Inhibitor of z-alpha1-antitrypsin polymerization to treat alpha-1-antitrypsin (A1AT) deficiency in specific patient populations

Abstract

Methods of treating AATD liver disease using N-(1-(2-chloro-3-fluorophenyl)-1-hydroxypentan-2-yl)-7-fluoro-2-oxoindoline-4-carboxamide and methods of identifying subjects likely to respond to treatment based on altered proteomic profile.

Description

Attorney Ref. No. 11808-584-228SMALL MOLECULES TO TREAT ALPHA- 1 -ANTITRYPSIN (A1AT) DEFICIENCY IN SPECIFIC PATIENT POPULATIONS1. CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 720,669, filed November 14, 2024, and U.S. Provisional Patent Application No.63 / 819,434, filed June 6, 2025, the disclosure of each of which is incorporated by reference herein in its entirety.2. SEQUENCE LISTING

[0002] This application contains an electronic Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “11808-584-228_SEQ_LISTING.xml”, was created on November 11, 2025, and is 5,165 bytes in size.3. FIELD OF THE INVENTION

[0003] The present disclosure relates generally to methods of using N-(l-(2-chloro-3-fluorophenyl)-l-hydroxypentan-2-yl)-7-fluoro-2-oxoindoline-4-carboxamide (Compound 1) to treat alpha- 1 antitrypsin deficiency (AATD) liver disease in subjects with altered expression levels of certain proteins.4. BACKGROUND

[0004] Alpha- 1 antitrypsin deficiency (AATD) is caused by mutations in the SERPINA1 gene which encodes alpha-1 antitrypsin (A1AT), a serine protease inhibitor secreted from the liver into the bloodstream (see, e.g., Seixas S et al., Appl Clin Genet., 2021, 14:173-94).

[0005] The normal allele of SERPINA1 is the “M” allele (PiM). The “Z” allele (PiZ) contains the E342K mutation that produces the “Z” form of AAT (Z-AAT) (see, e.g., Seixas et al. and Lomas DA et al., Thorax., 2004, 59:529-35.)

[0006] Z-AAT monomers form toxic polymers that accumulate in the liver and induce intracellular injury cascades that lead to fibrosis (see, e.g., Karatas E. et al., Eur Med J., 2019, 7:65-79; and Schneider CV et al., Gastroenterol., 2020, 159:534-48).

[0007] Currently, there are limited non-invasive biomarkers for AATD liver disease. Circulating biomarkers could assist in the identification of liver disease and broaden our1NAI-5005346498vlunderstanding of disease progression and response to treatment, such as treatment with N-(l-(2-chloro-3-fluorophenyl)-l-hydroxypentan-2-yl)-7-fluoro-2-oxoindoline-4-carboxamide, as described, e.g., in US Patent No. 11,919,856 B2 or PCT / US2024 / 044680, each of which is incorporated herein by reference in its entirety.5. SUMMARY

[0008] As disclosed herein, certain biomarkers (e.g, Coagulation Factor VII (FVII), Programmed Cell Death 6 (PDCD6), certain apolipoproteins, and others that are described herein) have been identified as having significantly altered expression levels in subjects with AATD, such as those with a PiZ or PiM genotype. As disclosed herein, these biomarkers can serve as a basis to provide a less invasive and more comprehensive method for identifying subjects having AATD liver disease (e.g, as opposed to genotyping or just measuring Al AT levels in the blood) and serve as a predictor of AATD subjects that could be effectively treated with the compounds, e.g., Compound 1-A, disclosed herein.

[0009] Accordingly, in one aspect, the present disclosure provides a method of treating a subject having alpha- 1 antitrypsin deficiency (AATD) liver disease, the method comprising:a. identifying a subject as having an altered blood protein expression level of one or more of Coagulation Factor VII (FVII), Programmed Cell Death 6 (PDCD6), apolipoprotein C-l (APOCI), apolipoprotein C-III (APOC3), and N-terminal pro-peptide of type III collagen (PRO-C3), relative to a control; andb. administering to the subject a therapeutically effective amount of Compound 1-A:

[0010] In certain embodiments, the method may comprise identifying a subject as having an altered blood protein expression level of one or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3, relative to a control.2NAI-5005346498vl

[0011] In another aspect, the present disclosure provides a method of treating a subject having alpha- 1 antitrypsin deficiency (AATD) liver disease, the method comprising administering to the subject a therapeutically effective amount of Compound 1-A:wherein the subject has been identified as having an altered blood protein expression level of one or more of Coagulation Factor VII (FVII), Programmed Cell Death 6 (PDCD6), apolipoprotein C-l (APOCI), apolipoprotein C-III (APOC3), and N-terminal pro-peptide of type III collagen (PRO-C3), relative to a control.

[0012] In certain embodiment, the subject has been identified as having an altered blood protein expression level of one or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3, relative to a control.

[0013] In another aspect, the present disclosure provides a method of treating a subject having alpha- 1 antitrypsin deficiency (AATD) liver disease, the method comprising:a. selecting a subject who has been determined as having an altered blood protein expression level of one or more of Coagulation Factor VII (FVII), Programmed Cell Death 6 (PDCD6), apolipoprotein C-l (APOCI), apolipoprotein C-III (APOC3), and N-terminal pro-peptide of type III collagen (PRO-C3), relative to a control; andb. administering to the subject a therapeutically effective amount of Compound 1-A:

[0014] In certain embodiment, the method comprises selecting a subject who has been determined as having an altered blood protein expression level of one or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3, relative to a control.3NAI-5005346498vl

[0015] In another aspect, the present disclosure provides a method of treating a subject having alpha- 1 antitrypsin deficiency (AATD) liver disease, the method comprising administering a therapeutically effective amount of Compound 1-A:wherein the subject has been selected based on an altered blood protein expression level of one or more of Coagulation Factor VII (FVII), Programmed Cell Death 6 (PDCD6), apolipoprotein C-l (APOCI), apolipoprotein C-III (APOC3), and N-terminal pro-peptide of type III collagen (PRO-C3), relative to a control.

[0016] In certain embodiments, the subject has been selected based on an altered blood protein expression level of one or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3, relative to a control.

[0017] In another aspect, the present disclosure provides a method of treating a subject identified as having alpha- 1 antitrypsin deficiency (AATD) liver disease, the method comprising:a. determining a blood protein expression level of one or more of Coagulation Factor VII (FVII), Programmed Cell Death 6 (PDCD6), apolipoprotein C-l (APOCI), apolipoprotein C-III (APOC3), and N- terminal pro-peptide of type III collagen (PRO-C3) in a sample obtained from the subject;b. comparing the blood protein expression level of the one or more of FVII, PDCD6, APOCI, APOC3, and PRO-C3 to a control, wherein an alteration in the blood protein expression level of the one or more of FVII, PDCD6, APOCI, APOC3, and PRO-C3 in the subject sample relative to the control identifies the subject as having the AATD liver disease; andc. administering to the subject a therapeutically effective amount of Compound 1-A:4NAI-5005346498vl

[0018] In certain embodiments, blood protein expression level of one or more of F VII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3 is determined in a sample obtained from the subject, which is compared to the blood protein expression level of the one or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3 in a control, wherein an alteration in the blood protein expression level of the one or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3 in the subject sample relative to the control identifies the subject as having the AATD liver disease.

[0019] In another aspect, the present disclosure provides a method of treating a subject having alpha- 1 antitrypsin deficiency (AATD) liver disease, the method comprising administering to the subject a therapeutically effective amount of Compound 1-A:wherein the subject has been determined to have an altered blood protein expression level of one or more of Coagulation Factor VII (FVII), Programmed Cell Death 6 (PDCD6), apolipoprotein C-l (APOCI), apolipoprotein C-III (APOC3), and N-terminal pro-peptide of type III collagen (PRO-C3) compared to the blood protein expression level of the one or more of FVII, PDCD6, APOCI, APOC3, and PRO-C3 in a control.

[0020] In certain embodiments, the subject has been determined to have an altered blood protein expression level of one or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3, compared to a control.

[0021] In another aspect, the present disclosure provides a method for identifying a subject having alpha- 1 antitrypsin deficiency (AATD) liver disease who is (a) likely to, or (b) less likely to, respond to treatment with Compound 1-A:5NAI-5005346498vlwherein the method comprises determining whether the subject has (a) an altered blood protein expression level or (b) an unaltered blood protein expression level of one or more of Coagulation Factor VII (FVII), Programmed Cell Death 6 (PDCD6), apolipoprotein C-l (APOCI), apolipoprotein C-III (APOC3), and PRO-C3, as compared to a control, wherein the altered blood protein expression level identifies that the subject is (a) likely to respond to treatment with Compound 1-A, and the unaltered blood protein expression level indicates that the subject is (b) less likely to respond to treatment with Compound 1-A.

[0022] In certain embodiments, the method comprises determining whether the subject has (a) an altered blood protein expression level or (b) an unaltered blood protein expression level of one or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3, as compared to a control, wherein the altered blood protein expression level identifies that the subject is (a) likely to respond to treatment with Compound 1-A, and the unaltered blood protein expression level indicates that the subject is (b) less likely to respond to treatment with Compound 1-A.

[0023] In certain embodiments of any of the aforementioned methods, wherein the blood protein expression level of FVII, PDC6, APOCI, M30, M65, and APOC3 is measured using shotgun liquid chromatography -tandem mass spectrometry and wherein the blood protein expression level of PRO-C3 is measured by enzyme-linked immunosorbent assay (ELISA).

[0024] In certain embodiments, the altered blood protein expression level is of two or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3. In certain embodiments, the altered blood protein expression level is of three or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3. In certain embodiments, the altered blood protein expression level is of four or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PROCS. In certain embodiments, the altered blood protein expression level is of five or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3. In certain embodiments, the altered blood protein expression level is of six or each of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3. In certain embodiments, the altered blood protein expression level is of each of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3.6NAI-5005346498vl

[0025] In certain embodiments, the altered blood protein expression level of one or more of APOCI, APOC3, and FVII is a decreased blood protein expression level. In certain embodiments, the blood protein expression level is decreased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5 as compared to the control.

[0026] In certain embodiments, the altered blood protein expression level of PDCD6 is an increased blood protein expression level. In certain embodiments, the blood protein expression level is increased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5 as compared to the control.

[0027] In certain embodiments, the altered blood protein expression level of M30 is an increased blood protein expression level. In certain embodiments, the blood protein expression level is increased by a factor of about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, or about 7.5.

[0028] In certain embodiments, the altered blood protein expression level of M65 is an increased blood protein expression level. In certain embodiments, the blood protein expression level is increased by a factor of about 1.5, about 1.75, about 2.0, about 2.25, about 2.5, about 2.75, about 3.0, about 3.25, about 3.5, about 3.75, about 4.0, about 4.25, or about 5.0.

[0029] In another aspect, the present disclosure provides a method of treating a subject having alpha- 1 antitrypsin deficiency (AATD) liver disease, the method comprising administering to the subject a therapeutically effective amount of Compound 1-A,wherein:a. the blood protein expression level of one or more of apolipoprotein C-l (APOCI), apolipoprotein C-III (APOC3), and Coagulation Factor VII (FVII) in the subject is increased following the administering; and / orb. the blood protein expression level of one or both of Programmed Cell Death 6 (PDCD6) and PRO-C3 in the subject is decreased following the administering.7NAI-5005346498vl

[0030] In certain embodiments, the blood expression level of one or more of PDCD6, M30, M65, and PRO-C3 in the subject is decreased following the administering.

