Methods for treating TTR amyloidosis using AG10

Abstract

Described herein are methods for treating transthyretin (TTR) amyloidosis in a subject. These methods include specific dosing regimens that are highly effective in treating the subject and are well tolerated by the subject.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) to 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," a table, or a computer program listing appendix 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] 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, and therefore targeted protein-protein interactions (PPIs) are 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 stabilizing 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 shown 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 the Mayo Clinic, the largest amyloid referral center in the United States, noted 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 exists 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, there is provided a method of treating transthyretin (TTR) amyloidosis in a subject in need thereof, comprising administering to a subject a compound having the formula: [ka] or a pharmaceutically 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 daily. In some embodiments, Compound 1 is administered twice daily.

[0012] In some embodiments, there is provided a method of treating transthyretin (TTR) amyloidosis in a subject in need thereof, comprising administering to a subject a compound having the formula: [ka] or a pharmaceutically 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 pharmaceutically 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 explanation 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] [Figure 2] Figure 2 shows the PK profiles of fed versus fasted subjects in single-dose escalation cohort 3 (300 mg oral dose of AG10·HCl).

[0018] [Figure 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 escalation cohort 3 (300 mg oral AG10·HCl), depicting the target association rate as a function of time. In this cohort, six subjects receive Compound 1, while two subjects receive placebo. The placebo group includes subjects 1 and 4.

[0020] [Figure 5]Figure 5 shows the time course of the FPE assay for single-dose escalation cohort 4 (800 mg oral AG10·HCl), depicting the target engagement rate as a function of time. In this cohort, 6 subjects receive Compound 1, while 2 subjects receive placebo. The placebo group includes 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 (300 mg oral AG10·HCl).

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

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

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

[0027] [Figure 12] FIG. 12 shows the peak, mean, and trough target engagement 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-escalation cohort 3 (800 mg oral AG10·HCl 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, while subject 2 received placebo.

[0029] [Figure 14]FIG. 14 shows the aggregated pharmacokinetic and pharmacodynamic data from the single-dose ascending cohort and the multiple-dose ascending cohort, in which there is a predicted 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] Figures 16A-C show 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 the blue bar, ΔH is the green bar, and -TΔS is the red bar. (b) Fluorescence change caused by modification of TTR (2.5 μM) in buffer by the FPE probe monitored in the presence of probe alone (control DMSO) or TTR stabilizer (2.5 μM; stabilizer to TTR ratio 1:1). (c) 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 with 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]Figures 17A-D show the effectiveness of stabilizers in occupying and stabilizing TTR in human serum. (a) Representative Western blot images of the 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; and tolcapone (100 mg, tid), 20 μM. (b) Bar graph representation of the stabilization data obtained from the Western blot experiments. Error bars indicate SD (n = 3). (c) Fluorescence changes caused by modification of TTR in human serum by the 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 compared to probe alone, measured after 3 hours of incubation. 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] Figures 18A-D show crystal structures highlighting the similar interactions induced by the T119M mutation and AG10 binding to TTR. (a) The quaternary structure of AG10 bound to V122I-TTR (PDB: 4HIQ) is shown as a ribbon representation with individually colored monomers. A close-up of one of the two identical T4 binding sites is shown, with ribbons of different colors for the two monomers of the tetramer that make up the binding site. The key hydrogen bond between the pyrazole ring of AG10 and S117 / 117' is highlighted by a dashed line. (b) The crystal structure of the stabilized T119M-TTR variant (PDB: 1FHN) with a dashed line highlighting the key interaction between the hydroxyl groups of S117 and S117'. (c) The crystal structure of TTRwt (PDB: 3CFM). (d) The crystal structure of the thermodynamically stabilized R104H-TTR (PDB: 1X7T).

[0034] [Figure 19] Figures 19A–E illustrate 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, which cannot form a salt bridge with K15 / 15′. 3 is the methyl-pyrazole form of AG10, which can potentially form only one hydrogen bond with either K15 or K15′. 4 is the diethyl-pyrazole analog of AG10, which affects both hydrogen bonds with S117 / S117′. (b) Interaction of TTR with the analogs assessed by ITC. Thermodynamic data: ΔG is the blue bar, ΔH is the green bar, and −TΔS is the red bar. (c) Fluorescence change caused by modification of TTR (2.5 μM) in buffer by the FPE probe, monitored in the presence of the probe alone (control DMSO) or a TTR stabilizer (2.5 μM; stabilizer to TTR ratio 1:1). (d) Bar graph representation of the occupancy of TTR in buffer by the stabilizer in the presence of the FPE probe, measured after 3 hours of incubation, compared with the probe alone. Error bars indicate SD (n = 3). (e) Bar graph representation of Western blot data for the stabilization of TTR in human serum by the analog (10 μM; stabilizer to TTR ratio 2:1). Error bars indicate SD (n = 4). Significance was 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 assay 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 the 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)] (gray 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) Percent TTR occupancy in buffer by AG10 in the presence of the FPE probe or other serum proteins measured after 3 h of incubation compared to probe alone. (e, f) The same experiment described for AG10 was performed with 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]Figures 21A-D show the activity of AG10 and tafamidis in FPE and Western blot assays performed with pooled dog serum. (a) Fluorescence changes caused by modification of canine TTR in commercial beagle dog serum by the FPE probe monitored in the presence of probe alone (control DMSO, closed circle), AG10 (10 μM), or tafamidis (10 μM). (b) Occupancy of canine TTR in dog serum by AG10 and tafamidis in the presence of the FPE probe, measured after 3 hours of incubation compared with 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 escalating doses of AG10 (QD for 7 days). Circles (●) indicate pre-dose on Day 1, squares (■) indicate pre-dose on Day 7 (AG10 concentration at Cmin), and triangles (▲) indicate post-dose on Day 7 (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); and (iv) 200 mg / kg (n = 12, 6 males / 6 females). (b) Bar graph showing 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 the % 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 three replicates.

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

[0040] [Figure 25] FIG. 25 shows a standard calibration curve generated using Aviva Systems Biology's Prealbumin ELISA Kit (Human).

[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 change was 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 serum TTR concentrations in blood for all placebo- and AG10·HCl-treated cohorts (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).

[0044] [Figure 29] Figure 29 plots blood serum TTR concentrations at baseline and day 12 in all placebo- and AG10-treated cohorts (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).

[0045] [Figure 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] [Figure 31] Figure 31 shows that AG10 and the TTR stabilizers tafamidis and diflunisal all increase TTR serum concentrations. Reported TTR serum concentrations for the AG10 cohort are at 28 days after treatment. Reported tafamidis TTR serum concentrations are at day 28, interpolated based on week 2 and week 6 values. Reported diflunisal TTR serum concentrations are at 1 year after treatment.

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

[0048] [Figure 33] Figure 33 plots the distribution of baseline serum TTR concentrations for individuals participating in the Phase 2 trial.

[0049] [Figure 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), individuals with WT-TTR (center column), and individuals with mutant TTR (right column). Error bars provided are standard error of the mean.

[0050] [Figure 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 (checkerboard columns).

[0051] [Figure 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; if the fluorescent probe cannot bind to TTR due to AG10 occupancy, no fluorescence is measured.

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

[0053] [Figure 38] Figure 38 plots the relative fluorescence units measured in the fluorescent probe assay at the indicated time points. The y-axis represents the mean relative fluorescence units at 60 minutes corrected for background. Pre-dose is the trough level, while 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 mean relative fluorescence units at 60 minutes, corrected for background. Pre-dose is the trough level, while 1 hour after dosing is the peak level. Data presented is from individuals receiving placebo treatment.

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

[0056] [Figure 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 AG10·HCl treatment group at 800 mg BID with TTR proteins harboring the V30M mutation.

[0057] [Figure 42] Figures 42A-D plot random effects (ETA) versus categorical covariate plots in the population PK model. Panels A and B plot the effect on clearance, while 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 patients in the 400 mg BID group and 16 patients 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] [Figure 43]Figure 43 plots trough levels of AG10 on day 12 from MAD 3 (Study AG10-001) compared to trough levels in ATTR-CM patients (AG10-201) dosed with 800 mg BID. 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. The line indicates the median, and + indicates the mean. Both studies used 200 mg AG10 tablets for dosing.

[0059] [Figure 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 and widest whiskers relative to the highest value in each group. The line indicates the median, and + indicates the 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] [Figure 45] Figure 45 shows a summary of the trial design for the Phase 3 clinical trial in subjects with ATTR-CM.

