analysis PK parameters of PB and metabolites (phenylacetate [PAA]), TURSO and metabolites (UDCA and GUDCA) in plasma were estimated by noncompartmental analysis using Phoenix® WinNonlin software (v8.0 Certara USA, Inc., USA) and / or SAS® version 9.4 (SAS Institute, Inc., Cary, North Carolina, USA) for each enrolled subject and period, where possible and appropriate. Actual time since dosing was used for final plasma PK parameter calculations after database lock.

[0235] PK parameters for PB and PAA were derived from actual concentrations, i.e., non-baseline corrected concentrations.

[0236] Prior to derivation of PK parameters for TURSO and metabolites (UDCA and GUDCA), plasma concentrations were baseline corrected for each treatment period in SAS (version 9.4) using time-matched baseline concentrations from day −1 of period 1.

[0237] PK parameters were not determined at non-baseline corrected concentrations of TURSO and the metabolites (UDCA and GUDCA).

[0238] Where possible, the following parameter estimates were estimated: Tmax: time of highest observed concentration Cmax: maximum observed concentration AUC(0-last): area under the curve from time 0 to the last measurable concentration AUC(0-inf): Area under the curve extrapolated from time 0 to infinity AUCextrap: Percentage (%) of AUC(0-inf) extrapolated beyond the last measurable concentration T1 / 2: Apparent elimination half-life λ-z: apparent elimination phase gradient CL / F: Apparent plasma clearance calculated after a single oral dose, where F (fraction of the dose absorbed) is unknown (parent only) Vz / F: Apparent volume of distribution based on the terminal phase calculated after a single extravascular dose, where F (fraction of dose absorbed) is unknown (parent only) Frel Cmax: Relative bioavailability based on Cmax Frel AUC(0-last): Relative bioavailability based on AUC(0-last) Frel AUC(0-inf): Relative bioavailability based on AUC(0-inf) MR Cmax: Metabolite-to-parent ratio based on Cmax MR AUC(0-last): Ratio of metabolite to parent based on AUC(0-last) MR AUC(0-inf): Ratio of metabolite to parent based on AUC(0-inf)

[0239] Summary statistics (i.e., n, mean, standard deviation [SD], coefficient of variation [CV%], median, minimum, maximum, geometric n, geometric mean, geometric SD, and geometric CV%) were calculated for plasma concentrations by treatment (i.e., dietary condition) and time point, when possible.

[0240] Summary statistics (i.e., n, mean, SD, CV%, median, min, max) were calculated for all plasma PK parameters using the PK analysis population / subpopulations by treatment (i.e., food status). Geometric n, geometric mean, geometric SD, and geometric CV% were presented for all PK parameters (except Tmax).

[0241] Summary statistics for plasma concentrations were calculated and presented for the following: PB and PAA: Actual concentrations TURSO, UDCA and GUDCA: Actual concentrations (including day -1) and time-matched baseline-corrected concentrations

[0242] Additional summaries of plasma concentrations were presented by sex.

[0243] Arithmetic mean plasma concentration versus time curves were constructed for each treatment on a linear / linear scale, and error bars of ± arithmetic SD were included on the plots.

[0244] The geometric mean plasma concentration versus time curve is log 10 per treatment on a linear scale. Error bars are included in these plots, where the error bars are (geometric mean × / geometric SD).

[0245] Spaghetti plots by analyte and treatment displayed one line per subject per profile using actual sampling times after dosing. Plots were split into linear / linear and log 10 Both plots were generated on a linear scale. Legends identifying individual subject profiles were displayed above the plots.

[0246] Individual Cmax, AUC(0-last), AUC(0-inf) and Tmax values ​​were plotted on a linear / linear scale for each individual in the associated PK analysis data, i.e., fed and fasted regimens represent the x-axis. Individual points for each subject were connected by a line. Separate plots were provided for each analyte.

[0247] Statistical analysis of pharmacokinetic parameters Assessment of food effect: Cmax, AUC(0-last) and AUC(0-inf) To assess the effect of food, statistical analyses were performed on the PK parameters Cmax, AUC(0-last) and AUC(0-inf) of PB and PAA (based on actual concentrations), and TURSO, UDCA and GUDCA (based on time-matched baseline-corrected concentrations). The null hypothesis tested was that there was no difference between fed and fasted treatments.

[0248] PK parameters were natural log transformed and analyzed using mixed-effects modeling techniques. The full model included treatment (i.e., fed or fasted), period, sex, the interaction of treatment and sex, and sequence, all fitted as fixed effects, and the term of interest within sequence as a random effect.

[0249] If the treatment x gender interaction term was not significant at the 5% level (i.e. p>0.05), it was dropped from the model, a reduced model was used, and only results for the two genders combined were presented. If the interaction term was significant at the 5% level (i.e. p≦0.05), results were presented for the two genders combined and for males and females separately.

[0250] All other statistical tests related to PK parameters were two-sided and were performed using a 10% significance level resulting in 90% (two-sided) CIs. The adjusted means with differences and associated 90% confidence intervals (CIs) for fasted / fed comparisons obtained from the models were back-transformed on a log scale to obtain adjusted geometric mean ratios (GMRs) and 90% CIs for the ratios. These were presented together with the p-values ​​and within-subject variability values ​​(denoted as CVw in the results tables) from the fasted / fed comparisons.

[0251] Statistical analyses were performed using the treatments actually received and the planned sequence detailed in the random allocation schedule. Models were fitted using the SAS software procedure PROC MIXED, the method was specified as restricted maximum likelihood, and the denominator degrees of freedom for fixed effects were calculated using the Kenward-Roger method.

[0252] Assessment of food effect: Tmax Comparison of Tmax values ​​between treatment groups was investigated using non-parametric analysis. The difference in periods (period 1 minus period 2) was derived for each subject. The Hodges-Lehman estimation method was used to estimate the median difference in Tmax between treatments. The associated 90% CI and p-value were also derived. The null hypothesis was that the difference in medians between treatment groups was equal to zero. The alternative hypothesis was that the difference in medians between treatment groups was not equal to zero. A 90% CI that did not include zero, supported by a p-value <0.10, is an indication of a statistically significant difference in medians between treatments with respect to Tmax.

[0253] This procedure was carried out in the SAS software procedure PROC NPAR1WAY.

[0254] The median differences were presented along with the median Tmax values ​​and n for the two treatments, along with associated 90% CI and p-values.

[0255] Safety parameters Assessment of safety parameters included analysis of AEs, clinical laboratory variables, vital signs, ECGs, and physical examination findings. AEs and medications were coded using the Medical Dictionary for Regulatory Activities (MedDRA; v22.0) and the World Health Organization Drug Dictionary Enhanced (2019 Q1), respectively.

[0256] Treatment-emergent AEs (TEAEs) (i.e., those that began after dosing with study drug) were summarized for each treatment by system organ class (SOC) and within SOCs by event type, severity, and association with IMPs. If AE severity or IMP association was missing, the severity / association was tabulated as missing in the summary tables. TEAEs and pre-treatment AEs are listed.

[0257] Laboratory parameters, including changes from baseline, were summarized for each planned time point by treatment. Shift tables (from baseline to each post-baseline time point) are also presented.

[0258] Vital signs and ECG data, including changes from baseline, were summarized for each scheduled time point for each study part. The number of subjects with a "substantial" increase or decrease from baseline in systolic BP (>20 mmHg), diastolic BP (>10 mmHg), and HR (>15 bpm) were summarized. The number and percentage of subjects with the following ECG characteristics were also summarized: QTcF ≦450 ms, 451-480 ms, 481-500 ms, >500 ms; increases from baseline in QTcF interval of <30 ms, 30-60 ms, and >60 ms; and QT interval of ≦500 ms and >500 ms.

[0259] All clinical laboratory parameters, vital signs, ECG data and abnormal physical examination findings were listed for each time point; values ​​outside reference ranges were flagged where appropriate.

[0260] Statistical analysis of ECG data (exposure response analysis) Exposure-response analyses were performed using the combined safety-PK analysis population.

[0261] Exposure-response analyses were performed to evaluate the association between time-matched baseline-corrected QTcF (ΔQTcF) values ​​and plasma concentrations for each of the parent drug and related metabolites.

[0262] The time-matched change from baseline (i.e., ΔQTcF), based on the average of triplicate values, was calculated for each subject at each post-dose time point as follows: ΔQTcF = (relevant post-baseline QTcF) - (relevant baseline QTcF)

[0263] All concentrations below the limit of quantification were assigned a value of zero.

[0264] The association between ΔQTcF and AMX0035 plasma concentrations for each analyte (i.e., PB, PAA, TURSO, UDCA, and GUDCA) was analyzed using linear mixed models with plasma concentrations for each analyte fitted as covariates, time-matched baseline QTcF as a continuous covariate, and ΔQTcF as the dependent variable. Subject-specific random effects were added to intercept and slope parameters using an unstructured covariance matrix. If an unstructured covariance matrix was not supported by the data, other simplified or reduced structures were explored (e.g., variance components).

[0265] Models were fitted using PROC MIXED in SAS, the method was specified as restricted maximum likelihood, and denominator degrees of freedom for fixed effects were calculated using the Kenward-Roger method.

[0266] Models were initially fitted with all analytes. Models were then iteratively fitted, removing at each step the analyte with the least significant slope for the model (i.e., the largest p-value associated with the null hypothesis that slope=0 for each analyte) until a single analyte remained. As a result, a total of five models were fitted, where five is the number of analytes. Using standard PROC MIXED model fit diagnostics, the model with the best fit was selected as the main one, i.e., the model with the smallest Akaike Information Criterion (AIC).

[0267] The primary model fit (i.e., best fit) was used to identify the median Tmax for each analyte included in the model. The geometric mean concentration value for each analyte with corresponding Tmax was identified and used to estimate the effect on ΔQTcF (90% CI), i.e., if three analytes were included in the model, the effect on ΔQTcF was determined using three sets of data based on the geometric mean concentrations corresponding to the Tmax of analyte 1, as well as the Tmax of analyte 2 and the Tmax of analyte 3. To conclude that AMX0035 does not significantly affect the change in QTcF, the upper limit of the two-sided 90% CI of the predicted mean change should be less than 10 ms at each geometric mean concentration as above (i.e., one per analyte).

