HK40081935A

HK40081935A - Treatment of amyotrophic lateral sclerosis

Info

Publication number: HK40081935A
Application number: HK62023068556.7A
Authority: HK
Inventors: 约书亚·科恩; 贾斯廷·克莱
Original assignee: 艾米利克斯制药公司
Priority date: 2019-12-16
Filing date: 2020-08-28
Publication date: 2023-06-02

Description

Treatment of amyotrophic lateral sclerosis

Priority requirement

This application claims the benefit of U.S. provisional patent application serial No. 62/948,770, filed on 12, 16, 2019. The entire contents of the aforementioned application are incorporated herein by reference.

Technical Field

The present disclosure relates generally to compositions and methods for preparing a variety of conditions.

Background

Amyotrophic Lateral Sclerosis (ALS) is the most common progressive motor neuron disease. ALS causes progressive degeneration of motor neurons, leading to rapidly progressive muscle weakness and atrophy, which ultimately leads to partial or complete paralysis. Median survival after onset of symptoms is 2 to 3 years, with respiratory failure being the leading cause of death. ALS treatment is currently focused on symptom management. Only two FDA-approved drugs are currently available for ALS, namely riluzole (riluzole) and edaravone (edaravone). Thus, there is a need for improved therapies for treating ALS.

Disclosure of Invention

The present disclosure provides methods of treating at least one symptom of ALS in a subject (e.g., a subject diagnosed with or at risk of developing ALS), the method comprising administering to the subject about 10mg/kg to about 50mg/kg body weight of a bile acid or a pharmaceutically acceptable salt thereof, and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound.

In one aspect, provided herein is a method of treating at least one symptom of ALS in a human subject, the method comprising administering to the human subject about 10mg/kg to about 50mg/kg body weight of a bile acid or a pharmaceutically acceptable salt thereof, and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound, wherein the human subject: (a) has been diagnosed with ALS for about 24 months or less; (b) has shown one or more symptoms of ALS for about 24 months or less; (c) has an ALS disease progression rate (Δ FS) of about 0.50 or greater; (d) has an ALSFRS-R score of 40 or less; (e) has a mutation in SOD1, C9ORF72, ANG, TARDBP, VCP, VAPB, SQSTM1, DCTN1, FUS, UNC13A, ATXN2, HNRNPA1, CHCHHD 10, MOBP, C21ORF2, NEK1, TUBA4A, TBK1, MATR3, PFN1, UBQLN2, TAF15, OPTN or TDP-43; (f) cerebrospinal fluid (CSF) or blood levels of a phosphorylated neurofilament protein heavy chain (pNF-H) of about 300pg/mL or greater; (g) a CSF or blood level with a neurofilament protein light chain of about 50pg/mL or greater; or (h) an ALSFRS-R score of about 0.8 to about 2 lost on average each month over the preceding 3-12 months; thereby treating at least one symptom of ALS in the human subject. In some embodiments, prior to administration, the method comprises the step of determining whether the human subject has at least one of the characteristics of (a) - (h). In some embodiments, the human subject has been diagnosed with ALS for about 24 months or less. In some embodiments, the human subject has been diagnosed with ALS for about 18 months or less. In some embodiments, the human subject has been diagnosed with ALS for about 12 months or less. In some embodiments, the human subject has exhibited one or more symptoms of ALS for about 24 months or less. In some embodiments, the human subject has exhibited one or more symptoms of ALS for about 18 months or less. In some embodiments, the human subject has exhibited one or more symptoms of ALS for about 12 months or less. In some embodiments, the human subject has a rate of progression of ALS disease (Δ FS) of about 0.50 or greater. In some embodiments, the human subject has a rate of progression of ALS disease (Δ FS) of about 0.90 or greater. In some embodiments, the human subject has a rate of progression of ALS disease (Δ FS) of about 1.20 or greater. In some embodiments, the human subject has an ALSFRS-R score of 40 or less. In some embodiments, the human subject has an ALSFRS-R score of 38 or less. In some embodiments, the human subject has an ALSFRS-R score of 30 or less. In some embodiments, the human subject has a CSF or blood level of phosphorylated neurofilament protein heavy chain (pNF-H) of about 300pg/mL or greater. In some embodiments, the human subject has a CSF or blood level of pNF-H of about 1000pg/mL or more. In some embodiments, the human subject has been diagnosed with clear ALS based on revised EL escoral criteria.

In another aspect, provided herein is a method of reducing the rate of progression of an ALS disease in a human subject having one or more symptoms of ALS, the method comprising: administering to the human subject about 10mg/kg to about 50mg/kg body weight of a bile acid or a pharmaceutically acceptable salt thereof, and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound in a dosage regimen sufficient to reduce the monthly average loss of ALSFRS-R score in the human subject by at least about 0.2 as compared to a control subject that has not received the administration. In some embodiments, the average monthly loss ALSFRS-R score of the human subject is reduced by at least about 0.4 as compared to the control subject.

In another aspect, provided herein is a method of reducing muscle strength deterioration, maintaining muscle strength, or improving muscle strength in a human subject having one or more symptoms of ALS, the method comprising: administering to the human subject about 10mg/kg to about 50mg/kg body weight of a bile acid or a pharmaceutically acceptable salt thereof, and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound, thereby reducing muscle strength deterioration, maintaining muscle strength, or improving muscle strength in the human subject. In another aspect, provided herein is a method, the muscular strength is a lower limb strength, an upper limb strength, or a grasping strength. In some embodiments, the muscle strength is of quadriceps femoris, biceps, hamstring tendon, triceps, or tibialis anterior. In some embodiments, the muscle strength is assessed by a hand-held dynamometer (HHD), a hand-held dynamometer, a freehand muscle strength test (MMT), an electrical impedance Electromyogram (EIM), a Maximum Voluntary Isometric Contraction Test (MVICT), a motor unit estimate (muscle), an accurate isometric strength test for a limb (ATLIS), or a combination thereof, before, during, and/or after administration. In some embodiments, the muscle strength is assessed by ATLIS.

In another aspect, provided herein is a method of reducing respiratory muscle function deterioration, maintaining respiratory muscle function, or improving respiratory muscle function in a human subject with one or more symptoms of ALS, the method comprising: administering to the human subject about 10mg/kg to about 50mg/kg body weight of a bile acid or a pharmaceutically acceptable salt thereof, and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound, thereby reducing respiratory muscle function deterioration, maintaining respiratory muscle function, or improving respiratory muscle function in the human subject. In some embodiments, the subject is treated by evaluating the subject's Vital Capacity (VC), maximum mid-expiratory flow rate (MMERF), Forced Vital Capacity (FVC), Slow Vital Capacity (SVC), forced expiratory volume for 1 second (FEV), before, during, and/or after administration ₁ ) Or a combination thereof, to evaluate respiratory muscle function in the human subject. In some embodiments, respiratory muscle function in the human subject is assessed by evaluating SVC in the subject.

In another aspect, provided herein is a method of preventing or reducing constipation in a human subject having one or more symptoms of ALS, the method comprising: administering to the human subject about 10mg/kg to about 50mg/kg body weight of a bile acid or a pharmaceutically acceptable salt thereof, and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound, thereby preventing or reducing constipation in the human subject.

In another aspect, provided herein is a method of preventing or reducing at least one severe adverse event in a human subject with one or more symptoms of ALS, the method comprising: administering to the human subject about 10mg/kg to about 50mg/kg body weight of a bile acid or a pharmaceutically acceptable salt thereof, and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound, thereby preventing or reducing at least one serious adverse event in the human subject. In some embodiments, the at least one serious adverse event is a respiratory adverse event, a fall, or a laceration.

In another aspect, provided herein is a method of reducing, maintaining, or improving fine motor skills in a human subject having one or more symptoms of ALS, the method comprising: administering to the human subject about 10mg/kg to about 50mg/kg body weight of a bile acid or a pharmaceutically acceptable salt thereof, and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound, thereby reducing, maintaining, or improving fine motor skill deterioration in the human subject. In some embodiments, the fine motor skills are assessed using ALSFRS-R.

In another aspect, provided herein is a method of slowing progression of ALS disease in a human subject having one or more symptoms of ALS, the method comprising: administering to the subject about 10mg/kg to about 50mg/kg body weight of a bile acid or a pharmaceutically acceptable salt thereof, and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound, thereby slowing progression of ALS disease in the human subject.

In another aspect, provided herein is a method of increasing survival time in a human subject having one or more symptoms of ALS, the method comprising: administering to the subject about 10mg/kg to about 50mg/kg body weight of a bile acid or a pharmaceutically acceptable salt thereof, and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound, thereby increasing the survival time of the human subject.

In another aspect, provided herein is a method of treating at least one symptom of bulbar ALS in a human subject, the method comprising administering to the subject about 10mg/kg to about 50mg/kg body weight of a bile acid or pharmaceutically acceptable salt thereof, and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound, thereby treating at least one symptom of bulbar ALS in the human subject.

In another aspect, provided herein is a method of treating at least one symptom of Benign Fasciculation Syndrome (BFS) or spasm-fasciculation syndrome (CFS) in a human subject, the method comprising: administering to a human subject diagnosed with BFS or CFS about 10mg/kg to about 50mg/kg body weight of a bile acid or a pharmaceutically acceptable salt thereof, and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound, thereby treating at least one symptom of BFS or CFS in the human subject.

In another aspect, provided herein are methods comprising: administering to a human subject at risk of developing ALS about 10mg/kg to about 50mg/kg body weight of a bile acid or a pharmaceutically acceptable salt thereof and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound, thereby preventing or delaying the onset of ALS. In some embodiments, the subject is determined to be at risk of developing ALS by evaluating the level of a biomarker in a biological sample obtained from the subject. In some embodiments, the biomarker is pNF-H, a neurofilament light chain, S100-beta, cystatin C, chitotriosidase, p75ECD, a ketone, or creatinine. The biological sample is CSF, urine or blood. In some embodiments, the subject is determined to be at risk of developing ALS by identifying a mutation in one or more genes selected from the group consisting of: OD1, C9ORF72, ANG, TARDBP, VCP, VAPB, SQSTM1, DCTN1, FUS, UNC13A, ATXN2, HNRNPA1, CHCHCHD 10, MOBP, C21ORF2, NEK1, TUBA4A, TBK1, MATR3, PFN1, UBQLN2, TAF15, OPTN and TDP-43.

In some embodiments of any of the methods described herein, the bile acid is Tauroglycol (TURSO), ursodeoxycholic acid (UDCA), chenodeoxycholic acid, cholic acid, hyodeoxycholic acid, lithocholic acid, or glycoursodeoxycholic acid. In some embodiments of any of the methods described herein, the phenylbutyrate compound is 4-phenylbutyric acid (4-PBA), glycerol tris- (4-phenylbutyrate), phenylacetic acid, 2- (4-methoxyphenoxy) acetic acid (2-POAA-OMe), 2- (4-nitrophenoxy) acetic acid (2-POAA-NO2), 2- (2-naphthyloxy) acetic acid (2-NOAA), or a pharmaceutically acceptable salt thereof. In some embodiments of any of the methods described herein, the method comprises administering to the human subject about 10mg/kg to about 30mg/kg body weight of the bile acid. In some embodiments of any of the methods described herein, the method comprises administering to the human subject about 10mg/kg to about 100mg/kg body weight of the phenylbutyrate compound. In some embodiments of any of the methods described herein, the method comprises administering to the human subject about 30mg/kg to about 100mg/kg body weight of the phenylbutyrate compound. In some embodiments of any of the methods described herein, the bile acid and the phenylbutyrate compound are administered separately. In some embodiments of any of the methods described herein, the bile acid and the phenylbutyrate compound are administered concurrently. In some embodiments of any of the methods described herein, the bile acid and the phenylbutyrate compound are administered daily. In some embodiments of any of the methods described herein, the bile acid and the phenylbutyrate compound are administered once daily, twice daily, or three times daily. In some embodiments of any of the methods described herein, the bile acid and the phenylbutyrate compound are administered once daily for 60 days or less. In some embodiments of any of the methods described herein, the bile acid and the phenylbutyrate compound are administered once daily for 30 days or less. In some embodiments of any of the methods described herein, the bile acid and the phenylbutyrate compound are administered twice daily for 60 days or less. In some embodiments of any of the methods described herein, the bile acid and the phenylbutyrate compound are administered twice daily for 30 days or less. In some embodiments of any of the methods described herein, the bile acid and the phenylbutyrate compound are administered twice daily for 60 days or more. In some embodiments of any of the methods described herein, the bile acid and the phenylbutyrate compound are administered twice daily for 120 days or more. In some embodiments of any of the methods described herein, the bile acid and the phenylbutyrate compound are administered once daily for at least 14 days, followed by two times daily for at least 30 days. In some embodiments of any of the methods described herein, the bile acid and the phenylbutyrate compound are administered once daily for about 21 days, followed by twice daily for at least 30 days.

In some embodiments of any of the methods described herein, the bile acid and the phenylbutyrate compound are administered orally. In some embodiments of any of the methods described herein, the bile acid and the phenylbutyrate compound are administered via a feeding tube. In some embodiments of any of the methods described herein, the bile acid and the phenylbutyrate compound are administered by bolus injection. In some embodiments of any of the methods described herein, each of the bile acid and the phenylbutyrate compound is formulated as a solution. In some embodiments of any of the methods described herein, the bile acid and the phenylbutyrate compound are formulated in a single solution. In some embodiments of any of the methods described herein, the bile acid and the phenylbutyrate compound are formulated as a powder. In some embodiments of any of the methods described herein, the bile acid and the phenylbutyrate compound are formulated as a single powder formulation. In some embodiments of any of the methods described herein, the bile acid is TURSO. In some embodiments of any of the methods described herein, the TURSO is administered in an amount of about 0.5 to about 5 grams per day. In some embodiments of any of the methods described herein, the TURSO is administered in an amount of about 1.5 to about 2.5 grams per day. In some embodiments of any of the methods described herein, the TURSO is administered in an amount of about 1 gram twice daily. In some embodiments of any of the methods described herein, the phenylbutyrate compound is a pharmaceutically acceptable salt of 4-PBA. In some embodiments of any of the methods described herein, the pharmaceutically acceptable salt of 4-PBA is sodium phenylbutyrate. In some embodiments of any of the methods described herein, the sodium phenylbutyrate is administered in an amount of about 0.5 to about 10 grams per day. In some embodiments of any of the methods described herein, the sodium phenylbutyrate is administered in an amount of about 4.5 to about 8.5 grams per day. In some embodiments of any of the methods described herein, the sodium phenylbutyrate is administered in an amount of about 3 grams twice daily.

In some embodiments of any of the methods described herein, the method further comprises administering to the human subject one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents are selected from the group consisting of: combination of dextromethorphan (dextromethorphan) and quinidine (quinidine), riluzole, edaravone, mexiletine (mexilitine), anticholinergic drugs (anticholinergic drugs), and psychiatric drugs (psychotropic drugs). In some embodiments, the one or more additional therapeutic agents is riluzole. In some embodiments, the one or more additional therapeutic agents is edaravone. In some embodiments, the human subject has been previously treated with one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent is riluzole. In some embodiments, the human subject has been previously treated with riluzole for at least 30 days. In some embodiments, the additional therapeutic agent is edaravone. In some embodiments, the human subject has been previously treated with edaravone for at least 30 days. In some embodiments, the additional therapeutic agent is mexiletine. In some embodiments, the human subject has been previously treated with mexiletine at a dose less than or equal to 300 mg/day. In some embodiments of any of the methods described herein, the method further comprises administering to the human subject a plurality of food products comprising a solid food or a liquid food. In some embodiments, the human subject is about 18 years of age or older. In some embodiments, the human subject is about 18 to about 50 years old. In some embodiments, the subject is about 18 to about 40 years old.

In another aspect, provided herein is a method of treating at least one symptom of ALS or preventing the onset of ALS in a human subject, the method comprising administering to the human subject an effective amount of (a) a bile acid or a pharmaceutically acceptable salt thereof; (b) a phenylbutyrate compound; (c) riluzole; and (d) edaravone, thereby treating at least one symptom of ALS or preventing the onset of ALS in the human subject.

In another aspect, provided herein is a method of treating at least one symptom of ALS or preventing the onset of ALS in a human subject, the method comprising administering TURSO and sodium phenylbutyrate to the human subject according to a first regimen followed by a second regimen, wherein the first regimen comprises administering about 1 gram of TURSO once per day for at least 14 days and about 3 grams of sodium phenylbutyrate once per day, and the second regimen comprises administering about 1 gram of TURSO twice per day for at least 30 days and about 3 grams of sodium phenylbutyrate twice per day.

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

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

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

Drawings

Figure 1A shows treatment-dependent decline rates in terms of overall scores for ALSFRS-R estimated in an improved intent-to-treat (mITT) population in a preliminary analysis.

Figure 1B shows the treatment-dependent decline rate in the overall score of ALSFRS-R estimated in the drug-administered population in the preliminary analysis.

Figure 2 shows an overview of a clinical trial study.

FIG. 3 is a graphical and tabular summary of primary and secondary observation (outgome) results.

Figure 4 shows results from post hoc analysis of all successive observations in the mITT population.

Fig. 5 shows the results from the sensitivity analysis.

FIG. 6 shows the results for the various subfields of the ALSFRS-R.

Figure 7A shows the treatment-dependent decline rate in total ATLIS score in the mITT population.

Figure 7B shows the treatment-dependent decline rate in the mITT population at higher ATLIS scores.

Figure 7C shows the treatment-dependent decline rate in the mITT population at lower ATLIS scores.

Fig. 7D shows treatment-dependent decline rates in SVC in the mITT population.

FIG. 8 is a Kaplan-Meier plot of cumulative death, tracheostomy, and hospitalization events.

Fig. 9 is a graph showing the incidence of gastrointestinal adverse events by trial week.

FIG. 10 is a graph showing results from long-term survival analysis.

Detailed Description

Although the exact cause of ALS is not clear, ALS is strongly characterized by neuronal cell death and inflammation. These processes together form a toxic cycle that is a key driver of progressive neurological decline. The present disclosure provides methods of treating at least one symptom of ALS, methods of reducing progression of ALS disease; and methods of reducing deterioration, maintaining, or improving one or more bodily functions affected by ALS. Also provided are methods of preventing or reducing at least one serious adverse event associated with ALS or treatment thereof, and methods of increasing survival in a human subject having one or more symptoms of ALS. The methods described herein may also be used to treat or prevent, for example, constipation, or to ameliorate at least one symptom of Benign Fasciculation Syndrome (BFS) or spastic fasciculation syndrome (CFS). These methods comprise administering a bile acid or pharmaceutically acceptable salt thereof and a phenylbutyrate compound.

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

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

Certain ranges are presented herein as numerical values prefixed by the term "about". The term "about" is used herein to provide literal support for the exact number following it, as well as numbers that are near or approximate to the number following the term. In determining whether a number is near or approximate to a specifically enumerated number, a near or approximate noneenumerated number may be a number that provides a substantial equivalent of the specifically enumerated number in the context of its presentation.

Unless defined otherwise, all technical terms, symbols, and other scientific terms or words used herein are intended to have the meanings commonly understood by those of skill in the art to which this application belongs. In some instances, terms with commonly understood meanings are defined herein for clarity and/or for ease of reference, and such definitions contained herein should not necessarily be construed to represent substantial differences from the definitions generally understood in the art.

I. Composition comprising a metal oxide and a metal oxide

The present disclosure provides methods of treating at least one symptom of ALS in a human subject. Also provided herein are methods of slowing progression of an ALS disease (e.g., reducing the rate of progression of an ALS disease); and methods of reducing deterioration in muscle strength, respiratory muscle function or fine motor skills associated with ALS, and methods of maintaining and improving such functions and skills. The present disclosure also provides methods of preventing or reducing at least one serious adverse event associated with ALS or treatment thereof, and methods of increasing survival in a human subject having one or more symptoms of ALS. Also provided are methods of treating or preventing constipation, such as constipation associated with ALS, and methods of treating or preventing at least one symptom of Benign Fasciculation Syndrome (BFS) and/or spasm-fasciculation syndrome (CFS) in a human subject. Any of the methods described herein can include administering to the subject a bile acid or a pharmaceutically acceptable salt thereof (e.g., any bile acid or pharmaceutically acceptable salt thereof described herein or known in the art) and a phenylbutyrate compound (e.g., any phenylbutyrate compound described herein or known in the art).

As used herein, "bile acid" refers to a naturally occurring surfactant having a sterol nucleus derived from a sterol substituted by a 3 α -hydroxy group and optionally also substituted by other hydroxy groups (typically at the C6, C7 or C12 position of the sterol nucleus). Bile acid derivatives (e.g., water-soluble bile acid derivatives) and bile acids conjugated with amines are also encompassed by the term "bile acids". Bile acid derivatives include, but are not limited to, derivatives formed with other functional groups (including, but not limited to, halogen and amino groups) at the hydroxyl and carboxylic acid groups of the bile acid. The soluble bile acid may include an aqueous formulation of the free acid form of the bile acid in combination with one of HCl, phosphoric acid, citric acid, acetic acid, ammonia, or arginine. Suitable bile acids include, but are not limited to, Tauroglycol (TURSO), ursodeoxycholic acid (UDCA), chenodeoxycholic acid (also known as "chenediol" or "chenolic 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 analogs, derivatives, or prodrugs thereof.

In some embodiments, the bile acid of the present disclosure is a hydrophilic bile acid including, but not limited to, TURSO, UDCA, chenodeoxycholic acid, cholic acid, hyodeoxycholic acid, lithocholic acid, and glycoursodeoxycholic acid. Also contemplated are pharmaceutically acceptable salts or solvates of any bile acid disclosed herein. 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 trialkylamines, dicyclohexylamine; tributylamine; pyridine; n-methyl, N-ethylamine; diethylamine; triethylamine; mono-, di-or tri- (2-OH- (C1-C6) -alkylamine), such as N, N-dimethyl-N- (2-hydroxyethyl) amine or tri- (2-hydroxyethyl) amine; N-methyl-D-glucamine; morpholine; thiomorpholine; piperidine; a pyrrolidine; and amino acids such as arginine, lysine, and the like.

The terms "tauroursodeoxycholic acid" (TUDCA) and "tauroglycol" (TURSO) are used interchangeably herein.

The bile acid described herein may be TURSO (carbon labeled to aid in understanding where substitutions may be made) as shown in formula I.

The bile acid described herein may be UDCA (carbon labelled to aid in understanding where substitutions may be made) as shown in formula II.

Physiologically relevant bile acid derivatives are also contemplated herein. For example, any combination of substitution of hydrogen at the 3 or 7 position, stereochemical change of hydroxyl at the 3 or 7 position in the formula of TURSO or UDCA is suitable for use in the present compositions.

The "bile acid" may also be a bile acid conjugated with an amino acid. The amino acid in the conjugate can be, but is not limited to, taurine, glycine, glutamine, asparagine, methionine, or carboxycysteine. Other amino acids that may be conjugated to 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 beta-alanine and gamma-aminobutyric acid. One example of such a bile acid is a compound of formula III:

wherein

R is-H or C ₁ -C ₄ An alkyl group;

R ₁ is-CH ₂ -SO ₃ R ₃ 、CH ₂ COOH or CH ₂ CH ₂ COOH, and R ₂ is-H;

or R ₁ is-COOH and R ₂ is-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)NH ₂ 、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 group), CH ₂ (4-OH-phenyl) or-CH ₂ -S-CH ₂ -COOH; and is

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

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

wherein

R is-H or C ₁ -C ₄ An alkyl group;

R ₁ is-CH ₂ -SO ₃ R ₃ And R2 is-H;

or R ₁ is-COOH and R ₂ is-CH ₂ -CH ₂ -CONH ₂ 、-CH ₂ -CONH ₂ 、-CH ₂ -CH ₂ -SCH ₃ or-CH ₂ -S-CH ₂ -COOH; and is

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.

Taurine diol (TURSO)

TURSO is an amphiphilic bile acid and is a taurine conjugate form of UDCA. TURSO restores mitochondrial bioenergy deficiencies by incorporation into the mitochondrial membrane, reducing Bax translocation to the mitochondrial membrane, decreasing mitochondrial permeability, and increasing the apoptotic threshold of the cell (Rodrigues et al, Biochemistry 42, 10: 3070-3080, 2003). It is used to treat cholesterol gallstones, where long term treatment (e.g., 1 to 2 years) is typically required to obtain complete dissolution. It has been used to treat cholestatic liver diseases, including primary cirrhosis, pediatric familial intrahepatic cholestasis, as well as primary sclerosing cholangitis and cholestasis due to cystic fibrosis.

TURSO is contraindicated in subjects with biliary tract infections, frequent biliary colic, or subjects with difficulty in absorbing bile acids (e.g., ileal disease or resection). Known or theoretical drug interactions include with substances that inhibit bile acid absorption (e.g., cholestyramine) and with drugs that increase cholesterol elimination in bile (TURSO reduces bile cholesterol levels). Based on similar physicochemical properties, there is similarity between TURSO and UDCA in terms of drug toxicity and interaction. The most common adverse reactions reported using TURSO (. gtoreq.1%) were: abdominal discomfort, abdominal pain, diarrhea, nausea, itching and rash. There were some cases of pruritus and a limited number of cases of elevated liver enzymes.

UDCA

Ursodeoxycholic acid (UDCA) or bear diol, which is widely used for the treatment of gallstones, is endogenously produced and secreted by the liver as Taurine (TURSO) or Glycine (GUDCA) conjugates. Taurine conjugation increases the solubility of UDCA by making it more hydrophilic. TURSO is absorbed in the distal ileum in case of active transport and thus may have a slightly longer residence time in the intestine than UDCA absorbed more proximally in the ileum.

The treatment with ursodiol was not associated with liver damage. Lithocholic acid, a naturally occurring bile acid, is known to be a hepatotoxic metabolite. This bile acid is formed by the ursodiol in the gut in less effective and less amounts than seen from the goose diol. Previous studies have found that lithocholic acid is detoxified in the liver by sulfation, and while it may appear to be an effective sulfate-forming agent, it is likely that some subjects may be deficient in sulfation either congenital or acquired, thereby making them susceptible to lithocholate-induced liver injury. Liver enzyme abnormality Often harmonize with(ursodiol USP capsule) treatment is irrelevant, and in fact,it has been shown to reduce liver enzyme levels in liver disease. However, administration isThe subject of (a) should have sgot (ast) and sgpt (alt) measured at the beginning of treatment, then as indicated by the particular clinical situation. Ursodeoxycholic acid was tested in the previous 2 year oral carcinogenicity study in CD-1 mice and Sprague-Dawley rats at daily doses of 50, 250 and 1000 mg/kg/day. It is not tumorigenic in mice. In the rat study, it produced a statistically significant dose-related increased incidence of pheochromocytomas of the adrenal medulla in males (p ═ 0.014, Peto trend test) and females (p ═ 0.004, Peto trend test). A previous 78-week rat study using intrarectal infusion of lithocholic acid and metabolites of tauro-deoxycholic acid, ursodiol and chenediol has been performed. These bile acids alone do not produce any tumor. When both metabolites are co-administered with a carcinogen, tumor promoting effects of both of them are observed. The ursodiol was not mutagenic in the Amm's test (Ames test).

Previous studies have shown that bile acid sequestrants such as cholestyramine and colestipol (colestipol) can interfere with the action of bear diol by reducing its absorption. Aluminum-based antacids have been shown to adsorb bile acids in vitro and can be expected to interfere with ursolic glycol in the same way as bile acid sequestrants. Estrogens, oral contraceptives and clofibrate (and possibly other lipid lowering drugs) increase liver cholesterol secretion and promote the formation of cholesterol gallstones, and thus may counteract the effects of koala.

