COMPOUNDS AND THEIR USE FOR THE TREATMENT OF ALPHA1-ANTITRYPSIN DEFICIENCY

Abstract

The invention relates to specified carboxylic acid compounds of formula (1), and pharmaceutical compositions containing the compounds. The compounds may be inducers of α.sub.1-antitrypsin (A1AT) and may be used in the treatment of a disease or disorder such as α.sub.1-antitrypsin deficiency (A1AD or AATD).

Claims

1.-3. (canceled)

4. A pharmaceutical composition comprising a compound represented by the structure: ##STR00004## ##STR00005## or a pharmaceutically acceptable salt of any one thereof, and a pharmaceutically acceptable carrier.

5.-9. (canceled)

10. A method of inducing Z A1AT secretion in a subject in need thereof, comprising administering to the subject a compound represented by the structure: ##STR00006## ##STR00007## or a pharmaceutically acceptable salt of any one thereof.

11. (canceled)

12. A method of treating α.sub.1-antitrypsin deficiency (AATD), in a subject in need thereof, comprising administering to the subject a compound represented by the structure: ##STR00008## ##STR00009## or a pharmaceutically acceptable salt of any one thereof.

13.-14. (canceled)

15. The method of claim 10, wherein the method comprises administering a mixture of enantiomers selected from (a), (b), (c), or (d): ##STR00010##

16. The method of claim 10, wherein the compound is represented by: ##STR00011## ##STR00012## or a pharmaceutically acceptable salt of any one thereof.

17. The method of claim 16, wherein the compound is represented by: ##STR00013## or a pharmaceutically acceptable salt of any one thereof.

18. The method of claim 17, wherein the compound is represented by: ##STR00014## or a pharmaceutically acceptable salt thereof.

19. The method of claim 17, wherein the compound is represented by: ##STR00015## or a pharmaceutically acceptable salt thereof.

20. The method of claim 17, wherein the compound is represented by: ##STR00016## or a pharmaceutically acceptable salt thereof.

21. The method of claim 12, wherein the method comprises administering a mixture of enantiomers selected from (a), (b), (c), or (d): ##STR00017##

22. The method of claim 12, wherein the compound is represented by: ##STR00018## ##STR00019## or a pharmaceutically acceptable salt of any one thereof.

23. The method of claim 22, wherein the compound is represented by: ##STR00020## or a pharmaceutically acceptable salt of any one thereof.

24. The method of claim 23, wherein the compound is represented by: ##STR00021## or a pharmaceutically acceptable salt thereof.

25. The method of claim 23, wherein the compound is represented by: ##STR00022## or a pharmaceutically acceptable salt thereof.

26. The method of claim 23, wherein the compound is represented by: ##STR00023## or a pharmaceutically acceptable salt thereof.

Description

[0068] FIG. 1 is a graph showing the effect of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid on Z A1AT levels in mice expressing human Z A1AT (huZ mice). Mice were treated with vehicle, 5, 15 and 50 mg/kg of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid twice a day by oral gavage for 14 consecutive days. Blood was taken on days −12, −7 and −5 and plasma prepared to determine circulating basal levels of human Z A1AT. Plasma samples collected the last three days of the study (days 12, 13 and 14) were used to determine the effect of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid treatment on circulating human Z A1AT levels compared to basal levels. The x-axis is the treatment dose of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid in mg/kg; the y-axis is the mean percentage level of human Z A1AT compared to baseline levels for each treatment group (i.e. A1AT % Baseline).

EXPERIMENTAL

General Method

[0069] Compounds of formula 1 were prepared using the following synthetic procedure.

##STR00002##

[0070] The carboxylic acid (1 equivalent), potassium hydroxide (1 equivalent) and potassium carbonate (2 equivalents) were added to water and stirred. The sulfonyl chloride (1 equivalent) was added and the reaction was stirred at room temperature for 3 hours. The reaction was cooled to 0° C. and acidified with 2M hydrochloric acid to give a white precipitate. This precipitate was dried and triturated with n-pentane to give the compound of formula (1).