[0031] In certain embodiments, the blood protein expression level of APOCI is increased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5. In certain embodiments, the blood protein expression level of APOC3 is increased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5. In certain embodiments, the blood protein expression level of F VII is increased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5. In certain embodiments, the blood protein expression level of PDCD6 is decreased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5. In certain embodiments, the blood protein expression level of PRO-C3 is decreased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5.

[0032] In certain embodiments, the blood protein expression level of M30 is decreased by a factor of about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, or about 7.5. In certain embodiments, the blood protein expression level of M65 is decreased by a factor of about 1.5, about 1.75, about 2.0, about 2.25, about 2.5, about 2.75, about 3.0, about 3.25, about 3.5, about 3.75, about 4.0, about 4.25, or about 5.0.

[0033] In certain embodiments of any of the methods disclosed herein, the control is a sample having protein expression levels as follows: (1) a PRO-C3 blood protein expression level of 6.1 - 14.7 ng / mL, (2) an APOCI blood protein expression level of 0.45 - 0.75 mg / mL, (3) a APOC3 blood protein expression level of 10 - 15 mg / dL, (4) a FVII blood protein expression level of 0.5 - 8.4 ng / mL, and / or (5)a PDCD6 blood protein expression level of 2.2 pg / L, and / or a M30 blood protein expression level of about 95 ng / mL; and / or a M65 blood protein expression level of about 130 ng / mL.

[0034] In certain embodiments of any of the methods disclosed herein, the control is a sample having a M30 blood expression level of about 95 ng / mL. In certain embodiments of any of the methods disclosed herein, the control is a sample having a M65 blood expression level of about 230 ng / mL.8NAI-5005346498vl

[0035] In certain embodiments, the subject has or further has an altered blood protein expression level of one or more of TIMP-1, CO4A6, IGF2, IGF1R, IGFBP3, IGFBP5, LEPR, ICAM1, VC AMI, APOA5, APOC2, APOC4, APOF, APOL1, APOM, PDCD6IP, CHMP4B, VPS4B, PSMD5, PSMD9, PSDM10, OTUD6B, and PSMG1.

[0036] In certain embodiments of any of the methods disclosed herein, the subject has a Pi*Z genotype. In certain embodiments, the subject has a PiMZ genotype. In certain embodiments, the subject has metabolic dysfunction-associated steatohepatitis (MASH). In certain embodiments, the subject has cirrhosis of the liver. In certain embodiments, the subject has hepatitis. In certain embodiments, the subject has a PiZZ genotype.

[0037] In certain embodiments of any of the methods disclosed herein, the administering of the therapeutically effective amount of Compound 1-A to the subject is by oral administration. In certain embodiments, the administering of the therapeutically effective amount of Compound 1-A to the subject is by oral administration of one or more tablets. In certain embodiments, the administering of the therapeutically effective amount of Compound 1-A to the subject is once per day. In certain embodiments, the therapeutically effective amount of Compound 1-A is about 25 mg / day to about 750 mg / day of Compound 1-A. In certain embodiments, the therapeutically effective amount of Compound 1-A is about 25 mg / day to about 250 mg / day of Compound 1-A.6. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG. 1 depicts a graph demonstrating elevated protein levels of biomarker PROCS in subjects having a PiZZ genotype and cirrhosis / hepatitis.

[0039] FIG. 2 depicts protein expression levels of FVII in mice in controls, subjects treated with PiMZ and PiZZ genotypes and subjects with PiZZ genotypes administered 100 mg / kg / day or 200 mg / kg / day of Compound 1-A.

[0040] FIG. 3 depicts protein expression levels of PDCD6 in mice in controls, subjects treated with PiMZ and PiZZ genotypes and subjects with PiZZ genotypes administered 100 mg / kg / day or 200 mg / kg / day of Compound 1-A.

[0041] FIG. 4 depicts protein expression levels of APO1 in mice in controls, subjects treated with PiMZ and PiZZ genotypes and subjects with PiZZ genotypes administered 100 mg / kg / day or 200 mg / kg / day of Compound 1-A.

[0042] FIG. 5 depicts protein expression levels of APO3 in mice in controls, subjects treated with PiMZ and PiZZ genotypes and subjects with PiZZ genotypes administered 100 mg / kg / day or 200 mg / kg / day of Compound 1-A.9NAI-5005346498vl

[0043] FIG. 6 depicts protein expression levels of coagulation factor VII (FVII) in human clinical samples in controls, subjects with PiMZ and PiZZ genotypes, and subjects with PiZZ genotypes with comorbid conditions, such as cirrhosis / hepatitis or other liver diseases.

[0044] FIG. 7 depicts protein expression levels of Programmed Cell Death 6 (PDCD6) in human clinical samples in controls, subjects with PiMZ and PiZZ genotypes, and subjects with PiZZ genotypes with comorbid conditions, such as cirrhosis / hepatitis or other liver diseases.

[0045] FIG. 8 depicts protein expression levels of apolipoprotein C-l (APO1) in human clinical samples in controls, subjects with PiMZ and PiZZ genotypes, and subjects with PiZZ genotypes with comorbid conditions, such as cirrhosis / hepatitis or other liver diseases.

[0046] FIG. 9 depicts protein expression levels of apolipoprotein C-III (APO3) in human clinical samples in controls, subjects with PiMZ and PiZZ genotypes, and subjects with PiZZ genotypes with comorbid conditions, such as cirrhosis / hepatitis or other liver diseases.7. DETAILED DESCRIPTION

[0047] Provided herein are methods of treating AATD liver disease with certain compounds in subjects having an altered blood proteomic profile. The altered proteomic profile may advantageously help diagnose the AATD liver disease in the subject and help predict response to treatment thereof.

[0048] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art of the present disclosure. As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0049] In some embodiments, chemical structures are disclosed with a corresponding chemical name. In case of conflict, the chemical structure controls the meaning, rather than the name.

[0050] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context otherwise, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.

[0051] The term “alkyl” refers to monovalent aliphatic hydrocarbyl groups that lack a double or triple bonds between carbon atoms, and that preferably have from one to six carbon atoms, such as one to 10, one to 6, one to 5, one to 4, or one to three carbon atoms. This term includes, by way of example, linear, branched, and cyclic hydrocarbyl groups such as methyl 10NAI-5005346498vl(-CHs), ethyl (-CH2CH3), n-propyl (-CH2CH2CH3), isopropyl (CH(CH3)2), cyclopropyl, n-butyl (-CH2CH2CH2CH3), isobutyl (-CH2CH(CH3)2), sec-butyl (CH(CH3)(CH2CH3)), t-butyl (-C(CH3)3), n-pentyl (-CH2CH2CH2CH2CH3), and neopentyl (CH2C(CH3)3).

[0052] As used herein and unless otherwise specified, “stereoisomer” refer to a compound that has the same chemical formula and whose atoms have the same connectivity, but which has a different orientation in space. In some cases, stereoisomers can be isolated from mixtures by methods known to those skilled in the art. For analytical purposes, chiral high pressure liquid chromatography (HPLC) can be used, though this method may not provide enough material for industrial or commercial use. In some cases, chiral salts can be separated and crystallized; however, this requires asymmetric conditions that are often impractical for use on a commercial or industrial scale. In particular methods of this disclosure, stereoisomers can be prepared by asymmetric syntheses that provides the desired stereoisomer in a form that is substantially free of other, undesired stereoisomers. Particular compounds as described herein may be present in pharmaceutical compositions as individual stereoisomers that are substantially free of other stereoisomers, or alternatively, as mixtures of various stereoisomers.

[0053] The term “subject” to which administration is contemplated includes, but is not limited to, humans ( / .<?., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult)) and / or other primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, rats, mice, and / or dogs; and / or birds, including commercially relevant birds such as chickens, ducks, geese, quails, and / or turkeys. In certain embodiments, the subject is a human.

[0054] As used herein and unless otherwise specified, the terms “treatment” and “treating” refer to therapeutic or palliative measures. Beneficial or desired clinical results include, but are not limited to, alleviation, in whole or in part, of symptoms associated with a disease, disorder, or condition, diminishment of the extent of disease, stabilized ( / .<?., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state (e.g., one or more symptoms of the disease), and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. In one embodiment, “treatment” comprises administration of a therapeutic after manifestation of the unwanted condition ( / .<?., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof). In some embodiments, “treatment” can mean 11NAI-5005346498vlreducing the rate at which Al AT polymers accumulate in the liver, or preferably halting the accumulation of Al AT polymers in the liver, or more preferably reducing the amount of Al AT polymers in the liver (“amount” in this context is usually measured as a concentration but might also refer to weight.) In some embodiments, “treatment” can mean increasing the secretion of Al AT from the liver, usually as measured by an increase in the concentration of unpolymerized Al AT outside of the liver, such as in the plasma. In some embodiments, “treatment” can mean increasing the neutrophil elastase inhibition activity in the blood of the subject, as evidence of circulating (e.g., not polymerized) Z-AAT / M-AAT. In some embodiments, “treatment” can mean slowing the progression, halting progression (e.g., stabilization), or reversing liver fibrosis.

[0055] Measuring neutrophil elastase activity can be measured by methods well known to those skilled in the art, for example, by fluorometric assays using specific substrates, enzyme-linked immunosorbent assays (ELISAs), and activity-based probes.

[0056] A “therapeutically effective amount,” as used herein, refers to an amount that is sufficient to achieve a desired therapeutic effect. In some embodiments, “effective amount” can mean an amount effective for reducing the rate at which Al AT polymer accumulate in the liver, or preferably effective for halting the accumulation of Al AT polymer in the liver, or more preferably effective for reducing the amount of Al AT polymer in the liver (“amount” in this context is usually measured as a concentration but might also refer to weight.) In some embodiments, “therapeutically effective amount” can mean an amount effective for increasing the secretion of Al AT from the liver, usually as measured by an increase in the concentration of Al AT outside of the liver, such as in the blood or plasma.

[0057] As used herein, and unless otherwise specified, the terms “about” and “approximately,” when used in connection with doses, amounts, or weight percent of ingredients of a composition or a dosage form, mean a dose, amount, or weight percent that is recognized by one of ordinary skill in the art to provide a pharmacological effect equivalent to that obtained from the specified dose, amount, or weight percent. In certain embodiments, the terms “about” and “approximately,” when used in this context, contemplate a dose, amount, or weight percent within 30%, within 20%, within 15%, within 10%, or within 5%, of the specified dose, amount, or weight percent.