Phenylbutyrate compound (also referred to as phenylbutyric acid-based compound)

Phenylbutyrate compounds are defined herein to encompass phenylbutyrate, 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, co-crystals, polymorphs, hydrates, solvates, conjugates, derivatives, or prodrugs thereof. The phenylbutyrate compounds described herein also encompass analogs of 4-PBA, including, but not limited to, glycerol tri- (4-phenylbutyrate), phenylacetic acid (which is the active metabolite of PBA), 2- (4-methoxyphenoxy) acetic acid (2-POAA-OMe), 2- (4-nitrophenoxy) acetic acid (2-POAA-NO2), and 2- (2-naphthyloxy) acetic acid (2-NOAA), and 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 structure of 4-PBA. Other HDAC2 inhibitors are contemplated herein as alternatives for phenylbutyrate compounds.

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

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

phenylbutyrate is a pan-HDAC inhibitor and can ameliorate ER stress by upregulating the primary partner modulator DJ-1 and by recruiting other chaperones (see, e.g., Zhou et al, J Biol chem.286: 14941-. A large increase in chaperone production reduces activation of canonical ER stress pathways, folding misfolded proteins, and has been shown to increase survival in vivo models, including the G93A SOD1 mouse model of ALS (see, e.g., Ryu, H et al, J neurohem.93: 1087-.

A safety profile for phenylbutyrate administration is derived in large part from studies on subjects suffering from urea cycle disorders.Details of the safety profile may be in the phenylbutyrate tablet labelFound above. In female subjects, the most common clinical adverse event reported was amenorrhea/menstrual dysfunction (irregular menstrual cycle), which occurred in 23% of menstrual subjects. Appetite reduction occurred in 4% of all subjects. Each of 3% of the subjects reported body odor (probably from the metabolite phenylacetate [ PAA ] ]Cause) and off-taste or off-taste.

Other adverse events reported in 2% or less of subjects were:

gastrointestinal tract: abdominal pain, gastritis, nausea and vomiting; constipation, rectal bleeding, peptic ulcer disease and pancreatitis, respectively, occurred in one subject.

Hematology: aplastic anemia and ecchymosis, each occurred in one subject.

Cardiovascular: arrhythmia and edema, each occurred in one subject.

Renal: renal tubular acidosis

Psychiatry: depression

Skin: rash

Others: headache, fainting and weight gain

Phenyl butyrate has been evaluated in dose escalation studies in ALS subjects over 20 cycles and found to be generally safe and tolerable (see, e.g., Cudkowicz et al, Amyotrophic dental Sclerosis. 10: 2, 99-106, 2009). This study evaluated daily doses of 9 to 21 grams of phenylbutyrate. In particular, the most common adverse events include falls or other accidental injuries, dizziness, diarrhea, edema, dry mouth, headache, nausea, and rash. In addition to headache, the incidence of these adverse events was higher compared to the control placebo group. There were no clinically significant changes in laboratory values, EKG, or vital signs. No mortality or related serious adverse events occurred. Subjects taking riluzole in addition to phenylbutyrate did not experience significant adverse events more frequently than subjects taking phenylbutyrate alone. Neurotoxicity was reported in cancer subjects receiving intravenous phenylacetate (250-300 mg/kg/day for 14 days, repeated at 4-week intervals). Mainly manifested as somnolence, fatigue and dizziness; has low frequency of headache, dysgeusia, hearing loss, disorientation, impaired memory and deterioration of original neuropathy. The severity of these adverse events is primarily mild. The acute onset and reversibility when phenylacetate infusion was stopped indicated drug action.

In some embodiments, the combination of a bile acid (e.g., TURSO) or a pharmaceutically acceptable salt thereof and a phenylbutyrate compound (e.g., sodium phenylbutyrate), for example, when administered in a specific ratio (e.g., any ratio described herein), has synergistic efficacy in treating one or more symptoms associated with a neurodegenerative disease (e.g., ALS). For example, by simultaneously inhibiting endoplasmic reticulum stress and mitochondrial stress, by linear modeling (see, e.g., U.S. Pat. No. 9,872,865 and U.S. Pat. No. 10,251,896) in a strong oxidative damage model (H) ₂ O ₂ -mediated toxicity), the combination can induce a mathematical synergistic increase in neuronal viability.

Diagnosis and subject selection

In one aspect, provided herein is a method of treating at least one symptom of ALS in a human subject. Also provided are methods of reducing the rate of progression of ALS disease; a method of improving, maintaining or slowing ALS-associated deterioration in muscle strength, respiratory muscle function or fine motor skills; methods of preventing or reducing severe adverse events associated with ALS or treatment thereof; and methods of increasing survival in a human subject having one or more symptoms of ALS. Also provided herein are methods of treating or preventing constipation, such as constipation associated with ALS, and methods of treating or preventing at least one symptom of Benign Fasciculation Syndrome (BFS) or spasm-fasciculation syndrome (CFS) in a human subject.

Any human 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, and/or at risk of developing, ALS.

Some embodiments of any of the methods described herein may further comprise determining that the human subject has, or is at risk of developing, ALS, diagnosing the human subject as having, or at risk of developing, ALS, or selecting a human subject having, or at risk of developing, ALS. Likewise, some embodiments of any of the methods described herein may further comprise determining that the human subject has or is at risk of developing BFS or CFS, diagnosing the human subject as having or at risk of developing BFS or CFS, or selecting the human subject as having or at risk of developing BFS or CFS.

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

The order and type of ALS symptoms exhibited by a subject may depend on which motor neurons in the body are first injured, and thus which muscles in the body are first injured. For example, ALS with bulbar attacks, limb attacks, or respiratory attacks may present similar or different symptoms. Generally, ALS symptoms may include muscle weakness or atrophy (e.g., affecting the upper body, lower body, and/or speech), muscle fasciculations (twitching), cramping, or affected muscle stiffness. Early symptoms of ALS may include those in the arms or legs, difficulty speaking clearly, or swallowing (e.g., in bulbar onset ALS). Other symptoms include loss of tongue mobility, difficulty breathing or lung dysfunction, difficulty chewing, and/or difficulty walking (e.g., causing a trip). The subject may have respiratory muscle weakness as the initial manifestation of symptoms of ALS. Such subjects may have a very poor prognosis, and in some cases, a median survival of about two months after diagnosis. In some subjects, the time of onset of respiratory muscle weakness can be used as a prognostic factor.

ALS symptoms can also be classified according to the portion of the neuronal system that degenerates (i.e., upper motor neurons or lower motor neurons). Lower motor neuron degeneration manifests itself, for example, as weakness or wasting in one or more of the medulla oblongata, cervical, thoracic, and/or lumbosacral regions. Upper motor neuron degeneration may include increased tendinosis, spasticity, pseudobulbar features, Hoffmann reflexes, extensor plantar responses, and hyperreflexes including hyperactive vomiting reflexes. The progression of neuronal degeneration or muscle weakness is a hallmark of the disease. Accordingly, some embodiments of the present disclosure provide a method of ameliorating at least one symptom of lower motor neuron degeneration, at least one symptom of upper motor neuron degeneration, or at least one symptom from each of lower motor neuron degeneration and upper motor neuron degeneration. In some embodiments of any of the methods described herein, the symptom onset can be determined based on information from the subject and/or a family member of the subject. In some embodiments, the median time from symptom onset to diagnosis is about 12 months.

In some cases, the human 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, a subject may have been diagnosed with ALS for 1 week or less, or diagnosed with ALS on the same day as administration of a treatment disclosed herein. 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 of diagnosing ALS are known in the art. For example, a subject can be diagnosed based on clinical history, family history, physical or neurological examination (e.g., signs of degeneration of lower or upper motor neurons). The subject may be identified or identified as having ALS, for example, by a healthcare professional. Multiple parties may be involved in the diagnostic process. For example, where a sample is obtained from a subject as part of a diagnosis, a first party may obtain the sample from the subject and a second party may test the sample. In some embodiments of any of the human subjects described herein, the subject is diagnosed, selected, or referred to by a medical practitioner (e.g., a general practitioner).

In some embodiments, the subject meets the El escoral criteria for likely or established ALS, i.e., the subject exhibits:

1. examination by clinical, electrophysiological, or neuropathology of signs of Lower Motor Neuron (LMN) degeneration;

2. by clinical examination, there was a sign of Upper Motor Neuron (UMN) degeneration; and

3. progressive propagation of signs (sign) within one region or to other regions, while not:

electrophysiological evidence of other disease processes that may explain signs of LMN and/or UMN degradation; and

neuroimaging evidence of other disease processes that might explain observed clinical and electrophysiological signs.

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

A. clinically clear ALS, defined only by the presence of UMN in three areas and clinical evidence of LMN signs.

B. Clinically likely ALS, is defined only by clinical evidence of UMN and LMN signs (some of which must be located above (above) the LMN signs) in at least two areas.

C. Clinically likely ALS-is supported by the laboratory, defined when clinical signs of UMN and LMN dysfunction are in one area only or when there are individual signs of UMN in one area and LMN signs defined by EMG standards in at least two limbs (with neuroimaging and clinical laboratory protocols applied appropriately to exclude other causes).

D. Clinically probable ALS, defined when clinical signs of both UMN and LMN dysfunction are found in only one area or when signs of UMN are found separately in two or more areas; or LMN signs were found to be superior to those of UMN and clinically likely diagnosis-defined when supported by the laboratory.

In some embodiments, the subject has clinically definite ALS (e.g., based on the El escolar criteria).

The subject can be evaluated and/or diagnosed using the revised amyotrophic lateral sclerosis functional rating scale (ALSFRS-R). ALSFRS-R is an ordinal rating scale (ratings 0-4) used to determine a subject's assessment of his ability and independence in 12 ALS-associated functional activities. The ALSFRS-R score calculated at diagnosis can be compared to the score over time to determine the rate of progression. Changes in the ALSFRS-R score may be correlated with changes in intensity over time, and may be correlated with quality of life indicators and predicted survival. ALSFRS-R exhibits a linear average slope and can be used as a prognostic indicator (see, e.g., Berry et al, Amyotrop Lateral Screwanterior temporal Degener 2014 (amyotrophic Lateral sclera Frontotemporal lobe degeneration 2014); 15: 1-8; Traynor et al, Neurology 63: 1933-

In ALSFRS-R, functions mediated by cervical, trunk, lumbosacral and respiratory muscles were evaluated by 3 items, respectively. Each scored from 0-4, with 4 reflecting no disease involvement and 0 reflecting maximal involvement. The term scores are added to give a total score. The total score reflects the impact of ALS, with the following exemplary classifications:

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

For example, the subject's ALSFRS-R score (e.g., 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 less (e.g., 21, 22, 23, 24, 25, 26, 27, 28, or 29). In some embodiments of any of the methods described herein, the subject has an ALSFRS-R score (e.g., a baseline ALSFRS-R score) of 40 or less (e.g., 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 or less). In some embodiments, the subject has an ALSFRS-R score (e.g., a baseline ALSFRS-R score) of 20 or less (e.g., 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or less).

Since ALS is a progressive disease, all patients will typically progress over time. However, there is a large degree of inter-subject variability in the rate of progression, as some subjects die within months or require respiratory support, while others have a relatively long survival. The subject described herein may have rapidly progressing ALS or slowly progressing ALS. The rate of functional decline in subjects with ALS can be measured by the change in the monthly ALSFRS-R score. For example, the score may decrease by about 1.02 (+ -2.3) points per month.

One predictor of patient progression is the patient's previous rate of disease progression (Δ FS), which can be calculated as: Δ FS ═ 48-ALSFRS-R score at assessment)/duration from development to time of assessment (month). The Δ FS score represents the number of ALSFRS-R scores lost monthly since symptom onset and can be an important predictor of progression and/or survival in subjects with ALS (see, e.g., Labra et al, J neuro Neurosurg Psychiatry 87: 628-. The subject may have had a rate of disease progression (Δ FS) of about 0.50 or less (e.g., about 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, or 0.10 or less); about 0.50 to about 1.20 (inclusive) (e.g., about 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00, 1.05, 1.10, or 1.15); or about 1.20 or more (e.g., about 1.25, 1.30, 1.35, 1.40, 1.45, 1.50, 1.55, 1.60, 1.75, 1.80, 1.85, 1.90, 1.95, or 2.00 or more). In some embodiments of any of the methods described herein, the subject may have a rate of progression of ALS disease (Δ FS) of 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). It should be noted, however, that the Δ FS score is a predictor of patient progression and, once evaluated, may underestimate or overestimate patient progression.

In some embodiments, the subject loses an ALSFRS-R score 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) on average per month within 3-12 months from the initial assessment. In some embodiments, the subject loses an ALSFRS-R score of more than about 1.2 on average per month within 3-12 months from initial evaluation. A subject may have at least a 3 point drop in ALSFRS-R score (e.g., at least 4, 6, 8, 10, 12, 14, 16, 20, 24, 28, or 32 points) within 3-12 months from initial assessment. In some embodiments, the subject has lost an ALSFRS-R score 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) on average per month over the previous 3-12 months. In some embodiments, the subject loses an ALSFRS-R score of more than about 1.2 (e.g., more than about 1.5, 1.8, 2.0, 2.5, or 3) on average per month over the previous 3-12 months.

In some embodiments of any of the methods described herein, a marker (e.g., the presence or level of a marker) in a sample obtained from a subject can be used for ALS diagnosis or prognosis, and for tracking disease activity and treatment response. Suitable samples include, for example, cell, tissue, bodily fluids such as blood, urine, and/or cerebrospinal fluid (CSF) samples. For example, the level of phosphorylated neurofilament protein heavy subunit (pNF-H) or neurofilament protein light chain (NfL) in CSF and/or blood can be used as a biomarker for ALS diagnosis, prognosis, or tracking disease activity or observation of treatment. pNF-H is a major component of the cytoskeleton of neurons and is released into the cerebrospinal fluid and blood stream in the event of neuronal injury. The level of pNF-H may be correlated with axonal loss levels and/or the burden of motor neuron dysfunction (see, e.g., De Schaepdryver et al, Journal of Neurology, Neurosurgery & Psychiatry 2018; 89: 367-.

In some embodiments, the concentration of pNF-H in the CSF and/or blood of a subject with ALS is significantly increased at an early stage of the disease. The higher pNF-H levels in plasma, serum and/or cerebrospinal fluid may be associated with faster ALS progression (e.g., a faster decline in ALSFRS-R) and/or shorter survival. The concentration of pNF-H in plasma may be higher in ALS subjects with bulbar seizures than those with spinal cord seizures. In some cases, an imbalance between the relative expression levels of the neurofilament protein heavy and light chain subunits may be used to diagnose ALS, prognose, or track disease progression.

pNF-H and NfL can be detected in cerebrospinal fluid, plasma and/or serum using methods known in the art, such as, but 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, Amyotoph latex Frontotemporal Degener 14 (amyotrophic Lateral sclera temporal lobe degeneration) 146 (2): 146-9, 2013; De Schaepdryver et al, Annals of Clinical and transmural neural identification 6 (Clinical and transmural annual identification) (1971:. 1979, 2019; Willer et al, Clin Chem Lab 57: 10) 1556, Poisson 1554, Neurot 2019; Neurot et al, Neurot 1987: 2018: (Neuro kouchi) 1037: 2018; Neuro et al, Pro 7: 1039; Neuro et al, Neuro Op # 1039, Neuro et al, Neuro. 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 produced by Quanterix, can also be used (see, e.g., Thouvenot et al, European Journal of Neurology 27: 251, 257, 2020). Factors that affect the levels of pNF-H and NfL or their detection in serum and/or plasma associated with the disease process may differ from those in CSF. The levels of neurofilament proteins (e.g., pNF-H and/or NfL) in CSF and serum may be related (see, e.g., Wilke et al, Clin Chem Lab Med57 (10): 1556-1564, 2019).

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

A subject of the present disclosure may have a CSF or blood level of NfL of about 50pg/mL or higher (e.g., about 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250pg/mL or higher). In some embodiments, the serum NfL level of the subject in the methods described herein can be about 50 to about 300pg/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 of a subject in the methods described herein may be about 2000 to about 40,000pg/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 6000, about 10,000, about 40,000, about 6000 to about 6000, about 6000 to about 8000, about 6000 to about 35,000, about 6000 to about 8000, about 30,000, about 6000 to about 10,000, about 8000, about 10,000, about 8000, about 10,000, about 10 to about 8000, about 10,000, about 6000 to about 8000, about 10,000, about 30,000, about 6000 to about 8000, about 30,000, about 8000, about 6000 to about 10,000, about 8000, about 10,000, about 30,000, about 10,000, about 6000 to about 10,000, about 30,000, about 8000, about 6000 to about 8000, about 30,000, about 10,000, about 30,000, about 8000, about 30,000, about 10,000, about 2000 to about 10,000, about 8000, about 10,000, about 30,000, about 2000 pg/mL, about 2000 to about 10,000, about 2000 pg/mL, about 2000 to about 2000 pg/mL, about 2000 to about 10,000, about 2000 to about 2000 pg/mL, about 10,000, about 2000 to about 10,000, about 2000, about 10,000, about 2000 to about 10,000, about 2000 to about 10,000, about 10,000, About 10,000 to about 25,000, about 10,000 to about 20,000, about 10,000 to about 15,000, about 15,000 to about 40,000, about 15,000 to about 35,000, about 15,000 to about 30,000, about 15,000 to about 25,000, about 15,000 to about 20,000, about 20,000 to about 40,000, about 20,000 to about 35,000, about 20,000 to about 30,000, about 20,000 to about 25,000, about 25,000 to about 40,000, about 25,000 to about 35,000, about 25,000 to about 30,000, about 30,000 to about 40,000, about 30,000 to about 35,000, or about 35,000 to about 40,000 pg/mL).

Additional biomarkers that may be useful in the diagnosis, prognosis, and monitoring of disease progression of ALS are contemplated herein, including, but not limited to, CSF levels of S100-beta, 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 biomarkers for the prognosis of ALS (see, e.g., Atassi et al, Neurology 83 (19): 1719-1725, 2014). Akt phosphorylation can also be used as a biomarker for prognosis of ALS (see, e.g., WO 2012/160563). In some embodiments, the urine levels of p75ECD and ketones can be used as biomarkers for ALS diagnosis (see, e.g., shepherd et al, Neurology 88: 1137-. Serum and urine levels of creatinine may also be used as biomarkers. Other blood, CSF, neurophysiological and neuroradiological biomarkers useful for ALS are described, for example, in Turner et al, Lancet Neurol 8: 94-109, 2009. Any of the markers described herein can be used to diagnose a subject as having ALS, or to determine that a subject is at risk of developing ALS.

Based on the genetic analysis, the subject may also be identified as having ALS or as being at risk of developing ALS. Genetic variants associated with ALS are known in the art (see, e.g., Taylor et Al, Nature 539: 197-. In some embodiments of any of the methods described herein, the subject may carry a mutation 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 description of ALS-associated genes 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 progression of ALS in a subject, the pharmacokinetics of the compound administered in the subject, and/or the efficacy of the administered compound in the subject.

The subject may have a mutation in the gene encoding CuZn-superoxide dismutase (SOD 1). Mutations result in the SOD1 protein aggregating more readily, resulting in the deposition of cellular contents 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 inherited ALS, many of which result in single amino acid substitutions in proteins. In some embodiments, the SOD1 mutation is A4V (i.e., a valine substituted for alanine at position 4). SOD1 mutations are described, for example, in Rosen et al, hum.mol.Genet.3, 981-: 59-62, 1993. In some embodiments, the subject has a mutation in the C9ORF72 gene. Repeated amplification in the C9ORF72 gene is a common cause of ALS, with loss of function of C9ORF72 and acquisition of repeated toxic function both implicated in ALS (see, e.g., Balendra and Isaacs, Nature Reviews Neurology 14: 544-558, 2018). The methods described herein may include detecting a SOD1 mutation and/or a C9ORF72 mutation in the subject prior to administration of the bile acid and the phenylbutyrate compound. Methods for screening for mutations are well known in the art. Suitable methods include, but are not limited to, gene sequencing. See, e.g., Hou et al, Scientific Reports 6: 32478, 2016; and Vajda et al, Neurology 88: 1-9, 2017.

The skilled practitioner will appreciate that certain factors may influence the bioavailability and metabolism of the compound administered to the subject, and may be adjusted accordingly. These include, but are not limited to, liver function (e.g., liver enzyme levels), kidney function, and gallbladder function (e.g., ion absorption and secretion, cholesterol transporter levels). The level of exposure, level of excretion, and pharmacokinetics of the compound in the treated subjects may vary from subject to subject of the administered compound (e.g., bile acid and phenylbutyrate compounds). Any of the factors described herein may affect the drug exposure of a subject. For example, decreased clearance of a compound may lead to increased drug exposure, while improved kidney 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 the treatment.

The subject may be, e.g., greater than 18 years old (e.g., 18-100, 18-90, 18-80, 18-70, 18-60, 18-50, 18-40, 18-30, 18-25, 25-100, 25-90, 25-80, 25-70, 25-60, 25-50, 25-40, 25-30, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-100, 50-90, 50-80, 50-70, 50-60, 60-100, 60-90, 60-80, 60-70, 70-100, 70-90, 40-50, 50-100, 50-90, 50-80, 50-70-60, 60-90, 60-80, 60-70, 70-100, 70-90, etc, 70-80, 80-100, 80-90, or 90-100 years old). The subject's BMI may be 18.5-30kg/m ² (e.g., 18.5-28, 18.5-26, 18.5-24, 18.5-22, 18.5-20, 20-30, 20-28, 20-26, 20-24, 20-22, 22-30, 22-28, 22-26, 22-24, 24-30, 24-28, 24-26, 26-30, 26-28, or 28-30kg/m ² ). Having a mutation in or exhibiting any of the ALS-associated genes described hereinAny biomarker may indicate that the subject is at risk of developing ALS. Such subjects can be treated with the methods provided herein for prophylactic and preventative purposes.

In some embodiments, the subject has one or more symptoms of Benign Fasciculation Syndrome (BFS) and/or spasm-fasciculation syndrome (CFS). BFS and CFS are peripheral hyperexcitable disorders and can cause fasciculations, spasticity, pain, fatigue, muscle stiffness and paresthesia. Methods of identifying subjects suffering from these conditions are known in the art, such as by clinical examination and electromyography.

Methods of treatment

The present disclosure provides methods of treating ALS in a subject, or ameliorating at least one symptom of ALS in a subject, or prophylactically treating a subject at risk of developing ALS (e.g., a subject with a family history of ALS) or suspected to be developing ALS (e.g., a subject exhibiting at least one symptom of ALS, a symptom of upper motor neuron degeneration, and/or a symptom of lower motor neuron degeneration, but at the time the symptoms are insufficient to support a comprehensive diagnosis of ALS).

Also provided are methods of ameliorating at least one symptom of lower motor neuron degeneration, at least one symptom of upper motor neuron degeneration, or at least one symptom from each of lower motor neuron degeneration and upper motor neuron degeneration in a subject.

Some embodiments of the present disclosure provide methods of slowing progression of ALS disease (e.g., reducing the rate of progression of ALS disease); and methods of reducing deterioration in muscle strength, respiratory muscle/lung function and/or fine motor skills, and methods of maintaining or improving muscle strength, respiratory muscle/lung function and/or fine motor skills.

Also provided herein are methods of preventing or reducing constipation, such as constipation associated with ALS; methods of preventing or reducing at least one adverse event (e.g., a severe adverse event) associated with ALS or treatment thereof; and methods of increasing survival in a human subject having one or more symptoms of ALS.

The present disclosure further provides methods of treating at least one symptom of bulbar attack ALS in a human subject. Also provided are methods of ameliorating at least one symptom of benign fasciculation syndrome or spastic fasciculation syndrome.

