Combination of taurine diol and sodium phenylbutyrate for treatment of amyotrophic lateral sclerosis

Abstract

Provided herein are methods and compositions for the treatment of neurodegenerative diseases (e.g., ALS). The methods may comprise administering to the subject a bile acid or a pharmaceutically acceptable salt thereof and a phenylbutyric acid compound.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Application No. 17 / 746,458, filed on May 17, 2022, which is incorporated herein by reference in its entirety. Technical Field

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

[0004] Amyotrophic lateral sclerosis (ALS) is the most common progressive motor neuron disease. ALS causes progressive degeneration of motor neurons, resulting in rapidly progressive muscle weakness and atrophy, which ultimately leads to partial or complete paralysis. The median survival from symptom onset is 2 to 3 years, with respiratory failure being the leading cause of death. ALS treatment currently focuses on symptom management. Currently available are only two FDA-approved drugs for ALS, i.e. riluzole and edaravone. Therefore, there is a need for improved therapies for the treatment of ALS. Summary of the invention

[0005] Provided herein are methods of treating at least one symptom of ALS in a subject, the methods 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) determining that the subject has (1) a C of about 3 to about 425 µg / mL for sodium phenylbutyrate; max , and / or (2) about 5 to about 50 µg / mL of C for phenylacetate max ; And (c) administering to the subject an additional dose of the composition. In some embodiments, the method further comprises determining that the subject has a C of about 90 to about 170 μg / mL for sodium phenylbutyrate. max In some embodiments, the method further comprises determining that the subject has a C of about 110 to about 150 µg / mL for sodium phenylbutyrate. max In some embodiments, the method further comprises determining that the subject has a C of about 10 to about 45 µg / mL for phenylacetic acid. max In some embodiments, C max is the steady state C max .

[0006] Also provided herein are methods 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 gram of TURSO and about 3 grams of sodium phenylbutyrate; (b) determining that the subject has (1) an AUC for sodium phenylbutyrate of about 20 to about 550 μg*h / mL 0-最后 , and / or (2) an AUC of about 20 to about 160 μg*h / mL for phenylacetic acid 0-最后 ; And (c) administering an additional dose of the composition to the subject. In some embodiments, the method further comprises determining that the subject has an AUC of about 140 to about 300 μg*h / mL for sodium phenylbutyrate. 0-最后 In some embodiments, the method further comprises determining that the subject has an AUC for phenylacetic acid of about 40 to about 80 μg*h / mL. 0-最后 .

[0007] Also provided herein are methods 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 gram of TURSO and about 3 grams of sodium phenylbutyrate; (b) determining that the subject has (1) an AUC for sodium phenylbutyrate of about 25 to about 545 μg*h / mL 0-∞ , and / or (2) an AUC of about 21 to about 155 μg*h / mL for phenylacetic acid 0-∞ ; And (c) administering an additional dose of the composition to the subject. In some embodiments, the method further comprises determining that the subject has an AUC of about 140 to about 300 μg*h / mL for sodium phenylbutyrate. 0-∞ In some embodiments, the method further comprises determining that the subject has an AUC for phenylacetic acid of about 40 to about 80 μg*h / mL. 0-∞ .

[0008] Also provided herein are methods of administering TURSO and sodium phenylbutyrate to a subject having one or more symptoms of ALS, the methods comprising: (a) administering to the subject one or more doses of a composition comprising about 1 gram of taurine diol (TURSO) and about 3 grams of sodium phenylbutyrate; (b) determining that the subject has (i) a C of about 3 to about 425 µg / mL for sodium phenylbutyrate; max , or about 5 to about 50 µg / mL for phenylacetic acid max , (ii) AUC of about 20 to about 550 μg*h / mL for sodium phenylbutyrate 0-最后 , or an AUC of about 20 to about 160 μg*h / mL for phenylacetic acid 0-最后 , or (iii) an AUC of about 25 to about 545 μg*h / mL for sodium phenylbutyrate 0-∞, or an AUC of about 21 to about 155 μg*h / mL for phenylacetic acid 0-∞ ; and (c) administering an additional dose of the composition to the subject.

[0009] In some embodiments, in any of the above methods, step (a) includes applying the composition once a day or twice a day for about 1 day to about 40 weeks. In some embodiments, in any of the above methods, step (a) includes applying the composition once a day or twice a day for about 10 weeks to about 26 weeks. In some embodiments, in any of the above methods, step (a) includes applying the composition twice a day for about 9 weeks to about 21 weeks. In some embodiments, in any of the above methods, step (a) includes applying the composition once a day for about 3 weeks, followed by applying the composition twice a day for about 9 weeks to about 21 weeks. In some embodiments, in any of the above methods, step (b) includes obtaining a blood sample from a subject about one hour after the last dose of the composition. In some embodiments, in any of the above methods, step (b) includes obtaining a blood sample from a subject about four hours after the last dose of the composition.

[0010] Also provided herein is a method of treating at least one symptom of ALS in a subject or a method of administering TURSO and sodium phenylbutyrate to a subject having one or more symptoms of ALS, the method comprising: (a) administering one or more doses of a composition comprising about 1 gram of TURSO and about 3 grams of sodium phenylbutyrate to the subject; (b) determining the plasma concentration of one or more bile acids selected from TURSO, UDCA or GUDCA in the subject; and (c) administering another dose of the composition to the subject. In some embodiments, the plasma concentration is a steady-state plasma concentration. In some embodiments, step (a) comprises administering the composition once or twice a day for about 1 day to about 40 weeks. In some embodiments, step (a) comprises administering the composition once 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 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, step (b) comprises determining the plasma concentration about one hour after the last dose of the composition. In some embodiments, step (b) comprises determining the plasma concentration about four hours after the last dose of the composition.

[0011] In some embodiments, step (b) includes determining the plasma concentration of TURSO in the subject about one hour after the last dose of the composition, wherein the plasma concentration of TURSO is about 20 to about 2570 ng / mL. In some embodiments, wherein the plasma concentration of TURSO is about 20 to about 1045 ng / mL. In some embodiments, wherein the plasma concentration of TURSO is about 88 to about 540 ng / mL. In some embodiments, wherein step (b) includes determining the plasma concentration of TURSO in the subject about four hours after the last dose of the composition, wherein the plasma concentration of TURSO is about 20 to about 3250 ng / mL. In some embodiments, wherein the steady-state plasma concentration of TURSO is about 20 to about 1125 ng / mL. In some embodiments, wherein the steady-state plasma concentration of TURSO is about 155 to about 785 ng / mL.

[0012] In some embodiments, step (b) includes determining the plasma concentration of UDCA in the subject about one hour after the last dose of the composition, wherein the plasma concentration of UDCA is about 20 to about 6020 ng / mL. In some embodiments, wherein 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) includes determining the plasma concentration of UDCA in the subject about four hours after the last dose 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.

[0013] In some embodiments, step (b) comprises determining the plasma concentration of GUDCA in the subject about one hour after the last dose 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 determining the plasma concentration of GUDCA in the subject about four hours after the last dose 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.

[0014] In some embodiments, the method further comprises determining the baseline plasma concentration of bile acids in the subject prior to step (a). In some embodiments, the method comprises determining the baseline plasma concentration of TURSO in the subject, and 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.

[0015] In some embodiments, the method includes determining a baseline plasma concentration of UDCA in a subject, and 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.

[0016] In some embodiments, the method includes determining a baseline plasma concentration of GUDCA in a subject, and 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.

[0017] In some embodiments of any of the methods mentioned above, step (a) comprises administering a dose of the composition more than two hours after the subject has ingested food or more than one hour before the subject has ingested food.

[0018] Also provided herein is a method for increasing the plasma concentration of bile acids in a subject, the method comprising administering to the subject one or more doses of a composition comprising about 1 gram of TURSO and about 3 grams of sodium phenylbutyrate, wherein the bile acid is selected from TURSO, UDCA or GUDCA, wherein the plasma concentration is about 20 to about 3250 ng / mL when the bile acid is TURSO, wherein the plasma concentration is about 20 to about 7340 ng / mL when the bile acid is UDCA, and wherein the plasma concentration is about 20 to about 5290 ng / mL when the bile acid is GUDCA. In some embodiments, the plasma concentration is a steady-state plasma concentration. In some embodiments, the method comprises administering the composition once or twice a day for about 1 day to about 40 weeks. In some embodiments, the method comprises administering the composition once or twice a day for about 10 weeks to about 26 weeks. In some embodiments, the method comprises administering the composition twice a day for about 9 weeks to about 21 weeks. In some embodiments, the method 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, the method comprises determining the plasma concentration of bile acids about one hour after the last dose of the composition. In some embodiments, the method comprises determining the plasma concentration of bile acids about four hours after the last dose of the composition.

[0019] In some embodiments of any of the methods mentioned above, the composition is administered orally. In some embodiments of any of the methods mentioned above, the composition is administered by a feeding tube. In some embodiments of any of the methods mentioned above, the composition is administered by bolus injection. In some embodiments of any of the methods mentioned above, the composition is a powder formulation.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.

[0021] It should be understood that certain features of the present disclosure 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 disclosure described in the context of a single embodiment for brevity may also be provided individually or in any suitable sub-combination. All combinations of embodiments related to the present disclosure are specifically encompassed by the present disclosure and disclosed herein, just as each and every combination is individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically encompassed by the present disclosure and disclosed herein, just as each and every such sub-combination is individually and explicitly disclosed herein.

[0022] All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail. In addition, materials, methods and embodiments are merely illustrative and are not intended to be restrictive. Other features and advantages of the present invention will be apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a flow chart of the study sequence followed in Example 1.

[0024] Figure 2 It shows that in the Log 10 / Graph of the geometric mean (× / ÷geometric SD) plasma concentrations of PB following a single oral dose of AMX0035 in the fasted and fed states on a linear scale.

[0025] Figure 3 It shows that in the Log 10 / Graph of the geometric mean (× / ÷geometric SD) plasma concentrations of PAA following a single oral dose of AMX0035 in the fasted and fed states on a linear scale.

[0026] Figure 4 It shows that in the Log 10 / Graph of the geometric mean (× / ÷geometric SD) plasma concentrations of TURSO following a single oral dose of AMX0035 in the fasted and fed states on a linear scale.

[0027] Figure 5 It shows that in the Log 10 / Graph of the geometric mean (× / ÷geometric SD) plasma concentrations of UDCA following a single oral dose of AMX0035 in the fasted and fed states on a linear scale.

[0028] Figure 6 It shows that in the Log 10 / Graph of the geometric mean (× / ÷geometric SD) plasma concentrations of GUDCA following a single oral dose of AMX0035 in the fasting and fed states on a linear scale.

[0029] Figure 7 is a TUDCA boxplot of visit groups pooled by gender.

[0030] Figure 8 is a boxplot of GUDCA for visit groups clustered by gender.

[0031] Fig. 9 is a UDCA boxplot of visit groups clustered by age category.

[0032] Fig.10 is a GUDCA boxplot of visit groups clustered by age category.

[0033] Fig.11 is a TUDCA boxplot of visit groups clustered by antibiotic use.

[0034] Fig.12 is a box plot of UDCA by visit group clustered by antibiotic use.

[0035] Fig.13 is a box plot of GUDCA for visit groups clustered by antibiotic use.

[0036] Fig.14 is a TUDCA boxplot of visit groups clustered by GFR category.

[0037] Fig.15 is a box plot of UDCA for visit groups clustered by glomerular filtration rate category.

[0038] Fig.16 is a box plot of GUDCA for visit groups clustered by glomerular filtration rate category.

[0039] Fig.17 is a schematic diagram showing pathway visualization (week 12) of changes in CC based on ANOVA results.

[0040] Fig.18 is a schematic diagram showing pathway visualization (week 24) of changes in CC based on ANOVA results. DETAILED DESCRIPTION

[0041] In one aspect, provided herein are methods of treating at least one symptom of ALS in a subject, or administering TURSO and sodium phenylbutyrate to a subject having one or more symptoms of ALS, the methods comprising (a) administering to the subject one or more doses of a composition comprising about 1 gram of taurine diol (TURSO) and about 3 grams of sodium phenylbutyrate; (b) determining that the subject has (1) a C of about 3 to about 425 µg / mL for sodium phenylbutyrate; max , and / or (2) about 5 to about 50 µg / mL of C for phenylacetic acid max ; and (c) administering to the subject an additional dose of the composition. In another aspect, provided herein are methods of treating at least one symptom of ALS in a subject, or administering TURSO and sodium phenylbutyrate to a subject having one or more symptoms of ALS, the method comprising: (a) administering to the subject one or more doses of a composition comprising about 1 gram of TURSO and about 3 grams of sodium phenylbutyrate; (b) determining that the subject has (1) an AUC for sodium phenylbutyrate of about 20 to about 550 μg*h / mL; 0-最后 , and / or (2) an AUC of about 20 to about 160 μg*h / mL for phenylacetic acid 0-最后 ; and (c) administering to the subject an additional dose of the composition. In another aspect, provided herein are methods of treating at least one symptom of ALS in a subject, or administering TURSO and sodium phenylbutyrate to a subject having one or more symptoms of ALS, the method comprising: (a) administering to the subject one or more doses of a composition comprising about 1 gram of TURSO and about 3 grams of sodium phenylbutyrate; (b) determining that the subject has (1) an AUC for sodium phenylbutyrate of about 25 to about 545 μg*h / mL; 0-∞ , and / or (2) an AUC of about 21 to about 155 μg*h / mL for phenylacetic acid 0-∞ ; and (c) administering an additional dose of the composition to the subject.

[0042] Additional aspects of the present disclosure relate to methods of treating at least one symptom of ALS in a subject, or methods of administering TURSO and sodium phenylbutyrate to a subject having one or more symptoms of ALS, the methods comprising: (a) administering to the subject one or more doses of a composition comprising about 1 gram of TURSO and about 3 grams of sodium phenylbutyrate; (b) determining the plasma concentration of one or more bile acids selected from TURSO, UDCA, or GUDCA in the subject; (c) administering to the subject an additional dose of the composition.

[0043] An additional aspect of the present disclosure relates to a method of increasing the plasma concentration of a bile acid in a subject, the method comprising administering to the subject one or more doses of a composition comprising about 3 grams of sodium phenylbutyrate and about 1 gram of TURSO, wherein the bile acid is selected from TURSO, UDCA, and GUDCA, wherein in the case of the bile acid is TURSO, the plasma concentration is about 20 to about 3250 ng / mL, wherein in the case of the bile acid is UDCA, the plasma concentration is about 20 to about 7340 ng / mL, and wherein in the case of the bile acid is GUDCA, the plasma concentration is about 20 to about 5290 ng / mL.

[0044] Where a range of values ​​is provided, it is understood that, unless the context clearly dictates otherwise, each intermediate value (to one tenth of the unit of the lower limit) between the upper and lower limits of the range and any other stated or intermediate values ​​within the stated range are included in the present disclosure. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also included in the present disclosure, except for any specifically excluded limits within the stated ranges. Where the stated range includes one or two of the limits, the range excluding any one or two of those included limits is also included in the present disclosure. Throughout the specification, it should be noted that 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 also used interchangeably.

[0045] Certain ranges are given herein where a numerical value is preceded by the term "about". The term "about" is used herein to provide literal support for the exact number that follows it, as well as for numbers that are close to or approximately the number that follows the term. In determining whether a number is close to or approximately a specifically listed number, the close or approximate unlisted number may be a number that is substantially equivalent to the specifically listed number in the context in which it is presented.

[0046] Unless otherwise defined, all technical terms, symbols and other scientific terms or expressions used herein are intended to have the meanings commonly understood by those skilled in the art to which this application belongs. In some cases, for clarity and / or for ease of reference, terms with commonly understood meanings are defined herein, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from definitions commonly understood in the art.

[0047] I. Amyotrophic lateral sclerosis (ALS)

[0048] The terms "amyotrophic lateral sclerosis" and "ALS" are used interchangeably herein and include all classifications of ALS known in the art, including but not limited to classic ALS (e.g., ALS that affects lower and upper motor neurons), primary lateral sclerosis (PLS, such as those that affect only upper motor neurons), progressive bulbar palsy (PBP or bulbar onset, a form of ALS that typically begins with difficulty swallowing, chewing, and speaking), and progressive muscular atrophy (PMA, typically affecting only lower motor neurons). These terms include sporadic and familial (hereditary) ALS, ALS at any rate of progression (e.g., rapid, non-slow, or slowly progressive), and ALS at any stage (e.g., pre-onset, onset, and late stage of ALS).

[0049] The subject in the methods described herein may exhibit one or more symptoms associated with ALS, or have been diagnosed with ALS. In some embodiments, the subject may be suspected of having ALS, and / or be at risk of developing ALS.

[0050] The subject in the methods described herein may exhibit one or more symptoms associated with benign fasciculation syndrome (BFS) or cramps-fasciculation syndrome (CFS).

[0051] Some embodiments of any method described herein may also include determining that a subject suffers from ALS or is at risk of developing ALS, diagnosing a subject suffering from ALS or being at risk of developing ALS, or selecting a subject suffering from ALS or being at risk of developing ALS. Similarly, some embodiments of any method described herein may also include determining that a subject suffers from benign fasciculation syndrome or spastic fasciculation syndrome or is at risk of developing benign fasciculation syndrome or spastic fasciculation syndrome, diagnosing a subject suffering from BFS or CFS or being at risk of developing BFS or CFS, or selecting a subject suffering from BFS or CFS or being at risk of developing BFS or CFS.

[0052] In some embodiments of any of the methods described herein, the subject has displayed one or more symptoms of ALS for about 24 months or less (e.g., about 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 month, or 1 week or less). In some embodiments, the subject has displayed one or more symptoms of ALS for about 36 months or less (e.g., about 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, or 25 months or less).

[0053] The order and type of ALS symptoms shown by the subject may depend on which motor neurons in the body are first damaged, and therefore which muscles in the body are first damaged. For example, bulbar onset, limb onset or respiratory onset ALS may present similar or different symptoms. In general, ALS symptoms may include muscle weakness or atrophy (for example, affecting the upper body, lower body and / or speech), fasciculations (twitches), spasms or stiffness of the affected muscles. The early symptoms of ALS may include symptoms of the arms or legs, difficulty speaking clearly or dysphagia (for example, in bulbar onset ALS). Other symptoms include loss of tongue mobility, dyspnea, dyspnea or abnormal lung function, dyschewing and / or difficulty walking (for example, causing falls). The subject may have respiratory muscle weakness as the initial manifestation of ALS symptoms. Such a subject may have a very poor prognosis, and in some cases has an average survival period of about two months from diagnosis. In some subjects, the onset time of respiratory muscle weakness can be used as a prognostic factor.

[0054] ALS symptoms can also be classified according to the part (i.e. upper motor neuron or lower motor neuron) in which the nervous system degenerates. Lower motor neuron degeneration, for example, is manifested as weakness or emaciation in one or more of the medulla oblongata, cervical, thoracic and / or lumbosacral regions. Upper motor neuron degeneration can include tendon reflex enhancement, spasm, pseudobulbar features, Hoffmann reflex, extensor plantar response and hyperreflexia (hyperreflexia) (including overactive gagging reflex). The progress of neuronal degeneration or muscle weakness is the sign of the disease. Therefore, some embodiments of the present disclosure provide a method for improving at least one symptom of lower motor neuron degeneration, at least one symptom of upper motor neuron degeneration or at least one symptom of each of lower motor neuron degeneration and upper motor neuron degeneration. In some embodiments of any method described herein, symptom onset can be determined based on information from experimenter and / or experimenter family members. In some embodiments, the median time from symptom onset to diagnosis is about 12 months.

[0055] In some cases, the subject has been diagnosed with ALS. For example, the subject may have been diagnosed with ALS for about 24 months or less (e.g., about 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 month or less). For example, the subject may have been diagnosed with ALS for 1 week or less, or may have been diagnosed with ALS on the same day of administering the disclosed treatment. The subject may have been diagnosed with ALS for more than about 24 months (e.g., more than about 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76 or 80 months). Methods for diagnosing ALS are known in the art. For example, the subject may be diagnosed based on clinical history, family history, physical or neurological examination (e.g., signs of lower motor neuron or upper motor neuron degeneration). The subject can be confirmed or identified as having ALS, for example, by a healthcare professional. Multiple parties can be involved in the diagnostic process. For example, where a sample is obtained from a subject as part of the diagnosis, a first party can obtain a sample from the subject, and a second party can test the sample. In some embodiments of any human subject described herein, the subject is diagnosed, selected, or referred by a medical practitioner (e.g., a general practitioner).

[0056] In some embodiments, the subject meets the El Escorial criteria for possible or definite ALS, i.e., the subject exhibits:

[0057] 1. Signs of lower motor neuron (LMN) degeneration found by clinical, electrophysiological or neuropathological examination;

[0058] 2. Signs of upper motor neuron (UMN) degeneration detected by clinical examination; and

[0059] 3. Progressive spread of signs in one area or to other areas in the absence of:

[0060] Electrophysiological evidence for other disease processes that may explain the signs of LMN and / or UMN degeneration; and

[0061] Neuroimaging evidence of other disease processes that may explain the observed clinical and electrophysiological signs.

[0062] According to the El Escorial criteria, signs of LMN and UMN degeneration in four regions of the central nervous system (including the brainstem, cervical, thoracic, and lumbosacral spinal cord) were evaluated. Subjects could be determined to fall into one of the following categories:

[0063] A. Clinically definite ALS, defined based on clinical evidence only by the presence of UMN and LMN signs in three areas.

[0064] B. Clinically probable ALS, defined based on clinical evidence only by UMN and LMN signs in at least two areas, with some UMN signs necessarily being superior to (above) the LMN signs.

[0065] C. Clinically probable ALS - laboratory-supported, defined when clinical signs of UMN and LMN dysfunction are present in only one region or when isolated UMN signs are present in one region and LMN signs defined by EMG criteria are present in at least two limbs, with appropriate application of neuroimaging and clinical laboratory protocols to exclude other causes.

[0066] D. Clinically probable ALS, defined when clinical symptoms of UMN and LMN dysfunction are found together in only one area or when UMN signs are found alone in two or more areas; or when LMN symptoms are found on the upper side of UMN symptoms and the diagnosis is clinically probable-laboratory supported.

[0067] In some embodiments, the subject has clinically confirmed ALS (eg, based on El Escorial criteria).

[0068] Subjects can be evaluated and / or diagnosed using the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R). The ALSFRS-R is an ordinal rating scale (score 0-4) that determines the subject's assessment of their ability and independence in 12 functional activities related to ALS. The ALSFRS-R score calculated at the time of diagnosis can be compared to scores over time to determine the rate of progression. Changes in ALSFRS-R scores can be correlated with changes in strength over time and can be correlated with quality of life measures and predict survival. The ALSFRS-R shows a linear mean 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).

[0069] In the ALSFRS-R, the functions mediated by the cervical muscles, trunk muscles, lumbosacral muscles, and respiratory muscles are each assessed by 3 items. Each item is scored from 0 to 4, where 4 reflects no involvement with the disease and 0 reflects maximum involvement. The item scores are added to give a total score. The total score reflects the impact of ALS, with an exemplary classification as follows:

[0070] >40 (minimal to mild); 39-30 (mild to moderate); <30 (moderate to severe); <20 (advanced disease).

[0071] For example, a subject's ALSFRS-R score (e.g., a baseline ALSFRS-R score) can be 40 or higher (e.g., at least 41, 42, 43, 44, 45, 46, 47, or 48), 30 to 39 (inclusive) (e.g., 31, 32, 33, 34, 35, 36, 37, or 38), or 30 or lower (e.g., 21, 22, 23, 24, 25, 26, 27, 28, or 29). In some embodiments of any of the methods described herein, the subject has an ALSFRS-R score (e.g., a baseline ALSFRS-R score) of 40 or less (e.g., 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or less). In some embodiments, the subject has an ALSFRS-R score (e.g., a baseline ALSFRS-R score) of 20 or less (e.g., 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or less).