[0061] [Figure 46] DETAILED DESCRIPTION OF THE INVENTION

[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 may be employed in practicing the invention.

[0064] The section headings used herein are for general information purposes only and are not to be construed as limiting the scope of the disclosure. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, manuals, and treatises, 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 one of ordinary skill 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 used frequently herein and are not intended to limit the scope of the present disclosure.

[0066] "Compound 1" refers to 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 pharmaceutically acceptable salt thereof. When referring to a specific amount of Compound 1 administered to a patient, the application refers to the amount of the HCl salt of Compound 1 administered. Those skilled 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 "have" 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] As used herein, the term "about" 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 the 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 with the underlying disorder. Treatment includes causing a delay in the progression of clinical symptoms of the disease by administering a 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 a composition after the first appearance of symptoms; and / or palliating the disease, i.e., causing a regression of clinical symptoms by administering a 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 by reducing the symptoms of TTR amyloidosis.

[0071] An "effective amount" or "pharmaceutically effective amount" is an amount sufficient to achieve the stated purpose (e.g., achieve the effect for which it is administered, treat a disease, reduce enzyme activity, or 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 a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over the 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 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, the method comprising administering to a patient a compound having the formula: [ka] or a pharmaceutically acceptable salt thereof to a subject in need thereof, wherein the therapeutically effective amount is a total daily dose of about 10 mg to 2,000 mg. In some embodiments, the total daily dose 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 dose of Compound 1 is about 10 mg to 50 mg. In some embodiments, the total daily dose of Compound 1 is about 50 mg to 300 mg. In some embodiments, the total daily dose of Compound 1 is 50 mg to 150 mg. In some embodiments, the total daily dose of Compound 1 is 150 mg to 800 mg. In some embodiments, the total daily dose of Compound 1 is 800 mg to 1,600 mg. In some embodiments, the total daily dose of Compound 1 is 800 mg to 2,000 mg. It is understood that in this disclosure, the amounts of Compound 1 listed are the amounts of the HCl salt of Compound 1 administered. Those skilled in the art will recognize that minor adjustments to the total amount administered may be necessary to administer the same amount of Compound 1 as 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, the method comprising administering to a patient a compound having the formula: [ka] or a pharmaceutically 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 concentrations 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 concentrations 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 concentrations of transthyretin (TTR) of at least about 25% compared to baseline levels after 28 days of treatment. In some embodiments, subjects before treatment have TTR blood serum levels below baseline serum (ser) TTR concentrations (20 mg / dL TTR). In some embodiments, subjects receiving an effective amount of Compound 1 for 28 days experience increased blood serum TTR levels, such that blood serum TTR levels are above baseline levels. In some embodiments, the subject experiencing increased TTR levels is a subject diagnosed with transthyretin amyloidosis (ATTR) cardiomyopathy.

[0081] There are a variety of 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, a subject with leptomeningeal amyloidosis has a transthyretin protein with an aspartic acid to glycine mutation at position 18 (D18G). In some embodiments, a subject with leptomeningeal amyloidosis has a transthyretin protein with a glycine to arginine mutation at position 53 (G53R). A wild-type transthyretin (TTR) protein is provided herein as SEQ ID NO: 1.

[0083] In some embodiments, a subject with leptomeningeal amyloidosis has 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, a subject with leptomeningeal amyloidosis has 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, TTR amyloidosis is characterized by a TTR protein containing a threonine to alanine mutation at position 60 (T60A). In some embodiments, TTR amyloidosis is characterized by a TTR protein containing a proline to serine mutation at position 24 (P24S). In some embodiments, TTR amyloidosis is characterized by a TTR protein containing an aspartic acid to alanine mutation at position 38 (D38A). In some embodiments, TTR amyloidosis is characterized by a TTR protein containing a leucine to histidine mutation at position 58 (L58H). Patients with these mutations often exhibit 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. Instead, ATTRwt-CM is generally age-related in its progression. In some embodiments, the ATTR cardiomyopathy is ATTRwt-CM. In some embodiments, the 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 exhibit 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 mutations in the TTR protein, whereas ATTRwt-PN does not have 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 containing a valine to methionine mutation at position 30 (V30M). In some embodiments, ATTRm-PN is characterized by a TTR protein containing a phenylalanine to leucine mutation at position 64 (F64L). In some embodiments, ATTRm-PN is characterized by a TTR protein containing 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, improvement in New York Heart Association (NYHA) functional class, Kansas City Cardiomyopathy Questionnaire (KCCQ) response, improvement in 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 methods provided herein improve, stabilize, or delay the progression of the subject's New York Heart Association (NYHA) functional class. 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 because 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 excessive shortness of breath, fatigue, or palpitations. Class III: Marked limitation of physical activity. Comfortable at rest, but less than usual physical activity results in excessive shortness of breath, fatigue, or palpitations. Class IV: Impossibility of performing any physical activity without discomfort. Symptoms may be present at rest. Any physical activity increases discomfort.

[0091] In some embodiments, administration of a therapeutically effective amount of Compound 1 reduces the subject's New York Heart Association (NYHA) functional class. In some embodiments, the 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, the NYHA functional class is reduced from class IV to class III. In some embodiments, the NYHA functional class is reduced from class IV to class II. In some embodiments, the NYHA functional class is reduced from class III to class II. In some embodiments, the NYHA functional class is reduced from class III to class I. In some embodiments, the 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 a subject's Kansas City Cardiomyopathy Questionnaire (KCCQ) classification. In some embodiments, the methods described herein provide an improved score on 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 measure of disease-specific health-related quality of life.

[0093] The KCCQ questionnaire asks subjects 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). In some embodiments, the subject has an average improvement of at least one level for all questions on the questionnaire after treatment with Compound 1 (e.g., from severely limited to very limited, very limited to moderately limited, or moderately limited to slightly limited).

