Methods of treating TTR amyloidosis using ag10

Abstract

To provide methods for treating transthyretin (TTR) amyloidosis in a subject.SOLUTION: A method comprises administering a therapeutically effective amount of the compound illustrated below, or a pharmaceutically acceptable salt thereof, to a subject in need thereof, wherein the therapeutically effective amount is a total daily dosage of about 10 mg to 2,000 mg.SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Nos. 62 / 647,411, filed March 23, 2018; 62 / 765,096, filed August 17, 2018; 62 / 731,629, filed September 14, 2018; 62 / 758,235, filed November 9, 2018; and 62 / 810,651, filed February 26, 2019, the disclosures of each of which are incorporated herein by reference in their entirety.

[0002] Statement of Rights to Inventions Made Under Federally Sponsored Research and Development Not applicable.

[0003] Reference to a "Sequence Listing," Table, or Computer Program Listing Exhibit submitted on a Compact Disc This application hereby incorporates by reference in its entirety the Sequence Listing, which is submitted in computer readable form herewith. The file name for this application is: "Sequence Listing - 051418-505". [Background technology]

[0004] 2. Background of the Invention Aberrant protein interactions and aggregation, either through protein misfolding or overactivation of signaling pathways, are the underlying causes of many human degenerative diseases, therefore targeting protein-protein interactions (PPIs) is of therapeutic interest.

[0005] One such example of abnormal protein aggregation is the soluble protein transthyretin (TTR or prealbumin). TTR is a 55 kDa homotetrameric protein present in blood and cerebrospinal fluid. When dissociated from its homotetrameric form, TTR dimers can misfold into amyloidogenic monomers. This is observed with wild-type TTR as well as over 100 different mutated variants. Studies have shown that stabilization of the tetrameric form of TTR inhibits the misfolding of amyloidogenic monomers and subsequent TTR amyloid formation and deposition.

[0006] A benzoxazole derivative called tafamidis (2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid) has been described to inhibit abnormal TTR aggregation and fibrillogenesis and is undergoing clinical trials for the treatment of cardiomyopathy in familial and wild-type TTR patients. Tafamidis is still under evaluation by the FDA, the primary drug registration agency.

[0007] Despite ongoing efforts in managing and improving the treatment of subjects with abnormal TTR aggregation and fibrillogenesis, there has been little progress. For example, a recent retrospective review from Mayo Clinic, the largest amyloid referral center in the United States, noted that there was no discernible change in overall mortality in subjects with TTR amyloid cardiomyopathy (ATTR-CM) between 1965 and 2013 (Grogan et al., J Am Coll Cardiol 2016; 68(10): 1014-20).

[0008] Thus, there is a need in the art to provide methods for treating abnormal TTR aggregation and fibrillogenesis. The present disclosure addresses these needs and further provides related advantages. Summary of the Invention

[0009] Brief invention summary In some embodiments, a method of treating transthyretin (TTR) amyloidosis in a subject in need thereof comprises administering to a subject a compound having the formula: [ka] or a pharma- ceutical acceptable salt thereof to a subject in need thereof, wherein the therapeutically effective amount is a total daily dose of about 50 mg to 2,000 mg.

[0010] In some embodiments, the total daily dose of Compound 1 is about 800 mg. In some embodiments, the total daily dose of Compound 1 is about 1,600 mg. In some embodiments, the HCl salt form of Compound 1 is administered.

[0011] In some embodiments, Compound 1 is administered once a day. In some embodiments, Compound 1 is administered twice a day.

[0012] In some embodiments, a method of treating transthyretin (TTR) amyloidosis in a subject in need thereof comprises administering to a subject a compound having the formula: [ka] or a pharma- ceutical acceptable salt thereof to a subject in need thereof, wherein the therapeutically effective amount of Compound 1 maintains a desired trough blood plasma concentration of Compound 1.

[0013] In an additional embodiment, compound 1 has the formula: [ka] or a pharma- ceutical acceptable salt thereof, a single unit dosage of about 10 to 1,000 mg is provided herein.

[0014] In some embodiments, the single unit dosage form contains 200 mg of Compound 1. In some embodiments, the single unit dosage form contains 400 mg of Compound 1. In some embodiments, the single unit dose contains the HCl salt of Compound 1.

[0015] Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description and drawings. [Brief description of the drawings]

[0016] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1 shows the pharmacokinetic (PK) profiles of single-dose ascending cohorts 1-4 at oral doses of 50 mg, 150 mg, 300 mg, and 800 mg of AG10·HCl.

[0017] [Diagram 2] Figure 2 shows the PK profiles of fed vs. fasted subjects in single-dose escalation cohort 3 (300 mg oral dose of AG10·HCl).

[0018] [Diagram 3] Figure 3 shows the PK profiles of multiple-dose ascending cohorts 1-3 at oral doses of 100 mg, 300 mg, and 800 mg of AG10·HCl every 12 hours over 12 days.

[0019] [Figure 4] Figure 4 shows the time course of the fluorescent probe exclusion (FPE) assay for single-dose ascending cohort 3 (AG10·HCl 300 mg orally) depicting the target association rate as a function of time. In this cohort, 6 subjects receive compound 1 while 2 subjects receive placebo. The placebo group is subjects 1 and 4.

[0020] [Diagram 5]Figure 5 shows the time course of the FPE assay for single dose ascending cohort 4 (800 mg oral AG10·HCl) depicting the target association rate as a function of time. In this cohort, 6 subjects receive compound 1 while 2 subjects receive placebo. The placebo group is subjects 1 and 7.

[0021] [Figure 6] FIG. 6 shows the mean target engagement rate for each single-dose ascending cohort as a function of time.

[0022] [Figure 7A] Figure 7A shows Western blot data for single-dose escalation cohort 3 (300 mg of AG10·HCl). Arrows on the side of the gel indicate the location of the TTR tetramer based on molecular weight and recognition by a TTR-specific antibody. [Figure 7B] Figure 7B shows Western blot data for single-dose escalation cohort 3 (300 mg of AG10·HCl). Arrows on the side of the gel indicate the location of the TTR tetramer based on molecular weight and recognition by a TTR-specific antibody. [Figure 7C] Figure 7C shows Western blot data for single-dose escalation cohort 3 (300 mg of AG10·HCl). Arrows on the side of the gel indicate the location of the TTR tetramer based on molecular weight and recognition by a TTR-specific antibody. [Figure 7D] Figure 7D shows Western blot data for single-dose escalation cohort 3 (300 mg of AG10·HCl). Arrows on the side of the gel indicate the location of the TTR tetramer based on molecular weight and recognition by a TTR-specific antibody.

[0023] [Figure 8] Figure 8 shows the correlation between Western blot and FPE data for single-dose escalation cohort 3 (AG10·HCl 300 mg orally).

[0024] [Figure 9]Figure 9 shows the time course of the FPE assay for repeat dose titration cohort 1 (AG10·HCl 100 mg orally q12h) over 12 days. In this cohort, 6 subjects received compound 1, whereas 2 subjects received placebo. The placebo group is subjects 1 and 8.

[0025] [Figure 10] Figure 10 shows the time course of the FPE assay for repeat dose titration cohort 2 (AG10·HCl 300 mg orally q12h) over 12 days. In this cohort, 6 subjects received compound 1, whereas 2 subjects received placebo. The placebo group is subjects 3 and 5.

[0026] [Figure 11] Figure 11 shows the time course of the FPE assay for repeat dose titration cohort 3 (AG10·HCl 800 mg orally q12h) over 12 days. In this cohort, 6 subjects received compound 1, whereas 2 subjects received placebo. The placebo group is subjects 1 and 7.

[0027] [Figure 12] FIG. 12 shows peak, mean, and trough target association rates for cohort 3 (AG10·HCl 800 mg orally q12h) over 12 days using the FPE assay.

[0028] [Figure 13] Figure 13 shows Western blot data for repeat-dose escalation cohort 3 (AG10·HCl 800 mg orally q12h). Arrows on the side of the gel indicate the location of TTR tetramers based on molecular weight and recognition by TTR-specific antibodies. Subjects 1 and 3 received AG10, whereas subject 2 received placebo.

[0029] [Figure 14]FIG. 14 shows the aggregated pharmacokinetic and pharmacodynamic data from the single-dose ascending and multiple-dose ascending cohorts, in which there is an expected dose-responsive PD effect.

[0030] [Figure 15] Figure 15 shows the synthesis of AG10 analogs 1, 2, 3, and 4. a) 5a, i. acetylacetone, DBU, benzene, rt, 3 days; ii. hydrazine hydrate, ethanol, 90°C, 4 h; iii. NaOH, MeOH / water, 50°C, 14 h; b) 5b, i. acetylacetone, DBU, benzene, rt, 3 days; ii. hydrazine hydrate, ethanol, 90°C, 4 h; c) i. NaH, MeI, DMF, rt, 12 h; ii. NaOH, MeOH / water, 50°C, 14 h; d) 5b, i. 3,5-heptanedione, DBU, benzene, rt, 3 days; ii. hydrazine hydrate, ethanol, 90°C, 4 h; e) NaOH, MeOH / water, 50°C, 14 h.

[0031] [Figure 16] Figure 16A-C shows the binding affinity and efficacy of stabilizers for TTR in buffer. (a) Interaction of TTR with stabilizers assessed by ITC. Thermodynamic data (summarized in Table 5); ΔG is blue bar, ΔH is green bar, and -TΔS is red bar. (b) Fluorescence change caused by modification of TTR (2.5 μM) in buffer by FPE probe monitored in the presence of probe alone (control DMSO) or TTR stabilizer (2.5 μM; 1:1 ratio of stabilizer to TTR). (c) Bar graph representation of occupancy of TTR in buffer by stabilizer in the presence of FPE probe measured after 3 hours of incubation compared to probe alone. Error bars indicate SD (n=3). Significant differences were determined by one-way ANOVA followed by Tukey's multiple comparison test (*p≦0.05; ***p≦0.001).

[0032] [Figure 17]Figure 17A-D shows the efficacy of stabilizers in occupying and stabilizing TTR in human serum. (a) Representative Western blot images of stabilization of TTR in human serum subjected to acid-mediated (pH 4.0) denaturation in the presence of AG10 (10 μM) and other stabilizers tested at their estimated mean clinical Cmax at steady state when administered at the indicated doses: diflunisal (250 mg, BID, 200 μM); tafamidis (80 mg, qd), 20 μM; tolcapone (100 mg, tid), 20 μM. (b) Bar graph representation of stabilization data obtained from Western blot experiments. Error bars indicate SD (n=3). (c) Fluorescence changes caused by modification of TTR in human serum by FPE probe monitored in the presence of probe alone (control DMSO), AG10 (10 μM), or TTR stabilizers (at their estimated mean clinical steady state Cmax). (d) Bar graph representation of the occupancy of TTR in buffer by stabilizer in the presence of FPE probe measured after 3 hours of incubation compared to the probe alone. Error bars indicate SD (n=4). Significant differences were determined by one-way ANOVA followed by Tukey's multiple comparison test (ns, not significant; *p≦0.05; **p≦0.01; ***p≦0.001).

[0033] [Figure 18] Figure 18A-D shows crystal structures highlighting the similar interactions caused by the T119M mutation and the binding of AG10 to TTR. (a) The quaternary structure of AG10 bound to V122I-TTR (PDB:4HIQ) shown as a ribbon representation with the monomers colored separately. A close-up of one of the two identical T4 binding sites with ribbons of different colors for the two monomers of the tetramer that make up the binding site. The important hydrogen bond between the pyrazole ring of AG10 and S117 / 117' is highlighted by a dashed line. (b) Crystal structure of the stabilized T119M-TTR variant (PDB:1FHN) with dashed lines highlighting the important interaction between the hydroxyl groups of S117 and S117'. (c) Crystal structure of TTRwt (PDB:3CFM). (d) Crystal structure of the thermodynamically stabilized R104H-TTR (PDB:1X7T).

[0034] [Figure 19] Figure 19A-E illustrates that the hydrogen bond between the pyrazole ring of AG10 and S117 / S117' of TTR is important for effective binding to TTR. (a) Chemical structures and in silico docking studies of synthesized AG10 analogs 1, 2, 3, and 4. Cocrystals of AG10 bound to TTR used for docking experiments. 1 is the iodo-analogue of AG10. 2 is the methyl-ester form of AG10 that cannot form a salt bridge with K15 / 15'. 3 is the methyl-pyrazole form of AG10 that can potentially form only one hydrogen bond with either K15 or K15'. 4 is the diethyl-pyrazole analog of AG10 that affects both hydrogen bonds with S117 / S117'. (b) Interaction of TTR with the analogs assessed by ITC. Thermodynamic data; ΔG is blue bar, ΔH is green bar, and -TΔS is red bar. (c) Fluorescence change caused by modification of TTR (2.5 μM) in buffer by FPE probe monitored in the presence of probe alone (control DMSO) or TTR stabilizer (2.5 μM; 1:1 ratio of stabilizer to TTR). (d) Bar graph representation of occupancy of TTR in buffer by stabilizer in the presence of FPE probe measured after 3 h incubation compared to probe alone. Error bars indicate SD (n=3). (e) Bar graph representation of Western blot data for stabilization of TTR in human serum by analogs (10 μM; 2:1 ratio of stabilizer to TTR). Error bars indicate SD (n=4). Significant differences were determined by one-way ANOVA followed by Tukey's multiple comparison test (ns, not significant; *p≦0.05; **p≦0.01; ***p≦0.001).

[0035] [Figure 20]Figures 20A-F illustrate that AG10 has high selectivity for TTR binding over albumin or other abundant human serum proteins. (a) Gel filtration and dialysis assays comparing AG10 and tafamidis (30 μM each) incubated with purified human serum albumin (600 μM). The concentration of tafamidis bound to albumin after gel-filtration (i.e., dialysis time 0 h) was normalized to 100%. Error bars indicate SD (n=3). (b) 24-h time course for dialysis of AG10 (10 μM) incubated with purified human TTR (5 μM). Error bars indicate SD (n=3). (c) Fluorescence change due to modification of purified human TTR (5 μM) by FPE probe monitored for 6 h in the presence of probe alone (black circle), probe + albumin (600 μM) (black triangle), probe + all [fibrinogen (5 μM), albumin (600 μM), IgG (70 μM), transferrin (25 μM)] (grey triangle); probe and AG10 (10 μM) (red square), or probe and AG10 + albumin (green diamond), probe and AG10 + all [fibrinogen (5 μM), albumin (600 μM), IgG (70 μM), transferrin (25 μM)] (blue circle). (d) Percentage of TTR occupancy in buffer by AG10 in the presence of FPE probe or other serum proteins measured after 3 h incubation compared to probe alone. (e, f) The same experiment as described for AG10 was performed for tafamidis. Error bars indicate SD (n = 3). Significant differences were determined by one-way ANOVA followed by Tukey's multiple comparison test (ns, not significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001).

[0036] [Figure 21]Figure 21A-D shows the activity of AG10 and tafamidis in FPE and Western blot assays performed with pooled dog serum. (a) Fluorescence change caused by modification of canine TTR in commercial beagle dog serum by FPE probe monitored in the presence of probe alone (control DMSO, black circle), AG10 (10 μM) or tafamidis (10 μM). (b) Occupancy of canine TTR in dog serum by AG10 and tafamidis in the presence of FPE probe measured after 3 hours of incubation compared to probe alone. Error bars indicate SD (n=4). (c) Western blot images of stabilization of TTR in pooled dog serum against acid-mediated denaturation in the presence of AG10 (10 μM) and tafamidis (10 μM). Serum samples were incubated with DMSO or test compounds in acetate buffer (pH 4.0) for the desired times (0 and 72 hours) before cross-linking and immunoblotting. (d) Bar graph representation of stabilization data obtained from Western blot experiments. Error bars indicate SD (n=3). Significant differences were determined by one-way ANOVA followed by Tukey's multiple comparison test (ns, not significant; *p≦0.05; **p≦0.01; ***p≦0.001).

[0037] [Figure 22]Figures 22A-D show that orally administered AG10 is effective in binding and stabilizing TTR in dogs. (a and b) TTR occupancy in beagle dogs after oral administration of increasing doses of AG10 (QD for 7 days). Circles (●) indicate day 1 pre-dosing, squares (■) indicate day 7 pre-dosing (AG10 concentration at Cmin), and triangles (▲) indicate day 7 post-dosing (AG10 concentration at Cmax). Four groups of animals were dosed: (i) 0 mg / kg (n=12, 6 males / 6 females); (ii) 50 mg / kg (n=4, 2 males / 2 females); (iii) 100 mg / kg (n=4, 2 males / 2 females); (iv) 200 mg / kg (n=12, 6 males / 6 females). (b) Bar graphs representing TTR occupancy at 3 hours. Error bars indicate SD (n=3). (c and d) Pharmacokinetic-pharmacodynamic (PK-PD) analysis of AG10 in dogs receiving single oral doses of (c) 5 mg / kg and (d) 20 mg / kg of AG10·HCl. Scatter plots of concentration [AG10] versus % TTR occupancy in serum samples obtained from dogs at various time points (n=4, 2 males / 2 females per dose group). Error bars indicate SD (n=3). Significant differences were determined by one-way ANOVA followed by Tukey's multiple comparison test (ns, not significant; *p≦0.05; **p≦0.01; ***p≦0.001).

[0038] [Figure 23] Figure 23 illustrates % TTR occupancy in serum from three male cynomolgus monkeys at 3 hours after oral dosing of 5 mg / kg of AG10·HCl. Data are the mean ± SD of triplicates.

[0039] [Figure 24] FIG. 24 illustrates the dose-dependent relationship between orally administered AG10 and TTR stabilization in monkeys.

[0040] [Diagram 25] FIG. 25 shows a standard calibration curve generated using the Prealbumin ELISA Kit (Human) from Aviva Systems Biology.

[0041] [Figure 26] Figure 26 shows the relative change in TTR concentration over time for each MAD cohort (total number of medicated healthy volunteers = 24; placebo:active = 1:3; MAD1 cohort = 100 mg Q12h for 12 days; MAD2 cohort = 300 mg Q12h for 12 days; MAD3 cohort = 800 mg Q12h for 12 days). The changes were calculated by normalizing to baseline values.

[0042] [Figure 27] FIG. 27 illustrates the mean percent change in blood serum TTR concentrations from baseline to day 12 in all placebo- and AG10-treated MAD subjects.

[0043] [Figure 28] Figure 28 plots baseline and day 12 blood serum TTR concentrations in all placebo- and AG10·HCl-treated cohorts (total number of healthy volunteers dosed=24; placebo:active=1:3; MAD1 cohort=100 mg Q12h for 12 days; MAD2 cohort=300 mg Q12h for 12 days; MAD3 cohort=800 mg Q12h for 12 days).

[0044] [Figure 29] FIG. 29 plots blood serum TTR concentrations at baseline and day 12 in all placebo- and AG10-treated cohorts (total number of healthy volunteers medicated=24; placebo:active=1:3; MAD1 cohort=100 mg Q12h for 12 days; MAD2 cohort=300 mg Q12h for 12 days; MAD3 cohort=800 mg Q12h for 12 days).

[0045] [Diagram 30] Figure 30 illustrates the dose-response changes in serum TTR levels for subjects in each treatment group. Data are reported as percentage change from baseline to day 28.

[0046] [Diagram 31] Figure 31 shows that AG10 and the TTR stabilizers tafamidis and diflunisal all increase TTR serum concentrations. TTR serum concentrations reported for the AG10 cohort are at 28 days after treatment. TTR serum concentrations reported for tafamidis are at day 28, interpolated based on week 2 and week 6 values. TTR serum concentrations reported for diflunisal are at 1 year after treatment.

[0047] [Diagram 32] Figure 32 shows that AG10 treatment restores low TTR levels to the normal range in ATTR-CM patients. The percentage of ATTR-CM patients in each treatment group (placebo, 400 mg BID, and 800 mg BID) exhibiting low and normal levels of serum TTR concentrations is reported before treatment (left) and 28 days after treatment (right).

[0048] [Diagram 33] FIG. 33 plots the distribution of baseline serum TTR concentrations for individuals participating in the Phase 2 study.

[0049] [Diagram 34] Figure 34 illustrates the percent stabilization of TTR determined by Western blot assay to assess stabilization of tetrameric TTR. Data provided are for all individuals in the study (left column), WT-TTR individuals (middle column), and mutant TTR individuals (right column). Error bars provided are standard error of the mean.

[0050] [Diagram 35] Figure 35 illustrates the occupancy of AG10 as determined by fluorescent probe assay at trough (pre-dose) and peak (1 hour post-dose) on day 28. Data provided are for all individuals in the study (solid columns), WT-TTR individuals (open columns), and mutant TTR individuals (checkered columns).

[0051] [Diagram 36] Figure 36 plots the relationship between fluorescent probe binding and blood plasma AG10 concentration. If the fluorescent probe is bound to TTR, probe fluorescence is measured, and if the fluorescent probe cannot bind to TTR due to AG10 occupancy, no fluorescence is measured.

[0052] [Figure 37] FIG. 37 plots the relative fluorescence units measured in the fluorescent probe assay at the indicated time points. Pre-dose is the trough level versus the peak level 1 hour post-dose. The y-axis represents the mean 60 minute relative fluorescence units corrected for background. Data presented are from an individual receiving 400 mg of AG10·HCl salt BID.