[0072] Because ALS is a progressive disease, all patients will usually progress over time. However, there is a large degree of inter-subject variability in the rate of progression, because some subjects die or need respiratory support within a few months, while other subjects have a relatively extended survival time. The subject described herein can have a rapidly progressive ALS or a slowly progressive ALS. The functional decline rate of the subject suffering from ALS can be measured by the change of the monthly ALSFRS-R score. For example, the score can drop by about 1.02 (± 2.3) points per month.

[0073] One predictor of patient progression is the patient's previous disease progression rate (ΔFS), which can be calculated as: ΔFS = (48 - ALSFRS-R score at assessment) / duration from onset to assessment time (months). The ΔFS score represents the ALSFRS-R score lost per month since symptom onset and can serve as an important predictor of progression and / or survival in patients 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) can 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 (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 greater (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 greater). In some embodiments of any of the methods described herein, the subject's ALS disease progression rate (ΔFS) can 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 may underestimate or overestimate a patient's progression when evaluated.

[0074] In some embodiments, the subject loses an average of about 0.8 to about 2 (e.g., about 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9) ALSFRS-R points per month over a 3-12 month period since the initial assessment. In some embodiments, the subject loses an average of more than about 1.2 ALSFRS-R points per month over a 3-12 month period since the initial assessment. The subject may have lost at least 3 points (e.g., at least 4, 6, 8, 10, 12, 14, 16, 20, 24, 28, or 32 points) in ALSFRS-R score over a 3-12 month period since the initial assessment. In some embodiments, the subject has lost an average of about 0.8 to about 2 (e.g., about 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9) ALSFRS-R points per month over the past 3-12 months. In some embodiments, the subject has lost an average of more than about 1.2 (e.g., more than about 1.5, 1.8, 2.0, 2.5, or 3) ALSFRS-R points per month over the past 3-12 months.

[0075] In some embodiments of any method described herein, the presence or level of a marker in a sample obtained from a subject can be used for ALS diagnosis or prognosis, or for tracking disease activity and therapeutic response. Suitable samples include, for example, cells, tissues or body fluids (e.g., blood, urine or cerebrospinal fluid (CSF) samples). For example, the level of phosphorylated neurofilament heavy subunit (pNF-H) or neurofilament light chain (NfL) in CSF and / or blood can be used as a biomarker for ALS diagnosis, prognosis, or for tracking disease activity or treatment outcome. pNF-H is the main component of the neuron cytoskeleton and is released into CSF ​​and bloodstream along with neuronal damage. The level of pNF-H may be related to 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).

[0076] The concentration of pNF-H in the CSF and / or blood of patients with ALS may increase significantly in the early disease stage. The higher levels of pNF-H in plasma, serum and / or CSF may be related to faster ALS progression (e.g., faster ALSFRS-R decline) and / or shorter survival time. The pNF-H concentration in plasma may be higher than those ALS patients with spinal onset in ALS patients with bulbar onset. In some cases, the imbalance between the relative expression levels of neurofilament heavy chain subunits and light chain subunits can be used for ALS diagnosis, prognosis or tracking disease progression.

[0077] Methods for detecting pNF-H and NfL (e.g., pNF-H and NfL in cerebrospinal fluid, plasma, or serum) are known in the art and include, but are not limited to, ELISA and Simoa assays (see, e.g., Shaw et al., Biochemical and Biophysical Research Communications 336:1268-1277, 2005; Ganesalingam et al., Amyotroph Lateral Scler Frontotemporal Degener 14(2):146-9, 2013; DeSchaepdryver et al., Annals of Clinical and Translational Neurology 6(10):1971-1979, 2019; Wilke et al., Clin Chem Lab Med 57(10):1556-1564, 2019; Poesen et al., Front Neurol 9:1167, 2018; Pawlitzki et al., Front. Neurol. 9:1037, 2018; Gille et al., Neuropathol Appl Neurobiol 45(3):291-304, 2019). Commercial pNF-H detection assays, such as those developed by EnCor Biotechnology, BioVendor, and Millipore-EMD, can also be used. Commercial NfL assay kits based on Simoa technology, such as those manufactured by Quanterix, can also be used (see, e.g., Thouvenot et al., European Journal of Neurology 27:251-257, 2020). Factors affecting pNF-H and NfL levels or their detection in serum or plasma associated with disease processes may be different from those in CSF. The levels of neurofilaments (e.g., pNF-H and / or NfL) in CSF and serum may be associated (see, e.g., Wilke et al., Clin Chem Lab Med 57(10):1556-1564, 2019).

[0078] The CSF or blood pNF-H level in a subject as described herein can be about 300 pg / mL or more (e.g., about 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 180 ... 50, 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 can be about 70 to about 1200 pg / mL (e.g., about 70 to about 1000, about 70 to about 800, about 80 to about 600, or about 90 to about 400 pg / mL). In some embodiments, CSF pNF-H levels can be about 1000 to about 5000 pg / mL (eg, about 1500 to about 4000 or about 2000 to about 3000 pg / mL).

[0079] The subject may have a CSF or blood NfL level of about 50 pg / mL or more (e.g., about 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 pg / mL or more). In some embodiments, serum NfL levels can be about 50 to about 300 pg / mL (e.g., about 50 to about 280, about 50 to about 250, about 50 to about 200, about 50 to about 150, about 50 to about 100, about 100 to about 300, about 100 to about 250, about 100 to about 200, about 100 to about 150, about 150 to about 300, about 150 to about 250, about 150 to about 200, about 200 to about 300, about 200 to about 250, or about 250 to about 300 pg / mL).In some embodiments, the CSF NfL level can be 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 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 3 0,000, 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 pg / mL).

[0080] Additional biomarkers useful for ALS diagnosis, prognosis, and disease progression monitoring are contemplated herein, including but not limited to CSF ​​levels of S100-β, cystatin C, and chitotriosidase (CHIT) (see, e.g., Chen et al., BMC Neurol 16:173, 2016). Serum levels of uric acid can be used as a biomarker for ALS prognosis (see, e.g., Atassi et al., Neurology 83(19):1719-1725, 2014). Akt phosphorylation can also be used as a biomarker for ALS prognosis (see, e.g., WO2012 / 160563). Urine levels of p75ECD and ketones can be used as biomarkers for ALS diagnosis (see, e.g., Shepheard et al., Neurology 88: 1137-1143, 2017). Serum and urine levels of creatinine can also be used as biomarkers. Other useful blood, CSF, neurophysiological, and neuroradiological biomarkers for ALS are described, e.g., in Turner et al., Lancet Neurol 8: 94-109, 2009. Any of the markers described herein can be used to diagnose whether a subject has ALS, or to determine whether a subject is at risk of developing ALS.

[0081] Subjects can also be identified as suffering from ALS or at risk of developing ALS based on gene analysis.Genetic variants associated with ALS are known in the art (see, for example, Taylor et al., Nature 539:197-206, 2016; Brown and Al-Chalabi N Engl J Med 377:162-72, 2017; and http: / / alsod.iop.kcl.ac.uk). Subjects described herein may carry mutations in one or more genes associated with familial and / or sporadic ALS.Exemplary genes associated with ALS include, but are not limited to: ANG, TARDBP, VCP, VAPB, SQSTM1, DCTN1, FUS, UNC13A, ATXN2, HNRNPA1, CHCHD10, MOBP, C21ORF2, NEK1, TUBA4A, TBK1, MATR3, PFN1, UBQLN2, TAF15, OPTN, TDP-43 and DAO. Additional descriptions of genes associated with ALS can be found in Therrien et al., Curr Neurol Neurosci Rep 16:59-71, 2016; Peters et al., J Clin Invest 125:2548, 2015, and Pottier et al., J Neurochem, 138:Suppl 1:32-53, 2016. Genetic 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.

[0082] The subject may have a mutation in the gene encoding CuZn-superoxide dismutase (SOD1). The mutation causes the SOD1 protein to be more prone to aggregation, resulting in the deposition of cellular inclusions containing misfolded SOD1 aggregates (see, e.g., Andersen et al., Nature Reviews Neurology 7:603-615, 2011). More than 100 different mutations in SOD1 are associated with hereditary ALS, many of which result in single amino acid substitutions in the protein. In some embodiments, the SOD1 mutation is A4V (i.e., alanine at position 4 is substituted with valine). SOD1 mutations are further described in, e.g., Rosen et al., Hum. Mol. Genet. 3, 981-987, 1994 and Rosen et al., Nature 362:59-62, 1993. In some embodiments, the subject has a mutation in the C9ORF72 gene. Repeat expansion in the C9ORF72 gene is a common cause of ALS, where both loss of C9ORF72 function and gain of repeated toxic function are associated with ALS (see, e.g., Balendra and Isaacs, Nature Reviews Neurology 14:544-558, 2018). The methods described herein may include detecting SOD1 mutations and / or C9ORF72 mutations in a subject prior to administering bile acid and phenylbutyric acid compounds. Methods for screening mutations are well known in the art. Suitable methods include, but are not limited to, gene sequencing. See, e.g., Hou et al., Scientific Reports 6:32478, 2016; and Vajda et al., Neurology 88:1-9, 2017.

[0083] Skilled practitioners will appreciate that certain factors may affect the bioavailability and metabolism of the administered compound for a subject and may be adjusted accordingly. These include, but are not limited to, liver function (e.g., levels of liver enzymes), renal function, and gallbladder function (e.g., levels of ion absorption and secretion, cholesterol transporters). There may be variability in the exposure level of each subject to the administered compound (e.g., bile acids and phenylbutyric acid compounds), there may be differences in excretion levels, and there may be differences in the pharmacokinetics of the compound in the treated subjects. Any of the factors described herein may affect the drug exposure of the subject. For example, reduced compound clearance may result in increased drug exposure, while improved renal function may reduce actual drug exposure. The extent of drug exposure may be related to the subject's response to the administered compound and the outcome of treatment.

[0084] The age of the subject can be, for example, greater than about 18 years (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 The subject may have a BMI of about 18.5-30 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 28-30 kg / m 2 ). Having a mutation in any ALS-related gene described herein or presenting any biomarker described herein can indicate that the subject is at risk of developing ALS. Such subjects can be treated using the methods provided herein for preventive and prophylactic purposes.

[0085] In some embodiments, the subject has one or more symptoms of benign fasciculation syndrome (BFS) or cramp-fasciculation syndrome (CFS). BFS and CFS are peripheral nerve overexcitation disorders and can cause fasciculation, cramps, pain, fatigue, muscle stiffness and paresthesia. Methods for differentiating subjects suffering from these disorders are known in the art, such as by clinical examination and electromyography.

[0086] II. Composition

[0087] The present disclosure provides methods of treating at least one symptom of ALS in a subject, the methods comprising administering to the subject a bile acid or a pharmaceutically acceptable salt thereof and a phenylbutyrate compound. In some embodiments, the methods comprise administering to the subject a composition comprising TURSO and sodium phenylbutyrate.

[0088] Bile acid

[0089] As used herein, "bile acid" refers to a naturally occurring surfactant having a core derived from cholanic acid substituted with 3α-hydroxyls and optionally also substituted with other hydroxyls (typically at the C6, C7 or C12 position of the sterol core). Bile acid derivatives (e.g., water-soluble bile acid derivatives) and bile acids conjugated to amines are also covered by the term "bile acid". Bile acid derivatives include but are not limited to derivatives formed at the carbon atom to which the hydroxyl and carboxylic acid groups of bile acids are connected using other functional groups (including but not limited to halogens and amino groups). Soluble bile acids can include an aqueous preparation of a free acid form of bile acid combined with one of HCl, phosphoric acid, citric acid, acetic acid, ammonia or arginine. Suitable bile acids include, but are not limited to, taurodiol (TURSO), ursodeoxycholic acid (UDCA), chenodeoxycholic acid (also known as "chenodiol" or "chenic acid"), cholic acid, hyodeoxycholic acid, deoxycholic acid, 7-oxolithocholic acid, lithocholic acid, iododeoxycholic acid, iodocholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, glycoursodeoxycholic acid, taurocholic acid, glycocholic acid, or an analog, derivative, or prodrug thereof.

[0090] In some embodiments, the bile acid of the present disclosure is a hydrophilic bile acid. Hydrophilic bile acids include, but are not limited to, TURSO, UDCA, chenodeoxycholic acid, cholic acid, hyodeoxycholic acid, lithocholic acid, and glycoursodeoxycholic acid. Pharmaceutically acceptable salts or solvates of any bile acid disclosed herein are also contemplated. In some embodiments, bases commonly used to form pharmaceutically acceptable salts of bile acids of the present disclosure include hydroxides of alkali metals (including sodium, potassium and lithium); hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals such as aluminum and zinc; ammonia, organic amines such as unsubstituted or hydroxy-substituted mono-, di- or tri-alkylamines, dicyclohexylamine; tributylamine; pyridine; N-methylamine, N-ethylamine; diethylamine; triethylamine; mono-, di- or tri-(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.

[0091] The terms "tauroursodeoxycholic acid" (TUDCA) and "taurine diol" (TURSO) are used interchangeably herein.

[0092] The bile acid described herein can be TURSO, as shown in Formula I (with labeled carbons to aid in understanding where substitutions can be made).

[0093]

[0094] or a pharmaceutically acceptable salt thereof.

[0095] The bile acid described herein may be UDCA, as shown in Formula II (with labeled carbons to aid in understanding where substitutions may be made).

[0096]

[0097] or a pharmaceutically acceptable salt thereof.

[0098] The bile acid derivatives of the present disclosure may be physiologically relevant bile acid derivatives. For example, in the formula of TURSO or UDCA, any combination of substitution of hydrogen at position 3 or 7, stereochemical shift of the hydroxyl group at position 3 or 7 is suitable for use in the present composition.

[0099] "Bile acid" can also be a bile acid conjugated to an amino acid. The amino acid in the conjugate can be, but is not limited to, taurine, glycine, glutamine, asparagine, methionine or carboxymethylcysteine. Other amino acids that can be conjugated to the bile acids of the present disclosure include arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, cysteine, proline, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine and tryptophan as well as β-alanine and γ-aminobutyric acid. An example of such a bile acid is a compound of formula III:

[0100] ,

[0101] in

[0102] R is -H or C 1 -C 4 alkyl;

[0103] R 1 Yes-CH 2 -SO 3 R 3 , CH 2 COOH or CH 2 CH 2 COOH, and R 2 Yes - H;

[0104] or R 1 is -COOH and R 2 Yes-CH 2 -CH 2 -CONH 2 、-CH 2 -CONH 2 、-CH 2 -CH 2 -SCH 3 , CH 2 CH 2 CH 2 NH(C=NH)NH2 , CH 2 (Imidazolyl), CH 2 CH 2 CH 2 CH 2 NH 2 , CH 2 COOH, CH 2 CH 2 COOH, CH 2 OH, CH(OH)CH 3 , CH 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; and

[0105] R 3 is -H or the residue of an amino acid, or a pharmaceutically acceptable analog, derivative, prodrug or mixture thereof. An example of an amino acid is a basic amino acid. Other examples of amino acids include glycine, glutamine, asparagine, methionine, carboxymethylcysteine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, cysteine, proline, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine and tryptophan as well as β-alanine and γ-aminobutyric acid.

[0106] Another example of a bile acid of the present disclosure is a compound of Formula IV:

[0107]

[0108] in

[0109] R is -H or C 1 -C 4 alkyl;

[0110] R 1 Yes-CH 2 -SO 3 R 3 , and R 2 Yes - H;

[0111] or R 1 is -COOH and R 2 Yes-CH 2 -CH 2 -CONH 2 、-CH 2 -CONH 2 、-CH2 -CH 2 -SCH 3 or -CH 2 -S-CH 2 -COOH; and

[0112] R 3 is -H or a basic amino acid residue, or a pharmaceutically acceptable analog, derivative, prodrug or mixture thereof. Examples of basic amino acids include lysine, histidine and arginine.

[0113] In some embodiments, bile acid is TURSO. TURSO is an amphipathic bile acid and is a taurine conjugate form of UDCA. TURSO restores mitochondrial bioenergetic defects (Rodrigues et al., Biochemistry 42, 10: 3070-3080, 2003) by incorporating into mitochondrial membranes, reducing Bax translocation to mitochondrial membranes, reducing mitochondrial permeability, and increasing the apoptosis threshold of cells. It is used to treat cholesterol gallstones, where long-term treatment (e.g., 1 to 2 years) is generally required to obtain complete dissolution. It has been used to treat cholestatic liver diseases, including primary cirrhosis, familial intrahepatic cholestasis in children, and primary sclerosing cholangitis, as well as cholestasis caused by cystic fibrosis. TURSO is contraindicated in subjects with biliary infection, frequent biliary colic, or subjects with bile acid absorption difficulties (e.g., ileal disease or resection). Drug interactions may include interactions with substances that inhibit bile acid absorption, such as cholestyramine, and with drugs that increase cholesterol elimination in bile (TURSO reduces bile cholesterol content). Based on similar physicochemical properties, there are similarities in drug toxicity and interactions between TURSO and UDCA. The most common adverse reactions (≥1%) reported with TURSO were: abdominal discomfort, abdominal pain, diarrhea, nausea, pruritus, and rash. There were some cases of pruritus and a limited number of cases of elevated liver enzymes.

[0114] In some embodiments, bile acid is UDCA.UDCA or ursodeoxycholic acid have been used to treat gallstones, and are endogenously produced and secreted by the liver in the form of taurine (TURSO) or glycine (GUDCA) conjugates.Taurine conjugation increases its solubility by making UDCA more hydrophilic.TURSO is absorbed in the distal ileum under active transport, and therefore may have a slightly longer intestinal residence time than UDCA absorbed more proximally in the ileum.Ursodeoxycholic acid therapy is unrelated to liver damage.The abnormality of liver enzymes is unrelated to Actigall (ursodeoxycholic acid USP capsules) therapy, and Actigall has been confirmed to reduce liver enzyme levels in liver disease.However, give Actigall The subject should have SGOT (AST) and SGPT (ALT) measured at the beginning of therapy and as indicated by specific clinical situations afterwards. Previous studies have shown that bile acid sequestrants (such as cholestyramine and colestipol) can interfere with the action of ursodeoxycholic acid by reducing its absorption. Aluminum-based antacids have been shown to adsorb bile acids in vitro and may be expected to interfere with ursodeoxycholic acid in the same manner as bile acid sequestrants. Estrogens, oral contraceptives, and clofibrate (and possibly other lipid-lowering drugs) increase hepatic cholesterol secretion and promote cholesterol gallstone formation, and therefore may counteract the effectiveness of ursodeoxycholic acid.

[0115] Phenylbutyric acid compounds

[0116] Phenylbutyrate compounds are defined herein to encompass phenylbutyric acid (a low molecular weight aromatic carboxylic acid) as the free acid (4-phenylbutyrate, 4-PBA, 4-phenylbutyric acid or phenylbutyric acid) and pharmaceutically acceptable salts, cocrystals, polymorphs, hydrates, solvates, conjugates, derivatives or prodrugs thereof. Phenylbutyrate compounds described herein also encompass analogs of 4-PBA, including but not limited to tri-(4-phenylbutyrate)glyceride, phenylacetic acid (which is an active metabolite of PBA), 2-(4-methoxyphenoxy)acetic acid (2-POAA-OMe), 2-(4-nitrophenoxy)acetic acid (2-POAA-NO2) and 2-(2-naphthyloxy)acetic acid (2-NOAA) and pharmaceutically acceptable salts thereof. Phenylbutyrate compounds also encompass physiologically relevant 4-PBA species, such as, but not limited to, any substitution of hydrogen with deuterium in the 4-PBA structure. Other HDAC2 inhibitors are considered herein as alternatives to phenylbutyrate compounds.

[0117] Physiologically acceptable salts of phenylbutyric acid include, for example, 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.

[0118] In some embodiments of any of the methods described herein, the phenylbutyric acid compound is sodium phenylbutyrate. Sodium phenylbutyrate has the formula:

[0119] .

[0120] Phenylbutyrate is a pan-HDAC inhibitor and can improve ER stress by upregulating the master chaperone regulator DJ-1 and by recruiting 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 reduces activation of the classic ER stress pathway, folds misfolded proteins, and has been shown to improve 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).

[0121] In some embodiments, the combination of a bile acid (e.g., TURSO) or a pharmaceutically acceptable salt thereof and a phenylbutyric acid compound (e.g., sodium phenylbutyrate) has synergistic efficacy in treating one or more symptoms associated with ALS when administered at a specific ratio (e.g., any ratio described herein). The combination can be, for example, synergistically effective in treating one or more symptoms associated with ALS by simultaneously inhibiting endoplasmic reticulum stress and mitochondrial stress through linear modeling in a strong oxidative damage model (H 2 O 2 mediated toxicity) (see, e.g., U.S. Pat. No. 9,872,865 and U.S. Pat. No. 10,251,896).

[0122] preparation

[0123] Bile acid and phenylbutyric acid compounds described herein can be formulated as pharmaceutical compositions or used in pharmaceutical compositions. For example, methods described herein can include compositions comprising TURSO and sodium phenylbutyrate that use an effective amount. As used herein, the term "effective amount" refers to the amount or concentration of one or more drugs that effectively cause a desired effect or physiological outcome in the context of its use over a period of time (including acute or chronic administration and regular or continuous administration). Compositions can include TURSO of about 5% to about 15% w / w (for example, 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) and sodium phenylbutyrate of about 15% to about 45% w / w (for example, about 20% to about 40%, about 25% to about 35%, about 28% to about 32% or about 29% to about 30%, such as about 29.2% w / w). In some embodiments, the composition comprises about 9.7% w / w TURSO and 29.2% w / w sodium phenylbutyrate.

[0124] 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 between sodium phenylbutyrate and TURSO is about 3:1.

[0125] The compositions described herein may include any pharmaceutical carrier, adjuvant and / or vehicle. The term "pharmaceutical carrier or adjuvant" refers to a carrier or adjuvant that can be administered to a patient together with the compounds disclosed herein, and that does not destroy its pharmacological activity and is nontoxic when administered at a dose sufficient to deliver a therapeutic amount of the compound. As used herein, the term "pharmaceutical carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents compatible with drug administration. The pharmaceutical composition may contain any conventional nontoxic pharmaceutical carrier, adjuvant, or vehicle. In some cases, the pH of the preparation may be adjusted with a pharmaceutical acid, base, or buffer to enhance the stability of the formulated compound or its delivery form.

[0126] The compositions of the present disclosure may include about 8% to about 24% w / w dextrates (e.g., about 9% to about 23%, about 10% to about 22%, about 10% to about 20%, about 11% to about 21%, about 12% to about 20%, about 13% to about 19%, about 14% to about 18%, about 14% to about 17%, about 15% to about 16%, or about 15.6% w / w dextrates). Both anhydrous and hydrated dextrates are contemplated herein. The dextrates of the present disclosure may include a mixture of sugars produced by controlled enzymatic hydrolysis of starch. Some embodiments of any of the compositions described herein include a hydrated dextrate (e.g., NF grade, obtained from JRS Pharma, Colonial Scientific, or Quadra).

[0127] The composition of the present disclosure may include about 1% to about 6% w / w of sugar alcohol (e.g., about 2% to about 5%, about 3% to about 4% or about 3.9% w / w of sugar alcohol). Sugar alcohol can be derived from sugar and contain a hydroxyl group (-OH) connected to each carbon atom. Disaccharides and monosaccharides can form sugar alcohol. Sugar alcohol can be natural or produced by hydrogenation of sugar. Exemplary sugar alcohols include but are not limited to sorbitol, xylitol and mannitol. In some embodiments, the composition includes about 1% to about 6% w / w (e.g., about 2% to about 5%, about 3% to about 4% or about 3.9% w / w) of sorbitol.