[0094] In some embodiments, the methods provided herein improve, stabilize, or delay the deterioration of a subject's EuroQoL-5 dimension (EQ-5D-5L) score. The EQ-5D-5L is a brief, self-administered general health instrument that takes approximately 5 minutes to complete. This instrument includes two parts. In the first part, respondents are asked to rate their current health status along 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-rating of their current health status on a visual analog scale (EQ VAS) with endpoints marked "best possible health state" (score 100) and "worst possible health state" (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 a subject's performance on the 6-minute walk test. The 6-minute walk test is a 6-minute, self-paced, timed walk to assess a subject's level of functional capacity. The subject can stop and rest during the test if the level of effort exceeds their comfort level. The pre-treatment, post-treatment, and treatment assessments are relatively easy to assess and consist of measuring the distance the subject walks in a 6-minute period. Thus, in some embodiments, a subject increases the total distance covered in the 6-minute walk test after treatment with Compound 1. In some embodiments, a subject walks at least 25 meters more than the baseline distance measured before treatment with Compound 1. In some embodiments, a subject walks at least 30 meters more than the baseline distance measured before treatment with Compound 1. In some embodiments, a subject walks at least 50 meters more than the baseline distance measured before treatment with Compound 1. In some embodiments, a subject walks at least 75 meters more than the baseline distance measured before treatment with Compound 1. In some embodiments, the subject walked at least 100 m more than the baseline distance measured before treatment with Compound 1. In some embodiments, subjects receiving the treatment methods described herein experienced a reduction in the delay in 6-minute walk distance. For example, in some embodiments, the subject maintained approximately the same 6-minute walk distance as before treatment. In some embodiments, the subject covered 10 m less in the 6-minute walk test. In some embodiments, the 6-minute walk test was used to compare a treated group with a non-treated group. In some embodiments, the mean change between groups from baseline was at least 10 m. In some embodiments, the mean change between groups from baseline was at least 20 m. In some embodiments, the mean change between groups from baseline was at least 30 m. In some embodiments, the treatment methods provided herein reduced 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 the 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 in 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 in 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, hi some embodiments, patients experience, on average, at least 0.5, 1, 1.5, 2, 3, 4, or 5 fewer cardiovascular-related hospitalizations per year 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, hi 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 the 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 ATTR polyneuropathy with clinical improvement. Clinical improvement includes, but is not limited to, improvement in the Neuropathy Score (NIS) or modified Neuropathy Score +7 (mNIS+7), improvement on the Norfolk Quality of Life Diabetic Neuropathy (QOL-DN) questionnaire, improvement on the Composite Autonomic Symptom Score (COMPASS-31), 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 scores (NIS). NIS refers to a scoring system that measures weakness, sensation, and reflexes. The NIS score evaluates standard muscle groups for weakness (1 being 25% reduction, 2 being 50% reduction, 3 being 75% reduction, 3.25 being movement against gravity, 3.5 being movement with gravity removed, 3.75 being muscle twitching without movement, and 4 being paralysis), standard muscle stretch reflex groups (0 being normal, 1 being reduced, and 2 being absent), and touch, vibration, joint position and movement, and pain (pinprick) sensation (all graded on the index finger and thumb: 0 being normal, 1 being reduced, and 2 being absent). The evaluation is adjusted 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 after 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 measures of small and large nerve fiber function (NCS and QST) and autonomic function (postural blood pressure). The mNIS+7 score is a modified NIS+7 score (representing the NIS+7 tests). The NIS+7 analyzes weakness and muscle stretch reflexes. Five of the seven tests include nerve conduction characteristics: 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 variations in age, sex, height, and weight. The remaining two of the seven tests include vibration detection threshold and heart rate reduction after deep breathing. The mNIS+7 score modifies 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, as well as 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 test 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 after 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 an improved score on the Norfolk Quality of Life Diabetic Neuropathy (QOL-DN) questionnaire, which is a validated questionnaire well known to those skilled in the art 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 dysautonomia. In one embodiment, the methods of the present invention provide a subject with an improvement in their 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 disease progression 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 declines, e.g., slow the rate at which the COMPASS-31 score declines in subjects treated with AG10 compared to the rate at which the COMPASS-31 score declines 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 of the present disclosure provide a subject with an improvement in mBMI relative to baseline. Such an 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 disclosure slow the rate at which the mBMI score increases, e.g., slow the rate at which the mBMI score increases in subjects treated with AG10 compared to the rate at which the 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 disability associated with peripheral neuropathy is graded. In determining whether a patient has difficulty or inability to perform specific tasks or activities of daily living, more than patient report is used; 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 neuropathic 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 normal activities of daily living, work or leisure activities, and to fulfill their normal family and social responsibilities. * Neuropathic symptoms: NSS ≥ 1 muscle weakness, atrophy, or spasms; negative or positive neuropathic sensory symptoms (N-NNS, P-NSS); or neuropathic autonomic symptoms. - Usual activities of daily living, work, leisure, and social and domestic activities: Despite neuropathic symptoms, patients are able to work at their usual activities, maintain their usual 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, such as athletics, feats of endurance, etc. 4. Symptomatic neuropathy (as defined in 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 of usual* work, usual or leisure activities, family or social obligations* due to 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 "normal" activities of daily living, leisure activities, and fulfill social and family responsibilities, the use of a cane or orthotics would probably place the patient in this (or higher) category (they would then be placed in a lower category). 5. Symptomatic neuropathy (as defined in 3) limiting activities of daily living, work, and leisure activities. Assistance* from other caregivers (<2.5 hours / day) is required. Patients would normally score 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 administration of pain medications or opiates, or to assist with the management of autonomic dysfunction that the patient is unable to do adequately and safely for themselves. 6. Symptomatic neuropathy (as defined in 3) requiring caregiver assistance as described in 5 for ≥ 2.5 hours to < 8 hours / day. 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 numerous factors, including the particular disease being treated. For example, particularly in transthyretin (TTR) amyloidosis diseases or conditions, there is a genetic component that necessitates chronic (i.e., continuous, long-term) administration. However, in some embodiments, administration of Compound 1 to a subject with a genetic TTR amyloidosis disease continues for a set period of time while the subject manifests or experiences symptoms associated with a TTR amyloidosis disease or condition, or after a specific endpoint is achieved (e.g., reduction or complete elimination of symptoms). If TTR amyloidosis disease symptoms 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-term 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 a TTR amyloidosis disease or condition, or after a specific endpoint has been achieved (e.g., reduction 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 comprises 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, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 1 In some embodiments, Compound 1 is administered for 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. 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, or 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 did not alter the exposure of AG10 during treatment. Thus, patients receiving diuretic therapy can be administered AG10 without a modified or specific dosing regimen. As a result, in some embodiments, subjects receiving AG10 also receive 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 Composition 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 pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0120] Pharmaceutical compositions for administering Compound 1 can be conveniently presented in unit dosage form and can be prepared by any method known in the art 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 compositions are prepared by uniformly and intimately bringing the active ingredient into association with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product into the desired formulation.

[0121] Suitable formulations for use in the present invention can be found in "Remington: The Science and Practice of Pharmacy," 21st ed., 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 pharmaceutically 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, the 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, for example, as 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. Additionally, emulsions can be prepared with water-immiscible ingredients such as oils and stabilized with surfactants such as mono-diglycerides, PEG esters, and the like.

[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, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum and acacia gum; dispersing agents or wetting agents can be natural phosphatides, for example, lecithin, condensation products of alkylene oxides with fatty acids, for example, polyoxyethylene stearate, 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, for example, sweeteners, flavorings, and coloring agents, may also be present.

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

[0127] In some embodiments, the present disclosure provides Compound 1 having the formula: [ka] or a pharmaceutically 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, a 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 kits comprising Compound 1 or a pharmaceutically acceptable salt thereof. Some of the kits described herein include a label that describes a method for administering Compound 1. Some of the kits described herein include a label that describes a method for treating transthyretin (TTR) amyloidosis. In some embodiments, the kits described herein include a label that describes a method for treating wild-type transthyretin amyloid cardiomyopathy (ATTR-CM, also known as senile systemic amyloidosis). In some embodiments, the kits described herein include a label that describes a method for treating familial amyloid cardiomyopathy (ATTR-mCM). In some embodiments, the kits described herein include a label that describes a method for treating familial amyloid polyneuropathy (ATTR-PN, also known 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 pharmaceutically acceptable salt thereof. The package or dispenser may also include 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, a 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 notices or instructions describing the methods of use described herein.

[0135] Packaging systems such as blister packs include thermoformable rigid films or PVC suitable for pharmaceutical packaging and extrusion of a lid type. The lid 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 that can be manufactured in various sizes (e.g., 75cc, 100cc, 200cc, etc.) and can generally be manufactured from polypropylene. In some embodiments, a pharmaceutical dosage form of Compound 1 is packaged in a 75cc bottle with a child-resistant seal. Those skilled in the art will easily prepare the bottle systems described herein.

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

[0137] In some embodiments, the total daily dose of Compound 1 is 800 mg, which means that 400 mg is administered at the first dose and 400 mg is administered at the second dose.In some embodiments, two unit doses containing 200 mg of Compound 1 are administered at the first dose, and two unit doses containing 200 mg of Compound 1 are administered at the second dose.In some embodiments, one unit dose containing 400 mg of Compound 1 is administered at the first dose, and one unit dose containing 400 mg of Compound 1 is administered at 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 1,600 mg, which means that 800 mg is administered at the first dose and 800 mg is administered at the second dose.In some embodiments, four unit doses containing 200 mg of Compound 1 are administered at the first dose, and four unit doses containing 200 mg of Compound 1 are administered at the second dose.In some embodiments, two unit doses containing 400 mg of Compound 1 are administered at the first dose, and two unit doses containing 400 mg of Compound 1 are administered at the second dose.In some embodiments, the HCl salt form of Compound 1 is administered. [Example]

[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 relative to the peak area of ​​the product ion of the AG10-D6 internal standard. Quantitation was performed using a weighted 1 / χ2 correlation coefficient generated from calibration standards prepared on the day of extraction. 2 Linear least squares regression analysis was used.