Example 1: 1-(Quinolin-8-ylsulfonyl)piperidine-4-carboxylic acid

[0071] The compound of Example 1 was prepared using the general method and 1-quinolin-8-ylsulfonylchloride and piperidine-4-carboxylic acid.

[0072] .sup.1H NMR (400 MHz, d6 DMSO) δ 12.26 (1H, s), 9.07 (OH, s), 8.54 (1H, d), 8.36 (1H, d), 8.30 (1H, d), 7.74 (1H, m), 7.70 (OH, m), 3.81 (2H, m), 2.82 (2H, m), 2.31 (1H, m), 1.82 (2H, m), 1.44 (0H, m).

Example 2: 1-((2-Chlorophenyl)sulfonyl)piperidine-4-carboxylic acid

[0073] The compound of Example 2 was prepared using the general method and 1-(2-chlorophenyl)sulfonylchloride and piperidine-4-carboxylic acid.

[0074] .sup.1H NMR (400 MHz, d6 DMSO) δ 12.38 (1H, br s), 7.97 (1H, d), 7.88 (2H, m), 7.56 (1H, m), 3.60 (2H, m), 2.83 (2H, t), 2.40 (1H, m), 1.86 (2H, m), 1.48 (2H, m).

Example 3: 1-((3-Chlorophenyl)sulfonyl)piperidine-4-carboxylic acid

[0075] The compound of Example 3 was prepared using the general method and 1-(3-chlorophenyl)sulfonylchloride and piperidine-4-carboxylic acid.

[0076] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.77 (1H, s), 7.76 (1H, d), 7.63 (1H, m), 7.60 (1H, m), 3.68 (2H, m), 2.55 (2H, t), 2.37 (1H, m), 2.02 (2H, m), 1.86 (2H, m).

Example 4: 1-((2,3-Dichlorophenyl)sulfonyl)piperidine-4-carboxylic acid

[0077] The compound of Example 4 was prepared using the general method and 1-(2,3-dichlorophenyl)sulfonylchloride and piperidine-4-carboxylic acid.

[0078] .sup.1H NMR (400 MHz, d6 DMSO) δ 12.36 (1H, br s), 7.97 (2H, d), 7.59 (1H, m), 3.64 (2H, m), 2.90 (2H, t), 2.43 (1H, m), 1.80 (2H, m), 1.54 (2H, m).

Example 5: (S)-1-((3-Fluorophenyl)sulfonyl)piperidine-3-carboxylic acid

[0079] The compound of Example 5 was prepared using the general method and 1-(3-fluorophenyl)sulfonylchloride and (S)-piperidine-3-carboxylic acid.

[0080] .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.52 (2H, m), 7.48 (1H, m), 7.44 (1H, m), 3.73 (1H, d), 3.53 (1H, d), 2.60 (3H, m), 1.97 (1H, m), 1.81 (1H, m), 1.59 (1H, m), 1.50 (1H, m).

Example 6: (S)-1-((3-Chlorophenyl)sulfonyl)piperidine-3-carboxylic acid

[0081] The compound of Example 6 was prepared using the general method and 1-(3-chlorophenyl)sulfonylchloride and (S)-piperidine-3-carboxylic acid.

[0082] .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.79 (1H, s), 7.68 (2H, m), 7.62 (1H, m), 3.72 (1H, d), 3.51 (1H, d), 2.60 (3H, m), 1.97 (1H, m), 1.81 (1H, m), 1.58 (1H, m), 1.51 (1H, m).

Example 7: (R)-1-((3-Fluorophenyl)sulfonyl)piperidine-3-carboxylic acid

[0083] The compound of Example 7 was prepared using the general method and 1-(3-fluorophenyl)sulfonylchloride and (R)-piperidine-3-carboxylic acid.

[0084] .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.52 (2H, m), 7.48 (1H, m), 7.44 (1H, m), 3.73 (1H, d), 3.53 (1H, d), 2.60 (3H, m), 1.97 (1H, m), 1.81 (1H, m), 1.59 (1H, m), 1.50 (1H, m).