[0128] The composition of the present disclosure can include about 22% to about 35% w / w of maltodextrin (e.g., about 22% to about 33%, about 24% to about 31%, about 25% to about 32%, about 26% to about 30% or about 28% to about 29% w / w, such as about 28.3% w / w of maltodextrin). Maltodextrin can form a flexible spiral, thereby enabling active ingredients (e.g., any phenylbutyric acid compounds described herein and bile acids) to be captured when dissolved in a solution, thereby masking the taste of the active ingredient. Maltodextrin produced by any suitable source is contemplated herein, including but not limited to pea, rice, cassava, corn and potato. In some embodiments, maltodextrin is pea maltodextrin. In some embodiments, the composition includes about 28.3% w / w of pea maltodextrin. For example, the pea maltodextrin obtained from Roquette (KLEPTOSE® LINECAPS) can be used.

[0129] The compositions described herein may also include a sugar substitute (e.g., sucralose). For example, the composition may include about 0.5% to about 5% w / w sucralose (e.g., about 1% to about 4%, about 1% to about 3%, or about 1% to about 2%, such as about 1.9% w / w sucralose). Other sugar substitutes contemplated herein include, but are not limited to, aspartame, neotame, acesulfame potassium, saccharin, and advantame.

[0130] In some embodiments, the composition includes one or more flavoring agents. The composition can include about 2% to about 15% w / w of flavoring agent (for example, about 3% to about 13%, about 3% to about 12%, about 4% to about 9%, about 5% to about 10% or about 5% to about 8%, such as about 7.3% w / w). Flavoring agent can include a material that imparts another material flavor or changes the composition characteristics by affecting its taste. Flavoring agent can be used to cover unpleasant taste without affecting physical and chemical stability, and can be selected based on the taste of the medicine to be mixed. Suitable flavoring agents include but are not limited to natural flavoring substances, artificial flavoring substances and imitation flavoring agents. The blend of flavoring agent can also be used. For example, the composition described herein can include two or more (for example, two, three, four, five or more) flavoring agents. Flavoring agent can be soluble and stable in water. The selection of suitable flavoring agent can be based on taste testing. For example, a variety of different flavoring agents can be added to the composition respectively, and taste testing is performed on it. Exemplary flavoring agents include any fruit flavor powder (e.g., peach, strawberry, mango, orange, apple, grape, raspberry, cherry, or mixed berry flavor powder). The compositions described herein may include about 0.5% to about 1.5% w / w (e.g., about 1% w / w) of mixed berry flavor powder and / or about 5% to about 7% w / w (e.g., about 6.3% w / w) of masking flavor. Suitable masking flavors can be obtained, for example, from Firmenich.

[0131] The compositions described herein may also include silicon dioxide (or silica). Adding silicon dioxide to the composition may prevent or reduce the aggregation of the components in the composition. Silicon dioxide may be used as an anti-caking agent, adsorbent, disintegrant, or glidant. In some embodiments, the compositions described herein include about 0.1% to about 2% w / w porous silicon dioxide (e.g., about 0.3% to about 1.5%, about 0.5% to about 1.2%, or about 0.8% to about 1%, such as 0.9% w / w). Porous silicon dioxide may have a higher H 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) compared to fumed silica. 2 O absorption capacity and / or higher porosity. The porous silica may have a H absorption capacity of about 5% to about 40% by weight (e.g., about 20% to about 40% or about 30% to about 40%) at a relative humidity of about 50%. 2 O absorption capacity.Compared with fumed silica, porous silica can have higher porosity at a relative humidity of about 20% or higher (e.g., about 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher).In some embodiments, the average particle size of porous silica is from about 2 μm to about 10 μm (e.g., from about 3 μm to about 9 μm, from about 4 μm to about 8 μm, from about 5 μm to about 8 μm or about 7.5 μm).In some embodiments, the average pore volume of porous silica is from about 0.1 cc / gm to about 2.0 cc / gm (e.g., from about 0.1 cc / gm to about 1.5 cc / gm, from about 0.1 cc / gm to about 1 cc / gm, from about 0.2 cc / gm to about 0.8 cc / gm, from 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 from about 50 g / L to about 700 g / L (e.g., from about 100 g / L to about 600 g / L, from about 200 g / L to about 600 g / L, from about 400 g / L to about 600 g / L, from about 500 g / L to about 600 g / L, from about 540 g / L to about 580 g / L, or about 560 g / L). In some embodiments, the compositions described herein comprise from about 0.05% to about 2% w / w (e.g., any subrange of this range described herein) of Syloid® 63FP (WR Grace).

[0132] Compositions described herein can also include one or more buffers.For example, compositions can include about 0.5% to about 5% w / w buffer (for example, about 1% to about 4% w / w, about 1.5% to about 3.5% w / w or about 2% to about 3% w / w, for example, about 2.7% w / w buffer). Buffer can include weak acid or weak base that the acidity or pH of compositions is maintained near the selected value after adding another acid or base.Suitable buffer is known in the art.In some embodiments, the buffer in compositions provided herein is phosphate, such as sodium phosphate (for example, anhydrous disodium hydrogen phosphate).For example, compositions can include about 2.7% w / w disodium hydrogen phosphate.

[0133] Composition can also include one or more lubricants.For example, composition can include about 0.05% to about 1% w / w lubricant (for example, about 0.1% to about 0.9%, about 0.2% to about 0.8%, about 0.3% to about 0.7% or about 0.4% to about 0.6%, for example, about 0.5% w / w lubricant). Exemplary lubricants include but are not limited to sodium stearyl fumarate, magnesium stearate, stearic acid, metal stearate, talcum, wax and glyceride with high melting point, colloidal silicon dioxide, polyethylene glycol, alkyl sulfate, glyceryl behenate and hydrogenated oil.Other lubricants are known in the art.In some embodiments, composition includes about 0.05% to about 1% w / w sodium stearyl fumarate (for example, any sub-range of this scope described herein).For example, composition can include about 0.5% w / w sodium stearyl fumarate.

[0134] 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 flavoring, about 0.9% w / w silicon dioxide, about 2.7% w / w sodium phosphate (e.g., disodium hydrogen phosphate), and about 0.5% w / w sodium stearyl fumarate.

[0135] The composition can include about 3000 mg of sodium phenylbutyrate, about 1000 mg of TURSO, about 1600 mg of dextrates, about 400 mg of sorbitol, about 200 mg of sucralose, about 97.2 mg of silicon dioxide, about 2916 mg of maltodextrin, about 746 mg of flavoring (e.g., about 102 mg of mixed berry flavor and about 644 mg of masking flavor), about 280 mg of sodium phosphate (e.g., disodium hydrogen phosphate), and about 48.6 mg of sodium stearyl fumarate.

[0136] The composition can also include other suitable sweeteners or taste masking agents, such as but not limited to xylose, ribose, glucose, mannose, galactose, fructose, dextrose, sucrose, maltose, steviol glycosides, partially hydrolyzed starch and corn syrup solids. Water-soluble artificial sweeteners are contemplated herein, such as soluble saccharin salts (e.g., sodium or calcium saccharin salts), cyclamate salts, acesulfame potassium (acesulfame K) and sweeteners such as L-aspartame-phenylalanine methyl ester, Alitame® or Neotame® based on saccharin and aspartame in free acid form. The amount of sweetener or taste masking agent can vary with the desired amount of sweetener or taste masking agent selected for a particular final composition.

[0137] In addition to those mentioned above, pharmaceutical binders are also contemplated. Examples include: cellulose derivatives, including microcrystalline cellulose, low-substituted hydroxypropyl cellulose (e.g., LH 22, LH 21, LH 20, LH 32, LH 31, LH30); starch, including potato starch; cross-linked carboxymethyl cellulose sodium (i.e., cross-linked carboxymethyl cellulose sodium salt; e.g., Ac-Di-Sol®); alginic acid or alginates; insoluble polyvinyl pyrrolidone (e.g., Polyvidon® CL, Polyvidon® CL-M, Kollidon® CL, Polyplasdone® XL, Polyplasdone® XL-10); and sodium carboxymethyl starch (e.g., Primogel® and Explotab®).

[0138] Additional fillers, diluents or binders may be incorporated, such as 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 Tai® and Solka-Floc®), hydroxypropyl cellulose, L-hydroxypropyl cellulose (low substituted) (e.g., L-HPC-CH31, L-HPC-LH11, LH 22, LH 21, LH 20, LH 32, LH 31, LH30), dextrin, maltodextrin (such as Lodex® 5 and Lodex® 10), starch or modified starch (including potato starch, corn starch and rice starch), sodium chloride, sodium phosphate, calcium sulfate and calcium carbonate.

[0139] The compositions described herein can be formulated or adjusted to be administered to a subject via any route (e.g., any route approved by the U.S. Food and Drug Administration (FDA)). Exemplary methods are described in the FDA's CDER Data Standards Manual (Revision No. 004) (which can be obtained at fda.give / cder / dsm / DRG / drg00301.html).

[0140] The pharmaceutical composition is typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (subcutaneous, intradermal, intravenous, intradermal, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques), oral (e.g., inhaled or by feeding tube), transdermal (topical), transmucosal, and rectal administration.

[0141] The pharmaceutical composition can be in the form of a solution or powder for inhalation and / or nasal administration. In some embodiments, the pharmaceutical composition is formulated as a powder filled sachet. Suitable powders may include those that are substantially soluble in water. The pharmaceutical composition can be prepared using suitable dispersants or wetting agents (such as, for example, Tween 80) and suspending agents according to techniques known in the art. Sterile injection preparations can also be sterile injection solutions or suspensions in nontoxic parenteral acceptable diluents or solvents, for example, as solutions in 1,3-butanediol. Among the acceptable excipients and solvents that can be used are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids (such as oleic acid and its glyceride derivatives) can be used to prepare injections, as can natural medicinal oils, such as olive oil or castor oil, especially in their polyoxyethylated forms. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant or carboxymethyl cellulose or similar dispersants, which are commonly used in the preparation of pharmaceutical dosage forms such as emulsions and / or suspensions. Other commonly used surfactants (such as Tweens or Span) and / or other similar emulsifiers or bioavailability enhancers commonly used in the manufacture of pharmaceutical solid, liquid or other dosage forms may also be used for formulation purposes.

[0142] The composition can be orally administered in any oral acceptable dosage form, including but not limited to powders, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of powders for oral administration, the powder can be fully dissolved in water before administration. In the case of tablets for oral use, conventional carriers include lactose and corn starch. Lubricants such as magnesium stearate can be added. For oral administration in the form of capsules, available diluents include lactose and dry corn starch. When aqueous suspensions and / or emulsions are administered orally, the active ingredient can be suspended or dissolved in the oil phase (in combination with emulsifiers and / or suspending agents). If desired, some sweeteners and / or flavorings and / or coloring agents can be added.

[0143] Alternatively or additionally, the composition may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons and / or other solubilizing or dispersing agents known in the art.

[0144] In some embodiments, the therapeutic compositions disclosed herein can be formulated for sale in the United States, imported into the United States, and / or exported from the United States. The pharmaceutical composition can be contained in a container, package, or dispenser together with instructions for administration. In some embodiments, the present invention provides a kit including bile acid and phenylbutyric acid compounds. The kit can also include instructions for doctors and / or patients, syringes, needles, boxes, bottles, vials, etc.

[0145] III. Treatment Methods / Pharmacokinetics

[0146] In one aspect, provided herein is a method of treating at least one symptom of ALS in a subject, or a method of administering TURSO and sodium phenylbutyrate to a subject having at least one symptom of ALS, wherein the method comprises: (a) administering to the subject one or more doses of a composition comprising about 1 gram of taurine diol (TURSO) and about 3 grams of sodium phenylbutyrate; (b) determining that the subject has a particular level of C for sodium phenylbutyrate; max , AUC 0-最后 and / or AUC 0-∞ , and / or for the metabolite phenylacetic acid, specific levels of C max , AUC 0-最后 and / or AUC 0-∞ ; and (c) administering an additional dose of the composition to the subject.

[0147] The method can include determining that the subject has a C for sodium phenylbutyrate of 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). max .

[0148] Sodium phenylbutyrate can be rapidly cleared by metabolism (β-oxidation in the liver and kidneys) to the major metabolite phenylacetic acid (PAA). Thus, the method may include determining that the subject has a C for phenylacetic acid of 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 In some embodiments of any of the methods described herein, C max is the steady state C max (For example, the steady-state average C max ).

[0149] The method may include determining that the subject has an AUC for sodium phenylbutyrate of 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-最后 The method may also include determining that the subject has an AUC for phenylacetic acid of about 20 to about 160 μg*h / mL (e.g., about 30 to about 150, about 40 to about 120, or about 40 to about 80 μg*h / mL). 0-最后 In some embodiments of any of the methods described herein, AUC 0-最后 is the steady-state AUC 0-最后 (For example, steady-state average AUC 0-最后 ).

[0150] In some cases, the method comprises determining that the subject has (1) an AUC of about 25 to about 545 μg*h / mL for sodium phenylbutyrate 0-∞ , and / or (2) an AUC of about 21 to about 155 μg*h / mL for phenylacetic acid 0-∞ For example, the method may include determining that the subject has an AUC for sodium phenylbutyrate of 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-∞ The method may also include determining that the subject has an AUC for phenylacetic acid of about 30 to about 150, about 40 to about 120, or about 40 to about 80 μg*h / mL. 0-∞ In some embodiments of any of the methods described herein, AUC 0-∞ is the steady-state AUC 0-∞ (For example, steady-state average AUC 0-∞ ).

[0151] In some embodiments of any of the methods described herein, step (a) can include administering the composition once 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 cases, the composition is administered once a day for about 3 weeks, followed by administration of the composition twice a day for about 9 weeks to about 21 weeks.

[0152] Step (b) of the methods described herein can include obtaining a blood sample from the subject about 30 minutes to about 8 hours (eg, about 1, 2, 3, 4, 5, 6, or 7 hours) after the last dose of the composition.

[0153] In another aspect, provided herein is a method for treating at least one symptom of ALS in a subject, or a method for administering TURSO and sodium phenylbutyrate to a subject having 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 TURSO and about 3 grams of sodium phenylbutyrate; (b) determining the plasma concentration of one or more bile acids in the subject; and (c) administering to the subject another dose of the composition. For example, the method may include determining the 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 taurodeoxycholic acid (TDCA). In some embodiments, the plasma concentration is a steady-state plasma concentration. In some embodiments, step (b) may include determining the plasma concentration from about 30 minutes to about 8 hours (e.g., about 1, 2, 3, 4, 5, 6, or 7 hours) after the last dose of the composition.

[0154] In some embodiments of the above aspects, the method comprises determining the plasma concentration of TURSO in the subject about one hour after the last dose of the composition, wherein 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 can also be determined about 4 hours after the last dose of the composition, wherein 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).

[0155] In some embodiments of the above aspects, the method comprises determining the plasma concentration of UDCA in the subject about one hour after the last dose of the composition, wherein 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 can also be determined about 4 hours after the last dose of the composition, wherein 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).

[0156] In some embodiments of the above aspects, the method comprises determining the plasma concentration of GUDCA in the subject about one hour after the last dose of the composition, wherein 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 dose of the composition, wherein 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).

[0157] In some embodiments of the above aspects, the method also includes determining the baseline plasma concentration of bile acids in the subject before step (a). For example, the method may include determining the baseline plasma concentration of TURSO in the subject, wherein 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 may include determining the baseline plasma concentration of UDCA in the subject, wherein 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, wherein 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).

[0158] In some embodiments of any of the methods described herein, the subject has fasted for a period of time before receiving a dose of the composition. For example, step (a) of the method may include administering a dose of the composition more than two hours after the subject has ingested food or more than one hour before the subject has ingested food. In some embodiments, the subject has ingested food within two hours (e.g., within one hour or within 30 minutes) of receiving administration of the composition.

[0159] In another aspect, provided herein is a method for increasing the plasma concentration of bile acids (e.g., any bile acids described herein) in a subject, the method comprising administering to the subject one or more doses of a composition comprising about 3 grams of sodium phenylbutyrate and about 1 gram of TURSO. The composition can be administered according to any 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 administering the composition is about 20 to about 3250 ng / mL (e.g., any sub-range within this range described herein). The bile acid can be UDCA, and the plasma concentration of UDCA after administering the composition is about 20 to about 7340 ng / mL (e.g., any sub-range within this range described herein). The bile acid can also be GUDCA, and the plasma concentration of GUDCA after administering the composition is about 20 to about 5290 ng / mL (e.g., any sub-range within this range described herein).

[0160] Also provided herein is a method of administering a composition comprising about 1 gram of taurine diol (TURSO) and about 3 grams of sodium phenylbutyrate to a subject (e.g., a healthy subject) who has fasted for 24 hours and measuring one or more pharmacokinetic parameters (such as T max , C max and AUC 0-最后 ). In some embodiments, the subject has a T for sodium phenylbutyrate of about 0.25 hours to about 0.50 hours. max In some embodiments, the subject has a C of about 64.4 µg / mL to about 260 µg / mL for sodium phenylbutyrate. max In some embodiments, the subject has an AUC for sodium phenylbutyrate of about 73.5 µg*h / mL to about 423 µg*h / mL. (0-最后) In some embodiments, the subject has an AUC for sodium phenylbutyrate of about 74.8 µg*h / mL to about 425 µg*h / mL. (0-∞) In some embodiments, the subject has a T for TURSO of about 1.50 hours to about 10.00 hours. max In some embodiments, the subject has a C for TURSO of about 0.219 µg / mL to about 1.74 µg / mL. max In some embodiments, the subject has an AUC for TURSO of about 1.18 µg*h / mL to about 11.6 µg*h / mL. (0-最后) In some embodiments, the subject has an AUC for TURSO of about 1.80 µg*h / mL to about 6.67 µg*h / mL. (0-∞) In any of the above embodiments, T max Can be steady-state T max (For example, the steady-state average T max In any of the above embodiments, C max Can be steady-state C max (For example, the steady-state average C max ). In any of the above embodiments, AUC (0-最后) It can be the steady-state AUC (0-最后) (For example, steady-state average AUC (0-最后) ). In any of the above embodiments, AUC (0-∞) It can be the steady-state AUC (0-∞) (For example, steady-state average AUC (0-∞) ).

[0161] Also provided herein are methods of administering a composition comprising about 1 gram of taurine diol (TURSO) and about 3 grams of sodium phenylbutyrate to a subject (e.g., a healthy subject) who has fasted for 24 hours and measuring one or more pharmacokinetic parameters (such as TURSO) of one or more metabolites of sodium phenylbutyrate and / or TURSO. max , C max and AUC 0-最后 ) method. Specifically, phenylacetate is believed to be a metabolite of sodium phenylbutyrate. In addition, under physiological conditions, enterohepatic recirculation results in active deconjugation of TURSO to UDCA by the intestinal flora, and reconjugation of UDCA to glycine or taurine (GUDCA and TURSO, respectively) in the liver. In some embodiments, the subject has a T for phenylacetate of about 1.50 hours to about 3.50 hours. max In some embodiments, the subject has a C of about 13.3 μg / mL to about 42.3 μg / mL for phenylacetate. max In some embodiments, the subject has an AUC for phenylacetic acid of about 43.8 µg*h / mL to about 141 µg*h / mL. (0-最后) In some embodiments, the subject has an AUC for phenylacetic acid of about 45.2 µg*h / mL to about 142 µg*h / mL. (0-∞) In some embodiments, the subject has a T for UDCA of about 0.25 hours to about 20.00 hours. max In some embodiments, the subject has a C for UDCA of about 195 ng / mL to about 1380 ng / mL. max In some embodiments, the subject has an AUC for UDCA of about 1970 ng*h / mL to about 14900 ng*h / mL. (0-最后) In some embodiments, the subject has an AUC for UDCA of about 11300 ng*h / mL. (0-∞) In some embodiments, the subject has a T for GUDCA of about 6.00 hours to about 20.00 hours. max In some embodiments, the subject has a C for GUDCA of about 143 ng / mL to about 1420 ng / mL. max In some embodiments, the subject has an AUC for GUDCA of about 1840 ng*h / mL to about 12000 ng*h / mL. (0-最后) In any of the above embodiments, T max Can be steady-state T max (For example, the steady-state average T max In any of the above embodiments, C max Can be steady-state C max(For example, the steady-state average C max In any of the above embodiments, AUC (0-最后) It can be the steady-state AUC (0-最后) (For example, steady-state average AUC (0-最后) In any of the above embodiments, AUC (0-∞) It can be the steady-state AUC (0-∞) (For example, steady-state average AUC (0-∞) ).

[0162] Also provided herein is a method of administering a composition comprising about 1 gram of taurine diol (TURSO) and about 3 grams 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 (such as T max , C max and AUC 0-最后 ). In some embodiments, the subject has a T for sodium phenylbutyrate of about 0.25 hours to about 1.50 hours. max In some embodiments, the subject has a C of about 12.9 µg / mL to about 70.5 µg / mL for sodium phenylbutyrate. max In some embodiments, the subject has an AUC for sodium phenylbutyrate of about 34.6 µg*h / mL to about 198 µg*h / mL. (0-最后) In some embodiments, the subject has an AUC for sodium phenylbutyrate of about 76.2 µg*h / mL to about 200 µg*h / mL. (0-∞) In some embodiments, the subject has a T for TURSO of about 4.50 hours to about 10.00 hours. max In some embodiments, the subject has a C for TURSO of about 0.435 µg / mL to about 2.16 µg / mL. max In some embodiments, the subject has an AUC for TURSO of about 2.45 µg*h / mL to about 22.4 µg*h / mL. (0-最后) In some embodiments, the subject has an AUC for TURSO of about 3.09 µg*h / mL to about 5.83 µg*h / mL. (0-∞) In any of the above embodiments, T max Can be steady-state T max (For example, the steady-state average T max In any of the above embodiments, C max Can be steady-state C max (For example, the steady-state average C max In any of the above embodiments, AUC (0-最后) It can be the steady-state AUC (0-最后)(For example, steady-state average AUC (0-最后) ). In any of the above embodiments, AUC (0-∞) It can be the steady-state AUC (0-∞) (For example, steady-state average AUC (0-∞) ).

[0163] Also provided herein are methods of administering a composition comprising about 1 gram of taurine diol (TURSO) and about 3 grams 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 (such as TURSO) of one or more metabolites of sodium phenylbutyrate and / or TURSO. max , C max and AUC 0-最后 ). In some embodiments, the subject has a T for phenylacetic acid of about 2.00 hours to about 4.50 hours. max In some embodiments, the subject has a C of about 8.39 µg / ml to about 36.5 µg / mL for phenylacetate. max In some embodiments, the subject has an AUC for phenylacetic acid of about 30.6 µg*h / mL to about 128 µg*h / mL. (0-最后) In some embodiments, the subject has an AUC for phenylacetic acid of about 31.3 µg*h / mL to about 130 µg*h / mL. (0-∞) In some embodiments, the subject has a T for UDCA of about 6.00 hours to about 24.00 hours. max In some embodiments, the subject has a C for UDCA of about 181 ng / mL to about 6250 ng / mL. max In some embodiments, the subject has an AUC for UDCA of about 1590 ng*h / mL to about 33400 ng*h / mL. (0-最后) In some embodiments, the subject has an AUC for UDCA of about 8580 ng*h / mL. (0-∞) In some embodiments, the subject has a T for GUDCA of about 0.50 hours to about 24.00 hours. max In some embodiments, the subject has a C for GUDCA of about 114 ng / mL to about 2430 ng / mL. max In some embodiments, the subject has an AUC for GUDCA of about 900 ng*h / mL to about 18400 ng*h / mL. (0-最后) In any of the above embodiments, T max Can be steady-state T max (For example, the steady-state average T max In any of the above embodiments, Cmax Can be steady-state C max (For example, the steady-state average C max ). In any of the above embodiments, AUC (0-最后) It can be the steady-state AUC (0-最后) (For example, steady-state average AUC (0-最后) ). In any of the above embodiments, AUC (0-∞) It can be the steady-state AUC (0-∞) (For example, steady-state average AUC (0-∞) ).