[0053] [Figure 38] FIG. 38 plots the relative fluorescence units measured in the fluorescent probe assay at the indicated time points. The y-axis represents the mean value of 60 min relative fluorescence units corrected for background. Pre-dose is the trough level, whereas 1 hour post-dose is the peak level. Data presented is from an individual receiving 800 mg of AG10·HCl salt BID.

[0054] [Figure 39] Figure 39 plots the relative fluorescence units measured in the fluorescent probe assay at the indicated time points. The y-axis represents the average relative fluorescence units at 60 minutes corrected for background. Pre-dosing is the trough level, whereas 1 hour after dosing is the peak level. Data presented is from individuals receiving placebo treatment.

[0055] [Diagram 40]Figure 40 illustrates the occupancy of AG10 as determined by fluorescent probe assay at day 14 trough (pre-dose) and day 14 peak (0.5 hours post-dose). Data provided are for individuals in the 800 mg BID dose group of the AG10·HCl treatment group with a TTR protein harboring the V30M mutation.

[0056] [Diagram 41] Figure 41 illustrates the percent stabilization of TTR as determined by Western blot assay to assess stabilization of tetrameric TTR. Data provided are for individuals in the 800 mg BID AG10·HCl treatment group with TTR protein harboring the V30M mutation.

[0057] [Diagram 42] Figures 42A-D plot the random effects (ETA) versus categorical covariate plots in the population PK model. Panels A and B plot the effect on clearance, whereas Panels C and D plot the effect on volume. In the plots labeled "DIS" (Panels A and C), 0 = healthy volunteers, 1 = diseased subjects. In the plots labeled "ConMed1" (Panels B and D), 0 = subjects who received either Furosemide or Torsemide, 1 = patients who received neither Furosemide nor Torsemide. DIS: 0 (n=42) = all AG10-treated healthy adult volunteers from AG10-001 (SAD and MAD). 1 (n=32) = all active ATTR-CM patients from AG10-201 who were included in the population PK analysis. 16 in the 400 mg BID group and 16 in the 800 mg BID group. ConMed1: 0 (n=49) = subjects not receiving concomitant diuretics: furosemide or torasemide (42 healthy adult volunteers, 7 ATTR-CM patients), 1 (n=25) = subjects receiving either furosemide or torasemide.

[0058] [Diagram 43]Figure 43 plots the trough levels of AG10 on day 12 from MAD 3 (Study AG10-001) compared to trough levels in ATTR-CM patients dosed with 800 mg BID (AG10-201). Box and whisker plots show the 25th to 75th percentiles of results with the smallest and widest whiskers relative to the highest value in each group. Lines indicate median and + indicates mean. Both studies used 200 mg AG10 tablets for dosing.

[0059] [Diagram 44] Figure 44 plots trough levels of AG10 in healthy subjects treated with 400 mg tablets compared to trough levels in ATTR-CM patients dosed with 400 mg BID from AG10-201. Box and whisker plots show the 25th to 75th percentiles of results with the smallest spreading whiskers relative to the highest value in each group. Lines indicate median and + indicates mean. The healthy volunteer study (AG10-003) used 400 mg AG10 tablets for dosing and the Phase 2 study AG10-201 used 200 mg tablets for dosing.

[0060] [Diagram 45] FIG. 45 shows a summary of the study design for the Phase 3 clinical trial in subjects with ATTR-CM.

[0061] [Figure 46] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] Detailed Description of the Invention I. General Described herein are methods for treating transthyretin (TTR) amyloidosis in a subject. These methods involve specific dosing regimens that are highly effective in treating the subject and are well tolerated by the subject.

[0063] II. Definition While various embodiments and aspects of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various modifications to the embodiments of the invention described herein can be utilized in the practice of the invention.

[0064] The section headings used herein are for general information purposes only and are not to be construed as limiting the purposes set forth. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, manuals, and papers, are expressly incorporated herein by reference in their entirety for any purpose.

[0065] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., J. Wiley & Sons, Inc. (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, Inc. (Cold Spring Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of the present invention. The following definitions are provided to facilitate understanding of certain terms frequently used herein and are not meant to limit the scope of the present disclosure.

[0066] "Compound 1" is the chemical compound 3-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)-4-fluorobenzoic acid (AG10), having the formula: [ka] (Compound 1) or a pharma- ceutically acceptable salt thereof. When referring to a particular amount of Compound 1 administered to a patient, the application refers to the amount of the HCl salt of Compound 1 administered. One of ordinary skill in the art will recognize that minor adjustments to the total amount administered may be necessary to administer the same amount of Compound 1 in the free base or a different salt form.

[0067] As used herein, the terms "a" or "an" mean one or more.

[0068] The terms "comprise," "include," and "have" are used interchangeably herein as inclusive open-ended terms. For example, the use of "comprise," "including," and "having" refers to not only the elements encompassed by the subject of the clause containing the verb, regardless of whether the elements comprise, have, or are included.

[0069] The term "about" as used herein refers to a range of values ​​that includes the specified value, which those skilled in the art would consider to be reasonably similar to the specified value.In some embodiments, the term "about" refers to within standard deviation using measurements generally accepted in the art.In some embodiments, about refers to a range that extends to ±10% of the specified value.In some embodiments, about refers to the specified value.

[0070] As used herein, "treatment" or "treating" or "alleviating" or "ameliorating" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results, including, but not limited to, therapeutic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. A therapeutic benefit is also achieved by eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in a subject, even if the subject is still afflicted by the underlying disorder. Treatment includes causing a delay in the progression of clinical symptoms of the disease by administering the composition; suppressing the disease, i.e., causing a reduction in clinical symptoms of the disease; inhibiting the disease, i.e., halting the progression of clinical symptoms by administering the composition after the first appearance of symptoms; and / or relieving the disease, i.e., causing a regression of clinical symptoms by administering the composition after their first appearance. For example, certain methods described herein treat transthyretin (TTR) amyloidosis by reducing or attenuating the appearance or progression of TTR fibril formation; or treat TTR amyloidosis by reducing the symptoms of TTR amyloidosis.

[0071] An "effective amount" or a "therapeutically effective amount" is an amount sufficient to achieve a stated purpose (e.g., to achieve the effect for which it is administered, to treat a disease, to reduce enzyme activity, to alleviate one or more symptoms of a disease or condition). An example of an "effective amount" is an amount sufficient to contribute to the treatment or alleviation of one or more symptoms of a disease, which is also referred to as a "therapeutically effective amount". "Alleviation" of one or more symptoms (and grammatical equivalents of this phrase) means a decrease in the severity or frequency of the symptom(s), or the elimination of the symptom(s). Efficacy can also be expressed as a "-fold" increase or decrease. For example, a therapeutically effective amount can have at least 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more efficacy over a control.

[0072] "Patient" or "subject" or "subject in need thereof" refers to a living organism suffering from or prone to a disease or condition that can be treated by using the methods provided herein. The term does not necessarily indicate that the subject has been diagnosed with a specific disease, but typically refers to an individual under medical supervision. Non-limiting examples include humans, other mammals, cows, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammalian animals. In some embodiments, the patient, subject, or subject in need thereof is a human.

[0073] III. Detailed Description of the Preferred Embodiments method In one aspect, provided herein is a method of treating transthyretin (TTR) amyloidosis, comprising administering to a patient a compound having the formula: [ka] or a pharma- ceutical acceptable salt thereof to a subject in need thereof, wherein the therapeutically effective amount is a total daily amount of about 10 mg to 2,000 mg. In some embodiments, the total daily amount of Compound 1 is about 10 mg to 50 mg, 50 mg to 300 mg, 50 mg to 150 mg, 150 mg to 800 mg, 800 mg to 1,600 mg, or 800 mg to 2,000 mg. In some embodiments, the total daily amount of Compound 1 is about 10 mg to 50 mg. In some embodiments, the total daily amount of Compound 1 is about 50 mg to 300 mg. In some embodiments, the total daily amount of Compound 1 is 50 mg to 150 mg. In some embodiments, the total daily amount of Compound 1 is 150 mg to 800 mg. In some embodiments, the total daily amount of Compound 1 is 800 mg to 1,600 mg. In some embodiments, the total daily amount of Compound 1 is 800 mg to 2,000 mg. It is understood that in this disclosure, the amounts of Compound 1 recited are the amounts of the HCl salt of Compound 1 administered. One of skill in the art will recognize that minor adjustments to the total amount administered may be necessary to administer the same amount of Compound 1 in the free base or a different salt form.

[0074] In some embodiments, the total daily dose of Compound 1 is about 10 mg. In some embodiments, the total daily dose of Compound 1 is about 25 mg. In some embodiments, the total daily dose of Compound 1 is about 50 mg. In some embodiments, the total daily dose of Compound 1 is about 1000 mg. In some embodiments, the total daily dose of Compound 1 is about 150 mg. In some embodiments, the total daily dose of Compound 1 is about 200 mg. In some embodiments, the total daily dose of Compound 1 is about 300 mg. In some embodiments, the total daily dose of Compound 1 is about 600 mg. In some embodiments, the total daily dose of Compound 1 is about 800 mg. In some embodiments, the total daily dose of Compound 1 is about 1,6000 mg.

[0075] Compound 1 can be administered once (SID or qd), twice (BID or q12h), three times (TID), or four times (QID) per day. In some embodiments, Compound 1 is administered once per day. In some embodiments, Compound 1 is administered twice per day. In some embodiments, Compound 1 is administered three times per day. In some embodiments, Compound 1 is administered four times per day.

[0076] In some embodiments, about 50 mg of compound 1 is administered once a day. In some embodiments, about 150 mg of compound 1 is administered once a day. In some embodiments, about 300 mg of compound 1 is administered once a day. In some embodiments, about 800 mg of compound 1 is administered once a day.

[0077] In some embodiments, about 100 mg of compound 1 is administered twice a day. In some embodiments, about 300 mg of compound 1 is administered twice a day. In some embodiments, about 400 mg of compound 1 is administered twice a day. In some embodiments, about 800 mg of compound 1 is administered twice a day.

[0078] In another aspect, provided herein is a method of treating transthyretin (TTR) amyloidosis, comprising administering to a patient a compound having the formula: [ka] or a pharma- ceutical acceptable salt thereof to a subject in need thereof, wherein the therapeutically effective amount of Compound 1 maintains a trough blood plasma concentration of Compound 1 of at least 5 μM. In some embodiments, the therapeutically effective amount of Compound 1 maintains a trough blood plasma concentration of Compound 1 of at least 6 μM. In some embodiments, the therapeutically effective amount of Compound 1 maintains a trough blood plasma concentration of Compound 1 of at least 7.5 μM. In some embodiments, the therapeutically effective amount of Compound 1 maintains a trough blood plasma concentration of Compound 1 of at least 8 μM.

[0079] In some embodiments, a therapeutically effective amount of Compound 1 maintains a trough blood plasma concentration of Compound 1 between 5 and 30 μM. In some embodiments, a therapeutically effective amount of Compound 1 maintains a trough blood plasma concentration of Compound 1 between 5 and 25 μM. In some embodiments, a therapeutically effective amount of Compound 1 maintains a trough blood plasma concentration of Compound 1 between 6 and 20 μM. In some embodiments, a therapeutically effective amount of Compound 1 maintains a trough blood plasma concentration of Compound 1 between 7.5 and 15 μM. In some embodiments, a therapeutically effective amount of Compound 1 maintains a trough blood plasma concentration of Compound 1 between 7.5 and 10 μM.

[0080] In some embodiments, subjects receiving a therapeutically effective amount of Compound 1 experience an increase in blood serum concentration of transthyretin (TTR) compared to baseline levels. In some embodiments, subjects receiving a therapeutically effective amount of Compound 1 experience an increase in blood serum concentration of transthyretin (TTR) of at least about 10, 15, 20, 25, 30% or more compared to baseline levels after 28 days of treatment. In some embodiments, subjects receiving a therapeutically effective amount of Compound 1 experience an increase in blood serum concentration of transthyretin (TTR) of at least about 25% compared to baseline levels after 28 days of treatment. In some embodiments, subjects prior to treatment have TTR blood serum levels below baseline serum (seru) TTR concentration (20 mg / dL TTR). In some embodiments, subjects receiving an effective amount of Compound 1 for 28 days experience an increased blood serum TTR level such that blood serum TTR level is above baseline levels. In some embodiments, the subject experiencing increased TTR levels is a subject diagnosed with transthyretin amyloidosis (ATTR) cardiomyopathy.

[0081] There are various diseases or disorders associated with transthyretin (TTR) amyloidosis, including, but not limited to, familial amyloid polyneuropathy, familial amyloid cardiomyopathy, senile systemic amyloidosis, central amyloidosis, ocular amyloidosis, cerebral leptomeningeal amyloidosis, ocular leptomeningeal amyloidosis, vitreous amyloidosis, gastrointestinal amyloidosis, neurogenic amyloidosis, non-neurogenic amyloidosis, non-hereditary amyloidosis, reactive / secondary amyloidosis, and cerebral amyloidosis.

[0082] In some embodiments, the disease or disorder associated with transthyretin (TTR) amyloidosis is leptomeningeal amyloidosis. In some embodiments, the subject with leptomeningeal amyloidosis has a transthyretin protein with an aspartic acid to glycine mutation at position 18 (D18G). In some embodiments, the subject with leptomeningeal amyloidosis has a transthyretin protein with a glycine to arginine mutation at position 53 (G53R). Wild-type transthyretin (TTR) protein is provided herein as SEQ ID NO:1.

[0083] In some embodiments, subjects with leptomeningeal amyloidosis have a transthyretin protein with a tyrosine to cysteine ​​mutation at position 114 (Y114C). Subjects with a tyrosine to cysteine ​​mutation at position 114 (Y114C) may exhibit ATTRm-PN symptoms, leptomeningeal amyloidosis symptoms, or a combination of both.

[0084] In some embodiments, subjects with leptomeningeal amyloidosis have a transthyretin protein with a threonine to proline mutation at position 49 (T49P). Subjects with a threonine to proline mutation at position 49 (T49P) may exhibit ATTRm-CM symptoms, leptomeningeal amyloidosis symptoms, or a combination of both.

[0085] In some embodiments, the transthyretin (TTR) amyloidosis disease is transthyretin amyloidosis (ATTR) cardiomyopathy or ATTR polyneuropathy. In some embodiments, the TTR amyloidosis is characterized by a TTR protein containing a threonine to alanine mutation at position 60 (T60A). In some embodiments, the TTR amyloidosis is characterized by a TTR protein containing a proline to serine mutation at position 24 (P24S). In some embodiments, the TTR amyloidosis is characterized by a TTR protein containing an aspartic acid to alanine mutation at position 38 (D38A). In some embodiments, the TTR amyloidosis is characterized by a TTR protein containing a leucine to histidine mutation at position 58 (L58H). Patients with these mutations often have a combination of symptoms of both ATTR cardiomyopathy and ATTR polyneuropathy. The wild-type transthyretin (TTR) protein is provided herein as SEQ ID NO:1.

[0086] In some embodiments, the transthyretin (TTR) amyloidosis disease is transthyretin amyloidosis (ATTR) cardiomyopathy. In some embodiments, the transthyretin (TTR) amyloidosis disease is transthyretin amyloidosis (ATTR) polyneuropathy.

[0087] ATTR cardiomyopathy includes wild-type ATTR cardiomyopathy (ATTRwt-CM) and hereditary (familial) ATTR cardiomyopathy (ATTRm-CM). ATTRm-CM is caused by a mutation in the TTR protein, whereas ATTRwt-CM is not caused by a mutation. Instead, ATTRwt-CM is generally age-related in its course. In some embodiments, ATTR cardiomyopathy is ATTRwt-CM. In some embodiments, ATTR cardiomyopathy is ATTRm-CM. In some embodiments, a subject with ATTRm-CM has a valine to isoleucine mutation at position 122 (V122I) in the TTR protein. In some embodiments, a subject with ATTRm-CM has a threonine to proline mutation at position 49 (T49P). A subject with a threonine to proline mutation at position 49 (T49P) may show ATTRm-CM symptoms, leptomeningeal amyloidosis symptoms, or a combination of both. The wild-type transthyretin (TTR) protein is provided herein as SEQ ID NO:1.

[0088] ATTR polyneuropathy includes both wild-type and inherited (familial) ATTR polyneuropathy. As discussed for cardiomyopathies, ATTRm-PN is caused by a mutation in the TTR protein, whereas ATTRwt-PN does not include a genetic component. In some embodiments, ATTR-PN is ATTRwt-PN. In some embodiments, ATTR-PN is ATTRm-PN. In some embodiments, ATTRm-PN is characterized by a TTR protein that includes a valine to methionine mutation at position 30 (V30M). In some embodiments, ATTRm-PN is characterized by a TTR protein that includes a phenylalanine to leucine mutation at position 64 (F64L). In some embodiments, ATTRm-PN is characterized by a TTR protein that includes a tyrosine to cysteine ​​mutation at position 114 (Y114C). Subjects with a tyrosine to cysteine ​​mutation at position 114 (Y114C) may exhibit ATTRm-PN symptoms, leptomeningeal amyloidosis symptoms, or a combination of both. The wild-type transthyretin (TTR) protein is provided herein as SEQ ID NO:1.

[0089] ATTR cardiomyopathy (both wild type and familial) is a slowly progressing disease that causes heart failure and death in affected subjects. The disclosed methods provide clinical improvement in subjects with ATTR cardiomyopathy by halting or slowing the accumulation of TTR fibrils in the myocardium. Through this process, the presently described methods provide clinical improvement in subjects with ATTR cardiomyopathy. Clinical improvement includes, but is not limited to, improved New York Heart Association (NYHA) functional class, Kansas City Cardiomyopathy Questionnaire (KCCQ) response, improved EuroQoL-5 dimensions (EQ-5D-5L), improvement in 6-minute walk test performance, improvement in markers related to cardiac health such as troponin T, troponin I, brain natriuretic peptide (BNP), and N-terminal pro-BNP, reduced frequency of cardiovascular-related hospitalizations, and / or reduced mortality.

[0090] In some embodiments, the method provided herein improves, stabilizes, or delays the progression of the New York Heart Association (NYHA) functional class of a subject.The NYHA functional class grades the severity of heart failure symptoms into one of four functional classes.The NYHA functional class is widely used in clinical practice and research, as it provides a standard description of severity that can be used to assess response to treatment and guide management.The NYHA functional class is based on the severity of symptoms and physical activity limitations: Class I: No limitation of physical activity. Ordinary physical activity does not cause undue shortness of breath, fatigue, or palpitations. Class II: Slight limitation of physical activity. Comfortable at rest, but ordinary physical activity results in undue shortness of breath, fatigue, or palpitations. Class III: Marked limitation of physical activity. Comfortable at rest, but less than normal physical activity results in excessive shortness of breath, fatigue, or palpitations. Class IV: Impossibility to perform any physical activity without discomfort. Symptoms may be present at rest. If any physical activity is performed, discomfort increases.

[0091] In some embodiments, administering a therapeutically effective amount of Compound 1 reduces the New York Heart Association (NYHA) functional class of the subject. In some embodiments, NYHA functional class is reduced from class IV to class III, from class IV to class II, or from class IV to class I. In some embodiments, NYHA functional class is reduced from class IV to class III. In some embodiments, NYHA functional class is reduced from class IV to class II. In some embodiments, NYHA functional class is reduced from class III to class II. In some embodiments, NYHA functional class is reduced from class III to class I. In some embodiments, NYHA functional class is reduced from class II to class I.

[0092] In some embodiments, the methods provided herein improve, stabilize, or delay progression of the Kansas City Cardiomyopathy Questionnaire (KCCQ) classification of the subject. In some embodiments, the methods described herein provide improved scores in the Kansas City Cardiomyopathy Questionnaire (KCCQ) (Green CP et al., (2000) Journal of the American College of Cardiology 35: 1245-55), the contents of which are incorporated herein by reference for all purposes. The KCCQ includes specific questions related to cardiac health and provides a validated, reliable and sensitive measurement of disease-specific health-related quality of life.

[0093] The KCCQ questionnaire asks the subject to rate how limited they are in carrying out normal aspects of their life (e.g., severely limited, very limited, moderately limited, slightly limited, or not limited at all, etc.). In some embodiments, the subject has an average improvement of at least one level (e.g., from severely limited to very limited, very limited to moderately limited, moderately limited to slightly limited) for all questions on the questionnaire after treatment with Compound 1.

[0094] In some embodiments, the methods provided herein improve, stabilize, or delay the deterioration of EuroQoL-5 Dimensions (EQ-5D-5L) scores in a subject. The EQ-5D-5L is a brief, self-administered, general health status instrument that takes about 5 minutes to complete. This instrument includes two parts. In the first part, the respondent is asked to grade their current health status on five dimensions (mobility, self-care, usual activities, pain or discomfort, and anxiety or depression), with each dimension having five levels of functioning (1-no problems, 2-slight problems, 3-moderate problems, 4-significant problems, and 5-extreme problems). The second part is the respondent's self-grading of their current health status on a visual analog scale (EQ VAS) with endpoints marked "best possible health status" (score 100) and "worst possible health status" (score 0). Scores from the five dimensions can be used to calculate a single index value, also known as the utility score. The EQ-5D-5L questionnaire is in the public domain and is available from EuroQoL.