Example 8: (R)-1-((3-Chlorophenyl)sulfonyl)piperidine-3-carboxylic acid

[0085] The compound of Example 8 was prepared using the general method and 1-(3-chlorophenyl)sulfonylchloride and (R)-piperidine-3-carboxylic acid.

[0086] .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.79 (1H, s), 7.68 (2H, m), 7.62 (1H, m), 3.73 (1H, d), 3.53 (1H, d), 2.60 (3H, m), 1.97 (1H, m), 1.81 (1H, m), 1.59 (1H, m), 1.50 (1H, m).

Example 9: 1-((4-Chlorophenyl)sulfonyl)piperidine-4-carboxylic acid

[0087] The compound of Example 9 was prepared using the general method and 1-(4-chlorophenyl)sulfonylchloride and piperidine-4-carboxylic acid.

[0088] .sup.1H NMR (400 MHz, d6 DMSO) δ 12.32 (1H, s), 7.73 (4H, m), 3.47 (2H, m), 2.44 (2H, m), 2.28 (1H, m), 1.86 (2H, m), 1.57 (2H, m).

Example 10: 1-((2-(Trifluoromethyl)phenyl)sulfonyl)piperidine-4-carboxylic acid

[0089] The compound of Example 10 was prepared using the general method and 1-(2-(trifluoromethyl)phenyl)sulfonylchloride and piperidine-4-carboxylic acid.

[0090] .sup.1H NMR (400 MHz, d6 DMSO) δ 12.36 (1H, s), 8.03 (2H, m), 7.90 (2H, m), 3.61 (2H, m), 2.85 (2H, m), 2.41 (1H, m), 1.90 (2H, m), 1.55 (2H, m).

Example 11: 1-((3-(Trifluoromethyl)phenyl)sulfonyl)piperidine-4-carboxylic acid

[0091] The compound of Example 11 was prepared using the general method and 1-(3-(trifluoromethyl)phenyl)sulfonylchloride and piperidine-4-carboxylic acid.

[0092] .sup.1H NMR (400 MHz, d6 DMSO) δ 12.33 (1H, s), 8.13 (1H, m), 8.07 (1H, m), 7.95 (1H, m), 7.90 (1H, m), 3.53 (2H, m), 2.45 (2H, m), 2.32 (1H, m), 1.89 (2H, m), 1.57 (2H, m).

Example 12: 1-((4-(Trifluoromethyl)phenyl)sulfonyl)piperidine-4-carboxylic add

[0093] The compound of Example 12 was prepared using the general method and 1-(4-(trifluoromethyl)phenyl)sulfonylchloride and piperidine-4-carboxylic acid.

[0094] .sup.1H NMR (400 MHz, d6 DMSO) δ 8.00 (2H, m), 7.92 (2H, m), 3.20 (2H, m), 2.54 (2H, m), 1.70 (3H, m), 1.57 (2H, m).

Example 13: 1-((2,5-Bis(trifluoromethyl)phenyl)sulfonyl)piperidine-4-carboxylic acid

[0095] The compound of Example 13 was prepared using the general method and 1-(2,5-bis(trifluoromethyl)phenyl)sulfonylchloride and piperidine-4-carboxylic acid.

[0096] .sup.1H NMR (400 MHz, d6 DMSO) δ 12.36 (1H, s), 8.30 (2H, m), 8.24 (1H, s), 3.65 (2H, m), 2.87 (2H, m), 2.43 (1H, m), 1.89 (2H, m), 1.54 (2H, m).

Example 14: 1-((2-(Trifluoromethoxy)phenyl)sulfonyl)piperidine-4-carboxylic acid

[0097] The compound of Example 14 was prepared using the general method and 1-(2-(trifluoromethoxy)phenyl)sulfonylchloride and piperidine-4-carboxylic acid.

[0098] .sup.1H NMR (400 MHz, d6 DMSO) δ 12.33 (1H, s), 7.93 (1H, m), 7.83 (1H, m), 7.62 (2H, m), 3.56 (2H, m), 2.71 (2H, m), 2.36 (1H, m), 1.87 (2H, m), 1.52 (2H, m).