[0058] As used herein and unless otherwise specified, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit / risk ratio.12NAI-5005346498vlPharmaceutically acceptable salts are well known in the art. For example, Berge et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66:1-19. In certain embodiments, pharmaceutically acceptable salts include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain embodiments, pharmaceutically acceptable salts include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-m ethylglucamine, hydrabamine, IH-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, l-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, dicyclohexylammonium, and zinc salts. In certain embodiments, pharmaceutically acceptable salts include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts. Pharmaceutically acceptable anionic salts include, but are not limited to, acetate, aspartate, benzenesulfonate, benzoate, besylate, bicarbonate, bitartrate, bromide, camsylate, carbonate, chloride, citrate, decanoate, edetate, esylate, fumarate, gluceptate, gluconate, glutamate, glycolate, hexanoate, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl sulfate, mucate, napsylate, nitrate, octanoate, oleate, pamoate, pantothenate, phosphate, polygalacturonate, propionate, salicylate, stearate, acetate, succinate, sulfate, tartrate, teoclate, and tosylate. Pharmaceutically acceptable salts, such as those described herein, can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.

[0059] As used herein, unless otherwise specified “fibrosis” refers to liver fibrosis (and not to other forms of fibrosis, such as myocardial fibrosis), which can be measured by an art-recognized scale, e.g., the METAVIR scoring system, Knodell score / Histological Activity Index (HAI), or the Batts and Ludwig system. In a typical scoring system, fibrosis severity is indicated by a stage, usually stage 0 to stage 4, wherein stage 0 indicates a healthy liver ( / .<?., a liver essentially free of fibrosis), stage 4 indicates cirrhosis of the liver (the most severe form of fibrosis), and stages 1-3 indicate progressively more severe intermediate forms of fibrosis that have not progressed to cirrhosis (see, e.g., Chowdhury, A.B. and Mehta, K.J. Clin Exp Med 2023; 23:273-285).

[0060] As used herein, a Pi*Z genotype refers to a genotype, wherein the individual has at least one PiZ allele. Examples of genotypes within the Pi*Z genotype are the PiZZ13NAI-5005346498vlgenotype, wherein the subject has two PiZ alleles, and the PiMZ genotype, wherein the individual has one PiZ allele and one normal PiM allele.

[0061] As used herein, AATD liver disease refers to a disease or condition of the liver that is caused by or characterized by a deficiency in alpha- 1 antitrypsin. Such disease or condition may be characterized by the presence of liver fibrosis which can, over time, lead to inflammation and cell injury of the liver. Non-limiting examples of Al AT-associated liver diseases include hepatitis, hepatic fibrosis, hepatic cirrhosis, cholestasis, metabolic dysfunction-associated steatohepatitis (MASH), liver cancer, or liver disease caused in part by or exacerbated by alcohol use. The AATD liver disease may be in a stage not yet severe enough to receive a disease diagnosis or may already have been diagnosed.

[0062] The terms “expression” and “expression level” in general are used interchangeably and generally refer to the amount of an amino acid product or protein in a biological sample. "Expression" generally refers to the process by which gene-encoded information is converted into the protein structures present and operating in the cell.Fragments of the translated protein, or the post-translationally modified protein shall also be regarded as expressed if they originate from a post-translational processing of the protein, e.g., by proteolysis.

[0063] Protein expression levels may be protein expression levels in any biological sample collected from the subject. In certain embodiments, the protein expression level is described herein as being a “blood” protein expression level, which may be a protein level measured in a whole blood sample of the subject or any processed derivative thereof, e.g., blood plasma. One of ordinary skill in the art will be familiar with methods by which blood can or should be processed to accurately measure the expression level of the proteins disclosed herein.

[0064] The disclosure can be understood more fully by reference to the following detailed description and illustrative examples, which are intended to exemplify non-limiting embodiments.

[0065] The methods disclosed herein are based on the finding that certain proteins are differently expressed, with a greater than 1.5-fold change in some instances, in subjects having AATD liver disease and that such subjects may be advantageously treated with a therapeutically effective amount of a compound of Formula (I):14NAI-5005346498vlor a pharmaceutically acceptable salt thereof, wherein:Ri is selected from the group consisting of F, Cl, Br, I, and C1-6 alkyl;R2 is C1-6 alkyl;each R is independently selected from the group consisting of F, Cl, Br, I, CN, and C1-6 alkyl; andn is an integer from 0-5.

[0066] In certain embodiments, the subject is treated with a therapeutically effective amount of a compound of Formula (I- A):wherein Ri, R2, R, and n are as defined with respect to Formula (I).

[0067] In certain embodiments, the subject is treated with a therapeutically effective amount of a compound of Formula (I- A'):wherein Ri, R2, R, and n are as defined with respect to Formula (I). In certain embodiments of Formula (I-A1), Ri is F, R2 is propyl, one of R' and R" is F, and one of R' and R" is Cl.

[0068] In certain embodiments, the subject is treated with a therapeutically effective amount of N-(l-(2-chl oro-3 -fluorophenyl)- 1 -hydroxypentan-2 -yl)-7-fluoro-2-oxoindoline-4-15NAI-5005346498vlcarboxamide (Compound 1, shown below), or a pharmaceutically acceptable salt or solvate thereof, to treat the AATD liver disease.Compound 1

[0069] In one embodiment, the subject with AATD liver disease and an altered blood protein expression level may be advantageously treated with a therapeutically effective amount of N-[(lR)-l-[(S)-(2-chloro-3-fluorophenyl)hydroxymethyl]butyl]-7-fluoro-2,3-dihydro-2-oxo-lH-indole-4-carboxamide (Compound 1-A, shown below), or a pharmaceutically acceptable salt or solvate thereof, which is one stereoisomer of Compound 1:Compound 1-A

[0070] These findings also provide less invasive and more comprehensive methods of identifying subjects that may benefit from treatment according to the methods, as disclosed herein, as compared to currently used methods in the art.

[0071] Compound 1 and its stereoisomers, including Compound 1-A may be synthesized according to methods known in the art, for example, as described in any one of US Patent No.11,919,856 B2 or PCT / US2024 / 044680, each of which is incorporated herein by reference in its entirety.

[0072] In one embodiment, the altered protein expression level is an altered protein expression level in the blood of the subject. In one embodiment, the altered blood protein expression level is of one or more of FVII, PDCD6, apolipoprotein C-l (APOCI), apolipoprotein C-III (APOC3), and N-terminal pro-peptide of type III collagen (PRO-C3). In certain embodiments, the subject further has an altered blood protein expression level of one or more of tissue inhibitor of metalloproteinase 1 (TIMP-1), collagen type 4, alpha 616NAI-5005346498vl(CO4A6), insulin-like growth factor 2 (IGF2), insulin-like growth factor 1 receptor (IGF1R), insulin-like growth factor-binding protein 3 (IGFBP3), insulin-like growth factor-binding protein 5 (IGFBP5), leptin receptor (LEPR), intercellular adhesion molecule-1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), apolipoprotein A5 (AP0A5), apolipoprotein C-IVI (AP0C2), apolipoprotein C-IV (AP0C4), apolipoprotein F (APOF), apolipoprotein LI (AP0L1), apolipoprotein M (APOM), PDCD6 interacting protein (PDCD6IP), charged multivesicular body protein 4B (CHMP4B), vacuolar protein sorting-associated protein 4B (VPS4B), proteasome 26S subunit, non-ATPase 5 (PSMD5), proteasome 26S subunit, non-ATPase 9 (PSMD9), proteasome 26S subunit, non-ATPase 10 (PSDM10), ovarian-tumor domain-containing protein 6B (OTUD6B), and proteasome assembly chaperone 1 (PSMG1). In certain embodiments, the protein expression level is elevated by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5 as compared to a control. In certain embodiments, the protein expression level is decreased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5 as compared to a control.

[0073] In another embodiment, the altered protein expression level is an altered protein expression level in the blood of the subject. In one embodiment, the altered blood protein expression level is of one or more of FVII, PDCD6, APOCI, APOC3, M30 (a fragment of the cytokeratin 18 protein), M65 (another fragment of the cytokeratin 18 protein), and PROCS. In certain embodiments, the protein expression level is elevated by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5 as compared to a control. In certain embodiments, the protein expression level is decreased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5 as compared to a control.

[0074] For example, in certain embodiments, the subject with AATD liver disease that can advantageously be treated with a therapeutically effective amount of compound of Formula (I), e.g., Compound 1-A, according to the methods disclosed herein, may have an elevated protein expression level of PDCD6. In certain embodiments, the subject also has an elevated protein expression level of PRO-C3. In certain embodiments, the protein expression level, e.g., of PDCD6 is elevated by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5 as compared to a control.17NAI-5005346498vl

[0075] In another example, in certain embodiments, the subject with AATD liver disease that can advantageously be treated with a therapeutically effective amount of compound of Formula (I), e.g., Compound 1-A, according to the methods disclosed herein, may have an elevated protein expression level of one or both of M30 and M65. In certain embodiments, the subject also has an elevated protein expression level of PRO-C3 and / or PDCD6. In certain embodiments, the protein expression level of M30 is elevated by a factor of about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5 or even higher, such as up to 8.0, as compared to a control. In certain embodiments, the protein expression level of M65 is elevated by a factor of about 1.5, about 1.75, about 2.0, about 2.25, about 2.5, about 2.75, about 3.0, about 3.25, about 3.5, about 3.75, about 4.0, about 4.25, or even higher, such as up to 5.0, as compared to a control.

[0076] In another example, in certain embodiments, the subject with AATD liver disease that can advantageously be treated with a therapeutically effective amount of compound of Formula (I), e.g., Compound 1-A, according to the methods disclosed herein, may have a decreased protein expression level of one or more of APOCI, APOC3, and FVII. In certain embodiments, the protein expression level, e.g., of APOCI, APOC3, and / or FVII is decreased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5 as compared to a control.

[0077] In certain embodiments, the subject with AATD liver disease that can advantageously be treated with a therapeutically effective amount of compound of Formula (I), e.g., Compound 1-A, according to the methods disclosed herein, may have both a) an elevated protein expression level of one or both of PDCD6 and PRO-C3; and b) a decreased protein expression level of one or more of APOCI, APOC3, and FVII.

[0078] In other embodiments, the subject with AATD liver disease that can advantageously be treated with a therapeutically effective amount of compound of Formula (I), e.g., Compound 1-A, according to the methods disclosed herein, may have both a) an elevated protein expression level of one or more of PDCD6, PRO-C3, M30, and M65; and b) a decreased protein expression level of one or more of APOCI, APOC3, and FVII.

[0079] Examples of methods encompassing the treatment of such subjects with a therapeutically effective amount of the compounds disclosed herein are further detailed below.18NAI-5005346498vl

[0080] For example, in one embodiment, the present disclosure provides a method of treating a subject having AATD liver disease, the method comprising:a. identifying a subject as having an altered blood protein expression level of one or more of F VII, PDCD6, APOCI, APOC3, and PRO-C3, relative to a control; andb. administering to the subject a therapeutically effective amount of a compound of Formula (I).

[0081] In one embodiment, the present disclosure provides a method of treating a subject having AATD liver disease, the method comprising:a. identifying a subject as having an altered blood protein expression level of one or more of F VII, PDCD6, APOCI, APOC3, and PRO-C3, relative to a control; andb. administering to the subject a therapeutically effective amount of Compound 1-A.

[0082] In another embodiment, the present disclosure provides a method of treating a subject having AATD liver disease, the method comprising:a. identifying a subject as having an altered blood protein expression level of one or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3, relative to a control; andb. administering to the subject a therapeutically effective amount of Formula (I), such as Compound 1-A.