[0164] Example

[0165] The present invention will be described in more detail by specific examples. The following examples are provided only 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, which can be changed or modified to produce substantially the same results. It should be understood that some features of the present invention described in the context of a separate embodiment for clarity may also be provided in combination in a single embodiment. On the contrary, the various features of the present invention described in the context of a single embodiment for brevity may also be provided separately or in any suitable sub-combination.

[0166] Example 1: Pharmacokinetic study

[0167] The pharmacokinetics of AMX0035 are based on plasma concentration data from two studies: a single-dose PK study in healthy volunteers and sparse sampling of patients in clinical efficacy and safety studies.

[0168] plan

[0169] 1. Protocol AMX3500: Evaluation of the safety, tolerability, efficacy, and activity of AMX0035, a fixed combination of phenylbutyric acid (PB) and tauroursodeoxycholic acid (TUDCA), for the treatment of amyotrophic lateral sclerosis (ALS).

[0170] •A 28-week, multicenter, randomized, double-blind, placebo-controlled Phase II trial to investigate the safety, tolerability, efficacy, pharmacokinetics and biological activity of AMX0035.

[0171] • Population: 132 men or women aged 18 to 80 years with sporadic or familial ALS, vital capacity > 60% predicted, and ALS symptom onset within the past 18 months.

[0172] • Treatment: Subjects were randomized in a 2:1 ratio to receive oral (or feeding tube) AMX0035 (approximately 88 subjects) or placebo (approximately 44 subjects). Treatment was administered as one sachet per day for the first 3 weeks, then increased to one sachet twice per day if tolerated. The sachets in the active treatment group contained 1 g TUDCA and 3 g PB.

[0173] • Pharmacokinetic sampling: Single blood samples were collected at the baseline visit (pre-dose) and at the 12-week and 24-week treatment visits for analysis of PB, phenylacetic acid (PAA), TUDCA, UDCA, and GUDCA plasma concentrations. Sampling was randomized to occur at either 1 or 4 hours post-dose at the 12-week visit and at another time at the 24-week visit.

[0174] • Duration of treatment: 24 weeks.

[0175] 2. Protocol A35-002: A Phase I study of a single oral dose of AMX0035 in healthy volunteers in the fasting and fed states to evaluate plasma pharmacokinetics.

[0176] • A Phase I, open-label, two-period crossover single-dose trial of AMX-0035 in healthy adult volunteers, examining the pharmacokinetics of PB, TUDCA, and major metabolites following single oral dose administration of AMX-0035 with and without food.

[0177] • Population: 14 healthy male or female volunteers aged 40 to 65 years with a body mass index of 18.5 to 32 kg / m2.

[0178] • Treatment: Subjects received a single dose of one sachet (1 g TUDCA and 3 g PB) of AMX0035 under fasting (overnight and 4 hours post-dose) and fed (standard high-fat breakfast 30 minutes before dosing) conditions, with a minimum 4-day washout between treatments. The order of administration with or without food was randomized.

[0179] • Pharmacokinetic Sampling: Blood samples were collected prior to dosing and on both study days 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 for analysis of PB, PAA, TUDCA, UDCA, and GUDCA plasma concentrations. Additionally, serial plasma samples were obtained one day prior to the first dose of AMX0035 to characterize endogenous concentrations of TUDCA and metabolites.

[0180] Pharmacokinetic Data Characterization

[0181] • Protocol AMX3500: 87 patients with ALS were sparsely sampled. Approximately 2 samples were collected from each patient at 1 or 4 hours after dosing after 12 and 24 weeks of treatment.

[0182] • Protocol A35-002: Abundant sampling of 14 healthy adults. Approximately 21 samples were collected over 24 hours for each subject on two occasions, once after a high-fat meal and once under fasting conditions.

[0183] result

[0184] The major metabolite of sodium phenylbutyrate (PB) is phenylacetic acid (PAA). PAA occurs endogenously as a metabolite of phenylalanine. Population PK analysis showed that PAA has nonlinear (saturable) elimination.

[0185] Population PK analysis has shown an inverse relationship between PAA exposure and body weight. There is a potential additive effect of fasting to low body weight with respect to increased PAA exposure.

[0186] TURSO, UDCA (ursodiol) and GUDCA (glycoursodeoxycholic acid) are naturally occurring hydrophilic bile acids and represent a small fraction of the total bile acid pool in the human body. All three substances undergo extensive enterohepatic circulation. Oral administration of TURSO results in increased plasma levels of all three substances.

[0187] The pharmacokinetic parameters for phenylbutyric acid and the major metabolite PAA following a single dose of ALBRIOZA in healthy subjects under fasting conditions are shown in Table 1 and for ursodoxicoltaurine (ursodoxicoltaurine) and its metabolites are shown in Table 2.

[0188] Table 1: Geometric Mean (CV%) Pharmacokinetic Parameters of Phenylbutyric Acid (PB) and Phenylacetic Acid (PA) Following a Single Oral Dose of ALBRIOZA (3 g Phenylbutyric Acid and 1 g Ursodeoxycholeyl Taurine) in n=14 Healthy Subjects Under Fasting Conditions

[0189]

[0190] Cmax = maximum plasma concentration; Tmax = time to maximum concentration; T1 / 2 = half-life; AUC(0-inf) = area under the concentration-time curve from time 0 to infinity; CL / F = oral clearance; Vz / F = apparent volume of distribution.

[0191] *Median (range).

[0192] **Terminal T1 / 2, metabolites appear to have non-linear (saturable) elimination based on population PK analysis.

[0193] NA: Not applicable.

[0194] Table 2: Geometric Mean (CV%) Pharmacokinetic Parameters of Ursodeoxycholetyltaurate (TURSO) and Derivatives UDCA and GUDCA Following a Single Oral Dose of ALBRIOZA (1 g UDTS and 3 g Phenylbutyric Acid) in n=14 Healthy Subjects Under Fasting Conditions*

[0195]

[0196] Cmax = maximum plasma concentration; Tmax = time to maximum concentration; T1 / 2 = half-life; AUC(0-last) = area under the concentration-time curve from time 0 to the last measured concentration (samples were collected 24 hours after dosing); CL / F = oral clearance; Vz / F = apparent volume of distribution.

[0197] *PK parameters derived from time-matched, baseline-corrected plasma concentrations to account for endogenous levels.

[0198] **Median (range).

[0199] ***The number of subjects contributing to the PK parameter values ​​was reduced due to an insufficient number of samples collected during the terminal elimination phase.

[0200] NC = not calculated due to insufficient number of samples collected during the terminal elimination phase.

[0201] NA = not applicable.

[0202] Table 3 shows steady-state plasma concentrations of ursodeoxycholetyltaurate, UDCA, and GUDCA following twice daily administration of ALBRIOZA in ALS patients. Oral administration of TURSO resulted in elevated plasma levels of all three substances.

[0203] Table 3: Steady-state plasma concentrations of TURSO, UDCA, and GUDCA following twice daily administration of AMX0035 in ALS patients

[0204]

[0205] *Plasma levels recorded as below measurable levels (<20 ng / ml) were set at 20 ng / ml. For all plasma concentration values ​​except pre-dose (baseline): the maximum number of samples with below measurable levels was 10.

[0206] absorb:

[0207] Following oral administration of a single dose of AMX0035 in healthy subjects under fasting conditions (as described in Example 2 above), sodium phenylbutyrate was rapidly absorbed and reached a mean C of 188 μg / mL by a median time of 1 hour. max (range 64.4 to 260). Based on population PK, the estimated steady-state mean C of sodium phenylbutyrate in ALS patients max was 131 µg / mL (range 3.8 to 423).

[0208] Under physiological conditions, bile acids in the intestine are absorbed primarily in the distal part (ileum) via passive diffusion (unconjugated bile acids) and active absorption (conjugated bile acids) through the apical sodium-dependent transporter (ASBT). Specific transporters in the intestinal enterocytes ensure that bile acids are directed to the liver via the portal vein, where they undergo extensive enterohepatic recirculation. Bile acids can circulate 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 reached a mean C of 871 ng / mL after a median time of 4.5 hours. max (Range 219 to 1740). TURSO plasma concentration profiles in many subjects had 2 to 3 peaks consistent with bile acid storage and release following a meal / snack.

[0209] Effects of food

[0210] Administration of a single dose of 3 g sodium phenylbutyrate and 1 g TURSO to healthy volunteers in the presence of a high-fat, high-calorie meal (approximately 800-1000 calories: 500-600, 250, and 150 calories from fat, carbohydrates, and protein, respectively) reduced the rate and extent of absorption of sodium phenylbutyrate (75% and 55% decreases in Cmax and AUC, respectively). A high-fat, high-calorie meal did not affect the Cmax of TURSO, but exposure (AUC) was increased by 46%. In the Phase II safety and efficacy studies, patients were advised to take the medication before a meal.

[0211] distributed

[0212] When co-administered in vitro, the plasma protein binding of sodium phenylbutyrate and ursodeoxycholetyltaurate was 82% and 98%, respectively.

[0213] Under physiological conditions, less than 10% of the total bile acid pool reaches the systemic circulation because the liver removes most of it from the hepatic circulation for reuse. Serum concentrations of bile acids reflect the balance between intestinal input and hepatic extraction, where hepatic extraction of conjugated bile acids is known to be more efficient than that of unconjugated bile acids.

[0214] metabolism:

[0215] Sodium phenylbutyrate is rapidly cleared by metabolism (β-oxidation in the liver and kidneys) to the major metabolite phenylacetic acid (PAA), which may be pharmacologically active. In all 13 dosed subjects, plasma concentrations of the metabolite PAA in healthy subjects were evident from 0.25 hours onwards, reaching a mean C of 26.44 µg / mL after a median time of 2.5 hours. max (range 13.3 to 42.3). Based on population PK analysis, the estimated steady-state mean C max was 24.1 µg / mL (range, 9.08 to 46.3).

[0216] Phenylacetate is rapidly conjugated to glutamine via acetylation in the liver and kidney to form phenylacetylglutamine, which is excreted by the kidney.

[0217] Under physiological conditions, enterohepatic recirculation results in active deconjugation of TURSO to UDCA by the intestinal flora, and reconjugation of UDCA with glycine or taurine in the liver (GUDCA and ursodeoxycholeyltaurine, respectively); approximately 95% of bile acids in the intestine are (re)absorbed into the enterohepatic circulation. All metabolites attributed to TURSO detected in vitro in human hepatocytes were also detected in rat and minipig hepatocytes.

[0218] eliminate

[0219] Phenylacetic acid exhibits nonlinear pharmacokinetics characterized by saturable metabolism. Within 6 hours after administration, both phenylbutyrate and phenylacetic acid were eliminated from the systemic circulation with estimated terminal half-lives of 0.46 h and 0.81 h, respectively. Based on this rapid elimination of PB and PA, there is no accumulation in plasma after once or twice daily dosing in patients. Other minor metabolites of phenylbutyrate have been identified. Most (~80-100%) of the administered sodium phenylbutyrate is excreted in the urine within 24 hours as the conjugated product phenylacetylglutamine.

[0220] The terminal half-life of bile acids could not be reliably determined in most subjects, primarily due to insufficient sampling duration during the terminal elimination phase (see also Tables 6 and 7 above). Comparisons across studies (single-dose pharmacokinetic studies, and sparse sampling data from Phase II safety and efficacy studies) indicated that accumulation of ursodeoxycholetyltaurine appeared to be minimal following twice daily dosing, whereas accumulation of UDCA and GUDCA was considerable.

[0221] Bile acids that are not (re)absorbed in the intestine (5% under physiological conditions) are further modified by bacteria and then excreted mainly in the feces. Bile acids found in feces are unconjugated and consist mainly of a complex mixture of the hydrophobic secondary bile acids lithocholic acid and deoxycholic acid as well as other components. In case of administration of ursodeoxycholyltaurine, UDCA levels in feces are known to increase.

[0222] Special groups and conditions

[0223] • Geriatrics

[0224] Model-based analyses showed that there were no significant differences in the pharmacokinetics of sodium phenylbutyrate or its metabolite phenylacetic acid between ALS patients aged 65 to 76 years and those younger than 65 years (n=18 vs. n=39, respectively). There were no differences in the steady-state plasma concentrations of ursodeoxycholetyltaurate or its major derivatives UDCA and GUDCA between ALS patients aged 65 to 76 years and those younger than 65 years (n=24-27 vs. n=78-82, respectively).

[0225] •gender

[0226] Following a single oral administration of ALBRIOZA to healthy volunteers, differences in exposure to sodium phenylbutyrate and phenylacetic acid were observed between males (N=8) and females (N=6) that were consistent under both fed and fasted conditions, with exposure levels in females being approximately 35% and 31% higher, respectively, when compared to males. However, due to moderate variability between subjects, this difference was not found to be statistically significant. Similarly, in a cross-study pharmacometry analysis in healthy subjects and ALS patients (N=26 females and 56 males contributed to the dataset), sex was not identified as a significant covariate affecting the PK parameters of sodium phenylbutyrate or phenylacetic acid.

[0227] Following a single oral administration of ALBRIOZA to healthy volunteers, differences in exposure to ursodeoxycholetyltaurine and UDCA were observed between males (N=8) and females (N=6) that were consistent under both food conditions: females were seen to have approximately 55% higher exposure to ursodeoxycholetyltaurine, while females had approximately 29% lower exposure to UDCA when compared to males. This effect was statistically significant only for ursodeoxycholetyltaurine AUC(0-last). No such effect was seen for GUDCA. In ALS patients (N=34 females; 75 males), there was no consistent discernible effect of gender on steady-state plasma concentrations of ursodeoxycholetyltaurine, UDCA, and GUDCA.

[0228] • Hepatic insufficiency

[0229] Since sodium phenylbutyrate and ursodeoxycholetyltaurate are metabolized in the liver and kidneys, plasma levels are expected to increase in the case of liver damage. Ursodeoxycholetyltaurate and its derivatives (such as UDCA and GUDCA) are bile salts and are circulated and stored in the gallbladder by enterohepatic recirculation. 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, it is reported that serum levels of lithocholic acid increase in the case of TURSO administration, and it is expected that liver dysfunction promotes potential harm.

[0230] • Renal insufficiency

[0231] The Phase II safety and efficacy study included 32 ALS patients with mild renal impairment (estimated glomerular filtration rate (eGFR) of 60-90 mL / min). Model-based analysis showed that there was no significant correlation in the pharmacokinetics of sodium phenylbutyrate or its metabolite phenylacetic acid in healthy subjects and ALS patients with normal renal function and mild renal insufficiency (eGFR greater than 60 mL / min). There was no discernible difference in the steady-state plasma concentrations of ursodeoxycholetyltaurate or its major metabolites UDCA and GUDCA between ALS patients with mild renal insufficiency (eGFR greater than 60 mL / min and less than 90 mL / min = 71-76). However, these data are inconclusive because i) a limited number of patients (N=8) had eGFR<75, and ii) eGFR was an overestimation of renal function in ALS patients.

[0232] The major terminal metabolite, phenylacetylglutamine, is excreted via the kidneys, and additionally phenylbutyrate and its major metabolite, phenylacetic acid, are metabolized in the kidneys and liver. Therefore, elevated plasma levels of phenylbutyrate and metabolites are expected in the setting of renal impairment. In addition, due to the high protein binding of ALBRIOZA (~99%), renal impairment may alter the distribution and efficacy of TURSO.

[0233] Example 2: Phase 1 Study of a Single Oral Dose of AMX0035 in Healthy Volunteers in the Fasted and Fed States

[0234] Purpose

[0235] The primary objective of the study was to determine the plasma levels and PK parameters of sodium phenylbutyrate (sodium PB), TURSO, and major metabolites following a single oral dose of AMX0035 in a population of male and female subjects aged >40 years.

[0236] The secondary objectives of the study were to evaluate the effect of a high-fat standardized breakfast on the extent and rate of absorption of PB, TURSO, and the active metabolite in healthy subjects; to evaluate the effect of sex on the extent and rate of absorption of PB, TURSO, and the active metabolite in healthy subjects; and to quantify the relationship between plasma concentrations of PB and TURSO and the active metabolite and the QT / corrected QT interval (QTc) prolongation and proarrhythmic potential of a single dose of AMX0035 in healthy subjects.

[0237] Overall study design and planning

[0238] This is a Phase I, open-label, 2-sequence, 2-period, crossover single-dose trial of AMX0035 in healthy subjects. A total of 14 healthy subjects were recruited so that no fewer than 12 subjects completed the trial. A predefined gender combination was selected. Subjects received a single oral dose of AMX0035 under two conditions, with a washout period of no less than 4 days between periods, either under fasting conditions or after a high-fat standardized meal, in a balanced two-period crossover design.

[0239] Prior to treatment period 1, 24 h matched sampling was performed at several time points to confirm the endogenous level of TURSO in each subject.

[0240] Each study period followed the same research 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 during Study Day -2 of Period 1 and Study Day -1 of Period 2, and were dosed on the morning of Day 1. Subjects in the fasting / fed or fed / fasting sequence fasted for a minimum of 10 hours prior to dosing the night before Period 1.

[0241] Subjects remained in the clinical unit according to the study protocol and did not leave until discharge on day 2. The minimum washout period between sessions was 4 days. A telephone follow-up was performed 7 days after the last dose to ensure the subject's continued well-being.

[0242] Each subject received the following treatments (Table 4):

[0243] Table 4 Dosage regimen

[0244]

[0245] Study Design

[0246] AMX0035 is a fixed-dose combination of two small molecules, PB and TURSO, designed to block neuronal death and neurotoxic inflammation by simultaneously inhibiting ER and mitochondrial cell stress. PB and TURSO act on two independent cellular pathways with a unique mode of action.

[0247] The current study was designed to determine the plasma levels and PK parameters of PB and TURSO and major metabolites of AMX0035, as well as to evaluate the effects of food and gender on the extent and rate of absorption. The AMX0035 formulation is expected to target both ER stress and mitochondrial bioenergetics pathways.

[0248] Selection of study population

[0249] Subjects were selected from a panel of volunteers recruited by Quotient Sciences and were screened for study inclusion up to 28 days prior to dosing.

[0250] Inclusion criteria

[0251] To be eligible for entry into the study, subjects must meet all of the following criteria:

[0252] 1. Healthy male or female subjects aged 40 to 65 years (inclusive).

[0253] 2. Body mass index is 18.5 to 32.0 kg / m 2 (inclusive).

[0254] 3. Women who are infertile due to surgery (bilateral tubal ligation, hysterectomy + / - oophorectomy, bilateral oophorectomy, bilateral salpingectomy) or postmenopausal (no menstrual period for at least 12 months, and menopause confirmed by follicle-stimulating hormone level >40 IU / L at screening).

[0255] 4. All subjects agree to use an acceptable contraceptive method during the duration of the study and within 30 days after the last dose. Acceptable contraceptive methods are:

[0256] a. Subjects were surgically sterilized (vasectomy, in cases of documented azoospermia);

[0257] b. The subject's female partner uses a non-hormonal intrauterine contraceptive device (IUD) with an annual failure rate of less than 1%, inserted by a qualified physician at least one month before study drug administration and maintained in place for at least 30 days after the last dose of study drug;

[0258] c. Barrier methods, such as male condoms or caps, diaphragms, or sponges with spermicide,

[0259] d. Infertile men (at least 3 months after vasectomy) and those who truly abstain from heterosexual intercourse or lead a purely homosexual lifestyle do not need to use two methods of contraception, but are required to maintain this lifestyle for the duration of the study and for 30 days after the last dose.

[0260] 5. Negative test for hepatitis B surface antigen (HBsAg), hepatitis C antibody (HCV Ab), or human immunodeficiency virus (HIV) 1 and 2 antibodies at screening.

[0261] 6. The subjects have been provided with written informed consent before admission into this study.

[0262] In addition to the above standards, the subject agrees to the following restrictions:

[0263] •Avoid alcohol consumption from 48 hours before dose administration until 24 hours after discharge.

[0264] •Avoid food or beverages containing caffeine or xanthine products (e.g., coffee, tea, cola drinks, energy drinks, and chocolate) from 24 hours before dose administration until 24 hours after discharge.

[0265] •Avoid consuming food or drinks containing grapefruit, grapefruit juice, Seville orange, Seville orange marmalade, Seville orange juice, or other products containing grapefruit or Seville orange from 7 days before admission until 24 hours after discharge.

[0266] • No unaccustomed strenuous exercise during the period 72 hours prior to dosing until 24 hours after discharge from the study.

[0267] •Advise subjects not to donate blood or plasma for at least 3 months after the last dose.

[0268] •Subjects complied with all contraceptive requirements based on their individual circumstances.

[0269] Exclusion criteria

[0270] Subjects were excluded from the study if one or more of the following statements applied:

[0271] 1. Subjects have a clinically significant persistent disease or condition, including, for example: cardiovascular disease; hypertension; cancer or tumor; diabetes; liver, endocrine, metabolic, respiratory, renal, gastrointestinal (except appendectomy) (including biliary disease or cholecystectomy), hematological, or first or second axial psychiatric disorder.

[0272] 2. Clinically significant laboratory test abnormalities considered by the investigator at screening or entry.

[0273] 3. Clinically significant infection or inflammation as determined by the investigator at screening or entry.

[0274] 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.

[0275] 5. The subject has a mean QT interval (QTcF) corrected by the Fridericia formula > 450 milliseconds from the screening and first admission electrocardiogram (ECG), or any clinically significant QTcF prolongation at any time point thereafter.

[0276] 6. Any current or previous illegal use of Class A drugs such as opioids, cocaine, ecstasy, LSD, and amphetamines (Class B). Subjects who admitted to occasional past use of marijuana were not excluded as long as they tested negative for drug abuse at screening and entry and had been abstinent for at least 3 months.

[0277] 7. Weekly alcohol intake greater than 14 units or unwilling to stop drinking during the study duration. Note: 1 unit = 8 g 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]).

[0278] 8. Use of medications (including over-the-counter [OTC] drugs) within 14 days of admission or within 5 elimination half-lives of the drug under consideration, whichever is longer.

[0279] 9. Use of prescribed centrally active or psychoactive drugs within 28 days from admission.

[0280] 10. Use of drugs with enzyme-inducing properties, such as St John's Wort, within 3 weeks prior to the first administration of the investigational product.

[0281] 11. Requirement to continue taking any medication during the study.

[0282] 12. Use of any investigational drug within 3 months before the start of the study, or plan to receive any investigational drug during the course of the study.

[0283] 13. Participated in another clinical study within 30 days before screening.

[0284] 14. History of plasma / blood donation in the past 2 months.

[0285] 15. History of severe allergic reactions or multiple adverse drug reactions, including penicillin and cephalosporin.

[0286] 16. Any condition that impairs the ability to give informed consent or communicate with the researchers required to complete the study.

[0287] 17. Unwillingness to comply with all lifestyle considerations and restrictions set forth in the Agreement.

[0288] 18. The subject has previously taken AMX0035 in this study.

[0289] 19. The subject has consumed grapefruit, grapefruit juice, Seville orange, Seville orange jam and Seville orange juice or other products containing grapefruit or Seville orange within 7 days prior to admission.

[0290] Removal of Subjects from the Study

[0291] If subjects wish to leave the study at any time, they are permitted to do so. Quotient Sciences will make every reasonable effort to complete the final evaluation / discharge process. Quotient Sciences will notify the Sponsor of any subject's withdrawal from the study.