Example 15: (S)-1-((2-(Trinfluoromethyl)phenyl)sulfonyl)pyrrolidine-3-carboxylic acid

[0099] The compound of Example 15 was prepared using the general method and 1-(2-(trifluoromethyl)phenyl)sulfonylchloride and (S)-pyrrolidine-3-carboxylic acid.

[0100] .sup.1H NMR (400 MHz, d6 DMSO) δ 12.62 (1H, br s), 8.05 (2H, m), 7.90 (2H, m), 3.48 (2H, m), 3.38 (2H, m), 3.15 (1H, m), 2.11 (2H, m).

Example 16: (R)-1-((2-(Trifluoromethyl)phenyl)sulfonyl)pyrrolidine-3-carboxylic acid

[0101] The compound of Example 16 was prepared using the general method and 1-(2-(trifluoromethyl)phenyl)sulfonylchloride and (R)-pyrrolidine-3-carboxylic acid

[0102] .sup.1H NMR (400 MHz, d6 DMSO) δ 12.62 (1H, br s), 8.05 (2H, m), 7.90 (2H, m), 3.48 (2H, m), 3.38 (2H, m), 3.15 (1H, m), 2.11 (2H, m).

Example 17: (S)-1-((2-(Trinfluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid

[0103] (S)-1-((2-(Trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid was prepared using the following synthesis procedure.

##STR00003##

[0104] (S)-Piperidine-3-carboxylic acid (1 g, 7.7 mmol), potassium hydroxide (434 mg, 7.7 mmol) and potassium carbonate (2.14 g, 15.4 mmol) were added to water (20 ml) and stirred. 2-(Trifluoromethyl)benzenesulfonyl chloride (1.89 g, 7.7 mmol) was added and the reaction was stirred at room temperature for 3 hours. The reaction was cooled to 0° C. and acidified with 2M hydrochloric acid to give a white precipitate. This precipitate was dried and triturated with n-pentane to give (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid.

[0105] Tlc Rf 0.3 70% ethyl acetate in hexane.

[0106] m/z: 337.98 (calc 338.03)

[0107] .sup.1H NMR (400 MHz, d6 DMSO) δ 12.33 (1H, s), 8.04 (2H, m), 7.90 (2H, m), 3.69 (1H, dd), 3.50 (1H, dd), 2.93 (1H, m), 2.81 (1H, m), 1.91 (1H, m), 1.72 (1H, m), 1.50 (2H, m).

Example 18: (R)-1-((2-(Trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid

[0108] (R)-1-((2-(Trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid was prepared in the same manner as (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid, but using (R)-piperidine-3-carboxylic acid.

[0109] Tlc Rf 0.3 70% ethyl acetate in hexane.

[0110] m/z: 338.03 (calc 338.03)

[0111] 1H NMR (400 MHz, d6 DMSO) δ 12.53 (1H, s), 8.04 (2H, m), 7.90 (2H, m), 3.69 (1H, dd), 3.49 (1H, dd), 2.93 (1H, m), 2.81 (1H, m), 1.90 (1H, m), 1.72 (1H, m), 1.49 (2H, m).

Example 19: Activity of the Compounds of Examples 1-18 in an A1AT Cell Secretion Assay Using HEK-Z Cells

Methods

[0112] HEK-Z cells, a human embryonic kidney cell line stably transfected with the human Z A1AT gene, were plated into 96 well plates (3.0×10.sup.5 cells/ml with 200 μl of media/well) overnight at 37° C. in a humidified atmosphere containing 5% CO.sub.2. Following incubation cells were washed with 200 μl serum-free media three times and media was replaced with treatments in quadruplicate using serum-free media containing vehicle, 10 μM suberanilohydroxamic acid (SAHA) or the compounds of Examples 1-18 (at concentrations of 10, 33, 100 and 333 nM) for 48 h in a 37° C. incubator in a final volume of 200 μl. At the end of the incubation step the supernatants were removed from the wells, centrifuged at 1000×g at 4° C. for 10 min and were assayed for human A1AT levels by ELISA (Human Serpin A1/α.sub.1-antitrypsin duo set ELISA, R& D Systems, DY1268) per manufacturer's instructions.