[0083] In another embodiment, the present disclosure provides a method of treating a subject having AATD liver disease, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), wherein the subject has been identified as having an altered blood protein expression level of one or more of FVII, PDCD6, APOCI, APOC3, and PRO-C3, relative to a control.

[0084] In another embodiment, the present disclosure provides a method of treating a subject having AATD liver disease, the method comprising administering to the subject a therapeutically effective amount of Compound 1-A, wherein the subject has been identified as having an altered blood protein expression level of one or more of FVII, PDCD6, APOCI, APOC3, and PRO-C3, relative to a control.

[0085] The present disclosure provides a method of treating a subject having AATD liver disease, the method comprising administering to the subject a therapeutically effective amount of Formula (I), such as Compound 1-A, wherein the subject has been identified as 19NAI-5005346498vlhaving an altered blood protein expression level of one or more of F VII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3, relative to a control.

[0086] In another embodiment, the present disclosure provides a method of treating a subject having AATD liver disease, the method comprising:a. selecting a subject who has been determined as having an altered blood protein expression level of one or more of F VII, PDCD6, APOCI, APOC3, and PROCS, relative to a control; andb. administering to the subject a therapeutically effective amount of a compound of Formula (I).

[0087] In another embodiment, the present disclosure provides a method of treating a subject having AATD liver disease, the method comprising:a. selecting a subject who has been determined as having an altered blood protein expression level of one or more of F VII, PDCD6, APOCI, APOC3, and PROCS, relative to a control; andb. administering to the subject a therapeutically effective amount of Compound 1-A.

[0088] In another embodiment, the present disclosure provides a method of treating a subject having AATD liver disease, the method comprising:a. selecting a subject who has been determined as having an altered blood protein expression level of one or more of F VII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3, relative to a control; andb. administering to the subject a therapeutically effective amount of a compound of Formula (I), such as Compound 1-A.

[0089] In another embodiment, the present disclosure provides a method of treating a subject having AATD liver disease, the method comprising administering a therapeutically effective amount of a Compound of Formula (I), wherein the subject has been selected based on an altered blood protein expression level of one or more of F VII, PDCD6, APOCI, APOC3, and PRO-C3, relative to a control.

[0090] In another embodiment, the present disclosure provides a method of treating a subject having AATD liver disease, the method comprising administering a therapeutically effective amount of Compound 1-A, wherein the subject has been selected based on an altered blood protein expression level of one or more of F VII, PDCD6, APOCI, APOC3, and PRO-C3, relative to a control.20NAI-5005346498vl

[0091] In yet another embodiment, the present disclosure provides a method of treating a subject having AATD liver disease, the method comprising administering a therapeutically effective amount of Formula (I), such as Compound 1-A, wherein the subject has been selected based on an altered blood protein expression level of one or more of F VII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3, relative to a control.

[0092] In another embodiment, the present disclosure provides a method of treating a subject identified as having AATD liver disease, the method comprising:a. determining a blood protein expression level of one or more of FVII, PDCD6, APOCI, APOC3, and PRO-C3 in a sample obtained from the subject; b. comparing the blood protein expression level of the one or more of FVII, PDCD6, APOCI, APOC3, and PRO-C3 to a control, wherein an alteration in the blood protein expression level of the one or more of FVII, PDCD6, APOCI, APOC3, and PRO-C3 in the subject sample relative to the control identifies the subject as having the AATD liver disease; andc. administering to the subject a therapeutically effective amount of a compound of Formula (I).

[0093] In another embodiment, the method comprises:a. determining a blood protein expression level of one or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3 in a sample obtained from the subject;b. comparing the blood protein expression level of the one or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3 to a control, wherein an alteration in the blood protein expression level of the one or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3 in the subject sample relative to the control identifies the subject as having the AATD liver disease; andc. administering to the subject a therapeutically effective amount of a compound of Formula (I).

[0094] In another embodiment, the present disclosure provides a method of treating a subject having AATD liver disease, the method comprising administering a therapeutically effective amount of a compound of Formula (I), wherein the subject has been selected based on an altered blood protein expression level of one or more of FVII, PDCD6, APOCI, APOC3, and PRO-C3, relative to a control.21NAI-5005346498vl

[0095] In another embodiment, the present disclosure provides a method of treating a subject having AATD liver disease, the method comprising administering a therapeutically effective amount of Compound 1-A, wherein the subject has been selected based on an altered blood protein expression level of one or more of F VII, PDCD6, APOCI, APOC3, and PRO-C3, relative to a control.

[0096] In yet another embodiment, the present disclosure provides a method of treating a subject having AATD liver disease, the method comprising administering a therapeutically effective amount of Formula (I), such as Compound 1-A, wherein the subject has been selected based on an altered blood protein expression level of one or more of F VII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3, relative to a control.

[0097] In another embodiment, the present disclosure provides a method of treating a subject identified as having AATD liver disease, the method comprising:a. determining a blood protein expression level of one or more of FVII, PDCD6, APOCI, APOC3, and PRO-C3 in a sample obtained from the subject; b. comparing the blood protein expression level of the one or more of FVII, PDCD6, APOCI, APOC3, and PRO-C3 to a control, wherein an alteration in the blood protein expression level of the one or more of FVII, PDCD6, APOCI, APOC3, and PRO-C3 in the subject sample relative to the control identifies the subject as having the AATD liver disease; andc. administering to the subject a therapeutically effective amount of Compound 1-A.

[0098] In another embodiment, the present disclosure provides a method of treating a subject identified as having AATD liver disease, the method comprising:a. determining a blood protein expression level of one or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3 in a sample obtained from the subject;b. comparing the blood protein expression level of the one or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3 to a control, wherein an alteration in the blood protein expression level of the one or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3 in the subject sample relative to the control identifies the subject as having the AATD liver disease; andc. administering to the subject a therapeutically effective amount of Compound (I), e.g., Compound 1-A.22NAI-5005346498vl

[0099] In another aspect, the present disclosure provides a method of treating a subject having AATD liver disease, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), wherein the subject has been determined to have an altered blood protein expression level of one or more of F VII, PDCD6, APOCI, APOC3, and PRO-C3 compared to the blood protein expression level of the one or more of F VII, PDCD6, APOCI, APOC3, and PRO-C3 in a control.

[0100] In another aspect, the present disclosure provides a method of treating a subject having AATD liver disease, the method comprising administering to the subject a therapeutically effective amount of Compound 1-A, wherein the subject has been determined to have an altered blood protein expression level of one or more of F VII, PDCD6, APOCI, APOC3, and PRO-C3 compared to the blood protein expression level of the one or more of FVII, PDCD6, APOCI, APOC3, and PRO-C3 in a control.

[0101] In yet another aspect, the present disclosure provides a method of treating a subject having AATD liver disease, the method comprising administering to the subject a therapeutically effective amount of Formula (I), e.g., Compound 1-A, wherein the subject has been determined to have an altered blood protein expression level of one or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3 compared to the blood protein expression level of the one or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3 in a control.

[0102] In another embodiment, the present disclosure provides a method for identifying a subject having AATD liver disease who is (a) likely to, or (b) less likely to, respond to treatment with a compound of Formula (I), wherein the method comprises determining whether the subject has (a) an altered blood protein expression level or (b) an unaltered blood protein expression level of one or more of FVII, PDCD6, APOCI, APOC3, and PRO-C3, as compared to a control, wherein the altered blood protein expression level identifies that the subject is (a) likely to respond to treatment with the compound of Formula (I), and the unaltered blood protein expression level indicates that the subject is (b) less likely to respond to treatment with the compound of Formula (I). In certain embodiments, the method is for identifying a subject having AATD liver disease who is (a) likely to, or (b) less likely to, respond to treatment with the compound of Formula (I).

[0103] In another embodiment, the present disclosure provides a method for identifying a subject having AATD liver disease who is (a) likely to, or (b) less likely to, respond to treatment with Compound 1-A, wherein the method comprises determining whether the subject has (a) an altered blood protein expression level or (b) an unaltered blood protein 23NAI-5005346498vlexpression level of one or more of F VII, PDCD6, APOCI, APOC3, and PRO-C3, as compared to a control, wherein the altered blood protein expression level identifies that the subject is (a) likely to respond to treatment with Compound 1-A, and the unaltered blood protein expression level indicates that the subject is (b) less likely to respond to treatment with Compound 1-A. In certain embodiments, the method is for identifying a subject having AATD liver disease who is (a) likely to, or (b) less likely to, respond to treatment with Compound 1-A.

[0104] In another embodiment, the present disclosure provides a method for identifying a subject having AATD liver disease who is (a) likely to, or (b) less likely to, respond to treatment with Compound 1-A, wherein the method comprises determining whether the subject has (a) an altered blood protein expression level or (b) an unaltered blood protein expression level of one or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3, as compared to a control, wherein the altered blood protein expression level identifies that the subject is (a) likely to respond to treatment with Formula (I), e.g., Compound 1-A, and the unaltered blood protein expression level indicates that the subject is (b) less likely to respond to treatment with Compound 1-A. In certain embodiments, the method is for identifying a subject having AATD liver disease who is (a) likely to, or (b) less likely to, respond to treatment with Formula (I), e.g., Compound 1-A.

[0105] In any of the methods disclosed herein the altered blood protein expression level may be of one or more of FVII, PDCD6, APOCI, APOC3, and PRO-C3. In certain embodiments, the altered blood protein expression level is of two or more of FVII, PDCD6, APOCI, APOC3, and PRO-C3. In certain embodiments, the altered blood protein expression level is of three or more of FVII, PDCD6, APOCI, APOC3, and PRO-C3. In certain embodiments, the altered blood protein expression level is of four or each of FVII, PDCD6, APOCI, APOC3, and PRO-C3. In certain embodiments, the altered blood protein expression level is of each of FVII, PDCD6, APOCI, APOC3, and PRO-C3. In certain embodiments, the subject further has an altered blood protein expression level of one or more of TIMP-1, CO4A6, IGF2, IGF1R, IGFBP3, IGFBP5, LEPR, ICAM1, VCAM1, APOA5, APOC2, APOC4, APOF, APOL1, APOM, PDCD6IP, CHMP4B, VPS4B, PSMD5, PSMD9, PSDM10, OTUD6B, and PSMG1. In certain embodiments, the subject further has an altered blood protein expression level of two or more of TIMP-1, CO4A6, IGF2, IGF1R, IGFBP3, IGFBP5, LEPR, ICAM1, VC AMI, APOA5, APOC2, APOC4, APOF, APOL1, APOM, PDCD6IP, CHMP4B, VPS4B, PSMD5, PSMD9, PSDM10, OTUD6B, and PSMG1. In certain embodiments, the subject further has an altered blood protein expression level of three 24NAI-5005346498vlor more of TIMP-1, CO4A6, IGF2, IGF1R, IGFBP3, IGFBP5, LEPR, ICAM1, VCAM1, AP0A5, APOC2, APOC4, APOF, APOL1, APOM, PDCD6IP, CHMP4B, VPS4B, PSMD5, PSMD9, PSDM10, OTUD6B, and PSMG1. In certain embodiments, the subject further has an altered blood protein expression level of four or more of TIMP-1, CO4A6, IGF2, IGF1R, IGFBP3, IGFBP5, LEPR, ICAM1, VCAM1, AP0A5, APOC2, APOC4, APOF, APOL1, APOM, PDCD6IP, CHMP4B, VPS4B, PSMD5, PSMD9, PSDM10, OTUD6B, and PSMG1.