In some embodiments of any of the methods described herein, the method comprises administering to the subject a bile acid or a pharmaceutically acceptable salt thereof, and a phenylbutyrate compound. In some embodiments, the methods described herein comprise administering to the subject about 10mg/kg to about 50mg/kg (e.g., about 10mg/kg to about 48mg/kg, about 10mg/kg to about 46mg/kg, about 10mg/kg to about 44mg/kg, about 10mg/kg to about 42mg/kg, about 10mg/kg to about 40mg/kg, about 10mg/kg to about 38mg/kg, about 10mg/kg to about 36mg/kg, about 10mg/kg to about 34mg/kg, about 10mg/kg to about 32mg/kg, about 10mg/kg to about 30mg/kg, about 10mg/kg to about 28mg/kg, about 10mg/kg to about 26mg/kg, about 10mg/kg to about 24mg/kg, about 10mg/kg to about 22mg/kg, about 10mg/kg to about 20mg/kg, about 10mg/kg to about 18mg/kg, about 10mg/kg to about 16mg/kg, about 10mg/kg to about 14mg/kg, about 10mg/kg to about 12mg/kg, about 12mg/kg to about 50mg/kg, about 12mg/kg to about 48mg/kg, about 12mg/kg to about 46mg/kg, about 12mg/kg to about 44mg/kg, about 12mg/kg to about 42mg/kg, about 12mg/kg to about 40mg/kg, about 12mg/kg to about 38mg/kg, about 12mg/kg to about 36mg/kg, about 12mg/kg to about 34mg/kg, about 12mg/kg to about 32mg/kg, about 12mg/kg to about 30mg/kg, about 12mg/kg to about 28mg/kg, About 12mg/kg to about 26mg/kg, about 12mg/kg to about 24mg/kg, about 12mg/kg to about 22mg/kg, about 12mg/kg to about 20mg/kg, about 12mg/kg to about 18mg/kg, about 12mg/kg to about 16mg/kg, about 12mg/kg to about 14mg/kg, about 14mg/kg to about 50mg/kg, about 14mg/kg to about 48mg/kg, about 14mg/kg to about 46mg/kg, about 14mg/kg to about 44mg/kg, about 14mg/kg to about 42mg/kg, about 14mg/kg to about 40mg/kg, about 14mg/kg to about 38mg/kg, about 14mg/kg to about 36mg/kg, about 14mg/kg to about 34mg/kg, about 14mg/kg to about 32mg/kg, About 14mg/kg to about 30mg/kg, about 14mg/kg to about 28mg/kg, about 14mg/kg to about 26mg/kg, about 14mg/kg to about 24mg/kg, about 14mg/kg to about 22mg/kg, about 14mg/kg to about 20mg/kg, about 14mg/kg to about 18mg/kg, about 14mg/kg to about 16mg/kg, about 16mg/kg to about 50mg/kg, about 16mg/kg to about 48mg/kg, about 16mg/kg to about 46mg/kg, about 16mg/kg to about 44mg/kg, about 16mg/kg to about 42mg/kg, about 16mg/kg to about 40mg/kg, about 16mg/kg to about 38mg/kg, about 16mg/kg to about 36mg/kg, about 16mg/kg to about 34mg/kg, About 16mg/kg to about 32mg/kg, about 16mg/kg to about 30mg/kg, about 16mg/kg to about 28mg/kg, about 16mg/kg to about 26mg/kg, about 16mg/kg to about 24mg/kg, about 16mg/kg to about 22mg/kg, about 16mg/kg to about 20mg/kg, about 16mg/kg to about 18mg/kg, about 18mg/kg to about 50mg/kg, about 18mg/kg to about 48mg/kg, about 18mg/kg to about 46mg/kg, about 18mg/kg to about 44mg/kg, about 18mg/kg to about 42mg/kg, about 18mg/kg to about 40mg/kg, about 18mg/kg to about 38mg/kg, about 18mg/kg to about 36mg/kg, about 18mg/kg to about 34mg/kg, About 18mg/kg to about 32mg/kg, about 18mg/kg to about 30mg/kg, about 18mg/kg to about 28mg/kg, about 18mg/kg to about 26mg/kg, about 18mg/kg to about 24mg/kg, about 18mg/kg to about 22mg/kg, about 18mg/kg to about 20mg/kg, about 20mg/kg to about 50mg/kg, about 20mg/kg to about 48mg/kg, about 20mg/kg to about 46mg/kg, about 20mg/kg to about 44mg/kg, about 20mg/kg to about 42mg/kg, about 20mg/kg to about 40mg/kg, about 20mg/kg to about 38mg/kg, about 20mg/kg to about 36mg/kg, about 20mg/kg to about 34mg/kg, about 20mg/kg to about 32mg/kg, About 20mg/kg to about 30mg/kg, about 20mg/kg to about 28mg/kg, about 20mg/kg to about 26mg/kg, about 20mg/kg to about 24mg/kg, about 20mg/kg to about 22mg/kg, about 22mg/kg to about 50mg/kg, about 22mg/kg to about 48mg/kg, about 22mg/kg to about 46mg/kg, about 22mg/kg to about 44mg/kg, about 22mg/kg to about 42mg/kg, about 22mg/kg to about 40mg/kg, about 22mg/kg to about 38mg/kg, about 22mg/kg to about 36mg/kg, about 22mg/kg to about 34mg/kg, about 22mg/kg to about 32mg/kg, about 22mg/kg to about 30mg/kg, about 22mg/kg to about 28mg/kg, About 22mg/kg to about 26mg/kg, about 22mg/kg to about 24mg/kg, about 24mg/kg to about 50mg/kg, about 24mg/kg to about 48mg/kg, about 24mg/kg to about 46mg/kg, about 24mg/kg to about 44mg/kg, about 24mg/kg to about 42mg/kg, about 24mg/kg to about 40mg/kg, about 24mg/kg to about 38mg/kg, about 24mg/kg to about 36mg/kg, about 24mg/kg to about 34mg/kg, about 24mg/kg to about 32mg/kg, about 24mg/kg to about 30mg/kg, about 24mg/kg to about 28mg/kg, about 24mg/kg to about 26mg/kg, about 26mg/kg to about 50mg/kg, about 26mg/kg to about 48mg/kg, About 26mg/kg to about 46mg/kg, about 26mg/kg to about 44mg/kg, about 26mg/kg to about 42mg/kg, about 26mg/kg to about 40mg/kg, about 26mg/kg to about 38mg/kg, about 26mg/kg to about 36mg/kg, about 26mg/kg to about 34mg/kg, about 26mg/kg to about 32mg/kg, about 26mg/kg to about 30mg/kg, about 26mg/kg to about 28mg/kg, about 28mg/kg to about 50mg/kg, about 28mg/kg to about 48mg/kg, about 28mg/kg to about 46mg/kg, about 28mg/kg to about 44mg/kg, about 28mg/kg to about 42mg/kg, about 28mg/kg to about 40mg/kg, about 28mg/kg to about 38mg/kg, About 28mg/kg to about 36mg/kg, about 28mg/kg to about 34mg/kg, about 28mg/kg to about 32mg/kg, about 28mg/kg to about 30mg/kg, about 30mg/kg to about 50mg/kg, about 30mg/kg to about 48mg/kg, about 30mg/kg to about 46mg/kg, about 30mg/kg to about 44mg/kg, about 30mg/kg to about 42mg/kg, about 30mg/kg to about 40mg/kg, about 30mg/kg to about 38mg/kg, about 30mg/kg to about 36mg/kg, about 30mg/kg to about 34mg/kg, about 30mg/kg to about 32mg/kg, about 32mg/kg to about 50mg/kg, about 32mg/kg to about 48mg/kg, about 32mg/kg to about 46mg/kg, About 32mg/kg to about 44mg/kg, about 32mg/kg to about 42mg/kg, about 32mg/kg to about 40mg/kg, about 32mg/kg to about 38mg/kg, about 32mg/kg to about 36mg/kg, about 32mg/kg to about 34mg/kg, about 34mg/kg to about 50mg/kg, about 34mg/kg to about 48mg/kg, about 34mg/kg to about 46mg/kg, about 34mg/kg to about 44mg/kg, about 34mg/kg to about 42mg/kg, about 34mg/kg to about 40mg/kg, about 34mg/kg to about 38mg/kg, about 34mg/kg to about 36mg/kg, about 36mg/kg to about 50mg/kg, about 36mg/kg to about 48mg/kg, about 36mg/kg to about 46mg/kg, About 36mg/kg to about 44mg/kg, about 36mg/kg to about 42mg/kg, about 36mg/kg to about 40mg/kg, about 36mg/kg to about 38mg/kg, about 38mg/kg to about 50mg/kg, about 38mg/kg to about 48mg/kg, about 38mg/kg to about 46mg/kg, about 38mg/kg to about 44mg/kg, about 38mg/kg to about 42mg/kg, about 38mg/kg to about 40mg/kg, about 40mg/kg to about 50mg/kg, about 40mg/kg to about 48mg/kg, about 40mg/kg to about 46mg/kg, about 40mg/kg to about 44mg/kg, about 40mg/kg to about 42mg/kg, about 42mg/kg to about 50mg/kg, about 42mg/kg to about 48mg/kg, about, About 42mg/kg to about 46mg/kg, about 42mg/kg to about 44mg/kg, about 44mg/kg to about 50mg/kg, about 44mg/kg to about 48mg/kg, about 44mg/kg to about 46mg/kg, about 46mg/kg to about 50mg/kg, about 46mg/kg to about 48mg/kg or about 46mg/kg to about 50mg/kg) body weight of a bile acid (e.g., any bile acid described herein or known in the art, such as TURSO) or a pharmaceutically acceptable salt thereof, and about 10mg/kg to about 400mg/kg (e.g., about 10mg/kg to about 380mg/kg, about 10mg/kg to about 360mg/kg, about 10mg/kg to about 340mg/kg, about 10mg/kg to about 320mg/kg, about 10mg/kg to about 300mg/kg, About 10mg/kg to about 280mg/kg, about 10mg/kg to about 260mg/kg, about 10mg/kg to about 240mg/kg, about 10mg/kg to about 220mg/kg, about 10mg/kg to about 200mg/kg, about 10mg/kg to about 180mg/kg, about 10mg/kg to about 160mg/kg, about 10mg/kg to about 140mg/kg, about 10mg/kg to about 120mg/kg, about 10mg/kg to about 100mg/kg, about 10mg/kg to about 80mg/kg, about 10mg/kg to about 60mg/kg, about 10mg/kg to about 40mg/kg, about 10mg/kg to about 20mg/kg, about 20mg/kg to about 400mg/kg, about 20mg/kg to about 380mg/kg, about 20mg/kg to about 360mg/kg, About 20mg/kg to about 340mg/kg, about 20mg/kg to about 320mg/kg, about 20mg/kg to about 300mg/kg, about 20mg/kg to about 280mg/kg, about 20mg/kg to about 260mg/kg, about 20mg/kg to about 240mg/kg, about 20mg/kg to about 220mg/kg, about 20mg/kg to about 200mg/kg, about 20mg/kg to about 180mg/kg, about 20mg/kg to about 160mg/kg, about 20mg/kg to about 140mg/kg, about 20mg/kg to about 120mg/kg, about 20mg/kg to about 100mg/kg, about 20mg/kg to about 80mg/kg, about 20mg/kg to about 60mg/kg, about 20mg/kg to about 40mg/kg, about 40mg/kg to about 400mg/kg, About 40mg/kg to about 380mg/kg, about 40mg/kg to about 360mg/kg, about 40mg/kg to about 340mg/kg, about 40mg/kg to about 320mg/kg, about 40mg/kg to about 300mg/kg, about 40mg/kg to about 280mg/kg, about 40mg/kg to about 260mg/kg, about 40mg/kg to about 240mg/kg, about 40mg/kg to about 220mg/kg, about 40mg/kg to about 200mg/kg, about 40mg/kg to about 180mg/kg, about 40mg/kg to about 160mg/kg, about 40mg/kg to about 140mg/kg, about 40mg/kg to about 120mg/kg, about 40mg/kg to about 100mg/kg, about 40mg/kg to about 80mg/kg, about 40mg/kg to about 60mg/kg, About 60mg/kg to about 400mg/kg, about 60mg/kg to about 380mg/kg, about 60mg/kg to about 360mg/kg, about 60mg/kg to about 340mg/kg, about 60mg/kg to about 320mg/kg, about 60mg/kg to about 300mg/kg, about 60mg/kg to about 280mg/kg, about 60mg/kg to about 260mg/kg, about 60mg/kg to about 240mg/kg, about 60mg/kg to about 220mg/kg, about 60mg/kg to about 200mg/kg, about 60mg/kg to about 180mg/kg, about 60mg/kg to about 160mg/kg, about 60mg/kg to about 140mg/kg, about 60mg/kg to about 120mg/kg, about 60mg/kg to about 100mg/kg, about 60mg/kg to about 80mg/kg, About 80mg/kg to about 400mg/kg, about 80mg/kg to about 380mg/kg, about 80mg/kg to about 360mg/kg, about 80mg/kg to about 340mg/kg, about 80mg/kg to about 320mg/kg, about 80mg/kg to about 300mg/kg, about 80mg/kg to about 280mg/kg, about 80mg/kg to about 260mg/kg, about 80mg/kg to about 240mg/kg, about 80mg/kg to about 220mg/kg, about 80mg/kg to about 200mg/kg, about 80mg/kg to about 180mg/kg, about 80mg/kg to about 160mg/kg, about 80mg/kg to about 140mg/kg, about 80mg/kg to about 120mg/kg, about 80mg/kg to about 100mg/kg, about 100mg/kg to about 400mg/kg, About 100mg/kg to about 380mg/kg, about 100mg/kg to about 360mg/kg, about 100mg/kg to about 340mg/kg, about 100mg/kg to about 320mg/kg, about 100mg/kg to about 300mg/kg, about 100mg/kg to about 280mg/kg, about 100mg/kg to about 260mg/kg, about 100mg/kg to about 240mg/kg, about 100mg/kg to about 220mg/kg, about 100mg/kg to about 200mg/kg, about 100mg/kg to about 180mg/kg, about 100mg/kg to about 160mg/kg, about 100mg/kg to about 140mg/kg, about 100mg/kg to about 120mg/kg, about 120mg/kg to about 400mg/kg, about 120mg/kg to about 380mg/kg, about 120mg/kg to about 360mg/kg, About 120mg/kg to about 340mg/kg, about 120mg/kg to about 320mg/kg, about 120mg/kg to about 300mg/kg, about 120mg/kg to about 280mg/kg, about 120mg/kg to about 260mg/kg, about 120mg/kg to about 240mg/kg, about 120mg/kg to about 220mg/kg, about 120mg/kg to about 200mg/kg, about 120mg/kg to about 180mg/kg, about 120mg/kg to about 160mg/kg, about 120mg/kg to about 140mg/kg, about 140mg/kg to about 400mg/kg, about 140mg/kg to about 380mg/kg, about 140mg/kg to about 360mg/kg, about 140mg/kg to about 340mg/kg, about 140mg/kg to about 320mg/kg, about 140mg/kg to about 300mg/kg, About 140mg/kg to about 280mg/kg, about 140mg/kg to about 260mg/kg, about 140mg/kg to about 240mg/kg, about 140mg/kg to about 220mg/kg, about 140mg/kg to about 200mg/kg, about 140mg/kg to about 180mg/kg, about 140mg/kg to about 160mg/kg, about 160mg/kg to about 400mg/kg, about 160mg/kg to about 380mg/kg, about 160mg/kg to about 360mg/kg, about 160mg/kg to about 340mg/kg, about 160mg/kg to about 320mg/kg, about 160mg/kg to about 300mg/kg, about 160mg/kg to about 280mg/kg, about 160mg/kg to about 260mg/kg, about 160mg/kg to about 240mg/kg, about 160mg/kg to about 220mg/kg, About 160mg/kg to about 200mg/kg, about 160mg/kg to about 180mg/kg, about 180mg/kg to about 400mg/kg, about 180mg/kg to about 380mg/kg, about 180mg/kg to about 360mg/kg, about 180mg/kg to about 340mg/kg, about 180mg/kg to about 320mg/kg, about 180mg/kg to about 300mg/kg, about 180mg/kg to about 280mg/kg, about 180mg/kg to about 260mg/kg, about 180mg/kg to about 240mg/kg, about 180mg/kg to about 220mg/kg, about 180mg/kg to about 200mg/kg, about 200mg/kg to about 400mg/kg, about 200mg/kg to about 380mg/kg, about 200mg/kg to about 360mg/kg, about 200mg/kg to about 340mg/kg, About 200mg/kg to about 320mg/kg, about 200mg/kg to about 300mg/kg, about 200mg/kg to about 280mg/kg, about 200mg/kg to about 260mg/kg, about 200mg/kg to about 240mg/kg, about 200mg/kg to about 220mg/kg, about 220mg/kg to about 400mg/kg, about 220mg/kg to about 380mg/kg, about 220mg/kg to about 360mg/kg, about 220mg/kg to about 340mg/kg, about 220mg/kg to about 320mg/kg, about 220mg/kg to about 300mg/kg, about 220mg/kg to about 280mg/kg, about 220mg/kg to about 260mg/kg, about 220mg/kg to about 240mg/kg, about 240mg/kg to about 400mg/kg, about 240mg/kg to about 380mg/kg, About 240mg/kg to about 360mg/kg, about 240mg/kg to about 340mg/kg, about 240mg/kg to about 320mg/kg, about 240mg/kg to about 300mg/kg, about 240mg/kg to about 280mg/kg, about 240mg/kg to about 260mg/kg, about 260mg/kg to about 400mg/kg, about 260mg/kg to about 380mg/kg, about 260mg/kg to about 360mg/kg, about 260mg/kg to about 340mg/kg, about 260mg/kg to about 320mg/kg, about 260mg/kg to about 300mg/kg, about 260mg/kg to about 280mg/kg, about 280mg/kg to about 400mg/kg, about 280mg/kg to about 380mg/kg, about 280mg/kg to about 360mg/kg, about 280mg/kg to about 340mg/kg, From about 280mg/kg to about 320mg/kg, from about 280mg/kg to about 300mg/kg, from about 300mg/kg to about 400mg/kg, from about 300mg/kg to about 380mg/kg, from about 300mg/kg to about 360mg/kg, from about 300mg/kg to about 340mg/kg, from about 300mg/kg to about 320mg/kg, from about 320mg/kg to about 400mg/kg, from about 320mg/kg to about 380mg/kg, from about 320mg/kg to about 360mg/kg, from about 320mg/kg to about 340mg/kg, from about 340mg/kg to about 400mg/kg, from about 340mg/kg to about 380mg/kg, from about 340mg/kg to about 360mg/kg, from about 360mg/kg to about 400mg/kg, from about 360mg/kg to about 380mg/kg, or from about 380mg/kg to about 400mg/kg) body weight of a phenylbutyrate compound (e.g., any phenylbutyrate compound described herein or known in the art, e.g., sodium phenylbutyrate).

In some embodiments, the bile acid (e.g., TURSO) is administered in an amount of about 10mg/kg, about 15mg/kg, about 20mg/kg, about 25mg/kg, about 30mg/kg, about 35mg/kg, about 40mg/kg, about 45mg/kg, about 50mg/kg, about 55mg/kg, about 60mg/kg, about 65mg/kg, or about 70mg/kg of body weight. In some embodiments, the phenylbutyrate compound (e.g., sodium phenylbutyrate) is administered in an amount of about 10mg/kg, about 20mg/kg, about 30mg/kg, about 40mg/kg, about 50mg/kg, about 60mg/kg, about 70mg/kg, about 80mg/kg, about 90mg/kg, about 100mg/kg, about 120mg/kg, about 140mg/kg, about 160mg/kg, about 180mg/kg, about 200mg/kg, about 220mg/kg, about 240mg/kg, about 260mg/kg, about 280mg/kg, about 300mg/kg, about 320mg/kg, about 340mg/kg, about 360mg/kg, about 380mg/kg, or about 400mg/kg body weight.

The bile acid or pharmaceutically acceptable salt thereof and the phenylbutyrate compound may be administered separately or concurrently, including as part of a treatment regimen. These compounds may be administered daily, weekly, monthly or quarterly. In some embodiments, these compounds are administered once daily, twice daily, or three or more times daily. These compounds may be administered over a period of weeks, months or years. For example, the compounds can be used over a period of at least about 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, or at least about 5 years or more. The bile acid and phenylbutyrate compound may be administered, for example, once daily or twice daily for 60 days or less (e.g., 55 days, 50 days, 45 days, 40 days, 35 days, 30 days, or less). Alternatively, the bile acid and phenylbutyrate compound may be administered once daily or twice daily for more than 60 days (e.g., more than 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 180, 200, 250, 300, 400, 500, 600 days).

In some embodiments of any of the methods described herein, the bile acid is TURSO. TURSO may be administered to a subject at a dose of about 0.5 grams to about 10 grams per day (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, or 9 grams per day). For example, TURSO may be present in an amount of about 0.5 to about 5 grams (e.g., about 0, 5 to about 4.5, about 0.5 to about 4, about 0.5 to about 3.5, about 0.5 to about 3, about 0.5 to about 2.5, about 0.5 to about 2, about 0.5 to about 1.5, about 0.5 to about 1, about 1 to about 5, about 1 to about 4.5, about 1 to about 4, about 1 to about 3.5, about 1 to about 3, about 1 to about 2.5, about 1 to about 2, about 1 to about 1.5, about 1.5 to about 5, about 1.5 to about 4.5, about 1.5 to about 4, about 1.5 to about 3.5, about 1.5 to about 3, about 1.5 to about 2.5, about 1.5 to about 2, about 2 to about 5, about 3.5 to about 3, about 3.5 to about 2, about 3.5, about 3 to about 5, about 3.5 to about 2, about 3, about 5 to about 5, about 5 to about 2.5, about 5, about 3, about 3.5 to about 5, about 5 to about 5, about 3.5, about 3, about 5 to about 5, about 3.5, about 5 to about 5, about 3.5 to about 3, about 5 to about 3.5 to about 3, about 5, about 3.5, about 3, about 5 to about 3.5 to about 5, about 3.5, about 5 to about 3, about 5, about 3.5 to about 5, about 3.5, about 3, about 5, about 3.5, about 5, about 3.5 to about 3, about 5 to about 3, about 3.5 to about 3.5, about 5, about 3, about 5 to about 5, about 5 to about 3.5 to about 5 to about 3.5 to about 3, about 5 to about 3, about 3.5, about 5 to about 5, about 3, about 3.5 to about 3.5, about 3, about 5, about 3, about 3.5 to about 5, about 5 to about 3, about 3.5 to about 5, about 5 to about 5, about 3.5 to about 5 to about 3.5 to about 3, about, About 3.5 to about 4, about 4 to about 5, about 4 to about 4.5, or about 4.5 to about 5 grams per day. In some embodiments, TURSO is administered to the subject in an amount of about 1 gram per day. In some embodiments, TURSO is administered to the subject in an amount of about 2 grams per day. For example, TURSO may be administered in an amount of about 1 gram twice daily.

In some embodiments of any of the methods described herein, the phenylbutyrate compound is sodium phenylbutyrate. Sodium phenylbutyrate may be administered in an amount of about 1 gram to about 30 grams per day (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 grams per day). For example, sodium phenylbutyrate may be present in an amount of about 0.5 to about 10 grams (e.g., about 0.5 to about 9.5, about 0.5 to about 9, about 0.5 to about 8.5, about 0.5 to about 7.5, about 0.5 to about 7, about 0.5 to about 6.5, about 0.5 to about 6, about 0.5 to about 5.5, about 0.5 to about 5, about 0.5 to about 4.5, about 0.5 to about 4, about 0.5 to about 3.5, about 0.5 to about 3, about 0.5 to about 2.5, about 0.5 to about 2, about 0.5 to about 1.5, about 0.5 to about 1, about 1 to about 10, about 1 to about 9.5, about 1 to about 9, about 1 to about 8.5, about 1 to about 8, about 1 to about 7.5, about 1 to about 1.5, about 1 to about 1, about 1.5, about 1 to about 5, about 1.5, about 1 to about 1, about 5, about 1 to about 5, about 1.5, about 1 to about 5, about 1 to about 5, about 1.5, about 5, about 1 to about 5, about 5 to about 1 to about 5, about 1 to about 5, about 5 to about 5, about 1.5 to about 1 to about 1.5, about 5, about 1.5, about 1 to about 5, about 1 to about 5, about 1 to about 5, about 5 to about 1 to about 5, about 5 to about 1 to about 5 to about 1.5, about 5, about 1 to about 5, about 5 to about 5, about 5 to about 1.5, about 1 to about 1.5, about 5, about 1.5, about 1 to about 5, about 1 to about 5, about 5 to about 1.5 to about 1 to about 1.5, about 1 to about 5, about 1.5, about 1 to about 5, about 5 to about 1 to about 1.5, about 1 to about 5 to about 1.5, about 1 to about 5, about 5 to about 1 to about, About 1.5 to about 8, about 1.5 to about 7.5, about 1.5 to about 7, about 1.5 to about 6.5, about 1.5 to about 6, about 1.5 to about 5.5, about 1.5 to about 5, about 1.5 to about 4.5, about 1.5 to about 4, about 1.5 to about 3.5, about 1.5 to about 3, about 1.5 to about 2.5, about 1.5 to about 2, about 2 to about 10, about 2 to about 9.5, about 2 to about 9, about 2 to about 8.5, about 2 to about 8, about 2 to about 7.5, about 2 to about 7, about 2 to about 6.5, about 2 to about 6, about 2 to about 5.5, about 2 to about 5, about 2 to about 4.5, about 2 to about 4, about 2 to about 3.5, about 2 to about 5.5, about 5, about 2 to about 5.5, about 5, about 5.5, about 2 to about 5, about 5.5, about 5, about 2 to about 5, about 5.5, about 2 to about 5, about 5.5, about 5, about 2 to about 5, about 5.5, about 5, about 2 to about 5.5, about 5, about 5.5, about 5, about 2 to about 5.5, about 5, about 5.5, about 5, about 2 to about 5, about 5.5, about 5, about 5.5, about 2 to about 5, about 5.5, about 5, about 2 to about 5, about 5.5.5, about 5.5.5.5, about 5, about 5.5, about 5, about 5.5, about 5, about 2 to about 5, about 5.5, about 5, about 5.5, about 5, about 5.5.5, about 5.5.5.5, about 5, about 2.5 to about 3, about 3 to about 10, about 3 to about 9.5, about 3 to about 9, about 3 to about 8.5, about 3 to about 8, about 3 to about 7.5, about 3 to about 7, about 3 to about 6.5, about 3 to about 6, about 3 to about 5.5, about 3 to about 5, about 3 to about 4.5, about 3 to about 4, about 3 to about 3.5, about 3.5 to about 10, about 3.5 to about 9.5, about 3.5 to about 9, about 3.5 to about 8.5, about 3.5 to about 8, about 3.5 to about 7.5, about 3.5 to about 7, about 3.5 to about 6.5, about 3.5 to about 6, about 3.5 to about 5, about 3.5 to about 4.5, about 4 to about 4.5, about 4 to about 5, about 4.5, about 4 to about 5, about 4.5, about 5 to about 5, about 4.5, about 4, about 4.5 to about 5, about 4, about 5 to about 5, about 4.5, about 5, about 4.5 to about 5, about 5 to about 4.5, about 4, about 5 to about 5, about 4, about 5 to about 5, about 5 to about 4.5, about 5, about 4.5, about 5 to about 4.5, about 5, about 4.5 to about 5, about 5 to about 5, about 5 to about 5, about 5 to about 5, about 5 to about 5, about 5 to about 5, about 4.5, about 5 to about 5, about 5 to about 5, about 5 to about 5, about 5 to about 5, about 5 to about 5, about 5 to about 5, about 5.5, about 5, about, About 4.5 to about 8.5, about 4.5 to about 8, about 4.5 to about 7.5, about 4.5 to about 7, about 4.5 to about 6.5, about 4.5 to about 6, about 4.5 to about 5.5, about 4.5 to about 5, about 5 to about 10, about 5 to about 9.5, about 5 to about 9, about 5 to about 8.5, about 5 to about 8, about 5 to about 7.5, about 5 to about 7, about 5 to about 6.5, about 5 to about 6, about 5 to about 5.5, about 5.5 to about 10, about 5.5 to about 9.5, about 5.5 to about 9, about 5.5 to about 8.5, about 5.5 to about 8, about 5.5 to about 7.5, about 5.5 to about 7, about 5.5 to about 6.5, about 5 to about 6.5, about 6 to about 6, about 6.5 to about 6, about 5 to about 6.5, about 6, about 5 to about 6.5, about 6, about 5 to about 6.5, about 6, about 5 to about 6, about 5 to about 6.5, about 6, about 5 to about 5, about 6, about 6.5 to about 6, about 5 to about 6, about 6.5, about 6, about 5, about 6, about 5 to about 5, about 5 to about 5.5, about 5 to about 6.5, about 6, about 5, about 6, about 5 to about 5, about 6, about 5 to about 6, about 5, about 6, about 5, about 6, about 5, about 6, about 5, about 6.5, about 6, about 5, about 6, about 5, about 6, about 5, about 6.5, about 6, about 6.5, about 6, about 5, about 6, about 7 to about 9.5, about 7 to about 9, about 7 to about 8.5, about 7 to about 8, about 7 to about 7.5, about 7.5 to about 10, about 7.5 to about 9.5, about 7.5 to about 9, about 7.5 to about 8.5, about 7.5 to about 8, about 8 to about 10, about 8 to about 9.5, about 8 to about 9, about 8 to about 8.5, about 8.5 to about 10, about 8.5 to about 9.5, about 8.5 to about 9, about 9 to about 10, about 9 to about 9.5, or about 9.5 to about 10 grams per day. In some embodiments, sodium phenylbutyrate is administered in an amount of about 3 grams per day. In some embodiments, sodium phenylbutyrate is administered in an amount of about 6 grams per day. For example, sodium phenylbutyrate may be administered in an amount of about 3 grams twice daily. In some embodiments, the bile acid and the phenylbutyrate compound are administered in a weight ratio of about 2.5: 1 to about 3.5: 1 (e.g., about 3: 1).

In some embodiments of any of the methods described herein, the method comprises administering TURSO and sodium phenylbutyrate to the subject according to a first regimen followed by a second regimen, wherein the first regimen comprises administering about 1 gram TURSO per day and about 3 grams sodium phenylbutyrate per day for at least 14 days (e.g., at least 16, 18, 21, 24, 27, 30, 35, or 40 days), and the second regimen comprises administering about 1 gram TURSO per day and about 3 grams sodium phenylbutyrate per day twice per day for at least 30 days (e.g., at least 35, 40, 45, 50, 60, 80, 100, 120, 150, 180, 250, 300, or 400 days).

In some embodiments of any of the methods described herein, the subject is diagnosed with, at risk of developing ALS, or suspected of having ALS. The subject may have been diagnosed with ALS, for example, for 24 months or less (e.g., any subrange within this range described herein). For example, a subject may have been diagnosed with ALS for 1 week or less, or diagnosed with ALS on the same day as administration of a treatment disclosed herein. The subject may have exhibited one or more symptoms of ALS for 24 months or less (e.g., any subrange within this range described herein), have a rate of progression of ALS disease (Δ FS) of about 0.50 or greater (e.g., any subrange within this range described herein), have an ALSFRS-R score of 40 or less (e.g., any subrange within this range described herein), have an average loss of about 0.8 to about 2 (e.g., any subrange within this range described herein) per month over the preceding 3-12 months, have a mutation in one or more genes selected from the group consisting of: SOD1, C9ORF72, ANG, TARDBP, VCP, VAPB, SQSTM1, DCTN1, FUS, UNC13A, ATXN2, HNRNPA1, chchchhd 10, MOBP, C21ORF2, NEK1, TUBA4A, TBK1, MATR3, PFN1, UBQLN2, TAF15, OPTN, and TDP-43, and/or have a CSF or blood level of pNF-H of about 300pg/mL or more (e.g., about 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, pg, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 2500, 1650, 1700, 1750, 1800, 2100, 1900, 1950, 2100, 250, 2700, 3800, 2700, 1000, 3800, 1000, 2800, 3800, 2350, 1000, 28050, 1000 mL, 22550, 1000 mL, 2650, 3000, 1000, 2650, 1000 mL, 2650, 3000, or more). In some embodiments, the subject's serum pNF-H level in the methods described herein may be about 70 to about 1200pg/mL (e.g., about 70 to about 1000, about 70 to about 800, about 80 to about 600, or about 90 to about 400 pg/mL). In some embodiments, the CSF pNF-H level of the subject in the methods described herein may be about 1000 to about 5000pg/mL (e.g., about 1500 to about 4000 or about 2000 to about 3000 pg/mL). The subject may have a CSF or blood level of NfL of about 50pg/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 250pg/mL or more). In some embodiments, the subject's serum NfL level in the methods described herein may be about 50 to about 300pg/mL (e.g., any subrange within this range described herein). In some embodiments, the level of CSF NfL in the subject in the methods described herein can be about 2000 to about 40,000pg/mL (e.g., any subrange within this range described herein).