[0113] Briefly, a 96 well plate was coated with human A1AT capture antibody overnight at room temperature (1:180 dilution from stock, 100 μl final volume/well). The capture antibody was then removed and wells washed three times with 300 μl wash buffer (0.05% Tween 20 in PBS) and then 200 μl reagent diluent (25% Tween 20 in PBS) was incubated in each well for 1 h at room temperature. Diluted samples, standards (125, 250, 500, 1000, 2000, 4000 and 8000 pg/ml A1AT) or blanks were then added to each well in duplicate and the plates were covered with a plate sealer and left at room temperature for 2 h. At the end of the sample incubation step, samples were removed and all wells washed as previously and 100 μl detection antibody (1:180 dilution from stock) was added to each well and incubated for a further 2 h at room temperature. Following incubation with detection antibody, supernatant was removed and wells were washed as previously and 100 μl streptavidin-HRP solution (1:200 dilution from stock) was added to each well for 20 min in the dark. After which, 50 μl stop solution (2M H.sub.2SO.sub.4) was added and optical density (OD) of each well was read at 450 nm with 570 nm blank subtracted from each well using a microplate reader. A 4 parameter logistic curve was constructed using GraphPad Prism 7 and A1AT concentrations were determined in each sample by interpolation from a standard curve and multiplying by the appropriate dilution factor.

Results

[0114] The amount of human A1AT secreted from transfected HEK-EBNA cells into the media was measured by ELISA. SAHA at 10 μM was used a positive control for all in vitro A1AT secretion experiments.

[0115] The data in Table 1 show that the compounds of Examples 1-18 increase the secretion of human Z A1AT from HEK-Z cells in a dose dependent manner as measured by ELISA.

TABLE-US-00001 TABLE 1 Median A1AT increase over vehicle at Example 300 nM 1 247 2 235 3 166 4 147 5 220 6 240 7 280 8 260 9 200 10 235 11 215 12 200 13 213 14 227 15 230 16 200 17 310 18 200

Example 20: Activity of the Compounds of Examples 1-18 in an A1AT Cell Secretion Assay Using HEK-M Cells

Methods

[0116] HEK-M cells, a human embryonic kidney cell line stably transfected with M A1AT, were plated into 96 well plates (3.0×10.sup.5 cells/ml with 200 μl of media/well) overnight at 37° C. in a humidified atmosphere containing 5% CO.sub.2. Following incubation cells were washed with 200 μl serum-free media three times and media was replaced with serum-free media containing vehicle, 10 μM suberanilohydroxamic acid (SAHA) or a compound of Examples 1-16 in replicates of six for 48 h in a 37° C. incubator in a final volume of 200 μl. At the end of the incubation step the supernatants were removed from the wells, centrifuged at 1000×g at 4° C. for 10 min and were assayed for human A1AT levels by ELISA (Human Serpin A1/α.sub.1 antitrypsin duo set ELISA, R& D Systems, DY1268) per manufacturer's instructions.

[0117] Briefly, a 96 well plate was coated with human A1AT capture antibody overnight at room temperature (1:180 dilution from stock, 100 μl final volume/well). The capture antibody was then removed and wells washed three times with 300 μl wash buffer (0.05% Tween 20 in PBS) and then 200 μl reagent diluent (25% Tween 20 in PBS) was incubated in each well for 1 h at room temperature. Diluted samples, standards (125, 250, 500, 1000, 2000, 4000 and 8000 pg/ml A1AT) or blanks were then added to each well in duplicate and the plates were covered with a plate sealer and left at room temperature for 2 h. At the end of the sample incubation step, samples were removed and all wells washed as previously and 100 μl detection antibody (1:180 dilution from stock) was added to each well and incubated for a further 2 h at room temperature. Following incubation with detection antibody, supernatant was removed and wells were washed as previously and 100 μl streptavidin-HRP solution (1:200 dilution from stock) was added to each well for 20 min in the dark. After which, 50 μl stop solution (2M H.sub.2SO.sub.4) was added and optical density (OD) of each well was read at 450 nm with 570 nm blank subtracted from each well using a microplate reader. A 4 parameter logistic curve was constructed using GraphPad Prism 7 and A1AT concentrations were determined in each sample by interpolation from a standard curve and multiplying by the appropriate dilution factor.