[0106] In any of the methods disclosed herein the altered blood protein expression level may be of one or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3. In certain embodiments, the altered blood protein expression level is of two or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3. In certain embodiments, the altered blood protein expression level is of three or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3. In certain embodiments, the altered blood protein expression level is of four or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3. In certain embodiments, the altered blood protein expression level is of five or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3. In certain embodiments, the altered blood protein expression level is of six or each of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3In certain embodiments, the altered blood protein expression level is of each of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3. In certain embodiments, the subject further has an altered blood protein expression level of one or more of TIMP-1, CO4A6, IGF2, IGF1R, IGFBP3, IGFBP5, LEPR, ICAM1, VCAM1, APOA5, APOC2, APOC4, APOF, APOL1, APOM, PDCD6IP, CHMP4B, VPS4B, PSMD5, PSMD9, PSDM10, OTUD6B, and PSMG1. In certain embodiments, the subject further has an altered blood protein expression level of two or more of TIMP-1, CO4A6, IGF2, IGF1R, IGFBP3, IGFBP5, LEPR, ICAM1, VC AMI, APOA5, APOC2, APOC4, APOF, APOL1, APOM, PDCD6IP, CHMP4B, VPS4B, PSMD5, PSMD9, PSDM10, OTUD6B, and PSMGL In certain embodiments, the subject further has an altered blood protein expression level of three or more of TIMP-1, CO4A6, IGF2, IGF1R, IGFBP3, IGFBP5, LEPR, ICAM1, VCAM1, APOA5, APOC2, APOC4, APOF, APOL1, APOM, PDCD6IP, CHMP4B, VPS4B, PSMD5, PSMD9, PSDM10, OTUD6B, and PSMGL In certain embodiments, the subject further has an altered blood protein expression level of four or more of TIMP-1, CO4A6, IGF2, IGF1R, IGFBP3, IGFBP5, LEPR, ICAM1, VCAM1, APOA5, APOC2, APOC4, APOF, APOL1, APOM, PDCD6IP, CHMP4B, VPS4B, PSMD5, PSMD9, PSDM10, OTUD6B, and PSMGL

[0107] In certain embodiments, the subject has altered blood protein expression level of PDCD6, APOCI, APOC3, M30, M65, and PRO-C3. In certain embodiments, the subject has 25NAI-5005346498vlaltered blood protein expression level of FVII, AP0C1, AP0C3, M30, M65, and PR0-C3. In certain embodiments, the subject has altered blood protein expression level of FVII, PDCD6, AP0C3, M30, M65, and PR0-C3. In certain embodiments, the subject has altered blood protein expression level of FVII, PDCD6, AP0C1, M30, M65, and AP0C3. In certain embodiments, the subject has altered blood protein expression level of FVII, PDCD6, AP0C1, AP0C3, M65, and PR0-C3. In certain embodiments, the subject has altered blood protein expression level of FVII, PDCD6, AP0C1, AP0C3, M30, and PR0-C3.

[0108] In certain embodiments, the subject has altered blood protein expression level of APOCI, APOC3, M30, M65, and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of FVII, APOC3, M30, M65, and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of FVII, PDCD6, M30, M65, and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of FVII, PDCD6, APOCI, M65, and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of FVII, PDCD6, APOCI, APOC3, and M30. In certain embodiments, the subject has altered blood protein expression level of PDCD6, APOC3, M30, M65, and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of FVII, APOCI, M30, M65, and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of FVII, PDCD6, APOC3, M65, and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of FVII, PDCD6, APOCI, M30, and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of FVII, PDCD6, APOCI, APOC3, and M65. In certain embodiments, the subject has altered blood protein expression level of PDCD6, APOCI, M30, M65, and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of FVII, APOCI, APOC3, M65, and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of FVII, PDCD6, APOC3, M30, and PROCS. In certain embodiments, the subject has altered blood protein expression level of FVII, PDCD6, APOCI, M30, and M65. In certain embodiments, the subject has altered blood protein expression level of PDCD6, APOCI, APOC3, M65, and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of FVII, APOCI, APOC3, M30, and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of FVII, PDCD6, APOC3, M30, and M65. In certain embodiments, the subject has altered blood protein expression level of FVII, PDCD6, APOCI, APOC3, M30, and PRO-C3. In certain embodiments, the subject has altered blood protein expression level26NAI-5005346498vlof F VII, AP0C1, APOC3, M30, and M65. In certain embodiments, the subject has altered blood protein expression level of F VII, PDCD6, AP0C1, APOC3, M30, and M65.

[0109] In certain embodiments, the subject has altered blood protein expression level of PDCD6, APOCI, APOC3, and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of F VII, APOCI, APOC3, and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of F VII, PDCD6, APOC3, and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of F VII, PDCD6, APOCI, and APOC3.

[0110] In certain embodiments, the subject has altered blood protein expression level of APOCI, APOC3, and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of FVII, APOC3, and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of FVII, APOCI, and APOC3. In certain embodiments, the subject has altered blood protein expression level of PDCD6, APOC3, and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of FVII, APOC3, and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of FVII, PDCD6, and APOC3. In certain embodiments, the subject has altered blood protein expression level of PDCD6, APOCI, and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of FVII, APOCI, and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of FVII, PDCD6, and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of FVII, PDCD6, and APOCI. In certain embodiments, the subject has altered blood protein expression level of PDCD6, APOCI, and APOC3. In certain embodiments, the subject has altered blood protein expression level of FVII, APOCI, and APOC3. In certain embodiments, the subject has altered blood protein expression level of FVII, PDCD6, and APOC3.

[0111] In certain embodiments, the subject has altered blood protein expression level of FVII and PDCD6. In certain embodiments, the subject has altered blood protein expression level of FVII and APOCI. In certain embodiments, the subject has altered blood protein expression level of FVII and APOC3. In certain embodiments, the subject has altered blood protein expression level of FVII and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of PDCD6 and APOCI. In certain embodiments, the subject has altered blood protein expression level of PDCD6 and APOC3. In certain embodiments, the subject has altered blood protein expression level of PDCD6 and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of APOCI and APOC3. In certain embodiments, the subject has altered blood protein expression level of APOCI and 27NAI-5005346498vlPR0-C3. In certain embodiments, the subject has altered blood protein expression level of AP0C3 and PRO-C3.

[0112] In certain embodiments, the subject has altered blood protein expression level of PDCD6. In certain embodiments, the subject has altered blood protein expression level of APOCI. In certain embodiments, the subject has altered blood protein expression level of APOC3. In certain embodiments, the subject has altered blood protein expression level of FVII.

[0113] In certain embodiments, the subject has altered blood protein expression level of FVII and one or both of M30 or M65. In certain embodiments, the subject has altered blood protein expression level of PDCD6 and one or both of M30 or M65. In certain embodiments, the subject has altered blood protein expression level of APOCI and one or both of M30 or M65. In certain embodiments, the subject has altered blood protein expression level of APOC3 and one or both of M30 or M65. In certain embodiments, the subject has altered blood protein expression level of one or both of M30 or M65 and PRO-C3. In certain embodiments, the subject has altered blood protein expression level of one or both of M30 or M65. In certain embodiments, the subject has altered blood protein expression level of M30. In certain embodiments, the subject has altered blood protein expression level of M65.

[0114] As used herein, “altered” protein expression level is altered, e.g., increased or decreased, compared to a control. For example, in any of the methods disclosed herein, the altered blood protein expression level of one or more of APOCI, APOC3, and FVII is decreased compared to a control. In certain embodiments, the blood protein expression level is decreased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5 as compared to the control. In certain embodiments, the blood protein expression level is decreased by a factor of about 1.5 as compared to the control.

[0115] In one embodiment, the altered blood protein expression level of APOCI is decreased compared to a control. In certain embodiments, the blood protein expression level of APOClis decreased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5 as compared to the control. In certain embodiments, the blood protein expression level of APOCI is decreased by a factor of about 1.5 as compared to the control.

[0116] In one embodiment, the altered blood protein expression level of APOC3 is decreased compared to a control. In certain embodiments, the blood protein expression level of APOC3 is decreased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5,28NAI-5005346498vlabout 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5 as compared to the control. In certain embodiments, the blood protein expression level of APOC3 is decreased by a factor of about 1.5 as compared to the control.

[0117] In one embodiment, the altered blood protein expression level of F VII is decreased compared to a control. In certain embodiments, the blood protein expression level of FVII is decreased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5 as compared to the control. In certain embodiments, the blood protein expression level of FVII is decreased by a factor of about 1.5 as compared to the control.

[0118] In another example, in any of the methods disclosed herein, the altered blood protein expression level of one or both of PDCD6 and PRO-C3 is increased compared to a control. In certain embodiments, the blood protein expression level is increased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5 as compared to the control. In certain embodiments, the blood protein expression level is increased by a factor of about 1.5 as compared to the control.

[0119] In another example, in any of the methods disclosed herein, the altered blood protein expression level of one or both of M30 and M65 is increased compared to a control. In certain embodiments, the protein expression level of M30 is elevated by a factor of about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5 or even higher, such as up to 8.0, as compared to a control. In certain embodiments, the protein expression level of M65 is elevated by a factor of about 1.5, about 1.75, about 2.0, about 2.25, about 2.5, about 2.75, about 3.0, about 3.25, about 3.5, about 3.75, about 4.0, about 4.25, or even higher, such as up to 5.0, as compared to a control.

[0120] In another example, in any of the methods disclosed herein, the altered blood protein expression level of PDCD6 is increased compared to a control. In certain embodiments, the blood protein expression level of PDCD6 is increased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5 as compared to the control. In certain embodiments, the blood protein expression level of PDCD6 is increased by a factor of about 1.5 as compared to the control.

[0121] In another example, in any of the methods disclosed herein, the altered blood protein expression level of PRO-C3 is increased compared to a control. In certain29NAI-5005346498vlembodiments, the blood protein expression level of PRO-C3 is increased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5 as compared to the control. In certain embodiments, the blood protein expression level of PRO-C3 is increased by a factor of about 1.5 as compared to the control.

[0122] In another example, in any of the methods disclosed herein, the altered blood protein expression level of M30 is increased compared to a control. In certain embodiments, the protein expression level of M30 is elevated by a factor of about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5 or even higher, such as up to 8.0, as compared to a control.

[0123] In another example, in any of the methods disclosed herein, the altered blood protein expression level of M65 is increased compared to a control. In certain embodiments, the protein expression level of M65 is elevated by a factor of about 1.5, about 1.75, about 2.0, about 2.25, about 2.5, about 2.75, about 3.0, about 3.25, about 3.5, about 3.75, about 4.0, about 4.25, or even higher, such as up to 5.0, as compared to a control.