[0095] In some embodiments, patients receiving the methods of treatment described herein have a mean improvement in EuroQoL-5 Dimensions (EQ-5D-5L) utility score of at least 1, 2, 3, 4, 5, 6, 7, 8 (either), 9, or 10 points. In some embodiments, patients receiving the methods of treatment described herein have a mean improvement in EuroQoL-5 Dimensions (EQ-5D-5L) utility score of at least 5 points.

[0096] In some embodiments, the methods described herein improve the subject's performance in the 6-minute walk test. The 6-minute walk test is a 6-minute, self-paced, timed walk to assess the subject's level of functional capacity. The subject can stop and rest during this test if the level of work exceeds his / her comfort level. The pre-treatment, post-treatment and during-treatment assessments are relatively easy to assess and consist of measuring the distance the subject has walked in a 6-minute period. Thus, in some embodiments, the subject increases the total distance covered in the 6-minute walk test after treatment with Compound 1. In some embodiments, the subject walks at least 25m more than the baseline distance measured before treatment with Compound 1. In some embodiments, the subject walks at least 30m more than the baseline distance measured before treatment with Compound 1. In some embodiments, the subject walks at least 50m more than the baseline distance measured before treatment with Compound 1. In some embodiments, the subject walks at least 75m more than the baseline distance measured before treatment with Compound 1. In some embodiments, the subject walks at least 100m more than the baseline distance measured before treatment with Compound 1. In some embodiments, the subject receiving the treatment methods described herein has a reduction in lag in 6-minute walk distance. For example, in some embodiments, the subject maintains about the same 6-minute walk distance as before treatment. In some embodiments, the subject covers 10m less in the 6-minute walk test. In some embodiments, the 6-minute walk test is used to compare treated groups with non-treated groups. In some embodiments, the average change between groups from baseline is at least 10m. In some embodiments, the average change between groups from baseline is at least 20m. In some embodiments, the average change between groups from baseline is at least 30m. In some embodiments, the treatment methods provided herein reduce the decrease in 6-minute walk distance compared to individuals not receiving treatment.

[0097] Troponin T, troponin I, brain natriuretic peptide (BNP), and N-terminal pro-BNP are polypeptides elevated in blood serum of subjects with poor myocardial health. In some embodiments, the levels of troponin T, troponin I, BNP, and / or N-terminal pro-BNP are decreased after treatment with Compound 1. In some embodiments, the levels of troponin T, troponin I, BNP, and / or N-terminal pro-BNP are decreased by about 10% compared to the baseline levels of troponin T, troponin I, BNP, and / or N-terminal pro-BNP of the subject before treatment with Compound 1. In some embodiments, the levels of troponin T, troponin I, BNP, and / or N-terminal pro-BNP are decreased by about 15% compared to the baseline levels of troponin T, troponin I, BNP, and / or N-terminal pro-BNP of the subject before treatment with Compound 1.

[0098] As discussed above, the clinical improvement provided in some embodiments of the disclosed methods is a reduction in the rate of cardiovascular-related hospitalizations in subjects receiving the treatment compared to subjects not receiving the treatment. In some embodiments, patients have at least 0.5, 1, 1.5, 2, 3, 4, 5 fewer cardiovascular-related hospitalizations per year on average compared to those not receiving the treatment.

[0099] An additional clinical benefit provided in some embodiments of the methods disclosed herein is a reduction in mortality compared to individuals not receiving the treatment. In some embodiments, mortality is reduced by about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30% or more compared to subjects not receiving the treatment.

[0100] ATTR polyneuropathy is a disease in which deposition of TTR amyloid (ATTR) impairs or otherwise impairs normal nerve function. ATTR polyneuropathy is a progressive disease that causes cachexia and death in affected subjects. The disclosed methods provide clinical improvement in subjects with ATTR polyneuropathy by halting or slowing the accumulation of TTR fibrils. Through this process, the methods described herein provide subjects with clinical improvement in ATTR polyneuropathy. Clinical improvement includes, but is not limited to, improvement in neuropathy score (NIS) or modified neuropathy score +7 (mNIS+7), improvement in Norfolk Quality of Life Diabetic Neuropathy (QOL-DN) questionnaire, improvement in Composite Autonomic Symptom Score (COMPASS-31) score, improved nutritional status as measured by modified body mass index (mBMI), and / or improvement in the subject's 10-meter walk test.

[0101] In some embodiments, the methods described herein provide improved neuropathy score (NIS). NIS refers to a scoring system that measures weakness, sensation and reflexes. NIS score evaluates the standard muscle groups for weakness (1 is 25% reduction, 2 is 50% reduction, 3 is 75% reduction, 3.25 is movement against gravity, 3.5 is movement with gravity removed, 3.75 is muscle twitching without movement, and 4 is paralysis), the standard group of muscle stretch reflex (0 is normal, 1 is reduced, 2 is absent), and touch, vibration, joint position and movement, and pinprick (all graded on index finger and thumb: 0 is normal, 1 is reduced, 2 is absent). Evaluation is corrected for age, sex, and physical strength.

[0102] In some embodiments, the methods described herein slow disease progression such that the rate of increase in NIS score is reduced compared to subjects not receiving Compound 1. In some embodiments, the methods described herein halt disease progression such that there is no change in NIS score following treatment with Compound 1.

[0103] In some embodiments, the methods described herein reduce the NIS score after treatment with Compound 1. In some embodiments, the methods described herein reduce the NIS score by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60% compared to the baseline level measured before treatment with Compound 1. In some embodiments, the methods described herein reduce the NIS score by at least 5% compared to the baseline level measured before treatment with Compound 1. In some embodiments, the methods described herein reduce the NIS score by at least 10% compared to the baseline level measured before treatment with Compound 1. In some embodiments, the methods described herein reduce the NIS score by at least 15% compared to the baseline level measured before treatment with Compound 1.

[0104] In some embodiments, the methods described herein provide an improved modified neuropathy score (mNIS+7). mNIS+7 refers to a clinical trial-based assessment of neurological dysfunction (NIS) combined with electrophysiological measurements of small and large nerve fiber function (NCS and QST) and measurements of autonomic function (postural blood pressure). mNIS+7 score is a modified NIS+7 score (which represents NIS+7 tests). NIS+7 analyzes weakness and muscle stretch reflexes. Five of the seven tests include nerve conduction characteristics. These characteristics are peroneal nerve compound muscle action potential amplitude, motor nerve conduction velocity and motor nerve distal latency (MNDL), tibial MNDL, and sural sensory nerve action potential amplitude. These values ​​are corrected for age, sex, height, and weight variations. The remaining two of the seven tests include vibration detection threshold and heart rate reduction after deep breathing. The mNIS+7 score amends the NIS+7 to account for the use of smart quantitative somatosensory testing, novel autonomic assessments, and compound muscle action potentials of the ulnar, peroneal, and tibial nerve amplitudes, and sensory nerve action potentials of the ulnar and sural nerves (Suanprasert, N. et al., Retrospective study of a TTR FAP cohort to modify NIS+7 for therapeutic trials, J. Neurol. Sci., 2014. 344(1-2): 121-128). Further details of the mNIS+7 study can be found in US2017 / 0307608, the contents of which are incorporated herein by reference, for all purposes.

[0105] In some embodiments, the methods described herein slow disease progression such that the rate of increase in mNIS+7 score is reduced compared to subjects not receiving Compound 1. In some embodiments, the methods described herein halt disease progression such that there is no change in mNIS+7 score following treatment with Compound 1.

[0106] In some embodiments, the methods described herein reduce the mNIS+7 score after treatment with Compound 1. In some embodiments, the methods described herein reduce the mNIS+7 score by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60% compared to the baseline level measured before treatment with Compound 1. In some embodiments, the methods described herein reduce the mNIS+7 score by at least 5% compared to the baseline level measured before treatment with Compound 1. In some embodiments, the methods described herein reduce the mNIS+7 score by at least 10% compared to the baseline level measured before treatment with Compound 1. In some embodiments, the methods described herein reduce the mNIS+7 score by at least 15% compared to the baseline level measured before treatment with Compound 1.

[0107] In some embodiments, the methods described herein provide improved scores on the Norfolk Quality of Life Diabetic Neuropathy (QOL-DN) questionnaire, which is a well-known and validated questionnaire that captures pain associated with large fiber, small fiber, and autonomic neuropathy. The questionnaire includes items related to symptoms experienced by the subject and questions related to the impact of neuropathy on the subject's daily activities.

[0108] In some embodiments, the methods described herein slow the progression of the disease such that the rate of decline in Norfolk QOL-DN score is reduced compared to subjects not receiving Compound 1. In some embodiments, the methods described herein halt the progression of the disease such that there is no change in Norfolk QOL-DN score after treatment with Compound 1. In some embodiments, the methods described herein slow the progression of the disease such that there is no change in Norfolk QOL-DN score after treatment with Compound 1.

[0109] In some embodiments, the methods described herein improve the Norfolk QOL-DN score after treatment with Compound 1. In some embodiments, the methods described herein improve the Norfolk QOL-DN score by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60% compared to the baseline level measured before treatment with Compound 1. In some embodiments, the methods described herein improve the Norfolk QOL-DN score by at least 5% compared to the baseline level measured before treatment with Compound 1. In some embodiments, the methods described herein improve the Norfolk QOL-DN score by at least 10% compared to the baseline level measured before treatment with Compound 1. In some embodiments, the methods described herein improve the Norfolk QOL-DN score by at least 15% compared to the baseline level measured before treatment with Compound 1. In some embodiments, the methods described herein provide a change in the subject's Norfolk QOL-DN of about -1.5, -2.0, -2.5, -3.0, -3.5, -4.0, -4.5, -5.0, -5.5, -6.0, -6.7, -7.0, -7.5, -8.0, -8.5, -9.0, -9.5, or -10.0 compared to the subject's baseline score.

[0110] In some embodiments, the methods disclosed herein provide a composite autonomic symptom score (COMPASS-31). The composite autonomic symptom score (COMPASS-31) is a patient questionnaire that assesses symptoms of autonomic dysbiosis. In one embodiment, the methods of the present invention provide the subject with an improvement in the COMPASS-31 score relative to baseline. Such improvement can take the form of an increase in the subject's COMPASS-31 score of at least 0.1 points, e.g., at least 0.2, at least 0.3, at least 0.4, or at least 0.5 points, e.g., 0.1, 0.2, 0.3, 0.4, or 0.5. In some embodiments, these methods slow the progression of the disease such that there is no change in the COMPASS-31 score. In yet another embodiment, the methods of the present invention slow the rate at which the COMPASS-31 score decreases, e.g., slow the rate at which the COMPASS-31 score decreases in subjects treated with AG10 compared to the rate at which the COMPASS-31 score decreases in subjects not treated with AG10.

[0111] In some embodiments, the methods disclosed herein provide improved nutritional status as measured by modified body mass index (mBMI), which is determined by multiplying an individual's BMI by their serum albumin level. The calculation of mBMI takes into account the contribution of edema to total body weight. In one embodiment, the methods disclosed herein provide a subject with an improvement in mBMI relative to baseline. Such improvement can take the form of a reduction in mBMI score of about 2, 5, 7, 10, 12, 15, 20, or about 25. In other embodiments, these methods stop the mBMI index score from increasing, e.g., these methods result in a 0% increase in mBMI score. In yet another embodiment, the methods of the present invention slow the rate at which mBMI score increases, e.g., slow the rate at which mBMI score increases in subjects treated with AG10 compared to the rate at which mBMI score increases in subjects not treated with AG10.

[0112] In some embodiments, the method disclosed herein provides improvement in 10m walking test.This test measures the walking speed of an individual over 10m.In one embodiment, the method disclosed herein provides the subject with an increase from baseline in 10m walking test.In some embodiments, the increase from baseline in 10m walking test is about 0.025, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or about 5.0m / s.

[0113] In some embodiments, the methods disclosed herein provide an improved Dyck / Rankin score. Dyck / Rankin scores are known in the art and are assigned by physicians after evaluating the patient's symptoms, neuropathy, test results, and verifying the patient's ability to perform activities of daily living. Only the disability associated with peripheral neuropathy is graded. In determining whether a patient has difficulty or inability to perform a specific task or perform an activity of daily living, more than the patient's report is used in the judgment; physicians must use objective criteria. The stages (0-8) are outlined below: 0. No neuropathy No symptoms (NSS<1), no signs (NIS<2 points); or no abnormal neuropathy tests (e.g., 7 tests <97.5). 1. Minimal neuropathy (only one of A, B, or C is abnormal) a) the test is the only abnormality (e.g., 7 tests > 97.5); or b) Neuropathic signs are the only abnormality (e.g., NIS > 2 points); or c) Neuropathic symptoms are the only abnormality (e.g., NSS>1) 2. Minimal neuropathy: 1a+1b 3. Symptomatic neuropathy: 1a, 1a+1c; 1a+1c or 1b+1c. Patients are able to carry on with their usual activities of daily living, work or leisure activities, and are able to fulfill their usual family and social responsibilities. * Neuropathic symptoms: NSS≧1 symptoms of muscle weakness, atrophy or spasm; negative or positive, neuropathic sensory symptoms (N-NNS, P-NSS); or, neuropathic autonomic symptoms. - Normal activities of daily living, work, leisure and social and domestic activities: Despite the neuropathic symptoms, patients are able to work at their normal activities, maintain their normal household duties and participate in leisure activities. Generally, patients are able to continue despite some motor, sensory or autonomic symptoms. Patients are unable to perform extraordinary activities, e.g. athletics, feats of endurance, etc. 4. Symptomatic neuropathy (defined as 3) that interferes with and limits work, usual activities of daily living, leisure activities, or family and social obligations, but independent function is possible without assistance. With this score, there is a clear limitation in usual* work, usual or leisure activities, family or social obligations* because of the neuropathy. * The degree of motor, sensory, or autonomic symptoms or impairment is sufficient to limit the ability to work, perform usual activities of daily living, leisure activities, or fulfill family and usual social responsibilities. Unless the patient is able to perform the "usual" activities of daily living, leisure activities, and fulfill social and family responsibilities, the use of a can or orthotics would probably place the patient in this (or higher) category (they would then be in a lower category). 5. Symptomatic neuropathy (as defined in 3) limiting activities of daily living, work, and leisure activities. Assistance* from other caregiver (<2.5 hrs / day) is required. Patients would normally be scored on this or higher (>5) if wheelchair use is essential for daily loving activities, leisure activities, or social and family responsibilities. * A family member or visiting nurse is required to provide activities of daily living (bathing, shaving, brushing teeth, eating, etc.), daily management of pain medications or opiates, or to assist with management of autonomic dysfunction that the patient cannot adequately and safely do themselves. 6. Symptomatic neuropathy (as defined in 3) requiring caregiver assistance for ≥2.5 to <8 hours / day as described in 5. 7. Symptomatic neuropathy (as defined in 3) requiring caregiver assistance for >8 hours / day, but not continuously as in stage 8. 8. Symptomatic neuropathy (as defined in 3) requiring constant nursing care in an intensive care unit.

[0114] The duration of administration will vary depending on a number of factors, including the specific disease being treated. For example, there is a genetic component, particularly in transthyretin (TTR) amyloidosis disease or condition, that requires chronic (i.e., continuous, long-term) administration. However, in some embodiments, administration of Compound 1 to a subject with genetic TTR amyloidosis disease continues for a set period of time while the subject is manifesting or experiencing symptoms associated with TTR amyloidosis disease or condition, or after a specific endpoint is achieved (e.g., alleviation or complete elimination of symptoms). If symptoms of TTR amyloidosis disease recur or begin to re-emerge, administration of Compound 1 is resumed.

[0115] For subjects with ungenetically linked TTR amyloidosis disease, numerous administration options are available and will depend on the severity of the disease and the clinical symptoms presented. In some embodiments, long-term administration of Compound 1 is required. In some embodiments, shorter or acute administration of Compound 1 is required. In some embodiments, administration of Compound 1 to subjects with ungenetically linked TTR amyloidosis disease continues for a set period of time while the subject is manifesting or experiencing symptoms associated with TTR amyloidosis disease or pathology, or after a specific endpoint is achieved (e.g., alleviation or complete elimination of symptoms). If symptoms of TTR amyloidosis disease recur or begin to re-emerge, administration of Compound 1 is resumed.

[0116] In some embodiments, Compound 1 is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 108, 109, 109, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 1 3, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 days or more are administered. In some embodiments, compound 1 is administered for 7, 14, 21, 28, 35, 42, 49, or 56 days. In some embodiments, compound 1 is administered for 28 days. In some embodiments, compound 1 is administered for 56 days. In some embodiments, compound 1 is administered for 84 days.

[0117] In some embodiments, compound 1 is administered for at least 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, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 months. In some embodiments, compound 1 is administered for 10, 15, 20, 25, 30, 35, 40, 45, or 50 months. In some embodiments, compound 1 is administered for 6 months. In some embodiments, compound 1 is administered for 12 months. In some embodiments, compound 1 is administered for 18 months. In some embodiments, compound 1 is administered for 24 months. In some embodiments, Compound 1 is administered for 30 months. In some embodiments, Compound 1 is administered for 36 months. In some embodiments, Compound 1 is administered for 42 months.

[0118] Advantageously, the drugs used in diuretic therapy do not change the exposure of AG10 during treatment. Thus, patients receiving diuretic therapy can be administered AG10 without modified or specific dosing regimen. As a result, in some embodiments, the subject receiving AG10 also receives additional diuretic therapy drugs. Diuretic therapy drugs include, but are not limited to, ethacrynic acid, bumetanide, furosemide, and torasemide. In some embodiments, the diuretic is selected from the group consisting of furosemide or torasemide.

[0119] Pharmaceutical Compositions Compound 1 can be formulated into a variety of compositions suitable for delivery to a subject. Compositions suitable for administration to a subject typically contain Compound 1, or a pharma- ceutically acceptable salt thereof, and a pharma-ceutically acceptable excipient.

[0120] The pharmaceutical composition for administration of Compound 1 can be conveniently presented in unit dosage form and can be prepared by any method known in the field of pharmacy and drug delivery.All methods include the step of bringing the active ingredient into association with a carrier that contains one or more accessory ingredients.In general, the pharmaceutical composition is prepared by bringing the active ingredient into uniform and intimate association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into desired formulation.

[0121] Suitable formulations for use in the present invention can be found in "Remington: The Science and Practice of Pharmacy", 21st Edition, edited by Gennaro, Lippincott Williams & Wilkins (2003), which is incorporated herein by reference. The pharmaceutical compositions described herein can be manufactured in a manner known to those skilled in the art, i.e., by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. The following methods and excipients are merely illustrative and are in no way limiting.

[0122] Compound 1 can be incorporated into various preparations for therapeutic administration. More specifically, compound 1 can be formulated into pharmaceutical compositions, either together or separately, by formulation with suitable pharma- ceutical acceptable carriers or diluents, and can be formulated into preparations in solid, semi-solid, liquid or gaseous form, such as tablets, capsules, pills, powders, granules, dragees, gels, slurries, ointments, liquids, suppositories, injections, inhalants and aerosols. Thus, administration of the compounds of the present invention can be achieved in various ways, including oral, buccal, parenteral, intravenous, intradermal (e.g., subcutaneous, intramuscular), transdermal, etc. Furthermore, compound 1 can be administered in a local rather than systemic manner, such as, for example, a depot or sustained release formulation.

[0123] Formulations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil, etc. Additionally, emulsions can be prepared with non-water-miscible ingredients such as oils and stabilized with surfactants such as mono-diglycerides, PEG esters, etc.

[0124] Aqueous suspensions contain the active substance mixed with excipients suitable for the manufacture of aqueous suspensions.Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing agents or wetting agents can be natural phosphatides, for example, lecithin, condensation products of alkylene oxides with fatty acids, for example, polyoxy-ethylene stearates, or condensation products of ethylene oxide with long-chain aliphatic alcohols, for example, heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitols, for example, polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitol anhydrides, for example, polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0125] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, such as sweeteners, flavorings and coloring agents, may also be present.

[0126] Pharmaceutical dosage form The present disclosure includes pharmaceutical dosage forms of Compound 1, or pharma- ceutically acceptable forms thereof. The dosage forms described herein are suitable for oral administration to a subject. The dosage forms may be any form suitable for oral administration, including, but not limited to, capsules or tablets.

[0127] In some embodiments, the present disclosure provides Compound 1 having the formula: [ka] or a pharma- ceutical acceptable salt thereof in the form of a capsule or tablet in a single unit dosage form containing 10 to 1,000 mg.

[0128] In some embodiments, the amount of Compound 1 is about 100-800 mg. In some embodiments, the amount of Compound 1 is about 150-600 mg. In some embodiments, the amount of Compound 1 is about 200-400 mg. In some embodiments, the amount of Compound 1 is about 200 mg. In some embodiments, the amount of Compound 1 is about 400 mg. In some embodiments, the single dose capsule or tablet contains the HCl salt of Compound 1.

[0129] In some embodiments, the single unit dosage form of Compound 1 is a tablet.

[0130] In some embodiments, the single unit dosage form of Compound 1 is a capsule.

[0131] In some embodiments, the single unit dosage form is a size #0, #1, #2, #3, #4, or #5 capsule. In some embodiments, the single unit dosage form is a size #0 capsule. In some embodiments, the single unit dosage form is a size #1 capsule. In some embodiments, the single unit dosage form is a size #2 capsule. In some embodiments, the single unit dosage form is a size #3 capsule. In some embodiments, the single unit dosage form is a size #4 capsule. In some embodiments, the single unit dosage form is a size #5 capsule.