[0142] Fluorescent probe exclusion assay (FPE) The 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] Aliquots of each serum sample are placed in a 96-well plate. The change in fluorescence (λ) after addition of the probe is measured. 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] At both time points, 0 and 72 hours, blood plasma samples from subjects were diluted with an acidified buffer (sodium acetate, KCl, EDTA, DTT, pH 4.0). The 0 hour sample was directly cross-linked with glutaraldehyde and then quenched. The 72 hour sample was incubated at room temperature for 72 hours, then cross-linked and quenched using the same protocol. All samples were then denatured by adding SDS gel loading buffer and boiling before loading. Each sample was 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 was quantified using an infrared LICOR imaging system or a fluorescent imaging system, and normalization of the IgG bands was reported 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 solution, followed by KCO (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 equivalent) was dissolved in ethanol (1 L). Hydrazine hydrate (87 g, 1738 mmol, 2.75 equivalents) and concentrated HCl (4.6 mL, 0.2 equivalents) were added to the solution at room temperature. The reaction mixture was heated to 75±5° C. and stirred at this temperature for 3 hours. 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 portionwise over 30 minutes at room temperature. The reaction mixture was heated to 75±5°C and stirred at this temperature for 3 hours. After completion of the reaction by TLC (50% ethyl acetate:n-hexane, visualized with iodine) and observation of a 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 vigorously stirred 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-hydroxybenzoic acid methyl ester (VIIa), potassium carbonate, and anhydrous DMSO was transferred to the mixture. The DMSO solution of the alkyl bromide was slowly added to maintain an internal temperature of 55.0±5°C. The addition was complete after 6 hours, and the mixture was vigorously stirred 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. 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 (30%) in heptane. The isolated solid was dried under a stream of nitrogen. The solid was poured 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 a 30% ethyl acetate / heptane solvent mixture and dried in a vacuum oven at 55°C, yielding VIIIa in >99.5% purity.

[0151] [ka] A jacketed glass vessel was charged with the compound of Formula VIIIa (1.0 equivalent) and methanol. The mixture was cooled with stirring to 10±5°C, and aqueous sodium hydroxide (3 equivalents) was charged over 20 minutes. The mixture was aged with stirring at 20±5°C for NLT 2 hours, 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 NLT 99.5% purity.

[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 in an overall 3:1 ratio to receive 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 3:1 to receive AG10 or placebo for 12 days of treatment. In total, four cohorts of eight healthy subjects each (32 subjects total, 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-dose ascending 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 had a mean T of 1 hour or less. max The results demonstrate rapid oral absorption, with an elimination half-life of approximately 22 to 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] Additionally, as shown in Figure 2 (showing Cohort 3), the food effect portion of the SAD study revealed minimal food effect on the pharmacokinetics of AG10, with 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] Multiple ascending doses Pharmacokinetic plots for MAD Cohort 1 (healthy subjects receiving 100 mg of AG10·HCl every 12 hours for 12 days), Cohort 2 (healthy subjects receiving 300 mg of AG10·HCl every 12 hours for 12 days), and Cohort 3 (healthy subjects receiving 800 mg of 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, the 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 a fluorescent probe exclusion (FPE) assay or a Western blot assay (described previously), 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 titration cohort as a function of time; the data demonstrate that escalating 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 300 mg dose of AG10·HCl. Results from the FPE and Western blot assays for SAD Cohort 3 showed a significant improvement in R 2 There is a good correlation with a coefficient >0.9 (Figure 8). Both pharmacological activity measures correlate well with each other.

[0160] Additionally, 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 engagement at steady state, with mean stabilization of TTR at 12 hours post-dose on the final 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 engagement rates over 12 days for the 800 mg q12h dose of AG10·HCl.

[0161] Figure 13 shows TTR Western blots from three subjects in Cohort 3 treated with 800 mg AG10·HCl q12h. Subjects 1 and 3 received AG10, and subject 2 received placebo. The lane labeled 0 hours on day 12 pre-dose contains a sample collected from the subject at trough levels of AG10 after 11 days of dosing (22 doses total). The lane labeled day 12 post-dose shows a sample collected at peak levels after the 23rd dose of AG10. Stabilization of tetrameric TTR protein was detected using acidification for 72 hours, followed by cross-linking, SDS-PAGE, and immunoblotting, as previously described. Subject 2 shows no residual 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 the predictable and 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 multiple 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 on a stable background of 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 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 TTR was fully saturated with ligand. Calorimetric data were plotted and fitted using a standard single-site binding model. As a control, we examined the enthalpy change caused by titrating stock DMSO in buffer to TTR; the resulting binding enthalpy was <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 previously reported (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 were determined by their ability to compete with the binding of a fluorescent probe exclusion (FPE) probe to TTR in buffer and human serum. The FPE probe is a thioester TTR ligand that is not itself fluorescent but covalently modifies lysine 15 (K15) upon binding to the T4 binding site of TTR, resulting in a fluorescent conjugate. Ligand binding to the T4 site of TTR reduces FPE probe binding, as observed by less fluorescence. The FPE assay was also adapted to use canine 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 spectrophotometer (SpectraMax M5). FPE by TTR in human and dog serum A 98 μL aliquot of pooled human serum (prepared from human male type AB plasma, Sigma; catalog no. H4522; TTR concentration 5 μM) or dog serum (Innovative Research, catalog no. IBG-SER; TTR concentration 4.6 μM) was mixed with 1 μL of test compound (all compounds 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 with 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). These samples were incubated at 37°C for 2 hours, after which 10 μL of sample was diluted 1:10 with 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 at room temperature for 72 hours, 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 canine 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 (ranging from 10% to 35% remaining TTR). The tetramer stabilization rate was calculated as 100 × [(tetramer and tetramer + RBP concentration, 72 h) / (tetramer and tetramer + RBP concentration in DMSO, time 0)].

[0169] In silico structural and modeling studiesAnalysis of the TTR crystal structure was performed on four TTR crystal structures obtained from the RCSB PDB site. A biological assembly of the TTR tetramer was constructed using the X-ray crystallographic unit cell information provided 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 geometry optimization 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, confirming that it had no virtual frequencies. The dock6 program was used for 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 encompassed the T4 binding site. The same receptor and grid were used for all docking experiments. Free ligand docking was performed to allow rotation 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 derived from human serum; Sigma-Aldrich, catalog number: 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 HSA and AG10 or tafamidis mixture in assay buffer was subjected to gravity gel filtration on a PD Minitrap G25 column (GE Life Sciences, catalog number 45-001-529), and fractions containing HSA were identified by NanoDrop™. The concentration of HSA (i.e., the zero-time concentration) 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 test compounds in these fractions (i.e., concentrations at time zero) were assessed using HPLC (based on a calibration curve of test compounds with known concentrations). 500 μL of each HSA / test compound sample was then added to a Slide-A-Lyzer G2 dialysis cassette (3.5K MWCO, Thermo Scientific, catalog number 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 HSA 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, catalog number P1742) 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 each AG10 / TTR solution was then added to a Slide-A-Lyzer G2 dialysis cassette. The 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 a 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 to the TTR and FPE mixture either individually or in combination [fibrinogen (5 μM), albumin (600 μM), IgG (70 μM), transferrin (25 μM)], and fluorescence was monitored for 6 h as previously described. The percentage of FPE probe binding 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 16 female (F) beagle dogs were divided into four treatment groups, and a total of 32 dogs were orally dosed by gavage with vehicle (6M / 6F at 0 mg / kg) or AG10 in a 0.5% methylcellulose formulation (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 administration of AG10 to dogs to determine the exposure-effect (PK-PD) relationship for binding to 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 for AG10 binding to and stabilization of TTR to obtain concurrent pharmacokinetic (PK) and pharmacodynamic (PD) data. 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 using 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 Unless otherwise noted, all reactions were carried out under an argon atmosphere with dry solvents under anhydrous conditions. 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) performed on 0.20 mm POLYGRAM® SIL silica gel plates (Art.-Nr. 805 023) with fluorescent indicator UV254 using UV light as the visualization agent. Normal-phase flash column chromatography was performed 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 an 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, and 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 of 200-400 nm and quantified using Agilent Chemstation software. HPLC analysis was performed at ambient temperature on both Waters™ XBridge C18 columns equipped with L1 packing (4.6 × 250 mm, 5 μm) and Symmetric™ C4 (2.1 × 150 mm, 5 μm) packings, with 50 μl injections of blank buffer, standard, and / or sample. The mobile phase consisted of solvent A (5:95, v / v) containing 0.1% formic acid in methanol-water, and solvent B (95:5, v / v) containing 0.1% formic acid in methanol-water. The HPLC program was a gradient method, linearly increasing solvent B from 0% to 100% over 20 min, followed by a 30-min hold at 100% solvent B.