[0268] Changes in the conduct of the study or planned analyses Planned changes in analysis Because 24-h baseline (day -1) ECG monitoring was performed in the fasted state (i.e., no high-fat breakfast was provided on day -1 at a time consistent with the time of dosing), the baseline did not truly represent the fed state in drug naive. Thus, exposure-response analyses were performed twice: one primary analysis using only fasted data and one exploratory analysis using only fed data.

[0269] Test subjects Fourteen subjects were enrolled in the study, seven subjects each randomly assigned to either the fasting / fed or fed / fasting treatment sequence. All 14 subjects were dosed, and 13 (92.9%) subjects completed the study. One (14.3%) subject discontinued from the fed / fasting treatment sequence at the investigator's discretion.

[0270] Protocol Deviation The study was conducted according to the clinical protocol with no major protocol deviations and minor deviations from protocol occurred (missed or delayed safety or clinical laboratory assessments / third of three ECGs inadvertently missed at 3 hours on Period 1, Day -1; data not shown).

[0271] It was the opinion of the PI and sponsor that the protocol deviations did not affect the overall integrity or quality of the study results and did not pose safety concerns for the subjects.

[0272] All subjects signed a study-specific ICF before study procedures were performed and met the inclusion and exclusion criteria; data not shown.

[0273] Analyzed population Overall, all 14 subjects received at least one dose of IMP; therefore, all subjects were included in the safety population.

[0274] All subjects received at least one dose of IMP and had at least one profile with no missing samples, invalid post-dose analysis results, relevant protocol deviations, or AEs potentially affecting PK endpoints (see Analyzed Populations). Thus, all 14 subjects were included in the PK population.

[0275] The subjects included in the safety, PK and safety-PK analysis populations for each treatment were: Fed-state AMX0035 consisted of 14 subjects AMX0035 in the fasted state consisted of only 13 subjects. One subject withdrew between periods 1 and 2 due to significant comorbidities or need for prohibited medications (TEAE of musculoskeletal pain that led to discontinuation of IMP) and therefore only received AMX0035 in the fed state.

[0276] Finally, 13 subjects were included in the PK analysis subpopulation; 1 subject was excluded from this subpopulation because he did not complete the fasting treatment.

[0277] Demographic and other baseline characteristics Demographic variables Demographic and lifestyle details of individual subjects are not provided, but a brief summary is provided below.

[0278] Fourteen healthy men (8 [57.1%]) and women (6 [42.9%]) aged 45-64 years participated in the study (Table 8). The majority of subjects were white (12 [85.7%]), and two (14.3%) subjects were black or African American. All subjects were of Hispanic or Latino ethnicity. All subjects were within the reference range for body mass index required by the protocol (18.5-32.0 kg / m 2 All 14 subjects were nonsmokers and none consumed alcohol.

[0279] [Table 8]

[0280] There were no clinically important differences between treatment sequences with regard to demographic characteristics, except for the fed / fasted sequence, which had only white subjects, and the fasted / fed sequence, which had two black or African American subjects (28.6%).

[0281] Other baseline characteristics Details of the medical and surgical history of individual subjects did not preclude enrollment in the study.

[0282] None of the subjects reported taking any medications prior to dosing.

[0283] One subject reported a predose AE of skin abrasions on the left foot and upper arm. This event resolved without the need for medication.

[0284] All urine drug screens, urine alcohol screens, and serum pregnancy tests were negative. Serological tests were unreactive, and coagulation test results were within reference ranges. Per protocol, all subjects were postmenopausal (FSH >40 IU / L), except for one subject who was bilaterally ligated and unable to bear children.

[0285] Measuring adherence to treatment During all clinical phases of the study, subjects were observed by study staff to ensure compliance with all study procedures, including drug administration.

[0286] The date and time each subject was dosed was recorded in the subject's source workbook. Details of individual subject dosing and subject compliance with dietary requirements were all recorded but not provided in this specification.

[0287] Pharmacokinetic evaluation Pharmacokinetics of PB and PAA PB The mean plasma concentration versus time profiles of PB were calculated using log 10 / linear scale, shown in Figure 2 for each dietary condition.

[0288] Key PK parameters for PB for all subjects in the PK analysis population are presented in Table 9.

[0289] [Table 9]

[0290] Following a single oral dose of AMX0035 to healthy male and female subjects in the fasted state, concentrations of PB were evident from 0.25 hours onwards in all 13 dosed subjects. Peak plasma concentrations occurred between 0.25 and 0.50 hours post-dose. Concentrations then declined in a generally monophasic manner and remained quantifiable until 3.00 to 5.00 hours post-dose. Terminal slopes were reliably determined for all subjects, and elimination half-lives obtained ranged from 0.40 to 0.71 hours. The geometric mean (geometric CV%) half-life was 0.461 hours (15.1%).

[0291] The geometric mean (geometric CV%) apparent volume of distribution and apparent plasma clearance were 8.4 L (45.8%) and 211 mL / min (44.9%), respectively.

[0292] Following a single oral dose of AMX0035 in the fed state, concentrations of PB were evident from 0.25 hours onwards in all 14 subjects. Peak plasma concentrations occurred between 0.25 and 1.50 hours post-dose. Concentrations then declined in a generally monophasic manner and remained quantifiable until 3.50 to 6.00 hours post-dose. Terminal slopes were reliably determined for 13 subjects, with resulting elimination half-lives ranging from 0.36 to 1.12 hours. The geometric mean (geometric CV%) half-life was 0.599 hours (41.4%). When the terminal slope could not be reliably determined, this resulted in an unacceptable coefficient of determination (i.e., adjusted R 2 <0.9). One subject showed decreased exposure to PB (with respect to Cmax, AUC(0-last) and AUC(0-inf)) following administration of AMX0035 in both the fasted and fed state compared to the rest of the group.

[0293] The geometric mean (geometric CV%) apparent volume of distribution and apparent plasma clearance were 21.3 L (67.0%) and 410 mL / min (28.6%), respectively.

[0294] PAA The mean plasma concentration versus time profiles of PAA were calculated using log 10 / linear scale, shown in Figure 3 for each dietary condition.

[0295] Key PK parameters for PAA for all subjects in the PK analysis population are presented in Table 10.

[0296] [Table 10]

[0297] Following a single oral dose of AMX0035 to healthy male and female subjects in the fasted state, concentrations of the metabolite PAA were evident from 0.25 hours onwards in all 13 dosed subjects. Peak plasma concentrations occurred between 1.50 and 3.50 hours post-dose. Concentrations then declined in a monophasic / biphasic manner and remained quantifiable until 6.00-8.00 hours post-dose. Terminal slopes were reliably determined for all subjects, and elimination half-lives obtained ranged from 0.69 to 0.95 hours. The geometric mean (geometric CV%) half-life was 0.813 hours (11.5%).

[0298] The geometric mean (geometric CV%) PAA / PB metabolite to parent ratios after dosing in the fasted state were 0.168 (40.3%) for Cmax, 0.416 (38.9%) for AUC(0-last), and 0.421 (38.5%) for AUC(0-inf).

[0299] Following a single oral dose of AMX0035 in the fed state, concentrations of the metabolite PAA were evident from 0.25 to 0.50 hours onwards in all 14 subjects. Peak plasma concentrations occurred between 2.00 and 4.50 hours post-dose. Concentrations then declined in a monophasic / biphasic manner and remained quantifiable until 6.00 to 8.00 hours post-dose. Terminal slopes were reliably determined for all subjects, and elimination half-lives obtained ranged from 0.60 to 1.04 hours. The geometric mean (geometric CV%) half-life was 0.780 hours (16.3%).

[0300] The geometric mean (geometric CV%) PAA / PB metabolite to parent ratios after dosing in the fed state were 0.407 (45.6%), 0.626 (49.2%) and 0.598 (46.8%) for Cmax, AUC(0-last) and AUC(0-inf), respectively.

[0301] Pharmacokinetics of TURSO, UDCA and GUDCA TURSO The mean plasma concentration versus time profiles of TURSO were calculated using log 10 / linear scale, shown in Figure 4 for each dietary condition.

[0302] Key PK parameters for TURSO for all subjects in the PK analysis population are presented in Table 11.

[0303] [Table 11]

[0304] After a single oral dose of AMX0035 to healthy male and female subjects in the fasted state, time-matched baseline-corrected peak plasma concentrations for TURSO occurred between 1.50 and 10.00 hours post-dose. Terminal slopes were reliably determined for five subjects, and the resulting elimination half-lives ranged from 2.51 to 8.71 hours. The geometric mean (geometric CV%) half-life was 4.337 hours (49.7%). When the terminal slope could not be reliably determined, this resulted in an unacceptable coefficient of determination (i.e., adjusted R 2 <0.9).

[0305] The geometric mean (geometric CV%) apparent volume of distribution and apparent plasma clearance were 1600 L (22.1%) and 4260 mL / min (58.9%), respectively.

[0306] After a single oral dose of AMX0035 in the fed state, time-matched baseline-corrected peak plasma concentrations for TURSO occurred between 4.50 and 10.00 hours post-dose. Terminal slopes were reliably determined for six subjects, with resulting elimination half-lives ranging from 1.53 to 5.36 hours, and a geometric mean (geometric CV%) of 3.359 hours (45.1%). When the terminal slope could not be reliably determined, this resulted in an unacceptable coefficient of determination (i.e., adjusted R 2 <0.9).

[0307] The geometric mean (geometric CV%) apparent volume of distribution and apparent plasma clearance were 1000 L (41.2%) and 3440 mL / min (24.1%), respectively.

[0308] U.D.C.A. The mean plasma concentration versus time profiles of UDCA were calculated using log 10 / linear scale, shown in Figure 5 for each dietary condition.

[0309] Key PK parameters for UDCA for all subjects in the PK analysis population are presented in Table 12.

[0310] [Table 12]

[0311] Following a single oral dose of AMX0035 to healthy male and female subjects in the fasted state, time-matched baseline-corrected peak plasma concentrations of the metabolite UDCA occurred between 0.25 and 20.00 hours post-dose. The terminal slope was reliably determined for two subjects, and the resulting elimination half-lives ranged from 3.01 to 7.58 hours. When the terminal slope could not be reliably determined, this was interpreted as an unacceptable coefficient of determination (i.e., adjusted R 2 <0.9) or insufficient data after Cmax. The geometric mean (geometric CV%) UDCA / TURSO metabolite to parent ratios after dosing in the fasted state were 1.099 (119.9%) for Cmax and 1.620 (149.3%) for AUC(0-last).