[0132] kit The present disclosure also encompasses kits that include the pharmaceutical compositions and dosage forms of the present invention.

[0133] In some embodiments, the present invention provides a kit comprising Compound 1 or a pharma- ceutically acceptable salt thereof. Some of the kits described herein include a label that describes how to administer Compound 1. Some of the kits described herein include a label that describes how to treat transthyretin (TTR) amyloidosis. In some embodiments, the kits described herein include a label that describes how to treat wild-type transthyretin amyloid cardiomyopathy (ATTR-CM, also referred to as senile systemic amyloidosis). In some embodiments, the kits described herein include a label that describes how to treat familial amyloid cardiomyopathy (ATTR-mCM). In some embodiments, the kits described herein include a label that describes how to treat familial amyloid polyneuropathy (ATTR-PN, also referred to as FAP).

[0134] Compositions of the invention include, but are not limited to, compositions containing Compound 1 in a bottle, jar, vial, ampoule, tube, blister pack, or other container closure system approved by the U.S. Food and Drug Administration (FDA) or other regulatory agency, which may provide one or more unit doses containing Compound 1 or a pharma- ceutically acceptable salt thereof. The package or dispenser may also bear a notice associated with the container in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, a notice indicating approval by the agency. In certain embodiments, the kit may include a formulation or composition described herein, a container closure system containing the formulation or one or more unit dosage forms containing the formulation, and a notice or instructions describing the method of use described herein.

[0135] Packaging systems such as blister packs include thermoformable rigid film or PVC, suitable for pharmaceutical packaging and extrusion of type lid. Lids can include foil, made of primer / aluminum / heat seal-coating, or can be paper-based. Those skilled in the art will easily prepare blister packs containing Compound 1. The bottle systems described herein include a child-resistant seal, which can be made in various sizes (e.g., 75cc, 100cc, 200cc, etc.), and generally can be made of polypropylene. In some embodiments, pharmaceutical dosage forms of Compound 1 are packaged in 75cc bottles with child-resistant seals. Those skilled in the art can easily prepare the bottle systems described herein.

[0136] In some embodiments, the disclosure provides kits for twice-daily dosing. These kits provide one or more unit doses containing Compound 1 for each administration.

[0137] In some embodiments, the total daily dose of compound 1 is 800mg, which means that 400mg is administered in the first dose and 400mg is administered in the second dose.In some embodiments, two unit doses containing 200mg of compound 1 are administered in the first dose, and two unit doses containing 200mg of compound 1 are administered in the second dose.In some embodiments, one unit dose containing 400mg of compound 1 is administered in the first dose, and one unit dose containing 400mg of compound 1 is administered in the second dose.In some embodiments, the HCl salt form of compound 1 is administered.

[0138] In some embodiments, the total daily dose of compound 1 is 1,600mg, meaning that 800mg is administered in the first dose and 800mg is administered in the second dose.In some embodiments, four unit doses containing 200mg of compound 1 are administered in the first dose, and four unit doses containing 200mg of compound 1 are administered in the second dose.In some embodiments, two unit doses containing 400mg of compound 1 are administered in the first dose, and two unit doses containing 400mg of compound 1 are administered in the second dose.In some embodiments, the HCl salt form of compound 1 is administered. EXAMPLES

[0139] IV. Working Examples The following examples are offered to illustrate, but not to limit, the claimed invention.

[0140] Materials and Methods The following general materials and methods were used as specified or may be used in the examples below.

[0141] Determination of AG10 blood plasma concentration Human plasma containing AG10 and the internal standard AG10-D6 was extracted using protein precipitation and analyzed by a Sciex API4000 LC-MS-MS equipped with an HPLC column. The peak area of ​​the AG10 product ion was measured against the peak area of ​​the product ion of the AG10-D6 internal standard. Quantification was performed using a weighted 1 / χ2 ratio generated from calibration standards prepared on the day of extraction. 2 Linear least squares regression analysis was used.

[0142] Fluorescent Probe Exclusion Assay (FPE) Occupancy of AG10 in the thyroxine-binding pocket of tetrameric TTR is determined by the ability of the fluorescent probe to covalently bind to free tetrameric TTR binding sites in serum over a 6 hour reaction period.

[0143] An aliquot of each serum sample is placed in a 96-well plate. The change in fluorescence (λ) after addition of the probe is ex = 328 nm and λ em = 384 nm) is monitored every 15 min using a fluorescence capable microplate reader for 6 h at RT.

[0144] Western blot for assessment of tetrameric TTR stabilization Stabilization of TTR tetramers by AG10 is determined by comparing the amount of tetrameric TTR protein remaining after 72 hours of acid denaturation to the initial amount of tetrameric TTR protein, as determined by densitometry of Western blot gels.

[0145] Blood plasma samples from subjects at both time points 0 and 72 hours are diluted with acidified buffer (sodium acetate, KCl, EDTA, DTT, pH approx. 4.0). Time 0 samples are cross-linked directly with glutaraldehyde and then quenched. The 72 hour samples are incubated at room temperature for 72 hours and then cross-linked and quenched by the same protocol. All samples are then denatured by adding SDS gel loading buffer and boiling before loading the gel. Each sample is separated on an SDS-PAGE gel and analyzed by immunoblotting using anti-TTR antiserum (polyclonal rabbit anti-human prealbumin, DAKO, catalog number A0002). The density of all TTR bands is quantified using an infrared LICOR imaging system or a fluorescent imaging system and reported with normalization of the IgG bands using LICOR 925-32232 or Invitrogen 84546.

[0146] In the experiments, tables, and figures discussed in more detail below, nominal times of blood collection are used for all pharmacokinetic and pharmacodynamic data.

[0147] Example 1: Preparation of AG10·HCl [ka] The compound of formula IIIa (100 g, 495 mmol, 1.0 equiv.) was dissolved in acetone (1 L). The compound of formula II (49.59 g, 495 mmol, 1.0 equiv.) was added to the previous solution, followed by K2CO3 (82.14 g, 594.38 mmol, 1.2 equiv.) and KI (41.11 g, 247 mmol, 0.5 equiv.) at room temperature with stirring. The reaction mixture was heated to 60±5° C. and stirred at this temperature for 40 hours. The reaction mixture was filtered and then concentrated under reduced pressure to provide the compound of formula IV (102 g) as a viscous orange liquid.

[0148] [ka] The compound of formula IV (100 g, 632 mmol, 1.0 equiv.) was dissolved in ethanol (1 L). Hydrazine hydrate (87 g, 1738 mmol, 2.75 equiv.) and concentrated HCl (4.6 mL, 0.2 equiv.) were added to the previous solution at room temperature. The reaction mixture was heated to 75±5° C. and stirred at this temperature for 3 h. After completion of the reaction by TLC (70% ethyl acetate:n-hexane, visualized with iodine) and observation of the product peak by mass spectrometry, the reaction mixture was concentrated under reduced pressure to provide the compound of formula V (70 g) as a colorless liquid syrup, which was used directly in the next step.

[0149] [ka] The compound of formula V (35 g, 227 mmol, 1.0 equiv) was dissolved in 1,2-dichloroethane (525 mL). PBr3 (64.67 mL, 681 mmol, 3 equiv) was added in portions over 30 min at room temperature. The reaction mixture was heated to 75±5° C. and stirred at this temperature for 3 h. After completion of the reaction by TLC (50% ethyl acetate:n-hexane, visualized with iodine) and observation of the product peak by mass spectrometry, the reaction mixture was diluted with dichloromethane (350 mL) and quenched with saturated NaHCO3 solution until pH=7-8. Both the organic and aqueous layers were separated and collected. The organic layer was dried over MgSO4 and filtered. The filtrate was concentrated under reduced pressure to provide the compound of formula VIa (38 g) as a viscous orange liquid.

[0150] [ka] 4-(3-Bromopropyl)-3,5-dimethyl-1H-pyrazole hydrobromide (VIa) and DMSO were charged to a vessel and stirred vigorously at 20±10° C. for 10 minutes. The mixture was then heated to 55±5° C. with stirring. A stirred solution containing 4-fluoro-3-hydroxy-benzoic acid methyl ester (VIIa), potassium carbonate, and anhydrous DMSO was transferred to the mixture. The DMSO solution of the alkyl bromide was transferred slowly to maintain an internal temperature of 55.0±5° C. The addition was complete after 6 hours and the mixture was stirred vigorously at 55.0±5° C. for an additional hour. The mixture was cooled to 25±5° C. over 30 minutes and water was added while maintaining the temperature below 25° C. The mixture was extracted with ethyl acetate and the returned aqueous layer was extracted with ethyl acetate. The pooled ethyl acetate solution was washed with brine. The combined ethyl acetate washes were concentrated under vacuum to a minimum volume and heptane was added, which precipitated VIIIa. The mixture was heated to 75±5° C. and stirred for 1 hour and aged. The mixture was cooled to 25±5° C. over 2 hours and the resulting solid was collected by filtration. The filter cake was washed with ethyl acetate in heptane (30%). The isolated solid was dried with a stream of nitrogen. The solid was dumped into a vessel and combined with ethyl acetate and heptane. The resulting mixture was heated to 75±5° C. to dissolve the solid. The solution was cooled to 25±5° C. over 2 hours and the resulting solid was collected by filtration. The solid was washed with 30% ethyl acetate / heptane solvent mixture and dried in a vacuum oven at 55° C. to yield VIIIa in >99.5% purity.

[0151] [ka] A jacketed glass vessel was charged with the compound of formula VIIIa (1.0 equiv.) and methanol. The mixture was cooled with stirring to 10±5° C. and aqueous sodium hydroxide (3 equiv.) was charged over 20 min. The mixture was aged by stirring at 20±5° C. for NLT 2 h, at which point the reaction was complete. Stirring was stopped and water was added. Methanol was then removed by vacuum distillation at an internal temperature of NMT 35° C. The resulting concentrated clear aqueous solution was cooled to 10° C. and concentrated HCl was added until the pH was below 1.4-1.6 (pH meter) to precipitate the HCl salt. The solid was collected by filtration, washed with 0.2 N HCl and dried under vacuum at 50° C. to yield the compound of formula Ia in purity NLT 99.5%.

[0152] Example 2: Phase 1 Clinical Trial Study AG10-001 was a two-part, randomized, double-blind, placebo-controlled, single- and multiple-ascending dose, first-in-human study conducted in healthy adult volunteers to evaluate the safety, tolerability, PK, and PD of AG10 after single and multiple doses. The study was also designed to evaluate the effect of food on the PK of AG10.

[0153] Part A was a single ascending dose (SAD) design in which four cohorts of eight healthy men and / or women were randomized 3:1 overall to AG10 or matching placebo.

[0154] Part B was a multiple ascending dose (MAD) design in which three cohorts of eight healthy men and / or women were randomized in a 3:1 ratio to receive AG10 or its placebo for 12 days of dosing. In total, four cohorts of eight healthy subjects each (total of 32 subjects, 24 receiving AG10·HCl and 8 receiving matching placebo) received ascending doses of 50 mg, 150 mg, 300 mg, and 800 mg of blinded study medication and completed the SAD portion of the study. One of these SAD cohorts received two 300 mg doses, one under fasting conditions, and the other followed a high-fat test meal after an appropriate washout period. Three cohorts of eight healthy subjects each (24 subjects total, 18 receiving AG10·HCl and 6 receiving matching placebo) received 100 mg, 300 mg, or 800 mg of blinded study medication every 12 hours for 12 days and completed the MAD portion of the trial.

[0155] Single Ascending Dose The PK profile observed in healthy subjects dosed with 50 mg AG10·HCl (SAD cohort 1), 150 mg AG10·HCl (SAD cohort 2), 300 mg AG10·HCl (SAD cohort 3), and 800 mg AG10·HCl (SAD cohort 4) showed that AG10 showed a mean T of 1 hour or less. max The results demonstrate that sirolimus has rapid oral absorption with an elimination half-life of approximately 22-27 hours. Table 1 lists the geometric means of PK parameters for SAD cohorts 1-4. Furthermore, as shown in Figure 1, there was moderate intersubject variability in the PK of SAD cohorts 1-4, with C max The %CV for ranged from 10.0% to 41.9%, as well as the AUC inf The %CV for ranges from 12.5% ​​to 40.4%. [Table 1]

[0156] In addition, as shown in Figure 2 (showing cohort 3), the food effect portion of the SAD study revealed minimal food effects on the pharmacokinetics of AG10, with no significant difference in C max A slight decrease in and a slightly longer T max However, AUC 0-24 There were no overall significant changes in exposure as defined by PK data for Cohort 3 are shown in Table 2 below. [Table 2]

[0157] Repeated dose escalation Pharmacokinetic plots for MAD cohort 1 (healthy subjects receiving 100 mg AG10·HCl every 12 hours for 12 days), cohort 2 (healthy subjects receiving 300 mg AG10·HCl every 12 hours for 12 days), and cohort 3 (healthy subjects receiving 800 mg AG10·HCl every 12 hours for 12 days) are shown in Figure 3 and PK parameters are listed in Table 3. For example, the C max Even if some intersubject variability was observed in the values, AUC 0-12 Values ​​were remarkably similar with %CV values ​​ranging from 8% to 22.2%. No significant accumulation was observed over the 12 days of dosing. [Table 3]

[0158] AG10 pharmacodynamics The pharmacodynamic (PD) properties of AG10 were assessed using fluorescent probe exclusion (FPE) assays or Western blot assays (described above), both of which are established assays of TTR target association and TTR stabilization.

[0159] As shown in Figures 4 and 5, data from the FPE assay confirmed target association at single doses of 300 mg and 800 mg, complete stabilization of TTR at peak concentrations, and sustained stabilization for up to 12 hours, ranging from 29% to 62% at 300 mg and 56% to 82% at 800 mg. Figure 6 shows the average percent target association for each single-dose ascending cohort as a function of time; the data demonstrate that ascending doses increase stabilization. Similarly, Western blot assays (Figures 7A and 7B) confirmed complete stabilization of TTR at peak concentrations, and sustained stabilization for up to 12 hours with a single dose of 300 mg of AG10·HCl. The FPE and Western blot assay results for SAD cohort 3 showed that R 2 There is good correlation, with coefficients >0.9 (Figure 8). Both pharmacological activity measures correlate well with each other.

[0160] In addition, as shown in Figures 9, 10, and 11, after 12 consecutive days of dosing with 100 mg q12h, 300 mg q12h, and 800 mg q12h of AG10·HCl, data from the FPE assay confirmed sustained target association at steady state, with mean stabilization of TTR at 12 hours post-dose on the last day of dosing ranging from 33% for the 100 mg q12h dose to 89% for the 800 mg q12h dose. Figure 12 shows the peak, mean, and trough TTR target association rates over 12 days for the 800 mg q12h dosing cohort with AG10·HCl.

[0161] FIG. 13 shows TTR Western blots from three subjects in cohort 3 treated with 800 mg AG10·HCl q12h. Subjects 1 and 3 were dosed with AG10 and subject 2 was dosed with placebo. The lane marked 0 hours, day 12 pre-dose, contains a sample collected from the subject at trough levels of AG10 after 11 days of dosing (total of 22 doses). The lane marked day 12 post-dose shows a sample collected at peak levels after the 23rd dose of AG10. Stabilization of tetrameric TTR protein is detected using acidification for 72 hours followed by cross-linking, SDS-PAGE and immunoblotting as described above. Subject 2 shows no remaining TTR tetramers detected by this experimental protocol. In contrast, subjects 1 and 3 show complete stabilization of tetrameric TTR at both trough and peak levels of AG10.

[0162] The mean PK-PD data from the SAD and MAD cohorts shown in Figure 14 demonstrate predictable, dose-responsive PD effects of AG10 as measured by the FPE assay in human subjects dosed with AG10.

[0163] summary The data provided herein for AG10 confirms target engagement, with complete stabilization of TTR at peak concentrations following both single and repeated doses, with stabilization continuing for up to 12 hours.

[0164] Example 3: Planned Phase 2 Clinical Trial The Phase 2 study is designed as a randomized, multicenter, double-blind, parallel-group, placebo-controlled, dose-ranging study to evaluate the safety, tolerability, PK, and PD of AG10 in patients with ATTR-CM with a background of stable heart failure therapy. Screening and randomization will be followed by a 28-day blinded, placebo-controlled treatment period. Approximately 45 subjects are planned to enroll in the Phase 2 study. A summary of this Phase 2 study is provided in Table 4. [Table 4]

[0165] Example 4: Enthalpy-driven stabilization of transthyretin by AG10 mimics natural generic variations that protect against transthyretin amyloidosis The Examples below illustrate the correlation between the enthalpic binding of AG10 and its enhanced efficacy in stabilizing multiprotein complexes.

[0166] Materials and Methods Isothermal titration calorimetry (ITC) Binding experiments were performed using a MicroCal PEAQ-ITC at 25°C. Ligand solutions (25 μM in PBS (pH 7.4), 100 mM KCl, 1 mM EDTA, 2.5% DMSO) were prepared and titrated into an ITC cell containing 2 μM TTR in the same buffer. Nineteen injections of ligand (2.0 μL each) were injected into the ITC cell (at 25°C) until the point at which TTR was fully saturated with ligand. Calorimetric data were plotted and fitted using a standard single-site binding model. For control, we tested the enthalpy change caused by titrating a bank of DMSO in buffer into TTR, resulting in binding enthalpies of <0.4 kcal / mol. We also used the ITC to titrate tafamidis and AG10 against human serum albumin (HSA). The Kd value for tafamidis (2.3 μM) was similar to that reported previously (Kd=2.5 μM; EMA Assessment Report EMA / 729083 / 2011). The binding affinity of AG10 was calculated to be approximately 8 μM, which also fits our data in Figure 20, where AG10 has lower binding to albumin compared to tafamidis.

[0167] FPE assay for TTR binding in buffer and human or dog serumThe binding affinity and selectivity of AG10 and other stabilizers to TTR in buffer and serum was determined by their ability to compete with the binding of fluorescent probe exclusion (FPE probe) which binds to TTR in buffer and human serum. The FPE probe is a thioester TTR ligand that is not itself fluorescent but which upon binding to the T4 binding site of TTR covalently modifies lysine 15 (K15) resulting in a fluorescent conjugate. Ligands that bind to the T4 site of TTR reduce FPE probe binding as observed by less fluorescence. The FPE assay was also adapted to use dog serum. FPE with TTR in buffer A 98 μL aliquot of TTR in PBS (pH 7.4, final concentration: 2.5 μM) was mixed with 1 μL of test compound (2.5 μM) and 1 μL of FPE probe (0.18 mM stock solution in DMSO: final concentration: 1.8 μM). The change in fluorescence (λex=328 nm and λem=384 nm) was monitored for 6 h at rt using a microplate spectrophotometric reader (SpectraMax M5). FPE by TTR in human and dog serum A 98 μL aliquot of pooled human serum (prepared from human male AB plasma, Sigma; Cat. No. H4522; TTR concentration 5 μM) or dog serum (Innovative Research, Cat. No. IBG-SER; TTR concentration 4.6 μM) was mixed with 1 μL of test compound [all compounds were prepared as 10 mM stock solutions in DMSO and diluted with DMSO as follows (final concentrations in serum: AG10 10 μM; diflunisal 200 μM; tafamidis 20 μM; tolcapone 20 μM)] and 1 μL of FPE probe (0.36 mM stock solution in DMSO; final concentration: 3.6 μM). For dog serum (after oral treatment with AG10), 1 μL of FPE probe and 1 μL of DMSO were added to each well and mixed with 98 μL of the appropriate dog serum sample. The change in fluorescence (λex=328 nm and λem=384 nm) was monitored for 6 h at rt using a microplate spectrophotometric reader (SpectraMax M5).

[0168] Stability test of TTR in serum by immunoblottingWestern blotting was performed as previously reported. All compounds were prepared as 10 mM stock solutions in DMSO and diluted in DMSO as follows (final concentrations in serum: AG10 10 μM; diflunisal 200 μM; tafamidis 20 μM; tolcapone 20 μM). 2 μL of each compound was added to 98 μL of human serum (TTR concentration 5 μM). The samples were incubated at 37° C. for 2 hours, after which 10 μL of sample was diluted 1:10 in acidified buffer (pH 4.0, 100 mM sodium acetate, 100 mM KCl, 1 mM EDTA, 1 mM DTT). Western blot assays were also performed in urea buffer (pH 7.4) as previously reported. Samples were incubated for 72 hours at room temperature, cross-linked with glutaraldehyde (final concentration 2.5%) for 5 minutes, and then quenched with 10 μL of 7% sodium borohydride solution in 0.1 M NaOH. All samples were denatured by adding 100 μL of SDS gel loading buffer and boiling for 5 minutes. 10 μL of each sample was separated on a 12% SDS-PAGE gel and analyzed by immunoblotting using anti-TTR antiserum (DAKO A0002, diluted 1:10,000 for human serum and 1:2,000 for dog serum). The combined intensity of the TTR bands (TTR tetramer and tetramer bound to RBP) was quantified using an Odyssey IR imaging system (LI-COR Bioscience) and reported as the percentage of TTR tetramer relative to the TTR tetramer concentration in the DMSO control at time 0 (considered 100% stabilization) and 72 h (range of 10%-35% remaining TTR). Percentage tetramer stabilization was calculated as 100 × [(concentration of tetramer and tetramer + RBP at 72 h) / (concentration of tetramer and tetramer + RBP in DMSO at time 0)].