[0177] Purity of key compounds HPLC analysis was performed on both C18 and C4 reverse-phase columns. Purity of all key compounds was >95%. A description of the purity analysis is included in the experimental section. Detailed HPLC information (traces, retention times, and % purity) of key compounds is included in the supplemental 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) was added hydrazine hydrate (0.28 mL, 5.25 mmol, 2.5 equiv.) 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 / CHCl) 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] Methyl 3-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)-4-fluorobenzoate (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 hours. 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; Found 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 eq.) and 1,3-dibromopropane (3.0 mL, 29.4 mmol, 5 eq.) in DMF (15 mL) was added K2CO3 (0.98 g, 7.1 mmol, 1.2 eq.). The reaction mixture was stirred at room temperature for 16 hours. 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] Methyl 3-(3-(3,5-diethyl-1H-pyrazol-4-yl)propoxy)-4-fluorobenzoate (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 / hexanes) 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 hours. The reaction was concentrated and purified by flash column chromatography (silica gel, 1-5% MeOH / EtOAc) to provide compound 6 (75 mg, 65% yield over two steps). 1H 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). C 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 underlying mechanism of molecular interaction for 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, and as a result, binding of the first ligand will dominate the overall binding energy and stabilization activity. Although slight differences in cooperativity can be observed in the ITC thermograms, these differences will likely have little impact 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 affinities of AG10 and tafamidis to TTR in buffer (Kd = 4.8 ± 1.9 and 4.4 ± 1.3 nM, respectively) were 4-fold higher than tolcapone (Kd = 20.6 ± 3.7 nM) and ~100-fold higher than diflunisal (Kd = 407 ± 35 nM). The Kd values ​​for compounds 1-4 ranged from 90 to 1250 nM, and these results are summarized in Table 5. The Kd for the binding of a stabilizer 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 assess 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 in the system and vibrational frequencies). 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 enthalpy-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 than ligands with lower ΔH effects. While this study describes a correlation between enthalpic force 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 covalently modifies lysine 15 (K15) upon binding to the T4 binding site of TTR, resulting in a fluorescent conjugate. Ligand binding to the T4 site of TTR will reduce FPE probe binding, as evidenced 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 efficacy of stabilizers for binding and stabilizing TTR in buffer (stabilizer to TTR tetramer ratios of 1:1 were tested; Figure 16b, c and Table 5). The order of efficacy of stabilizers for TTR in buffer was AG10 > tolcapone > tafamidis > diflunisal.

[0187] Next, we evaluated the efficacy (representing both potency and selectivity) of stabilizers (10 μM) in occupying and stabilizing TTR in human serum (TTR concentration 5 μM) using FPE and Western blot assays (Table 5). The Western blot assay measures the amount of intact TTR tetramer after 72 hours of acid treatment in the presence and absence of stabilizers. The order of efficacy of stabilizers in human serum was similar to that observed for TTR in buffer (AG10 > tolcapone > tafamidis > diflunisal; Table 5). Diflunisal had the lowest efficacy and potency compared to all other stabilizers (20- to 80-fold lower affinity than the other stabilizers), as predicted based on its significantly lower binding affinity to TTR (Kd = 407 ± 35 nM). Surprisingly, there was no correlation between the Kd values ​​of the three other stabilizers and their efficacy and potency in occupying and stabilizing TTR in both buffer and human serum. For example, despite the fact that the binding affinity of tolcapone to TTR (Kd = 20.6 ± 3.7 nM) was slightly lower than that of tafamidis (Kd = 4.4 ± 1.3 nM), the potency and efficacy of tolcapone were higher than that of tafamidis. 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 indicates that the enthalpic binding of AG10 and tolcapone to TTR (discussed in detail below) is the primary driver of their efficient stabilization of TTR compared with 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 stabilization: 95.4 ± 4.8%); other compounds were significantly lower than their reported clinical efficacy. 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). Therefore, the ionization rate of the carboxylic acid group of the stabilizer may vary at pH 4. This may affect the strength of the electrostatic interaction between the carboxylic acid group and the ε-amino groups of lysine 15 (K15) and K15' at the apex of the T4 binding site, which may affect the efficacy of the stabilizer. To address this concern, we performed Western blot assays using urea buffer (pH 7.4). 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 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) (Figure 17c, d).

[0189] The binding interaction between AG10 and S117 / S117' of TTR mimics the molecular interaction within the disease-protective T119M mutation We investigated the correlation between binding enthalpy and TTR stabilization by comparing reported cocrystal structures of TTR and stabilizers to those 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 enthalpy-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; the ideal distance for a hydrogen bond 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 positioned in the halogen-binding pocket (HBP) 3 and 3', where they interact with TTR primarily through hydrophobic interactions. In addition to the electrostatic interaction between the carboxylic acid moiety of AG10 and K15 / K15', AG10 also forms two hydrogen bonds (distance ∼2.8 Å) with the hydroxyl side chain serine 117 (S117) and S117' of an adjacent monomer within a low-dielectric large molecule within the T4 binding site (Figure 18a). These additional hydrogen bonds likely contribute to the driving force for the primary 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] The two S117 side chain hydroxyl groups of monomers A and B in the 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) between these two dimers within 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 in TTRwt (the average dimer-dimer distance is ∼5.6 Å, Figure 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 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, highlighting closer dimer-dimer contacts in both the T119M-TTR (distance ∼4.8 Å) and AG10-V122I-TTR (distance ∼4.66 Å) crystal structures compared with 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; Figure 15) and evaluated their ability to bind and stabilize TTR (Figure 19). AG10 binds to TTR with unfavorable entropy (TΔS = −2.26 kcal / mol). The fluorine atom of AG10 is positioned 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 showed that the iodine of 1 fits in the HBP1 of TTR (where the iodine of T4 binds), suggesting that displacing more water molecules from HBP1 would improve entropic binding (Figure 19a). Compound 1a exhibited significantly lower binding affinity (Kd = 90 ± 14 nM) to TTR in buffer compared with AG10 (Kd = 4.8 ± 1.9 nM). ITC analysis indicated that the entropic interaction of 1 with TTR was more favorable than that of AG10 (TΔS = -0.21 kcal / mol and -2.26 kcal / mol, respectively), but there was 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 a decreased strength of the salt bridge between the carboxylic acid moiety of 1 and K15 / K15' (~4.7 Å compared with ~2.8 Å for AG10). Compound 1a also showed reduced potency for 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 partially shield it from solvent. We synthesized a methyl-ester analog of AG10 (compound 2, Figure 19a) and tested its effect of modifying the two salt bridges that AG10 forms at the periphery of the T4-binding site. Compound 2 exhibited significantly lower affinity for TTR in buffer (Kd = 258 ± 17 nM) compared with AG10 (Kd = 4.8 ± 1.9 nM), which is explained by the lower strength of the potential hydrogen bond between the ester group of 2 and K15 / K15' (ΔH = -6.49 kcal / mol) compared with the salt bridge in AG10 (Figure 19b). Compound 2 also showed reduced potency relative to AG10 and compound 1a for TTR in buffer and human serum (Figure 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, which has an N-methylpyrazole. The N-methyl group constrains the pyrazole ring of 3 and will 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 prevents these molecules from reaching deep within 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) exhibited greatly reduced binding affinity to TTR in buffer. This decreased affinity translated into significantly reduced potency for TTR in buffer and human serum, particularly 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 have observed with clinical TTR stabilizers, the potencies of AG10 and compounds 1, 2, 3, and 4 in occupying and stabilizing TTR correlated very well (R2 = 0.98) with the binding enthalpies 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 examine the role of enthalpy in the selectivity of AG10 for TTR over other abundant serum proteins, we examined the concentration-effect relationships of AG10 and tafamidis in whole human serum in an FPE assay. We tested AG10 and tafamidis because their binding affinities for TTR in buffer are very similar (Kd = 4.8 ± 1.9 nM and 4.4 ± 1.3 nM, respectively), but their thermodynamics for TTR binding, particularly the enthalpic component, are significantly different. Therefore, data obtained in serum will largely reflect selectivity. AG10 showed increasing concentration-dependent occupancy, with complete occupancy achieved at AG10 concentrations ≥ 10 μM. Even at substoichiometric concentrations, AG10 can occupy and stabilize a large portion of TTR (69.2% TTR occupancy by FPE and 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 than tafamidis (18.3 ± 0.98 μM vs. 24.1 ± 1.1 μM; Figure 20a). After 24 hours 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 than 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 at a 1:1 molar ratio throughout the 24 h incubation (Figure 20b).