[0124] Methods of determining blood protein expression level in a subject will be routine and well-known by one of skill in the art. For example, blood protein expression levels, e.g., of F VII, PDCD6, APOCI, and / or APOC3 may be measured using liquid chromatographytandem mass spectrometry, e.g., shotgun liquid chromatography -tandem mass spectrometry. Blood protein expression level of PRO-C3 may be measured, e.g., by enzyme-linked immunosorbent assay (ELISA).

[0125] These above-noted methods may also be used, as will be understood by one of skill in the art, to measure blood protein expression levels of M30 and M65. Other methods that may be used include the chemiluminescent enzyme immunoassay method disclosed, e.g., in Yamada M, et al, Sci Rep. 2021 Sep 14; 11(1): 18187, which is incorporated by reference herein in its entirety.

[0126] As used herein, a control may be a sample, e.g., blood sample, obtained from a control subject in which protein expression levels of the biomarkers described herein may be measured. A control subject may be a subject who does not have or has not been diagnosed with AATD liver disease. In certain embodiments, the control subject is not deficient for AAT. In certain embodiments, the control subject does not have a PiZ allele. In certain embodiments, the control subject has two PiM alleles. In certain embodiments, the control subject is homozygous for PiM. In certain embodiments, the control subject is not heterozygous for PiM / PiZ (e.g., does not have the genotype PiMZ). In certain embodiments,30NAI-5005346498vlthe control subject is not homozygous for PiZ (e.g., does not have the genotype PiZZ). In certain embodiments, the control subject has normal liver function according to one or more liver function tests, e.g., serum bilirubin test and / or serum albumin test. In certain embodiments, the control subject is age-matched to the subject being treated. In certain embodiments, the control is a protein expression level reported as within a normal or a healthy range, such as disclosed in, e.g., Zhao et al. Analytica Chimica Acta 2016, 904, 65-75 and Pememalm et al., eLife 2019, 8, e41608. For example, in certain embodiments, the control or normal value of PRO-C3 in the blood may be about 6.1 ng / mL to about14.7 ng / mL (see, e.g., Erhardtsen et al., JHEP Rep., 2021, 3:100317). In certain embodiments, the control or normal value of APOCI in the blood may be about 0.45 mg / mL to about 0.75 mg / mL (see, e.g., Fuior and Gafencu, Int J Mol Sci., 2019 Nov 26, 20(23): 5939). In certain embodiments, the control or normal value of APOC3 in the blood may be about 10 mg / dL to about 15 mg / dL (see, e.g., Gaudet et al., N Engl J Med, 2014, 371:2200-2206). In certain embodiments, the control or normal value of FVII in the blood may be about 0.5 ng / mL to about 8.4 ng / mL (see, e.g., Morrissey et al., Blood, 1993, 81 (3):734-744). In certain embodiments, the control or normal value of PDCD6 in the blood may be about 2.2 pg / L (see, e.g., The Human Protein Atlas for PDCD6). Methods of determining blood protein expression level in a control subject will be routine and well-known by one of skill in the art, as described herein.

[0127] In certain embodiments, the control or normal value of M30 in the blood may be about 95 ng / mL (see, e.g., Dai et al., Medicine (Baltimore), 2024 Jun 7, 103(23):e38342), which is incorporated herein by reference in its entirety), or less, for example, 95 ng / mL ± 20 ng / mL. In certain embodiments, the control or normal value of M65 in the blood may be about 230 ng / mL or less, such as 230 ng / mL ± 50 ng / mL.

[0128] The altered blood protein expression level or levels of a subject treated according to the methods disclosed herein may, as a result of the treating, normalize (e.g., decrease or increase to match more closely those of the control). For example, in one embodiment, the present disclosure provides a method of treating a subject having AATD liver disease, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), wherein:a. the blood protein expression level of one or more of APOCI, APOC3, and FVII in the subject is increased following the administering; and / or b. the blood protein expression level of one or both of PDCD6 and PRO-C3 in the subject is decreased following the administering.31NAI-5005346498vl

[0129] The altered blood protein expression level or levels of a subject treated according to the methods disclosed herein may, as a result of the treating, normalize (e.g., decrease or increase to match more closely those of the control). For example, in one embodiment, the present disclosure provides a method of treating a subject having AATD liver disease, the method comprising administering to the subject a therapeutically effective amount of Compound 1-A, wherein:a. the blood protein expression level of one or more of APOCI, APOC3, and FVII is increased following the administering; and / orb. the blood protein expression level of one or both of PDCD6 and PRO-C3 is decreased following the administering.

[0130] In another embodiment, the present disclosure provides a method of treating a subject having AATD liver disease, the method comprising administering to the subject a therapeutically effective amount of Compound 1-A, wherein:a. the blood protein expression level of one or more of APOCI, APOC3, and FVII is increased following the administering; and / orb. the blood protein expression level of one or more of PDCD6, M30, M65 and PRO-C3 is decreased following the administering.

[0131] In certain embodiments, the blood protein expression level of APOCI is increased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5 after administering the therapeutically effective amount of a compound of Formula (I), e.g., Compound 1-A. In certain embodiments, the blood protein expression level of APOCI of a subject administered the therapeutically effective amount of a compound of Formula (I), e.g., Compound 1-A increases to a level that is within the expression level range of APOCI in the control.

[0132] In certain embodiments, the blood protein expression level of APOC3 is increased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5 after administering the therapeutically effective amount of a compound of Formula (I), e.g., Compound 1-A. In certain embodiments, the blood protein expression level of APOC3 of a subject administered the therapeutically effective amount of a compound of Formula (I), e.g., Compound 1-A increases to a level that is within the expression level range of APOC3 in the control.32NAI-5005346498vl

[0133] In certain embodiments, the blood protein expression level of F VII is increased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5 after administering the therapeutically effective amount of a compound of Formula (I), e.g., Compound 1-A. In certain embodiments, the blood protein expression level of F VII of a subject administered the therapeutically effective amount of compound of Formula (I), e.g., Compound 1-A increases to a level that is within the expression level range of FVII in the control.

[0134] In certain embodiments, the blood protein expression level of PDCD6 is decreased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5 after administering the therapeutically effective amount of compound of Formula (I), e.g., Compound 1-A. In certain embodiments, the blood protein expression level of PDCD6 of a subject administered the therapeutically effective amount of compound of Formula (I), e.g., Compound 1-A decreases to a level that is within the expression level range of PDCD6 in the control.

[0135] In certain embodiments, the blood protein expression level of M30 is decreased by a factor of about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5 or even more, such as up to about 8.0, after administering the therapeutically effective amount of compound of Formula (I), e.g., Compound 1-A. In certain embodiments, the blood protein expression level of M30 of a subject administered the therapeutically effective amount of compound of Formula (I), e.g., Compound 1-A decreases to a level that is within the expression level of M30 range in the control.

[0136] In certain embodiments, the blood protein expression level of M65 is decreased by a factor of about 1.5, about 1.75, about 2.0, about 2.25, about 2.5, about 2.75, about 3.0, about 3.25, about 3.5, about 3.75, about 4.0, about 4.25, or even further, such as up to a factor of 5.0, after administering the therapeutically effective amount of compound of Formula (I), e.g., Compound 1-A. In certain embodiments, the blood protein expression level of M65 of a subject administered the therapeutically effective amount of compound of Formula (I), e.g., Compound 1-A decreases to a level that is within the expression level of M65 range in the control.

[0137] In certain embodiments, the blood protein expression level of PRO-C3 is decreased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 33NAI-5005346498vl1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5 after administering the therapeutically effective amount of compound of Formula (I), e.g., Compound 1-A. In certain embodiments, the blood protein expression level of PRO-C3 of a subject administered the therapeutically effective amount of compound of Formula (I), e.g., Compound 1-A decreases to a level that is within the expression level of PRO-C3 range in the control.

[0138] In certain embodiments, administration of Compound 1-A to a subject does not cause development of lung disease. In certain embodiments, administration of Compound 1-A to a subject does not cause major pulmonary adverse events in the subject. In certain embodiments, administration of Compound 1-A to a subject does not result in forced expiratory volume in 1 second (FEV1) to decline by 15% or more in the subject.

[0139] In addition to having an altered blood protein expression level of any of the proteins as disclosed herein, a subject that may be advantageously treated with a therapeutically effective amount of compound of Formula (I), e.g., Compound 1-A to treat the AATD liver disease may have at least one mutated allele of SERPINA11. For example, in one embodiment, the subject has a Pi*Z genotype. In one embodiment, the subject has a PiMZ genotype. In one embodiment, the subject has a PiZZ genotype. In one embodiment, the subject has a PiZZ genotype with an elevated PRO-C3 blood protein expression level. In one embodiment, the subject has a PiZZ genotype with altered FVII, PDCD6, APO1, and APO3 blood protein expression levels.

[0140] In one embodiment, the subject has a PiMZ genotype with MASH and an elevated PRO-C3 blood protein expression level. In one embodiment, the subject has a PiMZ genotype and MASH with altered FVII, PDCD6, APO1, and APO3 blood protein expression levels.

[0141] In other embodiment, the subject has a PiZZ genotype with altered FVII, PDCD6, APO1, APO3, M30, and M65 blood protein expression levels. In other embodiment, the subject has a PiMZ genotype with MASH with altered FVII, PDCD6, APO1, APO3, and M30 blood protein expression levels.

[0142] A subject that may be advantageously treated with a therapeutically effective amount of compound of Formula (I), e.g., Compound 1-A to treat the AATD liver disease may further have one or more comorbid conditions, such as metabolic dysfunction-associated steatohepatitis (MASH), cirrhosis of the liver, or hepatitis.

[0143] The therapeutically effective amount of compound of Formula (I), e.g., Compound 1-A may be administered to the subject as part of a pharmaceutical composition 34NAI-5005346498vlcomprising the therapeutically effective amount of compound of Formula (I), e.g., Compound 1-A, or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable excipients.

[0144] In one embodiment, the one or more pharmaceutically acceptable excipients comprises at least one polymer. In one embodiment, the at least one polymer comprises an acidic polymer.

[0145] In one embodiment, the at least one polymer comprises polyethylene oxide, a polymethacrylate-based copolymer, ethylene-vinyl acetate (EVA), a polyolefin, a polyamide, a polyester, a styrene block copolymer, polyethylene, ethyl ene-methyl acrylate (EMA), ethylene n-butyl acrylate (EnBA), one or more of hydroxypropyl methylcellulose (HPMC), a copolymer comprising HPMC, one or more esters of HPMC, a copolymer comprising one or more esters of HPMC, an enteric polymer or copolymer, polyvinylpyrrolidone, or a copolymer comprising polyvinylpyrrolidone; preferably a HPMC, a copolymer comprising HPMC, ester or esters of HPMC, or a copolymer comprising ester or esters of HPMC.

[0146] In one embodiment, the at least one polymer comprises one or more of hydroxypropyl methylcellulose (HPMC), a copolymer comprising HPMC, one or more esters of HPMC, a copolymer comprising one or more esters of HPMC, an enteric copolymer, polyvinylpyrrolidone, or a copolymer comprising polyvinylpyrrolidone. In one embodiment, the at least one polymer comprises HPMC, a copolymer comprising HPMC, ester or esters of HPMC, or a copolymer comprising ester or esters of HPMC. In yet another embodiment, the at least one polymer comprises one or more esters of HPMC. In yet a further embodiment, the at least one polymer comprises one or more esters of HPMC wherein the esters of HPMC comprise one or more of acetate ester and succinate ester. In one embodiment, the esters of HPMC comprise hydroxypropyl methylcellulose acetate succinate (HPMCAS).