[0169] In silico structure and modeling studiesAnalysis of the crystal structure of TTR was performed on four TTR crystal structures obtained from the RCSB PDB site. The biological assembly of the TTR tetramer was constructed using the X-ray crystallographic unit cell information given in the pdb file. When multiple models were suggested, the first selected model was used. The initial geometry of AG10 and its four derivatives (1, 2, 3, and 4) constructed by Molden38 was used, and optimization of the geometry was performed at the hybrid concentration function B3LYP level with the 6-311+G(d) basis set using the Gaussian09 program package (Wallingford, CT, USA: Gaussian, Inc., 2009). Vibrational frequency calculations were performed on the optimized geometry to confirm that they had no virtual frequencies. The dock6 program was used for the docking experiments. The crystal structure of the V122I mutant TTR complex with AG10 (pdb id:4HIQ) was used as the receptor. Tetrameric TTR was constructed using crystallographic data, solvent and other heteroatoms were removed, and one large docking grid was selected that included the T4 binding site. The same receptor and grid were used for all docking experiments. Free ligand docking was performed to allow rotations around torsion angles. The UCSF Chimera package was used for visualization and 3D structure analysis.

[0170] Binding of AG10 and tafamidis to human serum albumin.Test compounds (AG10 or tafamidis; both at 30 μM) were incubated with human serum albumin (HSA; 600 μM; albumin from human serum; Sigma Aldrich, Catalog No.: A3782) in assay buffer (10 mM sodium phosphate, 100 mM KCl, and 1 mM EDTA, pH 7.6) for 1 hour at 37° C. 500 μL of the solution of HAS and AG10 or tafamidis mixture in assay buffer was subjected to gel filtration on a PD Minitrap G25 column (GE Life Sciences, Catalog No. 45-001-529) by gravity, and fractions containing HSA were identified by NanoDrop™. The concentration of HSA (i.e., concentration at time zero) was also determined using NanoDrop™ (based on a calibration curve of known HAS concentrations). The HSA concentration was 351 μM for the tafamidis sample and 345 μM for the AG10 sample. The concentrations of the test compounds in these fractions (i.e., concentrations at time 0) were assessed using HPLC (based on a calibration curve of test compounds of known concentrations). 500 μL of each HSA / test compound sample was then added to a Slide-A-Lyzer dialysis cassette G2 (3.5K MWCO, Thermo Scientific, Cat. No. PI87722). The dialysis cassette was placed in 100 ml of assay buffer and stirred at room temperature. After 24 hours, the samples were removed from the dialysis cassette and the volume was measured. The concentrations of HAS and test compounds were determined using NanoDrop™ and HPLC as previously described.

[0171] Dialysis of AG10:TTR complexAG10 (10 μM) was incubated with human wild-type TTR (5 μM; purified from human plasma; Sigma Aldrich, Cat. No. P1742) in assay buffer (10 mM sodium phosphate, 100 mM KCl, and 1 mM EDTA, pH 7.6) for 1 hour at 37° C. Then 500 μL of each AG10 / TTR solution was added to a Slide-A-Lyzer dialysis cassette G2. The dialysis cassette was placed in 100 ml of assay buffer and stirred at room temperature. Samples were taken from the dialysis buffer at different time points (0, 0.5, 1, 2, 6, and 24 hours). After 24 hours, samples were removed from the dialysis cassette, the volume was measured, and the results were normalized. The concentrations of TTR and AG10 obtained from the assay buffer were determined using NanoDrop™ and LCMS, respectively.

[0172] Selectivity of AG10 and tafamidis for TTR compared with other serum proteins The FPE assay was modified and performed with purified human TTRwt (5 μM). Other serum proteins were added either individually or in combination [fibrinogen (5 μM), albumin (600 μM), IgG (70 μM), transferrin (25 μM)] to the mixture of TTR and FPE, and fluorescence was monitored for 6 h as described previously. The percentage of FPE probe bound to TTR in the presence of serum proteins measured after 3 h of incubation was used to calculate TTR occupancy.

[0173] Repeated oral administration of AG10 to dogs for 7 days Sixteen male (M) and sixteen female (F) beagle dogs were divided into four treatment groups and a total of 32 dogs were orally dosed by force with vehicle (6M / 6F at 0 mg / kg) or AG10 in 0.5% methylcellulose formulations (2M / 2F at 50 mg / kg, 2M / 2F at 100 mg / kg, and 6M / 6F at 200 mg / kg). Blood (approximately 1.5 mL) was collected from the jugular vein into serum separator tubes on study day 1 (pre-dose D1), the day 7 pre-dose study (pre-dose D7), and the day 7 1-hour post-dose study (post-dose D7). These serum samples were analyzed for their TTR occupancy using the FPE assay described previously.

[0174] Single oral dose of AG10 in dogs to determine exposure-effect (PK-PD) relationships for binding and stabilization of TTR Four male and four female beagle dogs were divided into two treatment groups (n=2 / sex / group) and a total of eight dogs were evaluated to obtain concurrent pharmacokinetic (PK) and pharmacodynamic (PD) data for AG10 binding and stabilization to TTR. Each animal received a single oral gavage (PO) dose of AG10 at a single dose of either 5 or 20 mg / kg in 0.5% methylcellulose. Blood was collected and analyzed pre-dose and at 2, 4, 6, 8, 12 and 24 hours post-dose. The concentration of AG10 in these serum samples was analyzed by LCMS and TTR occupancy was analyzed by FPE assay.

[0175] statistical analysis All results were expressed as mean ± SD. All statistical analyses were performed with GraphPad PRISM software. Significant differences were determined by one-way ANOVA followed by Tukey's multiple comparison test (ns, not significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001).

[0176] General Chemistry All reactions were carried out under an argon atmosphere with dry solvents under anhydrous conditions unless otherwise noted. Solvents used were Fisher ACS grade. Reagents were purchased from Aldrich and Fisher and used without further purification. Reactions were monitored by thin layer chromatography (TLC) carried out on 0.20 mm POLYGRAM® SIL silica gel plates (Art.-Nr. 805 023) with fluorescent indicator UV254 using UV light as visualization agent. Normal phase flash column chromatography was carried out using Davisil® silica gel (100-200 mesh, Fisher). 1 H NMR and 13C NMR spectra were recorded on a Jeol JNM-ECA600 spectrometer and calibrated using residual native solvent as internal standard. Coupling constants (J) were expressed in Hertz. The following abbreviations were used to describe multiplicity: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. High resolution mass spectrometry (HRMS) was recorded on a JEOL DART AccuTOF (real-time direct analysis). HPLC analyses were performed on an Agilent 1100 series HPLC system coupled to a diode array detector operating in the UV range 200-400 nm and quantified using Agilent Chemstation software. HPLC analysis was performed on both Waters™ XBridge C18 columns with L1 packing (4.6×250 mm, 5 μm) and Symmetric™ C4 (2.1×150 mm, 5 μm) at ambient temperature upon injection of 50 μl each of blank buffer, standard and / or sample to obtain chromatograms. The mobile phase consisted of solvent A consisting of methanol-water (5:95, v / v) containing 0.1% formic acid, and solvent B consisting of methanol-water (95:5, v / v) containing 0.1% formic acid. The HPLC program was a gradient separation method with a linear increase of solvent B from 0% to 100% over 0-20 min, followed by a hold at 100% solvent B for 30 min.

[0177] Purity of important compounds HPLC analysis was performed on both C18 and C4 reversed-phase columns. Purity of all key compounds was >95%. Description of purity analysis is included in the experimental section. Detailed HPLC information (traces, retention times, and % purity) of key compounds is included in the supplementary information of the revised manuscript.

[0178] Synthesis procedure AG10 and tafamidis were synthesized as previously reported. Tolcapone and diflunisal were purchased from Fisher. All AG10 analogs were prepared as described below.

[0179] 3-(3-(3,5-Dimethyl-1H-pyrazol-4-yl)propoxy)-4-iodobenzoic acid (1a)A solution of methyl 3-(3-bromopropoxy)-4-iodobenzoate (5a) (834 mg, 2.1 mmol, 1 equiv.) in benzene (3 ml) was added dropwise to a solution of acetylacetone (0.43 ml, 4.2 mmol, 2 equiv.) and DBU (0.627 ml, 4.2 mmol, 2 equiv.) in benzene (7 ml). The reaction mixture was stirred at room temperature for 3 days. The mixture was filtered and concentrated. To a solution of this intermediate in ethanol (5 ml), hydrazine hydrate (0.28 ml, 5.25 mmol, 2.5 equiv.) was added and the reaction was heated under reflux for 4 hours. The reaction was concentrated and purified by flash column chromatography (silica gel, 1-20% MeOH / CH2Cl2) to provide the methyl ester of compound 1a; sodium hydroxide (79 mg, 1.98 mmol, 2 equiv) in water (2.5 ml) was added to a solution of the ester intermediate (412 mg, 0.99 mmol) in methanol (10 ml) and the reaction was heated under reflux (50° C.) for 4 h. The reaction was concentrated and purified by flash column chromatography (silica gel, 1-5% MeOH / EtOAc) to provide compound 1a (183 mg, 22% yield over three steps); (98.3% purity by HPLC): tR(column)(C18)=25.72 min; tR(C4)=16.06 min. 1 H NMR (CD3OD, 600 MHz) δ 7.86 (d, 1H, J=8.4 Hz), 7.41 (d, 1H, J=1.2 Hz), 7.34 (dd, 1H, J=1.2 Hz and 8.4 Hz), 4.0 (t, 2H, J=6.0 Hz), 2.67 (t, 2H, J=7.2 Hz), 2.13 (s, 6H), 1.97-1.93 (m, 2H). 13 (HRMS (DART) m / z:C 15 H 17 IN2O3+ H + Calculated value 401.0362; Found value 401.0347 (M+H + ).

[0180] 3-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)-4-fluorobenzoate methyl (2) To a solution of methyl 3-(3-bromopropoxy)-4-fluorobenzoate (5b) (780 mg, 2.69 mmol, 1 equiv.) in benzene (3 ml) was added dropwise a solution of acetylacetone (0.552 ml, 5.38 mmol, 2 equiv.) and DBU (0.804 ml, 5.38 mmol, 2 equiv.) in benzene (7 ml). The reaction mixture was stirred at room temperature for 3 days. The mixture was filtered and concentrated. The residue was purified by flash column chromatography (silica gel, 1-10% EtOAc / hexanes) to provide the alkylated intermediate, which was used directly in the next step. To a solution of this intermediate in ethanol (5 ml) was added hydrazine hydrate (0.36 ml, 6.73 mmol, 2.5 equiv.) and the reaction was heated to reflux for 4 h. The reaction was concentrated and purified by flash column chromatography (silica gel, 1-20% MeOH / CH2Cl2) to provide compound 2 (288 mg, 35% yield); (96.3% purity by HPLC): tR(column)(C18)=25.11 min; tR(C4)=14.03 min. 1 H NMR (CD3OD, 600 MHz) δ 7.63-7.58 (m, 2H), 7.19-7.15 (m, 1H), 4.00 (t, 2H, J=6.0 Hz), 3.86 (s, 3H), 2.58 (t, 2H, J=7.2 Hz), 2.12 (s, 6H), 1.97-1.92 (m, 2H). 13 C NMR (CD3OD, 600 MHz) δ 168.1, 158.4, 156.7, 148.9, 128.5, 124.6, 117.6, 117.0, 115.6, 69.4, 53.3, 31.1, 20.2, 10.9;HRMS(DART) m / z:C 16 H 19 FN2O3+ H + Calculated value 307.1458; Found value 307.1463 (M+H + ).

[0181] 4-Fluoro-3-(3-(1,3,5-trimethyl-1H-pyrazol-4-yl)propoxy)benzoic acid (3)To a solution of 2 (21 mg, 0.07 mmol, 1 equiv) in DMF (3 ml) was added sodium hydride (5 mg, 0.21 mmol, 3 equiv) and methyl iodide (17 μl, 0.28 mmol, 4 equiv). The reaction mixture was stirred at room temperature for 2 h. The mixture was extracted with brine, filtered and concentrated. The residue was purified by flash column chromatography (silica gel, 0.5-2% MeOH / EtOAc) to provide the alkylated intermediate, which was used directly in the next step. Sodium hydroxide (5.6 mg, 0.14 mmol, 2 equiv) in water (0.5 ml) was added to a solution of the alkylated intermediate in methanol (2 ml) and the reaction was heated to reflux (50° C.) for 4 h. The reaction was concentrated and purified by flash column chromatography (silica gel, 1-5% MeOH / EtOAc) to provide compound 3 (11 mg, 52% yield over two steps); (97.8% purity by HPLC): tR(column)(C18)=25.25 min; tR(C4)=15.71 min. 1 H NMR (CD3OD, 600 MHz) δ 7.58-7.51 (m, 2H), 7.10-7.06 (m, 1H), 3.92 (t, 2H, J=6.0 Hz), 3.56 (s, 3H), 2.49 (t, 2H, J=7.2 Hz), 2.05 (s, 3H), 2.01 (s, 3H), 1.83-1.88 (m, 2H). 13 C NMR (CD3OD, 600 MHz) δ 168.1, 154.6, 146.8, 145.3, 137.2, 128.1, 122.8, 115.5, 115.4, 114.8, 67.4, 34.3, 29.4, 18.8, 10.1, 7.9;HRMS (DART) m / z:C 16 H 19 FN2O3+ H + Calculated value 307.1458; Found value 307.1449 (M+H + ).

[0182] 3-(3-(3,5-diethyl-1H-pyrazol-4-yl)propoxy)-4-fluorobenzoic acid (4)Sodium hydroxide (3.2 mg, 0.08 mmol, 2 equiv) in water (0.5 ml) was added to a solution of 6 (13 mg, 0.04 mmol, 1 equiv) in methanol (2 ml) and the reaction was heated to reflux (50° C.) for 4 h. The reaction was concentrated and purified by flash column chromatography (silica gel, 1-5% MeOH / EtOAc) to provide compound 4 (10 mg, 80% yield); (96.0% purity by HPLC): tR(column)(C18)=25.16 min; tR(C4)=15.56 min. 1 H NMR (CD3OD, 600 MHz) δ 7.57-7.49 (m, 2H), 7.08-7.04 (m, 1H), 3.94 (t, 2H, J=6.0 Hz), 2.51-2.43 (m, 6H), 1.87-1.82 (m, 2H), 1.06 (t, 6H, J=7.8 Hz). 13 C NMR (CD3OD, 600 MHz) δ 169.8, 157.9, 156.3, 149.3, 148.5, 124.6, 117.2, 117.1, 114.1, 69.4, 31.8, 20.1, 19.9, 14.7;HRMS(DART) m / z:C 17 H 21 FN2O3+ H + Calculated value 321.1614; Measured value 321.1601 (M+H + ).

[0183] Methyl 3-(3-bromopropoxy)-4-fluorobenzoate (5) Compound 5 was synthesized as previously reported. To a solution of methyl 4-fluoro-3-hydroxybenzoate (1.0 g, 5.87 mmol, 1 equiv.) and 1,3-dibromopropane (3.0 ml, 29.4 mmol, 5 equiv.) in DMF (15 ml), K2CO3 (0.98 g, 7.1 mmol, 1.2 equiv.) was added. The reaction mixture was stirred at room temperature for 16 h. The mixture was diluted with EtOAc (500 ml), washed with brine (3×200 ml), and dried over Na2SO4. The solution was filtered and concentrated. The residue was purified by flash column chromatography (silica gel, 1-10% EtOAc / hexane) to provide compound 5 (1.3 g, 76% yield); 1H NMR (CD3OD, 600 MHz) δ 7.67-7.61 (m, 2H), 7.14-7.07 (m, 1H), 4.21 (t, 2H, J=5.89 Hz), 3.89 (s, 3H), 3.62 (t, 2H, J=6.38 Hz), 2.38-2.31 (m, 2H);(ESI+) m / z:C 11 H 12 BrFO3+H + Calculated value 290.00; Measured value 290.01 (M + H + ).

[0184] 3-(3-(3,5-diethyl-1H-pyrazol-4-yl)propoxy)-4-fluorobenzoate methyl (6) : A solution of 5b (100 mg, 0.35 mmol, 1 equiv) in benzene (2 ml) was added dropwise to a solution of 3,5-heptanedione (0.095 ml, 0.7 mmol, 2 equiv) and DBU (0.104 ml, 0.7 mmol, 2 equiv) in benzene (5 ml). The reaction mixture was stirred at room temperature for 3 days. The mixture was filtered and concentrated. The residue was purified by flash column chromatography (silica gel, 1-10% EtOAc / Hexane) to provide the alkylated intermediate, which was used directly in the next step. Hydrazine hydrate (0.047 ml, 0.875 mmol, 2.5 equiv) was added to the alkylated intermediate in ethanol (4 ml) and the reaction was heated to reflux for 4 h. The reaction was concentrated and purified by flash column chromatography (silica gel, 1-5% MeOH / EtOAc) to provide compound 6 (75 mg, 65% yield for two steps); 11H NMR (CD3OD, 600 MHz) δ 7.59 - 7.54 (m, 2H), 7.15 - 7.11 (m, 1H), 3.98 (t, 2H, J = 6.0 Hz), 3.81 (s, 3H), 2.56 - 2.47 (m, 6H), 1.91 - 1.86 (m, 2H), 1.13 (t, 6H, J = 7.8 Hz). 13C NMR (CD3OD, 600 MHz) δ 167.9, 156.6, 156.2, 148.8, 148.7, 124.4, 117.5, 117.3, 116.9, 113.9, 69.5, 53.1, 31.8, 20.1, 14.7; HRMS (DART) m / z: C 18 H 23 FN2O3 + H + calculated value 335.1771: measured value 335.1773 (M + H + ).

[0185] result Determination of the binding affinity and thermodynamics of the interaction between stabilizers and TTRWe used isothermal titration calorimetry (ITC) to determine the binding affinity (Kd) and the mechanism underlying the molecular interactions of all TTR stabilizers in clinical development (i.e., AG10, tafamidis, diflunisal, and tolcapone) and AG10 analogs 1, 2, 3, and 4. Most of the reported TTR ligands bind to the two identical T4 binding sites of TTR with strong negative cooperativity, so that the binding of the first ligand will dominate the overall binding energy and stabilization activity. Although some differences in cooperativity can be observed in the ITC thermograms, these differences will have a small effect on the binding energy and stabilization activity. Therefore, the Kd values ​​reported in Table 5 were based on data fitted to an independent single-site binding model. The binding affinity of AG10 and tafamidis to TTR in buffer (Kd=4.8±1.9 and 4.4±1.3 nM, respectively) was 4-fold higher than tolcapone (Kd=20.6±3.7 nM) and ∼100-fold higher than diflunisal (Kd=407±35 nM). The Kd values ​​of compounds 1-4 ranged from 90 to 1250 nM, and these results are summarized in Table 5. The Kd for the binding of the stabilizers to TTR is expressed as the difference in Gibbs binding free energy (ΔG), where ΔG=ΔH-TΔS. By analyzing the thermodynamic signature of each molecule, we can evaluate the relative contributions of enthalpic forces (ΔH; representing the formation or breaking of chemical bonds) and entropic forces (ΔS; governed by the release of water molecules bound by hydrophobic interactions and related to the amount of disorder of the system and vibrational frequencies favored). Despite the similar binding affinities of AG10 and tafamidis to TTR in buffer (i.e., similar ΔG values), their binding energetics to TTR are significantly different: AG10 binding (ΔH=-13.60 kcal / mol and TΔS=-2.26 kcal / mol) is enthalpidically driven, whereas tafamidis binding is approximately 50% entropic and 50% enthalpic (ΔH=-5.00 kcal / mol and TΔS=6.39 kcal / mol) (Figure 16a and Table 5).The binding of tolcapone (ΔH=-10.1 kcal / mol and TΔS=0.4 kcal / mol) and diflunisal (ΔH=-8.38 kcal / mol and TΔS=0.34 kcal / mol) is entropically favorable but primarily driven by enthalpic interactions. The unfavorable entropic binding energy of AG10 for TTR (TΔS=-2.26 kcal / mol) may be due to its higher polarity and / or conformational flexibility compared to other TTR stabilizers. The thermodynamics of the binding interactions between compounds 1-4 and TTR are discussed below. [Table 5]

[0186] Enthalpic forces predict the potency of TTR stabilizers in buffers and their efficacy in human serumRecent studies with diflunisal and other nonsteroidal anti-inflammatory drugs (NASAIDs) have found that ligands with favorable (i.e., more negative) ΔH have proportionally greater TTR selectivity compared to ligands with lower ΔH impact. Although this study describes a correlation between enthalpic power and selectivity of TTR stabilizers, no correlation has yet been reported between the binding enthalpy of a ligand and its efficacy in stabilizing TTR or, to our knowledge, any other multimeric protein. To evaluate the efficacy of stabilizers in occupying and stabilizing TTR in buffer, we used a fluorescent probe exclusion (FPE) assay. The FPE assay uses a fluorogenic probe (FPE probe), which is a thioester TTR ligand that is not itself fluorescent but which, upon binding to the T4 binding site of TTR, covalently modifies lysine 15 (K15), resulting in a fluorescent conjugate. Ligands that bind to the T4 site of TTR will reduce FPE probe binding, as seen by less fluorescence. A linear correlation has been reported between the degree of fluorescence in the FPE assay and the stabilization of TTR. Therefore, we first used the FPE assay to measure the potency of stabilizers for binding and stabilizing TTR in buffer (stabilizer to TTR tetramer ratios of 1:1 were tested; Fig. 16b, c and Table 5). The order of potency of stabilizers for TTR in buffer was AG10>tolcapone>tafamidis>diflunisal.