[0312] Following a single oral dose of AMX0035 in the fed state, time-matched baseline-corrected peak plasma concentrations of the metabolite UDCA occurred between 6.00 and 24.00 hours post-dose. The terminal slope was reliably determined for one subject, and the resulting elimination half-life was 5.312 hours. When the terminal slope could not be determined, this was interpreted as an unacceptable coefficient of determination (i.e., adjusted R 2 <0.9) or insufficient data after Cmax. The geometric mean (geometric CV%) UDCA / TURSO metabolite to parent ratios were 1.093 (169.5%) and 1.406 (148.8%) for Cmax and AUC(0-last), respectively.

[0313] GUDCA The mean plasma concentration versus time profiles of GUDCA were calculated for all subjects in the PK population using log 10 / linear scale, shown in Figure 6 for each dietary condition.

[0314] Key PK parameters for GUDCA for all subjects in the PK analysis population are presented in Table 13.

[0315] [Table 13]

[0316] Following a single oral dose of AMX0035 to healthy male and female subjects in the fasted state, time-matched baseline-corrected peak plasma concentrations of the metabolite GUDCA occurred between 6.00 and 20.00 hours post-dose. The terminal slope was reliably determined for one subject, and the resulting elimination half-life was 12.744 hours. When the terminal slope could not be reliably determined, this was interpreted as an unacceptable coefficient of determination (i.e., adjusted R 2 <0.9) or insufficient data after Cmax. The geometric mean (geometric CV%) GUDCA / TURSO metabolite to parent ratios after dosing in the fasted state were 0.571 (107.4%) for Cmax and 1.056 (114.8%) for AUC(0-last).

[0317] Following a single oral dose of AMX0035 in the fed state, time-matched baseline-corrected peak plasma concentrations of the metabolite GUDCA occurred between 0.50 and 24.00 hours post-dose. Terminal slopes were reliably determined for two subjects, with resulting elimination half-lives of 11.03 and 24.71 hours. When terminal slopes could not be determined, this was associated with an unacceptable coefficient of determination (i.e., adjusted R 2 <0.9) or insufficient data after Cmax. The geometric mean (geometric CV%) GUDCA / TURSO metabolite to parent ratios were 0.675 (92.9%) and 0.918 (103.4%) for Cmax and AUC(0-last), respectively.

[0318] Statistics and Analysis PB The results of the statistical analysis of Cmax, AUC(0-last) and AUC(0-inf) for the assessment of the effect of food on PB are presented in Table 14.

[0319] [Table 14]

[0320] One subject had an unacceptable coefficient of determination for feeding status [i.e., adjusted R 2 <0.9], we did not have reliable estimates of AUC(0-inf) for both fed and fasted treatments and therefore they were excluded from the statistical analysis of AUC(0-inf).

[0321] The treatment x sex interaction term was not significant at the 5% level for each of Cmax (p=0.52), AUC(0-last) (p=0.21) and AUC(0-inf) (p=0.21) and therefore this term was dropped from the respective models.

[0322] For the comparison of fed versus fasted treatments, point estimates of the GMR associated with peak (Cmax) and overall (AUC(0-last) and AUC(0-inf)) exposure levels varied from 24.35% to 45.98%, i.e., the levels of exposure for the fed treatments were on average about 24% to 46% of those seen for the fasted treatments. Each of the upper limits associated with the GMR was less than 51%, and therefore mean levels of exposure for the fed treatments exceeding 51% of the mean levels of exposure seen for the fasted treatments can be confidently excluded.

[0323] The differences between fed and fasted treatments associated with each of the PK parameters were statistically significant at the 10% level with p-values ​​<0.001 for each of Cmax, AUC(0-last) and AUC(0-inf). As a result, there is statistical evidence to reject the null hypothesis of no difference between fed and fasted treatments.

[0324] No statistically significant effect of gender was observed (i.e., p=0.17, p=0.11 and p=0.14 for Cmax, AUC(0-last) and AUC(0-inf), respectively), however there was evidence of higher PB exposure levels in women compared to men, which was consistent across fed and fasted regimens (data not shown).

[0325] The median Tmax for the fasted treatment was 0.500 hours and 1.000 hours for the fed treatment. The Hodges-Lehman estimate of the difference in fed and fasted medians (i.e., fed minus fasting) was 0.500 hours with a 90% CI (0.125, 0.750). The slight increase in Tmax for the fed condition when compared to the fasted condition was statistically significant at the 10% level (p=0.007) (Table 15).

[0326] [Table 15]

[0327] PAA The results of the statistical analysis of Cmax, AUC(0-last) and AUC(0-inf) for the assessment of the effect of food on PAA are presented in Table 16.

[0328] [Table 16]

[0329] The treatment x sex interaction term was not significant at the 5% level for Cmax (p=0.46), AUC(0-last) (p=0.77) and AUC(0-inf) (p=0.77), respectively, and therefore this term was dropped from the respective models.

[0330] For the comparison of fed versus fasted treatments, the point estimates of the GMR associated with peak (Cmax) and overall (AUC(0-last) and AUC(0-inf)) exposure levels varied from 60.34% to 70.54%, i.e., fed treatment levels of exposure were, on average, approximately 60% to 71% of those seen in the fasted treatments. Each of the upper limits associated with the GMR was less than 77%, and therefore, mean levels of exposure in the fed treatment that exceeded 77% of the mean level of exposure seen in the fasted treatments can be confidently excluded.

[0331] The differences between fed and fasted treatments associated with each of the PK parameters were statistically significant at the 10% level with p-values ​​<0.001 for each of Cmax, AUC(0-last) and AUC(0-inf). As a result, there is statistical evidence to reject the null hypothesis of no difference between fed and fasted treatments.

[0332] No statistically significant effect of gender was observed (i.e., p=0.24, p=0.18, and p=0.18 for Cmax, AUC(0-last), and AUC(0-inf), respectively), but there was evidence of higher PAA exposure levels in women compared to men, which was consistent across fed and fasted regimens (data not shown).

[0333] The median Tmax for both the fasted and fed treatments was 2.500 hours. The Hodges-Lehman estimate of the difference in medians between fed and fasted treatments was 0.250 hours with a 90% CI (0.000, 0.750). The slight increase in Tmax for the fed condition compared to the fasted condition was statistically significant at the 10% level (p=0.058) (Table 17).

[0334] [Table 17]

[0335] TURSO The results of statistical analysis of time-matched baseline-corrected concentration-based Cmax and AUC(0-last) for the assessment of food effect on TURSO are presented in Table 18.

[0336] [Table 18]

[0337] The number of subjects with robust estimates of AUC(0-inf) for both periods was less than seven; therefore, formal statistical analysis of AUC(0-inf) was not performed.

[0338] The treatment x sex interaction term was not significant at the 5% level for each of Cmax (p=0.74) and AUC(0-last) (p=0.96), and therefore this term was dropped from the respective models.

[0339] The fed / fasted GMRs (90%CI) were 101.56% (78.52%, 131.36%) and 138.81% (105.39%, 182.83%) for Cmax and AUC(0-last), respectively, i.e., the mean levels of exposure as measured by Cmax and AUC(0-last) for the fed treatment were, on average, approximately 2% and 39%, respectively, higher than those seen for the fasted treatment. However, these estimates should be viewed with caution because of the width of the corresponding 90%CIs; for example, mean increases in AUC(0-last) as low as 5% and as high as 83% cannot be confidently excluded.

[0340] The difference between fed and fasted treatments with respect to Cmax was not statistically significant at the 10% level, i.e., p=0.92. For the analysis of overall exposure over the entire sampling period, i.e., AUC(0-last), the difference between fed and fasted treatments was statistically significant at the 10% level (p=0.056), i.e., there was no statistical evidence to reject the null hypothesis of no effect of food on Cmax, and there was evidence to reject the null hypothesis of no effect of food on AUC(0-last).

[0341] There was evidence of higher TURSO exposure levels in women compared to men, which was consistent across fed and fasted regimens; this effect was significant for AUC(0-last) at the 10% level (p=0.060), but not statistically significant for Cmax (p=0.13) (data not shown).

[0342] The median Tmax based on time-matched baseline-corrected concentrations for the fasted treatment was 4.500 hours and 5.000 hours for the fed treatment. The Hodges-Lehman estimate of the difference between fed and fasted medians was 2.000 hours with a 90% CI (0.250, 2.758). The increase in Tmax for the fed state when compared to the fasted state was statistically significant at the 10% level (p=0.052) (Table 19).

[0343] [Table 19]

[0344] U.D.C.A. The results of statistical analysis of time-matched baseline-corrected concentration-based Cmax and AUC(0-last) for the assessment of food effect on UDCA are presented in Table 20.

[0345] [Table 20]

[0346] The number of subjects with robust estimates of AUC(0-inf) for both periods was less than seven; therefore, formal statistical analysis of AUC(0-inf) was not performed.

[0347] The treatment x sex interaction term was not significant at the 5% level for each of Cmax (p=0.45) and AUC(0-last) (p=0.86), and therefore this term was dropped from the respective models.

[0348] The fed / fasted GMRs (90%CI) were 112.17% (65.80%, 191.21%) and 136.34% (88.53%, 209.98%) for Cmax and AUC(0-last), respectively, i.e., the mean levels of exposure as measured by Cmax and AUC(0-last) for the fed treatment were, on average, approximately 12% and 36%, respectively, higher than those seen for the fasted treatment. However, these estimates should be considered with caution because of the width of the corresponding 90%CIs, e.g., mean decreases in AUC(0-last) as low as 11% and mean increases in AUC(0-last) as large as 110% (i.e., more than 2-fold) cannot be confidently excluded.

[0349] The differences between fed and fasted treatments associated with each of the PK parameters were not statistically significant at the 10% level, i.e., p=0.71 for Cmax and 0.22 for AUC(0-last). As a result, there is no statistical evidence of a difference between fed and fasted treatments, i.e., there is no evidence to reject the null hypothesis of no effect of food on each of Cmax and AUC(0-last).