Results

[0118] The amount of human M A1AT secreted from transfected HEK-EBNA cells into the media was measured by ELISA. SAHA at 10 μM was used a positive control for all in vitro A1AT secretion experiments. The compounds of Examples 1, 3, 4, 10, 17 and 18 did not lead to an increase in secretion of human M A1AT from HEK-M cells at 10 μM.

Example 21: Activity of the Compounds of Examples 1 and 17 in an A1AT Cell Secretion Assay Using HEK-Siiyama Cells

[0119] The rare Siiyama mutation (Ser 53 to Phe, mature A1AT numbering) was identified in a Japanese male with AATD (Seyama et al J Biol Chem (1991) 266:12627-32). Ser53 is one the conserved serpin residues and is thought to be important for the organization of the internal core of the A1AT molecule. The change from an uncharged polar to a large nonpolar amino acid on the conserved backbone of the protein affects the folding and intracellular processing of Siiyama A1AT.

Methods

[0120] HEK-Siiyama cells, a human embryonic kidney cell line stably transfected with the human Siiyama A1AT gene, were plated into 96 well plates (3.0×10.sup.5 cells/ml with 200 μl of media/well) overnight at 37° C. in a humidified atmosphere containing 5% CO.sub.2. Following incubation cells were washed with 200 μl serum-free media three times and media was replaced with serum-free media containing vehicle, 10 μM suberanilohydroxamic acid (SAHA) or a compound of Example 1 (at 1 and 10 μM) in replicates of eight for 48 h in a 37° C. incubator in a final volume of 200 μl. At the end of the incubation step the supernatants were removed from the wells, centrifuged at 1000×g at 4° C. for 10 min and were assayed for human A1AT levels by ELISA (Human Serpin A1/α.sub.1-antitrypsin duo set ELISA, R& D Systems, DY1268) per manufacturer's instructions.

[0121] Briefly, a 96 well plate was coated with human A1AT capture antibody overnight at room temperature (1:180 dilution from stock, 100 μl final volume/well). The capture antibody was then removed and wells washed three times with 300 μl wash buffer (0.05% Tween 20 in PBS) and then 200 μl reagent diluent (25% Tween 20 in PBS) was incubated in each well for 1 h at room temperature. Diluted samples, standards (125, 250, 500, 1000, 2000, 4000 and 8000 pg/ml A1AT) or blanks were then added to each well in duplicate and the plates were covered with a plate sealer and left at room temperature for 2 h. At the end of the sample incubation step, samples were removed and all wells washed as previously and 100 μl detection antibody (1:180 dilution from stock) was added to each well and incubated for a further 2 h at room temperature. Following incubation with detection antibody, supernatant was removed and wells were washed as previously and 100 μl streptavidin-HRP solution (1:200 dilution from stock) was added to each well for 20 min in the dark. After which, 50 μl stop solution (2M H.sub.2SO.sub.4) was added and optical density (OD) of each well was read at 450 nm with 570 nm blank subtracted from each well using a microplate reader. A 4 parameter logistic curve was constructed using GraphPad Prism 7 and A1AT concentrations were determined in each sample by interpolation from a standard curve and multiplying by the appropriate dilution factor.

Results

[0122] The amount of human Siiyama A1AT secreted from transfected HEK-EBNA cells into the media was measured by ELISA. SAHA at 10 μM was used a positive control for all in vitro A1AT human secretion experiments. The exemplar compounds of Example 1 and 17 did not stimulate secretion of Siiyama A1AT from HEK-Siiyama cells at 1 or 10 μM, as measured by ELISA. In contrast, the positive control 10 μM SAHA stimulated an increase in Siiyama A1AT secretion.