The methods described in this disclosure may include treating ALS itself, as well as treating one or more symptoms of ALS. "treating" ALS does not require 100% elimination of the disease or disease symptoms in a subject. Any alleviation or reduction of the severity of the symptoms or features of the disease is contemplated. "treating" ALS also refers to delaying the onset of symptoms (e.g., in prophylactic treatment) or delaying the progression of symptoms or delaying the loss of function associated with the disease. "treating" ALS also refers to eliminating or reducing one or more side effects of treatment (e.g., side effects caused by any therapeutic agent disclosed herein or known in the art for treating ALS). "treating" ALS also refers to eliminating or reducing one or more direct or indirect effects of ALS disease progression such as falls, tears, or an increase in the number of GI problems. The subject may not exhibit signs of ALS, but may be at risk of developing ALS. For example, a subject may carry a mutation in a gene associated with ALS, have a family history of having ALS, or have elevated levels of a biomarker indicative of a risk of developing ALS. The subject may exhibit early signs of disease or symptoms of established or progressive disease. The present disclosure contemplates any degree of delay in the onset of symptoms, alleviation of one or more symptoms of the disease, or delay in the progression of any one or more disease symptoms (e.g., any improvement measured by the ALSFRS-R, or maintenance of the ALSFRS-R assessment (signaling delays disease progression)). Any alleviation or reduction of the severity of the symptoms or features of benign fasciculation syndrome and spasm-fasciculation syndrome is also contemplated herein.

The treatment provided in the present disclosure may begin at any stage during the progression of the disease. For example, treatment may begin prior to onset (e.g., for a subject at risk of developing ALS), at the onset of symptoms or immediately after detection of ALS symptoms, after observing any one or more symptoms (e.g., muscle weakness, muscle fasciculation, and/or muscle spasm) that would cause a skilled practitioner to suspect that the subject may be developing ALS. The therapeutic power may be initiated at a later stage. For example, treatment may begin at the advanced stage of the disease, for example, when muscle weakness and atrophy spread to different parts of the body and the subject is experiencing increasing motion problems. At or before the onset of treatment, the subject may suffer from muscle tightness and stiffness (spasticity), hyperreflexia (hyperreflexia), muscle weakness and atrophy, muscle spasms, and/or transient twitches of the muscles that can be observed under the skin (fasciculations), difficulty swallowing (dysphagia), speech, or word formation (dysarthria).

Methods of treatment may include single administration, multiple administrations, and repeat administrations as needed to prevent or treat ALS or at least one symptom of ALS. The duration of prophylactic treatment can be a single dose or the treatment can be continued (e.g., multiple doses), e.g., for years or indefinitely over the life of the subject. For example, a subject at risk of ALS may be treated with the methods provided herein for days, weeks, months, or even years to prevent the disease from occurring or fulminating. In some embodiments, the method of treatment can include assessing the level of disease in the subject before, during, and/or after treatment. The treatment provided herein may be administered one or more times per day, or it may be administered weekly or monthly. In some embodiments, treatment can continue until a decrease in the subject's disease level is detected. In some embodiments, the methods provided herein can begin to exhibit efficacy (e.g., alleviate one or more symptoms of ALS, as measured by improvement in or maintenance of ALSFRS-R assessment) after less than 60 days (e.g., less than 50, 45, 40, 30, 25, 20, 15, or 10 days) after initial administration, or less than 60 administrations (e.g., less than 50, 45, 40, 35, 30, 25, 20, 15, or 10 administrations).

The terms "administering," "administering," or "administering," as used herein, refer to administering a drug described herein to a subject (regardless of form) using any method known in the art, such as ingestion, injection, implantation, absorption, or inhalation of the drug. In some embodiments, one or more compounds disclosed herein can be administered to a subject by oral and/or topical (e.g., nasal) ingestion. For example, the methods herein comprise administering an effective amount of a compound or compound composition to achieve a desired or specified effect. The specific dose and treatment regimen for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, disorder or condition, the subject's disposition to the disease, disorder or condition, and the judgment of the treating physician.

After administration, the subject can be evaluated to detect, assess or determine their disease level. In some embodiments, treatment can be continued until a change (e.g., a decrease) in the level of disease in the subject is detected.

After the patient's condition improves (e.g., a change (e.g., a decrease) in the level of disease in the subject), a maintenance dose of a compound, composition, or combination of the disclosure can be administered, if desired. Subsequently, the dose or frequency of administration, or both, can be reduced as a function of the symptoms to a level that maintains an improved condition. However, after any disease symptoms have recurred, the patient may require long-term intermittent treatment.

Mitochondrial dysfunction

Mitochondrial dysfunction is ubiquitous in neurodegenerative diseases. In Alzheimer's disease, the mitochondrial membrane potential of cells is significantly reduced, glucose metabolism by mitochondria is impaired, and permeability of mitochondria is increased. Mitochondria have been observed to mediate a variety of apoptotic pathways leading to neuronal death in alzheimer's disease.

PINK1 and Parkin are both mitochondrial quality control proteins. Mutations or deficiencies in these proteins are closely related to Parkinson's disease. MPTP, a molecule used to induce permanent symptoms of parkinson's disease, acts by disrupting mitochondrial complex I, causing mitochondrial dysfunction, alteration of cellular redox state, and apoptosis.

It has been directly shown in cell culture that the mutated Huntingtin gene (Huntingtin gene) and its produced protein, which is thought to be the major mediator of Huntington's disease, results in a loss of membrane potential and a decrease in expression of key oxidative phosphorylation genes in mitochondria. The pathology of huntington's disease is also associated with a reduction in the number of mitochondria present in the central nervous system.

Abnormal localization of mitochondria, impaired energy metabolism and apoptotic pathways are thought to mediate amyotrophic lateral sclerosis. Mitochondria from affected tissues also show an overproduction of reactive oxygen metabolites and leakage of them into the cytoplasm.

In many neurodegenerative diseases, mitochondria overproduce free radicals, leading to a decrease in energy metabolism, increased permeability, decreased membrane potential, decreased antioxidants, leakage of metal ions into cells, alteration of the redox state of cells and resulting in a decrease in pro-apoptotic pathways. Thus, there is a need for agents that alter and reduce the mechanisms of mitochondrial dysfunction.

Also included are methods of reducing mitochondrial dysfunction or treating at least one symptom associated with mitochondrial dysfunction, preventing the time of onset of, or slowing the progression of a disease or condition associated with mitochondrial dysfunction.

Symptom and observation indicators (also known as efficacy assessment)

Described herein are methods of evaluating symptoms, monitoring ALS progression, and/or evaluating a subject's response to a treatment method. Non-limiting examples include physical assessment by a physician, body weight, Electrocardiogram (ECG), ALS function rating scale (ALSFRS or ALSFRS-R) score, respiratory function, muscle strength, cognitive/behavioral function, quality of life, and speech analysis.

The respiratory function of a subject can be measured, for example, by spirometric capacity (including forced and slow spirometric capacity), maximum mid-expiratory flow rate (MMERF), forced spirometric capacity (FVC), forced expiratory volume for 1 second (FEV) ₁ ). Muscle strength can be evaluated by, for example, evaluating hand-held ergometers (HHDs), hand-held ergometers, bare-handed muscle strength tests (MMTs), electrical impedance Electromyograms (EIMs), Maximum Voluntary Isometric Contraction Tests (MVICTs), motor unit estimates (MUNEs), accurate testing of isometric muscle strength of limbs (ATLIS), or combinations thereof. Cognitive/behavioral functions can be assessed by, for example, the ALS depression scale (ADI-12), the becker depression scale (BDI), and the Hospital Anxiety Depression Scale (HADS) questionnaire. Quality of life can be assessed, for example, by the ALS assessment questionnaire (ALSAQ-40). Akt levels, Akt phosphorylation and/or paktdktt ratios can also be used to assess disease progression and response to treatment in a subject (see, e.g., WO 2012/160563).

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

Muscular strength

The muscle strength of a subject can be evaluated using methods known in the art. Quantitative force indicators generally show linear, predictable loss of force in ALS patients. Tuffs Quantitative Neuromuscular Examination (TQNE) can be used to provide quantitative measurements using a fixed strain gauge. TQNE measures isometric muscle strength of 20 muscle groups and produces interval strength data in both strong and weak muscles (see, e.g., Andres et al, Neurology 36: 937-. Hand-held ergometers (HHDs) test isometric muscle strength of specific muscles in the arm and leg and generate interval level data (see, e.g., Shefne JM, Neurothelietics 14: 154-.

An isometric Muscle strength Accurate Test for Limbs (ATLIS) can be used to measure both strong and weak Muscle groups using fixed wireless load cells (see, e.g., Andres et al, Muscle Nerve 56 (4): 710-. The force in the 12 muscle groups was evaluated in the ATLIS test, which reflects the strength in the lower and upper limbs of the subject and the grip of the subject. In some embodiments, the ATLIS test detects changes in muscle strength before any functional changes are observed.

As assessed by any suitable method described herein, the methods provided herein can improve, maintain, or slow the deterioration of muscle strength (e.g., lower limb strength, upper limb strength, or grip strength) in a subject. In some embodiments, these methods may result in an improvement in upper limb strength in the subject more significantly than in other muscle groups. For example, the effect on muscle strength may be reflected in one or more muscle groups selected from quadriceps femoris, biceps, hamstring tendon, triceps, and tibialis anterior.

In some embodiments of any of the methods of ameliorating, maintaining, or slowing muscle strength deterioration in a human subject having one or more symptoms of ALS described herein, the muscle strength is assessed by HHD, hand-dynamometer, MMT, EIM, MVICT, MUNE, ATLIS, or a combination thereof, before, during, and/or after administration of the bile acid or pharmaceutically acceptable salt thereof and the phenylbutyrate compound.

In some embodiments, muscle strength is assessed by ATLIS. Total ATLIS scores as well as upper and lower limb ATLIS scores can be assessed. The methods of the present disclosure can result in a rate of decline in the subject's total ATLIS score of about 3.50 PPN/month or less (e.g., about 3.45, 3.40, 3.35, 3.30, 3.25, 3.20, 3.15, 3.10, 3.05, 3.00 PPN/month or less). The methods of the present disclosure can also result in a decrease in the average rate of decrease in the total ATLIS score of the subject by at least about 0.2 PPN/month (e.g., at least about 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 PPN/month) as compared to a control subject that did not receive the administration. The average rate of decline of the upper limb ATLIS score of the subject can be reduced by at least about 0.50 PPN/month (e.g., at least about 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, or 0.90 PPN/month) compared to a control subject not receiving the administration described herein. The mean rate of decline of the lower limb ATLIS score of the subject may be reduced by at least about 0.20 PPN/month (e.g., at least about 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, or 0.60 PPN/month) compared to a control subject not receiving the administration described herein. In some embodiments, the improvement or maintenance of muscle strength in the subject may begin to occur less than 60 days (e.g., less than 55, 50, 45, 40, 30, 25, or 20 days) after the initial administration. PPN represents the percentage of predicted normal force based on age, gender, weight and height.

Pulmonary function

ALS is a progressive neurodegenerative disease that ultimately leads to respiratory failure and death. Pulmonary function tests, such as but not limited to spirometry(VC), maximum expiratory mid-flow rate (MMERF), Forced Vital Capacity (FVC), Slow Vital Capacity (SVC), forced expiratory volume for 1 second (FEV) ₁ ) May be used to monitor the progression of ALS and/or the response of a subject to treatment. On average, the rate of decline in respiratory function in ALS patients, as measured by Vital Capacity (VC), is about 2.24% of the monthly prediction (± 6.9). In some embodiments, the indicators from lung function tests correlate with survival (see, e.g., Moufavi et al, Iran J Neurol 13 (3): 131-. Additional indicators, such as maximum inspiratory and expiratory pressure, arterial blood gas measurements, and nocturnal blood oxygen saturation, can provide early evidence of dysfunction. Comparison of vital capacity in upright and supine positions may also provide an early indication of diminished respiratory muscle strength.

The methods provided herein can improve or maintain respiratory muscle and/or lung function in a subject, or slow deterioration of respiratory muscle and/or lung function in a subject. Respiratory muscle and/or lung function of a subject can be assessed by any suitable method described herein or known in the art. In some embodiments, respiratory muscle function of a human subject is assessed based on the SVC of the subject. In some embodiments of any of the methods of improving, maintaining, or slowing deterioration of respiratory muscle function in a human subject described herein, the treatment results in a mean rate of decline of SVC in the subject of about 3.50 PPN/month or less (e.g., about 3.45, 3.40, 3.35, 3.30, 3.25, 3.20, 3.15, 3.10, 3.05, or 3.00 PPN/month or less). In some embodiments, the treatment reduces the mean rate of decline of SVC in the subject by at least about 0.5 PPN/month (e.g., at least about 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1.00 PPN/month) compared to a control subject not receiving the treatment. In some embodiments, improvement or maintenance of lung function in a subject can begin to occur less than 60 days (e.g., less than 55, 50, 45, 40, 30, 25, or 20 days) after initial administration. In some embodiments, the progression of pulmonary function in the subject is less than expected less than 60 days after the initial administration.

Adverse events

A subject treated with any of the methods provided herein may exhibit fewer adverse events (e.g., any of the adverse events disclosed herein), or exhibit one or more adverse events to a lesser extent than an untreated control subject. Exemplary adverse events include gastrointestinal related adverse events (e.g., abdominal pain, gastritis, nausea and vomiting, constipation, rectal bleeding, peptic ulcer disease and pancreatitis); hematological adverse events (e.g., aplastic anemia and ecchymosis); cardiovascular adverse events (e.g., arrhythmia and edema); renal adverse events (e.g., renal tubular acidosis); psychiatric adverse events (e.g., depression); adverse skin events (e.g., rashes); and other adverse events (e.g., syncope and weight gain). In some embodiments, the methods provided herein do not cause or cause minimal symptoms of constipation, neck pain, headaches, falls, dry mouth, muscle weakness, falls, tears, and elevated alanine Aminotransferase (ALT). In some embodiments, the adverse event is a severe adverse event such as, but not limited to, a respiratory adverse event, a fall, or a tear.

In some embodiments, administration of a combination of a bile acid and a phenylbutyrate compound may result in fewer adverse events (e.g., any of the adverse events disclosed herein) or less severe adverse events than administration of the bile acid or the phenylbutyrate compound alone.

The average survival time of ALS patients may vary. Median survival time may be from about 30 to about 32 months after onset of symptoms, or from about 14 to about 20 months after diagnosis. A subject with bulbar attack ALS may have a survival time from about 6 months to about 84 months after onset of symptoms, with a median time of about 27 months. In some embodiments, the methods provided herein can increase survival of a subject with ALS by at least one month (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 28, 32, 36, 40, 50, 60, 70, 80, or 90 months). In some embodiments, the methods provided herein can delay the onset of ventilator dependence or tracheostomy for at least one month (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 28, 32, 36, 40, 50, 60, 70, 80, or 90 months).

The methods provided herein can reduce the rate of disease progression, wherein the subject's monthly average loss ALSFRS-R score is reduced by at least about 0.2 (e.g., at least about 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, or 1.5) compared to untreated control subjects. The methods provided herein can slow progression in one or more categories evaluated by the ALSFRS scale, including: speech, salivation, swallowing, handwriting, cutting food and handling appliances, dressing and hygiene, bed flipping and adjusting sheets, walking, climbing stairs, dyspnea, sitting up breathing, respiratory insufficiency. In some embodiments, the methods provided herein improve or slow the deterioration of fine motor function in a subject as assessed by one or more categories of the ALSFRS-R scale (e.g., handwriting, cutting food and handling utensils, or dressing and hygiene).

In some embodiments, the methods provided herein are more effective in treating a subject from about 18 to about 50 years old (e.g., from about 18 to about 45 years old, from about 18 to about 40 years old, from about 18 to about 35 years old, from about 18 to about 30 years old, from about 18 to about 25 years old, or from about 18 to about 22 years old) as compared to a subject over 50 years old (e.g., 55, 60, 65, 70, 75, or 80 years old or old). In some embodiments, the methods provided herein are more effective in treating a subject who has been diagnosed with ALS and/or who shows an onset of ALS symptoms for less than about 24 months (e.g., less than about 22, 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, or 1 month) as compared to a subject who has been diagnosed with ALS and/or who shows an onset of ALS symptoms for more than about 24 months (e.g., more than about 26, 28, 30, 32, 34, 36, 40, 45, 50, 55, or 60 months). In some embodiments, the methods provided herein are more effective in treating a subject who has been diagnosed with ALS and/or who shows an onset of symptoms of ALS for more than about 24 months (e.g., more than about 26, 28, 30, 32, 34, 36, 40, 45, 50, 55, or 60 months) as compared to a subject who has been diagnosed with ALS and/or who shows symptoms of ALS for less than about 24 months (e.g., less than about 22, 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, or 1 month).

In some embodiments, responsiveness to a treatment provided herein is gender-dependent. The methods provided herein may be more or less effective in treating female subjects as compared to male subjects. For example, a female subject may exhibit improvement earlier or later than a male subject when treated at a similar stage of disease progression (e.g., as measured by ALSFRS-R or any other observation described herein). In some embodiments, a female subject may exhibit greater or less improvement (e.g., as measured by ALSFRS-R or any other observation described herein) than a male subject when treated at a similar stage of disease progression. The pharmacokinetics of the bile acid and phenylbutyrate compounds may be the same or different in female and male subjects.

Pharmaceutical compositions and methods of administration

The bile acids or pharmaceutically acceptable salts thereof and phenylbutyrate compounds described herein may be formulated for use as or in a pharmaceutical composition. Such compositions may be formulated or adapted for administration to a subject via any route, such as any route approved by the U.S. Food and Drug Administration (FDA). An exemplary method is described in the FDA's CDER data standards Manual, version number 004 (which is available at FDA. give/CDER/dsm/DRG/drg00301. html). The pharmaceutical compositions may be formulated for oral, parenteral, or transdermal delivery.

The pharmaceutical composition may include an effective amount of a bile acid or a pharmaceutically acceptable salt thereof and/or a phenylbutyrate compound. As used herein, the term "effective amount" refers to an amount or concentration of one or more drugs that is effective to elicit the intended effect or physiological observation over a period of time (including acute or chronic administration as well as periodic or continuous administration) over the range of its administration.

In some embodiments, the pharmaceutical composition comprises a bile acid or a pharmaceutically acceptable salt thereof, and/or a phenylbutyrate compound, and any pharmaceutically acceptable carrier, adjuvant, and/or vehicle. The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that can be administered to a patient with a compound of the present invention and which does not destroy the pharmacological activity of the compound and is non-toxic when administered in a dosage sufficient to deliver a therapeutic amount of the compound. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

The pharmaceutical compositions may contain any conventional non-toxic pharmaceutically acceptable carrier, adjuvant or vehicle. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases, or buffers to enhance the stability of the formulated compound or its delivery form.

The pharmaceutical compositions are typically formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., by inhalation or through a feeding tube), transdermal (topical), transmucosal, and rectal administration. The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

The pharmaceutical compositions may be in the form of solutions or powders for inhalation and/or nasal administration. In some embodiments, the pharmaceutical composition is formulated as a powder-filled pouch. Suitable powders may include those that are substantially water soluble. The pharmaceutical compositions may be formulated according to the techniques known in the art using suitable dispersing or wetting agents (e.g. tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Acceptable vehicles and solvents that may be employed include mannitol, water, ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents, which are conventionally used in the formulation of pharmaceutical dosage forms such as emulsions and/or suspensions. Other commonly used surfactants such as tweens or spans and/or other similar emulsifying agents or bioavailability enhancers, which are commonly used in the preparation of pharmaceutical solid, liquid or other dosage forms, may also be used for formulation purposes.

The pharmaceutical compositions may be administered orally in any orally acceptable dosage form, including without limitation powders, capsules, tablets, emulsions, and aqueous suspensions, dispersions, and solutions. In the case of powders for oral administration, the powder may be substantially dissolved in water prior to administration. In the case of tablets for oral use, commonly used carriers include lactose and corn starch. Lubricating agents such as magnesium stearate may be added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions and/or emulsions are administered orally, the active ingredient may be suspended or dissolved in the oil phase, in combination with emulsifying and/or suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.

Alternatively or additionally, the pharmaceutical 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.

In some embodiments, the therapeutic compositions disclosed herein can be formulated for sale in the united states, import into the united states, and/or export from the united states. The pharmaceutical composition may be contained in a container, package or dispenser together with instructions for administration. In some embodiments, the present invention provides kits comprising a bile acid and a phenylbutyric acid compound. The kit may also include instructions for the physician and/or patient, syringes, needles, cassettes, bottles, vials, and the like.

Additional therapeutic agents and other combination therapies

Any of the pharmaceutical compositions described herein may further include one or more additional therapeutic agents in an amount effective for treating or effecting modulation of at least one symptom of ALS. Any known therapeutic agent for ALS known in the art may be used as the additional therapeutic agent. Exemplary therapeutic agents include riluzole (C) ₈ H ₅ F ₃ N ₂ OS, e.g. under the trade nameAndsold under the trade name) edaravone (e.g., trade name)Andsold as mexilil (e.g. sold under the trade names Mexitil and NaMuscla), a combination of dextromethorphan and quinidine (e.g. sold as Mexitil and NaMuscla)) Anticholinergic and psychiatric agents such as, but not limited to, antidepressants, antipsychotics, anxiolytics/hypnotics, mood stabilizers and stimulants.

Mexiletine can be used, for example, for cramps and fasciculations.Is a combination of dextromethorphan and quinidine, and can be used to treat pseudobulbar effects (improper laughing or crying). Anticholinergic drugs and antidepressants may be used, for example, to treat hypersalivation. Any known anticholinergic agent is contemplated herein, including, but not limited to, glycopyrrolate, scopolamine, atropine (Atropen), belladonna alkaloids (belladonna alkaloids), benztropine mesylate (cogenin), clidinium, cyclonexine, and glycopyrrolatePentetate (cycloglycol) (cyclogol), darifenacin (darifenacin) (Enablx), dicyclomine (dicylomine), fesoterodine (fetoterodine) (Toviaz), flavoxate (flavoate) (Urispas), glycopyrronium bromide, homatropine hydrobromide (homatropine hydrobromide), hyoscyamine (hyoscyamine) (Levsinex), ipratropium bromide (ipratropium) (Atrovent), oxyphenamine (oxyphenamine), oxybutynin (Ditropan XL), procarypsin (Pro-bathylline), scopolamine (dyposolamine), methscopolamine (metacolomide), metafenacin (sulfofenacin) (trospide), thiofenadine (sulfofenate) (Betrotrix), and triamcinolone (fenrium chloride). Any known antidepressant is contemplated herein, including but not limited to selective 5-hydroxytryptamine inhibitors, 5-hydroxytryptamine-norepinephrine reuptake inhibitors, 5-hydroxytryptamine modulators and stimulators, 5-hydroxytryptamine antagonists and reuptake inhibitors, norepinephrine-dopamine reuptake inhibitors, tricyclic antidepressants, tetracyclics, monoamine oxidase inhibitors, and NMDA receptor antagonists.

The methods of the present disclosure may comprise administering to the subject one or more additional therapeutic agents (e.g., any additional therapeutic agent disclosed herein or known in the art) in combination with a bile acid (e.g., any suitable bile acid described herein) or a pharmaceutically acceptable salt thereof and a phenylbutyrate compound (e.g., any suitable phenylbutyrate compound described herein). The additional one or more therapeutic agents may be administered for a duration of time prior to administration of an initial dose of a composition comprising a bile acid or pharmaceutically acceptable salt thereof (e.g., TURSO) and a phenylbutyrate compound (e.g., sodium phenylbutyrate), and/or for a duration of time after administration of a final dose of the composition. In some embodiments, the subject in the methods described herein has been previously treated with one or more additional therapeutic agents (e.g., any of the additional therapeutic agents described herein, such as riluzole, edaravone, and mexiletine). In some embodiments, a stable dose of one or more therapeutic agents (e.g., riluzole and/or edaravone) has been administered to a subject for at least 30 days (e.g., at least 40 days, 50 days, 60 days, 90 days, or 120 days) prior to administration of a composition of the present disclosure. In some embodiments, mexiletine has been administered to the subject at a dose of about 300 mg/day or less (e.g., about 250 mg/day, 200 mg/day, 150 mg/day, 100 mg/day, or 50 mg/day or less). Absorption, metabolism, and/or excretion of one or more additional therapeutic agents may be affected by the bile acid or pharmaceutically acceptable salt thereof and/or the phenylbutyrate compound. For example, co-administration of sodium phenylbutyrate with riluzole, edaravone, or mexiletine may increase exposure of a subject to riluzole, edaravone, or mexiletine. In some cases, co-administration of riluzole with a bile acid or a pharmaceutically acceptable salt thereof and a phenylbutyrate compound may improve tolerance to riluzole in a subject compared to riluzole alone.

The combination of a bile acid or pharmaceutically acceptable salt thereof, a phenylbutyrate compound, and one or more additional therapeutic agents may have a synergistic effect in treating ALS. When administered in combination with a bile acid or pharmaceutically acceptable salt thereof and a phenylbutyrate compound, a smaller dose of the additional therapeutic agent may be required to achieve the same pharmacological effect. In some embodiments, the amount of the one or more additional therapeutic agents administered in combination with the bile acid or pharmaceutically acceptable salt thereof and the phenylbutyrate compound may be reduced by at least about 10% (e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55%) as compared to the dose used when the one or more additional therapeutic agents are administered alone. Additionally or alternatively, the methods of the present disclosure may reduce the required frequency of administration of other therapeutic agents (e.g., other ALS therapeutic agents) to achieve the same pharmacological effect.

Some embodiments of the present disclosure provide a method of treating at least one symptom of ALS or preventing the onset of ALS in a human subject, the method comprising administering to the human subject an effective amount of (a) a bile acid or a pharmaceutically acceptable salt thereof (e.g., any of the bile acids or pharmaceutically acceptable salts thereof described herein); (b) a phenylbutyrate compound (e.g., any of the phenylbutyrate compounds described herein); (c) riluzole; (d) edaravone, thereby treating at least one symptom of ALS or preventing the onset of ALS in the human subject.

The bile acid or pharmaceutically acceptable salt thereof and the phenylbutyrate compound may be administered shortly after a meal (e.g., within two hours after a meal) or under fasting conditions. The subject may have ingested a food product (e.g., a solid food or a liquid food) less than 2 hours prior to administration of the bile acid or pharmaceutically acceptable salt thereof and/or the phenylbutyrate compound; or will be ingested less than 2 hours after administration of one or both compounds. The food product may affect the rate and extent of absorption of the bile acid or pharmaceutically acceptable salt thereof and/or the phenylbutyrate compound. For example, food can alter the bioavailability of a compound by delaying gastric emptying, stimulating bile flow, altering gastrointestinal pH, increasing visceral blood flow, altering luminal metabolism of a substance, or otherwise interacting physically or chemically with a dosage form or substance. The nutritional and caloric content of a meal, meal volume, and meal temperature can cause physiological changes in the GI in a manner that affects drug transit time, luminal dissolution, drug permeability, and systemic availability. In general, diets high in total calories and fat content are more likely to affect GI physiology and thus have a greater impact on the bioavailability of the drug. The methods provided herein can further comprise administering to the subject a plurality of food products, e.g., within less than 2 hours (e.g., less than 1.5 hours, 1 hour, or 0.5 hours), before or after administration of the bile acid or pharmaceutically acceptable salt thereof, and/or the phenylbutyrate compound.