[0196] Finally, the selectivity of AG10 and tafamidis for binding to TTR in human serum was assessed using a modified FPE assay in which human serum was replaced with 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. The 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; Figure 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; Figure 20e, f). The addition of all plasma proteins tested simultaneously yielded identical results for AG10. The higher selectivity of AG10 for TTR can be attributed to a number of properties, including enthalpic binding and 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. Therefore, we adopted an approach similar to that currently used in clinical settings to test the efficacy of AG10 versus other TTR kinetic stabilizers. The activity of TTR stabilizers in occupying and stabilizing TTR is typically assessed ex vivo in blood samples obtained from patients before and after administration of the stabilizer. To examine the in vivo activity of AG10, we used this same approach 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, to which 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 it to be similar to that in healthy humans. To confirm the suitability of the assays for use with human-based reagents for canine testing, the activities of AG10 and tafamidis were assessed in pooled canine serum using the same FPE and Western blot assays used in the previously described experiments. The concentration-effect relationships for in vitro TTR binding and stabilization of AG10 and tafamidis in both assays repeated with canine serum were similar to those observed in human serum (Figure 21). These characteristics made healthy dogs a suitable system for subsequent 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-dose data were collected on study day 1 (baseline), 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). 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 arm 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 The 7-day post-dosing study demonstrated complete (>97%) TTR occupancy. To determine the minimum 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-response) relationship. Eight dogs were divided into two active treatment groups receiving a single oral dose of either 5 or 20 mg / kg of AG10·HCl. These results demonstrated enhanced TTR occupancy in the 20 mg / kg vs. 5 mg / kg dose groups (Fig. 22c, d). max The % TTR occupancy at C for both doses 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 with 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 to monkeys by intravenous administration or oral gavage AG10·HCl was administered intravenously to three male cynomolgus monkeys at a dose level of 1 mg / kg or orally once at 5 mg / kg. A 2-week washout period was 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 Figure 23, the results from the FPE assay demonstrated that orally administered AG10 effectively bound to TTR in monkey serum.

[0201] The FPE assay 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 manufacturer's recommended calibration curve. The supplied TTR calibrator was dissolved in 1 mL of distilled water, resulting in a concentration of 8.85 μg / mL. The first additional standard was 1000 ng / mL, prepared by adding 178.4 μL of calibrator to 1400 μL of 1× diluent. The second additional standard was 200 ng / mL, prepared by adding 32.4 μL of calibrator to 1400 μL of 1× diluent. The third additional standard was 0.78125 ng / mL, 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 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 minutes. All samples were diluted 1:10,000 in two steps. First, 5 μL of serum was mixed with 995 μL of the 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. From this point on, the TTR test followed the manufacturer's protocol without further modification. Briefly, standards and serum samples were incubated in the ELISA plate at room temperature for 1 hour. The ELISA plate was washed four times with 1× wash buffer and then incubated with 1× horseradish peroxidase conjugate for 30 minutes at room temperature, protected from light. The ELISA plate was then washed four 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 the 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 four-parameter curve. The log (ng / mL) of pooled human serum and MAD serum samples was 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 testing.

[0207] Samples from healthy human volunteers from the three cohorts of the MAD study (100, 300, and 800 mg of AG10·HCl were administered twice daily for 12 consecutive days) as well as 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 is run 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-dose (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 patients (patent) were included instead 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 this 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 of 36% in serum TTR levels, and individuals receiving 800 mg of AG10·HCl twice daily had a mean increase of 50% in serum TTR levels. In comparison, the placebo-treated group had a mean decrease of 7% in serum TTR levels.

[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 (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 before treatment. After 28 days of treatment, the number of individuals without normal serum TTR concentrations increased to 31% of placebo individuals. See Figure 32. The baseline distribution of serum TTR concentrations for individuals in this study is shown in Figure 33.

[0216] Ex vivo Western blot analysis confirmed that AG10·HCl at dose 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 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 the relationship between circulating plasma concentration of AG10 and occupancy of the thyroxine-binding pocket of tetrameric TTR by AG10 was plotted, it was determined that significant target association (occupancy by AG10) occurred at a circulating plasma concentration of approximately 5 μM, and complete target association occurred at approximately 7.5 μM (see Figure 36).

[0219] Figures 37 and 38 plot the relative fluorescence units measured in the fluorescent probe assay for the 400 mg BID cohort (Figure 37) and the 800 mg BID cohort (Figure 38) 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). As can be seen in both figures, the fluorescent probe assay indicates that there is near complete target association by Day 14 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 previously mentioned time points in the placebo control group. This plot reveals a lack of target association for the placebo control group.

[0220] In summary, the data presented here indicated 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, and that AG10 completely stabilized TTR across both dose levels tested.

[0221] When examining wild-type and specific mutant TTR populations in the cohort studied, each active treatment group—regardless of TTR genotype—showed an increase in blood serum TTR concentrations at the end of treatment compared to starting levels. TTR blood serum data were analyzed using the ARUP prealbumin assay described in Example 6 and are 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 assay and Western blot assay, respectively. See Figures 40 and 41.

[0223] Example 8: Commonly used diuretics do not interfere with AG10 exposure Patients with ATTR-CM who have clinical evidence of heart failure often experience 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 trial in patients with ATTR-CM. See Table 9. [Table 9]

[0224] For the population PK analysis, a formal 3-step covariate selection process was used to examine their influence on the pharmacokinetics of AG10. 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 upon backward elimination at a significance level of α = 0.01. 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 subjects in Phase 2 who received AG10, 25 were treated with furosemide or torasemide. Of the 17 placebo subjects, 14 received furosemide or torasemide.

[0226] No differences in the 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 disease state of ATTR-CM. In the lower volume (Fig. 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 subjects receiving furosemide / torasemide compared with healthy adult volunteers. There was no difference between drug-disease interactions (i.e., the effect of chronic heart failure on volume of distribution) and drug-drug interactions (the effect of diuretics on volume of distribution). Variations in volume of distribution primarily affect the peak plasma concentration of the drug. Both a Phase 1 (AG10-001) study in healthy adult volunteers (MAD3, 800 mg Q12h) and a Phase 2 (AG10-201) study in patients with ATTR-CM demonstrated that a circulating trough concentration of ∼8 μM of AG10 is an appropriate target based on TTR stabilization. As shown in Figure 43, a dose of 800 mg BID of AG10 achieves optimal concentrations in both patients treated with furosemide or torasemide and in subjects not taking concomitant diuretics.

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

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

[0231] Example 9: Phase 3 Clinical Trial - ATTR-CM This prospective, randomized, multicenter, parallel-group study evaluated the efficacy and safety of AG10 compared with placebo in symptomatic subjects receiving 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 assessed 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 assessed by analysis of all-cause mortality and CV-related hospitalization.

[0232] There are currently no approved therapies indicated for the treatment of ATTR-CM. Other investigational therapies or therapies using off-label or non-prescription supplements are not permitted for the treatment of ATTR-CM. However, in the event that other therapies receive 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, several potential pathways exist for study subjects: 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 withdraw from the study at any time, they are encouraged to complete the final visit and related 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 their blinded study treatment, even after initiating therapy with that product. Subjects who initiate therapy with an approved, indicated product and remain in the study must attend an unscheduled study evaluation visit before initiating combination 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 either 800 mg of AG10 or a matching placebo orally twice daily. 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 (i.e., 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 subjects are enrolled in the study, such subjects with unknown TTR status will be stratified into the "wild-type TTR" stratum. Subjects were also randomly assigned to a randomized controlled trial (NCT02242626) to evaluate their renal function as defined by NT-proBNP levels (≤3000 vs. >3000 pg / mL) and eGFR (≥45 vs. <45 mL / min / 1.73 m ) at screening. 2 ) are also stratified according to

[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 study conduct will be monitored by an independent Data Monitoring Committee (DMC).

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

[0238] Part A of this study will determine the efficacy of AG10 in treating subjects with symptomatic transthyretin amyloid cardiomyopathy (ATTR-CM) by assessing 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 assessing 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 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 subjects are enrolled in the study, such subjects with unknown TTR status will be stratified into the "wild-type TTR" stratum. Subjects were also randomly assigned to a randomized controlled trial (NCT02242626) to evaluate their renal function as defined by NT-proBNP levels (≤3000 vs. >3000 pg / mL) and eGFR (≥45 vs. <45 mL / min / 1.73 m ) at screening. 2 ) are also stratified according to

[0241] Treatment duration Subjects will be treated with the investigational drug (AG10 or placebo) for 30 months unless it is not well tolerated. Eligible subjects who complete 30 months of treatment will continue in OLE to receive AG10 at the investigator's direction.