[0350] No statistically significant effect of gender was observed (i.e., p=0.13 and p=0.16 for Cmax and AUC(0-last), respectively), but there was evidence of higher UDCA exposure levels in men compared to women, which was consistent across fed and fasted regimens (data not shown).

[0351] The median Tmax based on time-matched baseline-corrected concentrations for the fasted treatment was 6,000 hours and 16,000 hours for the fed treatment. The Hodges-Lehman estimate of the difference between fed and fasted medians was 7,000 hours with a 90% CI (2,000, 11,500). The increase in Tmax for the fed state compared to the fasted state was statistically significant at the 10% level (p=0.022) (Table 21).

[0352] [Table 21]

[0353] GUDCA The results of statistical analysis of time-matched baseline-corrected concentration-based Cmax and AUC(0-last) for the assessment of food effect on GUDCA are presented in Table 22.

[0354] [Table 22]

[0355] The number of subjects with robust estimates of AUC(0-inf) for both periods was less than seven; therefore, formal statistical analysis of AUC(0-inf) was not performed.

[0356] The treatment x sex interaction term was not significant at the 5% level for each of Cmax (p=0.77) and AUC(0-last) (p=0.83), and therefore this term was dropped from the respective models.

[0357] The fed / fasted GMRs (90%CI) were 134.45% (95.95%, 188.41%) and 137.31% (105.64%, 178.49%) for Cmax and AUC(0-last), respectively, i.e., the mean levels of exposure as measured by Cmax and AUC(0-last) for the fed treatment were, on average, approximately 34% and 37%, respectively, higher than those seen for the fasted treatment. However, these estimates should be considered with caution because of the width of the corresponding 90%CIs; for example, mean decreases in Cmax as low as 4% and mean increases in AUC(0-last) as large as 78% cannot be confidently excluded.

[0358] The difference between fed and fasted treatments with respect to Cmax was not statistically significant at the 10% level (p=0.14), i.e. there was no evidence to reject the null hypothesis of no effect of food on Cmax. For the analysis of the overall exposure over the entire sampling period, i.e. AUC(0-last), the difference between fed and fasted treatments was statistically significant at the 10% level (p=0.053), i.e. there was evidence to reject the null hypothesis of no effect of food on AUC(0-last) (data not shown).

[0359] The median Tmax based on time-matched baseline-corrected concentrations for both fasted and fed treatments was 16,000 hours. The Hodges-Lehman estimate of the difference between fed and fasted regimens was 2,000 hours with a 90% CI (-4,000, 5,000). This difference was not statistically significant at the 10% level (p=0.66) (Table 23).

[0360] [Table 23]

[0361] statistical / analytical problems The distribution assumptions underlying the statistical analysis appear to have been met for the analysis of the various PK parameters, although there is evidence for a possible exception for Cmax in the PAA, i.e., the residuals from the model fit were not normally distributed. Further examination of the residuals did not indicate a specific reason for this deviation from the underlying assumptions, and the results are consistent with the PK parameters in the other PAAs.

[0362] There were no other statistically significant issues.

[0363] Pharmacokinetic and statistical conclusions PB and PAA Following a single oral dose of AMX0035, plasma exposure to PB based on Cmax, AUC(0-last) and AUC(0-inf) showed a statistically significant decrease of approximately 76%, 55% and 54%, respectively, in the fed state compared to the fasted state.

[0364] In fasted conditions, peak exposure to the metabolite PAA was approximately 14% of that in PB, whereas total exposure based on AUC(0-inf) was approximately 35% of that in PB. The PAA / PB metabolite-to-parent ratio was higher in the fed state, with peak exposure approximately 34% of that in PB and total exposure based on AUC(0-inf) approximately 48% of that in PB.

[0365] A significant increase in Tmax for PB and PAA was observed in the fed state when compared to the fasted state, however the increases were small, ie, median differences of less than 0.5 hours.

[0366] Following administration in the fed state, the geometric mean Cmax, AUC(0-last) and AUC(0-inf) for the metabolite PAA showed statistically significant decreases of approximately 40%, 30% and 29%, respectively, compared to the fasted state.

[0367] TURSO, UDCA and GUDCA Following a single oral dose of AMX0035, peak plasma exposure to TURSO based on Cmax was relatively unchanged in the fed compared to fasted state, but overall exposure based on AUC(0-last) showed a statistically significant increase of approximately 39%.

[0368] In fasted conditions, conversion of TURSO to UDCA was extensive, with peak exposure to the metabolite UDCA being approximately 86% in TURSO and total exposure based on AUC(0-last) being approximately 127% in TURSO, which did not change significantly in the fed state, with peak exposure being approximately 86% in TURSO and total exposure based on AUC(0-last) being approximately 110% in TURSO.

[0369] Following dosing in the fed state, the geometric mean Cmax and AUC(0-last) for the metabolite UDCA showed increases of approximately 12% and 36%, respectively, compared to the fasted state, however, due to high variability, these changes did not achieve statistical significance.

[0370] In fasted conditions, peak exposure to the metabolite GUDCA was approximately 51% in TURSO and total exposure based on AUC(0-last) was approximately 95% in TURSO, which did not change significantly in the fed state, with peak exposure approximately 61% in TURSO and total exposure based on AUC(0-last) approximately 83% in TURSO.

[0371] Following administration in the fed state, the geometric mean Cmax and AUC(0-last) for the metabolite GUDCA showed increases of approximately 34% and approximately 37%, respectively, in the fed state; however, only the difference in AUC(0-last) achieved statistical significance.

[0372] A significant increase in Tmax for UDCA based on time-matched baseline-corrected concentrations was observed for the fed state when compared to the fasted state. A similar trend, albeit a smaller increase, was seen for Tmax for TURSO and GUDCA, although only TURSO achieved statistical significance.

[0373] Discussion and Overall Conclusions PB and PAA Following a single oral dose of AMX0035 in the fasted state, PB was rapidly absorbed, with a median Tmax of 0.500 hours post-dose for all subjects. The resulting profile was characterized by a geometric mean volume of distribution and total clearance of 8.4 L and 211 mL / min, respectively. Terminal elimination was also rapid, with a geometric mean plasma half-life of 0.461 hours.

[0374] Exposure-related intersubject variability (measured by Cmax, AUC(0-last), and AUC(0-inf)) was moderate, with geometric mean CV% ranging from 39.0% to 45.2%.

[0375] Administration of AMX0035 with a standardized high-fat breakfast resulted in a slight delay in the absorption of PB, delaying the median Tmax by 0.500 hours to a median of 1.000 hours compared to the fasted state, likely a result of delayed gastric emptying after the meal. Peak and total plasma exposure based on Cmax, AUC(0-last) and AUC(0-inf) showed statistically significant decreases of approximately 76%, 55% and 54%, respectively, after a meal compared to the fasted state. Inter-subject variability related to exposure was consistent across meal conditions, with geometric mean CV% for Cmax, AUC(0-last) and AUC(0-inf) ranging from 28.6% to 45.4%. Terminal elimination of PB also appeared to be unchanged in the fed state, with a geometric mean terminal half-life of 0.599 hours.

[0376] In contrast, the volume of distribution and total clearance of PB increased to 21.3 L and 410 mL / min, respectively, when administered in the fed state, which, combined with the decreased bioavailability, may indicate linear kinetics. Exposure to PB in fed and fasted conditions showed slight differences between men and women; however, this was not conclusive due to moderate intersubject variability, which was observed to be higher in men than in women, and statistical analysis did not show statistically significant differences between men and women.

[0377] Following administration of AMX0035 under fasting conditions, peak exposure to the metabolite PAA was 0.168-fold that of PB, whereas total exposure based on AUC(0-inf) was 0.421-fold that of PB. The plasma half-life of PAA was comparable to the fast half-life observed for PB, suggesting that elimination of PAA may be rate-limited.

[0378] The time required to reach the maximum concentration of PAA did not change after administration of AMX0035 in the fed state, with a median of 2.500 hours for the two dietary conditions. However, in contrast, the adjusted geometric mean Cmax, AUC(0-last) and AUC(0-inf) showed statistically significant decreases of about 40%, about 30% and about 29%, respectively, compared to the fasted state. The reduction in exposure for PAA was less than that observed for the parent PB; this is reflected in the metabolite-to-parent ratios reported to be slightly higher in the fed state, with peak exposures at 0.407-fold that of PB and total exposures based on AUC(0-inf) at 0.598-fold that of PB. The estimated geometric mean half-life was 0.780 hours and did not vary significantly with dietary condition. As with PB, slight differences were observed between males and females in the two dietary conditions; however, these differences were not conclusive and statistical analysis showed that there were no statistically significant differences.

[0379] One subject showed decreased exposure to PB after dosing with AMX0035 compared to the rest of the group in both the fasted and fed states. Peak plasma PB was 2.92- and 3.63-fold lower than the group geometric mean in the fasted and fed states, respectively. Overall exposure based on AUC(0-last) and AUC(0-inf) was 3.2-fold lower than the group geometric mean in both the fasted state and 3.2- and 3.4-fold lower than the group geometric mean in the fed state, respectively. In addition, due to the emergence of a curve in one subject, their AUC(0-inf) values ​​were considered unreliable.

[0380] TURSO, UDCA and GUDCA Following a single oral dose of AMX0035 under fasting conditions, TURSO was absorbed with a median Tmax of 4.500 hours post-dose. The resulting profile was characterized by a geometric mean volume of distribution and total clearance of 1600 L and 4260 mL / min, respectively. The geometric mean plasma half-life of TURSO was 4.337 hours under fasting conditions.

[0381] Between-subject variability related to exposure (measured by Cmax, AUC(0-last) and AUC(0-inf)) was large, with geometric mean CV% ranging from 58.9% to 79.4%.

[0382] Administration of AMX0035 with a standardized high-fat breakfast resulted in a slight delay in the absorption of TURSO, as demonstrated by the median Tmax, which increased from 4.500 hours in the fasted state to 5.000 hours in the fed state. This delay is likely the result of slower gastric emptying after a meal. In the fed state, peak plasma exposure based on Cmax was unchanged; however, overall exposure based on AUC(0-inf) and AUC(0-last) showed an increase of approximately 47% and approximately 39%, respectively. However, statistical analysis could only be performed on AUC(0-last) due to the limited availability of AUC(0-inf) data. Inter-subject variability related to exposure was slightly reduced in the fed state, with geometric CV% ranging from 24.1% to 62.7% for Cmax, AUC(0-last) and AUC(0-inf). Terminal clearance of TURSO was unchanged in the fed state with a geometric mean terminal T1 / 2 of 3.359 hours.