[0147] In one embodiment, the one or more pharmaceutically acceptable excipients further comprise one or more excipients selected from the group consisting of one or more filler, one or more disintegrant, one or more glidant, one or more lubricant, one or more binder, or any combination thereof.

[0148] Examples of suitable fillers include, but are not limited to, starch, powdered cellulose, microcrystalline cellulose, silicified microcrystalline cellulose, calcium phosphate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, xylitol, dextrose, crystalline maltose, and maltodextrins. In one embodiment, the filler is mannitol. Examples of suitable disintegrants include, but are not limited to, crosslinked polyvinylpyrrolidone (PVP), crosslinked croscarmellose, croscarmellose sodium, sodium starch glycolate, and microcrystalline 35NAI-5005346498vlcellulose. In one embodiment, the disintegrant is croscarmellose sodium. Examples of suitable glidants include, but are not limited to, silicon dioxide, talc, starch, calcium silicate, magnesium silicate, and magnesium aluminometasilicate. In one embodiment, the glidant is silicon dioxide. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, glyceryl behenate, glyceryl palmitostearate, waxes (e.g., bees wax, carnauba wax, stearyl alcohol, and polyglycolized esters of fatty acids with registered names: GELUCIRE®, COMPRITOL® 888, PRECIROL® AT05, palmitic acid, myristic acid, polyethylene glycol (e.g, CARBOWAX™), sodium stearyl fumarate, magnesium lauryl sulfate, sodium lauryl sulfate, poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 407, and sodium benzoate. In one embodiment, the lubricant is stearyl fumarate or a salt thereof, such as sodium stearyl fumarate. In one embodiment, the one or more pharmaceutically acceptable excipients comprises or further comprises one or more compression excipients.

[0149] In one embodiment, the pharmaceutical composition used to treat a subject for AATD liver disease is manufactured into and provided in a dosage form as a capsule or a tablet. In one embodiment, the pharmaceutical composition is manufactured into and provided in the form of a tablet.

[0150] In one embodiment, the pharmaceutical composition (e.g, tablet, capsule) administered to the subject to treat AATD liver disease comprises compound of Formula (I), e.g., Compound 1-A in an amount of about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 200 mg, or about 250 mg. In one embodiment, a pharmaceutical composition comprises about 25 mg or about 125 mg of compound of Formula (I), e.g., Compound 1-A. In one embodiment, a pharmaceutical composition comprises about 25 mg to about 125 mg, about 25 mg to about 100 mg, about 25 mg to about 75 mg, about 25 mg to about 50 mg, about 50 mg to about 125 mg, about 50 mg to about 100 mg, about 50 mg to about 75 mg, about 75 mg to about 125 mg, about 75 mg to about 100 mg, or about 100 mg to about 125 mg of compound of Formula (I), e.g., Compound 1-A.

[0151] In one embodiment, the pharmaceutical composition administered to the subject to treat AATD liver disease comprises compound of Formula (I), e.g., Compound 1-A in an amount of about 23 mg to about 27 mg of compound of Formula (I), e.g., Compound 1-A. In certain embodiments, the pharmaceutical composition comprises about 25 mg of compound of Formula (I), e.g., Compound 1-A. In certain other embodiments, the dosage form comprises about 123 mg to about 127 mg of compound of Formula (I), e.g., Compound 1-A. In one embodiment, the pharmaceutical composition administered to the subject to treat 36NAI-5005346498vlAATD liver disease comprises compound of Formula (I), e.g., Compound 1-A in an amount of about 125 mg of compound of Formula (I), e.g., Compound 1-A.

[0152] The therapeutically effective amount of compound of Formula (I), e.g., Compound 1-A may be administered to the subject by any administration route. For example, in one embodiment, compound of Formula (I), e.g., Compound 1-A is administered to the subject is by oral administration, e.g., in one or more tablets.

[0153] In certain embodiments, the therapeutically effective amount of compound of Formula (I), e.g., Compound 1-A is administered to the subject once per day.

[0154] In certain embodiments, the therapeutically effective amount of compound of Formula (I), e.g., Compound 1-A is about 25 mg / day to about 750 mg / day. In certain embodiments, the therapeutically effective amount of compound of Formula (I), e.g., Compound 1-A is about 25 mg / day, about 50 mg / day, about 75 mg / day, about 100 mg / day, about 125 mg / day, about 200 mg / day, about 250 mg / day, about 375 mg / day, about 500 mg / day, about 625 mg / day, or about 750 mg / day. In certain embodiments, the therapeutically effective amount of compound of Formula (I), e.g., Compound 1-A is about 25 mg / day to about 250 mg / day. In certain embodiments, the therapeutically effective amount of compound of Formula (I), e.g., Compound 1-A is about 25 mg / day, about 50 mg / day, about 75 mg / day, about 100 mg / day, about 125 mg / day, about 200 mg / day, or about 250 mg / day.8. EXAMPLESExample 1: PRO-C3 Expression Levels in AATD Liver Disease

[0155] Plasma concentrations of PRO-C3 were measured in blood samples collected from the following subjects: healthy subjects, subjects with PiMZ genotype, subjects with PiZZ genotype, subjects with a PiZZ genotype also having cirrhosis or hepatitis, and subjects with a PiZZ genotype with other liver disease.

[0156] Protein expression levels of the liver fibrosis biomarker PRO-C3 were elevated in PiZZ subjects with cirrhosis / hepatitis, as shown in FIG. 1. In FIG. 1, box edges are the 25thand 75thpercentiles, the solid line is the median, + represents the mean, whiskers are the minimum and maximum values, and additional symbols are outliers. Dotted black lines represent the range (min, max) of the respective PRO-C3 levels for the F0 / 1 (least severe) and F4 (most severe) fibrosis stages as described by Erhardtsen et al. JHEP Rep.2021;3: 100317. Significance (versus healthy subjects) was determined using a one-way ANOVA Tukey’s multiple comparisons test. Double asterisks (**) represents P < 0.01.37NAI-5005346498vlExample 2: Normalization of Certain Biomarkers by Treatment with Compound 1-A

[0157] Female PiZ mice (transgenic mouse model with E342K mutation) or WT littermates were assigned to untreated baseline control groups or treatment groups at approximately 12 weeks of age. Baseline measurements of plasma FVII, PDCD6, APOCI, and APOC3 were measured in all control and treatment groups. Animals were dosed twice daily orally (gavage) with 0 (vehicle), 50 mg / kg / dose, or 100 mg / kg / dose BID (two times per day) with Compound 1-A (0, 100 mg / kg / dose, or 200 mg / kg / day). In addition to collection at baseline, blood samples were also collected on day 5, day 20, and day 30.

[0158] 20 pL plasma of each provided mouse plasma sample was depleted using a Mouse-3 Multiple Affinity Removal Spin Cartridge (Agilent) according to the manufacturer's instructions. Digestion was carried out on single filter units (Sartorius Vivacon500, 30'000 MWCO HY) following a modified FASP protocol (described by the Max Planck Institute of Biochemistry, Martinsried, Germany). The depleted samples were denatured with Biognosys' Denature Buffer and reduced / alkylated using Biognosys' Reduction / Alkylation Solution for 30 min at 37°C. Subsequently, digestion to peptides was carried out using 1 pg trypsin (Promega) per sample overnight at 37°C. Peptides were desalted using a Cl 8 MicroSpin plate (The Nest Group) according to the manufacturer's instructions and dried down using a SpeedVac system. Peptides were resuspended in 1% acetonitrile and 0.1% formic acid (FA) and spiked with Biognosys’ iRT kit calibration peptides. Peptide concentrations were determined using a UV / VIS Spectrometer at 280nm (SPECTROstar Nano, BMG Labtech). The stable-isotope standard (SIS) peptides shown below in Table 1 were spiked into the final peptide samples at known concentration (20 fmol).Table 1: SIS Peptides

[0159] HRP Fractionation: Peptides from each sample were pooled. Ammonium hydroxide was added to a pH value > 10. The fractionation was performed using a Dionex UltiMate 3000 RS pump (Thermo Scientific) on an Acquity UPLC CSH C18 1.7 pm, 2.1 x 150 mm column (Waters). The gradient was 1% to 40% solvent B in 20 min. Solvents were: A: 20 mM ammonium formate in water; and B: acetonitrile. Fractions were taken every 30 seconds and sequentially pooled to 8 fraction pools. These were dried down and resuspended 38NAI-5005346498vlin LC solvent A. Prior to mass spectrometric analyses, they were spiked with Biognosys' iRT kit calibration peptides. Peptide concentrations were determined using a UV NIS Spectrometer at 280 nm (SPECTROstar Nano).

[0160] Shotgun LC-MS / MS for spectral library generation: For data-dependent acquisition (DDA) LC-MS / MS measurements, 1 pg of peptides per sample was injected to an packed reversed phase column (PicoFrit emitter with 75 pm inner diameter, 60 cm length and 10 pm tip from New Objective, packed with 1.7 pm Charged Surface Hybrid Cl 8 particles from Waters) on a Thermo Scientific™ EASY-nLC™ 1200 nano-liquid chromatography system connected to a Thermo Scientific™ Q Exactive™ HF-X mass spectrometer equipped with a Nanospray Flex™ Ion Source. LC solvents were A: water with 0.1% FA and B: 20% water in acetonitrile with 0.1% FA. The nonlinear LC gradient was 1% - 59% solvent B in 85 minutes followed by 59- 90% B in 10 seconds, 90% B for 8 minutes, 90% - 1% B in 10 seconds and 1% B for 5 minutes at 60°C and a flow rate of 250 nL / min. A modified TOP15 method from Kelstrup was used (Kelstrup et al., Journal of Proteome Research 2018, 17, 727-738). The full range MSI scan covered the m / z range of 350-1650 with a resolution of 60'000 and was followed by 15 data dependent MS2 scans with a resolution of 15'000. The MS2 acquisition precursor isolation width was 4 m / z while normalized collision energy was centered at 27% and the default charge state was 2+

[0161] The mass spectrometric data were analyzed using Biognosys' search engine SpectroMine™ (version 1.0.15606), the false discovery rate on peptide and protein level was set to 1%. A mouse UniProt .fasta database (Mus musculus, 2020-07-01), a UniProt.fasta for the canonical human SERPINA1 sequence and a UniProt .fasta for the human z-mutant SERPINA1 sequence were used for the search engine, allowing for 2 missed cleavages and variable modifications (N-term acetylation, methionine oxidation, deamidation (NQ) and carb amyl ati on (KR)).