[0187] We next used FPE and Western blot assays to evaluate the effectiveness (representing both potency and selectivity) of the stabilizers (10 μM) in occupying and stabilizing TTR in human serum (TTR concentration 5 μM) (Table 5). The Western blot assay measures the amount of intact TTR tetramers after 72 hours of acid treatment in the presence and absence of stabilizers. The order of efficacy of the stabilizers in human serum was similar to what we observed for TTR efficacy in buffer (AG10>tolcapone>tafamidis>diflunisal; Table 5). The potency and efficacy of diflunisal was the lowest (20-80 times lower affinity than the other stabilizers) as predicted based on its significantly lower binding affinity to TTR (Kd=407±35 nM) compared to all other stabilizers. Surprisingly, there was no correlation between the Kd values ​​of the three other stabilizers and their potency and efficacy in occupying and stabilizing TTR in both buffer and human serum. For example, the potency and efficacy of tolcapone was higher than that of tafamidis, despite the fact that the binding affinity of tolcapone to TTR (Kd=20.6±3.7 nM) was slightly lower than that of tafamidis to TTR (Kd=4.4±1.3 nM). Interestingly, both the potency and efficacy of these stabilizers for TTR in buffer and serum correlated very well (R2=0.98) with their binding enthalpies (ΔH=-13.6, -10.1, and -5.0 kcal / mol for AG10, tolcapone, and tafamidis, respectively). This data points out that the enthalpic-driven binding of AG10 and tolcapone to TTR (discussed in detail below) is the primary driver of their efficient stabilization of TTR compared to other stabilizers.

[0188] We then used a Western blot assay to compare the efficacy of AG10 (10 μM) with other stabilizers at their reported mean maximum plasma concentrations in humans (C maxAG10 at 10 μM completely stabilized TTR in human serum (% TTR stabilized: 95.4 ± 4.8%); the other compounds met their reported clinical Cs. max At pH 4, AG10 stabilized 50-75% of tetrameric TTR (Fig. 17a,b). The pKa values ​​for AG10 (pKa=4.13) and tafamidis (pKa=3.73) were higher than that for diflunisal (pKa=2.94). Thus, the ionization rate of the carboxylic acid group of the stabilizer may vary at pH 4. This will affect the strength of the electrostatic interaction between the carboxylic acid group and the ε-amino group of lysine 15 (K15) and K15' at the apex of the T4 binding site, which will affect the efficacy of the stabilizer. To address this concern, we used urea buffer (pH 7.4) and performed Western blot assays. The TTR stabilization data in urea buffer were similar to those obtained from Western blots at acidic pH and from FPE assays at physiological pH. Consistent with the Western blot TTR stabilization assay data, T4 binding site occupancy by 10 μM AG10 in the FPE assay was essentially complete (TTR occupancy 96.6 ± 2.1%) and was consistent with their reported clinical C max Target occupancy for tolcapone at 20 μM (TTR occupancy 86 ± 3.2%) was higher than that of tafamidis and diflunisal (TTR occupancy ∼65% at 20 μM and 200 μM, respectively) (Fig. 17c, d).

[0189] Binding interactions between AG10 and S117 / S117' of TTR mimic the molecular interactions within the disease-protective T119M mutation We investigated the correlation between binding enthalpy and TTR stabilization by comparing reported co-crystal structures of TTR and stabilizers to crystal structures stabilizing TTR variants (T119M and R104H). We hypothesized that this would allow us to identify amino acid functional groups within the T4-binding site of TTR that are important for TTR binding and stabilization. The carboxylic acid moieties of AG10, tafamidis, and diflunisal, and the hydroxyl group on tolcapone all participate in electrostatic interactions with the ε-amino groups of lysine 15 (K15) and K15' at the apex of the T4-binding site. The enthalpic driven binding of AG10 and tolcapone to TTR is driven by additional hydrogen bonds that both molecules form within the T4-binding site. The carbonyl group of tolcapone forms one hydrogen bond with the hydroxyl side chain of T119 of TTRwt (distance ∼2.6 Å; ideal distance for hydrogen bonds is <3 Å). The longer distance between the carbonyl group of tolcapone and the hydroxyl side chain of T119' on the adjacent monomer (distance ∼7.6 Å) precludes the formation of a second hydrogen bond. Interestingly, this interaction is weaker between tolcapone and V122I-TTR (the distances between the carbonyl group of tolcapone and the hydroxyl side chains of T119 and T119' of V122I-TTR are ∼5.5 Å and ∼9.6 Å, respectively), which may explain the lower binding affinity (Kd = 56 nM) and potency of tolcapone for V122I-TTR compared to TTRwt. In the case of tafamidis, there are no hydrogen bonds at the bottom of the T4 pocket; instead, the chlorine atoms of the 3,5-dichloro ring are also located in the halogen-binding pockets (HBP) 3 and 3', where they interact with TTR primarily through hydrophobic interactions. In addition to the electrostatic interaction of the carboxylate moiety of AG10 with K15 / K15', AG10 also forms two hydrogen bonds (distance ∼2.8 Å) with the hydroxyl side chains of serine 117 (S117) and S117' of adjacent monomers within a low-dielectric macromolecule inside the T4 binding site (Figure 18a). These additional hydrogen bonds likely contribute to the driving force for the main enthalpic binding of AG10 to TTR.Surprisingly, a similar hydrogen bond has been reported within the internal cavity of the kinetically stabilizing trans-suppressor T119M-TTR variant (FIG. 18b).

[0190] Two S117 side chain hydroxyl groups of monomers A and B in T119M variant TTR form direct hydrogen bonds at a distance of 2.8 Å, which are not observed in TTRwt (the distance between these two S117 residues is ∼6.0 Å) (Figure 18c). These unique hydrogen bonds lead to closer contact (∼4.8 Å) of these two dimers in the TTR tetramer, and highlight the potential importance of these hydrogen bonds in the anti-amyloidotic and disease-protective effects of the T119M variant on the TTR tetramer. The role of S117 in stabilizing TTR has also been suggested by the binding of flavonoids, which can form a single hydrogen bond with one S117. Interestingly, the distance between the S117 and S117' residues in the thermodynamically stabilized R104H variant, which is not involved in the kinetic stabilization of the TTR tetramer, is similar to that of TTRwt (average dimer-dimer distance is ∼5.6 Å, Fig. 18c, d). The lack of hydrogen bonds between the hydroxyl groups of S117 and S117' in the R104H variant (which is a less potent trans-suppressor mutant than T119M) highlights the importance of these hydrogen bonds in anti-amyloidogenic disease, which suppresses the effect of the kinetic stabilization of the TTR tetramer in the T119M variant. By forming two direct hydrogen bonds with S117 and S117' in the TTR tetramer, AG10 forms an electrostatic bridge similar to that observed in the protective T119M variant. This data is supported by an analysis of 40 reported crystal structures that highlight closer dimer-dimer contacts in both the T119M-TTR (distance 4.8 Å) and AG10-V122I-TTR (distance 4.66 Å) crystal structures compared to TTRwt or TTRm (distance 5.5 Å) (Supplementary Table 1). It is important to note that other known TTR stabilizers do not interact with S117 / S117' of TTR.

[0191] Characterization of key functional groups of AG10 important for TTR stabilization To investigate the enthalpic contribution of each functional group of AG10 to TTR binding and stabilization, we synthesized and tested four AG10 analogs (compounds 1, 2, 3, and 4; FIG. 15) and evaluated their ability to bind and stabilize TTR (FIG. 19). AG10 binds to TTR with unfavorable entropy (TΔS=−2.26 kcal / mol). The fluorine atom of AG10 is located at the HBP1 of TTR, and therefore we hypothesized that the entropic binding of AG10 to TTR would be optimized by replacing the fluorine atom of AG10 with an iodine (compound 1a). Modeling studies suggest that the iodine of 1 fits in the HBP1 of TTR (where the iodine of T4 binds), which improves the entropic binding by replacing more water molecules from HBP1 (FIG. 19a). Compound 1a showed a significantly lower binding affinity (Kd=90±14 nM) to TTR in buffer compared to AG10 (Kd=4.8±1.9 nM). ITC analysis showed that the entropic interaction of 1 with TTR is more favorable compared to AG10 (TΔS=-0.21 kcal / mol and -2.26 kcal / mol, respectively), but there is a significant decrease in the enthalpic contribution to this binding (ΔH=-9.82 kcal / mol and -13.6 kcal / mol, respectively) (Figure 19b). As suggested by modeling, the decrease in binding enthalpy can be explained by the decrease in the strength of the salt bridge between the carboxylic acid moiety of 1 and K15 / K15' (~4.7 Å compared to ~2.8 Å distance for AG10). Compound 1a also showed reduced potency with respect to TTR in buffer (58.3±0.98%) and human serum (75.9±3.1%) compared to AG10 (FIGS. 19c-e and Table 5).

[0192] The carboxylic acid moiety of AG10 forms two salt bridges directly with the ε-amino groups of K15 and K15' at the periphery of the T4-binding site, which serve to access the T4 pocket surrounding AG10 and to partially shield it from the solvent. We synthesized a methyl-ester analog of AG10 (compound 2, FIG. 19a) to test its effect of modifying the two salt bridges that AG10 forms at the periphery of the T4-binding site. Compound 2 showed a significantly lower affinity for TTR in buffer (Kd=258±17 nM) compared to AG10 (Kd=4.8±1.9 nM), which is explained by the lower strength of the possible hydrogen bond between the ester group of 2 and K15 / K15' (ΔH=−6.49 kcal / mol) compared to the salt bridge in AG10 (FIG. 19b). Compound 2 also showed reduced potency for TTR in buffer and human serum compared to AG10 and compound 1a (Figures 19c-e and Table 5).

[0193] The 3,5-dimethyl-1H-pyrazole ring of AG10 is located deep within the internal cavity of the T4-binding site and forms two hydrogen bonds with S117 and S117' of the adjacent subunit. By blocking these interactions, we can effectively observe their enthalpic contributions using ITC and FPE assays, respectively. Therefore, we synthesized compound 3 with N-methylpyrazole. The N-methyl group will constrain the pyrazole ring of 3 and form only one hydrogen bond with one of the adjacent TTR subunits (Figure 19a). We also synthesized compound 4, in which the dimethylpyrazole of AG10 was replaced with diethylpyrazole. Modeling studies suggested that the majority of the diethyl group would prevent these molecules from reaching deep into the T4-binding site, thereby reducing their ability to potentially form any hydrogen bonds with S117 / S117' (Figure 19a). As predicted by modeling, both 3 (Kd=251±12 nM) and 4 (Kd=1253±79 nM) showed greatly reduced binding affinity to TTR in buffer. This reduced affinity translated into a significant decrease in potency for TTR in buffer and human serum, especially for compound 4. The order of potency for stabilizing TTR was similar in buffer and serum (1>2>3>4; Figure 19c-e and Table 5). As we observed with clinical ATTR stabilizers, the potency of AG10 and compounds 1, 2, 3, and 4 in occupying and stabilizing TTR correlated very well (R2=0.98) with the binding enthalpy of these molecules (ΔH=-13.6, -9.82, -6.49, -4.73, and -2.1 kcal / mol, respectively). Interestingly, despite the similar binding affinities of 2 and 3, their potencies were significantly different (Table 5). Error! Reference source not found. The higher potency of 2 compared to 3 could be explained by its favorable enthalpic binding (ΔH=-6.49 kcal / mol and -4.73 kcal / mol, respectively) (Figure 19b). This finding is similar to the data obtained for AG10 and tafamidis (i.e., similar Kd values ​​but significantly different potencies) (Table 5).These results highlight the important role played by the pyrazole ring and the importance of the hydrogen bonds it forms with two TTR dimers, which mimic the interactions in the protective T119M-TTR mutation and enhance the kinetic stability of the TTR tetramer.

[0194] Examination of the effect of enthalpy on the selectivity of AG10 for TTR. To test the role of enthalpy on the selectivity of AG10 for TTR over other abundant serum proteins, we examined the concentration-effect relationship of AG10 and tafamidis in whole human serum in the FPE assay. We tested AG10 and tafamidis because their binding affinities to TTR in buffer are very similar (Kd=4.8±1.9nM and 4.4±1.3nM, respectively), but their thermodynamics for TTR binding, especially the enthalpic component, are significantly different. Thus, data obtained in serum will largely reflect selectivity. AG10 showed an increasing concentration-dependent occupancy, with complete occupancy achieved at AG10 concentrations ≧10 μM. Even at substoichiometric concentrations, AG10 can occupy and stabilize most of TTR (69.2% TTR occupancy by FPE, 74.5% stabilization by Western blot at 5 μM). In contrast, at concentrations above 20 μM there was a smaller increment in either tafamidis occupancy or stabilization of activity. When AG10 activity was assessed, a good correlation (R2=1.0) was observed between TTR occupancy (by FPE) and TTR stabilization (by Western blot). For tafamidis there was a good correlation (R2=0.87) at concentrations up to 10 μM, but at higher concentrations there was a plateau in the FPE assay.

[0195] The selectivity of AG10 and tafamidis for TTR was further investigated by repeating these assays in buffer in the presence or absence of purified serum proteins. AG10 or tafamidis (30 μM) was preincubated with purified human serum albumin (at its physiological concentration of 600 μM) and then subjected to gel filtration followed by dialysis. At time 0 (immediately after gel filtration), less AG10 bound to albumin compared to tafamidis (18.3±0.98 μM vs. 24.1±1.1 μM; FIG. 20a). After 24 h of dialysis against buffer, the concentration of AG10 bound to HSA was lower than that of tafamidis (7.8±0.1 μM vs. 18.8±2.1 μM). These data indicate that AG10 has a lower binding affinity for albumin compared to tafamidis. In parallel, the binding of AG10 to TTR was also examined in this gel filtration / dialysis assay. AG10 (10 μM) was preincubated with an equimolar ratio of TTR (5 μM of tetrameric TTR represents 10 μM of TTR T4 binding sites). Dissociation of AG10 from TTR was slow during the first 6 h (AG10-TTR molar ratio ∼1.2:1) and maintained the 1:1 molar ratio over the 24 h incubation (Figure 20b).

[0196] Finally, the selectivity of AG10 and tafamidis for binding to TTR in human serum was evaluated using a modified FPE assay in which human serum was replaced by purified human TTR in buffer (PBS buffer, pH 7.4). In addition to purified TTR (5 μM), four individual representative and abundant plasma proteins were added to the FPE assay in buffer. Addition of albumin, transferrin, fibrinogen or immunoglobulin (IgG) did not affect TTR occupancy by AG10 (>97% TTR occupancy in the absence or presence of any of these proteins, Fig. 20c, d). Albumin, unlike the other serum proteins tested, interfered with TTR occupancy by tafamidis (41.5 ± 0.9% vs. 68.2 ± 0.1% in the absence of albumin; Fig. 20e, f). Addition of all plasma proteins tested simultaneously produced identical results for AG10. The higher selectivity of AG10 for TTR can be attributed to a number of properties, including enthalpic binding as well as the greater hydrophilicity of AG10 (ClogP=2.78) compared to the more lipophilic tafamidis (ClogP=4.2).

[0197] Healthy beagle dogs are a suitable experimental model for evaluating the efficacy of TTR stabilizers.We next investigated whether the high potency and selectivity of AG10 for TTR could be maintained in vivo. A transgenic animal model that faithfully recapitulates the pathology of human ATTR-CM is not yet available. We therefore adopted a similar approach currently used in the clinic to test the efficacy of AG10 versus other TTR kinetic stabilizers. The activity of TTR stabilizers in occupying and stabilizing TTR is usually evaluated ex vivo in blood samples obtained from patients before and after dosing with the stabilizer. To examine the in vivo activity of AG10, this same approach was used in healthy beagle dogs. Healthy beagle dogs were chosen as an experimental model for several reasons. All amino acids in the T4 binding site of TTR, where AG10 and other stabilizers bind, are conserved between dogs and humans. We also tested the concentration of TTR in dog serum (~4.6 μM) and found that it was similar to that of healthy humans. To verify the suitability of the assays using human-based reagents for dog testing, the activity of AG10 and tafamidis was evaluated in pooled dog serum using the same FPE and Western blot assays used in the previously described experiments. The concentration-effect relationships of in vitro TTR binding and stabilization of AG10 and tafamidis in both assays repeated with dog serum were similar to those observed in human serum (Figure 21). These characteristics made healthy dogs a suitable system for further studies.

[0198] AG10 potently and selectively binds to canine TTR after oral administration To investigate the in vivo pharmacokinetic-pharmacodynamic (PK-PD) relationship, AG10 was administered daily by oral gavage to healthy beagle dogs for 7 days. A total of 16 male (M) and 16 female (F) beagle dogs were divided into four treatment groups: (i) 6M / 6F at 0 mg / kg / day (vehicle control); (ii) 2M / 2F at 50 mg / kg / day; (iii) 2M / 2F at 100 mg / kg / day; and (iv) 6M / 6F at 200 mg / kg / day. Pre-dosing on Study Day 1 (baseline), pre-dosing on Study Day 7 (trough concentration at steady state, or C min), and 1 hour post-dose on study day 7 (representing peak concentration at steady state, or C max Serum samples were collected at time points (representing day 7 pre-dose; ∼81-94% TTR occupancy) of TTR binding sites by AG10. TTR binding occupancy by AG10 was assessed by FPE assay (Fig. 22a, b). All samples from dogs treated with vehicle alone and those collected from the active treatment arms prior to exposure to AG10 showed zero TTR occupancy. Serum from AG10-treated dogs showed a dose-proportional response in binding occupancy at steady-state trough (day 7 pre-dose; ∼81-94% TTR occupancy), and all AG10-treated groups showed a steady-state C max (7 days post-dosing) demonstrated complete (>97%) TTR occupancy. To determine the minimal effective dose of AG10 that still effectively binds and stabilizes TTR, lower doses of AG10 were subsequently tested to further explore the PK-PD (exposure-effect) relationship. Eight dogs were divided into two active treatment groups receiving a single oral dose of either 5 or 20 mg / kg AG10·HCl. These results showed enhanced TTR occupancy in the 20 mg / kg vs. 5 mg / kg dose groups (Fig. 22c, d). max The TTR occupancy rate at both doses was min It was significantly higher than that of the control group (p≦0.001). min There was significantly (p≦0.001) higher TTR occupancy for the 20 mg / kg dose compared to the 5 mg / kg dose at 100 mg / kg. The data also showed that circulating plasma concentrations of AG10 correlated well with TTR occupancy.

[0199] In summary, beagle dogs demonstrated that AG10, at specific dose levels, is orally available and achieves dose-dependent plasma concentrations that potently and selectively bind and stabilize tetrameric TTR.

[0200] Example 5: Single-dose study of AG10·HCl administered intravenously or by oral gavage to monkeys AG10·HCl was administered to three male cynomolgus monkeys at a dose level of 1 mg / kg intravenously or 5 mg / kg orally once. There was a 2-week washout period between these two phases. Blood samples were collected pre-dose and approximately 0.083 (IV only), 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, and 96 hours post-dose. Plasma samples were assayed for AG10 and AG10 acyl glucuronide, and serum was tested in the FPE assay. As shown in FIG. 23, the results from the FPE assay reveal that orally administered AG10 bound effectively to TTR in monkey serum.

[0201] FPE assays also revealed that orally administered AG10 stabilized TTR in a dose-dependent manner (FIG. 24).

[0202] Example 6: TTR blood serum concentrations are increased in healthy individuals treated with AG10 To measure blood serum TTR concentrations, a Prealbumin ELISA kit (human) from Aviva Systems Biology, catalog number OKIA00081-96W, lot number KC0699, was used.

[0203] The test was performed according to the protocol provided by the ELISA kit manufacturer. The method was modified by adding three standard concentrations to the calibration curve recommended by the manufacturer. The supplied TTR calibrator was dissolved in 1 mL of distilled water, resulting in a concentration of 8.85 μg / mL. The first added standard was 1000 ng / mL, which was prepared by adding 178.4 μL of calibrator to 1400 μL of 1× diluent. The second added standard was 200 ng / mL, which was prepared by adding 32.4 μL of calibrator to 1400 μL of 1× diluent. The third added standard was 0.78125 ng / mL, which was prepared by adding 600 μL of the 1.5625 ng / mL standard to 600 μL of 1× diluent. The ELISA kit utilized a goat polyclonal anti-TTR antibody for capture and detection. This antibody was raised against the native human TTR protein. Pooled human serum was purchased from Innovative Research (catalog number IPLA-SER, lot number 24453).