[0383] The distribution volume and total clearance of TURSO decreased to 1000 L and 3440 mL / min, respectively, when administered in a fed state; combined with the increased bioavailability observed for AUC, this may indicate linear kinetics. The inter-subject variability observed after exposure to TURSO showed little difference between men and women in both fed and fasted states. As a result, statistical analysis showed no statistically significant difference between men and women.

[0384] Following administration of AMX0035 under fasting conditions, peak exposure to the metabolite UDCA was 1.099-fold that of TURSO, and total exposure based on AUC(0-last) was 1.620-fold that of TURSO, indicating extensive and complete metabolism of TURSO to UDCA. The plasma half-life of UDCA was similar to that of TURSO, suggesting that elimination of UDCA may be rate-limited by its formation rate.

[0385] The time required to reach the maximum concentration of UDCA was delayed after administration of AMX0035 in the fed state, with a median of 16.000 hours compared to 6.000 hours in the fasted state. However, in contrast, the geometric mean Cmax and AUC(0-last) showed an increase of about 12% and about 36%, respectively. The increase in overall exposure for UDCA was approximately equal to that observed for the parent TURSO. This was reflected in the metabolite to parent ratio, which did not change in the fed state, with peak exposure of 1.093-fold in TURSO and total exposure based on AUC(0-last) of 1.406-fold in TURSO. The estimated geometric mean half-life was also unchanged in the fed condition, at 5.312 hours. The inter-subject variability observed after exposure to UDCA showed little difference between males and females in both the fed and fasted states. As a result, statistical analysis showed that there were no statistically significant differences between males and females.

[0386] After dosing with AMX0035 under fasting conditions, peak exposure to the metabolite GUDCA was 0.571-fold higher in TURSO and total exposure based on AUC(0-last) was 1.056-fold higher in TURSO, suggesting complete and extensive metabolism of TURSO to UDCA and then to GUDCA. The plasma half-life of GUDCA was prolonged compared to that of TURSO (12.744 h vs. 4.337 h), suggesting that elimination of GUDCA was not limited by its rate of formation.

[0387] The time required to reach the maximum concentration of GUDCA was unchanged with a median of 16.000 hours after administration of AMX0035 in the fed state. In contrast, the geometric mean Cmax and AUC(0-last) showed an increase of 34% and 37%, respectively, in the fed state. However, due to the large variation observed, it was considered statistically significant only for AUC(0-last). The increase in overall exposure for GUDCA was equal to that observed for the parent TURSO. This is reflected in the metabolite-to-parent ratio, which did not change significantly in the fed state, with peak exposure of 0.675-fold in TURSO and total exposure based on AUC(0-last) of 0.918-fold in TURSO.

[0388] Similar to TURSO and UDCA, the between-subject variability observed after exposure to GUDCA showed only slight differences between males and females in the two dietary conditions, and statistical analysis showed that there were no statistically significant differences between males and females.

[0389] Safety Considerations There were no safety concerns associated with administration of AMX0035 in the fasted or fed state.

[0390] There were no serious or severe AEs or ADRs. Overall, one subject experienced an unrelated moderate AE that led to discontinuation of IMP.

[0391] With the exception of one subject (7.1%) in the fed-treatment group, no subjects had an increase in QTcF from baseline of more than 30 ms, no subjects had a QTcF value of more than 450 ms at any time point, and no subjects had an increase in QTcF of more than 60 ms.

[0392] There is no evidence to suggest that administration of AMX0035 in either the fasted or fed state is associated with clinically relevant effects on QTcF within the observed range of plasma concentrations, i.e., up to approximately 200 μg / mL PB in the fasted state and up to approximately 40 μg / mL PAA in the fed state.

[0393] There were no clinically significant findings on laboratory tests, vital signs, ECG or physical examination.

[0394] Overall conclusion Pharmacokinetic Conclusions PB and PAA Following a single oral dose of AMX0035, plasma exposure to PB based on Cmax, AUC(0-last) and AUC(0-inf) showed a statistically significant decrease of approximately 76%, approximately 55% and approximately 54%, respectively, in the fed state compared to the fasted state.

[0395] In fasted conditions, peak exposure to metabolite PAA was approximately 14% of that in PB, while total exposure based on AUC(0-inf) was approximately 35% of that in PB. The PAA / PB metabolite-to-parent ratio was higher in the fed state, with peak exposure approximately 34% of that in PB and total exposure based on AUC(0-inf) approximately 48% of that in PB.

[0396] A significant increase in Tmax for PB and PAA was observed in the fed state when compared to the fasted state, however the increases were small, ie, median differences of less than 0.5 hours.

[0397] After administration in the fed state, the geometric mean Cmax, AUC(0-last), and AUC(0-inf) for the metabolite PAA showed statistically significant decreases of approximately 40%, approximately 30%, and approximately 29%, respectively, compared to the fasted state.

[0398] TURSO, UDCA and GUDCA Following a single oral dose of AMX0035, peak plasma exposure to TURSO based on Cmax was relatively unchanged in the fed compared to fasted state, whereas overall exposure based on AUC(0-last) showed a statistically significant increase of approximately 39%.

[0399] In fasted conditions, conversion of TURSO to UDCA was extensive, with peak exposure to the metabolite UDCA being approximately 86% in TURSO and total exposure based on AUC(0-last) being approximately 127% in TURSO, and this did not change significantly in the fed state, with peak exposure being approximately 86% in TURSO and total exposure based on AUC(0-last) being approximately 110% in TURSO.

[0400] Following dosing in the fed state, the geometric mean Cmax and AUC(0-last) for the metabolite UDCA showed increases of approximately 12% and 36%, respectively, compared to the fasted state, however, due to high variability, these changes did not achieve statistical significance.

[0401] In fasted conditions, peak exposure to the metabolite GUDCA was approximately 51% in TURSO and total exposure based on AUC(0-last) was approximately 95% in TURSO. This did not change significantly in the fed state, where peak exposure was approximately 61% in TURSO and total exposure based on AUC(0-last) was approximately 83% in TURSO.

[0402] Following administration in the fed state, the geometric mean Cmax and AUC(0-last) for the metabolite GUDCA showed increases of approximately 34% and approximately 37%, respectively, in the fed state; however, only the difference in AUC(0-last) achieved statistical significance.

[0403] A significant increase in Tmax for UDCA based on time-matched baseline-corrected concentrations was observed for the fed state compared to the fasted state. A similar trend, albeit a smaller increase, was seen for Tmax for TURSO and GUDCA, with only TURSO achieving statistical significance.

[0404] Safety Conclusion AMX0035 administered under fasted and fed conditions was well tolerated in the study conditions.

[0405] One (7.1%) subject reported one TEAE following administration of AMX0035 in the fed treatment arm: the AE of musculoskeletal pain was moderate in severity (grade 2), unrelated to AMX0035, and led to discontinuation of IMP.

[0406] No IMP-related AEs were reported during this study.

[0407] There is no evidence to suggest that administration of AMX0035 in either the fasted or fed state is associated with clinically relevant effects on QTcF within the observed range of plasma concentrations, i.e., up to approximately 200 μg / mL PB in the fasted state and up to approximately 40 μg / mL PAA in the fed state.

[0408] No clinically significant changes were reported in laboratory tests, vital signs, ECGs, or physical examinations.

[0409] Example 3 Population Pharmacokinetics of Phenylbutyrate (PB) and Phenylacetate (PAA) Following Administration of AMX0035 for the Treatment of ALS The objectives of the study were: (1) to develop a population pharmacokinetic (PK) model that describes the plasma concentrations of PB and its metabolite PAA in patients with ALS receiving AMX0035; (2) to identify demographic and clinical characteristics that explain the PK variability of PB and PAA; and (3) to perform simulations using the final population PK model to examine the effects of various covariates on PB and PAA exposure.

[0410] Pharmacokinetic data for population PK analysis were obtained from a phase 1 food effect study in healthy adult volunteers and a phase 2 clinical trial in patients with ALS. The phase 1 food effect study investigated the PK of PB, TUDCA and major metabolites following a single oral dose of AMX0035 with and without food in healthy subjects. Subjects received a single dose of one sachet of AMX0035 (1 g TUDCA and 3 g PB) under fasted (overnight and 4 hours after dosing) and fed (standard high-fat breakfast 30 minutes prior to dosing) conditions with a minimum of 4 days of washout between treatments. The order of dosing with and without food was randomly assigned. Serial blood samples for analysis of plasma concentrations of PB, PAA, TUDCA, UDCA and GUDCA were collected 24 hours after each dose. In addition, plasma samples were obtained the day before the first dose of AMX0035 to characterize endogenous concentrations of TUDCA and metabolites. The protocol is called A35-002.

[0411] The Phase 2 study investigated the safety, tolerability, efficacy and bioactivity of AMX0035. Subjects were randomly assigned in a 2:1 ratio to receive AMX0035 or placebo. Treatment was administered as one sachet of AMX0035 or placebo per day for the first 3 weeks, then increased to one sachet twice daily if tolerated. Subjects were encouraged to take the drug before meals. A sparse sampling strategy was used for the analysis of plasma concentrations of PB, PAA, TUDCA, UDCA and GUDCA, including plasma samples collected at the baseline visit (pre-dose), and at treatment visits at weeks 12 and 24. Protocol 3500 was used.

[0412] result summary A one-compartment model with first order absorption, first order metabolism of PB to PAA (100% conversion), and metabolic (transport) compartments to model the delayed appearance of metabolite plasma concentrations and the nonlinear elimination of PAA best explained the PB and PAA plasma concentration data. The final model included the effect of food intake on the oral absorption rate constant (Ka), the apparent metabolic clearance of PB to PAA (CLPB / F) or the apparent volume of distribution of the PB (V PB The effect of food intake on the maximum rate of PAA elimination (V max -PAA) and the effect of body weight on V max Volume of distribution of PAA and PAA (V PAA We included covariates accounting for the effect of ALS diagnosis on risk (RR 0.01, ... and RR 0.01, respectively). Inter-temporal variation (IOV) was not included.