[0242] Part A, Part B, and OLE will be reported separately. The study will be completed after the data of all subjects who have completed the final post-treatment visit have been included in the final database and the final report of the study has been submitted.

[0243] Treatment administered 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 (two 400mg AG10 tablets BID) Matching placebo BID, oral (two matching 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 (e.g., green tea extract, tauroursodeoxycholic acid [TUDCA] / ursodiol) used as unproven therapies for ATTR-CM 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 the subject has 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 performed 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: Filling out informed consent Verification of inclusion / exclusion criteria to ensure subjects are eligible Submission of source documentation 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 Clinical and laboratory evidence to exclude the diagnosis of AL amyloidosis.

[0248] Note: Subjects with concomitant monoclonal gammopathy of undetermined significance (MGUS) require confirmation of ATTR-CM by endomyocardial biopsy with mass spectrometry. Medical and surgical history evaluation NYHA Class Rating Physical examination including weight and height measurement Vital signs assessment ·Resting 12-lead ECG Resting transthoracic echocardiogram (ECHO) 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), and urinalysis Blood sample collection for exploratory testing of serum and plasma Urine pregnancy test for women of childbearing potential only ·Medication history assessment.

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

[0250] Day 1 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), and urinalysis Urine pregnancy test for women of childbearing potential only PD blood sample collection (pre-dose) for analysis of TTR stabilization in PopPK-PD pilot study PK blood sample collection (before administration) in PopPK-PD pilot study Prealbumin blood sample collection (before administration) Distribution / collection and administration of IMP by designated witnesses (i.e., facility personnel) Assessment of concomitant medication use AE / vital status assessment / hospitalization decision IMP adherence assessment (at all visits except Day 1). Day 28 (± 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), and 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 (before administration) Distribution / collection and administration of IMP by designated witnesses (i.e., facility personnel) Assessment of concomitant medication 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) 6-minute walk test (6MWT) Blood sample collection for hematology, serum chemistry (including circulating biomarkers), and urinalysis Urine pregnancy test for women of childbearing potential only PD blood sample collection (pre-dose) for analysis of TTR stabilization in PopPK-PD pilot study PK blood sample collection (before administration) in PopPK-PD pilot study Prealbumin blood sample collection (before administration) Distribution / collection and administration of IMP by designated witnesses (i.e., facility personnel) Assessment of concomitant medication use AE / vital status assessment / hospitalization decision -IMP medication adherence assessment. Monthly phone contact (±7 days) These phone 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: Assessment of concomitant medication use AE / vital sign assessment / hospitalization decision -IMP medication adherence assessment.

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

[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) 6-minute walk test (6MWT) Blood sample collection for hematology, serum chemistry (including circulating biomarkers), and urinalysis Urine pregnancy test for women of childbearing potential only PD blood sample collection (pre-dose) for analysis of TTR stabilization in PopPK-PD pilot study PK blood sample collection (before administration) in PopPK-PD pilot study Prealbumin blood sample collection (before administration) Distribution / collection and administration of IMP by designated witnesses (i.e., facility personnel) Assessment of concomitant medication 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), and urinalysis Urine pregnancy test for women of childbearing potential only PD blood sample collection (pre-dose) for analysis of TTR stabilization in PopPK-PD pilot study PK blood sample collection (before administration) in PopPK-PD pilot study Prealbumin blood sample collection (before administration) Distribution / collection and administration of IMP by designated witnesses (i.e., facility personnel) Assessment of concomitant medication 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), and urinalysis Urine pregnancy test for women of childbearing potential only PD blood sample collection (pre-dose) for analysis of TTR stabilization in PopPK-PD pilot study PK blood sample collection (before administration) in PopPK-PD pilot study Prealbumin blood sample collection (before administration) Distribution / collection and administration of IMP by designated witnesses (i.e., facility personnel) Assessment of concomitant medication use AE / vital status assessment / hospitalization decision -IMP medication adherence assessment.

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

[0254] PD blood collection schedule The PD characteristics of AG10 will be assessed in a subgroup of subjects at participating centers using 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 administration - 1 month after starting OLE and every 3 months during OLE: before administration ·ET.

[0255] Prealbumin blood sampling procedure Sampling for measuring prealbumin concentrations will be performed at the following times: Study visits days 1 and 28, and every 3 months: Pre-dose - 1 month after starting OLE and every 3 months during OLE: before administration ·ET.

[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% of two consecutive trials on different days. If the results of the two trials are not within 15%, an additional 6MWT will be repeated within 24 hours and 2 weeks of one of the 6MWTs. If the final 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 visit after completion of the KCCQ and EQ-5D-5L.

[0258] The 6MWT with Borg scale is performed according to the guidelines of the American Thoracic Society. Full 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, its impact on physical and social function, 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 procedure 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 completing the KCCQ. This instrument includes two parts. In the first part, respondents are asked to rate their current health status along 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-rating of their current health status on a visual analog scale (EQ VAS) with endpoints labeled "best possible health state" (score 100) and "worst possible health state" (score 0). Scores from the five dimensions can be used to calculate a single index value, also known as a utility score. Full details of the questionnaire 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 the reference ranges provided by the clinical laboratory, and subjects with abnormalities deemed 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 before 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 results of the PK-PD data, the 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 sub-investigator will review all ECG interpretations and interval periods 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, which will be completed by a physician or appropriately trained healthcare professional. Any abnormal physical examination finding that is 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 A cardiovascular-related hospitalization is defined as an unplanned 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 is CV-related; and submitting AE notifications 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 1:1 to receive AG10 800 mg orally BID or matching placebo. Subjects will be stratified at randomization based on a Screening Neuropathy Score (NIS) cutoff 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 study conduct 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 selected to define a population of subjects with disease advanced enough to demonstrate progression in the placebo group but not so advanced as to preclude detection of a change in disease status (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 will be 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 will be excluded (with the potential exception of tafamidis 20 mg / day, if available).

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

[0273] Treatment duration Subjects will be treated with study medication (AG10 or placebo) for 18 months.

[0274] Based on longitudinal 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 the 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 administered 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 (two 400mg AG10 tablets BID) Matching placebo BID, oral (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 poor tolerability of the study medication, 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 medication for the treatment of ATTR-PN (other than tafamidis at a dose of 20 mg) is prohibited during this study. 2. The 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 performed throughout this study is provided below.

[0278] Screening (Day -28 to Day -1) Screening will occur within 28 days prior to administration of the first dose of IMP. The following steps will be performed at screening: Filling out informed consent 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 separated by >24 hours and <1 week Blood sample collection for hematology, serum chemistry (including circulating biomarkers), and urinalysis Blood sample collection for exploratory testing of serum and plasma Urine pregnancy test for women of childbearing potential only ·Medication history assessment.

[0279] Treatment date Testing procedures are listed below by test day and ideally should be performed in the order listed below for each day.

[0280] Day 1 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), and 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 (before administration) Distribution / collection and administration of IMP by designated witnesses (i.e., facility personnel) Assessment of concomitant medication use AE / vital status assessment 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), and 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 (before administration) Distribution / collection and administration of IMP by designated witnesses (i.e., facility personnel) Assessment of concomitant medication use AE / vital status assessment -IMP medication adherence assessment. Monthly phone contact (±7 days) These phone calls will occur at months 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, and 17: -Evaluation of combined drug therapy use AE / Vital Sign Assessment -IMP medication adherence assessment.

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

[0282] Drug concentration measurement PK blood collection schedule PK samples will be collected at the following time points to determine AG10 plasma concentrations in a subgroup of subjects at participating centers: 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 using 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 measuring prealbumin concentrations will be performed at the following times: Study visits days 1 and 28, and every 3 months: 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 used 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 deficits (weakness, decreased reflexes, and sensory loss).

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

[0289] Composite Autonomic Symptom Score-31 (COMPASS-31) COMPASS-31 is used to quantify the impact 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 the reference ranges provided by the clinical laboratory, and subjects with abnormalities deemed 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 before dosing. The investigator or qualified subinvestigator will review all ECG interpretations and interval periods 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.

[0295] Physical examination Subjects will undergo a complete physical examination (PE), including mBMI, completed by a physician or appropriately trained healthcare professional. 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] Although the invention has been described in some detail by way of figures and examples for purposes of clarity of understanding, those skilled in the art will understand that certain changes and modifications may be made within the scope of the appended claims. Additionally, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference were individually incorporated by reference. In the event of a conflict between this application and the references provided therein, the present application shall control.