Example 22: Activity of the Compounds of Examples 1 and 17 in a Mouse Expressing Human Z (huZ Mouse)

[0123] The huZ mouse (also referred to as the PiZZ mouse) is a transgenic mouse strain that contains multiple copies of the Z variant of the human A1AT gene, developed by two separate groups (Dycaico et al Science (1988) 242:1409-12) and Carlson et al J. Clin Invest (1989) 83:1183-90). HuZ mice are on a C57Bl/6 background and express the human Z A1AT protein in liver tissue. The mice used in this study are from the progeny of Carlson and colleagues (transgenic line Z11.03). HuZ mice have been used as a tool to assess the effects of an exemplar compound of the invention on either increasing the circulating levels of Z A1AT in plasma or the effects of compound on the accumulation of Z A1AT polymers in the liver and associated liver pathology.

[0124] HuZ mice (n=4/group; male or female) with basal human Z A1AT plasma levels of between 200-600 μg/ml were treated with either vehicle or the compounds of Examples 1 or 17 at 5, 15 or 50 mg/kg twice a day by oral gavage for 14 consecutive days. Mice had access to food (standard mouse chow, SAFE diets) and water ad libitum. On study day 14, each mouse was dosed one hour prior to terminal procedures. Blood was taken from each mouse from the tail vein on pre-dosing days −12, −7 and −5, and dosing days 12, 13 and 14. Blood was collected into microvettes containing EDTA and plasma was prepared by centrifugation at 2700×g at 4° C. for 10 min. Plasma was aliquoted and stored at −80° C. for bioanalysis. Plasma samples from pre-dosing days −12, −7 and −5 were used for to determine mean basal levels of human Z A1AT for each mouse. Plasma samples collected on the last three dosing days of the study (days 12, 13 and 14) were used to determine the effect of the compound of Example 1 or 17 on human Z A1AT secretion by measuring human Z A1AT levels and comparing to basal levels for each mouse. Human Z A1AT levels in mouse plasma samples were measured by ELISA (Human Serpin A1/α.sub.1 antitrypsin duo set ELISA, R& D Systems, DY1268) per manufacturer's instructions.

[0125] Briefly, a 96 well plate was coated with human A1AT capture antibody overnight at room temperature (1:180 dilution from stock, 100 μl final volume/well). The capture antibody was then removed and wells washed three times with 300 μl wash buffer (0.05% Tween 20 in PBS) and then 200 μl reagent diluent (25% Tween 20 in PBS) was incubated in each well for 1 h at room temperature. Diluted samples, standards (125, 250, 500, 1000, 2000, 4000 and 8000 pg/ml A1AT) or blanks were then added to each well in duplicate and the plates were covered with a plate sealer and left at room temperature for 2 h. At the end of the sample incubation step, samples were removed and all wells washed as previously and 100 μl detection antibody (1:180 dilution from stock) was added to each well and incubated for a further 2 h at room temperature. Following incubation with detection antibody, supernatant was removed and wells were washed as previously and 100 μl streptavidin-HRP solution (1:200 dilution from stock) was added to each well for 20 min in the dark. After which, 50 μl stop solution (2M H.sub.2SO.sub.4) was added and optical density (OD) of each well was read at 450 nm with 570 nm blank subtracted from each well using a microplate reader. A 4 parameter logistic curve was constructed using GraphPad Prism 7 and A1AT concentrations were determined in each sample by interpolation from a standard curve and multiplying by the appropriate dilution factor.

Results

[0126] The effect of the compound of Example 1 or 17 on circulating levels of human Z A1AT was assessed in the huZ mouse model.

[0127] The compounds of Example 1 and 17 stimulated secretion of human Z A1AT compared to baseline levels in huZ mice. FIG. 1 shows the data at each treatment dose for the compound of Example 17.