[0413] Parameter estimates of the final model and bootstrap analysis are presented in Table 24. The PB terminal elimination half-life is estimated to be 0.45 hours based on representative values ​​of CLPB / F and VPB / F. When PB is administered under fed conditions, relative to fasting conditions, the final model predicted a 52.4% (95% CI: 48.5% to 56.3%) decrease in relative bioavailability and a 60% (95% CI: 56% to 64%) decrease in Ka.

[0414] For Phase 1 studies, predicted individual peak plasma concentrations (C max ) and the area under the plasma concentration versus time curve from time 0 to infinity (AUC 0-∞ ) are the corresponding observed individual C for both fed and fasted conditions. max and AUC 0-∞ closely matched.

[0415] This finding, together with the lack of accumulation after multiple doses, supports the use of this approach to estimate steady-state subject-specific exposure parameters in ALS patients. Simulations were performed to evaluate the effect of covariates included in the final model on systemic exposure to PB and PAA. The only covariate affecting PB exposure was drug administration in relation to food. When administered with food, the C in the model-predicted PB was max and the area under the plasma concentration versus time curve (AUC 0-last ) were 3.2- and 2.2-fold lower, respectively, than when administered in the fasted state.

[0416] Food intake, ALS diagnosis, and body weight influenced PAA exposure. Model-predicted C max and AUC 0-last were 1.5- and 1.3-fold lower, respectively, than when administered fasting. Absence of ALS diagnosis was associated with a lower C max and AUC 0-last The dose-dependent effects of PAA on the PAA exposure were minimal (<1.2-fold). Model-based simulations indicate that body weight had a significant effect on PAA exposure. Cmax for PAA was 1.45-fold lower at 50 kg body weight and 1.47-fold lower at 115 kg compared to 70 kg, and AUC0-last for PAA was 1.87-fold lower at 50 kg and 1.72-fold lower at 115 kg compared to 70 kg.

[0417] The results demonstrate that administration of AMX0035 with food reduces PAA exposure in PB and, to a lesser extent, plasma compared to administration in the fasted state. PK of PB and PAA is generally similar in ALS patients and healthy subjects. PB exposure is not significantly affected by body weight, whereas PAA exposure is inversely correlated with body weight.

[0418] [Table 24]

[0419] Drug concentration The large sampling design from protocol 35-002 allowed for a complete description of the plasma concentration profile in volunteers. After oral administration, PB was rapidly absorbed and converted to PAA. Plasma concentrations typically peaked at 0.5-1 h for PB and 2-3 h for PAA and fell below the lower limit of quantification (LLOQ) by 4-5 h for PB and 6-8 h for PAA after dosing. No accumulation of drug or metabolites occurred with bid dosing. Compared with fasting dosing, PB plasma concentrations in volunteers were substantially lower and had a more extended profile after dosing with a high-fat meal. Food-associated changes in PAA plasma concentration-time data were comparable.

[0420] The effect of food on sparsely collected plasma concentrations from ALS clinical trials was examined. PB administration in clinical trial patients was classified as being administered on an empty stomach (fasted administration) if drug administration occurred more than 2 hours after or more than 1 hour before a meal. There was a trend towards higher PB and PAA plasma concentrations at 1 hour, and similar or slightly lower concentrations at 4 hours were noted with PB administration on an empty stomach compared with that with a meal.

[0421] Post-hoc analysis derived PK parameters Table 25 shows the subject-specific Cs derived from individual Bayesian parameter estimates in patients with ALS. max and AUC 0-∞ To summarize:

[0422] [Table 25]

[0423] Example 4 TUDCA Pharmacokinetic Study The study was conducted to examine plasma concentration data in various subgroups of the protocol AMX3500 Phase 2 study. This example summarizes the plasma concentrations of TUDCA and two metabolites that are also related acids, UDCA, the deconjugated TUDCA metabolite, and GUDCA, the glycine conjugate of UDCA.

[0424] For each of the two treatment groups (treatment and placebo), subjects were further divided into two sample sequence groups for the schedule of pharmacokinetic measurements: 1. 1 hour post-dose at the Week 12 visit and 4 hours post-dose at the Week 24 visit 2. 4 hours post-dose at the Week 12 visit and 1 hour post-dose at the Week 24 visit

[0425] Antibiotics listed in the concomitant medications were the basis for comparing subjects who took antibiotics in line with the time of the visit to those who did not. 24 separate medication descriptions were identified as including antibiotics. The date of antibiotic use was compared to the date of the visit, and if the date of antibiotic use included the date of the visit, that visit was flagged in that subject's data for antibiotic use. However, this approach may not account for some antibiotics with long elimination half-lives, as it does not flag subjects who took antibiotics before the visit, but whose antibiotic administration ended before the visit. If this event occurred, these antibiotics could hypothetically remain in the subject's body. However, a few subjects were observed to have taken antibiotics on days close to but not overlapping with the date of the visit. In other cases, subjects took antibiotics on one visit date and not others. Thus, some subjects fall into both antibiotic use and non-use categories, but the subject's individual visits are not counted in both at the same time.

[0426] Subjects recorded as "Black" or "African American" were assessed according to the standard glomerular filtration rate (eGFR) assessment for that race. Other standard eGFR assessments used, sometimes called Caucasian standards, were applied to all subjects of other races. These filtration rates were mL / min / 1.73 m 2 Units were analysed as a continuum and as the following categories: 1.90mL / min / 1.73m 2 less than 2.90mL / min / 1.73m 2 End

[0427] Age was converted from a continuous variable into two categories: 1. Age 65 or older 2. Younger than 65 years old

[0428] All boxplot boxes have bounds at the 25th and 75th percentiles, whiskers at the 10th and 90th percentiles, beyond which outliers are points, black lines are medians, and red lines are means.

[0429] result The results show that plasma concentrations of TUDCA, UDCA, and GUDCA appeared to reach steady state by week 12, supporting the pooled plasma concentration data across study visits. Steady-state plasma concentrations of TUDCA, UDCA, and GUDCA following administration of AMX0035 in ALS patients were highly variable and generally at least an order of magnitude higher than endogenous levels, which were also highly variable.

[0430] The mean to standard deviation ratio (MSDR) is a simple metric to examine the variability in each group and compare the acids to the groups. Table 26 compares the MSDR of TUDCA levels at 1 hour post-dose for the AMX0035 group (82.1% vs. 66.3%) with the 12-week and 24-week groups and the 4-hour group (97.7% vs. 112.7%), with large variability observed in all of these groups. The same pattern of large variability is found for UDCA (86.7% vs. 89.6%, 102.7% vs. 69.1%) (Table 27) and GUDCA (106.7% vs. 105.9%, 138.1% vs. 134.3%) (Table 28). It can be seen that significant endogenous concentrations of UDCA and GUDCA are present in placebo-treated patients. Groups pooled by post-dose time have a similar pattern of large variability (Table 29). Figures 7-16 show that outliers contribute to the large variation found in the tables.

[0431] Figure 7 shows that the TUDCA levels of female subjects 4 hours post-dose have a more positively skewed distribution, with the 75th and 90th percentiles being much higher than the other three groups. All four groups in Figure 7 have similar distributions of UDCA levels. Figure 8 shows the three groups with similar distributions of GUDCA levels, but the male subjects in the 4 hours post-dose group have greater variation and higher 75th and 90th percentile scores.

[0432] Within age categories (1 hour post-dose (<65 vs. ≥65) and 4 hours post-dose (<65 vs. ≥65)), TUDCA levels show similar distribution patterns. As shown in FIG. 9, the ≥65 group at 4 hours post-dose shows a different pattern than the other three groups, with greater variation and higher levels overall, for UDCA. GUDCA levels in FIG. 10 show similar distribution patterns among the three groups, but the <65 group has greater variation and lower median values ​​compared to the ≥65 group at 4 hours post-dose.

[0433] For each acid, the mean levels are comparable between each group in the antibiotic use plots. However, the distributions can have higher medians and variability for TUDCA, UDCA, and GUDCA (Figures 11, 12, and 13, respectively). These higher median groups are composed of subjects who used antibiotics.

[0434] Visual inspection of the box plots found for TUDCA, UDCA, and GUDCA in Figures 14 to 16 reveals that the high GFR group (90 mL / min / 1.73 m) had a significantly higher GFR than the control group (90 mL / min / 1.73 m) at 1 hour after administration. 2 (>100) has a narrower distribution than the other three groups with lower plasma concentrations.

[0435] [Table 26]

[0436] [Table 27]

[0437] [Table 28]

[0438] [Table 29]

[0439] Example 5 Statistical Analysis of Bile Acids in PK Samples from ALS Patients from the CENTAUR Study The aim of the study was to evaluate the effect of AMX0035 on human plasma bile acid profile. TUDCA is a bile acid that can be produced endogenously in humans and its effect on the balance of the bile acid pathway may be an important factor in both therapeutic efficacy and potential toxicity. A targeted metabolomic approach was applied using the biocrates Absolute IDQ® Bile Acid Assay to measure 20 bile acids by LC-MS.

[0440] The following samples were used: The primary analysis or complete cases (CC) included samples exclusively from subjects for whom all three time points (baseline visit, week 12 and week 24) were available and no premature discontinuation occurred. A secondary analysis or partial case (PC) included samples from subjects with at least two time points available, i.e., baseline visit and week 12 or week 24 or all three time points. This analysis also included samples taken at premature discontinuation (ED) at either week 12 or week 24 to test for potentially related undesirable effects of treatment. The time-course data (TC, 270 samples) included the same samples as the CC data analysed with different group classifications to investigate interactions between treatment and treatment period. Strength of response data (SR, 108 samples) comprised a subset of the CC data set selected based on ALSFRS-R slope. The SRTC data included the same samples as the SR data analyzed with different group classifications to explore interactions between strength of response and duration of treatment.

[0441] Univariate statistics In the complete case primary analysis, an ANOVA model was used to compare the AMX0035 and placebo groups at three time points: baseline visit, week 12, and week 24. Figures 17 and 18 show the path visualization of the effect of AMX0035 treatment at weeks 12 and 24. There were no significant differences between the two groups at baseline.