Claims

1. 1. A pharmaceutical composition for treating transthyretin (TTR) amyloidosis in a subject, comprising Compound 1 having the formula: 【Chemistry 1】 or a therapeutically effective amount of a pharmaceutically acceptable salt thereof, wherein the therapeutically effective amount is a total daily dose of 1,600 mg of Compound 1 in HCl salt form or an equivalent amount in other salt form; A pharmaceutical composition to be administered orally twice daily.

2. 2. The pharmaceutical composition of claim 1, wherein Compound 1 is in the HCl salt form.

3. 3. The pharmaceutical composition according to claim 1, wherein the TTR amyloidosis is a disease or condition selected from the group consisting of familial amyloid polyneuropathy, familial amyloid cardiomyopathy, senile systemic amyloidosis, central amyloidosis, ocular amyloidosis, leptomeningeal amyloidosis, ocular leptomeningeal amyloidosis, vitreous amyloidosis, gastrointestinal amyloidosis, neurogenic amyloidosis, non-neurogenic amyloidosis, non-hereditary amyloidosis, reactive / secondary amyloidosis, and cerebral amyloidosis.

4. The pharmaceutical composition according to claim 3, wherein the TTR amyloidosis is leptomeningeal amyloidosis.

5. 5. The pharmaceutical composition of claim 4, wherein the leptomeningeal amyloidosis is characterized by a TTR protein containing an aspartic acid to glycine mutation at position 18 (D18G), a glycine to arginine mutation at position 53 (G53R), a tyrosine to cysteine ​​mutation at position 114 (Y114C), or a threonine to proline mutation at position 49 (T49P).

6. 3. The pharmaceutical composition according to claim 1, wherein the TTR amyloidosis is transthyretin amyloidosis (ATTR) cardiomyopathy or transthyretin amyloidosis (ATTR) polyneuropathy.

7. 7. The pharmaceutical composition of claim 6, wherein the TTR amyloidosis is characterized by a TTR protein containing a threonine to alanine mutation at position 60 (T60A), a proline to serine mutation at position 24 (P24S), an aspartic acid to alanine mutation at position 38 (D38A), or a leucine to histidine mutation at position 58 (L58H).

8. 3. The pharmaceutical composition according to claim 1, wherein the TTR amyloidosis is transthyretin amyloidosis (ATTR) cardiomyopathy.

9. The pharmaceutical composition according to claim 8, wherein the ATTR cardiomyopathy is wild-type ATTR cardiomyopathy (ATTRwt-CM).

10. The pharmaceutical composition according to claim 8, wherein the ATTR cardiomyopathy is familial ATTR cardiomyopathy (ATTRm-CM).

11. 11. The pharmaceutical composition of claim 10, wherein the ATTRm-CM is characterized by a TTR protein containing a valine to isoleucine mutation at position 122 (V122I) or a threonine to proline mutation at position 49 (T49P).

12. 3. The pharmaceutical composition according to claim 1, wherein the TTR amyloidosis is transthyretin amyloidosis (ATTR) polyneuropathy.

13. The pharmaceutical composition according to claim 12, wherein the ATTR polyneuropathy is wild-type ATTR polyneuropathy (ATTRwt-PN).

14. The pharmaceutical composition according to claim 12, wherein the ATTR polyneuropathy is familial ATTR polyneuropathy (ATTRm-PN).

15. 15. The pharmaceutical composition of claim 14, wherein the ATTRm-PN is characterized by a TTR protein containing a valine-to-methionine mutation at position 30 (V30M), a phenylalanine-to-leucine mutation at position 64 (F64L), or a tyrosine-to-cysteine ​​mutation at position 114 (Y114C).

16. 12. The pharmaceutical composition of any one of claims 8 to 11, wherein administration of the pharmaceutical composition in a subject with ATTR cardiomyopathy improves, stabilizes, or delays the progression of: (i) the subject's New York Heart Association (NYHA) functional class; (ii) the subject's Kansas City Cardiomyopathy Questionnaire (KCCQ) class; or (iii) the subject's EuroQoL-5 Dimensions (EQ-5D-5L) class.

17. The pharmaceutical composition according to any one of claims 8 to 11, wherein administration of the pharmaceutical composition reduces the decrease in 6-minute walking distance compared to a subject not receiving the pharmaceutical composition.

18. 12. The pharmaceutical composition of any one of claims 8 to 11, wherein administration of said pharmaceutical composition improves the subject's performance in a 6-minute walk test compared to a baseline distance measured before treatment with said pharmaceutical composition.

19. 19. The pharmaceutical composition of claim 17 or 18, wherein the mean change from baseline in a 6-minute walk test between subjects receiving the pharmaceutical composition and subjects not receiving the pharmaceutical composition is at least 10 m.

20. 19. The pharmaceutical composition of claim 17 or 18, wherein the mean change from baseline in a 6-minute walk test between subjects receiving the pharmaceutical composition and subjects not receiving the pharmaceutical composition is at least 20 m.

21. 19. The pharmaceutical composition of claim 17 or 18, wherein the mean change from baseline in a 6-minute walk test between subjects receiving the pharmaceutical composition and subjects not receiving the pharmaceutical composition is at least 30 m.

22. 20. The pharmaceutical composition of claim 18, wherein the subject walks at least 25 meters further than the baseline distance measured before treatment with the pharmaceutical composition.

23. The pharmaceutical composition according to any one of claims 8 to 11, wherein the subject maintains approximately the same 6-minute walking distance as before treatment with the pharmaceutical composition.

24. The pharmaceutical composition according to any one of claims 8 to 11, wherein administration of the pharmaceutical composition slows the progression of ATTR cardiomyopathy.

25. 12. The pharmaceutical composition according to any one of claims 8 to 11, wherein administration of the pharmaceutical composition in a subject with ATTR cardiomyopathy reduces blood serum levels of brain natriuretic peptide (BNP) and / or N-terminal pro-brain natriuretic peptide (N-terminal pro-BNP).

26. 12. The pharmaceutical composition of any one of claims 8 to 11, wherein administration of the pharmaceutical composition to a subject with ATTR cardiomyopathy reduces the frequency of cardiovascular-related hospitalizations compared to a subject not receiving the pharmaceutical composition.

27. The pharmaceutical composition of any one of claims 8 to 11, wherein administration of the pharmaceutical composition to a subject with ATTR cardiomyopathy reduces mortality compared to a subject not receiving the pharmaceutical composition.

28. 16. The pharmaceutical composition of any one of claims 12 to 15, wherein administration of the pharmaceutical composition in a subject with ATTR polyneuropathy improves the Neuropathy Score (NIS) or modified Neuropathy Score+7 (mNIS+7) in the subject.

29. 29. The pharmaceutical composition of claim 28, wherein the NIS score or mNIS+7 score is reduced by at least 10% compared to the baseline level measured before treatment with the pharmaceutical composition.

30. 16. The pharmaceutical composition of any one of claims 12 to 15, wherein administration of the pharmaceutical composition in a subject with ATTR polyneuropathy improves the subject's Norfolk Quality of Life Diabetic Neuropathy (QOL-DN) questionnaire score and / or Composite Autonomic Symptoms Score (COMPASS-31) score.

31. 31. The pharmaceutical composition of claim 30, wherein the Norfolk QOL-DN score is improved by at least 10% compared to the baseline level measured before treatment with the pharmaceutical composition.

32. 31. The pharmaceutical composition of claim 30, wherein the COMPASS-31 score is improved by at least 0.5 points compared to the baseline level measured before treatment with the pharmaceutical composition.

33. 16. The pharmaceutical composition of any one of claims 12 to 15, wherein administration of said pharmaceutical composition in a subject with ATTR polyneuropathy improves the subject's corrected body mass index (mBMI).

34. 16. The pharmaceutical composition of any one of claims 12 to 15, wherein administration of said pharmaceutical composition in a subject with ATTR polyneuropathy improves the subject's 10 meter walk test speed.

35. The pharmaceutical composition according to any one of claims 1 to 34, wherein the pharmaceutical composition is administered chronically.

36. The pharmaceutical composition according to any one of claims 1 to 34, wherein the pharmaceutical composition is administered for 28 days.

37. The pharmaceutical composition of any one of claims 1 to 36, wherein the subject is receiving a diuretic therapeutic agent.

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