Examples

Further embodiments are disclosed in more detail in the following examples, which are provided by way of illustration and are not intended at all to limit the scope of the disclosure or claims.

Example 1: evaluation of safety, tolerability, efficacy and activity of AMX0035 (a fixed combination of Phenylbutyrate (PB) and tauroursodeoxycholic acid (TUDCA)) for the treatment of ALS

1. Overview

1.1 study goals and endpoints

The present study is intended to demonstrate that AMX0035 can be a safe and effective therapeutic agent for adult subjects with ALS. The main strategic goals of this study are as follows.

The main observation indexes are as follows:

1. confirming the safety and tolerability of fixed dose combinations of PB and TUDCA in subjects with ALS over a 6 month period;

2. measuring the effect of treatment using the slope of progression using the revised amyotrophic lateral sclerosis functional rating scale (ALSFRS-R);

secondary objectives of the study were:

1. evaluating the effect of AMX0035 on the rate of decline of peer-long muscle strength as measured by the isometric strength Accurate Test (ATLIS) of the limb;

2. assessing the effect of AMX0035 on disease progression as measured by slow lung capacity (SVC) decline, tracheostomy time, and survival;

3. Assessing the effect of AMX0035 on biomarkers including levels of phosphorylated axonal neurofilament protein H business base (pNF-H) and uptake of 18kDa Translocator (TSPO);

4. a concentration response model of TUDCA and phenylbutyric acid at steady state after twice daily AMX0035 sachet administration was established.

5. The effect of AMX0035 on lifetime was measured.

1.2 study design

This is a multicenter, randomized, double-blind, placebo-controlled 28-week study to evaluate safety, tolerability, efficacy, pharmacokinetics and bioactivity of AMX 0035.

1.3 study population

This study was conducted in subjects with sporadic or familial ALS diagnosed as defined by the revised El escoral standard (example 3). Subjects must provide written informed consent prior to screening. At the time of screening, eligible subjects must be at least 18 years of age and less than 80 years of age and have a predictive power of VC ≧ 60% for age, height, and gender. The subject must have an onset of ALS symptoms, defined as the first appearance of weakness, within less than or equal to 18 months prior to the screening visit. Subjects taking stable doses of riluzole and those not taking riluzole, as well as fertile women at screening, are eligible for selection as long as they meet specific protocol requirements. There is no limitation on subjects taking Radicava (edaravone) at screening, or if they begin taking at study participation. Detailed standards are described in the protocol text.

2. Research and observation index

2.1 Main Observation indicators

The main observational indices of this study included:

safety and tolerability, defined as the proportion of subjects able to keep taking study medication until scheduled withdrawal.

Rate of decline (decline slope) in ALS function rating Scale (ALSFRS-R).

Safety and tolerability were evaluated by the procedure outlined in section 8. A revision of ALSFRS was created to increase the assessment of respiratory dysfunction (including dyspnea, orthopnea, and the need for ventilatory support). The revised ALSFRS (ALSFRS-R) has been shown to retain the characteristics of the original scale and to exhibit strong internal consistency and structural validity. End-of-life was defined as death, tracheostomy or permanent assisted ventilation (> 22 hours per day).

2.2 secondary Observation indicators

Assessing the effect of AMX0035 on the rate of decline of isometric muscle strength as measured by the isometric strength Accurate Test for Limbs (ATLIS);

assess the effect of AMX0035 on disease progression as measured by slow lung capacity (SVC) decline;

evaluation of the effect of AMX0035 on survival, hospitalization and tracheostomy;

assessing the effect of AMX0035 on biomarkers including levels of phosphorylated axonal neurofilament H subunit (pNF-H) and uptake of 18kDa Translocator (TSPO); and

Concentration-response model evaluation of TUDCA and phenylbutyrate at steady state following twice daily AMX00354 grams.

3. Design of research

3.1 Overall study design and planning

During the enrollment period, approximately 176 subjects were screened from approximately 25 northeast ALS alliance (NEALS) centers in the united states. One hundred thirty-seven (137) of these subjects were randomly assigned to sachets or matched placebo for oral (or feeding tube) twice daily active agent therapy at a 2: 1 ratio. The duration of treatment was twenty-four (24) weeks. Study medication was administered once daily for the first three weeks. If tolerated, the dose was increased to twice daily. Clinical visits were performed at screening, baseline, week 3 (day 21), week 6 (day 42), week 12 (day 84), week 18 (day 126), and week 24 (day 168). Telephone calls were made at weeks 9, 15, 21 and 28 (4 weeks after completion of treatment).

All visit windows were on consecutive calendar days and were calculated from the day the subject started study treatment (day 0, baseline visit day). Any change in this visit window is considered an out-of-window visit deviation. Those subjects who completed randomized double-blind studies were available for an Open Label Extension (OLE) study for one hundred thirty-two (132) weeks (see example 2).

3.2 duration of study

Subjects continued to remain on randomized, placebo-controlled, double-blind treatment until week 24 visit. Each randomized subject also received a follow-up telephone visit 28 days after completion of dosing to assess Adverse Events (AE), changes in concomitant medications, and ALSFRS-R administration. Including screening and follow-up, each subject lasted approximately 8 months in the study.

4. Study enrollment and withdrawal

4.1 inclusion and exclusion criteria

4.1.1 inclusion criteria

1. Male or female, age 18-80 years

2. Sporadic or familial ALS diagnosed as definite as defined by the El Escorial Standard revised by the world neurology Association

3. Less than or equal to 18 months after onset of ALS symptoms

4. Can provide informed consent and follow the test procedure

5. Geographically accessible on-site

6. Slow Vital Capacity (SVC) > 60% of the predicted values for gender, height and age at the time of screening visit

7. The subject must not take riluzole or a stable dose of riluzole for at least 30 days prior to the screening visit. Subjects who had not used riluzole were allowed in the study.

8. Women with fertility potential (e.g., who are not postmenopausal for at least one year or surgically sterilized) must agree to use adequate contraceptive regimens during the study and within 3 months after the last dose of study medication

a. Women were not scheduled for pregnancy during the study period and within 3 months after the last dose of study drug

9. The men must agree to take contraceptive measures during the study and within 3 months after the last dose of study drug

a. During the study period and within 3 months after the last dose of study medication, the male must not plan to breed children or provide sperm for donation

Acceptable methods of contraception used in this study were:

hormonal methods, e.g. contraceptives, patches, injections, vaginal rings or implants

Barrier methods (e.g. condoms or diaphragms) for use with spermicides (foams, creams or gels to kill sperm)

Intrauterine device (IUD)

Abstinence (non-sexual behavior)

Unique partner for surgical sterilization (male) or sterility (female)

Date of onset of ALS symptoms

For the purposes of this study, the date of symptom onset is defined as the date on which the subject first developed symptoms of their disease (i.e., weakness). To qualify for the present study, the date of symptom onset must not exceed the exact 18 months prior to the date of screening visit.

MR-PET collateral study

A subset of subjects received MR-PET and required the following additional inclusion criteria to be met:

1. the on-site investigator believes that the MR-PET program can lie safely for 90min

2. High or mixed affinity binding to TSPO proteins (genotypes Ala/Ala or Ala/Thr)

TSPO affinity assay

Venous blood for the TSPO affinity test was taken from all subjects indicated to be interested in participating in the MR-PET collateral study (via check boxes on the consent). Blood was drawn at screening to genotype the subject for the Ala147Thr TSPO polymorphism in the TSPO gene (rs 6971). About 10% of people exhibit low binding affinity for PBR28 (Zurcher et al, incorporated in viral activation in subjects with amyotrophic lateral sclerosis: isolated with [ ] ¹¹ C]PBR28 (increased glial cell activation in a patient with amyotrophic lateral sclerosis: [ use ] ¹¹ C]PBR28 for evaluation). High or mixed affinity binders (Ala/Ala or Ala/Thr) were considered eligible, while low affinity binders (Thr/Thr) were considered ineligible for MR-PET collateral studies. The subject may be eligible for the primary study but not for the MR-PET collateral study. However, if the subject is found to be ineligible for the primary study, he or she is also automatically ineligible for the MR-PET accessory study.

4.1.2 exclusion criteria

Study subjects meeting any of the following criteria during the screening evaluation were excluded from the study:

1. Presence tracheostomy

2. Exposure to PB, TUDCA or UDCA within 3 months prior to screening visit or scheduled use of these drugs during the course of the study

3. Known history of hypersensitivity to PB or bile salts

4. Liver function abnormality, defined as AST and/or ALT > 3 times upper limit of normal

5. Renal insufficiency, e.g. by eGFR < 60mL/min/1.73m ² And (4) defining.

6. Poor arterial hypertension control (SBP > 160mmHg or DBP > 100mmHg) at the time of screening visit

7. Pregnant woman or woman nursing

8. History of gallbladder excision

9. Biliary tract diseases (active cholecystitis, primary biliary cirrhosis, sclerosing cholangitis, gallbladder cancer, gallbladder polyp, gallbladder gangrene, and gallbladder abscess) for obstructing bile flow.

History of grade III/IV Heart failure (according to New York Heart Association) -NYHA)

11. Serious pancreatic or intestinal disorders that may alter enterohepatic circulation and TUDCA uptake (including biliary infection, pancreatitis and ileectomy)

12. At the discretion of the on-site investigator, there is an unstable mental disorder, cognitive impairment, dementia or drug abuse that would impair the ability of the subject to provide informed consent

13. A patient having a cancer other than: basal cell carcinoma or successfully treated squamous cell carcinoma of the skin; cervical cancer in situ; prostate cancer in situ; or other malignancies that have cured and no evidence of disease recurrence for at least 3 years.

14. Clinically significant unstable medical conditions (other than ALS) will impose a risk to the subject if they participate in the study

15. Actively participate in ALS clinical trials evaluating experimental small molecules within 30 days of screening visits. (for a current list of experimental small molecules, please refer to MOP section e.

16. Any cell and gene therapy (off-label or in-study) that is readily exposed to the subject under study for treatment of subjects with ALS

17. Exposure to the monoclonal antibody being studied for the treatment of ALS (used off-label or in study) was within 90 days after screening. If previously exposed to the monoclonal antibody being studied for the treatment of ALS, a 90 day washout period would be required prior to screening.

18. Implantation of a Diaphragm Pacing System (DPS)

19. Anything that the site investigator deems will put the subject at elevated risk or prevent the subject from fully following or completing the study

20. Exposure to any of the prohibited drugs listed below

MR-PET collateral study

A subset of study subjects received MR-PET. The following additional exclusion criteria apply to this subset:

1. screening for immunomodulatory drugs within 30 days after visit

2. Any contraindications for performing MRI studies, such as:

a. cardiac pacemaker or pacemaker lead history

b. In vivo metal particles

c. Vascular clamp in head

d. Artificial heart valve

e. Severe claustrophobia hindering ability to participate in imaging studies

3. Low affinity binders (Thr/Thr) for TSPO affinity assays

4. Radiation exposure beyond current guidelines in the field

The subject may be eligible for the primary study but not for the MR-PET collateral study. However, if the subject is found to be ineligible for the primary study, he or she is also automatically ineligible for the MR-PET accessory study.

Benzodiazepines for subjects in an MR-PET adjunctive studyQuasi drugs: if the MR-PET subject is taking benzodiazepines, he or she should not take benzodiazepines (other than lorazepam and clonazepam, which do not require withdrawal) for at least 1 day prior to the scan.

All subjects disabled medications include

HDAC inhibitors comprising:

valproic acid salts

O. Vorinostat (Vorinostat) (Zolinza)

O.Romidepsin (Romidepsin)

O.Sida Benamine (Chidamide)

Panobinostat (Panobinostat)

O lithium

O. butyric acid salt

Orchiramine (Suramin)

Probenecid (Probenecid)

A bile acid sequestrant comprising:

cholestyramine (Cholestyramine) and Light Cholestyramine (Cholestyramine Light)

O-hypo-cholestyramine (Questran) and Light hypo-cholestyramine (Questran Light)

○Welchol

Colestipol (Colestid) and Flavored colestipol (Colestid Flavored)

○Prevalite

Antacids within two hours after application of AMX0035

Antacids containing aluminum hydroxide or smectite (alumina) should not be taken within two hours after AMX0035 administration because they inhibit the absorption of TUDCA. These include:

○Alamag

alumina and magnesia

Antacid, antacid M and antacid suspension

○Gen-Alox

○Kudrox

○M.A.H.

Mallox HRF and Mallox TC

○Magnalox

○Madroxal

Mylanta and Mylanta Ultimate

○Ri-Mox

○Rulox

Mexiletine

Subjects participating in the mexiletine test over the past 30 days were excluded from the test. However, if the subject uses mexiletine for spasticity and fasciculations at a dose of less than or equal to 300 mg/day, the subject will not be excluded.

There is a possibility of interaction between AMX0035 and mexiletine. At the expected clinical concentration (C) _max ) At 20-fold higher, phenylacetate, the major metabolite of phenylbutyrate, has been shown to inhibit CYP 1a2 and CYP 2D6, the major enzyme responsible for mexiletine decomposition. Thus, it is possible to That is, co-administration of phenylbutyrate and mexiletine will increase exposure of the subject to mexiletine.

Thus, subjects administered AMX0035 concurrently with mexiletine should be monitored for mexiletine-related adverse events, and if these events are present, the field investigator should consider stopping or reducing the dose of mexiletine. Adverse events associated with mexiletine include, but are not limited to, arrhythmia, liver damage, and blood dyscrasias.

4.3 treatment Allocation procedure

Each subject meeting all eligibility criteria was randomly assigned to receive twice daily AMX0035 sachets (3g PB and 1g TUDCA) for treatment or matched placebo for 24 weeks of treatment. In the first three weeks of the study, subjects took only a single sachet per day and were instructed to increase to 2 sachets per day at week 3 visit.

4.4 Exit reason

Any clinical Adverse Event (AE), laboratory abnormality, need for concomitant medication, concurrent illness, or other medical condition or situation, such that the investigator deems continued participation in the study to be not in line with the subject's greatest benefit.

Subject non-compliance or loss of visit.

5. Treatment of administration

5.1.1 survey product description

AMX0035 is a combination therapy consisting of the two active pharmaceutical ingredients sodium Phenylbutyrate (PB) and tauroursodeoxycholic acid (TUDCA). Phenylbutyrate is a compound approved in the United states for the treatment of urea cycle disorders, and is administered in the United states And (4) marketing and selling. There is an existing USP monograph for this material. Drug substance PB was produced by Sri Krishna Pharmaceuticals, ltd. The manufacturing and control of PBA is described in Drug administration File No.019569 (Drug Master File No. 019569). PB specifications are the same as those of ph.

The chemical structure of PB is provided below.

Bulk drug TUDCA is currently marketed under the brand names Tudcabil and Taurolite. It can be used for treating cholesterol and cholelithiasis. It has been used to treat cholestatic liver diseases, including primary cirrhosis, pediatric familial intrahepatic cholestasis, primary sclerosing cholangitis, and cholestasis due to cystic fibrosis.

The chemical structure of TUDCA is provided below.

Bulk drug TUDCA was produced by Prodotti Chimici E Alimentaria s.p.a. The TUDCA specifications are the same as those used by the supplier.

Powder filled sachets are used as AMX0035 pharmaceuticals. The drug was filled in aluminum foil lined pouches under cGMP conditions.

A pouch containing an active ingredient comprising:

active ingredients:

·1g TUDCA

·3g PB

o excipients

Anhydrous disodium hydrogen phosphate

Dextrates (Dextrates), hydrates

Sorbitol

Syloid 63FP (colloidal silicon dioxide)

Sucralose (Sucralose)

Sodium stearyl fumarate

Weber Mixed Berry flavor (Weber Mixed Berry flavor)

Klepose Linecaps (maltodextrin)

5.1.2 placebo

Dose blinding was maintained using matched placebo. The placebo pouch used in this study matched the corresponding AMX0035 pouch in size, color and appearance. Administration of the matched placebo was the same as the subjects in the treatment group.

Placebo sachets contain:

o. adjuvant

The anhydrous disodium hydrogen phosphate was added to the reaction mixture,

dextrates, hydrates

Sorbitol

Syloid 63FP (colloidal silicon dioxide)

Sucralose

Sodium stearyl fumarate

Weber mixed berry seasoning

Klepose Linecaps (maltodextrin)

Denatonium Benzoate particles

5.2 product storage and stability

The drug supply in all studies was maintained at ambient temperature of 15-25 ℃. Subjects were asked to store the kit with the pouch away from moisture at room temperature. The stability of each individual active ingredient has been evaluated under ICH standards and accelerated conditions and found to be stable for five years. The drug product was subjected to stability testing periodically during the study to ensure that the product did not degrade.

5.3 dosage, preparation and administration of study intervention/investigational products

Study medication is recommended to be taken before meals. The subject should tear open the study drug pouch and add it to a cup or other container and add approximately 8 ounces (1 cup) of room temperature water and vigorously stir. The study drug mixture should be completely ingested within one hour after the pouch contents are mixed with water. Subjects can resume normal diet after taking study medication.

5.3.1 feeding tube study drug administration

For subjects with gastrostomy or nasogastric (feeding) tubes, the study drug can be dissolved in water according to the procedure outlined in section 5.3 above, and the study drug can be administered via a feeding tube.

5.4 treatment with and without therapy

Any small molecule therapy in the study used to treat ALS is banned from being used or evaluated 30 days prior to the screening visit and throughout the study. This includes, but is not limited to, the following:

pioglitazone (Pioglitazone)

Arilomo (Arimoglomol)

Olanzapine (Olanzapine)

Tamoxifen (Tamoxifen)

·NP001

Mexiletine

Rasagiline (Rasagiline)

Masitinib (Masitinib)

Dextrorotatory pramipexole (Dexpramixole)

·Tirasemtiv

Ibudilast (Ibudilast)

·TW001

Inosine (Inosine)

·RNS60

Acetyl L-Carnitine (Acetyl-L-Carnitine)

Mecobalamin (Methylcobalamine) (if administered at a dose equal to or greater than 25mg per week)

Any biological therapy used prior to the study excluded subjects from inclusion. This includes any cell or gene therapy being evaluated for the treatment of ALS, and includes, but is not limited to, the following:

·ISIS 333611

·Ionis SOD1R

·NurOwn

q-cells

·NSI-566

·GM604

·GSK 1223249

Treg cell therapy

5.4.1 contraindications and drugs

In the case of AMX0035, agents that may impair bile acid processing or renal function are contraindicated. Disabling drugs include, but are not limited to:

HDAC inhibitors comprising:

valproate salt

O. Vorinostat (Zollinza)

Romidicin

Cydariamine

Panobinostat

O lithium

O. butyric acid salt

Suramin O

Probenecid for potential renal interactions

Antacids containing aluminum hydroxide or smectite (alumina) are used within two hours after application of AMX 0035. These inhibit the absorption of TUDCA. These include:

○Alamag

alumina and magnesia

Antacid, antacid M and antacid suspension

○Gen-Alox

○Kudrox

○M.A.H.

Mallox HRF and Mallox TC

○Magnalox

○Madroxal

Mylanta and Mylanta Ultimate

○Ri-Mox

○Rulox

Bile acid sequestrants include:

cholestyramine (Cholestyramine) and Light Cholestyramine (Cholestyramine Light)

O-norgalanthamine (Questran) and lightweight norgalanthamine (Questran Light)

○Welchol

Colestipol (Colestid) and Flavored colestipol (Colestid Flavored)

○Prevalite

6. Study timetable

6.1 screening visit

The following procedures were performed at the outpatient visit to determine study eligibility for the subjects.

Obtaining written informed consent of the subject

Creation of Globally Unique Identifier (GUID)

Evaluation of inclusion and exclusion criteria

Acquiring medical history and demographic data

Review and record concomitant medications and therapies

Obtaining a diagnosis history of ALS

Management of the ALSFRS-R questionnaire

Performing a pulmonary function test, including Slow Vital Capacity (SVC)

Measuring isometric muscle force using ATLIS machine

Evaluation and recording of Adverse Events (AE) that occurred after subjects signed an Informed Consent Form (ICF)

Measuring vital signs (blood pressure, heart and respiration rate, body temperature)

Conducting a neurological examination

Carry out a comprehensive physical examination including height and weight

Performing a 12-lead ECG (Electrocardiogram)

[ post-other tests ] blood samples were collected for clinical laboratory evaluation, including hematology (CBC with triage), complete biochemical tests, liver function tests, and serum pregnancy tests (for fertility women [ WOCBP ])

MR-PET scan only subjects: TSPO affinity assay

Collecting urine samples for urinalysis

Scheduling baseline visits

MR-PET scan: for those subjects who agreed to participate in the study affiliated with the MR-PET scan, the scan was scheduled/performed prior to the baseline visit. At that time, blood was also collected for Peripheral Blood Mononuclear Cell (PBMC) storage and analysis.

6.2 visit 1 MR-PET (for patients in the MR-PET adjunctive study only)

The following procedure was performed to determine if the subjects were eligible for the MR-PET adjunctive study.

Get written informed consent

Evaluation of MR-PET inclusion and exclusion criteria

Complete MR-PET safety questionnaire

Performing MR-PET scans

O implementation Motor neuron burden on the (UMN-B) Scale

Measurement of vital signs (blood pressure, heart and respiratory rate, body temperature) and body weight

O. management of the ALSFRS-R questionnaire

Collecting blood for

O biomarker (PBMC) assay

Pregnancy test (for fertility women)

Review and record concomitant medications and therapies

Assessment and recording of Adverse Events (AE) that occurred after subjects signed an Informed Consent Form (ICF)

MR-PET follow-up phone: this visit will be made 24-48 hours after the 1 st MR-PET visit. The following procedure will be performed.

Evaluation and recording of AE directly related to MR-PET procedure

6.3 Baseline visit

This visit was conducted up to 42 days after the screening visit. The following procedure is performed.

Confirmation of still meeting qualification criteria

Randomized assignment of subjects using kit numbers from study drugs

Manage C-SSRS Baseline questionnaire

Management of the ALSFRS-R questionnaire

Performing a pulmonary function test, including Slow Vital Capacity (SVC)

Measuring isometric muscle force using ATLIS machine

Review and record concomitant medications and therapies

Review and record adverse events since last visit and study drug administration

Measuring vital signs

Collect blood samples [ after other tests ] for clinical laboratory evaluation, including hematology (CBC with classification test), complete blood biochemical examination, liver function test.

Collecting blood for biomarkers

Collection of pre-drug blood samples for pharmacokinetic analysis

Collect blood sample for optional DNA collection (note: if the baseline visit has passed or no blood sample for DNA is collected, then the blood sample should be collected at the next available visit)

Collecting urine samples for urinalysis

After all other visit activities are completed:

study drug dispensed for 6 weeks

The first dose of study drug was administered. The caregiver will advise the subject as to the appropriate administration (appendix VI). According to local institutional/state regulations, subjects will be observed on site for at least 60 minutes by appropriate medical personnel to assess medical conditions and any immediate response to the study drug.

Review and record any adverse events after the first dose of study drug

6.4 visit clinic at week 3

This visit occurred 21 ± 5 days after the baseline visit. The following procedure is performed.

Management of the ALSFRS-R questionnaire

Review and record concomitant medications and therapies

Review and evaluate adverse events

Measuring vital signs

Manage C-SSRS questionnaire

Blood samples were collected for clinical laboratory evaluation including hematology (CBC with differential test), complete blood biochemical examination, liver function test

Collecting urine samples for urinalysis

Performing a research drug accountability system

Subject is advised to increase dose levels from one to two sachets per day unless the drug is intolerant.

Scheduling the next study visit

6.5 visit at week 6

This visit occurred 42 ± 5 days after the baseline visit. The following procedure is performed.

Manage ALSFRS-R questionnaire

Performing a pulmonary function test, including Slow Lung Vital Capacity (SVC)

Measuring isometric muscle force using ATLIS machine

Review and record concomitant medications and therapies

Review and evaluate adverse events

Measuring vital signs

Manage C-SSRS questionnaire

[ after other tests ] blood samples were collected for clinical laboratory evaluation, including hematology (CBC with differential test), complete blood biochemical examination, liver function test

Collecting blood samples for biomarkers

Collecting urine samples for urinalysis

Performing a study drug accountability system and collecting all unused study drugs and empty containers

Dispense study drug for the next 6 weeks

Scheduling the next study visit

6.6 week 9 Call follow-up

This follow-up occurred 63 ± 5 days after the baseline visit. The following procedure is performed.

Manage ALSFRS-R questionnaire

Review and record concomitant medications and therapies

Evaluation and recording of AE

Ask about tolerance and compliance

Scheduling the next study visit

Remind subjects to bring study medication to visit at week 12

6.7 clinic visit at week 12

This visit occurred 84 ± 5 days after the baseline visit. Due to PK analysis, subjects had to take study medication on site after starting the visit. Since the medication is administered in the outpatient setting, this visit is recommended earlier in the day. The following procedure was performed:

record the date/time of prior study medication intake, including whether the subject missed the intake.

Notice the time of the last meal

Administration of study drug and recording the time of administration

Blood samples were collected for PK (i.e., 1 or 4 hours after dosing) as indicated at random dispensing time

Management of the ALSFRS-R questionnaire

Performing a pulmonary function test, including Slow Vital Capacity (SVC)

Measuring isometric muscle force using ATLIS machine

Review and record concomitant medications and therapies

Review and evaluate adverse events

Measuring vital signs

Conducting a neurological examination

Performing a comprehensive physical examination including body weight

Performing a 12-lead ECG (Electrocardiogram)

o manage C-SSRS questionnaire

[ after other tests ] blood samples were collected for clinical laboratory evaluation, including hematology (CBC with differential testing), complete blood biochemical examination, liver function testing

Collecting blood samples for biomarkers

Collecting urine samples for urinalysis

Dispense study drug for the next 6 weeks

Scheduling the next study visit

6.8 week 15 telephone follow-up

This follow-up occurred 105 ± 5 days after baseline visit. The following procedure is performed.

Manage ALSFRS-R questionnaire

Review and record concomitant medications and therapies

Evaluation and recording of AE

Ask about tolerance and compliance

Scheduling the next study visit

6.9 visit clinic at week 18

This visit occurred 126 ± 5 days after the baseline visit. The following procedure is performed.

Management of the ALSFRS-R questionnaire

Performing a pulmonary function test, including Slow Vital Capacity (SVC)

Measuring isometric muscle force using ATLIS machine

Review and record concomitant medications and therapies

Review and evaluate adverse events

Measuring vital signs

Manage C-SSRS questionnaire

Collecting blood samples for biomarkers

Collecting urine samples for urinalysis

Dispense study drug for the next 6 weeks

Scheduling the next study visit

6.10 week 21 Call follow-up

This follow-up occurred 147 ± 5 days after baseline visit. The following procedure is performed.

Manage ALSFRS-R questionnaire

Review and record concomitant medications and therapies

Evaluation and recording of AE

Query tolerance and compliance

Scheduling the next study visit

Remind subjects to bring study medication to clinic for week 24 visit

Scheduling MR-PET scans for subjects participating in an MR-PET collateral study

6.11 visit 2 MR-PET (for patients in the MR-PET adjunctive study only)

This visit occurred between study visits at week 12 and week 20.

Complete MR-PET safety questionnaire

Performing MR-PET scans

O Upper motoneuron load on execution (UMN-B) scale

Measuring vital signs (blood pressure, heart and respiratory rate, body temperature), height and weight

Management of the ALSFRS-R questionnaire

Collecting blood for

O biomarker (PBMC) assay

Pregnancy test (for fertility women)

Review and record concomitant medications and therapies

Evaluation and recording of Adverse Events (AE)

MR-PET follow-up phone: this follow-up occurred 24-48 hours after the 2 nd MR-PET visit. The following procedure is performed.