[0292] The early withdrawal date was defined as the date on which the decision was made to withdraw the subject from the study. The subject completion date was defined as the date on which the last procedure or last contact (ie, telephone call) was performed for that subject.

[0293] If a subject requests to leave the clinical unit earlier than planned discharge, e.g. due to unforeseen personal circumstances, but intends to return to the unit to complete the study, this should be recorded as a self-discharge of the subject and a protocol deviation. Subjects must complete the planned assessment / discharge procedures prior to discharge from the clinical unit and return as planned for the next study period / assessment.

[0294] Subjects were withdrawn from one or more study drugs for the following reasons:

[0295] • Experiencing serious or severe AEs, including but not limited to:

[0296] a. Clinically significant QTc(F)>500 msec or increase in QTc(F)>60 msec from baseline (confirmed after repeat ECG),

[0297] b. Baseline was considered to be the closest QTcF in the triplicate ECGs before dosing for each period,

[0298] c. Alanine aminotransferase (ALT) concentration >3× upper limit of reference range and total bilirubin >2× upper limit of reference range;

[0299] • Termination of the study by the sponsor, regulatory agency, or IRB;

[0300] •At the subject’s request (consent to withdraw);

[0301] •Major concurrent illness or need to use prohibited medications;

[0302] •Subjects’ non-compliance;

[0303] •At the discretion of the researcher.

[0304] For the purpose of exit criteria, baseline was considered to be the most recent predose assessment.

[0305] For subjects who withdraw due to IMP-related AEs, every effort should be made to ensure that the subject completes follow-up procedures. Any subject who withdraws or discontinues the study prematurely due to an IMP-related AE or study termination should be considered to have completed the study and will not be replaced.

[0306] Early termination due to any of the above reasons should be distinguished from the subject's consent to withdraw from any further activities.

[0307] Subjects who withdrew for other reasons may have been replaced at the discretion of the investigators and sponsors.

[0308] Investigational Medicinal Product (IMP) Administered

[0309] Subjects were supervised to mix thoroughly and consume the drink. It is normal for a small amount of powder to not dissolve. Subjects should consume the drink within 60 minutes of adding the powder to the water. Subjects were allowed to wash down the bitter taste of AMX0035 with a small amount of water (1 oz).

[0310] The following information is recorded in the clinical database:

[0311] • Date and time of administration

[0312] •Confirm that the entire dose is administered

[0313] Review of labeling of pharmaceutical products

[0314] AMX0035 was provided by the Sponsor to the on-site pharmacy as a carton containing 40 single-use sachets. The active pharmaceutical ingredients were TURSO and PB (AMX0035), which were provided by the Sponsor as a white powder in sachets. Each sachet contained 1 g TURSO and 3 g PB.

[0315] Authorized pharmacy personnel prepared subject study medication by opening one AMX0035 sachet, pouring the powder into a container, and adding approximately 240 mL of room temperature water. The solution was stirred until the powder was substantially dissolved.

[0316] Responsible records of use will be maintained throughout the study. Unused IMPs will be destroyed upon request by the sponsor.

[0317] Methods of assigning subjects to treatment groups

[0318] At enrollment (period 1), each enrolled subject was randomized to one of the two sequences in a 1:1 ratio, such that 7 subjects were randomized to sequence 1 and 7 subjects were randomized to sequence 2.

[0319] • Sequence 1: AMX0035 is administered in the fasted state in period 1 and in the fed state in period 2;

[0320] • Sequence 2: AMX0035 is administered in the fed state during period 1 and in the fasted state during period 2.

[0321] A treatment allocation list was generated prior to dosing using a randomization schedule.

[0322] The randomization schedule and treatment allocation list were generated according to Quotient Sciences' standard operating procedures (SOPs). The treatment allocation list provides the dosing team with information about which treatment to administer to each subject in each time period and is archived in the investigator site files.

[0323] Selection of the dose under study

[0324] PB has been evaluated in a dose escalation study in subjects with neurodegenerative diseases and was found to be generally safe and tolerable at doses significantly higher than those evaluated in that study. Specifically, the most common AEs included: falls or other unexpected injuries, dizziness, diarrhea, edema, dry mouth, headache, nausea, and rash. In addition to subject-onset headaches, these AEs occurred at a higher rate than the placebo cohort, and side effects from PB were expected. 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 between 9 and 21 g and 12 to 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. MovDisord. 2007;22(13):1962-1964).

[0325] Selection and timing of dose for each subject

[0326] AMX0035 was taken orally as a single dose in the morning of Day 1 in Periods 1 and 2. Any deviations from the intended regimen were recorded in the eCRF. All dosing was performed in the study center under the supervision of appropriately trained staff.

[0327] On day 1, meals are controlled by clinical staff. Meals are served at nominal times. The start and end times of meals are recorded in the source workbook.

[0328] For oral administration

[0329] Subjects were provided with a snack and then abstained from all food and drink (except water) until the next morning, when they were provided with a high-fat breakfast. Breakfast was consumed over a maximum period of 25 minutes, and dosing was performed 30 minutes after the start of breakfast. Subjects were encouraged to eat their meals evenly over a 25-minute period. For this trial, subjects were considered compliant if they consumed >80% of a high-fat standard breakfast.

[0330] For fasting medication

[0331] Subjects in the 24-hour baseline and in the fasting state period remained fasted for up to 4 hours after dosing and were provided a standardized meal for ingestion 4.00 to 5.00 hours after dosing. Subjects in the fed state fasted overnight and received a high-fat standardized breakfast 30 minutes before dosing and then remained fasted from 4.00 to 5.00 hours after dosing, after which they received a standardized meal.

[0332] No water was allowed between 1 hour before and 1 hour after dosing (except when study drug was administered as directed), except for a small amount (1 oz) to wash down the taste, and otherwise water was allowed ad libitum. Subjects were encouraged to drink water regularly during their stay in the room (including fasting periods), but no formal water guidelines were required.

[0333] Previous and concomitant therapy

[0334] No medication was allowed from 14 days before IMP administration or 5 half-lives (whichever was longer) until the 7-day follow-up telephone call after dosing and any medication deemed necessary by the investigator to treat the AE. Any medication used was recorded in the source data workbook.

[0335] Treatment compliance

[0336] During all clinical phases of the study, subjects were observed by study staff to ensure compliance with all study procedures, including dose administration.

[0337] Subjects should mix thoroughly and drink under supervision. It is normal for a small amount of powder to not dissolve. Subjects should consume within 60 minutes of adding the powder to the water. Subjects may use a small amount of water (1 oz) to wash down the bitter taste of AMX0035.

[0338] The date and time of dosing for each subject is recorded in the subject's original data, along with confirmation that the entire dose was taken. Any violation of compliance requires evaluation by the investigator and the sponsor to determine whether the subject can continue to participate in the study.

[0339] Pharmacokinetic and safety variables

[0340] The schedule of evaluations is shown in Tables 5-7 below, where the PK and safety procedures are shown.

[0341] When more than one program is scheduled at the same time, the order of precedence is as follows:

[0342]

[0343] When both measurements are scheduled at the same time point, vital signs are measured before the ECG. Other assessments such as physical examinations are performed within a specified time window. The ECG and blood sample should be collected within no more than 10 minutes of each other.

[0344] All safety assessments were timed and performed relative to the start of dosing.

[0345] Table 5. Schedule of assessments: 24 h baseline

[0346]

[0347]

[0348] Table 6 Evaluation schedule: Time period 1

[0349]

[0350] Table 7 Evaluation schedule: Time period 2

[0351]

[0352] Pharmacokinetic measurements

[0353] Sample collection

[0354] Venous blood samples were drawn via an indwelling cannula or by venipuncture according to the schedules presented in Tables 5, 6 and 7.

[0355] Approximately 4 mL of blood samples were collected into 4 mL BD Vacutainer plastic K2EDTA tubes with Lavender Hemogard caps and gently inverted approximately 8 times. These tubes were then immediately stored on ice and processed within 60 minutes after collection by centrifugation at 1500 g for 10 minutes at 2 to 8 ° C. The resulting plasma was divided into 2 aliquots and transferred to 2 mL of appropriately labeled primary and backup polypropylene cryovials. The samples were stored below -20 ° C until they were shipped to WWCT for analysis of AMX0035.

[0356] Analytical methods

[0357] Plasma concentrations of sodium PB, PAA, TURSO, ursodeoxycholic acid (UDCA), and glycoursodeoxycholic acid (GUDCA) were determined at Worldwide Clinical using validated analytical methods. The lower limit of quantification was 1.6 µg / mL for sodium PB, 0.8 µg / mL for PAA, 0.02 µg / mL for TURSO, and 20 ng / mL for UDCA and GUDCA.

[0358] Safety Measurement

[0359] Adverse Events

[0360] An adverse event (AE) is any untoward medical occurrence in a subject administered a medicinal product that is not necessarily causally related to this treatment. An AE may be any unfavorable or unexpected sign (including abnormal laboratory findings), symptom, or disease temporally associated with the administration of an IMP, whether or not considered related to the IMP. Existing conditions that worsen during the study should be reported as AEs. An adverse drug reaction (ADR) is any AE for which there is at least a reasonable possibility of a causal relationship to the IMP (possibly related or related).

[0361] Any clinically significant abnormality in laboratory parameters, vital signs, or ECGs, at the discretion of the PI, taking into account any relevant clinical signs and symptoms and pre-dose values, may be reported as an AE.

[0362] All AEs were recorded from the time written informed consent was provided until 30 days after the last dose of study drug. During each study visit, the subject was questioned and / or examined for evidence of AEs by the investigator or his / her designee. Each AE was recorded in the subject's source data workbook, indicating 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 life-threatening [grade 4]), actions taken, outcome, and the investigator's opinion on the relationship between the study treatment and the event. The investigator provided a diagnosis and final opinion on the relationship between the study treatment and the event (unrelated, possibly related, or related) at the end of the study.

[0363] Subjects who withdrew from the study due to AEs were followed up until the outcome was determined and a written report was provided by the investigator.

[0364] Serious adverse events

[0365] A serious adverse event is any adverse medical occurrence or effect that, at any dose, results in death, is life-threatening, requires or prolongs inpatient hospitalization, causes persistent or significant disability / incapacity, congenital anomaly / birth defect, or is determined by the PI to be a medically significant event.

[0366] A suspected unexpected serious adverse reaction (SUSAR) is any unanticipated reaction to an IMP (i.e., there is at least a reasonable possibility of a causal relationship between the IMP and the AE) at any dose that is both serious and inconsistent with the applicable product information. All SUSARs are the subject of expedited reporting.

[0367] Laboratory parameters

[0368] The following laboratory evaluations were performed at the time points specified in Tables 2, 3, and 4.

[0369] Hematology: Blood samples were collected in K2EDTA coated tubes (4 mL) with Lavender Hemogard caps. The following analyses were performed: hemoglobin, hematocrit, erythrocytes, erythrocyte distribution width, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, mean platelet volume, leukocytes, neutrophils, lymphocytes, monocytes, eosinophils, and basophils.

[0370] Clinical Chemistry: Blood samples were collected into serum separator tubes (7.5 mL) containing a coagulation activator and a 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, phosphorus (phosphate), glucose.

[0371] Coagulation: Blood samples were collected into sodium citrate tubes (2.7 mL). The following analyses were performed: prothrombin time (PT), prothrombin international normalized ratio, and activated partial thromboplastin time (aPTT).

[0372] Urinalysis: Urine samples (>20 mL) were analyzed using urine test strips for the following: pH, specific gravity, glucose, ketones, nitrites, leukocyte esterase, protein, urobilinogen, blood, and bilirubin.

[0373] Drug Screening: Urine samples (>20 mL) are tested for drugs, including drugs of abuse, at screening and admission.

[0374] Virology: Screening for HBsAg, HCV Ab, and HIV (1 and 2) was performed using clinical chemistry blood samples.

[0375] Alcohol Tests: These tests were performed at Screening and Entry for each study period.

[0376] In cases where laboratory findings were outside the normal range and the investigator believed that the results might be clinically significant, repeat sampling was requested, except in the case of positive virology results. If the abnormal findings were clinically significant, appropriate measures were taken, such as excluding the subject from the study or withdrawing the subject from the study. The subject was referred to his or her general practitioner or other appropriate provider (e.g., genitourinary medicine clinic) for further care. The same applied if the results of the HBsAg, HCVAb, or HIV tests were positive and the investigator ensured that adequate counseling was provided when needed.

[0377] Abnormal findings on subsequent evaluations also require repeat testing if the investigators believe the results may be clinically significant.

[0378] Vital signs

[0379] Vital signs (systolic and diastolic blood pressure [BP], heart rate, and oral temperature) were measured by automated recording after the subject had been in the supine position for a minimum of 5 minutes, using the arm opposite to the blood draw, according to the schedule presented in Tables 2, 3, and 4. The subject should rest for 5 minutes before BP and pulse were measured.

[0380] •Predose vital sign measurements were performed ≤ 2 hours before dosing.

[0381] •Post-dose vital sign measurements were performed at the nominal post-dose time point ± 15 minutes.

[0382] •Discharge vital signs measurements were performed at nominal time points ± 1 hour.

[0383] •For follow-up visits, vital signs were measured at the nominal follow-up time point ± 2 hours.

[0384] Electrocardiogram

[0385] Digital 12-lead ECGs were recorded after the subject had been in the supine position for a minimum of 15 minutes as detailed in Tables 2, 3, and 4. Triplicate ECGs were taken one to two minutes apart.

[0386] ECGs were evaluated for QT, QTcF, PR, QRS, RR, and HR intervals, and data collected from the clinical database. The ECGs were interpreted by investigators at Quotient.

[0387] •Predose ECG measurement was performed ≤ 2 hours before dosing.

[0388] •Post-dose ECG measurements were performed at the nominal post-dose time point ± 15 minutes.

[0389] •Discharge ECG measurements were performed at the nominal time point ± 1 hour.

[0390] •For follow-up visits, ECG measurements were performed at the nominal follow-up time point ± 2 hours.

[0391] Physical examination

[0392] Subjects underwent a complete physical examination, including a neurological examination, as detailed in Tables 2, 3, and 4. Any changes were recorded. Height was collected once, and weight was measured on the schedule of the assessment.

[0393] Pregnant

[0394] Male subjects agree to inform the PI if their partner becomes pregnant during the study. Any pregnancy will be followed up and the status of the mother and child will be reported to the Sponsor whenever possible after delivery.

[0395] Appropriateness of measurement

[0396] The nature and timing of the safety assessments as detailed in the final protocol were considered appropriate to assess the safety of the active drug, taking into account the properties of the compound and its route of administration.

[0397] The timing of blood sample and urine collection intervals was considered appropriate to assess the PK profile of active drug and metabolites.

[0398] Data Quality Assurance

[0399] All study data recorded in the source workbooks were transcribed into a validated database (InForm v5.0). Quality control, data validation, and query resolution were performed in accordance with Quotient Sciences SOPs. All raw files generated during the study were made available to the PI for review and raw data validation by representatives of the sponsor, regulatory agencies, and appropriate IRBs.

[0400] Statistical methods and sample size determination

[0401] Analyze groups

[0402] The safety population included all subjects who received any amount of IMP.

[0403] The safety analysis set should be defined on a treatment basis and include all relevant data from subjects included in the safety population who have received that treatment. For the purpose of developing subject propensity, demographic, and baseline tables, the safety analysis set should also be defined on a sequence basis (i.e., all subjects randomized to that sequence who received at least one treatment).

[0404] The PK population should include all subjects who received at least 1 dose of IMP and had no missing samples or invalid post-dose analytical results at critical time points (e.g., near Cmax), no relevant protocol deviations that could affect study objectives related to PK endpoints (e.g., if a subject consumed >80% of a high-fat standard breakfast, they were considered compliant), and no relevant AEs such as vomiting (indicating that a particular subject did not absorb the entire dose).

[0405] The PK analysis set should be a subset of the PK population and should be defined based on treatment and include all relevant data from subjects included in the PK population receiving that treatment. Individual subject characteristics (i.e., time periods) should be excluded from the PK analysis set where deemed appropriate, such as if subjects in the affected study time period do not meet the above criteria or other study-critical points relevant to the PK analysis or interpretation.

[0406] If necessary, the PK analysis subset should be determined at the same time as the PK population and based on the PK analysis set (if additional subjects need to be excluded from the statistical analysis) (see the "Analyzed Population" section).

[0407] The safety-PK population / analysis set should include all subjects included in the safety and PK population / analysis sets.

[0408] Pharmacokinetic analysis

[0409] Phoenix® WinNonlin software (v8.0 Certara USA, Inc., USA) and / or SAS ® PK parameters for PB and metabolites (phenylacetic acid [PAA]), TURSO and metabolites (UDCA and GUDCA) in plasma were estimated for each enrolled subject and time period, when possible and appropriate, using noncompartmental analysis using the SAS® software, version 9.4 (SAS Institute, Inc., Cary, North Carolina, USA). After database lock, actual elapsed time from dosing should be used for final plasma PK parameter calculations.

[0410] PK parameters for PB and PAA should be derived from actual concentrations (ie, not baseline-corrected concentrations).

[0411] Before PK parameters for TURSO and metabolites (UDCA and GUDCA) are derived, plasma concentrations for each treatment period should be baseline corrected in SAS (version 9.4) using the time-matched baseline concentrations from period 1, day -1.

[0412] PK parameters should not be determined on non-baseline corrected concentrations of TURSO and metabolites (UDCA and GUDCA).

[0413] The following parameter estimates should be estimated where possible:

[0414] Tma: time of maximum observed concentration;

[0415] Cmax: maximum observed concentration;

[0416] AUC(0-last): area under the curve from time 0 to the last measurable concentration;

[0417] AUC(0-inf): Area under the curve extrapolated from time 0 to infinity;

[0418] AUCextrap: Percentage of AUC(0-inf) extrapolated beyond the last measurable concentration;

[0419] T1 / 2: apparent elimination half-life;

[0420] λ-z: slope of the apparent elimination period;

[0421] CL / F: Apparent plasma clearance calculated after a single oral administration, where F (fraction of absorbed dose) is unknown (maternal only);

[0422] Vz / F: Apparent volume of distribution based on the terminal phase calculated after a single extravascular administration, where F (fraction of absorbed dose) is unknown (maternal only);

[0423] Frel Cmax: relative bioavailability based on Cmax;

[0424] Frel AUC(0-last): relative bioavailability based on AUC(0-last);

[0425] Frel AUC(0-inf): relative bioavailability based on AUC(0-inf);

[0426] MR Cmax: metabolite-to-parent ratio based on Cmax;

[0427] MR AUC(0-Last): metabolite to parent ratio based on AUC(0-Last);

[0428] MR AUC(0-inf): metabolite to parent ratio based on AUC(0-inf);

[0429] 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., food state) and time point when possible.

[0430] Summary statistics (i.e., n, mean, SD, CV%, median, minimum, maximum) will be calculated for all plasma PK parameters using the PK analysis set / subset by treatment (i.e., food status). Geometric n, geometric mean, geometric SD, and geometric CV% are presented for all PK parameters (except Tmax).

[0431] Summary statistics of plasma concentrations will be calculated and presented for:

[0432] • PB and PAA: actual concentrations;

[0433] • TURSO, UDCA, and GUDCA: actual concentrations (including day -1) and time-matched baseline-corrected concentrations.

[0434] Additional summary data on plasma concentrations by sex will be presented.

[0435] Arithmetic mean plasma concentration versus time curves will be generated by treatment on a linear / linear scale and error bars for ± the arithmetic SD will be included on the graphs.

[0436] The geometric mean plasma concentration versus time curve will be expressed as log 10 / Linear scale generated by treatment. Error bars are included on these graphs where error bars are (geometric mean × / ÷geometric SD).

[0437] Spaghetti plots by analyte and treatment will show one line for each characteristic for each subject using the actual sampling time after dosing. These plots will be presented in linear / linear and log 10 / Linear scale generation. A legend identifying the characteristics of the individual subjects will be displayed on the graph.

[0438] Individual Cmax, AUC(0-last), AUC(0-inf), and Tmax values ​​will be plotted for each individual within the relevant PK analysis data set on a linear / linear scale (i.e., the fed and fasted regimens will represent the x-axis). The individual points for each subject are connected by a line. A separate graph will be provided for each analyte.

[0439] Statistical analysis of pharmacokinetic parameters

[0440] Food effect assessment: Cmax, AUC(0-last) and AUC(0-inf)

[0441] Statistical analysis will be performed on the PK parameters Cmax, AUC(0-last) and AUC(0-inf) for PB and PAA (based on actual concentrations) and TURSO, UDCA and GUDCA (based on time-matched baseline-corrected concentrations) to assess the presence of a food effect. The null hypothesis to be tested is that there is no difference between the fed and fasted treatments.

[0442] PK parameters will be natural log transformed and will be analyzed using mixed effects modeling techniques. The full model will include the following terms: treatment (i.e., fed or fasted), time period, sex, treatment by sex interaction, and matched sequence as fixed effects and subject within sequence as a random effect.

[0443] If the treatment by sex interaction term is not significant at the 5% level (i.e. p>0.05), the term will be removed from the model and a reduced model will be used, and only the combined results for the two sexes will be presented. If the interaction term is significant at the 5% level (i.e. p<0.05), the combined results for the two sexes and the results for males and females separately will be presented.

[0444] All other statistical tests related to PK parameters are 2-sided and performed using a 10% significance level, resulting in 90% (2-sided) CIs. Adjusted means (including differences in fasting / fed comparisons and associated 90% confidence intervals (CIs) obtained from the model) will be back-transformed on a logarithmic scale to obtain adjusted geometric mean ratios (GMRs) and 90% CIs for the ratios. These will be presented together with p-values ​​from fasting / fed comparisons and intra-subject variability values ​​(expressed as CVw in the results tables).

[0445] Statistical analyses will be performed using the actual treatments received and the planned order as detailed on the randomization schedule. Models will be fit using the SAS software procedure PROC MIXED, the method will be specified as restricted maximum likelihood, and the denominator degrees of freedom for the fixed effects will be calculated using the Kenward and Roger method.

[0446] Food Effect Assessment: Tmax

[0447] Comparison of Tmax values ​​between treatment groups was investigated using nonparametric analysis. Time period differences (time period 1 minus time period 2) were obtained for each subject. The median difference of Tmax between treatments was estimated using the Hodges-Lehmann estimator. The relevant 90% CI and p value were also obtained. The null hypothesis was that the difference between the treatment group medians was equal to zero. The alternative hypothesis was that the difference between the treatment group medians was not equal to zero. The 90% CI supported by p value <0.10 without zero would indicate that there was a statistically significant difference between the treatment medians about Tmax.

[0448] This procedure will be performed in the SAS software program PROC NPAR1WAY.

[0449] The median difference and associated 90% CI and p-value will be presented, along with the median Tmax value and n for the two treatments.

[0450] Security parameters

[0451] Evaluation of safety parameters will include analysis of AEs, laboratory variables, vital signs, ECGs, and physical examination findings. AEs and drugs will be coded using the Medical Dictionary for Regulatory Activities (MedDRA; v 22.0) and the World Health Organization Drug Dictionary Enhanced (2019 Q1), respectively.

[0452] Treatment-emergent AEs (TEAEs) (i.e., those that began after dosing with study drug) will be summarized by system organ class (SOC) for each treatment and within the SOC by event type, severity, and relationship to IMP. If the severity or IMP relationship of the AE is missing, the severity / relationship should be listed as missing in the summary table. TEAEs and pre-dose AEs are listed.

[0453] Laboratory parameters (including changes from baseline) will be summarized by treatment for each scheduled time point. A table of changes (from baseline to each post-baseline time point) is also presented.