[0442] At weeks 12 and 24, TUDCA, UDCA, and GUDCA, as well as the hydrophilic / hydrophobic bile acid ratio, increased in AMX0035-treated subjects, most likely a direct effect of oral administration of TUDCA to these subjects. Indeed, UDCA is the precursor of TUDCA in physiological conditions, but can also be generated by deconjugation of taurine from TUDCA in the intestine. In addition, UDCA appears to be exclusively generated by the gut microbiota, which further supports its synthesis from orally administered TUDCA in these conditions. Finally, after taurine deconjugation, during the enterohepatic recycling of TUDCA, UDCA can be reconjugated with glycine to form GUDCA. Interestingly, in the correlation analysis, it was UDCA, but not TUDCA, that was found to be negatively correlated, albeit weakly, with the ALSFRS-R slope (Table 30). This suggests that high UDCA values ​​may be expected in subjects with rapid disease progression, whereas low UDCA values ​​are expected in subjects with slow disease progression.

[0443] At week 12, glycine and taurine conjugates (GCA (glycocholic acid), TCA (taurocholic acid), GDCA (glycodeoxycholic acid), TCDA) and indicators of glycine and taurine conjugation, CDCA (chenodeoxycholic acid), DCA (deoxycholic acid), and pBA, were all decreased in AMX0035-treated subjects. Similar trends remained at week 24, with the exception of the glycine conjugates GCA and GDCA, which were still decreased overall but not significantly. The pBA conjugation ratio, which reflects both glycine and taurine conjugation of CA and CDCA, also decreased at both time points. Interestingly, at week 12, the total conjugated primary bile acids was significantly decreased, whereas at week 24, it was the total unconjugated primary bile acids that increased.

[0444] CA (cholic acid) and CDCA levels decreased and increased overall, respectively, but the difference was significant only for CDCA at week 24. However, the CDCA / CA ratio increased significantly at both time points. Bile acid biosynthesis is classically described as the combined effect of the classical pathway generating CA and CDCA, and an alternative pathway that favors CDCA production.

[0445] At week 12, the sum of 12α-hydroxylated bile acids (CA, DCA and their conjugates) decreased. An increase in this indicator was associated with insulin resistance. Although a decrease was observed here, this could represent an effect related to diabetes, one of the confounding factors included in the analysis. At week 24, the sum of glycine-conjugated bile acids increased significantly, probably as a result of a more pronounced increase in GLCA at this time point.

[0446] Across CC, AMX0035 treatment (including a daily dose of TUDCA) caused an increase in TUDCA and related metabolites and indices at weeks 12 and 24. Interestingly, downstream metabolites of CA and DCA conjugated with glycine and taurine decreased at both time points, likely due to a shift in metabolic resources to accommodate higher levels of TUDCA and GUDCA. Similarly, an increase in CDCA, a precursor of UDCA, may be related to treatment-induced UDCA utilization.

[0447] Additional information from PC: This data differs from CC in that in addition to the 270 CC samples, it includes samples from subjects who participated through week 12 and subjects who provided samples at week 24 but were forced to discontinue treatment. The overall profile as shown in the univariate statistics file was similar to CC. Some differences are described below.

[0448] In PC, the trend towards increased CDCA levels in AMX0035-treated subjects was already significant from week 12 and continued until week 24. The total of conjugated pBA was also decreased but not significantly, whereas the increase in the total of glycine-conjugated bile acids was significant but only slightly above the p-value threshold in CC. A unique feature of PC at week 12 was the significant decrease in TDCA synthesis, as seen in the CA ratio (TDCA / CA), again a trend, but not significant in the other comparisons.

[0449] At 24 weeks, the main difference with CC was that the effects on GCA, GDCA and total 12α-hydroxylated bile acids (all decreased) persisted from week 12 but disappeared by week 24 in CC.

[0450] Time-course analysis (TC): This analysis focused on the effect of time (or here treatment period) on the effect of treatment. For this purpose, CC data were utilized with different "group" classifications combining all AMX0035 or placebo samples regardless of time point. The effects of the different time points were then tested in dedicated interaction tests.

[0451] Three interaction effects were tested here: (i) interactions between treatment and baseline and the 12-week time point, (ii) interactions between treatment and baseline and the 24-week time point, and (iii) interactions between treatment and the 12-week and 24-week time points.

[0452] Here again, when interactions between treatment and later and baseline time points were examined, the most striking effects were increases in TUDCA, UDCA, and GUDCA levels and increases in the hydrophilic / hydrophobic bile acid ratio. Increases in the CDCA / CA ratio, as well as decreases in glycine and taurine conjugates and related indices, were robustly present when compared to baseline.

[0453] Interestingly, the decrease in the TDCA / CA ratio was significant for interactions (i) and (ii). In CC, a trend toward a decrease was present at both 12 and 24 weeks, but was not significant. This suggests a global role for TDCA synthesis from CA by the gut microbiome, which may have been overlooked in the simple ANOVA analysis. Interestingly, this ratio also decreased at 12 weeks in the PC analysis. This ratio was strongly associated with cognitive decline in a study of serum bile acid profiles in Alzheimer's disease.

[0454] However, this time course analysis revealed no interaction effects between treatment and the 12-week and 24-week time points, and thus the effect of AMX0035 treatment on bile acid levels persisted overall between these two time points.

[0455] Correlation analysis Correlations of the concentration of metabolites and metabolic indices with the parameter "ALSFRS-R slope" were investigated for the most relevant subgroups of CC data. The ALSFRS-R (ALS Functional Rating Scale-Revised) is a widely used indicator of disease progression in ALS patients. Here, the slope of the 24-week study was used after correction and transformation to allow correlation analysis. The higher the ALSFRS-R slope, the slower the disease progression over 24 weeks. Thus, a positive correlation with the ALSFRS-R slope indicates that the value (metabolite or index) was higher in patients with slower disease progression and lower in patients with rapid disease progression. Conversely, a negative correlation with the ALSFRS-R slope corresponds to high values ​​in patients with rapid disease progression and low values ​​in patients with slow disease progression.

[0456] [Table 30]

[0457] Correlation analysis was performed using different subgroups of CC data at time point week 24 versus transformed ALSFRS-R slope. The only subgroup showing correlation with disease progression indicators was the complete group of AMX0035-treated patients at week 24 (n=61). As shown in Table 30, two metabolites had a weak negative correlation with ALSFRS-R slope (CDCA and UDCA). Metabolic indicators related to glycine and taurine conjugation showed a weak positive correlation, and the sum of unconjugated (primary) bile acids was weakly negatively correlated with ALSFRS-R slope.

[0458] When the group was divided into subgroups based on other ALS medications (edaravone, riluzole, both or none), metabolites / indicators did not correlate significantly with the transformed ALSFRS-R slope. Note that for these subgroups, the number of replicates was very small (maximum 23 in the riluzole-only group). Similar evaluations in samples from placebo subjects at week 24 showed no significant correlation with the ALSFRS-R slope for the entire population (n=29) and for the ALS treatment subgroups (again, with very small number of replicates).

[0459] Response Strength When AMX0035-treated subjects were divided into response strength groups based on ALSFRS-R slope, differences in ANOVA results could be observed between strong and weak responders only at week 24. CDCA and TDCA were the only metabolites that could distinguish the two groups at this time point. CDCA, which is produced by both the classical and alternative bile acid biosynthetic pathways and is also a precursor to UDCA, was higher in strong responders. TDCA and the TDCA / CA ratio, which has been found to be associated with cognitive decline, were both higher in weak responders.

[0460] Interestingly, the levels of TUDCA, UDCA, GUDCA or the hydrophilic / hydrophobic bile acid ratio did not appear to be decisive factors in distinguishing these two subgroups. Strong responders had significantly lower ratios for processes related to glycine and taurine conjugation, as well as higher levels of unconjugated bile acids and unconjugated pBA. In the time course analysis (SRTC), three interaction effects were tested: (i) interaction of response with baseline and 12-week time point, (ii) interaction of response with baseline and 24-week time point, and (iii) interaction of response with 12-week and 24-week time points. Interestingly, unlike what was observed in the time course analysis of CC against placebo, which combined all responders into one group, there was an interaction effect of the strength of response with the 12-week and 24-week time points, but not the other interactions tested, meaning that the difference between strong and weak responders continues to increase even if the overall effect of the treatment does not seem to show a large difference in the TC analysis. CDCA was the only significant difference in metabolite levels, but indices of bile acid conjugation and the TDCA / CA ratio still distinguished strong and weak responders in terms of changes that occurred between weeks 12 and 24.

[0461] conclusion CC analysis showed that in addition to the expected increase in circulating levels of TUDCA and related metabolites, a greater effect on bile acid conjugation could be observed. PC analysis, including subjects who had to discontinue treatment, showed a similar bile acid profile with no significant differences. TC analysis confirmed that the bile acid profile was already fixed at 12 weeks and was mostly sustained at 24 weeks.

[0462] The ALSFRS-R slopes of CC subjects allowed bile acid levels to be examined in the context of disease progression over the 24-week study. Only weak correlations could be found in AMX0035-treated subjects, including a weak negative correlation with UDCA. Correlation analyses were directed to CDCA (negative correlation) and effects on bile acid conjugation, which occurs primarily in the liver (positive correlation), but these results should be interpreted with caution due to the small correlations.

[0463] SR analysis revealed no differences between strong and weak responders to treatment at baseline or week 12. At week 24, levels of TUDCA, UDCA, and GUDCA, as well as the ratio of hydrophilic to hydrophobic bile acids, were similar in both groups. However, CDCA (a precursor of UDCA via metabolism by the gut microbiota), the TDCA / CA ratio (a proposed indicator of cognitive decline), and indicators of bile acid conjugation with glycine and taurine emerged as the main drivers in distinguishing strong and weak responders at the end of treatment.

[0464] SRTC analysis showed that differences in bile acid profiles still developed between weeks 12 and 24 when discriminating between strong and weak responders. Here again, CDCA, TDCA / CA and indices of bile acid conjugation were the main discriminators.