Evaluation and recording of AE directly related to MR-PET procedure

6.12 Final study visit (week 24)

This visit occurred 168 ± 5 days after the baseline visit. Due to PK analysis, subjects had to take study medication at the start of the visit. Since the medication is administered at an outpatient clinic, this visit is recommended earlier in the day. The following procedure was performed:

record the date/time of prior study medication intake, including whether the subject missed the intake

Record the time of the last meal

Administration of study drug and recording time of administration

As indicated at random assignment (week 24 only, not including subjects terminated prematurely), a single blood sample was collected for PK (i.e. 1 or 4 hours post-dose)

Management of the ALSFRS-R questionnaire

Performing a pulmonary function test, including Slow Vital Capacity (SVC)

Measuring isometric muscle force using ATLIS machine

Review and record concomitant medications and therapies

Review adverse events

Measuring vital signs

Performing a neurological examination

Performing a physical examination, including body weight

Performing a 12-lead ECG (Electrocardiogram)

Manage C-SSRS questionnaire

Exit questionnaire

Collecting blood samples for biomarkers

Collecting urine samples for urinalysis

6.13 last call visit (week 28)

Telephone follow-up was performed 28+5 days (not earlier than 28 days) after the subjects last dose of study drug. The following procedure is performed.

Completion of the ALSFRS-R questionnaire

Review and record concomitant medications and therapies

Evaluation and recording of AE

7. Clinical assessment and Observation indicators

7.1 clinical variables

Assessments were performed at designated time points throughout the study for clinical evaluation. In addition to the evaluation evaluated below, the subjects also provided information about their demographics, past medical history (including ALS and cardiac medical history), and concomitant drug use.

7.1.1 Vital signs, height and weight

Vital signs were obtained after the subject had been in a sitting position for several minutes. Vital signs were assessed at the prescribed visit, including systolic and diastolic blood pressure, pulse rate (radial)/minute, respiratory rate/minute, body temperature and body weight. Height was measured and recorded only at the screening visit.

7.1.2 clinical laboratory evaluation

Hematology with classification examination item (Hematology with differential panel): whole blood cell count with classification (hematocrit, hemoglobin, platelet count, RBC index, total RBC, total WBC, and WBC & classification)

Blood biochemical test/Liver Function Test (LFT): alanine aminotransferase (ALT (SGPT)), aspartate aminotransferase (AST (SGOT)), albumin, alkaline phosphatase, bicarbonate, blood urea nitrogen, calcium, chloride, creatinine, glucose, magnesium, phosphate, potassium, sodium, total bilirubin, and total protein

Urine analysis: albumin, bilirubin, blood, clarity, color, glucose, ketones, nitrate, pH, protein, specific gravity, urobilinogen and WBC screening

Serum human chorionic gonadotropin (hCG) from fertile Women (WOCBP) (collected only at the screening visit and as needed throughout the study)

7.1.3 biomarkers and pharmacokinetic analysis

Subjects draw blood at baseline visits to assess AMX0035 concentrations for pre-drug Pharmacokinetics (PK) and then re-draw blood at 1 or 4 hours (+ -10 minutes) post-drug at week 12 and week 24 visits.

In addition, blood was collected for biomarker analysis, including light and heavy neurofilament protein tests (NF-L and pNF-H, respectively). Neurofilament protein was used as a mechanical indicator of neuronal death. NF-L and pNF-H were tested at multiple time points to generate longitudinal datasets correlating neurofilament protein levels with observed clinical observations.

7.1.412-lead Electrocardiogram (ECG)

A standard 12-lead electrocardiogram is performed and recorded.

7.1.5 physical examination

A full physical examination was performed and recorded.

7.1.6 examination of nervous system

Nervous system examinations were performed and recorded. The examination includes assessment of mental state, cranial nerves, motor and sensory functions, reflexes, coordination and posture/gait.

7.1.7 Upper motor neuron load (UMN-B)

Motor neuron load on Penn (UMN-B) is the total number of pathological UMN signs at the time of examination, including pathologically active biceps, supinator, triceps, finger, knee and ankle reflexes, as well as bilaterally assessed extensor plantar responses and active facial and mandibular reflexes. The scale is a combination of the Ashworth, Reflexes (Reflexes) and Pseudobulbar emotion (Pseudobulbar Affect) scales (range score: 0-32). UMN also includes a score for the center of the neurological study instability scale (CNS-LS), which is a 7-item self-reporting scale that assesses false bulbar mood (PBA) by measuring the perceived frequency of PBA attacks (laughing or crying). Data were generated from clinical examinations and scored from 1-5, with the lowest score indicating normal health and the highest score indicating extreme spasticity.

7.1.8 Columbia suicide severity rating Scale (C-SSRS)

C-SSRS involves a series of exploratory questions to ask about possible suicidal thoughts and behaviors. At baseline visit, the C-SSRS baseline version was administered. This version was used to assess suicidality in a subject's lifetime. In all outpatient visits following the baseline visit, the since last visit version of C-SSRS was administered. This version of the scale assesses suicidal ideality since the subject's last visit.

7.1.9 adverse event

Adverse Events (AEs), if any, were recorded at each study visit, including the screening visit after the subjects had signed an informed consent, and at all study visits, including the last phone call at 28 days (+5 days) after the last dose of study medication. At each visit, information about adverse reactions and complications of study medication was determined by directly interrogating the subjects, reviewing concomitant medication and vital sign results.

7.2 Observation index

7.2.1 ALSFRS-R (amyotrophic lateral sclerosis functional rating Scale-revision)

ALSFRS-R is a rapid-administration (5 min) sequential rating scale (ratings 0-4) used to determine the subject's assessment of their ability and independence among 12 functional activities. All 12 activities were related to ALS. Initial effectiveness was established by noting that in ALS subjects, changes in the ALSFRS-R score, which correlate with changes in muscle strength over time, correlated closely with quality of life indicators, and predicted survival. The retest confidence for all test items was greater than 0.88. The advantages of ALSFRS-R are that these categories are related to ALS, that it is a sensitive and reliable tool for assessing the functional activities of daily life of patients suffering from ALS, and that it can be administered quickly. With appropriate training, the ALSFRS-R can be administered with high inter-rater and re-confidence. The ALSFRS-R can be administered by telephone with good inter-rater and re-confidence. The equivalence of telephone and face-to-face testing, and the equivalence of study subject and caregiver responses, have also recently been established. Therefore, ALSFRS-R can also be provided to the study subjects by telephone.

7.2.2 pulmonary function test-Chronic Vital Capacity (CVC)

VC (percent predicted normal) was determined using the upright slow Vital Capacity (VC) method. VC can be measured using a conventional spirometer that has been subjected to a calibration check prior to subject testing. For each test phase (session), three VC trials are required, however if the variability between the highest VC and the next highest VC of the first 3 trials is 10% or more, a maximum of 5 trials can be performed. Only 3 best tests were recorded on CRF. The highest VC recorded is used for qualification.

7.2.3 isometric muscle testing (accurate testing of isometric muscle strength of limbs, or ATLIS)

Isometric muscle strength was measured using an isometric limb muscle strength precision testing Apparatus (ATLIS) developed by doctor Patricia Andres at Massachusetts General Hospital (Massachusetts General Hospital). The device is specifically designed to alleviate the reproducibility problems of previous force measurements such as hand-held ergometers (HHDs). ATLIS is independent of experimenter strength and has measurement settings to ensure that subjects are in the same position at each time they test. ATLIS may detect a decrease in function before ALSFRS-R, which may have a ceiling effect, and may be able to detect a change in function with greater sensitivity to ALSFRS-R. This indicator does show little training and therefore includes an indicator at the time of the initial screening visit so that the subject can become familiar with the device.

7.2.4 MR-PET adjunctive study of neuroimaging

A subset of subjects underwent MR-PET scanning at the baseline visit and again between week 12 and week 21 visits. Prior to scanning, each subject of the MR-PET collateral study completed an MR-PET safety questionnaire.

7.2.5 survival assessment

The end of life is considered death, tracheostomy or permanent assisted ventilation.

8. Safety and adverse events

The Adverse Event (AE) definition and reporting procedures provided in this protocol are in compliance with all applicable U.S. Food and Drug Administration (FDA) regulations and international harmonization conference (ICH) guidelines. The site investigator will carefully monitor each subject for possible adverse events throughout the study. All AEs will be recorded on CRFs specifically designed for this purpose. It is also important to report all AEs, especially those adverse events (whether severe or not) that result in permanent withdrawal of the investigational product being studied.

8.1 definition of AES, suspected adverse drug reactions and SAES

8.1.1 adverse events and suspected adverse drug reactions

An Adverse Event (AE) is any adverse and unexpected sign (including, for example, clinically significant abnormal laboratory findings), symptom, or disease (whether or not considered related to the drug or device) temporally associated with the study, use, or use of the drug or device.

Adverse Drug Reactions (ADRs) are all harmful and unintended reactions to drugs associated with any dose. The phrase "response to a drug" means that the causal relationship between the drug and an adverse event is at least a reasonable probability, i.e. such relationship cannot be excluded. Thus, a subset of AEs may be classified as suspected ADRs if there is a causal relationship with the drug.

Examples of adverse events include: new conditions, worsening of the original condition, clinically significant signs of abnormal physical examination (i.e. rash, peripheral edema, etc.), or clinically significant abnormal test results (i.e. laboratory values or vital signs) with the exception of observation indicators that were not recorded as adverse events in this trial (they were being collected but analyzed separately). Stable chronic conditions (i.e. diabetes, arthritis) present before the study began and not worsened during the trial were not considered adverse events. Chronic disease that occurs more frequently (for intermittent conditions) or with greater severity will be considered worsening and will therefore be recorded as an adverse event.

Adverse events are typically detected in two ways:

clinical → symptoms reported by the subject or signs detected at the time of examination.

Adjuvant test → abnormalities in vital signs, laboratory tests and other diagnostic procedures (except for observational indices, whose results are not captured as AEs).

For the purposes of this study, symptoms of progression/worsening of ALS (including "normal" progression) will be recorded as adverse events. The following indicators of disease progression, even if they worsened, were not recorded as adverse events (they are being recorded and analyzed separately): vital capacity results, ALSFRS-R and ATLIS results.

If identifiable at the completion of the AE log, specific diseases or syndromes, rather than individual-related signs and symptoms, should be identified by the field investigator and recorded in the AE log. However, if the observed or reported signs, symptoms, or clinically significant laboratory abnormalities are not considered by the field investigator to be a component of a particular disease or syndrome, they should be recorded as a separate AE in the AE log. Clinically significant laboratory abnormalities, such as those requiring intervention, are those identified by field investigators as such.

Subjects will be monitored for adverse events from the time they sign the consent to their completion of participation in the study (defined as death, withdrawal of consent, loss of visit, premature termination of the study for other reasons or completion of the entire study).

An unexpected adverse event is any adverse event whose specificity or severity is inconsistent with current investigator manuals. An unexpected, suspected adverse drug reaction is any unexpected adverse event for which the site investigator or sponsor (or their designated personnel) believes there is a reasonable likelihood of the investigational product causing the event.

8.1.2 Severe adverse events

Severe Adverse Events (SAE) are defined as adverse events that meet any one of the following criteria:

1. resulting in death.

2. Life-threatening: i.e., pose an immediate death risk when the event occurs.

a. Study Subjects if deemed by the site investigator or sponsorAt the occurrence of AEThis severe criterion applies at risk of instant death. Not applicable if AE is assumed to be more severe and may cause death.

3. Requiring hospitalization or extending existing hospital stays.

a. Hospitalization for selective surgery (including selective PEG tube/g-tube/feeding tube placement) or regularly scheduled treatments is not an SAE according to this standard, as selective or scheduled "surgery" or "treatment" is not an adverse medical event.

4. Resulting in persistent or severe disability or incapacitation.

a. This severe criterion applies if the "disability" caused by the reported AE results in a severe disruption in the subject's ability to fulfill normal life functions.

5. Resulting in congenital abnormalities or birth defects in the offspring of the subject, whether the subject is male or female.

6. Medical or surgical intervention is required to prevent permanent damage to body functions or permanent damage to body structures.

7. Important medical events that may not result in death, be life threatening or require hospitalization may also be considered SAE (when they may be dangerous to the subject based on appropriate medical judgment and may require medical or surgical intervention to prevent one of the outcomes listed in this definition). Examples of such medical events include dyscrasia or convulsions (which do not lead to hospitalization, or the development of drug dependence or abuse).

Hospitalized patient admission without a sudden, ongoing clinical adverse event may meet "severity" criteria, but is not an adverse experience, and therefore will not be considered an SAE. An example of this would include social admission (subjects admitted to the hospital for reasons other than medical, e.g., staying far away from the hospital, with no place to sleep).

A severe, suspected adverse drug reaction (SUSAR) is an SAE for which the on-site investigator or sponsor believes there is a reasonable likelihood of investigating that the product will cause the event. The field investigator is responsible for classifying adverse events as severe or non-severe.

8.2 evaluation and recording of adverse events

The field investigator will carefully monitor each subject for possible AEs throughout the study. All AEs will be recorded on source document templates and ecrfs designed specifically for this purpose. All AEs will be collected and reported in an electronic data acquisition (EDC) system and compiled into reports for regular review by medical monitors. The medical supervisor should review all information relevant to the safety of the investigated product in a timely manner, including all Serious Adverse Events (SAE). Particular attention will be paid to those (whether severe or not) that lead to permanent withdrawal of the investigational product under study.

8.2.1 assessment of adverse events

At each visit (including a telephone interview), subjects will be asked if they have any problems or symptoms since the last visit to determine the occurrence of an adverse event. If the subject reported an adverse event, the investigator will further probe to determine:

1. type of event

2. Date of attack and regression (duration)

3. Severity (mild, moderate, severe)

4. Severity (whether the event meets the above definition of SAE)

5. Causal relationship to investigation of products and diseases

6. Action taken on inspecting product

7. Observation results (outcomem)

8.2.2 correlation of adverse events with investigational products

The relationship of AE to investigational products shall be specified by the site investigator using the following definitions:

1. irrelevant: concomitant diseases, accidents or incidents that are not reasonably associated with treatment.

2. It is unlikely that: there was little or no temporal stratification in response from the application of the investigational product, and/or there was a more likely alternative etiology.

3. Possibly related: the response follows a reasonable time sequence from administration of the investigational product and follows a known pattern of response to the suspect investigational product; the response may be due to an investigational product, or may be due to the clinical status of the subject or other mode of treatment administered to the subject. (suspected ADR).

4. It is likely that: the response follows a reasonable time sequence from administration of the investigational product; confirmed by stopping the investigation of the product or by re-challenge; and cannot be reasonably interpreted by known characteristics of the clinical state of the subject. (suspected ADR)

5. Specifically related: the response follows a reasonable time sequence from administration of the investigational product; it follows a known or expected pattern of response to the investigational product; and confirmed by the improvement of stopping or reducing the dose of the investigational product and the reoccurrence of the response after repeated exposures. (suspected adverse reaction)

8.2.3 adverse events in previous human experiences with each individual component

TUDCA

A small number (> 1%) of subjects receiving TUDCA experienced abdominal discomfort, abdominal pain, diarrhea, nausea, vomiting, itching and rash.

PB

Common adverse events include: irregular menstruation (23%), decreased appetite (4%), sweat-like body taste (3%) and bad taste (3%)

Rare effects (< 2%) included the gastrointestinal tract: abdominal pain, gastritis, nausea and vomiting; constipation, rectal bleeding, peptic ulcer disease and pancreatitis occurred in one subject, respectively.

Hematology: aplastic anemia and ecchymosis occurred in one subject, respectively.

Cardiovascular: arrhythmia and edema occurred in one subject, respectively.

Kidney: renal tubular acidosis

Psychiatric: depression

Skin: rash

O others: headache, fainting and weight gain

Hypoalbuminemia (hyperalburninimia), metabolic acidosis, alkalosis, hyperchloremia (hyperchloremia), hyperuricemia (hyperuricemia), hypokalemia (hypokalemia), hypophosphatemia (hypophosphatemia), hyperphosphatemia (hyperphosphatemia), and hypernatremia have been observed.

8.2.4 recording of adverse events

All clinical adverse events were recorded in the Adverse Event (AE) log in the subject study folder. AE logs should be filled and AE information entered into an Electronic Data Collection (EDC) system within 48 hours after the field learns a new AE or receives an update of an existing AE.

Serious Adverse Events (SAE) must be reported to the medical monitoring and coordination center within 24 hours after the site of SAE is informed.

Entries on the AE log (and EDC) will include the following: the name and severity of the event, the date of onset, the date of regression, the relationship to the investigational product, the action taken and the primary observation of the event.

8.3 adverse events and Severe adverse events-reportable events

The following events are considered reportable events and must be reported to the medical monitoring and coordination center within 24 hours after the event is notified onsite.

All events meeting the above criteria for Serious Adverse Events (SAE)

O dose Change (dose management)

Investigating product pauses, decreases or re-challenges

Product withdrawal in the survey

Key study events:

subject Final treatment

O feeding tube placement

Permanent Assisted Ventilation (PAV)

Tracheostomy

O. death

O. pregnancy

O septum pacing System (DPS) device implant

Emergency or unexpected blind-uncovering event

Permanent Assisted Ventilation (PAV) is defined as non-invasive mechanical ventilation of more than 22 hours per day for more than one week (7 days). The day of onset of PAV was the first of these seven days.

9. Statistical considerations

9.1 statistical methods

Analysis of PROACT and ceftriaxone (ceftriaxone) databases to identify subjects showed that statistical power could be significantly improved by recruiting subjects with ALS with symptom onset <1.5 years and unequivocally diagnosed according to the El Escorial criteria. The mixed effect modeling is used to account for differences between subjects and deviations of subjects from their average rate of decline.

The ability to consider safety and tolerability in three respects: the incidence of Adverse Events (AE), changes in ALFSR-R and ATLIS, and changes in biomarkers such as pNF-H. For 88 subjects receiving treatment, we will have an 80% probability of detecting any adverse event that is expected to occur in at least 2% of the subjects receiving treatment. Based on the single tail test at α ═ 0.05, we will have 80% ability to detect any 28 percentage point increase in the incidence of adverse events relative to placebo. If the proportion of treatment failures (discontinuation of study drug due to adverse events) is less than 40% (80% confidence, single tail), we will consider the dose tolerable. This would occur if 30 or fewer subjects taking AMX0035 failed to complete the 6-month study for 88 subjects receiving treatment. According to this criterion, if the true treatment failure rate is 30%, we will have 80% capacity to declare AMX0035 tolerable at the tested dose.

Preliminary analysis was performed using shared baseline, mixed effects analysis. Covariates for bulbar or other episodes and a second covariate for age at enrollment were included in the analysis. The mixed effect model takes into account both the differences between subjects and the deviations of the subjects from their average rate of decline. The same analysis was also used for clinical observations in this trial. Alpha of 0.05 was used for the test.

9.2 analysis for safety

Safety data were aggregated by treatment group. The therapeutic AE is encoded and ranked using the MedDRA ranking criteria. The incidence on each adverse event and the incidence of grade III/IV adverse events were compared for the treatment groups. Fisher's exact test was used to compare the total number of severe adverse events and abnormal laboratory tests among the groups. Withdrawal, abnormal laboratory tests, vital signs and concomitant drug use were evaluated to characterize the safety profile of the PB and TUDCA combination. Compliance data was determined for each visit and treatment group. The time of subject rejection was compared between treatment groups to better determine tolerability. This is accomplished using a life cycle analysis method that allows for information review due to death. Descriptive statistical data indicating changes from baseline to the last assessment visit with respect to key laboratory parameters and vital signs is also provided.

9.3 analysis for efficacy

An improved analysis of intent-to-treat was performed, including all randomized subjects who received at least one dose of study drug and had at least one primary efficacy assessment after randomization. The slope is derived from the available data and the point in time. Clinical characteristics at baseline and homogeneity of efficacy variables (baseline differences between groups) were assessed between two randomly assigned groups by analysis of variance on continuous variables and chi-squared test on discrete variables. All efficacy endpoints (differences between groups at the end of the study) between the two randomly assigned groups at the end of the study were compared by analysis of the covariance of the continuous variable, adjustment of baseline values and central effects, and by chi-square test on discrete variables. Survival time between treatments was compared by Kaplan-Meier survival analysis.

The primary analysis strategy used a shared baseline, mixed-effects model of the ALSFRS-R rate of progression. The mixed effect model takes into account both the differences between subjects and the deviations of the subjects from their average rate of decline. The same analysis was used for clinical observations in this trial. An alpha of 0.05 was used for the test. An effect magnitude (slowing of the ALSFRS-R slope) of greater than 30% was tested.

9.4 analysis of the population

The improved intent-to-treat (ITT) population included all study subjects who were randomly assigned and received at least one dose of study medication. The ITT population was considered for the primary analysis. For ITT analysis, subjects were grouped based on randomly assigned treatments (without regard to the actual treatments received).

Example 2: open label extension study

To determine the long-term safety of AMX0035 in subjects with ALS, an open label extension study was performed.

Study design and planning

This is a multi-center, open label extension, up to 132-week study to evaluate the long-term safety of AMX 0035. Up to 132 subjects participating in a randomly assigned double blind trial will be able to join the study. The subject will orally (or with a feeding tube) administer an active therapeutic agent pouch twice daily. From the screening/baseline visit, the treatment duration will be as long as one hundred thirty-two (132) weeks. Clinical visits will be made at screening/baseline, week 6 (day 42), week 12 (day 84), week 24 (day 168), week 36 (day 252) and week 52 (day 364), week 68 (day 476), week 84 (day 588), week 100 (day 700), week 116 (day 812), week 132 (day 924).

All visit windows are consecutive calendar days and are calculated from the day the subject started study treatment (day 0, the day of screening/baseline visits). The screening/baseline visit must be performed within 28 days after the 24 week visit of the primary study. If the screening/baseline visit is performed on the day of the 24 th visit or within 7 days after the visit, no assessment, laboratory and observation work is done. If the screening/baseline visit is performed on days 8-28, all assessment, laboratory and observation work needs to be done. The visit window for week 6 and week 12 visits will be +/-10 days, and the visit window for weeks 24, 36, 52, 68, 84, 100, 116 and 132 will be +/-28 days. Any change from this visit window will be considered an out-of-window visit deviation.

Object of study

The main objective of this study was to evaluate the long-term safety of AMX0035 administered orally (or via a feeding tube) via sachets (3g PB and 1g TUDCA) twice daily for use in the same situation.

The main observation indexes are:

1. confirmation of long-term safety of AMX0035 in subjects with ALS over a 132 week period

Secondary observation indicators would include:

1. incidence of key research events including tracheostomy, hospitalization, and death

Progression Rate on ALSFRS-R Scale

ATLIS Rate of progression

4. Rate of progression of slow lung capacity

Research population

This study will be conducted in subjects with sporadic or familial ALS that are diagnosed as unequivocal as defined by the revised El escolar criteria (see example 3). Subjects must provide written informed consent prior to screening. At screening/baseline, subjects must be fully enrolled in a randomized, double-blind trial.

Research registration

Inclusion criteria were:

1. all visits in the randomized, double-blind AMX0035 study were completed. Subjects receiving tracheostomy or PAV during the course of the main study will still be tracked as ITT until the 24 th week visit before registration in OLE.

2. Must be registered in OLE within 28 days after visit at week 24 of the main study.

3. The informed consent was signed to enter the open label extension phase.

Exclusion criteria:

1. study medication was prematurely discontinued in the double-blind phase of the study for reasons other than tracheostomy or PAV.

2. Exposure to or anticipation of the need for any of the non-approved drugs listed below.

3. Any adverse events that are occurring are considered by field investigators to be a clear contraindication for study medication.

4. Unstable hearts or other life-threatening diseases occur during randomized, double-blind studies.

5. The field investigator deems it to interfere with the study and place the subject in any significant medical condition that increases risk.

Subjects who received tracheostomy or PAV in randomized, double-blind trials may choose to be registered in the OLE as long as they completed all visits in the main study.

Drugs that were contraindicated for all subjects included:

HDAC inhibitors comprising:

valproic acid salt

Vorinostat (Vorinostat) (Zolinza)

Romidepsin (Romidepsin)

Xidabenamine (Chidamide)

Panobinostat (Panobinostat)

Lithium ion source

Butyric acid salt

Suramin (Suramin)

Probenecid (Probenecid)

A bile acid sequestrant comprising:

cholestyramine (Cholestyramine) and Light Cholestyramine (Cholestyramine Light)

Hypocholic acid (Questran) and mild hypocholic acid (Questran Light)

Welchol

Colestipol (Colestid) and flavoured colestipol (Colestid Flavored)

Prevalite

Antacids within two hours after study drug administration

Antacids containing aluminum hydroxide or smectite (aluminum oxide) cannot be taken within two hours after study drug administration because they inhibit absorption of TUDCA. These include: alamag, alumina and magnesia, antacids, antacid M and antacid suspensions, Gen-Alox, Kudrox, M.A.H., Malalox HRF and Malalox TC, Magnalox, Madroxal, Mylanta and Mylanta Ultimate, Ri-Mox and Rulox.

Study drug and therapeutic administration

New formulations were used for open label extension, which had been optimized for better taste. A powder filled pouch was used as the AMX0035 drug and the drug was filled in an aluminum foil lined pouch under cGMP conditions.

A pouch containing an active ingredient comprising:

active ingredients:

·1g TUDCA

3g PB (phenylbutyrate)

O excipients

Dextrates (Dextrates)

Sorbitol

Sucralose

Syloid 63FP (colloidal silicon dioxide)

Klepose Linecaps (maltodextrin)

Fragrance masking in Fenmeii (Firmenich)

Fenmeiyi blended berry flavor

Disodium hydrogen phosphate

Sodium stearyl fumarate

Variations from batches used in randomized, double-blind studies include different levels of sucralose, mixed berry flavor provided by new companies, and the addition of flavor masking agents. Study drugs will be provided at the outpatient clinic on the day of the screening/baseline visit and re-supplied at each subsequent visit. Throughout the study, subjects took 2 sachets, 1 sachet in the morning and 1 sachet in the afternoon daily.

Duration of treatment and follow-up

The subject will continue to receive treatment until week 132 or early withdrawal.

Study timetable

Screening/baseline outpatient visit:

the day 0 visit of the open label extension study may be the same as the week 24 visit of the primary study-so that if the examination and testing is completed before, no repetition is required.

The following procedure will be performed:

obtaining written informed consent of the subjects

Evaluation of inclusion and exclusion criteria

Review and record concomitant medications and therapies

O management C-SSRS (Baseline version)

O. management of the ALSFRS-R questionnaire

Lung function tests, including Slow Vital Capacity (SVC), were performed noting that height should be recorded from the primary study screening visit.

Using ATLIS machine to measure isometric muscle force

Assessment and recording of Adverse Events (AE) after Subjects signed an Informed Consent Form (ICF)

Measuring vital signs (blood pressure, heart and respiration rate, body temperature and weight)

Carrying out a 12-lead ECG (electrocardiogram)

O [ post-other tests ] blood samples were collected for clinical laboratory evaluation, including hematology (CBC with differential test), complete blood biochemical examination, liver function test, serum pregnancy test (for fertility women [ WOCBP ]), optional DNA analysis (if not completed during primary learning)

O collecting urine samples for urine analysis

Divide 2 study drugs in kit (12 weeks + 2 additional weeks)

Acquisition of Key research events

Scheduled visit at week 6

Week 6, week 12, week 24, week 36, week 52, week 68, week 84, week 100, week 116, week 132 or prior discontinuation/last safety clinic visit:

from the time specified in the activity schedule (table at the beginning of this section), visits at weeks 6 and 12 will be made within +/-10 days, and visits at weeks 24, 36, 52, 68, 84, 100, 116 and 132 will be made within +/-28 days.