[0454] Vital signs and ECG data (including changes from baseline) will be summarized for each scheduled time point in 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) will be summarized. The number and percentage of subjects with the following ECG characteristics will also be summarized: QTcF of ≤450 msec, 451 to 480 msec, 481 to 500 msec, >500 msec; increase in QTcF interval from baseline of <30 msec, 30 to 60 msec, and >60 msec; and QT interval of ≤500 msec and >500 msec.

[0455] All laboratory parameters, vital signs, ECG data, and abnormal physical examination findings will be listed for each time point; values ​​outside the reference range should be noted where appropriate.

[0456] Statistical analysis of ECG data (exposure-response analysis)

[0457] Exposure-response analyses will be performed using the pooled safety-PK analysis set.

[0458] Exposure-response analyses will be performed to assess the relationship between time-matched baseline-corrected QTcF (ΔQTcF) values ​​and plasma concentrations of each parent drug and relevant metabolites.

[0459] The time-matched change from baseline (ie, ΔQTcF) based on the mean of the triplicate values ​​will be calculated for each subject at each post-dose time point as follows:

[0460] ∆QTcF = (QTcF at relevant post-baseline time point) - (QTcF at relevant baseline time point)

[0461] All concentrations below the LOQ should be entered as zero.

[0462] The relationship between ΔQTcF and AMX0035 plasma concentration for each analyte (i.e., PB, PAA, TURSO, UDCA, and GUDCA) will be analyzed using a linear mixed model with plasma concentration of each analyte fitted as a covariate, time-matched baseline QTcF as a continuous covariate, and ΔQTcF as the dependent variable. Subject-specific random effects will be added on the intercept and slope parameters using an unstructured covariance matrix. If the unstructured covariance matrix is ​​not supported by the data, other simplified or reduced structures (e.g., variance components) should be investigated.

[0463] The models were fitted using PROC MIXED in SAS, the method was specified as restricted maximum likelihood, and the denominator degrees of freedom for the fixed effects were calculated using the Kenward and Roger method.

[0464] The model was initially fitted with all analytes. The model was then iteratively fitted, removing the analyte with the least significant slope (i.e., the maximum p-value associated with the null hypothesis of slope=0 based on each analyte) for the model in each step until only a single analyte remained. As a result, a total of 5 models were fitted, where 5 was the number of analytes. Using standard PROC MIXED model fit diagnostics, the model with the best fit was selected as the primary model, i.e., the model with the minimum Akaike Information Criterion (AIC).

[0465] Using the primary model fit (i.e., best fit), the median Tmax was identified for each analyte included in the model. The geometric mean concentration value at the corresponding Tmax for each analyte will be identified and used to estimate the effect on ΔQTcF (90% CI), i.e., if 3 analytes were included in the model, the effect on ΔQTcF would be determined using 3 sets of data based on the geometric mean concentration corresponding to the Tmax of analyte 1, and similarly for the Tmax of analyte 2 and the Tmax of analyte 3. To infer that AMX0035 has no significant effect on the change in QTcF, the upper limit of the two-sided 90% CI (predicted mean change) should be below 10 milliseconds for each geometric mean concentration as described above (i.e., one concentration for each analyte).

[0466] Changes in study execution or planned analysis

[0467] Changes in Plan Analysis

[0468] Because the 24-h baseline (Day -1) ECG monitoring was performed in the fasting state (i.e., no high-fat breakfast was provided at the time-matched time of dosing on Day -1), the baseline was not truly representative of the fed state when not taking medication. Therefore, the exposure-response analysis was performed in two conditions: once using only the fasting data, which was the primary analysis, and another time using only the fed data as an exploratory analysis.

[0469] Study participants

[0470] Fourteen subjects entered the study, of which seven subjects were each randomly assigned to 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 the fed / fasting treatment sequence at the discretion of the investigator.

[0471] Deviation from the plan

[0472] The study was conducted according to the clinical protocol with no major protocol deviations, but minor deviations from the protocol occurred (missed or late safety or laboratory assessments / inadvertent omission of the third of the triplicate ECGs during the 3 h time point on Day -1 of Period 1; data not shown).

[0473] The PI and the sponsor believe that this protocol deviation will not affect the overall integrity or quality of the study results or lead to safety issues for any subject.

[0474] All subjects signed a study-specific ICF before any study procedures and met the inclusion and exclusion criteria; data not shown.

[0475] Groups analyzed

[0476] Overall, all 14 subjects received at least 1 dose of IMP; therefore, all 14 subjects were included in the safety population.

[0477] All 14 subjects received at least 1 dose of IMP and had no missing samples, invalid post-dose analysis results, relevant protocol deviations, or AEs that could affect at least 1 characteristic of any PK endpoint (see the "Analysis Population" section). Therefore, all 14 subjects were included in the PK population.

[0478] The subjects included in the safety, PK, and safety-PK analysis sets for each treatment were as follows:

[0479] • AMX0035 in the fed state consisted of all 14 subjects

[0480] • AMX0035 in the fasted state consisted of only 13 subjects. One subject withdrew between Time Periods 1 and 2 due to major intercurrent illness or the need to use a contraindicated medication (TEAE of musculoskeletal pain leading to withdrawal of IMP) and therefore received administration of AMX0035 in the fed state only.

[0481] Finally, 13 subjects were included in the PK analysis subset; one subject was excluded from this subset because he did not complete the fasting treatment.

[0482] Demographics and other baseline characteristics

[0483] Demographic variables

[0484] Demographic and lifestyle details of individual subjects were not provided, but a brief summary is provided below.

[0485] Fourteen healthy male (8 [57.1%]) and female (6 [42.9%]) subjects aged 45 to 64 years entered the study (Table 8). The majority of subjects were Caucasian (12 [85.7%]) and 2 (14.3%) subjects were black or African American. All subjects were Hispanic or Latino. Body mass index for all subjects was within the reference range (18.5 to 32.0 kg / m 2 All 14 subjects were non-smokers and none of them were alcohol consumers.

[0486] Table 8: Demographic Characteristics: Safety Analysis Set

[0487]

[0488] There were no clinically significant differences between treatment sequences with respect to demographic characteristics, however, 2 (28.6%) subjects in the fasting / fed sequence were black or African American, unlike the fed / fasting sequence, which had only Caucasian subjects.

[0489] Other baseline features

[0490] Details of individual subjects' medical and surgical history did not preclude inclusion in the study.

[0491] No subject reported taking medication prior to dosing.

[0492] One subject reported a predose AE of skin abrasions on the left foot and upper arm. This event resolved without medication.

[0493] All urine drug screens, urine alcohol screens, and serum pregnancy tests were negative. Serologic tests were nonreactive, and none of the coagulation test results were outside the reference range. Per protocol, all subjects were postmenopausal (FSH >40 IU / L) except for one subject who had bilateral tubal ligation and was therefore non-fertile.

[0494] Measurement of treatment adherence

[0495] During all clinical phases of the study, subjects were observed by study staff to ensure compliance with all study procedures, including dose administration.

[0496] The date and time of dosing for each subject was recorded in the subject's source workbook. Details of dosing for individual subjects and subject compliance with meal requests were recorded but are not presented here.

[0497] Pharmacokinetic evaluation

[0498] Pharmacokinetics of PB and PAA

[0499] PB

[0500] for Figure 2 The mean plasma concentration of PB versus time profiles for all subjects in the PK analysis set are shown as log 10 / Linear scale shown by food condition.

[0501] The key PK parameters for PB for all subjects in the PK analysis set are presented in Table 9 below.

[0502] Table 9: Summary of plasma pharmacokinetic parameters in PB following a single oral dose of AMX0035 to healthy male and female subjects in the fasted and fed states: Pharmacokinetic Analysis Set

[0503]

[0504] After a single oral administration of AMX0035 to healthy male and female subjects in the fasting state, the concentration of PB was evident from 0.25 hours in all 13 subjects. The maximum plasma concentration occurred at 0.25 to 0.50 hours after administration. The concentration then decreased in a generally monophasic manner and remained quantifiable until 3.00 to 5.00 hours after administration. The terminal slope was reliably determined for all 13 subjects, and the resulting elimination half-life was in the range of 0.40 to 0.71 hours. The geometric mean (geometric CV%) half-life was 0.461 hours (15.1%).

[0505] 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.

[0506] After a single oral administration of AMX0035 in the fed state, the concentration of PB was evident in all 14 subjects from 0.25 hours onwards. The maximum plasma concentration occurred at 0.25 to 1.50 hours after administration. The concentration then decreased in a generally monophasic manner and remained quantifiable until 3.50 to 6.00 hours after administration. The terminal slope was reliably determined for 13 subjects, and the resulting elimination half-life was in the range of 0.36 to 1.12 hours. The geometric mean (geometric CV%) half-life was 0.599 hours (41.4%). In the case where the terminal slope could not be reliably determined, this was the result of an unacceptable coefficient of determination (i.e., adjusted R 2 <0.9). Following administration of AMX0035 in both the fasting and fed states, one subject showed reduced exposure to PB (in terms of Cmax, AUC(0-last) and AUC(0-inf)) compared to the other subjects in the group.

[0507] 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.

[0508] PAA

[0509] for Figure 3 The mean plasma concentration of PAA versus time profiles for all subjects in the PK analysis set are shown as log 10 / Linear scale shown by food condition.

[0510] The key PK parameters of PAA for all subjects in the PK analysis set are presented in Table 10 below.

[0511] Table 10: Summary of plasma pharmacokinetic parameters of PAAs following a single oral dose of AMX0035 to healthy male and female subjects in the fasted and fed states: Pharmacokinetic Analysis Set

[0512]

[0513] Following a single oral administration of AMX0035 to healthy male and female subjects in the fasting state, concentrations of the metabolite PAA were evident from 0.25 hours onwards in all 13 subjects dosed. Maximum 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 to 8.00 hours post-dose. The terminal slope was reliably determined for all 13 subjects, and the resulting elimination half-lives were in the range of 0.69 to 0.95 hours. The geometric mean (geometric CV%) half-life was 0.813 hours (11.5%).

[0514] Following administration in the fasted state, the geometric mean (geometric CV%) PAA / PB metabolite to parent ratios were 0.168 (40.3%) for Cmax, 0.416 (38.9%) for AUC(0-last), and 0.421 (38.5%) for AUC(0-inf).

[0515] Following a single oral administration of AMX0035 in the fed state, concentrations of the metabolite PAA were evident from 0.25 to 0.50 hours in all 14 subjects. Maximum plasma concentrations occurred from 2.00 to 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. The terminal slope was reliably determined for all 14 subjects, and the resulting elimination half-lives ranged from 0.60 to 1.04 hours. The geometric mean (geometric CV%) half-life was 0.780 hours (16.3%).

[0516] Following administration in the fed state, the geometric mean (geometric CV%) PAA / PB metabolite to parent ratios were 0.407 (45.6%), 0.626 (49.2%), and 0.598 (46.8%) for Cmax, AUC(0-last), and AUC(0-inf), respectively.

[0517] Pharmacokinetics of TURSO, UDCA, and GUDCA

[0518] TURSO

[0519] for Figure 4 The mean plasma concentration profiles of TURSO versus time for all subjects in the PK analysis set are shown as log 10 / Linear scale shown by food condition.

[0520] The key PK parameters of TURSO for all subjects in the PK analysis set are presented in Table 11 below.

[0521] Table 11: Summary of plasma pharmacokinetic parameters of TURSO following a single oral dose of AMX0035 to healthy male and female subjects in the fasted and fed states: Pharmacokinetic Analysis Set

[0522]

[0523] Following a single oral administration of AMX0035 to healthy male and female subjects in the fasting state, time-matched baseline-corrected maximum plasma concentrations of TURSO occurred between 1.50 and 10.00 hours post-dose. The terminal slope was reliably determined for 5 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%). In the absence of a reliable determination of the terminal slope, this was a result of an unacceptable coefficient of determination (i.e., adjusted R 2 <0.9).

[0524] 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.

[0525] Following a single oral administration of AMX0035 in the fed state, time-matched baseline-corrected maximum plasma concentrations of TURSO occurred between 4.50 and 10.00 hours post-dose. The terminal slope was reliably determined for 6 subjects, and the resulting elimination half-lives ranged from 1.53 to 5.36 hours with a geometric mean (geometric CV%) of 3.359 hours (45.1%). In the absence of a reliable determination of the terminal slope, this was a result of an unacceptable coefficient of determination (i.e., adjusted R 2 <0.9).

[0526] 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.

[0527] UDCA

[0528] for Figure 5 The mean plasma concentration of UDCA versus time profiles for all subjects in the PK analysis set are shown as log 10 / Linear scale shown by food condition.

[0529] The key PK parameters of UDCA for all subjects in the PK analysis set are presented in Table 12 below.

[0530] Table 12: Summary of plasma pharmacokinetic parameters of UDCA following a single oral dose of AMX0035 to healthy male and female subjects in the fasted and fed states: Pharmacokinetic Analysis Set

[0531]

[0532] Following a single oral administration of AMX0035 to healthy male and female subjects in the fasting state, time-matched baseline-corrected maximum plasma concentrations of the metabolite UDCA occurred between 0.25 and 20.00 hours post-dose. The terminal slope was reliably determined for 2 subjects, and the resulting elimination half-lives ranged from 3.01 to 7.58 hours. In the absence of a reliable determination of the terminal slope, this was a result of an unacceptable coefficient of determination (i.e., adjusted R 2 <0.9) or the result of insufficient number of data points after Cmax. After administration in the fasting state, the geometric mean (geometric CV%) of the UDCA / TURSO metabolite to parent ratios was 1.099 (119.9%) for Cmax and 1.620 (149.3%) for AUC(0-last).

[0533] Following a single oral administration of AMX0035 in the fed state, time-matched baseline-corrected maximum plasma concentrations of the metabolite UDCA occurred between 6.00 and 24.00 hours post-dose. The terminal slope was reliably determined for 1 subject, with a resulting elimination half-life of 5.312 hours. In cases where the terminal slope could not be determined, this was a result of an unacceptable coefficient of determination (i.e., adjusted R 2 <0.9) or the result of insufficient number of data points after Cmax. The geometric mean (geometric CV%) of the UDCA / TURSO metabolite to parent ratios was 1.093 (169.5%) and 1.406 (148.8%) for Cmax and AUC(0-last), respectively.

[0534] GUDCA

[0535] for Figure 6 The mean plasma concentration of GUDCA versus time profiles for all subjects in the PK analysis set are shown as log 10 / Linear scale shown by food condition.

[0536] The key PK parameters of GUDCA for all subjects in the PK analysis set are presented in Table 13 below.

[0537] Table 13: Summary of plasma pharmacokinetic parameters of GUDCA following a single oral dose of AMX0035 to healthy male and female subjects in the fasted and fed states: Pharmacokinetic Analysis Set

[0538]

[0539] Following a single oral administration of AMX0035 to healthy male and female subjects in the fasting state, time-matched baseline-corrected maximum plasma concentrations of the metabolite GUDCA occurred between 6.00 and 20.00 hours post-dose. The terminal slope was reliably determined for 1 subject, with the resulting elimination half-life of 12.744 hours. In cases where the terminal slope could not be reliably determined, this was a result of an unacceptable coefficient of determination (i.e., adjusted R 2 <0.9) or as a result of insufficient number of data points after Cmax. Following administration in the fasting state, the geometric mean (geometric CV%) ratios of the GUDCA / TURSO metabolites to the parent were 0.571 (107.4%) for Cmax and 1.056 (114.8%) for AUC(0-last).

[0540] Following a single oral administration of AMX0035 in the fed state, time-matched baseline-corrected maximum plasma concentrations of the metabolite GUDCA occurred between 0.50 and 24.00 hours post-dose. The terminal slope was reliably determined for 2 subjects, and the resulting elimination half-lives were 11.03 and 24.71 hours. In the absence of a terminal slope, this was a result of an unacceptable coefficient of determination (i.e., adjusted R 2 <0.9) or as a result of insufficient number of data points after Cmax. The geometric mean (geometric CV%) ratios of the GUDCA / TURSO metabolites to the parent were 0.675 (92.9%) and 0.918 (103.4%) for Cmax and AUC(0-last), respectively.

[0541] Statistical results and analysis

[0542] PB

[0543] Table 14 presents the results of the statistical analysis of Cmax, AUC(0-last) and AUC(0-inf) for assessing the food effect of PB.

[0544] Table 14: Results of statistical analysis to assess food effects in PB following a single oral dose of AMX0035 to healthy male and female subjects in the fasted and fed states: Pharmacokinetic analysis subset

[0545]

[0546] One subject did not have a reliable estimate of AUC(0-inf) for both the fed and fasted treatments (due to an unacceptable coefficient of determination for the fed state [i.e., adjusted R 2<0.9]) and were therefore excluded from the statistical analysis of AUC(0-inf).

[0547] The treatment by 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 the term was removed from the corresponding models.

[0548] For the comparison of the fed versus fasted treatments, point estimates of the GMR associated with peak (Cmax) and overall (AUC(0-last) and AUC(0-inf)) exposure levels ranged from 24.35% to 45.98%, i.e., exposure levels for the fed treatment averaged approximately 24% to 46% of those seen for the fasted treatment. Each upper bound associated with the GMR was less than 51%, and therefore mean exposure levels for the fed treatment greater than 51% of the mean exposure seen for the fasted treatment can be confidently excluded.

[0549] The differences between the fed and fasted treatments associated with each PK parameter 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 was statistical evidence to reject the null hypothesis that there was no difference between the fed and fasted treatments.

[0550] Although no statistically significant sex effects were observed (i.e., p=0.17, p=0.11, and p=0.14 for Cmax, AUC(0-last), and AUC(0-inf), respectively), there was some evidence of higher PB exposure levels in females when compared to males, and this was consistent for both fed and fasting regimens (data not shown).

[0551] The median value of the Tmax of fasting treatment is 0.500 hours, and the median value of the Tmax of feeding treatment is 1.000 hours. The Hodges-Lehmann estimated value of the difference between feeding and fasting median (i.e. feeding-fasting) is 0.500 hours, with 90%CI (0.125, 0.750). The slight increase of the Tmax of the fed state when compared with the fasting state has statistical significance (p=0.007) (table 15) on the 10% level.

[0552] Table 15: Plasma pharmacokinetic parameters using actual concentrations: PB

[0553]

[0554] PAA

[0555] Table 16 presents the results of the statistical analysis of Cmax, AUC(0-last) and AUC(0-inf) for evaluating the food effect of PAA.

[0556] Table 16: Results of statistical analysis to evaluate the food effect of PAA following a single oral dose of AMX0035 to healthy male and female subjects in the fasted and fed states: Pharmacokinetic analysis subset

[0557]

[0558] The treatment by sex interaction term was not significant at the 5% level for each of Cmax (p=0.46), AUC(0-last) (p=0.77), and AUC(0-inf) (p=0.77), and therefore the term was removed from the corresponding models.

[0559] For the comparison of the fed versus fasted treatments, point estimates of the GMR associated with peak (Cmax) and overall (AUC(0-last) and AUC(0-inf)) exposure levels ranged from 60.34% to 70.54%, i.e., exposure levels for the fed treatment averaged approximately 60% to 71% of those seen for the fasted treatment. Each of the upper bounds associated with the GMR was less than 77%, and therefore mean exposure levels for the fed treatment that were greater than 77% of the mean exposure level seen for the fasted treatment could be confidently excluded.

[0560] The differences between the fed and fasted treatments associated with each PK parameter 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 was statistical evidence to reject the null hypothesis that there was no difference between the fed and fasted treatments.

[0561] Although no statistically significant sex effects were observed (i.e., p=0.24, p=0.18, and p=0.18 for Cmax, AUC(0-last), and AUC(0-inf), respectively), there was some evidence of higher PAA exposure levels in females when compared to males, and this was consistent for both fed and fasting regimens (data not shown).

[0562] The median value of Tmax for both fasting and feeding treatment was 2.500 hours. The Hodges Lehmann estimated value of the difference in the median value between feeding and fasting treatment was 0.250 hours, with 90% CI (0.000, 0.750). The slight increase of Tmax in the fed state when compared with the fasting state had statistical significance (p=0.058) at the 10% level (Table 17).

[0563] Table 17: Plasma pharmacokinetic parameters using actual concentrations: PAA

[0564]

[0565] TURSO

[0566] Table 18 presents the results of the statistical analysis of Cmax and AUC(0-Last) based on time-matched baseline-corrected concentrations for assessing the food effect of TURSO.

[0567] Table 18: Results of statistical analyses to evaluate the food effect of TURSO following a single oral dose of AMX0035 to healthy male and female subjects in the fasted and fed states: Pharmacokinetic Analysis Subset

[0568]

[0569] The number of subjects with reliable estimates of AUC(0-inf) for both time periods was less than 7; therefore, formal statistical analysis of AUC(0-inf) was not performed.

[0570] The treatment by 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 the term was removed from the corresponding models.

[0571] The fed / fasted GMR (90% CI) for Cmax and AUC(0-Last) were 101.56% (78.52%, 131.36%) and 138.81% (105.39%, 182.83%), respectively, i.e., the mean exposure levels as measured by Cmax and AUC(0-Last) for the fed treatment were, on average, approximately 2% and 39% higher than the mean exposure levels seen for the fasted treatment, respectively. However, due to the width of the associated 90% CIs, these estimates should be viewed with caution, for example, mean increases in AUC(0-Last) as small as 5% and as large as 83% cannot be confidently excluded.

[0572] The difference between the fed and fasted treatments related to Cmax was not statistically significant at the 10% level, i.e., p = 0.92. For the analysis of overall exposure across the entire sampling period, i.e., AUC(0-Last), the difference between the 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 food effect for Cmax, and there was no evidence to reject the null hypothesis of no food effect for AUC(0-Last).

[0573] There was some evidence of higher TURSO exposure levels in females when compared to males, and this was consistent for both the fed and fasting regimens, i.e., this effect was significant at the 10% level for AUC(0-last) (p=0.060), but not statistically significant for Cmax (p=0.13) (data not shown).

[0574] The median value of Tmax based on time-matched baseline-corrected concentration was 4.500 hours for fasting treatment and 5.000 hours for feeding treatment. The Hodges-Lehmann estimated value of the difference between feeding and fasting median value was 2.000 hours, with 90%CI (0.250, 2.758). The increase of Tmax in fed state when compared with fasting state had statistical significance (p=0.052) at 10% level (Table 19).

[0575] Table 19: Plasma Pharmacokinetic Parameters Using Time-Matched Baseline Corrected Concentrations: TURSO

[0576]

[0577] UDCA

[0578] Table 20 presents the results of the statistical analysis of Cmax and AUC(0-last) based on time-matched baseline-corrected concentrations for assessing the food effect of UDCA.

[0579] Table 20: Results of statistical analysis to evaluate the food effect of UDCA following a single oral dose of AMX0035 to healthy male and female subjects in the fasted and fed states: Pharmacokinetic analysis subset

[0580]

[0581] The number of subjects with reliable estimates of AUC(0-inf) for both time periods was less than 7; therefore, formal statistical analysis of AUC(0-inf) was not performed.

[0582] The treatment by 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 the term was removed from the corresponding models.

[0583] The fed / fasted GMR (90% CI) for Cmax and AUC(0-Last) were 112.17% (65.80%, 191.21%) and 136.34% (88.53%, 209.98%), respectively, i.e., the mean exposure levels as measured by Cmax and AUC(0-Last) for the fed treatment were, on average, approximately 12% and 36% higher than those seen for the fasted treatment, respectively. However, due to the width of the associated 90% CIs, these estimates should be viewed with caution, for example, mean decreases in AUC(0-Last) as small as 11% and mean increases in AUC(0-Last) as large as 110% (i.e., more than two-fold) cannot be confidently excluded.

[0584] The differences between the fed and fasted treatments associated with each PK parameter were not statistically significant at the 10% level, i.e., p = 0.71 for Cmax and p = 0.22 for AUC(0-last). As a result, there was no statistical evidence for a difference between the fed and fasted treatments, i.e., there was no evidence to reject the null hypothesis of no food effect for each of Cmax and AUC(0-last).