[0204] Pooled human serum and MAD serum samples were thawed in a water bath at 37°C for 10 min. All samples were diluted 1:10000 in two steps. First, 5 μL of serum was mixed with 995 μL of 1× diluent provided in the ELISA kit. Second, 5 μL of this mixture was added to 245 μL of 1× diluent in a non-binding microplate. 100 μL of each final diluted sample was added to each well of the ELISA plate. All standards and samples were tested in duplicate. The TTR test followed the manufacturer's protocol from this point onwards without further modification. Briefly, standards and serum samples were incubated in the ELISA plate for 1 h at room temperature. The ELISA plate was washed 4 times with 1× wash buffer and then incubated with 1× horseradish peroxidase conjugate for 30 min at room temperature, protected from light. The ELISA plate was then washed 4 times. TMB substrate was added and allowed to develop for 10 minutes, after which stop solution was added. Finally, the absorbance at 450 nm was measured for each well.

[0205] Absorbance measurements were reference corrected by taking the average absorbance at 450 nm of duplicate 0 ng / mL wells and subtracting this from the total 450 nm absorbance of each well.

[0206] A standard curve was generated for each ELISA plate using GraphPad PRISM software. The log(ng / mL) of the standards was plotted on the X-axis and the reference-corrected 450 nm absorbance values ​​were plotted on the Y-axis. The data was fitted using a sigmoidal 4-parameter curve. The log(ng / mL) of the pooled human serum and MAD serum samples were interpolated from the standard curve. The log(ng / mL) values ​​were converted to serum TTR concentrations (mg / L) and corrected for sample dilution. The standard curve generated using the supplied calibrants was reproducible (Figure 25) and the kit successfully discriminated pooled human serum samples at increasing dilution ratios from 1:5000 to 1:20000. Therefore, the Aviva ELISA kit was used for the study.

[0207] Samples from healthy human volunteers from the three cohorts of the MAD study (100, 300, and 800 mg AG10·HCl dosed twice daily for 12 consecutive days) plus the placebo cohort were tested using the Aviva ELISA kit. The relative change in TTR concentration over time for each cohort was calculated by normalizing to baseline values ​​(FIG. 26). FIG. 27 shows the mean percent change in blood serum TTR concentration from baseline to day 12 in all placebo and AG10-treated cohorts. Figures 28 and 29 plot blood serum TTR concentrations at baseline and day 12 in all placebo and AG10 treatment cohorts (total number of healthy volunteers medicated=24; placebo:active=1:3; MAD1 cohort=100 mg Q12h for 12 days; MAD2 cohort=300 mg Q12h for 12 days; MAD3 cohort=800 mg Q12h for 12 days).

[0208] ARUP Prealbumin Assay (Immunoturbidimetric Assay) The ARUP prealbumin assay was also used to analyze TTR blood serum concentrations in the MAD cohort studied. The ARUP uses a prealbumin reagent kit provided by Roche Diagnostics and runs on a Roche Diagnostics c702 module to analyze samples for prealbumin. The lowest limit of measurement is 3 mg / dL.

[0209] Table 6 summarizes the baseline and post-dosing (24 hour) blood serum TTR concentrations measured for each cohort. In each study group, there is a measurable increase in TTR concentrations over the 24 hour measurement period. [Table 6]

[0210] Example 7: Phase 2 Clinical Trial Results - Individuals with ATTR-CM A Phase 2 clinical trial was conducted essentially as described in Example 3. A total of 49 patents were included in place of 45: 16 received 400 mg BID; 16 received 800 mg BID; and 17 received placebo.

[0211] The baseline characteristics of the individuals who participated in the study are shown in Table 7 below. [Table 7]

[0212] result TTR stabilization was measured ex vivo using the FPE and Western blot assays described at the beginning of the Examples section, and in vivo in study participants by monitoring TTR serum concentrations.

[0213] Figure 30 illustrates the dose-response changes in serum TTR levels for subjects in each treatment group. Data are reported as percentage change from baseline to day 28. Individuals receiving 400 mg of AG10·HCl twice daily had a mean increase in serum TTR levels of 36%, and individuals receiving 800 mg of AG10·HCl twice daily had a mean increase in serum TTR levels of 50%. In comparison, the placebo treatment group had a mean decrease in serum TTR levels of 7%.

[0214] The increase in serum TTR levels in the AG10·HCl dosing groups (both 400 mg and 800 mg BID) tracks with findings previously reported for previously tested TTR stabilizers. Figure 31 plots the mean percent change from baseline to day 28 of dosing for each treatment group in this study. Also plotted are the percent changes in serum TTR levels reported for the tafamidis phase 2 (FDA CDER Advisory Committee Meeting background package) and diflunisal follow-up studies (Hanson, JLS et al., Circ Hert Fail 2018 11:e004000). As can be seen in this figure, AG10, tafamidis, and diflunisal all increased serum TTR levels.

[0215] Prior to treatment, 40% of subjects in the 400 mg BID treatment group and 56% of subjects in the 800 mg BID treatment group had serum TTR levels below normal TTR levels (normal levels of TTR are 20-40 mg / dL (3.6-7.3 μM)). After 28 days of treatment, 100% of each active cohort had serum TTR concentrations within the normal range (i.e., all treated patients had normal serum TTR levels by the end of the 28-day treatment regimen). In comparison, 18% of placebo individuals had serum TTR levels below normal TTR levels prior to treatment. After 28 days of treatment, the number of those without normal levels of serum TTR concentrations increased to 31% of placebo individuals. See FIG. 32. The baseline distribution of serum TTR concentrations for individuals in this study is shown in FIG. 33.

[0216] Ex vivo Western blot analysis confirms that AG10·HCl at dosage levels of 400 mg BID and 800 mg BID effectively stabilized TTR. See Figure 34, which illustrates high levels of TTR stabilization at trough (h0) and peak (h1) time points on days 14 and 28 compared to low levels of stabilization at the pre-dose time point (D1h0). Error bars provided are standard error of the mean.

[0217] The Western blot results were further confirmed by a fluorescent probe assay, which showed high levels of TTR stabilization at the day 28 trough (pre-dose) and day 28 peak (1 hour post-dose) in individuals with both wild-type and mutant TTR. See Figure 35.

[0218] When plotting circulating plasma concentrations of AG10 versus occupancy by AG10 in the thyroxine-binding pocket of tetrameric TTR, it was determined that significant target association (occupancy by AG10) occurred at a circulating plasma concentration of around 5 μM, and complete target association occurred at approximately 7.5 μM. See FIG. 36.

[0219] Figures 37 and 38 plot the relative fluorescence units measured in the fluorescent probe assay at pre-dose on day 1, pre-dose on day 14 (trough), 1 hour post-dose on day 14 (peak), pre-dose on day 28 (trough), and 1 hour post-dose on day 28 (peak) for the 400 mg BID (Figure 37) and 800 mg BID (Figure 38) cohorts. As can be seen in both figures, the fluorescent probe assay indicates that by day 14, there is nearly complete target association in both the 400 mg BID and 800 mg BID cohorts, at both the trough and peak time points. In comparison, Figure 39 plots the relative fluorescence units measured at each of the time points mentioned above in the placebo control group. This plot reveals the lack of target association for the placebo control group.

[0220] In summary, the data presented here indicate that AG10 was well tolerated over 28 days in patients with symptomatic ATTR-CM, that AG10 increased serum TTR concentrations in a dose-dependent manner, that AG10 restored low TTR levels to normal levels, and that AG10 completely stabilized TTR across both dose levels tested.

[0221] When examining wild-type and specific mutant TTR populations in the cohorts studied, each active dosing group - regardless of TTR genotype - demonstrated an increase in blood serum TTR concentrations at the end of dosing compared to starting levels. TTR blood serum data was analyzed using the ARUP prealbumin assay described in Example 6 and is presented in Table 8 below. [Table 8]

[0222] Looking more closely at the data for individuals with the V30M TTR mutation, a highly prevalent mutation associated with familial ATTR polyneuropathy (ATTRm-PN), near complete target engagement at day 14 and near complete TTR stabilization at day 28 of treatment were observed for this patient population using the FPE and Western blot assays, respectively. See Figure 40 and Figure 41.

[0223] Example 8: Commonly used diuretics do not interfere with AG10 exposure ATTR-CM patients with clinical evidence of heart failure often present with signs and symptoms of fluid overload or elevated intracardiac pressure, which necessitates treatment with diuretics (Ruberg and Berk, Circulation 2012 126:1286-300). Diuretics such as furosemide or torasemide are the most common medications used in the AG10-201 Phase 2 study in ATTR-CM patients. See Table 9. [Table 9]

[0224] For the population PK analysis, a formal 3-step covariate selection process was used to examine their effect on AG10 pharmacokinetics. Although multiple covariates were determined to be significant upon forward addition, only disease state on the central volume of distribution was found to be insignificant at the α=0.01 significance level upon backward elimination. Therefore, the final model retained disease state on the central volume of distribution.

[0225] Population PK analysis was used to determine whether coadministration of a diuretic, such as furosemide, had any effect on the pharmacokinetics of AG10. Of the 32 Phase 2 subjects dosed with AG10, 25 were treated with furosemide or torasemide. Of the 17 placebo subjects, 14 received furosemide or torasemide.

[0226] No differences in clearance of AG10 were observed between healthy adult volunteers in Phase 1 and subjects in Phase 2 based on patient condition or diuretic (furosemide / torasemide) (FIG. 42A, B).

[0227] As mentioned previously, the central volume of distribution was affected by the ATTR-CM disease state. In the lower volume (FIGS. 42C,D), the following was observed: a) ATTR-CM patients in Phase 2 vs. healthy adult volunteers in Phase 1 b) ATTR-CM patients receiving furosemide or torasemide versus patients not receiving furosemide or torasemide.

[0228] Thus, the volume of distribution was lower in furosemide / torasemide subjects compared to healthy adult volunteers. There was no difference between drug-disease interactions (i.e., the effect of chronic heart failure on the volume of distribution) and drug-drug interactions (the effect of diuretics on the volume of distribution). Variations in the volume of distribution primarily affect the peak plasma concentration of the drug. Both a phase 1 (AG10-001) healthy adult volunteer study (MAD3, 800mg Q12h) and a phase 2 (AG10-201) study in ATTR-CM patients revealed that a circulating trough concentration of AG10 ~8μM is an appropriate target based on stabilization of TTR. As shown in Figure 43, dosing of 800mg BID of AG10 reaches suitable concentrations in both patients treated with furosemide or torasemide and subjects not taking concomitant diuretics.

[0229] Plasma AG10 C in healthy volunteers following repeated dosing max The mean accumulation ratios at 12 h ranged from 1.3 to 1.6. This accumulation ratio was used to predict the pharmacokinetic profile of dosing with AG10 400 mg tablets at steady state. Figure 44 compares the plasma levels of AG10 12 h after administration of a single 400 mg AG10 tablet, assuming an accumulation ratio of 1.45 (after repeated dosing of 400 mg tablets), with the estimated trough steady state circulating concentrations of AG10, and the actual circulating concentrations of AG10 in ATTR-CM patients dosed with AG10 400 mg BID in Phase 2. The estimated trough levels with the higher dose of the stronger tablet (400 mg) also matched the trough profile of the lower 400 mg BID daily dose group in Phase 2 (2 x 200 mg AG10 tablets twice daily), as did the results obtained with the 800 mg BID daily dose (4 x 200 mg AG10 tablets twice daily).

[0230] Therefore, commonly used diuretics do not interfere with the exposure of AG10.

[0231] Example 9: Phase 3 Clinical Trial - ATTR-CM This prospective randomized, multicenter, parallel-group study evaluates the efficacy and safety of AG10 compared to placebo in symptomatic subjects administered on a background of stable heart failure therapy. After screening and randomization, subjects received a total of 30 months of blinded, placebo-controlled treatment. At the end of 12 months of treatment (Part A), the efficacy of AG10 was evaluated by analysis of functional endpoints (6MWT) and health-related QoL endpoints (measured by the HF-specific measure KCCQ). At the end of 30 months of treatment (Part B), the efficacy of AG10 was further evaluated by analysis of all-cause mortality and CV-related hospitalization.

[0232] There are currently no approved therapies indicated for the treatment of ATTR-CM. Off-label or non-prescription supplemental other investigational therapies or therapies are not permitted for the treatment of ATTR-CM. However, there are several potential pathways for study subjects in the event that other therapies are given regulatory approval with a specific indication for the treatment of ATTR-CM in one or more geographies at the time of the conduct of this study: Subjects are encouraged to remain in the study for at least 12 months of blinded study treatment, regardless of the availability of any approved indicated product. If subjects choose to exit the study at any time, they are encouraged to complete the final visit and associated procedures early. Any subject who has already completed at least 24 months of blinded study therapy and subsequently gains access to an approved, indicated product is encouraged to remain in the study and continue blinded study treatment even after initiation of therapy with that product. Subjects who initiate therapy with an approved, indicated product and remain in the study must attend an unscheduled visit for study evaluations prior to initiation of concomitant therapy.

[0233] All subjects who complete the 30-month blinded study treatment and final evaluation of the double-blind treatment period will be eligible to participate in an open-label follow-on study of long-term AG10 treatment.

[0234] Eligible subjects will be randomized in a 2:1 ratio to receive AG10 800 mg or a matching placebo orally BID. Subjects will be stratified at randomization based on whether they have wild-type ATTR-CM (ATTRwt-CM) or mutant ATTR-CM (ATTRm-CM), with a goal of a minimum of 20% of subjects with ATTRm-CM. Every effort will be made to confirm TTR status (wild-type or variant) by genotyping. In exceptional circumstances (subjects refusing to undergo genetic testing), approval may be sought from the medical monitor or designee to enroll subjects without documented genotyping. If approved and the subject is enrolled in the study, such subjects with unknown TTR status will be stratified into the "wild-type TTR" stratum. Subjects were also randomized to receive 100 mg / kg / day of NT-proBNP (≦3000 vs. >3000 pg / mL) and 100 mg / kg / day of eGFR (≧45 vs. <45 mL / min / 1.73 m 2 ) are also stratified.

[0235] Samples for plasma PK and serum / plasma PD will be collected in the PopPK-PD pilot study.

[0236] Information on AEs and concomitant medications will be collected throughout the study. Safety and conduct of the study will be monitored by an independent Data Monitoring Committee (DMC).

[0237] An overview of the clinical trial design is shown in Figure 45.

[0238] Part A of the study will determine the efficacy of AG10 in treating subjects with symptomatic transthyretin amyloid cardiomyopathy (ATTR-CM) by evaluating the difference between the AG10 and placebo groups in the change from baseline in the 6-minute walk test (6MWT) after 12 months of treatment.

[0239] Part B of the study will determine the efficacy of AG10 in treating subjects with symptomatic ATTR-CM by evaluating the difference between the AG10 and placebo groups in a combined endpoint of all-cause mortality and cumulative frequency of cardiovascular (CV)-related hospitalizations over a 30-month period.

[0240] Target population and statistics Approximately 510 men and women aged 18 years or older and 90 years or younger with chronic, stable, symptomatic (NYHA class I-III) ATTR-CM will be randomized in a 2:1 ratio in this study (340 subjects to active treatment and 170 to matching placebo). Subjects will be stratified according to whether they have ATTRm-CM or ATTRwt-CM at the time of randomization, with a goal of a minimum of 20% of subjects with ATTRm-CM. Every effort will be made to confirm TTR status (wild type or variant) by genotyping. In exceptional circumstances (subjects refusing to undergo genetic testing), approval may be sought from the medical monitor or designee to enroll subjects without documented genotyping. If approved and the subject is enrolled in the study, such subjects with unknown TTR status will be stratified into the "wild type TTR" stratum. Subjects were also randomized to receive 100 mg / kg / day of NT-proBNP (≦3000 vs. >3000 pg / mL) and 100 mg / kg / day of eGFR (≧45 vs. <45 mL / min / 1.73 m 2 ) are also stratified.

[0241] Duration of treatment Subjects will be treated with the investigational product (AG10 or placebo) for 30 months unless not well tolerated. Eligible subjects who complete 30 months of treatment will continue in OLE to receive AG10 at the discretion of the investigator.

[0242] Parts A, B, and OLE will be reported separately. The study will be completed after the data of all subjects who completed the final post-treatment visit are included in the final database and the final report of the study is published.

[0243] Treatment given Subjects who meet the eligibility criteria will be randomized in a 2:1 fashion (AG10:placebo) to receive the following treatment arms in a double-blind fashion: 800mg AG10 BID, orally (2 x 400mg AG10 tablets, BID) Matched placebo BID, orally (2 matched placebo tablets BID).

[0244] In the event that the investigator determines a dose adjustment is warranted based on a subject's report of an AE indicating that the study medication is not well tolerated, the blinded dose may be reduced to 400 mg AG10 or matching placebo administered BID. This will be accomplished by study staff instructing the subject to take one tablet BID instead of two tablets of study medication. Any dose adjustments will be documented in the database. Contraindicated Drug Therapy 1. Use of patisiran, inotersen, tafamidis [see Notes below] or other investigational drugs for the treatment of ATTR-CM is prohibited during this study. 2. Diflunisal, doxycycline; the use of natural products or derivatives used as unproven therapies for ATTR-CM (e.g., green tea extract, tauroursodeoxycholic acid [TUDCA] / ursodiol) is prohibited. 3. The use of calcium channel blockers (e.g., verapamil, diltiazem) or digitalis is prohibited.

[0245] NOTE: If tafamidis is commercially available and subjects have access to it at the time of study entry, subjects will be permitted to begin therapy with tafamidis as concomitant medication upon completion of at least 24 months of blinded study therapy.

[0246] Test procedure Evaluation Schedule A description of the procedures carried out throughout this study is provided below. Screening (days -35 to -1)

[0247] Screening will occur within 35 days prior to administration of the first dose of IMP. The following steps will occur at screening: ·Completion of informed consent form Verification of inclusion / exclusion criteria to ensure subjects are eligible Submission of source documents required by the Diagnostic Confirmation Committee (DCC) must be completed as early as possible during the screening period and must include one of the following: 1. Endomyocardial biopsy report; or 2. Positive 99m Planar images of Tc-pyrophosphate or -bisphosphonate scans; and (serum and / or urine immunofixation electrophoresis (IFE), and Serum free light chain (sFLC) analysis both Laboratory evidence to exclude the diagnosis of AL amyloidosis (based on

[0248] NOTE: Subjects with concomitant monoclonal gammopathy of undetermined significance (MGUS) require confirmation of ATTR-CM via endomyocardial biopsy with mass spectrometry. Evaluation of medical and surgical history NYHA Class Rating Physical examination, including weight and height measurement Vital signs assessment ·Resting 12-lead ECG · Transthoracic echocardiogram (ECHO) at rest if LV wall (interventricular septum or LV posterior wall) thickness is not documented in the echocardiographic or CMR-based clinical history Six-minute walk test (6MWT), two assessments separated by >24 hours and ≤2 weeks Blood sample collection for hematology, serum chemistry (including circulating biomarkers), urinalysis Blood sample collection for exploratory testing of serum and plasma Urine pregnancy test for women of childbearing potential only Medication history evaluation.

[0249] Treatment date Test procedures are listed below by test day and ideally will be performed on each day in the order listed below.

[0250] 1st day and every 3 months (±7 days) These assessments will be performed at Day 1 and at Months 3, 6, 9, 15, 18, 21, 24, and 27: Verification of inclusion / exclusion criteria to ensure subjects are eligible (Day 1) Randomization of subjects to treatment arms and assignment of randomization numbers (Day 1) NYHA Class Rating Physical examination including weight measurement Vital signs assessment ·Resting 12-lead ECG Kansas City Cardiomyopathy Questionnaire (KCCQ) EuroQoL-5 Dimensions (EQ-5D-5L) Six-minute walk test (6MWT) at 6, 9, 18, and 24 months Blood sample collection for hematology, serum chemistry (including circulating biomarkers), urinalysis Urine pregnancy test for women of childbearing potential only PD blood sample collection (pre-dose) for analysis of TTR stabilization in the PopPK-PD pilot study PK blood sample collection (pre-dose) in PopPK-PD pilot study Prealbumin blood sample collection (pre-medication) Distribution / collection and administration of IMP by designated witnesses (i.e., facility personnel) -Evaluation of concomitant drug therapy use AE / vital status assessment / hospitalization decision -IMP adherence assessment (at all visits except Day 1). 28th day (± 3 days) NYHA Class Rating Physical examination including weight measurement Vital signs assessment Resting 12-lead ECG before and 1 hour after dosing Blood sample collection for hematology, serum chemistry (including circulating biomarkers), urinalysis Urine pregnancy test for women of childbearing potential only PD blood sample collection (pre-dose and 1 hour post-dose) for analysis of TTR stabilization in the PopPK-PD pilot study PK blood sample collection (pre-dose and 1 hour post-dose) in PopPK-PD pilot study Prealbumin blood sample collection (pre-medication) Distribution / collection and administration of IMP by designated witnesses (i.e., facility personnel) -Evaluation of concomitant drug therapy use AE / vital status assessment / hospitalization decision -IMP medication adherence assessment. 12th month (± 7 days) NYHA Class Rating Physical examination including weight measurement Vital signs assessment ·Resting 12-lead ECG Kansas City Cardiomyopathy Questionnaire (KCCQ) EuroQoL-5 Dimensions (EQ-5D-5L) Six-minute walk test (6MWT) Blood sample collection for hematology, serum chemistry (including circulating biomarkers), urinalysis Urine pregnancy test for women of childbearing potential only PD blood sample collection (pre-dose) for analysis of TTR stabilization in the PopPK-PD pilot study PK blood sample collection (pre-dose) in PopPK-PD pilot study Prealbumin blood sample collection (pre-medication) Distribution / collection and administration of IMP by designated witnesses (i.e., facility personnel) -Evaluation of concomitant drug therapy use AE / vital status assessment / hospitalization decision -IMP medication adherence assessment. Monthly phone contact (±7 days) These calls will occur at months 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, and 29: -Evaluation of concomitant drug therapy use AE / vital signs assessment / hospitalization decision -IMP medication adherence assessment.