The following procedure will be performed:

review and evaluation of adverse events

Measuring vital signs

Manage C-SSRS questionnaire (since last visit)

Management of the ALSFRS-R questionnaire

O performing pulmonary function tests, including Slow Lung Vital Qi (SVC)

Using ATLIS machine to measure isometric muscle force

Carrying out a 12-lead ECG (electrocardiogram)

Collect blood samples for clinical laboratory evaluation, including hematology (CBC with differential testing), complete blood biochemical examination, liver function testing, optional DNA analysis (if not completed during the main study period)

O collecting urine samples for urine analysis

O performing a study drug accountability system

Dispensing study medication (except at week 132/stop-ahead)

Acquisition of Key research events

Schedule the next study visit (except at week 132/stop at early days)

Laboratory testing

For safety reasons, the following laboratory tests will be performed:

hematology with classification check: whole blood cell count with classification (hematocrit, hemoglobin, platelet count, RBC index, total RBC, total WBC, and WBC & classification)

Blood biochemical examination/Liver Function Test (LFT): alanine aminotransferase (ALT (SGPT)), aspartate aminotransferase (AST (SGOT)), albumin, alkaline phosphatase, bicarbonate, blood urea nitrogen, calcium, chloride, creatinine, glucose, potassium, sodium, total bilirubin, and total protein

Urine analysis: bilirubin, blood, clarity, color, glucose, ketones, nitrate, pH, protein, specific gravity, urobilinogen and WBC screening

Serum human chorionic gonadotropin (hCG) (WOCBP) for fertile women (collected only at the screening visit and as needed throughout the study)

Example 3: el Escorial world neurological Association standards for ALS diagnosis

Information is obtained from the following websites: www.wfnals.org are provided. Diagnosis of amyotrophic lateral sclerosis [ ALS ] requires:

A-presence:

(A: 1) evidence of degeneration of Lower Motoneurons (LMN) was found by clinical, electrophysiological or neuropathological examination,

(A: 2) finding evidence of degeneration of Upper Motoneurons (UMN) by clinical examination, and

(A: 3) progressive transmission of symptoms or signs within one area or to other areas as determined by medical history or examination, along with

B-is absent:

(B: 1) electrophysiological and pathological evidence of other disease processes that may explain signs of LMN and/or UMN degradation, and

(B: 2) can explain neuroimaging evidence of other disease processes observed in clinical and electrophysiological signs.

Clinical study of the diagnosis of ALS

Careful medical history, physical and neurological examinations must look for clinical evidence of UMN and LMN signs in four areas of the central nervous system [ CNS ] [ brainstem, cervical, thoracic or lumbosacral spinal ] (see table 1). The test should be aided by the rational application of clinical instructions to exclude other disease processes. These would include electrical diagnostics, neurophysiology, neuroimaging and clinical laboratory studies. Clinical evidence for regression of LMN and UMN is required for the diagnosis of ALS. Without pathological confirmation, clinical diagnosis of ALS can be classified by clinical assessment alone into different levels of certainty, depending on the presence of both the UMN and LMN signs in the same anatomical region of the brain stem [ globo-cranial motor neurons ], cervical, thoracic or lumbosacral spinal cord [ anterior horn motor neurons ]. The terms clinically definite ALS and clinically likely ALS are used to describe the clinical diagnostic certainty of these categories based only on clinical criteria:

A. Clinically clear ALS is defined only by the presence of UMN in three areas and LMN signs, based on clinical evidence.

B. Clinically likely ALS is defined only by the signs of UMN and LMN in at least two areas according to clinical evidence, with some signs of UMN necessarily being on the upper side (above) of the signs of LMN.

C. Clinically likely ALS-laboratory supported is defined when clinical signs of UMN and LMN dysfunction are only in one area or when only UMN signs are present in one area and LMN signs defined by EMG standards are present in at least two limbs, with neuroimaging and clinical laboratory protocols applied appropriately to exclude other causes.

D. Clinically probable ALS is defined when clinical signs of both UMN and LMN dysfunction are found in only one area or when signs of UMN are found separately in two or more areas; or LMN signs were found to be on the top of the UMN signs, and the clinically likely-laboratory supported diagnosis of ALS cannot be confirmed by clinical evidence in combination with electrical diagnosis, neurophysiology, neuroimaging, or clinical laboratory studies. Other diagnoses must be excluded to accept a diagnosis of clinically likely ALS.

TABLE 1

Example 4: ALS function rating Scale-revision (ALSFRS-R)

Example 5: analysis of test results

Test participants

To improve statistical power to detect differences in the rate of decline in the amyotrophic Lateral sclerosis functional rating scale revision (ALSFRS-R), we defined inclusion criteria to recruit individuals diagnosed with clear ALS within 18 months after symptom onset and as described by the revised El Escorial criteria (i.e., clinical evidence of both upper and lower Motor Neuron signs in at least three body regions) (see, e.g., Brooks et al, amyotrophic later latex injector nerve disease 2000; 1: 293-9). These criteria were selected to select a population of participants with rapidly progressing ALS based on analysis of a historical cohort from a previously conducted clinical survey (section 2.1, below). Such a selection has two potential benefits: one is to reduce the heterogeneity of disease progression rates among participants, thereby improving statistical power, and two is to select for populations with disease progression faster than average, thereby allowing for more rapid efficacy analysis.

Additional eligibility criteria include age 18 to 80 years; slow Vital Capacity (SVC) exceeds 60% of the predicted value of an individual's age, gender and height; and either no riluzole was used at the beginning of the experiment or a stable dose of riluzole was used at least 30 days prior to screening. After edaravone was marketed in 2017 at 8 months, the protocol was modified to allow edaravone to be used before and during the trial.

Exclusion criteria included the presence of a tracheostomy or diaphragm pacing system, history of ALS clinical trials actively involved in evaluating experimental small molecules within 30 days post-screening, and any of the following exposures within 3 months prior to screening: sodium phenylbutyrate, tauroglycol or koala diol (or any of these individual agents previously planned for use during the course of the test); any investigational cell or gene therapy at any time; or monoclonal antibodies within 90 days prior to screening.

Assay intervention and procedure

Eligible participants were randomly assigned to receive sodium phenylbutyrate/taurogluciol (AMX 0035; 3g sodium phenylbutyrate and 1g taurogluciol per sachet) or matched placebo either once daily, orally or via a feeding tube for the planned 24 week duration (for detailed information on random assignment and drug administration, see sections 2.2 and 2, 3, respectively, below). The two drug combinations and placebo are provided in the form of single use sachets as powders dissolved in room temperature water prior to administration. These powders were constructed to be identical in appearance, dissolution and taste. Participants were instructed to take one sachet per day for the first 3 weeks, followed by two sachets per day if tolerated (one sachet in the morning and one sachet in the evening). Outpatient or telephone visits were made at baseline and every 3 to 24 weeks thereafter, with the last telephone follow-up at week 28 (table 2). Participants who completed the randomized double-blind trial were eligible to participate in an open label extension trial evaluating the long-term safety of sodium phenylbutyrate/tauroglycol (NCT03488524) for up to 132 weeks.

^a The baseline visit was set to be conducted no more than 42 days after the screening visit.

^b The last safety call was made 28 days (+5 days) after the participants took the last dose of test drug (whether or not the participants discontinued the test) to assess adverse events and concomitant drug changes and to manage ALSFRS-R. Only participants not registered in the OLE need this phone.

^c Vital signs include systolic and diastolic blood pressure in mm Hg, respiratory rate/min, heart rate/min and body temperature.

^d Standard neurological examination was used for all participants. The upper motor neuron load scale was included only for MR-PET adjunctive tests and was administered at the time of scanning.

^e Physical examination included height and weight. Height was measured only at the screening visit.

^f Safety laboratories include hematology (CBC with classification test), complete blood biochemical tests, liver function tests, and urinalysis. Serum pregnancy tests were performed at the time of the screening visit and, if necessary, during the course of the trial in WOCBP.

^g The C-SSRS baseline version is completed only at baseline visits. The C-SSRS is completed in all other visits since the last visit version.

^h Approximately 20 participants performed MR-PET scans at selected sites. The first scan occurred prior to the baseline visit (pre-dose) and the second scan occurred between trial visits at weeks 12 and 21. Participants who underwent MR-PET also provided a blood sample for peripheral blood mononuclear cell extraction prior to each MR-PET scan.

ⁱ Participants provided blood samples for biomarker testing and storage in biological repositories.

^j All participants provided blood samples for PK testing at baseline visit (pre-dose). Participants also provided blood samples at 1 or 4 hours post-dose (10 min window per time point) at week 12 and week 24 visits. PK times were randomly assigned such that each participant had a 1 hour draw at one visit and a 4 hour draw at another visit.

^k PK samples were not drawn for early terminated participants.

^l If a baseline visit has been made or no sample has been collected, DNA is obtained at the next available visit. This is a one-time collection.

^m Adverse events that occurred after signing the consent were recorded.

ⁿ For participants in the MR-PET adjunctive trial only, blood was drawn at the participants' sites during the screening visit for TSPO testing.

^o Random assignments were made at baseline visit. Random assignment requires the participant's kit number to be entered into the data acquisition system.

^p After all baseline visit procedures were completed, the first dose of test drug was administered at the outpatient clinic.

^q Subjects were instructed to increase from one to two sachets per day (if tolerated).

Observation of

The primary efficacy observation was the rate of decline (slope) of the total ALSFRS-R score from baseline to the end of the week 24 trial. ALSFRS-R consists of 12 items of four sub-domains related to body function (medulla oblongata, fine movement, gross movement and respiration), each of which is rated in order (0 ═ complete loss of function, 4 ═ no loss of function, max 48, lower scores indicate greater difficulty in function) (see, e.g., Cedarbaum et al, J Neurol Sci 1999; 169: 13-21). The scale has been verified to be administered in person or by telephone and has shown high inter-and intra-rater reliability. The rate of decline of the ALSFRS-R subdomain score was evaluated as exploratory efficacy observations. Secondary clinical efficacy observations (in hierarchical order) include the rate of decline in isometric muscle strength as measured by the limb isometric muscle strength Accurate Test (ATLIS) apparatus; the rate of SVC degradation; and the incidence of death or death-equivalent events (tracheostomy or permanent assisted ventilation (> 22 hours per day for > 7 days) over the duration of 24-week treatment, tracheostomy only and hospitalization (except for elective surgery) (see, Paganoni et al, Clin Investig (Lond) 2014; 4: 605-18). Pharmacokinetic analysis was also included as a pre-assigned secondary observation. Changes in blood levels of phosphorylated neurofilament heavy chain protein (biomarker of motor neuron degeneration) from baseline to week 24 were assessed as a secondary biological observation (see, Poesen et al, Front Neurol 2019; 9: 1167).

The ATLIS device was used to evaluate isometric muscle strength of six upper and six lower limb muscle groups, each muscle group being tested in triplicate. Raw values are normalized to a Percentage of Predicted Normal (PPN) force based on age, gender, weight, and height (see, e.g., Andres et al, Muscle Nerve 2013; 47: 177-82). The normalized PPN scores for the highest recorded force for each muscle group were averaged to yield a total score, an upper summary score, and a lower summary score (more detailed information about ATLIS is provided in section 2.4 below). Respiratory muscle function was assessed by SVC, with at least three trials measured in the upright position, or a maximum of five trials with the highest and next highest of the current three measurements differing by 10% or more, each assessment. SVC volume was normalized to PPN based on age, gender and height. The highest SVC score recorded from all attempts was utilized for analysis.

Safety was assessed via recording adverse events (TEAE) of treatment appearance at each trial visit. Symptoms of ALS progression (including symptoms consistent with disease progression) were recorded as TEAE. Any deterioration in disease progression indicators (i.e., ALSFRS-R, ATLIS and SVC) recorded and analyzed alone was not recorded as TEAE. Test drugs were considered tolerable if the proportion of participants who stopped the drug by TEAE was below 40% (with 80% confidence, single tail).

Compliance with the test drugs (adherence) was assessed by returning participants to their empty and unused pouches at each outpatient visit. Compliance is defined as taking more than 80% or less than 125% of the expected test medication as determined by the pouch count.

Table 3: test drug compliance

Mean ± SD.Compliance was calculated as number of empty pouches returned/total number of pouches (empty + unused).

The exit questionnaire was administered at the last trial visit (week 24 or early termination) to assess the blindness (blinding) of treatment distribution by participants and investigators by asking whether they considered the participants to be receiving active treatment or placebo.

Statistical analysis

To calculate the sample size, we analyzed the first 6 months of data from participants in a large historical trial (ceftriaxone trial) that met the criteria for rapid progression described previously. Assume that a shared baseline, mixed-effect regression model is used with no model covariates added. This analysis found that tracking approximately 131 participants over 6 months with a random distribution ratio between treatment and placebo of 2: 1, when tested at a bilateral alpha of 0.1, would provide 80% ability to test 30% treatment effect on the ALSFRS-R total score. It is expected that terms included in the model for pre-baseline ALSFRS-R slope and age (as covariates for slope over time), and increasing the frequency of the increased assessments (nine assessments in CENTAUR within 6 months versus four assessments in 6 months in ceftriaxone trial) would add additional capacity, allowing for the use of a pre-specified bilateral alpha level of 0.05.

Safety analyses were performed in a safety population consisting of all participants who received at least one dose of the test drug. The main population for efficacy analysis was the modified intent-to-treat (mITT) population, which consisted of all participants who received at least one dose of the test drug and had recorded at least one total ALSFRS-R score after randomized assignment. Post-hoc analyses were also performed on the intent-to-treat (ITT) population, including two participants in the active agent group who had not been evaluated for post-baseline efficacy and who were excluded from the mITT population. Additional pre-specified efficacy analyses were performed in drug-treated populations consisting of all participants in the mITT population, but excluding data from any trial visits occurring more than 30 days after termination or temporary interruption of the trial drug, and excluding one participant who could not confirm administration of any trial drug.

A hierarchy is prepared for secondary observations for inferential testing. ATLIS is the first secondary observation in the hierarchy and comprises three individual measurements (upper, lower and total scores), wherein no hierarchy is assigned for individual ATLIS measurements. Due to its lack of hierarchy, our post hoc decision was to report an unadjusted 95% confidence interval for the three ATLIS measurements.

Absolute scores for all sustained efficacy observations were analyzed using a random-slope, shared baseline, linear mixed model adjusted for age and pre-baseline ALSFRS-R slope (rate of decline of total ALSFRS-R score from onset of ALS symptoms to baseline) two covariates that have been shown to correlate with historical data (see, e.g., Labra et al, J neuro neurosurry 2016; 87: 628-32; Daghlas et al, amytoph latex front degenerator 2018; 19: 206-11; taylol et al, Ann Clin Transl Neurol 2016; 3: 866-75). Interaction terms between time and age and time and pre-baseline ALSFRS-R slope were included, reflecting our interest in slope differences. The analysis to validate the linear model is described in section 2.5 below. A post hoc hybrid model with classified visits instead of continuous time is performed to generate separate estimates at each time point in order to visualize the visit data over time (fig. 1A and 1B). These estimates assume that the mean levels of baseline covariates were the same for both treatment groups.

Figures 1A and 1B show the estimated rate of decline of the total ALSFRS-R score over 24 weeks (main observations). Figure 1A shows the estimated treatment-dependent decline rate of the total ALSFRS-R score in the main analyzed mITT population (solid line sodium phenylbutyrate/tauroglycol, dashed line placebo; lines directly above and below each line reflect standard errors of plus or minus one). Superimposed on the estimated slope from the primary analysis are visit specific estimates (and standard error bars) from a post-hoc shared baseline, repeat-index hybrid model that utilizes the same adjusted but unstructured covariance between the classification time and the repeat index. Figure 1B shows estimates from the same pair of models applied to the dosing population. In the main model, the mean slopes of the total ALSFRS-R score were-1.24 min/month and-1.66 min/month for the active drug (sodium phenylbutyrate/tauroglycol) and placebo, respectively (difference 0.42 min/month; 95% CI, 0.03 to 0.81; P0.03). The results were similar in the dosing analysis, where the mean slopes of the total ALSFRS-R score were-1.22 min/month and-1.68 min/month for the test drug and placebo, respectively (difference 0.46 min/month; 95% CI, 0.05 to 0.87; P0.03). ALSFRS-R stands for amyotrophic lateral sclerosis functional rating scale revision, ANOVA for ANOVA, and mITT for treatment of intent to improve.

The pre-assigned primary model assumed that the baseline scores were the same for the active and placebo groups. A post hoc analysis of changes from baseline was performed without making this assumption (fig. 4). This analysis was performed ex post facto for all consecutive observations in the mITT population. Only significant P values are reported according to a pre-specified observation hierarchy order.

In addition to assessing the rate of decline of the ALSFRS-R total score, as an alternative way of expressing functional gain, the relative percentage of functional retention based on time was assessed in a post hoc analysis. The functional retention was calculated using the following formula, which introduces the time required for the total ALSFRS-R score to drop by 1 point:

table 4: time-based function retention

Of the participants in the mITT population, the overall ALSFRS-R score after 24 weeks of treatment for those receiving sodium phenylbutyrate/tauroglycol was the same as for the placebo group at week 18, corresponding to a 6-week increase in retained function.

The Cox proportional hazard model was used to analyze the rate of death, death equivalent events (including tracheostomy) and hospitalization, using the covariates of the pre-baseline ALSFRS-R slope and age at baseline. The inferential test is based on a likelihood ratio (likelihood ratio) test.

The primary efficacy analysis used all available baseline and post-baseline data for all participants in the mITT samples, including those who stopped the trial drug but continued to participate in the trial. For these analyses, no interpolation was performed on missing data. Additional details regarding the processing of missing data are provided in section 2.5 below. In addition to the post-hoc ITT analysis described above, a pre-specified sensitivity analysis was also performed to evaluate the impact of all missing data, specifically data missing due to death or death-equivalent events, and the concomitant use of riluzole, edaravone, or both, on the primary analysis (section 2.5 below). A post-event joint-level analysis was performed in the safety population to introduce all survival events into a functional analysis (ALSFRS-R) to provide adjusted estimates that take into account potential bias due to death.

Analysis was performed using SAS (version 9.4, SAS Institute, Cary, NC). For a two-tailed P ≦ 0.05, the test was declared significant. Using chi-square statistics, the proportion of treatment (active, placebo or missing) estimated to be assigned by participants and investigators based on their exit questionnaire responses was compared within each treatment group. The main reasons for their estimates are also summarized proportionally.

Results

Test participants

A total of 177 were screened, of which 137 were randomly assigned to sodium phenylbutyrate/tauroglucenediol (n-89) or placebo (n-48) (fig. 2). All randomly assigned participants received their assigned medication and all but one confirmed the start of treatment. Two participants in the sodium phenylbutyrate/taurine diol group, who died soon after random assignment, were not evaluated for post-baseline efficacy and were excluded from the mITT population, but included in the safety population and post-ITT analysis. In summary, in the mITT population, 77% of the participants in the placebo group and 69% of the participants in the sodium phenylbutyrate/taurine diol group completed the test for the indicated drug (figure 2). However, one participant in the placebo group who stopped the test drug before the end of the trial and seven participants in the sodium phenylbutyrate/taurine diol group completed the planned 24-week follow-up and the mITT analysis included all of the data available to them.

Baseline demographics and disease characteristics are summarized in table 5. The pre-baseline mean ALSFRS-R slope with prognostic utility in ALS was 0.93 min/month in the placebo group and 0.95 min/month in the sodium phenylbutyrate/taurine diol group. The mean baseline ALSFRS-R total scores in the placebo and sodium phenylbutyrate/taurine diol groups were 36.7 and 35.7, respectively. Most (77%) of the participants received riluzole or edaravone at or before the start of the trial, with 28% of the participants receiving both; a larger proportion of the participants (50%) in the placebo group received edaravone at or before the start of the trial compared to the sodium phenylbutyrate/taurine diol group (25%). A larger proportion of participants in the sodium phenylbutyrate/taurine diol group had bulbar onset ALS (30%, relative to 21% in the placebo group).

Table 5: baseline demographics and disease characteristics (mITT population).)

Positive and negative values are mean ± SD.At or before the start of the test.The highest score for the total ALSFRS-R score was 48 points and the highest score for each subdomain was 12 points. Normalized to PPN strength based on gender, age, weight, and height. ALS means amyotrophic lateral sclerosis, ALSFRS-R means amyotrophic lateral sclerosis function rating scale revision, ATLIS means limb isometric accurate testing, BMI means body mass index, mITT means modification intent to treat, PPN means percentage of predicted normal value, and SVC means slow vital capacity.

Main observation

The estimated mean slopes of the total ALSFRS-R score in the mITT population were-1.24 min/month and-1.66 min/month for the active drug and placebo, respectively (difference 0.42 min/month; 95% confidence interval [ CI ], 0.03 to 0.81; P0.03) (fig. 1A, 1B, 3A and 3B). Pre-assigned medication analysis (excluding data from any visits occurring more than 30 days after cessation of the test drug or more than 30 days after temporary drug discontinuation) yielded similar results, namely-1.22 min/month and-1.68 min/month for the test drug and placebo, respectively (difference 0.46 min/month; 95% CI, 0.05 to 0.87; P0.03) (fig. 1A and 1B).

To support the primary mITT analysis in centraur, a post-hoc ITT analysis was performed, including two participants in the active group who did not receive a post-baseline efficacy assessment and were therefore excluded from the mITT population. The ITT analysis, including all 137 randomly assigned participants, yielded the same results as the primary mITT analysis within the rounding error (table 6). The secondary observation was the same within rounding errors for the ITT and mITT analyses, which, in addition to the time-to-live analysis, included participants in the sodium phenylbutyrate/taurine diol group that died shortly after random distribution.

Table 6: post hoc ITT primary observation analysis

LS represents the mean or difference adjusted for the items in the model.Only significant P values are reported according to the pre-specified hierarchical order of observations.

Only significant P values were reported according to the pre-specified hierarchical order of observations.

Post hoc joint grade analysis of function and survival was significant (P ═ 0.01), indicating that the primary observation analysis was not affected by death (fig. 5). Fig. 5 shows the results from the sensitivity analysis: combination grade, missing data, concurrent events and concomitant medication time. Mut population.LS represents the mean or difference adjusted for the terms in the model. The joint rank analysis results are reported here as rank divided by 8, so that the results will be on a similar scale as those presented for ALSFRS-R. Average week number of riluzole is 17.86.The average number of cycles of edaravone was 10.50. The average number of cycles of riluzole and edaravone is 8.79.

The primary analysis for all consecutive observations was a random slope, linear mixed model (adjusted for age and pre-baseline ALSFRS-R slope) that assumed a shared baseline between the active and placebo groups. Analysis of relative baseline changes without this assumption was performed post hoc on all consecutive results in the mITT population. The results of the analysis of post-hoc relative baseline changes for both the mITT and the drug-dosed population are shown in fig. 4. Only significant P values are reported according to the pre-specified hierarchical order of observations. The results in the mITT population were similar to the primary observation model (-1.21 points/month in the active group versus-1.74 points/month in the placebo group; difference 0.53 points/month; 95% CI, 0.13 to 0.93; P0.01), indicating that the primary observation analysis was not affected by the use of shared baseline. The results of the analysis of post-hoc time-based functional retention in the mITT and drug-dosed populations are presented in table 4.

The results for the various subfields of the ALSFRS-R are shown in FIG. 6. LS represents the mean or difference adjusted for terms in the model. The highest score for each subdomain was 12. Sensitivity analysis considering missing data, concurrent events, and time associated with riluzole, edaravone, or both is summarized in fig. 5.

Secondary observation

Figures 7A-7D show secondary observations for ATLIS and SVC. Fig. 7A-7C show the treatment-dependent decline rates of total score, upper and lower ATLIS scores in the mITT population, respectively, while fig. 7D shows similar results for SVC (solid line sodium phenylbutyrate/tauroglycol, dashed line placebo; lines directly above and below each line reflect standard errors of plus or minus one). Superimposed on the estimated slope from the primary analysis are visit specific estimates (and standard error bars) from a post-hoc shared baseline, repeat-index hybrid model with the same adjustments but also with unstructured covariance between the classification time and the repeat index. The mean reduction rate of total ATLIS score was-3.03 PPN/month and-3.54 PPN/month for active treatment and placebo, respectively (difference 0.51 PPN/month; 95% CI, -0.12 to 1.14) (fig. 3, fig. 7A). The difference between groups (active treatment minus placebo) in the mean reduction rate of upper and lower limb ATLIS scores were 0.77 PPN/month (95% CI, 0.03 to 1.52) and 0.38 PPN/month (95% CI, -0.40 to 1.16), respectively (fig. 3, fig. 7A).

The mean rates of decline in SVC were-3.10 PPN/month and-4.03 PPN/month (difference 0.93 PPN/month; 95% CI, -0.10 to 1.95) for active treatment and placebo, respectively (fig. 3, fig. 7D). The proportion of participants who underwent death, tracheostomy (the only equivalent event of death in the trial) and hospitalization is summarized graphically in figure 8. FIG. 8 is a Kaplan-Meier plot of cumulative deaths, tracheostomies, and hospitalization events. General observations were defined as death, death-equivalent events (which consisted of tracheostomy of only one participant in the trial) or hospitalization, whichever occurred first. Survival status was obtained for all participants at their respective week 24 visit; thus, none of the data presented in the figure has been pruned.

The cumulative risk ratio for any of these three events was 0.53 (95% CI, 0.27 to 1.05) in the active treated group relative to placebo (fig. 3). Similar to the primary observations, all secondary observations were also identical within rounding errors for the ITT and mITT analyses, which in addition to the survival analysis included participants in the sodium phenylbutyrate/taurine diol group described above who died shortly after random distribution (table 6, table 12).

Safety and tolerability

Almost all participants (sodium phenylbutyrate/tauroglycol, 96%; placebo, 97%) reported one or more TEAEs during the trial. Most did not result in modification or discontinuation of drug administration for the test drugs and were not considered treatment-related (Table 7; see Table 8 for a complete list of relevant TEAEs).

Table 7: summary of adverse events occurring during treatment

Safety population included all participants who received at least 1 dose of test drug.

AEs reported by investigators include ECG abnormalities and symptoms such as heart beat and palpitations. For more detailed information on the center reading of an ECG abnormality, see Table S6 in the supplementary appendix. AE denotes adverse events, ECG denotes electrocardiogram, MedDRA denotes medical supervision activity dictionary, SOC denotes system organ categories, TEAE denotes adverse events occurring during treatment.

Table 8: adverse events in treatment

Relative to the placebo group, occurred more than 2% frequently in the sodium phenylbutyrate/taurine diol group.

Relative to the sodium phenylbutyrate/taurine diol group, this occurred more than 2% frequently in the placebo group.

The events that occurred with greater (≧ 2%) frequency in the sodium phenylbutyrate/taurine diol group were mainly the gastrointestinal tract (i.e. diarrhea, nausea, hypersalivation and abdominal discomfort); except for hypersalivation, all are known adverse events associated with tauroglycol (one of the active compounds in sodium phenylbutyrate/tauroglycol). Gastrointestinal events in the sodium phenylbutyrate/taurine diol group were most frequently reported in the first 3 weeks, and then decreased to below the placebo group for the remainder of the trial (figure 9). The sodium phenylbutyrate/taurine diol group (3% and 9%, respectively) experienced a reduction in drug dose and withdrawal due to gastrointestinal events more frequently than in the placebo group (0% and 2%, respectively). The mean change in body weight over 24 weeks from baseline was not significant in any of the groups and there was no difference between groups. Digital electrocardiograms were collected at baseline and repeated at weeks 12 and 24, with central evaluation detecting asymptomatic electrocardiographic changes, which included left anterior branch block, left bundle branch block, and non-specific T-wave changes in a total of three (6%) participants in the placebo group and seven (8%) participants in the sodium phenylbutyrate/taurine diol group, with minimal clinical significance (table 9). The corrected QT interval remained stable and did not differ significantly between the active and placebo groups at any time point.

Table 9: summary of electrocardiographic results appearing in treatment

2 (4%) participants and 5 (6%) participants who received sodium phenylbutyrate/taurine diol in the placebo group developed fatal TEAEs. Two of these deaths (both in the sodium phenylbutyrate/taurine diol group) did not occur in the mITT population, as these deaths occurred without a second assessment of ALSFRS-R. No mortality was considered to be associated with the test drug. The most common cause of death in general is respiratory failure, accounting for 4 of 7 deaths, consistent with a natural history of ALS. One (2%) of the participants in the placebo group experienced a death equivalent event and none of the participants in the sodium phenylbutyrate/taurine diol group experienced a death equivalent event. Severe adverse events in the placebo group were more frequent than in the sodium phenylbutyrate/tauroglycol group (19% versus 12%, respectively), mainly due to a higher incidence of respiratory events (8% for placebo; 3% for sodium phenylbutyrate/tauroglycol).