[0585] Although no statistically significant sex effects were observed (i.e., p=0.13 and p=0.16 for Cmax and AUC(0-last), respectively), there was some evidence of higher UDCA exposure levels in males when compared to females, and this was consistent for both the fed and fasting regimens (data not shown).

[0586] The intermediate value of Tmax based on time matching baseline correction concentration is 6.000 hours for fasting treatment and 16.000 hours for feeding treatment. The Hodges-Lehmann estimated value of the difference between feeding and fasting intermediate value is 7.000 hours, with 90%CI (2.000,11.500). The increase of Tmax in fed state when compared with fasting state has statistical significance (p=0.022) on 10% level (table 21).

[0587] Table 21: Plasma pharmacokinetic parameters using time-matched baseline-corrected concentrations: UDCA

[0588]

[0589] GUDCA

[0590] Table 22 presents the results of the statistical analysis of Cmax and AUC(0-last) based on time-matched baseline-corrected concentrations for assessing the food effect of GUDCA.

[0591] Table 22: Results of statistical analysis to evaluate the food effect of GUDCA following a single oral dose of AMX0035 to healthy male and female subjects in the fasted and fed states: Pharmacokinetic analysis subset

[0592]

[0593] The number of subjects with reliable estimates of AUC(0-inf) for both time periods was less than 7; therefore, formal statistical analysis of AUC(0-inf) was not performed.

[0594] The treatment by 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 the term was removed from the corresponding models.

[0595] The fed / fasted GMR (90% CI) for Cmax and AUC(0-Last) were 134.45% (95.95%, 188.41%) and 137.31% (105.64%, 178.49%), respectively, i.e., the mean exposure levels as measured by Cmax and AUC(0-Last) for the fed treatment were, on average, approximately 34% and 37% higher than the mean exposure levels seen for the fasted treatment, respectively. However, due to the width of the associated 90% CIs, these estimates should be viewed with caution, for example, mean decreases in Cmax of as little as 4% and mean increases in AUC(0-Last) of as much as 78% cannot be confidently ruled out.

[0596] The difference between the fed and fasted treatments related 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 food effect for Cmax. For the analysis of overall exposure across the entire sampling period, i.e., AUC(0-Last), the difference between the fed and fasted treatments was statistically significant at the 10% level (p=0.053), i.e., evidence to reject the null hypothesis of no food effect for AUC(0-Last) (data not shown).

[0597] The median value of Tmax based on time-matched baseline-corrected concentration was 16.000 hours for both fasting and feeding treatments. The Hodges-Lehmann estimated value of the difference between the feeding and fasting regimens was 2.000 hours with 90% CI (-4.000, 5.000). This difference was not statistically significant at the 10% level (p=0.66) (Table 23).

[0598] Table 23: Plasma Pharmacokinetic Parameters Using Time-Matched Baseline Corrected Concentrations: GUDCA

[0599]

[0600] Statistical / Analytical Questions

[0601] The distributional assumptions underlying the statistical analyses appeared to be met for the analyses of the various PK parameters, with the possible exception of PAA Cmax, where there was some evidence that the residuals from the model fit were not normally distributed. Closer examination of the residuals indicated no particular reason for this departure from the underlying assumptions, and the results were consistent with the other PAA PK parameters.

[0602] There were no other statistical analysis issues.

[0603] Pharmacokinetic and statistical conclusions

[0604] PB and PAA

[0605] Following a single oral administration of AMX0035, plasma exposure to PB showed statistically significant decreases of approximately 76%, 55%, and 54% in the fed state relative to the fasted state based on Cmax, AUC(0-last), and AUC(0-inf), respectively.

[0606] Under fasting conditions, peak exposure to metabolite PAA was approximately 14% of peak exposure to PB, while total exposure based on AUC(0-inf) was approximately 35% of that to PB. The PAA / PB metabolite to parent ratio was higher in the fed state, with peak exposure being approximately 34% of peak exposure to PB, and total exposure based on AUC(0-inf) being approximately 48% of that to PB.

[0607] Significant increases in PB and PAA Tmax were noted for the fed state when compared to the fasted state, although the increases were slight, ie, the differences in median values ​​were 0.5 hours or less.

[0608] The geometric mean Cmax, AUC(0-last) and AUC(0-inf) of the metabolite PAA showed significant decreases of approximately 40%, 30% and 29%, respectively, after administration in the fed state compared to the fasted state.

[0609] TURSO, UDCA and GUDCA

[0610] Following a single oral administration of AMX0035, peak plasma exposure to TURSO based on Cmax was relatively unchanged, while overall exposure based on AUC(0-last) showed a statistically significant increase of approximately 39% in the fed state relative to the fasted state.

[0611] Under fasting conditions, the conversion of TURSO to UDCA is extensive, with peak exposure to the metabolite UDCA being approximately 86% of the peak exposure to TURSO and total exposure based on AUC(0-last) being approximately 127% of the total exposure to TURSO. This is essentially unchanged in the fed state, with peak exposure being approximately 86% of the peak exposure to TURSO and total exposure based on AUC(0-last) being approximately 110% of the total exposure to TURSO.

[0612] The geometric mean Cmax and AUC(0-last) of the metabolite UDCA showed an increase of approximately 12% and 36%, respectively, after administration in the fed state compared to the fasted state. However, these changes did not reach statistical significance due to high variability.

[0613] Under fasting conditions, the peak exposure to the metabolite GUDCA was approximately 51% of the peak exposure to TURSO, and the total exposure based on AUC(0-last) was approximately 95% of the total exposure to TURSO. This was essentially unchanged in the fed state, with the peak exposure being approximately 61% of the peak exposure to TURSO, and the total exposure based on AUC(0-last) being approximately 83% of the total exposure to TURSO.

[0614] Following administration in the fed state, the geometric mean Cmax and AUC(0-Last) of the metabolite GUDCA showed increases of approximately 34% and 37%, respectively, in the fed state; however, only the difference in AUC(0-Last) reached statistical significance.

[0615] A significant increase in UDCATmax based on time-matched baseline corrected concentrations was noted for the fed state when compared to the fasted state. Similar trends (although smaller increases) were observed for TURSO and GUDCA Tmax, but only this increase for TURSO reached statistical significance.

[0616] Discussion and overall conclusions

[0617] PB and PAA

[0618] After a single oral administration of AMX0035 in the fasting state, PB was rapidly absorbed, with a median Tmax occurring at 0.500 hours post-dose for all subjects. The resulting properties were 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.

[0619] Inter-subject variability associated with exposure (as measured by Cmax, AUC(0-last), and AUC(0-inf)) was moderate, with geometric mean CV% ranging from 39.0% to 45.2%.

[0620] Administration of AMX0035 with a standardized high-fat breakfast resulted in a slight delay in PB absorption compared to the fasted state, delaying the median Tmax by 0.500 hours to a median of 1.000 hours compared to the fasted state, suspected to be a result of slowed gastric emptying after food. Peak and total plasma exposures based on Cmax, AUC(0-last), and AUC(0-inf) showed statistically significant decreases after food of approximately 76%, 55%, and 54%, respectively, compared to the fasted state. Inter-subject variability associated with exposure was consistent between food conditions, with Cmax, AUC(0-last), and AUC(0-inf) geometric mean CV% ranging from 28.6% to 45.4%. Terminal elimination of PB also appeared unchanged in the fed state, with a geometric mean terminal half-life of 0.599 hours.

[0621] In contrast, when administered in the fed state, the volume of distribution and total clearance of PB increased to 21.3 L and 410 mL / min, respectively, which, when combined with the reduced bioavailability, may indicate linear kinetics. Exposure to PB under fed and fasted food conditions showed slight differences between males and females; however, due to moderate inter-subject variability (which was observed to be higher in males than in females), this was not conclusive, and statistical analysis indicated that there were no statistically significant differences between males and females.

[0622] Following administration of AMX0035 under fasting conditions, the peak exposure to the metabolite PAA was 0.168-fold that to PB, while the total exposure based on AUC(0-inf) was 0.421-fold that to PB. The plasma half-life of PAA was comparable to the rapid half-life noted for PB, suggesting that the elimination of PAA may be formation rate limited.

[0623] The time taken to achieve maximum concentrations of PAA following administration of AMX0035 in the fed state was unchanged, with a median of 2.500 hours for both food conditions. However, in contrast, the adjusted geometric mean Cmax, AUC(0-last), and AUC(0-inf) showed statistically significant decreases of approximately 40%, 30%, and 29%, respectively, compared to the fasted state. The reduction in exposure to PAA was less than that observed for the parent PB; this was reflected in the metabolite to parent ratio, which was reported to be slightly higher in the fed state, with peak exposure being 0.407 times that of PB and total exposure based on AUC(0-inf) being 0.598 times that of PB. The estimated geometric mean half-life was almost unchanged by food condition at 0.780 hours. Similar to PB, slight differences were noted between males and females under both food conditions; however, these differences were not conclusive, and statistical analysis indicated that no statistically significant differences were observed.

[0624] One subject showed reduced exposure to PB following administration of AMX0035 in both the fasted and fed states compared to the rest of the group. Maximum plasma PB was 2.92-fold 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 the fasted state, and 3.2-fold and 3.4-fold lower than the group geometric mean in the fed state, respectively. Additionally, due to curvature in one subject, their AUC(0-inf) value is considered unreliable.

[0625] TURSO, UDCA and GUDCA

[0626] After a single oral administration of AMX0035 in the fasting state, TURSO was absorbed with a median Tmax occurring at 4.500 hours post-dose. The resulting properties were 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 in the fasting state was 4.337 hours.

[0627] Inter-subject variability associated with exposure (as measured by Cmax, AUC(0-last), and AUC(0-inf)) was high, with geometric mean CV% ranging from 58.9% to 79.4%.

[0628] Administration of AMX0035 with a standardized high-fat breakfast resulted in a slight delay in the absorption of TURSO, as evidenced by an increase in the median Tmax from 4.500 hours in the fasted state to 5.000 hours in the fed state. This delay is suspected to be the result of slowed gastric emptying after food. In the fed state, peak plasma exposure based on Cmax did not change; however, overall exposure based on AUC(0-inf) and AUC(0-last) showed increases of approximately 47% and 39%, respectively. However, due to the limited availability of AUC(0-inf) data, only AUC(0-last) could be statistically analyzed. In the fed state, inter-subject variability associated with exposure was slightly reduced, with geometric CV% ranging from 24.1% to 62.7% for Cmax, AUC(0-last) and AUC(0-inf). The terminal elimination of TURSO did not change in the fed state, with a geometric mean terminal T1 / 2 of 3.359 hours.

[0629] When administered in the fed state, the volume of distribution and total clearance of TURSO decreased to 1000 L and 3440 mL / min, respectively; when combined with the increased bioavailability seen for AUC, this may indicate linear kinetics. The inter-subject variability noted after exposure to TURSO showed little difference between males and females in the fed and fasted states. As a result, statistical analysis indicated that there were no statistically significant differences between males and females.

[0630] After administration of AMX0035 under fasting conditions, the peak exposure to the metabolite UDCA was 1.099 times that to TURSO, and the total exposure based on AUC (0-last) was 1.620 times that to TURSO, indicating extensive and complete metabolism of TURSO to UDCA. The plasma half-life of UDCA was comparable to that of TURSO, indicating that the elimination of UDCA may be limited by the formation rate.

[0631] After AMX0035 is used in the fed state, the time delay spent on the maximum concentration of UDCA is obtained, wherein the median is 16.000 hours (relative to 6.000 hours in the fasted state). However, by contrast, geometric mean Cmax and AUC (0-last) show an increase of approximately 12% and 36% respectively. The increase in overall exposure for UDCA is roughly equivalent to the increase in overall exposure observed for the parent TURSO. This is reflected in metabolites and parent ratios, which remain unchanged in the fed state, wherein the peak exposure is 1.093 times the peak exposure to TURSO, and the total exposure based on AUC (0-last) is 1.406 times the total exposure to TURSO. The estimated geometric mean half-life is also not changed by food conditions at 5.312 hours. The inter-subject variability noted after exposure to UDCA shows almost no difference between males and females in the fed and fasted states. As a result, statistical analysis shows that statistically significant differences are not observed between males and females.

[0632] After administration of AMX0035 under fasting conditions, the peak exposure to the metabolite GUDCA was 0.571 times the peak exposure to TURSO, and the total exposure based on AUC (0-last) was 1.056 times the total exposure to TURSO, indicating complete and extensive metabolism of TURSO to UDCA and then to GUDCA. The plasma half-life of GUDCA is extended relative to that of TURSO (12.744 hours vs. 4.337 hours), indicating that the elimination of GUDCA is not limited by the formation rate.

[0633] After AMX0035 is administered in the fed state, the time spent for obtaining the maximum concentration of GUDCA does not change, wherein the median is 16.000 hours. However, by contrast, geometric mean Cmax and AUC (0-last) show an increase of 34% and 37% respectively in the fed state. However, due to the high variability observed, it is considered that only AUC (0-last) has statistical significance. The increase in the overall exposure of GUDCA is comparable to the increase in the overall exposure observed for the parent TURSO. This is reflected in terms of metabolites and parent ratios, which are substantially unchanged in the fed state, wherein the peak exposure is 0.675 times the peak exposure to TURSO, and the total exposure based on AUC (0-last) is 0.918 times the total exposure to TURSO.

[0634] Similar to TURSO and UDCA, the inter-subject variability noted after exposure to GUDCA showed only slight differences between males and females in both food conditions. As a result, statistical analysis indicated that no statistically significant differences were observed between males and females.

[0635] Security Discussion

[0636] There were no safety concerns associated with administration of AMX0035 in the fasting or fed state.

[0637] There were no serious or severe AEs or ADRs. Overall, 1 subject experienced an unrelated AE of moderate severity which led to withdrawal of the IMP.

[0638] With the exception of 1 (7.1%) subject in the fed treatment group, no subject had an increase in QTcF from baseline of >30 msec. No subject recorded a QTcF value ≥450 msec at any time point, and no subject had an increase in QTcF >60 msec.

[0639] There was no evidence that administration of AMX0035 in the fasting or fed state was associated with any clinically relevant QTcF effects within the observed plasma concentration ranges (i.e., up to approximately 200 µg / mL for PB in the fasting state and up to approximately 40 µg / mL for PAA in the fed state).

[0640] There were no clinically significant findings in any of the clinical laboratory evaluations, vital signs, ECGs, or physical examination findings.

[0641] Overall conclusion

[0642] Pharmacokinetic conclusions

[0643] PB and PAA

[0644] Following a single oral administration of AMX0035, plasma exposure to PB based on Cmax, AUC(0-last) and AUC(0-inf) showed statistically significant decreases of approximately 76%, 55% and 54%, respectively, in the fed state relative to the fasted state.

[0645] Under fasting conditions, peak exposure to metabolite PAA was approximately 14% of peak exposure to PB, and total exposure based on AUC(0-inf) was approximately 35% of that to PB. The PAA / PB metabolite to parent ratio was higher in the fed state, with peak exposure being approximately 34% of peak exposure to PB, and total exposure based on AUC(0-inf) being approximately 48% of that to PB.

[0646] Significant increases in PB and PAA Tmax were noted for the fed state when compared to the fasted state, although the increases were slight, ie, the differences in median values ​​were 0.5 hours or less.

[0647] The geometric mean Cmax, AUC(0-last) and AUC(0-inf) of the metabolite PAA showed statistically significant decreases of approximately 40%, 30% and 29%, respectively, after administration in the fed state compared to the fasted state.

[0648] TURSO, UDCA and GUDCA

[0649] Following a single oral administration of AMX0035, peak plasma exposure to TURSO based on Cmax was relatively unchanged in the fed state relative to the fasted state, while overall exposure based on AUC(0-last) showed a statistically significant increase of approximately 39%.

[0650] Under fasting conditions, the conversion of TURSO to UDCA is extensive, with peak exposure to the metabolite UDCA being approximately 86% of the peak exposure to TURSO and total exposure based on AUC(0-last) being approximately 127% of the total exposure to TURSO. This is essentially unchanged in the fed state, with peak exposure being approximately 86% of the peak exposure to TURSO and total exposure based on AUC(0-last) being approximately 110% of the total exposure to TURSO.

[0651] The geometric mean Cmax and AUC(0-last) of the metabolite UDCA showed an increase of approximately 12% and 36%, respectively, after administration in the fed state compared to the fasted state. However, these changes did not reach statistical significance due to high variability.

[0652] Under fasting conditions, the peak exposure to the metabolite GUDCA was approximately 51% of the peak exposure to TURSO, and the total exposure based on AUC(0-last) was approximately 95% of the total exposure to TURSO. This was essentially unchanged in the fed state, with the peak exposure being approximately 61% of the peak exposure to TURSO, and the total exposure based on AUC(0-last) being approximately 83% of the total exposure to TURSO.

[0653] Following administration in the fed state, the geometric mean Cmax and AUC(0-Last) of the metabolite GUDCA showed increases of approximately 34% and 37%, respectively, in the fed state; however, only the difference in AUC(0-Last) reached statistical significance.

[0654] A significant increase in UDCATmax based on time-matched baseline corrected concentrations was noted for the fed state when compared to the fasted state. Similar trends (although smaller increases) were observed for TURSO and GUDCA Tmax, but only this increase for TURSO reached statistical significance.

[0655] Safety Conclusion

[0656] Under the conditions of this study, AMX0035 administered in both the fasted and fed states was well tolerated.

[0657] One (7.1%) subject in the fed treatment group reported 1 TEAE following administration of AMX0035. The AE of musculoskeletal pain was of moderate (Grade 2) severity, unrelated to AMX0035, and led to withdrawal of the IMP.

[0658] No IMP-related AEs were reported during this study.

[0659] There was no evidence that administration of AMX0035 in the fasting or fed state was associated with any clinically relevant QTcF effects within the observed plasma concentration ranges (i.e., up to approximately 200 µg / mL for PB in the fasting state and up to approximately 40 µg / mL for PAA in the fed state).

[0660] No clinically significant changes in clinical laboratory evaluations, vital signs, ECGs, or physical examination findings were reported.

[0661] Example 3: Population Pharmacokinetics of Phenylbutyrate (PB) and Phenylacetic Acid (PAA) Following Administration of AMX0035 for Treatment of ALS

[0662] The objectives of the study were to (1) develop a population pharmacokinetic (PK) model to describe the plasma concentrations of PB and its metabolite PAA in ALS patients receiving AMX003; (2) identify demographic and clinical characteristics that explain the PK variability of PB and PAA; and (3) perform simulations using the final population PK model to examine the effects of different covariates on PB and PAA exposure.

[0663] The pharmacokinetic data of population PK analysis are obtained from Phase 1 food effect study in healthy adult volunteers and Phase 2 clinical trials in ALS patients. Phase 1 food effect study examined the PK of PB, TUDCA and major metabolites after single oral dose administration of AMX0035 in healthy subjects with and without food. Subjects received a single dose of 1 sachet (1 g TUDCA and 3 g PB) of AMX0035 under fasting (overnight and 4 hours after administration) and feeding (standard high-fat breakfast 30 minutes before administration) conditions, with a minimum washout period of 4 days between treatments. The order of administration with and without food is randomly assigned. Continuous blood samples are collected within 24 hours after each dose for analysis of PB, PAA, TUDCA, UDCA and GUDCA plasma concentrations. In addition, plasma samples are obtained the day before the first dose of AMX0035 to characterize the endogenous concentrations of TUDCA and metabolites. It is called Scheme A35-002.

[0664] Phase 2 trials have examined the safety, tolerability, efficacy and biological activity of AMX0035. Subjects are randomly assigned to receive AMX0035 or placebo at a ratio of 2: 1. Treatment is administered with 1 sachet of AMX0035 or placebo per day for the first 3 weeks, and then increased to 1 sachet twice a day if tolerated. Subjects are advised to take medication before meals. A sparse sampling strategy of a single plasma sample collected during a baseline visit (before dosing) and a 12-week and 24-week treatment visit is used to analyze the plasma concentrations of PB, PAA, TUDCA, UDCA and GUDCA. It is called Scheme 3500.

[0665] result

[0666] Overview

[0667] A one-compartment model with first-order absorption, first-order metabolism from PB to PAA (100% conversion), and a metabolic (transport) compartment to simulate the delay in the appearance of metabolite plasma concentrations and the nonlinear elimination of PAA best described the PB and PAA plasma concentration data. The final model included parameters describing the effect of food administration on the oral absorption rate constant (Ka), the apparent metabolic clearance (CL) from PB to PAA, and the effect of food administration on the metabolite plasma concentration. PB / F) or the apparent volume of distribution of PB (V PB / F), the effect of body weight on the maximum elimination rate of PAA (V max -PAA) and the impact of ALS diagnosis on V max PAA and the distribution volume of PAA (V PAA ) were not included. Inter-occasion variability (IOV) was not included.

[0668] Table 24 presents parameter estimates for the final model and bootstrap analysis. Based on typical values ​​for CLPB / F and VPB / F, the PB terminal elimination half-life was estimated to be 0.45 hours. The final model demonstrated a 52.4% (95% confidence interval: 48.5% to 56.3%) decrease in relative bioavailability and a 60% (95% confidence interval: 56% to 64%) decrease in Ka when PB was administered under fed versus fasted conditions.

[0669] For Phase 1 studies, individual maximum plasma concentrations (C max ) and the area under the plasma concentration versus time curve from time 0 to infinity (AUC 0-∞ ) values ​​and the corresponding observed individual C for fed and fasted conditions max and AUC 0-∞ The values ​​are very consistent.

[0670] This finding, along with the lack of accumulation after multiple dosing, supports the use of this approach to estimate subject-specific exposure parameters at steady state in ALS patients. Simulations were performed to evaluate the effects of the covariates included in the final model on the systemic exposure of PB and PAA. The only covariate that affected PB exposure was drug administration in relation to food. The model-predicted PB C was significantly higher when administered with food relative to when administered under fasting conditions. max and from time 0 to the last quantifiable plasma concentration (AUC 0-最后 ) were 3.2- and 2.2-fold lower in the plasma concentration versus time curve, respectively.

[0671] Food administration, ALS diagnosis, and body weight affect PAA exposure. Model-predicted PAA C was higher when administered with food relative to when administered fasting. max and AUC 0-最后 Lack of ALS diagnosis has an impact on PAA C max and AUC 0-最后 There was only a small effect (<1.2-fold). Model-based simulations showed that body weight had a significant effect on PAA exposure. PAA Cmax was 1.45-fold higher at 50 kg body weight and 1.47-fold lower at 115 kg compared to 70 kg, and PAAAUC0-last was 1.87-fold higher at 50 kg and 1.72-fold lower at 115 kg compared to 70 kg.

[0672] These results demonstrate that administration of AMX0035 with food reduces PB exposure and, to a lesser extent, PAA exposure in plasma relative to administration in the fasting state. PB and PAA PK are generally similar in ALS patients and healthy subjects. PB exposure is not significantly affected by body weight, whereas an inverse relationship exists between PAA exposure and body weight.

[0673] Table 24: Population pharmacokinetic parameter estimates from the final model and bootstrap analysis of the final model

[0674]

[0675] *Average estimates (relative standard error) of the final model;

[0676] **Average percent coefficient of variation (relative standard error) of the final model [shrinkage, %];

[0677] Corr = correlation coefficient between etas (relative standard error) of the final model; PB = phenylbutyric acid; PAA = phenylacetic acid;

[0678] CL PB / F = apparent (oral) metabolic clearance of phenylbutyrate to phenylacetate; V PB / F = apparent volume of distribution of phenylbutyric acid;

[0679] CL PAA = Elimination rate of phenylacetic acid; V PAA =Volume of distribution of PAA;

[0680] Ka fast = PB oral absorption rate constant under fasting conditions (or fasting for ALS patients); V max = maximum elimination rate; K m-PAA = the amount of PAA when the elimination rate is half maximum; mean transit time - the transit time of PAA from the metabolic compartment to the plasma compartment, indicating the delay in the appearance of PAA in plasma; F food = relative bioavailability of phenylbutyric acid when administered with food (relative to fasting); Ka food = oral absorption rate constant when phenylbutyric acid is administered with food.