[0251] If a subject discontinues study drug and study evaluations, every effort must be made to continue to follow the subject over the course of the study by completing monthly contacts regarding vital status, or until withdrawal of consent.

[0252] Month 30 (± 7 days) and start of open-label follow-up study Subjects who complete the 30-month double-blind treatment period will continue to receive AG10 in the OLE. NYHA Class Rating Physical examination including weight measurement Vital signs assessment ·Resting 12-lead ECG Kansas City Cardiomyopathy Questionnaire (KCCQ) EuroQoL-5 Dimensions (EQ-5D-5L) Six-minute walk test (6MWT) Blood sample collection for hematology, serum chemistry (including circulating biomarkers), urinalysis Urine pregnancy test for women of childbearing potential only PD blood sample collection (pre-dose) for analysis of TTR stabilization in the PopPK-PD pilot study PK blood sample collection (pre-dose) in PopPK-PD pilot study Prealbumin blood sample collection (pre-medication) Distribution / collection and administration of IMP by designated witnesses (i.e., facility personnel) -Evaluation of concomitant drug therapy use AE / vital status assessment / hospitalization decision -IMP medication adherence assessment. One month after the start of the open-label follow-up study (± 3 days) NYHA Class Rating Physical examination including weight measurement Vital signs assessment ·Resting 12-lead ECG Blood sample collection for hematology, serum chemistry (including circulating biomarkers), urinalysis Urine pregnancy test for women of childbearing potential only PD blood sample collection (pre-dose) for analysis of TTR stabilization in the PopPK-PD pilot study PK blood sample collection (pre-dose) in PopPK-PD pilot study Prealbumin blood sample collection (pre-medication) Distribution / collection and administration of IMP by designated witnesses (i.e., facility personnel) -Evaluation of concomitant drug therapy use AE / vital status assessment / hospitalization decision -IMP medication adherence assessment. Every 3 months (±7 days) after the start of the open-label follow-up study NYHA Class Rating Physical examination including weight measurement Vital signs assessment ·Resting 12-lead ECG Kansas City Cardiomyopathy Questionnaire (KCCQ) EuroQoL-5 Dimensions (EQ-5D-5L) Six-minute walk test (6MWT) every six months Blood sample collection for hematology, serum chemistry (including circulating biomarkers), urinalysis Urine pregnancy test for women of childbearing potential only PD blood sample collection (pre-dose) for analysis of TTR stabilization in the PopPK-PD pilot study PK blood sample collection (pre-dose) in PopPK-PD pilot study Prealbumin blood sample collection (pre-medication) Distribution / collection and administration of IMP by designated witnesses (i.e., facility personnel) -Evaluation of concomitant drug therapy use AE / vital status assessment / hospitalization decision -IMP medication adherence assessment.

[0253] Drug concentration measurement PK Blood Collection Schedule PK samples will be collected to determine AG10 plasma concentrations in a subgroup of subjects at participating centers at the following time points: Day 1 of study visit and every 3 months: Pre-dose Day 28: Before and 1 hour after dosing - 1 month after starting OLE and every 3 months during OLE: before administration · E.T.

[0254] PD Blood Collection Schedule The PD characteristics of AG10 will be assessed in a subgroup of subjects at participating centers by established assays of TTR stabilization, including fluorescent probe exclusion (FPE) assays and Western blots. To perform these PD assays, sampling will be performed at the following time points: Day 1 of study visit and every 3 months: Pre-dose Day 28: Before and 1 hour after dosing - 1 month after starting OLE and every 3 months during OLE: before administration · E.T.

[0255] Prealbumin Blood Sampling Procedure Sampling for measurement of prealbumin concentration will be performed at the following times: Days 1, 28, and every 3 months of study visit: Pre-dose - 1 month after starting OLE and every 3 months during OLE: before administration · E.T.

[0256] 6-minute walk test (6MWT) Prior to randomization, two 6MWTs were performed, separated by >24 hours and ≦2 weeks. Walking distance must be ≧150 m, and the distance walked must be within 15% for two consecutive tests on different days. If the results of the two tests are not within 15%, an additional 6MWT will be repeated within 24 hours and 2 weeks of one 6MWT. If the last attempt is still not within 15% of the other 6MWT, the subject will be ineligible to participate.

[0257] The 6MWT will be performed at the required clinic visits and after completion of the KCCQ and EQ-5D-5L.

[0258] The 6MWT with the Borg scale is performed according to the guidelines of the American Thoracic Society. Complete details regarding the 6MWT procedure are provided in the Test Procedure Manual (SPM).

[0259] Kansas City Cardiomyopathy Questionnaire (KCCQ) ) The KCCQ is a 23-item questionnaire developed to measure the health status and health-related quality of life of subjects with heart failure. Items include symptoms of heart failure, impact on physical and social functioning, and how the patient's heart failure affects their quality of life (QoL). It is to be completed by subjects prior to dosing. Full details are provided in the Study Procedures Manual.

[0260] EuroQoL-5 Dimensions (EQ-5D-5L) The EQ-5D-5L is a brief, self-administered, general health status instrument that takes approximately 5 minutes to complete and should be administered after the completion of the KCCQ. The instrument includes two parts. In the first part, the respondent is asked to rate his / her current health status on five dimensions (mobility, care for personal care, usual activities, pain or discomfort, and anxiety or depression), with each dimension having five levels of functioning (1-no problems, 2-slight problems, 3-moderate problems, 4-significant problems, and 5-extreme problems). The second part is the respondent's self-rating of his / her current health status on a visual analog scale (EQ VAS) with endpoints marked "best possible health status" (score 100) and "worst possible health status" (score 0). Using the scores from the five dimensions, a single index value can be calculated, also known as the utility score. Full details of the filling and scoring are provided in the study procedure manual.

[0261] Clinical Laboratory Measurements Blood and urine samples for clinical laboratory testing will be collected. At screening, the investigator will assess the clinical significance of any values ​​outside of the reference ranges provided by the clinical laboratory, and subjects with abnormalities deemed to be clinically significant will be excluded from the study. The following clinical laboratory tests will be performed: [Table 10]

[0262] Vital Signs Study site staff will assess vital signs before dosing and after 5 minutes of rest after dosing. Any abnormal vital signs that are deemed clinically significant (i.e., associated with symptoms and / or requiring medical intervention) will be recorded as an AE.

[0263] electro-cardiogram A standard 12-lead ECG will be assessed. The ECG will be performed in the supine position after 5 minutes of rest prior to dosing. An ECG will be performed 1 hour post-dose on Day 28 until additional Phase 1 data on the PK-PD relationship to QTc are collected and analyzed. Based on the outcome of the PK-PD data, a 1 hour post-dose ECG on Day 28 will no longer be necessary to reduce subject burden. All investigators will communicate this change through routine communications.

[0264] The investigator or qualified subinvestigator will review all ECG interpretations and interval periods between measurements for clinical significance. Any ECG interpretation that is deemed clinically significant (i.e., associated with symptoms and / or requiring medical intervention) will be reported as an AE.

[0265] Physical examination Subjects will undergo a complete physical examination (PE), including measurements of weight and height, completed by a physician or appropriately trained medical personnel. Any abnormal physical examination finding deemed clinically significant (i.e., associated with symptoms and / or requiring medical intervention) will be recorded as an AE.

[0266] Definition of CV-related hospitalization Cardiovascular-related hospitalization is defined as an unscheduled admission to an acute care setting for medical therapy resulting in a stay of at least 24 hours (or a change in dates if admission / discharge times are not available), or a hospital stay of less than 24 hours if the discharge diagnosis and intervention indicate that the purpose of the hospital stay was for intravenous diuretic therapy for the management of decompensated heart failure. The investigator is responsible for ensuring potential study endpoints, including admission and discharge dates, are collected and recorded; providing the investigator's assessment of whether the hospitalization was CV-related; and submitting AE notification for all adverse events resulting in death or hospitalization.

[0267] Example 10: Phase 3 Clinical Trial - ATTR-PN This prospective, randomized, multicenter, parallel-group study will evaluate the safety and efficacy of AG10 compared with placebo in symptomatic subjects with ATTR-PN. Screening and randomization will be followed by an 18-month double-blind, placebo-controlled treatment period.

[0268] Eligible subjects will be randomized in a 1:1 ratio to receive AG10 800 mg orally BID or matching placebo. Subjects will be stratified at randomization based on Screening Neuropathy Score (NIS), with cutoffs of <30 and >= 30 points, and according to whether they are currently taking tafamidis (Vyndaqel®, Pfizer) or not (in countries or regions where it is not available).

[0269] Information on AEs and concomitant medications will be collected throughout the study. Safety and conduct of the study will be monitored by an independent Data Monitoring Committee (DMC).

[0270] A summary of the study design is shown in Figure 46.

[0271] Target population and statistics Documentation of a positive genotype is required to confirm a definitive diagnosis of ATTR-PN. Key eligibility criteria were chosen to define a population of subjects with disease that is advanced enough to show progression in the placebo group, but not advanced enough to preclude detection of a change in disease state (e.g., PND score ≦IIIa and Karnofsky performance status score ≧60%). Because many ATTR-PN patients have cardiac involvement, subjects with NYHA class IV symptoms are excluded, given the high mortality associated with the degree of cardiomyopathy. To better define the efficacy and safety signals attributable to AG10, concomitant use of other therapies that alter transthyretin production or stability is excluded (with the possible exception of tafamidis 20 mg / day, if available).

[0272] The Neuropathy Score (NIS) is a relatively easy to perform neurological assessment that uses standard groups of muscle, reflex, and sensory modalities and specific sites to provide a summary of clinical neuropathy (weakness, decreased reflexes, and loss of sensation). It is calculated on a scale of 0 to 244, with higher scores indicating worsening disease. The NIS has been shown to correlate with other measures of disease severity and prognosis, and a median of ∼30 has been reported in a large cohort of ATTR-PN patients, so statistics on NIS scores (<30 and ≥30) at screening are included to mitigate potential disparities in severity of neuropathy across treatment arms (Adams, 2015). Subjects are also stratified according to whether they take tafamidis 20 mg / day, which is indicated for the treatment of ATTR-PN in some countries or regions where it is available.

[0273] Duration of treatment Subjects will be treated with study medication (AG10 or placebo) for 18 months.

[0274] Based on long-term assessments observed in several published cohorts, the rate of progression in this subject population at 18 months is expected to be ~12.5 to 17 points on the mNIS+7 (Adams, 2017; Berk, 2013). Because disease progression in placebo subjects as measured by mNIS+7 is gradual, a worsening of at least 12 points is expected over the 18-month trial period (Adams, 2017; Berk, 2013). Because AG10 is expected to halt disease progression by interfering with ongoing amyloid formation, 18 months is likely long enough to detect clinically meaningful placebo-adjusted changes in mNIS+7 scores.

[0275] Treatment given Subjects will be randomized in a double-blind fashion in a 1:1 fashion (AG10:placebo) to receive the following treatment arms: 800mg AG10 BID, orally (2 x 400mg AG10 tablets, BID) Matching placebo BID, orally (2 placebo tablets BID).

[0276] In the event that the investigator determines a dose adjustment is warranted based on a subject's report of an AE indicating that the study medication is not well tolerated, the blinded dose may be reduced to 400 mg AG10 or matching placebo administered BID. This will be accomplished by study staff instructing the subject to take one tablet of study medication BID instead of two. Any dose adjustments will be documented in the database. Contraindicated Drug Therapy 1. Use of patisiran, inotersen, or any other approved or investigational drugs for the treatment of ATTR-PN (other than tafamidis dose 20 mg) is prohibited during this study. 2. Use of approved products other than those indicated for the treatment of ATTR (e.g., diflunisal, doxycycline) or natural products or derivatives used as unproven therapies for ATTR (e.g., green tea extract, tauroursodeoxycholic acid [TUDCA] / ursodiol) is prohibited during the study.

[0277] Test procedure Evaluation Schedule A description of the procedures carried out throughout this study is provided below.

[0278] Screening (days -28 to -1) Screening will occur within 28 days prior to administration of the first dose of IMP. The following steps will occur at screening: ·Completion of informed consent form Verification of inclusion / exclusion criteria to ensure subjects are eligible Evaluation of medical and surgical history NYHA Class Rating Karnofsky Performance Status Physical examination including mBMI Vital signs assessment ·Resting 12-lead ECG PND score ·NIS mNIS+7 10 Meter Walk Test (10MWT), two assessments, >24 hours to <1 week apart Blood sample collection for hematology, serum chemistry (including circulating biomarkers), urinalysis Blood sample collection for exploratory testing of serum and plasma Urine pregnancy test for women of childbearing potential only Medication history evaluation.

[0279] Treatment date Test procedures are listed below by test date and ideally will be performed on each day in the order listed below.

[0280] 1st day and every 3 months (±7 days) These assessments will be performed on Day 1

[0001] and at Months 3, 6, 9, and 15: Verification of inclusion / exclusion criteria to ensure subjects are eligible (Day 1) Randomization of subjects to treatment arms and assignment of randomization numbers (Day 1) NYHA Class Rating Physical examination including mBMI Vital signs assessment ·Resting 12-lead ECG PND score Dyck / Rankin score mNIS+7 ·Norfolk QOL-DN COMPASS-31 · 10 Meter Walk Test (10MWT), Blood sample collection for hematology, serum chemistry (including circulating biomarkers), urinalysis Urine pregnancy test for women of childbearing potential only PD blood sample collection (pre-dose) for analysis of TTR stabilization PK blood sample collection (pre-dose) Prealbumin blood sample collection (pre-medication) Distribution / collection and administration of IMP by designated witnesses (i.e., facility personnel) -Evaluation of concomitant drug therapy use AE / vital status evaluation -IMP adherence assessment (at all visits except Day 1). 18th month (± 7 days) NYHA Class Rating Physical examination including mBMI Vital signs assessment ·Resting 12-lead ECG PND score Dyck / Rankin score mNIS+7 ·Norfolk QOL-DN COMPASS-31 · 10 Meter Walk Test (10MWT), Blood sample collection for hematology, serum chemistry (including circulating biomarkers), urinalysis Urine pregnancy test for women of childbearing potential only PD blood sample collection (pre-dose) for analysis of TTR stabilization PK blood sample collection (pre-dose) Prealbumin blood sample collection (pre-medication) Distribution / collection and administration of IMP by designated witnesses (i.e., facility personnel) -Evaluation of concomitant drug therapy use AE / vital status evaluation -IMP medication adherence assessment. Monthly phone contact (±7 days) These calls will occur at months 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, and 17: -Combination drug therapy use evaluation AE / Vital Sign Assessment -IMP medication adherence assessment.

[0281] If a subject discontinues study drug and study evaluations, every effort must be made to continue to follow the subject over the course of the study by completing monthly contacts regarding vital status, or until withdrawal of consent.

[0282] Drug concentration measurement PK Blood Collection Schedule PK samples will be collected to determine AG10 plasma concentrations in a subgroup of subjects at participating centers at the following time points: Day 1 of study visit and every 3 months: Pre-dose

[0283] PD Blood Collection Schedule The PD characteristics of AG10 will be assessed in a subgroup of subjects at participating centers by established assays of TTR stabilization, including fluorescent probe exclusion (FPE) assays and Western blots. To perform these PD assays, sampling will be performed at the following time points: Day 1 of study visit and every 3 months: pre-dose.

[0284] Prealbumin Blood Sampling Procedure Sampling for measurement of prealbumin concentration will be performed at the following times: Days 1, 28, and every 3 months of study visit: Pre-dose

[0285] evaluation Complete the following assessments: · 10 Meter Walk Test (10MWT), Neuropathy Score (NIS) Corrected Neurological Impairment Score (mNIS+7) COMPASS-31 Dyck / Rankin score · Nutritional status (calculated based on mBMI).

[0286] 10 Meter Walk Test (10MWT) The 10MWT is a behavioral measure used to assess walking speed in meters per second over a short distance. It is utilized to determine functional mobility, gait (git), and vestibular function. Two 10MWTs were performed >24 hours to <1 week apart prior to randomization.

[0287] Neuropathy Score (NIS) The NIS is a neurological assessment that uses a standard panel of muscle, reflex and sensory modalities and specific sites to provide a summary of clinical neurological impairment (weakness, decreased reflexes and sensory loss).

[0288] Corrected Neurological Impairment Score (mNIS+7) The mNIS+7 neurological test is, in part, a composite scale that assesses muscle weakness, sensory loss, and impaired muscle stretch reflexes.

[0289] Composite Autonomic Symptom Score-31 (COMPASS-31) COMPASS-31 is used to quantify the effect of TTR amyloidosis on each subject's autonomic symptoms.

[0290] Dyck / Rankin score Quality of life will be assessed using the Dyck / Rankin score.

[0291] Nutritional status Nutritional status is assessed based on changes in mBMI.

[0292] Clinical Laboratory Measurements Blood and urine samples for clinical laboratory testing will be collected. At screening, the investigator will assess the clinical significance of any values ​​outside of the reference ranges provided by the clinical laboratory, and subjects with abnormalities deemed to be clinically significant will be excluded from the study. The following clinical laboratory tests will be performed: [Table 11]

[0293] Vital Signs Study site staff will assess vital signs before dosing and after 5 minutes of rest after dosing. Any abnormal vital signs that are deemed clinically significant (i.e., associated with symptoms and / or requiring medical intervention) will be recorded as an AE.

[0294] electro-cardiogram A standard 12-lead ECG will be assessed. The ECG will be performed in the supine position after 5 minutes of rest prior to dosing. The investigator or qualified subinvestigator will review all ECG interpretations and interval periods between measurements for clinical significance. Any ECG interpretation deemed clinically significant (i.e., associated with symptoms and / or requiring medical intervention) will be reported as an AE.

[0295] Physical examination Subjects will undergo a complete physical examination (PE), including mBMI, completed by a physician or appropriately trained medical personnel. Any abnormal physical examination finding deemed clinically significant (i.e., associated with symptoms and / or requiring medical intervention) will be recorded as an AE. [Table 12] [Table 13]

[0296] The invention has been described in some detail with reference to figures and examples for purposes of clarity of understanding, but those skilled in the art will understand that certain changes and modifications may be made within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference. In the event of a conflict between this application and a reference provided therein, this application shall control.

Claims

1. A pharmaceutical composition for treating transthyretin amyloidosis (ATTR) cardiomyopathy in humans in need, The pharmaceutical composition comprises approximately 800 mg of hydrochloride-type compound 1 or an equivalent amount of free base or a different salt-type compound 1. Compound 1 has the following structure: 【Chemistry 1】 It has and The pharmaceutical composition is administered orally to the human being twice a day.

2. The pharmaceutical composition according to claim 1, wherein the compound 1 is in the form of a hydrochloride salt.

3. The pharmaceutical composition according to claim 2, comprising two tablets, each containing about 400 mg of hydrochloride-type compound 1.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein administration of the pharmaceutical composition to a human being maintains a trough blood plasma concentration of at least 6 μM of compound 1.

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein administration of the pharmaceutical composition to a human being maintains a trough blood plasma concentration of compound 1 of 7.5 to 10 μM.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein administration of the pharmaceutical composition to a human increases the transthyretin serum concentration of the human 28 days after administration of the pharmaceutical composition compared to the baseline level of the human.

7. The pharmaceutical composition according to claim 6, wherein 28 days after administration of the pharmaceutical composition, the human transthyretin serum concentration increases by at least 10% compared to the baseline level of the human.

8. The pharmaceutical composition according to claim 6, wherein 28 days after administration of the pharmaceutical composition, the human transthyretin serum concentration increases by at least 25% compared to the human baseline level.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the ATTR cardiomyopathy is wild-type ATTR cardiomyopathy (ATTRwt-CM).

10. The pharmaceutical composition according to any one of claims 1 to 8, wherein the ATTR cardiomyopathy is familial ATTR cardiomyopathy (ATTRm-CM).

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein administration of the pharmaceutical composition delays the exacerbation of the human Kansas City Cardiomyopathy Questionnaire (KCCQ) classification.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein administration of the pharmaceutical composition to a human being reduces the decrease in the 6-minute walking distance compared to a human being who has not received the pharmaceutical composition.

13. The pharmaceutical composition according to any one of claims 1 to 11, wherein the mean change from baseline in a 6-minute walk test between a person who has been administered the pharmaceutical composition and a person who has not been administered the pharmaceutical composition is at least 30 m after 30 months of treatment.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein administration of the pharmaceutical composition to a human being slows the progression of ATTR cardiomyopathy.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein administration of the pharmaceutical composition reduces the frequency of cardiovascular-related hospitalizations in a person after 30 months of treatment compared to a person who required the treatment but did not receive the treatment.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein administration of the pharmaceutical composition to a human reduces the mortality rate after 30 months of treatment compared to a human who required the treatment but did not receive the treatment.

17. A method for treating transthyretin amyloidosis cardiomyopathy in a human, comprising administering a pharmaceutical composition according to any one of claims 1 to 16 to a human being in need.

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