Nineteen percent of participants in the sodium phenylbutyrate/taurine diol group discontinued the test drug prematurely due to TEAE, compared to 8% in the placebo group. The most common (≧ 5%) TEAEs that led to discontinuation of the trial drug were diarrhea (6% sodium phenylbutyrate/tauroglycol; placebo, 0%) and respiratory failure (0% sodium phenylbutyrate/tauroglycol; placebo, 6%).

The test drug compliance data is summarized in table 3. The exit questionnaire outputs are summarized in table 10 and table 11. For participants taking active medications, the investigator correctly guessed the participant at 49.4% of the time to take the medication, while the participant correctly guessed the time at 43.8%. For participants who took placebo, the time for the investigator to guess correctly was 39.6%, while the time for the participants to guess correctly was 62.5%. Participants considered that the most common cause of their placebo was no improvement in symptoms or disease progression. The discriminatory power of participants and investigators for the estimated treatment groups was not statistically different between the active and control groups (P > 0.05, chi-square test).

Table 10: estimation of treatment allocation on exit questionnaire

Table 11: reason for quitting questionnaire response

TABLE 12

After 24 weeks, all patients may choose to continue the activity and 86% of the patients catch the opportunity. Long-term survival analysis showed that patients initially randomized to active (drug administration started 24 weeks ago) showed significant survival benefit (fig. 10).

The CENTAUR test showed that Treatment with a co-formulated fixed dose sodium phenylbutyrate/tauroglycol slowed the rate of decline in participants with ALS as assessed by the ALSFRS-R total score, a measure of function in daily activities (see Amyotrophic Crystal patients: development Drugs for Treatment-guidelines for Industry.) Washington, DC: US Food and Drug Administration, 2019 months). After 24 weeks, there was an absolute difference between the mean ALSFRS-R total scores of the two groups, estimated to be 2.32-points, when assuming equivalent baseline scores. Without this assumption made in the post analysis, the estimated difference between groups was 2.92 points. ALSFRS-R has been shown to be associated with survival and quality of life, and each point reduction represents a loss of ability for important daily functions. Notably, sodium phenylbutyrate/tauroglycol treatment resulted in a slowing of disease progression in a population in which many participants had received standard care approved therapy for ALS (riluzole, edaravone, or both) during their participation in CENTAUR.

In view of the variability of ALS disease progression, capturing any changes on ALSFRS-R requires large sample volumes and long follow-up durations to obtain sufficient statistical power (see, e.g., Rutkove Neurotherapeutics 2015; 12: 384-93). Thus, CENTAUR was designed to introduce two key inclusion criteria, namely explicit ALS by the revised El Escorial criteria and symptom onset within 18 months after the start of the trial, with the aim of improving statistical power by reducing heterogeneity and excluding those that are unlikely to progress during the trial. The mean decrease in total ALSFRS-R score in placebo group in CENTAUR was-1.66 min/month. In comparison, the mean reduction in overall ALSFRS-R score in placebo-treated participants ranged from-1.06 to-1.22 cents/month in other datasets not selected for the rapidly progressing population (see, e.g., Cudkowicz et al, Lancet Neurol 2014; 13: 1083-91; Cudkowicz et al, Lancet Neurol 2013; 12: 1059-67; van Eijk et al, Clin epidemol 2018; 10: 333-41), and the mean reduction in overall ALSFRS-R score ranged from-1.41 to-1.67 cents/month when selected for the rapidly progressing participants in these same datasets using the CENTAUR standard (see Archibald et al, Amyophtral dental front tissue der (amyotrophic Lateral degeneration) 2013; temporal lobe 2017).

Both the FDA and the revised Airlie House consensus guidelines (Airlie House consensus guidelines) identify functional scales such as ALSFRS-R as suitable primary observations in ALS trials (see, e.g., van den Berg et al, Neurology 2019; 92: e1610-e 23). However, there are a number of important considerations with regard to ALSFRS-R. Given the heterogeneity of progression in ALS, the decline in ALSFRS-R may not be linear. The primary model in the current trial is assumed to be linear over time based on historical clinical trial data (see, Proudfoot et al, Amyotroph Lateral Scler Frontotemporal Degenerer 2016; 17: 414-25). A pre-specified sensitivity analysis was performed to assess whether a linear hypothesis was required and whether the data met the criteria for applying this hypothesis. Finally, functional outcomes such as ALSFRS-R can also be confounded by data loss resulting from participant withdrawal or death. In the current trial, the joint rank test was performed as a comprehensive analysis of function and survival and did not show a bias in the estimates of primary function observations due to data loss resulting from participant death. Additional sensitivity analyses were performed to account for missing data and death or death-equivalent events, and yielded results similar to the primary analysis.

Treatment with sodium phenylbutyrate/tauroglycol resulted in a slower progression slope of the total ALSFRS-R score over 24 weeks with 0.42 min/month difference between groups in participants with rapidly progressing ALS. No significant inter-group differences in secondary observations were observed based on the predetermined hierarchy for these observations. Sodium phenylbutyrate/tauroglucinol is associated with a higher incidence of TEAE-related withdrawal.

Section 2.1 selection method for rapidly progressing population

ALS patients enrolled by the CENTAUR were within 18 months since symptom onset and were diagnosed as definite ALS as described by the revised El Escorial criteria (i.e., clinical evidence of upper and lower Motor Neuron signs in three body regions) (Brooks et al, amyotrophic late cutter Motor Neuron disease) 2000; 1: 293-9). This selection of participants was derived from analysis of data from the PRO-ACT (the largest available database of anonymous clinical trial records from more than 10,000 ALS patients; available at https:// nctu. partners. org/ProACT) and from the ceftriaxone trial in ALS (Cudkowicz et al, Lancet Neurol 2014; 13: 1083-91), which produced a rapid, predictable, and relatively evenly progressing cohort.

Section 2.2 randomization procedure

The randomization was generated by the blinder statist using a SAS (SAS Institute, Cary, NC) computer. Using a permutation block structure with three and six blocks and no additional stratification, eligible participants were randomly assigned at a 2: 1 ratio to receive either sodium phenylbutyrate/taurogluciol or a matched placebo. The test medication is dispensed from a central pharmacy in the form of a kit with a random four-digit identification number. The kits are delivered to the site in sequence as each new participant registers. Participants were assigned to treatment based on the kit received by the participants. Due to errors in the initial kit dispensing at the central pharmacy, the first 17 participants received the active drug, while the next 9 participants received the placebo. A sensitivity analysis is performed from which participants affected by the transport event are excluded; this analysis gave similar results to the pre-assigned primary analysis (mean ALSFRS-R slope difference between groups in the primary analysis was 0.46 versus 0.42, both P ═ 0.03). Treatment distribution after the first 26 participants followed the initial randomized schedule.

Section 2.3 test drug preparation and administration

The active drug had a bitter taste, while the placebo formulation was designed to have a matching bitter taste, appearance and dissolution profile to prevent blindness problems.

At the baseline visit, the following instructions regarding test drug preparation and administration were provided orally to the participants by the health care worker.

The test drug should be taken (or administered) before a meal.

Tear open the pouch of test drug and pour the contents into a cup or other container.

Add approximately 8 ounces of room temperature water and stir vigorously. (the test drug may require extensive stirring or gentle crushing to dissolve).

Ingested or administered completely via gastrostomy or nasogastric tube and within 1 hour after mixing the contents of the pouch with water. For oral administration, use is permitted

Antacids containing aluminum hydroxide or montmorillonite (aluminum oxide) are not taken or administered within 2 hours after administration of the test drug because they inhibit the absorption of taurine diol.

The normal diet was restored after taking the test drugs.

The participants were informed that the test drugs (active and placebo) had a strong bitter taste and were advised of strategies for making the drugs more palatable if taken orally, including:

listerine Pocket is used in large quantities immediately before and/or after the dose (stripe) or Listerione(sprays) for coating the oral cavity

Snacks or dinner taken after taking the drug

Milk immediately after administration

Avoid taking juice at the same time as the test drug, as this may make the taste worse

Section 2.4 detailed observations

ATLIS

The ATLIS device measures isometric Muscle forces of six upper and six lower limb Muscle groups with high reproducibility using a fixed wireless load sensor (a type of sensor) with a standard position (rather than relying on examiner strength) (see Andres et al, Muscle Nerve 2012; 45: 81-5). During each evaluation, two attempts were made per action, and the third attempt was added if the first two differences exceeded 15%. Raw values were normalized to PPN force based on gender, age, weight and height and expressed using average scores for upper, lower and total ATLIS PPN values (Andres et al, Muscle Nerve 2013; 47: 177-82). The ATLIS scores for each participant and visit were then submitted to the following steps to be used for analysis:

1. the baseline information (gender, age, weight and height) of the participants, as well as the coefficients and intercept estimates provided in the table below, were used to determine the predictive value for each of the 12 muscle groups.

ATLIS normalized coefficients and intercept

Based on the use of ATLIS version 2, coefficients and intercept are modified from the originally published values as needed.

For example, for a 41 year old woman 62 inches in height and 126 pounds in weight, the predicted value for the left fist making motion will be calculated as follows:

predicted value-28.91-0.15 age +0.16 weight +1.18 height

Predicted value-28.91-0.15 × 41+0.16 × 126+1.18 × 62

Predicted value 58.26

2. For each of the 12 muscle groups, a normalized ATLIS score was calculated by dividing the maximum observation score for each participant and visit combination by the prediction score. If the participant limb is not moving and therefore unable to test, the participant's observation score is recorded as 0 (also converted to a standardized score of 0). If the participant had limb movement but failed to complete the test for some other reason, the data is considered missing.

The "upper limb ATLIS" score was obtained by averaging 6 standardized upper limb muscle groups (left fist, right fist, left elbow flexion, right elbow flexion, left elbow extension, right elbow extension). The average score was calculated only if at least 4 of the 6 terms were observed.

The "lower limb ATLIS" score was obtained by averaging 6 standardized lower limb muscle groups (left knee extension, right knee extension, left knee flexion, right knee flexion, left ankle dorsiflexion, right ankle dorsiflexion). The average score was calculated only if 4 of the 6 terms were observed.

The "total ATLIS" score was obtained by averaging the upper and lower extremity ATLIS scores (items 3 and 4 above); both upper and lower extremity ATLIS scores are required to make this calculation.

The analysis uses the highest score from all attempts at a given action at each evaluation.

Detailed statistical methods in section 2.5

Confirmation of Linear assumptions in Primary ALSFRS-R analysis

To analyze potential non-linearities in the progression of the ALSFRS-R, the analysis plan includes testing a model that includes quadratic terms with respect to time since baseline and with respect to key covariates. In this analysis plan, if the quadratic term with respect to time is found to be significant (P < 0.10), a quadratic model will be used instead of a linear model. However, for both major and minor observations, the quadratic term with respect to time is not significant (P > 0.10); thus, only the linear term is retained for the final analysis.

And (3) sensitivity analysis: missing data, concurrent events and concomitant medication times

Three sensitivity models were performed to assess the impact of missing data, and three additional sensitivity models were performed to assess the impact of concomitant medication. The first sensitivity model was a joint ranking model that ranked participants according to time of death and then according to changes in the overall ALSFRS-R score. This ranking score is then analyzed as an analytical observation of a covariance model that includes the same covariates as the primary model, but with the ranking covariates substituted for the covariates. The other two sensitivity models for missing data are based on using the estimated data to create a dataset. The first model estimates a lower value than the previous score for each dead participant and is referred to as the post-mortem estimation model. The second model estimates missing data for all participants who have aborted for any reason and is referred to as the multiple estimation model for MNAR. For this model, estimates for the placebo arm were estimated from their linear trajectories (with errors) and estimates for the active arm were estimated from their linear trajectories after subtracting the difference in average slope between the active and placebo groups.

Three sensitivity models were used to assess the effect of concomitant use of riluzole, edaravone, or both on the observation of efficacy. The primary efficacy model is used as the basis for all three models, and terms are added to account for the time of concomitant medication or both. Positive or negative synergistic effects of the interaction terms between treatment and concomitant drug use were evaluated. For any of these three models, there is no evidence of synergy.

Claims

1. A method of treating at least one symptom of ALS in a human subject, the method comprising administering to the human subject about 10mg/kg to about 50mg/kg body weight of a bile acid or a pharmaceutically acceptable salt thereof, and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound, wherein the human subject:

(a) has been diagnosed with ALS for about 24 months or less;

(b) has shown one or more symptoms of ALS for about 24 months or less;

(c) has an ALS disease progression rate (Δ FS) of about 0.50 or greater;

(d) has an ALSFRS-R score of 40 or less;

(e) has a mutation in SOD1, C9ORF72, ANG, TARDBP, VCP, VAPB, SQSTM1, DCTN1, FUS, UNC13A, ATXN2, HNRNPA1, CHCHCHD 10, MOBP, C21ORF2, NEK1, TUBA4A, TBK1, MATR3, PFN1, UBQLN2, TAF15, OPTN or TDP-43;

(f) A cerebrospinal fluid (CSF) or blood level of a phosphorylated neurofilament protein heavy chain (pNF-H) of about 300pg/mL or greater;

(g) a CSF or blood level with a neurofilament protein light chain of about 50pg/mL or greater; or

(h) An ALSFRS-R score of about 0.8 to about 2 is lost on average each month over the previous 3-12 months,

thereby treating at least one symptom of at least ALS in the human subject.

2. The method of claim 1, wherein the method comprises the step of determining whether the human subject has at least one of the characteristics of (a) - (h) prior to administration.

3. The method of claim 1, wherein the human subject has been diagnosed with ALS for about 24 months or less.

4. The method of claim 3, wherein the human subject has been diagnosed with ALS for about 18 months or less.

5. The method of claim 3, wherein the human subject has been diagnosed with ALS for about 12 months or less.

6. The method of claim 1, wherein the human subject has exhibited one or more symptoms of ALS for about 24 months or less.

7. The method of claim 6, wherein the human subject has exhibited one or more symptoms of ALS for about 18 months or less.

8. The method of claim 6, wherein the human subject has exhibited one or more symptoms of ALS for about 12 months or less.

9. The method of claim 1, wherein the human subject has a rate of progression of ALS disease (Δ FS) of about 0.50 or greater.

10. The method of claim 9, wherein the human subject has a rate of progression of ALS disease (Δ FS) of about 0.90 or greater.

11. The method of claim 9, wherein the human subject has a rate of progression of ALS disease (Δ FS) of about 1.20 or greater.

12. The method of claim 1, wherein the human subject has an ALSFRS-R score of 40 or less.

13. The method of claim 12, wherein the human subject has an ALSFRS-R score of 38 or less.

14. The method of claim 12, wherein the human subject has an ALSFRS-R score of 30 or less.

15. The method of claim 1, wherein the human subject has a CSF or blood level of phosphorylated neurofilament protein heavy chain (pNF-H) of about 300pg/mL or greater.

16. The method of claim 15, wherein the human subject has a CSF or blood level of pNF-H of about 1000pg/mL or higher.

17. The method of any one of claims 1-16, wherein the human subject has been diagnosed with clear ALS based on revised EL Escorial criteria.

18. A method of reducing the rate of progression of an ALS disease in a human subject having one or more symptoms of ALS, the method comprising:

administering to the human subject about 10mg/kg to about 50mg/kg body weight of a bile acid or a pharmaceutically acceptable salt thereof, and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound in a dosage regimen sufficient to reduce the monthly average loss of ALSFRS-R score in the human subject by at least about 0.2 as compared to a control subject not receiving the administration.

19. The method of claim 18, wherein the average monthly lost ALSFRS-R score of the human subject is reduced by at least about 0.4 compared to the control subject.

20. A method of reducing worsening muscle strength, maintaining muscle strength, or improving muscle strength in a human subject with one or more symptoms of ALS, the method comprising:

administering to the human subject about 10mg/kg to about 50mg/kg body weight of a bile acid or a pharmaceutically acceptable salt thereof, and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound, thereby reducing muscle strength deterioration, maintaining muscle strength, or improving muscle strength in the human subject.

21. The method of claim 20, wherein the muscular strength is a lower limb strength, an upper limb strength, or a grasping strength.

22. The method of claim 20, wherein the muscular strength is of quadriceps femoris, biceps, hamstring tendon, triceps, or tibialis anterior.

23. The method of claim 20, wherein the muscle strength is assessed by a hand-held dynamometer (HHD), a hand-held dynamometer, a freehand muscle strength test (MMT), an electrical impedance Electromyography (EIM), a Maximum Voluntary Isometric Contraction Test (MVICT), a motor unit estimate (MUNE), an accurate isometric strength test for a limb (ATLIS), or a combination thereof, before, during, and/or after administration.

24. The method of claim 23, wherein the muscle strength is assessed by ATLIS.

25. A method of reducing deterioration, maintaining or improving respiratory muscle function in a human subject having one or more symptoms of ALS, the method comprising:

administering to the human subject about 10mg/kg to about 50mg/kg body weight of a bile acid or a pharmaceutically acceptable salt thereof, and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound, thereby reducing respiratory muscle function deterioration, maintaining respiratory muscle function, or improving respiratory muscle function in the human subject.

26. The method of claim 25, wherein respiratory muscle function in the human subject is determined by assessing the subject's Vital Capacity (VC), maximum mid-expiratory flow rate (MMERF), Forced Vital Capacity (FVC), Slow Vital Capacity (SVC), forced expiratory volume (l.s) 1, before, during and/or after administration(FEV ₁ ) Or a combination thereof.

27. The method of claim 26, wherein respiratory muscle function in the human subject is assessed by assessing the subject's SVC.

28. A method of preventing or reducing constipation in a human subject having one or more symptoms of ALS, the method comprising:

administering to the human subject about 10mg/kg to about 50mg/kg body weight of a bile acid or a pharmaceutically acceptable salt thereof, and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound, thereby preventing or reducing constipation in the human subject.

29. A method of preventing or reducing at least one severe adverse event in a human subject having one or more symptoms of ALS, the method comprising:

administering to the human subject about 10mg/kg to about 50mg/kg body weight of a bile acid or a pharmaceutically acceptable salt thereof, and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound, thereby preventing or reducing at least one serious adverse event in the human subject.

30. The method of claim 29, wherein the at least one serious adverse event is a respiratory adverse event, a fall, or a laceration.

31. A method of reducing fine motor skill deterioration, maintaining fine motor skills, or improving fine motor skills in a human subject having one or more symptoms of ALS, the method comprising:

administering to the human subject about 10mg/kg to about 50mg/kg body weight of a bile acid or a pharmaceutically acceptable salt thereof, and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound, thereby reducing, maintaining, or improving fine motor skill deterioration in the human subject.

32. The method of claim 31, wherein the fine motor skills are assessed using ALSFRS-R.

33. A method of slowing progression of ALS disease in a human subject having one or more symptoms of ALS, the method comprising:

administering to the subject about 10mg/kg to about 50mg/kg body weight of a bile acid or a pharmaceutically acceptable salt thereof, and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound, thereby slowing progression of ALS disease in the human subject.

34. A method of increasing survival time in a human subject having one or more symptoms of ALS, the method comprising:

Administering to the subject about 10mg/kg to about 50mg/kg body weight of a bile acid or a pharmaceutically acceptable salt thereof, and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound, thereby increasing the survival time of the human subject.

35. A method of treating at least one symptom of bulbar attack ALS in a human subject, the method comprising administering to the subject about 10mg/kg to about 50mg/kg body weight of a bile acid or a pharmaceutically acceptable salt thereof, and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound, thereby treating at least one symptom of bulbar attack ALS in the human subject.

36. A method of treating at least one symptom of Benign Fasciculation Syndrome (BFS) or spasm-fasciculation syndrome (CFS) in a human subject, the method comprising:

administering to a human subject diagnosed with BFS or CFS about 10mg/kg to about 50mg/kg body weight of a bile acid or a pharmaceutically acceptable salt thereof, and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound, thereby treating at least one symptom of BFS or CFS in the human subject.

37. A method, the method comprising:

administering to a human subject at risk of developing ALS about 10mg/kg to about 50mg/kg body weight of a bile acid or a pharmaceutically acceptable salt thereof and about 10mg/kg to about 400mg/kg body weight of a phenylbutyrate compound, thereby preventing or delaying the onset of ALS.

38. The method of claim 37, wherein the subject is determined to be at risk of developing ALS by evaluating the level of a biomarker in a biological sample obtained from the subject.

39. The method of claim 38, wherein the biomarker is pNF-H, a neurofilament light chain, S100- β, cystatin C, chitotriosidase, p75ECD, a ketone, or creatinine.

40. The method of claim 38, wherein the biological sample is CSF, urine or blood.

41. The method of claim 37, wherein the subject is determined to be at risk of developing ALS by identifying a mutation in one or more genes selected from the group consisting of: SOD1, C9ORF72, ANG, TARDBP, VCP, VAPB, SQSTM1, DCTN1, FUS, LUNC13A, ATXN2, HNRNPA1, CHCHCHD 10, MOBP, C21ORF2, NEK1, TUBA4A, TBK1, MATR3, PFN1, UBQLN2, TAF15, OPTN and TDP-43.

42. The method of any one of the preceding claims, wherein the bile acid is Taurogluciol (TURSO), ursodeoxycholic acid (UDCA), chenodeoxycholic acid, cholic acid, hyodeoxycholic acid, lithocholic acid, or glycoursodeoxycholic acid.

43. The method of any one of the preceding claims, wherein the phenylbutyrate compound is 4-phenylbutyric acid (4-PBA), glycerol tri- (4-phenylbutyrate), phenylacetic acid, 2- (4-methoxyphenoxy) acetic acid (2-POAA-OMe), 2- (4-nitrophenoxy) acetic acid (2-POAA-NO2), 2- (2-naphthyloxy) acetic acid (2-NOAA), or a pharmaceutically acceptable salt thereof.

44. The method of any one of the preceding claims, wherein the method comprises administering about 10mg/kg to about 30mg/kg body weight of the bile acid to the human subject.

45. The method of any one of the preceding claims, wherein the method comprises administering to the human subject about 10mg/kg to about 100mg/kg body weight of the phenylbutyrate compound.

46. The method of claim 45, wherein the method comprises administering to the human subject about 30mg/kg to about 100mg/kg body weight of the phenylbutyrate compound.

47. The method of any one of the preceding claims, wherein the bile acid and the phenylbutyrate compound are administered separately.

48. The method of any one of the preceding claims, wherein the bile acid and the phenylbutyrate compound are administered simultaneously.

49. The method of any one of the preceding claims, wherein the bile acid and the phenylbutyrate compound are administered daily.

50. The method of claim 49, wherein the bile acid and the phenylbutyrate compound are administered once daily, twice daily, or three times daily.

51. The method of claim 1, wherein the bile acid and the phenylbutyrate compound are administered once daily for 60 days or less.

52. The method of claim 1, wherein the bile acid and the phenylbutyrate compound are administered once daily for 30 days or less.

53. The method of claim 1, wherein the bile acid and the phenylbutyrate compound are administered twice daily for 60 days or less.

54. The method of claim 1, wherein the bile acid and the phenylbutyrate compound are administered twice daily for 30 days or less.

55. The method of claim 1, wherein the bile acid and the phenylbutyrate compound are administered twice daily for 60 days or more.

56. The method of claim 1, wherein the bile acid and the phenylbutyrate compound are administered twice daily for 120 days or more.

57. The method of claim 1, wherein the bile acid and the phenylbutyrate compound are administered once daily for at least 14 days, followed by twice daily for at least 30 days.

58. The method of claim 1 wherein the bile acid and the phenylbutyrate compound are administered once daily for about 21 days, followed by twice daily for at least 30 days.

59. The method of any one of the preceding claims, wherein the bile acid and the phenylbutyrate compound are administered orally.

60. The method of any one of the preceding claims, wherein the bile acid and the phenylbutyrate compound are administered via a feeding tube.

61. The method of any one of claims 1-58, wherein the bile acid and the phenylbutyrate compound are administered by bolus injection.

62. The method of any one of claims 1-61, wherein each of the bile acid and the phenylbutyrate compound is formulated as a solution.

63. The method of any one of claims 1-61, wherein the bile acid and the phenylbutyrate compound are formulated in a single solution.

64. The method of any one of claims 1-61, wherein each of the bile acid and the phenylbutyrate compound is formulated as a powder.

65. The method of any one of claims 1-61, wherein the bile acid and the phenylbutyrate compound are formulated as a single powder formulation.

66. The method of claim 1, wherein the bile acid is TURSO.

67. The method of claim 66, wherein the TURSO is administered in an amount of about 0.5 to about 5 grams per day.

68. The method of claim 66, wherein the TURSO is administered in an amount of about 1.5 to about 2.5 grams per day.

69. The method of claim 66, wherein the TURSO is administered in an amount of about 1 gram twice daily.

70. The method of any preceding claim, wherein the phenylbutyrate compound is a pharmaceutically acceptable salt of 4-PBA.

71. The method of claim 70 wherein the pharmaceutically acceptable salt of 4-PBA is sodium phenylbutyrate.

72. The method of claim 71 wherein the sodium phenylbutyrate is administered in an amount of from about 0.5 to about 10 grams per day.

73. The method of claim 71 wherein the sodium phenylbutyrate is administered in an amount of from about 4.5 to about 8.5 grams per day.

74. The method of claim 71, wherein the sodium phenylbutyrate is administered in an amount of about 3 grams twice daily.

75. The method of any one of the preceding claims, further comprising administering one or more additional therapeutic agents to the human subject.

76. The method of claim 75, wherein the one or more additional therapeutic agents are selected from the group consisting of: combination of dextromethorphan and quinidine, riluzole, edaravone, mexiletine, anticholinergic drugs, and psychotropic drugs.

77. The method of claim 76, wherein the one or more additional therapeutic agents is riluzole.

78. The method of claim 76, wherein the one or more additional therapeutic agents is edaravone.

79. The method of any one of the preceding claims, wherein the human subject has been previously treated with one or more additional therapeutic agents.

80. The method of claim 79, wherein the additional therapeutic agent is riluzole.

81. The method of claim 80, wherein the human subject has been previously treated with riluzole for at least 30 days.

82. The method of claim 79, wherein the additional therapeutic agent is edaravone.

83. The method of claim 82, wherein the human subject has been previously treated with edaravone for at least 30 days.

84. The method of claim 79, wherein the additional therapeutic agent is mexiletine.

85. The method of claim 84, wherein the human subject has been previously treated with mexiletine at a dose less than or equal to 300 mg/day.

86. The method of any one of the preceding claims, further comprising administering to the human subject a plurality of food products including solid or liquid foods.

87. The method of any one of the preceding claims, wherein the human subject is about 18 years of age or older.

88. The method of any one of the preceding claims, wherein the human subject is about 18 to about 50 years old.

89. The method of any one of the preceding claims, wherein the subject is about 18 to about 40 years old.

90. A method of treating at least one symptom of ALS or preventing the onset of ALS in a human subject, the method comprising administering to the human subject an effective amount of

(a) A bile acid or a pharmaceutically acceptable salt thereof;

(b) a phenylbutyrate compound;

(c) riluzole; and

(d) the edaravone is used for preparing the edaravone,

thereby treating at least one symptom of ALS or preventing the onset of ALS in the human subject.

91. A method of treating at least one symptom of ALS or preventing the onset of ALS in a human subject, the method comprising administering TURSO and sodium phenylbutyrate to the human subject according to a first regimen followed by a second regimen,

wherein the first regimen comprises administration of about 1 gram of TURSO per day and about 3 grams of sodium phenylbutyrate once per day for at least 14 days, and the second regimen comprises administration of about 1 gram of TURSO per day and about 3 grams of sodium phenylbutyrate twice per day for at least 30 days.