[0681] Drug concentration

[0682] The extensive sampling design from protocol 35-002 allowed for a complete characterization of plasma concentration profiles in volunteers. Following oral administration, PB was rapidly absorbed and converted to PAA. Plasma concentrations typically peaked at 0.5 to 1 hour for PB and 2 to 3 hours for PAA, and dropped below the lower limit of quantitation (LLOQ) at 4 to 5 hours post-dose for PB and 6 to 8 hours post-dose for PAA. Thus, no accumulation of drug or metabolites occurred with twice daily (bid) dosing. PB plasma concentrations in volunteers were significantly lower and the distribution profile was longer following administration with a high fat meal compared to fasting administration. Food-related changes in PAA plasma concentration-time data were comparable.

[0683] The effect of food on sparsely collected plasma concentrations from an ALS clinical trial was examined. In clinical trial patients, PB administration was classified as fasting administration (fasting administration) when drug administration was performed ≥2 hours after a meal or ≥1 hour before a meal. There was a trend toward higher PB and PAA plasma concentrations at 1 hour and similar or slightly lower concentrations at 4 hours between fasting administration of PB compared to with food.

[0684] PK parameters derived from post hoc analysis

[0685] Table 25 summarizes the subject-specific C values ​​derived from individual Bayesian parameter estimates in ALS patients. max and AUC 0-∞ .

[0686] Table 25

[0687]

[0688] Number - indicates the number of regimens in which phenylbutyrate was administered under fasting (>2 hours before a meal or >1 hour after a meal) or fed conditions; C max = maximum plasma concentration; AUC 0-最后 = Area under the plasma concentration-time curve from time zero to the last measurable plasma concentration; AUC 0-∞ = area under the plasma concentration-time curve from time zero to infinity; %CV = coefficient of variation expressed as a percentage.

[0689] Example 4: TUDCA Pharmacokinetic Study

[0690] A study was conducted to examine plasma concentration data in various subgroups of the Phase 2 study of regimen AMX3500. This example summarizes the plasma concentrations of TUDCA and 2 metabolites, namely the related acids, UDCA (deconjugated TUDCA metabolite) and GUDCA (glycine conjugate of UDCA).

[0691] For each of the 2 treatment groups (treatment and placebo), subjects were further divided into 2 sample sequence groups to arrange pharmacokinetic measurements:

[0692] 1. 1 hour post-dose for Week 12 visit and 4 hours post-dose for Week 24 visit

[0693] 2. 4 hours post-dose for Week 12 visit and 1 hour post-dose for Week 24 visit

[0694] The antibiotics listed in the concomitant medications were the basis for comparing subjects who took antibiotics when their visit times overlapped with those who did not. Twenty-four different drug descriptions were identified as containing antibiotics. The dates of antibiotic use were compared to the visit dates, and if the range of antibiotic use dates included the visit date, the visit was marked as antibiotic use data for that subject. However, this approach would not mark subjects who took antibiotics before the visit but ended antibiotic administration before the visit was made, and therefore may not account for certain antibiotics with long elimination half-lives. If this occurs, these antibiotics may hypothetically still be in the subject's body. However, it was observed that only a few subjects took antibiotics in cases where the visit dates were adjacent but not overlapping. In other cases, subjects took antibiotics on the day of one visit but not on the day of the other visit. Therefore, some subjects belonged to both the antibiotic use and antibiotic non-use categories, but the individual visits of the subjects were not counted in both categories.

[0695] Subjects whose race was recorded as "black" or "African American" were evaluated according to the standard glomerular filtration rate (eGFR) score for that race. Another standard eGFR score used (sometimes referred to as the standard for people who are Caucasian) is applicable to subjects of all other races. These rates are analyzed continuously in mL / min / 1.73m2 units and in the following categories:

[0696] 1. Less than 90 mL / min / 1.73m 2 ;

[0697] 2. Equal to or greater than 90 mL / min / 1.73m 2 .

[0698] Age was converted from a continuous variable into 2 categories:

[0699] 1. Aged 65 or above;

[0700] 2. Under 65 years old.

[0701] All box plots have boxes bounded by the 25th and 75th percentiles, whiskers at the 10th and 90th percentiles, extreme values ​​beyond that as points, the black line as the median, and the red line as the mean.

[0702] result

[0703] The results showed that TUDCA, UDCA, and GUDCA plasma concentrations appeared to have reached steady state by week 12, supporting the pooled plasma concentration data across study visits. In ALS patients, steady-state plasma concentrations of TUDCA, UDCA, and GUDCA following administration of AMX0035 were extremely highly variable and were generally at least an order of magnitude higher than endogenous levels, which are also highly variable.

[0704] Mean and standard deviation ratio (MSDR) is a simple measure for examining the variability in each group and comparing across acid and group. Table 26 shows the MSDR of TUDCA level of AMX0035 group (for 1 hour after administration, 12 week group and 24 week group (82.1% relative to 66.3%) were compared, and 4 hours group (97.7% relative to 112.7%) after administration, wherein all these groups have obvious high variability). For UDCA (86.7% relative to 89.6%, 102.7% relative to 69.1%) (table 27) and for GUDCA (106.7% relative to 105.9%, 138.1% relative to 134.3%) (table 28), the same high variability pattern was found. It can be noted that there is significant endogenous concentration of UDCA and GUDCA in the patient treated with placebo. The pooled group by the number of hours after administration has similar high variation pattern (table 29). Figure 7-16 This indicates that extreme values ​​contribute to the high variability found in the table.

[0705] Figure 7 It was shown that the TUDCA levels of female subjects 4 hours after dosing had a more positively skewed distribution, with the 75th and 90th percentiles being much higher than those of the other 3 groups. Figure 7 All 4 groups had similar distributions of UDCA levels. Figure 8 Three groups with similar distributions of GUDCA levels are shown, but male subjects in the 4 hour post-dose group had greater variability and higher 75th and 90th percentile scores.

[0706] TUDCA levels showed similar distribution patterns under age categories (1 hour after administration (age << 65 years vs. age >= 65 years) and 4 hours after administration (age << 65 years vs. age >= 65 years). In UDCA, the group over 65 years showed a different pattern from the other 3 groups for 4 hours after administration, with overall higher variability and higher levels, such as Fig. 9 shown. Fig.10 GUDCA levels in the 3 groups showed similar distribution patterns, but the group younger than 65 years had greater variability and lower median values ​​4 hours after administration compared with the group older than 65 years.

[0707] For each acid, the average levels across the groups in the antibiotic use graph were comparable. However, the distributions sometimes had higher medians and variability for TUDCA, UDCA, and GUDCA ( Fig.11 , 12 and 13). These higher median groups consisted of subjects who used antibiotics.

[0708] When visual inspection Figure 14-16 In the box plots for TUDCA, UDCA, and GUDCA, the high GFR group (greater than 90 mL / min / 1.73m 2 ) had a narrower distribution along with lower plasma concentrations compared to the other 3 groups.

[0709] Table 26: Summary Statistics of TUDCA Plasma Concentrations (ng / mL) at Each Visit

[0710]

[0711]

[0712] Table 27: Summary Statistics of UDCA Plasma Concentrations (ng / mL) at Each Visit

[0713]

[0714]

[0715] Table 28: Summary Statistics of GUDCA Plasma Concentrations (ng / mL) at Each Visit

[0716]

[0717]

[0718] Table 29: Summary Statistics of Plasma Concentrations (ng / mL) at Each Pooled Visit for Subjects in the AMX0035 Group

[0719]

[0720] Example 5: Statistical Analysis of Bile Acids in PK Samples from ALS Patients from the CENTAUR Trial

[0721] The purpose of the study was to evaluate the effect of AMX0035 on human plasma bile acid profiles. TUDCA is a bile acid that can be produced endogenously in humans, and its effect on the balance of bile acid pathways may be an important factor in both the efficacy and potential toxicity of treatment. A targeted metabolomics approach was used using the biocrates AbsoluteIDQ® Bile Acid Assay to measure 20 bile acids by LC-MS.

[0722] The following samples were used:

[0723] • The primary analysis, or complete cases (CC), included only samples from subjects for whom all 3 time points (baseline visit, Week 12, and Week 24) were available and in whom no premature discontinuation occurred.

[0724] • Secondary analysis or partial case (PC), including samples from subjects with at least 2 time points available (i.e., baseline visit and Week 12 or Week 24) or all 3 time points available. This analysis also includes samples collected after premature discontinuation (ED) at Week 12 or Week 24 to investigate potential undesired effects of treatment.

[0725] • The time course dataset (TC, 270 samples) includes the same samples as the CC dataset analyzed with different group categories to examine the interaction of treatment and treatment duration.

[0726] • The response strength dataset (SR, 108 samples), including a subset of the CC dataset selected based on its ALSFRS-R slope.

[0727] • The SRTC dataset, including the same samples as the SR dataset analyzed with different group categories, to examine the interaction of response intensity and treatment duration.

[0728] Univariate Statistics

[0729] In the primary analysis of complete cases, an ANOVA model was used to compare the AMX0035 group and the placebo group at three time points: baseline visit, week 12, and week 24. Fig.17 and 18 Pathway visualization showing the effect of AMX0035 treatment at Week 12 and Week 24. There were no significant differences between the two groups at baseline.

[0730] At the 12th and 24th weeks, TUDCA, UDCA and GUDCA and hydrophilic / hydrophobic bile acid ratios increased in subjects treated with AMX0035, which is most likely the direct effect of oral administration of TUDCA to these subjects. In fact, although it is the precursor of TUDCA under physiological conditions, UDCA can also be produced in the intestine by taurine from the deconjugation of TUDCA. In addition, UDCA is considered to be produced exclusively by intestinal flora, which further supports its synthesis from orally administered TUDCA under these conditions. Finally, after taurine is deconjugated, UDCA can be experienced during the enterohepatic recirculation of TUDCA and glycine is reconjugated to form GUDCA. Interestingly, UDCA (rather than TUDCA) is found to be negatively correlated with ALSFRS-R slope (although weakly negatively correlated) (Table 30) in correlation analysis. This shows that high UDCA values ​​can be expected in subjects with rapid disease progression, while low UDCA values ​​will be expected in subjects with slow disease progression.

[0731] At week 12, indices of glycine and taurine conjugates (GCA (glycocholic acid), TCA (taurocholic acid), GDCA (glycodeoxycholic acid), TCDA) as well as CDCA (chenodeoxycholic acid), DCA (deoxycholic acid), and glycine and taurine conjugation of pBA were all reduced in AMX0035 treated subjects. Similar trends were still present at week 24, with the exception of the glycine conjugates GCA and GDCA (which were still reduced overall, but not significantly). The pBA conjugation ratio, which reflects the glycine and taurine conjugation of CA and CDCA, also decreased at both time points. Interestingly, at week 12, the total amount of conjugated major bile acids was significantly reduced, while at week 24, the total amount of unconjugated major bile acids increased.

[0732] The levels of CA (cholic acid) and CDCA decreased and increased overall, respectively, although the difference was significant only for CDCA at week 24. However, the CDCA / CA ratio increased significantly at both time points. The biosynthesis of bile acids is typically described as a combination of the classical pathway that produces CA and CDCA and the alternative pathway that favors CDCA production.

[0733] At week 12, the total amount of 12-α-hydroxylated bile acids (CA, DCA and their conjugates) decreased. An increase in this index has been associated with insulin resistance. Even if a decrease was observed here, this could represent an effect related to diabetes, which was one of the confounders included in the analysis. At week 24, the total amount of glycine-conjugated bile acids increased significantly, probably due to a more significant increase in GLCA at this time point.

[0734] Overall, in CC, AMX0035 treatment (including daily doses of TUDCA) caused an increase in TUDCA and related metabolites and indices at both weeks 12 and 24. Interestingly, CA and DCA downstream metabolites conjugated to glycine and taurine decreased at both time points, which may be due to changes in metabolic resources to adapt to high levels of TUDCA and GUDCA. Similarly, the increase in UDCA precursor CDCA may be related to the acquisition of UDCA caused by treatment.

[0735] Additional information from PC: This dataset differs from CC in the fact that, in addition to the 270 CC samples, it includes samples from subjects who participated only up to 12 weeks, and samples from subjects who provided samples at week 24 but were forced to stop treatment. As shown in the univariate statistics file, the overall properties are similar to CC. Several differences are discussed below.

[0736] In PC, the trend of increased CDCA levels in AMX0035 treated subjects was significant from week 12 and continued to week 24. The total amount of conjugated pBAs also decreased, but not significantly, while the increase in the total amount of glycine conjugated bile acids was significant here, while it was slightly above the p-value threshold in CC. A unique feature of PC at week 12 was a significant decrease in the ratio of TDCA synthesis from CA (TDCA / CA), although this trend was also present in other comparisons here, but was not significant.

[0737] At week 24, the main difference from CC was that the effects on GCA, GDCA, and total 12-α-hydroxylated bile acids (all decreased) persisted from week 12, whereas in CC they had dissipated by week 24.

[0738] Time course analysis (TC): This analysis focuses on the effect of time (or here treatment duration) on the treatment effect. For this purpose, the CC dataset was used with different "group" categories, which combined all AMX0035 or placebo samples regardless of time point. The effects of different time points were then investigated in dedicated interaction tests.

[0739] Three interaction effects were investigated here: (i) the interaction between treatment and time point between baseline and week 12, (ii) the interaction between treatment and time point between baseline and week 24, and (iii) the interaction between treatment and time point between weeks 12 and 24.

[0740] Here again, when studying the interaction between treatment and time between later time points and baseline, increased TUDCA, UDCA, and GUDCA levels and increased hydrophilic / hydrophobic bile acid ratios were the most striking effects. An increase in the CDCA / CA ratio was also evident when compared to baseline, as were decreases in glycine and taurine conjugates and related indices.

[0741] Interestingly, the reduction in the TDCA / CA ratio was significant for interactions i and ii. In CC, there was a trend towards a reduction at both weeks 12 and 24, but it was not significant. This suggests an overall effect of TDCA synthesis from CA by the gut microbiota that may have been overlooked in the simple ANOVA analysis. Interestingly, in the PC analysis, this ratio was also reduced at week 12. In a study on serum bile acid profiles in Alzheimer's disease, this ratio was strongly associated with cognitive decline.

[0742] However, the time course analysis revealed no interaction effect between treatment and time point between weeks 12 and 24. Thus, the effect of AMX0035 treatment on bile acid levels was overall sustained between these two time points.

[0743] Correlation analysis

[0744] For the most relevant subgroups of the CC data set, the correlation of metabolite concentrations and metabolic indices with the parameter "ALSFRS-R slope" was examined. ALSFRS-R (ALS Functional Rating Scale-Revised) is a widely used indicator of disease progression in ALS patients. Here, the slope within the 24-week study was used after correction and transformation to allow for correlation analysis. The higher the ALSFRS-R slope, the slower the disease progression over the 24-week period. Therefore, a positive correlation with the ALSFRS-R slope indicates that the value (metabolite or indicator) is 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 higher values ​​in patients with rapid disease progression and lower values ​​in patients with slower disease progression.

[0745] Table 30: Metabolites / Indicators Significantly Correlated with Transformed ALSFRS-R Slope in the AMX0035 Group

[0746]

[0747] Correlation analysis was performed using different subgroups of the CC dataset at time point week 24 relative to the transformed ALSFRS-R slope. The only subgroup that showed correlation with disease progression indicators was the complete group of AMX0035 treated patients at week 24 (n=61). As shown in Table 30 above, two metabolites were weakly negatively correlated with the ALSFRS-R slope (CDCA and UDCA). Metabolic indicators related to glycine and taurine conjugation showed weak positive correlations, and the total amount of unconjugated (major) bile acids was weakly negatively correlated with the ALSFRS-R slope.

[0748] When the group was divided into subgroups based on other ALS medications (edaravone, riluzole, both, or none), no metabolites / indicators were significantly associated with the transformed ALSFRS-R slope. Note that for these subgroups, the number of replicates was very low (maximum n=23 in the riluzole only group). Similar evaluations in samples from placebo subjects at week 24 showed no significant correlation with ALSFRS-R slope for the entire group (n=29) and for the ALS treatment subgroups (again, with very low numbers of replicates).

[0749] Reaction intensity

[0750] When subjects treated with AMX0035 were divided into response intensity groups based on ALSFRS-R slope, differences were observed in the ANOVA results 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 via the classical and alternative bile acid biosynthetic pathways and is also a precursor to UDCA, was higher in strong responders. Both TDCA and the TDCA / CA ratio, which were found to be associated with cognitive decline, were higher in weak responders.

[0751] Interestingly, the levels of TUDCA, UDCA, GUDCA or the hydrophilic / hydrophobic bile acid ratio did not appear to be the key factors distinguishing the two subgroups.

[0752] Strong responders had significantly lower rates for processes associated with 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 investigated: (i) the interaction between response and the time point between baseline and week 12, (ii) the interaction between response and the time point between baseline and week 24, and (iii) the interaction between response and the time point between weeks 12 and 24. Interestingly, unlike what was observed in the time course analysis of CC where all responders were combined into one group (relative to placebo), there was an interaction effect of response intensity with the time point between weeks 12 and 24, which was absent in the other interactions tested, meaning that the difference between strong and weak responders continued to increase, even though the overall effect of the treatment did not appear to show a large difference in the TC analysis. CDCA was the only significant difference in metabolite levels, while indices of bile acid conjugation and the TDCA / CA ratio still distinguished strong from weak responders given the changes that occurred between weeks 12 and 24.

[0753] in conclusion

[0754] The CC analysis showed that in addition to the expected increase in circulating levels of TUDCA and related metabolites, a large effect on bile acid conjugation could be observed. The PC analysis (including subjects who had to stop treatment) showed similar bile acid profiles (without dramatic differences). The TC analysis confirmed that the bile acid profile had been set at week 12 and was largely maintained at week 24.

[0755] The ALSFRS-R slope in CC subjects allowed the study of bile acid levels in light of disease progression within the 24 weeks of the study. Only weak correlations could be found in AMX0035-treated subjects, including a weak negative correlation with UDCA. Correlation analyses pointed to CDCA (negative correlation) and an effect on bile acid conjugation (which occurs primarily in the liver) (positive correlation), but these results should be interpreted with caution due to the weak correlations.

[0756] SR analysis revealed no differences between strong and poor responders to treatment at baseline or week 12. Levels of TUDCA, UDCA, and GUDCA, as well as the hydrophilic to hydrophobic bile acid ratios, were similar in both groups at week 24. However, CDCA (a precursor of UDCA via metabolism by the gut microbiota), the TDCA / CA ratio (a suggestive indicator of cognitive decline), and indicators of bile acid conjugation to glycine and taurine appeared to be the main drivers that differentiated strong from poor responders at the end of treatment.

[0757] SRTC analysis showed that when differentiating between strong and poor responders, differences in bile acid profiles still evolved between weeks 12 and 24. Here again, CDCA, TDCA / CA and indicators of bile acid conjugation were the main discriminators.

Claims

1. A method of treating at least one symptom of amyotrophic lateral sclerosis (ALS) in a subject, the method comprising: (a) administering to the subject one or more doses of a composition comprising about 1 gram of taurine diol (TURSO) and about 3 grams of sodium phenylbutyrate; (b) determining that the subject has (i) For sodium phenylbutyrate, C is about 3 to about 425 µg / mL max , or about 5 to about 50 µg / mL for phenylacetic acid max , (ii) AUC of about 20 to about 550 μg*h / mL for sodium phenylbutyrate 0-最后 , or an AUC of about 20 to about 160 μg*h / mL for phenylacetic acid 0-最后 ,or (iii) AUC of about 25 to about 545 μg*h / mL for sodium phenylbutyrate 0-∞ , or an AUC of about 21 to about 155 μg*h / mL for phenylacetic acid 0-∞ ;as well as (c) administering to the subject an additional dose of the composition.

2. The method of claim 1, wherein step (b) comprises determining that the subject has a C of about 90 to about 170 μg / mL for sodium phenylbutyrate. max .

3. The method of claim 2, wherein step (b) comprises determining that the subject has a C of about 110 to about 150 μg / mL for sodium phenylbutyrate. max .

4. The method of claim 1, wherein step (b) comprises determining that the subject has a C of about 10 to about 45 μg / mL for phenylacetic acid. max .

5. The method of claim 1, wherein step (b) comprises determining that the subject has an AUC of about 140 to about 300 μg*h / mL for sodium phenylbutyrate. 0-最后 .

6. The method of claim 1, wherein step (b) comprises determining that the subject has an AUC for phenylacetic acid of about 40 to about 80 μg*h / mL. 0-最后 .

7. The method of claim 1, wherein step (b) comprises determining that the subject has an AUC of about 140 to about 300 μg*h / mL for sodium phenylbutyrate. 0-∞ .

8. The method of claim 1, wherein step (b) comprises determining that the subject has an AUC for phenylacetic acid of about 40 to about 80 μg*h / mL. 0-∞ .

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

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

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

12. The method of any one of claims 1-10, wherein 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.

13. The method of any one of claims 1-12, wherein step (b) comprises obtaining a blood sample from the subject about one hour after the last dose of the composition.

14. The method of any one of claims 1-12, wherein step (b) comprises obtaining a blood sample from the subject about four hours after the last dose of the composition.

15. 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 gram of TURSO and about 3 grams of sodium phenylbutyrate; (b) determining the plasma concentration of one or more bile acids selected from TURSO, UDCA or GUDCA in the subject; and (c) administering to the subject an additional dose of the composition.

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

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

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

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

20. The method of any one of claims 15-17, 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 15-20, wherein step (b) comprises determining the plasma concentration about one hour after the last dose of the composition.

22. The method of any one of claims 15-20, wherein step (b) comprises determining the plasma concentration about four hours after the last dose of the composition.

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

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

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

26. The method of any one of claims 15-22, wherein step (b) comprises determining the plasma concentration of TURSO in the subject about four hours after the last dose of the composition, wherein the plasma concentration of TURSO is about 20 to about 3250 ng / mL.

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

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

29. The method of any one of claims 15-22, wherein step (b) comprises determining the plasma concentration of UDCA in the subject about one hour after the last dose of the composition, wherein the plasma concentration of UDCA is about 20 to about 6020 ng / mL.

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

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

32. The method of any one of claims 15-22, wherein step (b) comprises determining the plasma concentration of UDCA in the subject about four hours after the last dose of the composition, wherein the plasma concentration of UDCA is about 20 to about 7340 ng / mL.

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

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

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

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

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

38. The method of any one of claims 15-22, wherein step (b) comprises determining the plasma concentration of GUDCA in the subject about four hours after the last dose of the composition, wherein the plasma concentration of GUDCA is about 20 to about 5290 ng / mL.

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

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

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

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

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

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

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

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

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

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

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

50. The method of any one of claims 1-49, wherein step (a) comprises administering a dose of the composition more than two hours after the subject has ingested food or more than one hour before the subject has ingested food.

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

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

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

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

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

56. The method of any one of claims 51-53, comprising 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. The method of any one of claims 51-56, comprising determining the plasma concentration of the bile acid about one hour after the last dose of the composition.

58. The method of any one of claims 51-56, comprising determining the plasma concentration of the bile acid about four hours after the last dose of the composition.

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

60. The method of any one of claims 1-58, wherein the composition is administered by feeding tube.

61. The method of any one of claims 1-58, wherein the composition is administered by bolus injection.

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

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