Claims

1. 1. A method of treating at least one symptom of amyotrophic lateral sclerosis (ALS) in a subject, comprising: (a) administering to the subject one or more doses of a composition comprising about 1 gram of taurursodiol (TURSO) and about 3 grams of sodium phenylbutyrate; (b) the said object (i) Sodium phenylbutyrate C ｍａｘ is about 3 to about 425 μg / mL, or the C of phenylacetic acid ｍａｘ is about 5 to about 50 μg / mL; (ii) AUC of sodium phenylbutyrate ０－ｌａｓｔ is about 20 to about 550 μg×h / mL, or the AUC of phenylacetic acid ０－ｌａｓｔ is about 20 to about 160 μg×h / mL; or (iii) AUC of sodium phenylbutyrate ０－∞ is about 25 to about 545 μg×h / mL, or the AUC of phenylacetic acid ０－∞ is about 21 to about 155 μg x h / mL; determining that (c) further administering said composition to said subject; A method comprising:

2. Step (b) is a step of subjecting the sodium phenylbutyrate to C ｍａｘ is about 90 to about 170 μg / mL.

3. Step (b) is a step of subjecting the sodium phenylbutyrate to C ｍａｘ is about 110 to about 150 μg / mL.

4. Step (b) is a step of isolating the target phenylacetic acid from C ｍａｘ is about 10 to about 45 μg / mL.

5. Step (b) comprises measuring the AUC of sodium phenylbutyrate in the subject. ０－ｌａｓｔ is about 140 to about 300 μg×h / mL.

6. Step (b) comprises measuring the AUC of phenylacetic acid in the subject. ０－ｌａｓｔ is about 40 to about 80 μg×h / mL.

7. Step (b) comprises measuring the AUC of sodium phenylbutyrate in the subject. ０－∞ is about 140 to about 300 μg×h / mL.

8. Step (b) comprises measuring the AUC of phenylacetic acid in the subject. ０－∞ is about 40 to about 80 μg×h / mL.

9. 9. The method of any one of claims 1 to 8, wherein step (a) comprises administering the composition once daily or twice daily for about 1 day to about 40 weeks.

10. 10. The method of any one of claims 1 to 9, wherein step (a) comprises administering the composition once daily or twice daily for about 10 weeks to about 26 weeks.

11. 11. The method of any one of claims 1 to 10, wherein step (a) comprises administering the composition twice daily for about 9 weeks to about 21 weeks.

12. 11. The method of any one of claims 1 to 10, wherein step (a) comprises administering the composition once daily for about 3 weeks, followed by administering the composition twice daily for about 9 weeks to about 21 weeks.

13. The method of any one of claims 1 to 12, wherein step (b) comprises drawing blood from the subject about 1 hour after the last administration of the composition.

14. The method of any one of claims 1 to 12, wherein step (b) comprises drawing blood from the subject about 4 hours after the last administration of the composition.

15. 1. A method of treating at least one symptom of ALS in a subject, comprising: (a) administering to the subject one or more times a composition comprising about 1 gram of TURSO and about 3 grams of sodium phenylbutyrate; (b) measuring the plasma concentration of one or more bile acids selected from TURSO, UDCA, or GUDCA in the subject; and (c) further administering said composition to said subject; A method comprising:

16. 16. The method of claim 15, wherein the plasma concentration is a steady state plasma concentration.

17. 17. The method of claim 15 or 16, wherein step (a) comprises administering the composition once daily or twice daily for about 1 day to about 40 weeks.

18. 18. The method of any one of claims 15 to 17, wherein step (a) comprises administering the composition once daily or twice daily for about 10 weeks to about 26 weeks.

19. 18. The method of any one of claims 15 to 17, wherein step (a) comprises administering the composition twice daily for about 9 weeks to about 21 weeks.

20. 18. The method of any one of claims 15 to 17, wherein step (a) comprises administering the composition once daily for about 3 weeks, followed by administering the composition twice daily for about 9 weeks to about 21 weeks.

21. The method of any one of claims 15 to 20, wherein step (b) comprises measuring the plasma concentration about 1 hour after the last administration of the composition.

22. The method of any one of claims 15 to 20, wherein step (b) comprises measuring the plasma concentration about 4 hours after the last administration of the composition.

23. 23. The method of any one of claims 15 to 22, wherein step (b) comprises measuring the plasma concentration of TURSO in the subject about 1 hour after the last administration of the composition, and the plasma concentration of TURSO is about 20 to about 2570 ng / mL.

24. 24. The method of claim 23, wherein the plasma concentration of TURSO is about 20 to about 1045 ng / mL.

25. 24. The method of claim 23, wherein the plasma concentration of TURSO is about 88 to about 540 ng / mL.

26. 21. The method of any one of claims 15 to 20, wherein step (b) comprises measuring the plasma concentration of TURSO in the subject about 4 hours after the last administration of the composition, and the plasma concentration of TURSO is about 20 to about 3250 ng / mL.

27. 27. The method of claim 26, wherein the steady state plasma concentration of TURSO is from about 20 to about 1125 ng / mL.

28. 27. The method of claim 26, wherein the steady state plasma concentration of TURSO is from about 155 to about 785 ng / mL.

29. 21. The method of any one of claims 15 to 20, wherein step (b) comprises measuring the plasma concentration of UDCA in the subject about 1 hour after the last administration of the composition, and the plasma concentration of UDCA is about 20 to about 6020 ng / mL.

30. 30. The method of claim 29, wherein the plasma concentration of UDCA is about 20 to about 1955 ng / mL.

31. 30. The method of claim 29, wherein the plasma concentration of UDCA is about 285 to about 1125 ng / mL.

32. 23. The method of any one of claims 15 to 22, wherein step (b) comprises measuring the plasma concentration of UDCA in the subject about 4 hours after the last administration of the composition, and the plasma concentration of UDCA is about 20 to about 7340 ng / mL.

33. 33. The method of claim 32, wherein the plasma concentration of UDCA is about 20 to about 2550 ng / mL.

34. 33. The method of claim 32, wherein the plasma concentration of UDCA is about 305 to about 1395 ng / mL.

35. 23. The method of any one of claims 15 to 22, wherein step (b) comprises measuring the plasma concentration of GUDCA in the subject about 1 hour after the last administration of the composition, and wherein the plasma concentration of GUDCA is about 20 to about 4600 ng / mL.

36. 36. The method of claim 35, wherein the plasma concentration of GUDCA is about 65 to about 2085 ng / mL.

37. 36. The method of claim 35, wherein the plasma concentration of GUDCA is about 340 to about 1635 ng / mL.

38. 23. The method of any one of claims 15 to 22, wherein step (b) comprises measuring the plasma concentration of GUDCA in the subject about 4 hours after the last administration of the composition, and wherein the plasma concentration of GUDCA is about 20 to about 5290 ng / mL.

39. 39. The method of claim 38, wherein the plasma concentration of GUDCA is about 320 to about 2315 ng / mL.

40. 39. The method of claim 38, wherein the plasma concentration of GUDCA is about 530 to about 1915 ng / mL.

41. 41. The method of any one of claims 15 to 40, wherein the method further comprises, prior to step (a), determining a baseline plasma concentration of the bile acid in the subject.

42. 42. The method of claim 41, wherein the method comprises determining a baseline plasma concentration of TURSO in the subject, the baseline plasma concentration of TURSO being from about 20 to about 577 ng / mL.

43. 43. The method of claim 42, wherein the baseline plasma concentration of TURSO is about 20 to about 125 ng / mL.

44. 42. The method of claim 41, wherein the method comprises determining a baseline plasma concentration of UDCA in the subject, the baseline plasma concentration of UDCA being from about 20 to about 5970 ng / mL.

45. 45. The method of claim 44, wherein the baseline plasma concentration of UDCA is from about 20 to about 825 ng / mL.

46. 45. The method of claim 44, wherein the baseline plasma concentration of UDCA is about 20 to about 53 ng / mL.

47. 42. The method of claim 41, wherein the method comprises determining a baseline plasma concentration of GUDCA in the subject, wherein the baseline plasma concentration of GUDCA is from about 20 to about 4540 ng / mL.

48. 48. The method of claim 47, wherein the baseline plasma concentration of GUDCA is from about 20 to about 755 ng / mL.

49. 48. The method of claim 47, wherein the baseline plasma concentration of GUDCA is about 25 to about 180 ng / mL.

50. 50. The method of any one of claims 1 to 49, wherein step (a) comprises administering the composition at least 2 hours after the subject ingests food or at least 1 hour before the subject ingests food.

51. 1. A method of increasing a plasma concentration of a bile acid in a subject, comprising administering to the subject one or more times a composition comprising about 1 g of TURSO and about 3 g of sodium phenylbutyrate, wherein the bile acid is selected from TURSO, UDCA, or GUDCA; When the bile acid is TURSO, the plasma concentration is about 20 to about 3250 ng / mL, when the bile acid is UDCA, the plasma concentration is about 20 to about 7340 ng / mL, and when the bile acid is GUDCA, the plasma concentration is about 20 to about 5290 ng / mL. method.

52. 52. The method of claim 51, wherein the plasma concentration is a steady state plasma concentration.

53. 53. The method of claim 51 or 52, wherein the method comprises administering the composition once daily or twice daily for about 1 day to about 40 weeks.

54. 54. The method of any one of claims 51-53, wherein the method comprises administering the composition once daily or twice daily for about 10 weeks to about 26 weeks.

55. 54. The method of any one of claims 51-53, wherein the method comprises administering the composition twice daily for about 9 weeks to about 21 weeks.

56. 54. The method of any one of claims 51-53, wherein the method comprises administering the composition once daily for about 3 weeks, followed by administering the composition twice daily for about 9 weeks to about 21 weeks.

57. 57. The method of any one of claims 51 to 56, wherein the method comprises measuring the plasma concentration of the bile acid about 1 hour after the last administration of the composition.

58. 57. The method of any one of claims 51 to 56, wherein the method comprises measuring the plasma concentration of the bile acid about 4 hours after the last administration of the composition.

59. The method of any one of claims 1 to 58, wherein the composition is administered orally.

60. 59. The method of any one of claims 1 to 58, wherein the composition is administered through a feeding tube.

61. The method of any one of claims 1 to 58, wherein the composition is injected as a bolus.

62. The method of any one of claims 1 to 61, wherein the composition is a powder.

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