[0162] HRM Mass Spectrometry: For data-independent acquisition (DIA) LC-MS / MS measurements, 1 pg of peptides per sample were injected to an in-house packed reversed phase column (PicoFrit emitter with 75 pm inner diameter, 60 cm length and IO pm tip from New Objective, packed with 1.7 pm Charged Surface Hybrid Cl 8 particles from Waters) on a Thermo Scientific™ EASY-nLC™ 1200 nano-liquid chromatography system connected to a Thermo Scientific™ Q Exactive™ HF-X mass spectrometer equipped with a Nanospray Flex™ Ion Source. LC solvents were A: water with 0.1% FA; B: 20% water in acetonitrile with 0.1% FA The nonlinear LC gradient was 1 - 59% solvent B in 85 minutes followed by 59 - 90% B in IO seconds, 90% B for 8 minutes, 90% - 1% B in 10 seconds and 1% B for 539NAI-5005346498vlminutes at 60°C and a flow rate of 250 nL / min. The DIA method consisted of one full range MSI scan and 29 DIA segments were adopted from Bruderer et al. (Bruderer et al., Molecular and Cellular Proteomics, 2017, 12:2296-2309).

[0163] HRM mass spectrometric data were analyzed using Spectronaut™ Pulsar software (Biognosys). The false discovery rate on peptide and protein level was set to 1%, data was filtered using row-based extraction. The assay library (protein inventory) generated in this project was used for the analysis. The HRM measurements analyzed with Spectronaut were normalized using local regression normalization (Callister et al., J Proteome Res 2006, 5(2):277-86). Signal processing and data analysis for the absolute quantification of SERPINAI peptides were carried out using SpectroDive™ 9.0 software (Biognosys) for multiplexed MRM / PRM data analysis based on mProphet (Reiter, et al., Nature Methods, 2011, 8:430-435).

[0164] Data Analysis: HRM mass spectrometric data were analyzed using Spectronaut™ Pulsar software (Biognosys). The false discovery rate on peptide and protein level was set to 1%, data was filtered using row-based extraction. The assay library (protein inventory) generated in this project was used for the analysis. The HRM measurements analyzed with Spectronaut were normalized using local regression normalization (Callister et al.). Signal processing and data analysis for the absolute quantification of SERPINAI peptides were carried out using SpectroDive™ 9.0 software (Biognosys) for multiplexed MRM / PRM data analysis based on mProphet.

[0165] Differential protein abundance was calculated for all combinations of sample groups and differentially regulated proteins were identified using a two sample Student’s t test having a threshold q-value less than 0.05 and an average fold change of greater than 1.5.

[0166] Changes in expression levels of F VII are shown in FIG. 2. FVII is a coagulation protein produced and secreted by the liver. As can be seen in FIG. 2, the PiZZ genotype was associated with significantly reduced FVII protein levels, but after treatment with Compound 1-A, these levels were significantly increased.

[0167] Changes in expression levels of PDCD6 are shown in FIG. 3. PDCD6 is a calcium-binding protein that regulates membrane trafficking and is involved in apoptosis and vesicular transport. As can be seen in FIG. 3, the PiZZ genotype was associated with significantly elevated PDCD6 protein levels, but after treatment with Compound 1-A, these levels were significantly reduced.

[0168] Changes in expression levels of APOCI are shown in FIG. 4. Changes in expression levels of APOC3 are shown in FIG. 5. The liver synthesizes and secretes40NAI-5005346498vlapolipoproteins that transport lipids through the blood. As can be seen in FIGs. 4 and 5, the PiZZ genotype was associated with lower levels of apolipoproteins, including AP0C1 and AP0C3. The PiZZ genotype was associated with significantly reduced AP0C1 and AP0C3 protein levels, but after treatment with Compound 1-A, these levels were significantly increased.

[0169] The normalization of biomarker protein expression levels after treatment with Compound 1-A was accompanied by an overall improvement in the disease state of the mouse, including significant reduction in Z-AAT polymer liver levels (and conversely, a higher level of Z-AAT monomer levels) and an improvement in fibrosis assessment.

[0170] As seen in FIGs. 2-5, PiZ mice had altered levels of F VII, PDCD6, APOCI, and APO3 as compared to WT controls and treatment with Compound 1-A normalized the expression levels of these biomarkers in the PiZ mice to match more closely that of the WT control. The altered plasma concentration of each of these biomarkers observed in PiZ mice was also observed in humans, as shown in FIGs. 5, 6, 7, and 8.

[0171] In each of FIGs. 2-7, significance (versus WT mice) was determined using a oneway ANOVA Dunnett’s multiple comparisons test. Protein intensity is a relative abundance unit that equals the sum of ion peaks (area under the curve) for the three most abundant peptides associated with FVII. Treatment with Compound 1-A lasted 30 days. Baseline “+” represents the samples taken at baseline. Asterisks have the following meanings: * = P < 0.05; ** = P < 0.01; *** = P < 0.001; and **** = P < 0.0001.

[0172] Other proteins that may be associated with AATD liver disease and that also had altered protein expression levels in the plasma of AATD individuals include TIMP-1, CO4A6, IGF2, IGF1R, IGFBP3, IGFBP5, LEPR, ICAM1, VCAM1, APOA5, APOC2, APOC4, APOF, APOL1, APOM, PDCD6IP, CHMP4B, VPS4B, PSMD5, PSMD9, PSDM10, OTUD6B, andPSMGl.Example 3: A Phase 1, Randomized, Double-Blind, Placebo-Controlled, Single Oral Dose Study Evaluating the Safety, Tolerability, Pharmacokinetics (PK), and Pharmacodynamics of Compound 1-A in Human Subjects with AATD Liver Disease

[0173] In this Phase 1, Randomized, Double-Blind, Placebo-Controlled, Single Oral Dose Study Evaluating the Safety, Tolerability, Pharmacokinetics (PK), and Pharmacodynamics of Compound 1-A in adult participants homozygous for the Z mutation of the alpha 1 antitrypsin (AAT) gene (hereafter referred to as protease inhibitor [Pi] ZZ [PiZZ]) or heterozygous for the Z mutation (hereafter referred to PiMZ) with presumed or biopsy-confirmed metabolic dysfunction-associated steatohepatitis (MASH).41NAI-5005346498vl

[0174] The primary, secondary, and exploratory objectives and corresponding endpoints are presented in Table 2 below.42NAI-5005346498vl

[0175] The study includes one dose cohort consisting of two groups: participants with the PiZZ genotype and participants with the PiMZ genotype and MASH. Participants enter the study following a 28 to 42-day screening period, are randomized, treated on Day 1, and followed up until Day 36.

[0176] The inclusion / exclusion criteria of the study set a liver elastography upper limit of 10 kPa to exclude participants with cirrhosis. It is important to note that there is no consensus on a single cut-off value for cirrhosis, and kPa values correlating with specific fibrosis stages vary. In one study, a 9.4 kPa cut-off was associated with > 90% sensitivity, specificity, and 94% accuracy in negatively predicting cirrhosis (see, e.g., Vigand M et al, Aliment Pharmacol Ther, 2011, 34:353-362). A meta-analysis reported higher cut-offs for cirrhosis (i.e., greater than 10 kPa; see, e.g., Kwok R et al., Aliment Pharmacol Ther, 2014, 39:254 269).

[0177] Inclusion Criteria: PiZZ Group

[0178] Participants who meet all of the following criteria are eligible to participate in the study:1. Participants must have confirmation of PiZZ genotype at screening and METAVIR fibrosis stage < 4 defined by one of the criteria below:a. Liver elastography with kPa < 10.b. Recent liver biopsy (within past 6 months) documenting METAVIR fibrosis stage < 4 (locally read).2. Females and males, of any race, 18 to 70 years of age, inclusive, at screening.3. Female participants of childbearing potential (i.e., ovulating, premenopausal, and not permanently surgically sterile) with male partners and all male participants with female partners of childbearing potential are included if they agree to use a highly effective contraceptive regimen during their participation in the study and for 30 days after the last administration of study drug.4. Female participants of childbearing potential must have a negative serum pregnancy test result at screening and a negative urine pregnancy test pre-dose (Day -1) and must not be pregnant, lactating, or planning to become pregnant during their participation in the study.43NAI-5005346498vl5. Female participants of nonchildbearing potential are included if they meet the following definition of nonchildbearing potential: are either surgically sterile (hysterectomy, bilateral salpingectomy, and / or bilateral oophorectomy at least 26 weeks before screening) or postmenopausal, defined as spontaneous amenorrhea for at least 1 year with follicle stimulating hormone in the postmenopausal range at screening, based on the central laboratory’s ranges.6. Participants must agree to abstain from sperm or egg donation through 90 days after administration of the last dose of study drug.7. Participants must have a body mass index (BMI) between 18.0 and 32.0 kg / m2, inclusive, at screening and body weight between 50 and 110 kg, inclusive, at screening.8. Participants must comprehend and sign an Informed Consent Form (ICF) in their language and abide by the study restrictions.9. Participants must be nonsmokers, defined as not using tobacco or nicotine-containing products for at least 6 months prior to screening.

[0179] Exclusion Criteria: PiZZ Group

[0180] Participants who meet any of the following criteria are excluded from participation in the study:1. International normalized ratio (INR) > 1.2.2. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels > 125 U7L at screening (one retest permitted).3. Total bilirubin > upper limit of normal (ULN) (normal range 0.2 to 1.2 mg / dL).Participants with Gilbert’s syndrome are allowed if they have a known history of Gilbert’s syndrome with a normal direct bilirubin value and normal reticulocyte count, and upon review by the Medical Monitor.4. Vibration-controlled transient elastography (VCTE) ( / .<?., FibroScan®) score > 10 kPa or presence of cirrhosis on liver biopsy.5. Estimated glomerular filtration rate < 90 mL / min / 1.73 m2based on Chronic Kidney Disease Epidemiology Collaboration.6. Current use of AAT augmentation therapy or recent use of AAT augmentation therapy defined as less than 5 half-lives from last AAT Augmentation therapy.7. Participants with abnormal spirometry according to 2021 European Respiratory Society / American Thoracic Society criteria (Stanojevic 2022). Abnormal spirometry is defined as post-bronchodilation FEV1 % predicted value below the lower limit of 44NAI-5005346498vlnormal (< 5th centile corresponding to a z-score of < -1.645) based on Global Lung Function Initiative reference equations for spirometry.8. Participants with recent (last 3 months) diagnosis of pneumonia.9. History of significant alcohol consumption for a period of more than 3 consecutive months within 1 year before screening. Significant alcohol consumption is defined by alcohol consumption of > 14 units per week for males and > 7 units per week for females. One unit of alcohol equals 12 oz (360 mL) beer, 1.5 oz (45 mL) liquor, or 5 oz (150 mL) wine.10. Human immunodeficiency virus (HIV) infection, as shown by the presence of anti-HIV antibody (seropositive).11. Any active hepatitis including those seropositive for hepatitis B virus (hepatitis B surface antigen positive at screening) or hepatitis C virus (HCV) (detectable HCV antibody at screening).12. Positive test result for severe acute respiratory syndrome coronavirus 2 at Day -1 (retests permitted)13. Clinically significant electrocardiogram (ECG) abnormalities or heart rate-corrected QT interval using Fridericia’s formula (QTcF) > 450 ms for males and > 470 ms for females at either screening or Baseline, or any prior history of QT abnormality.14. Significant history or clinical manifestation of any allergic, dermatological, renal, hematological, cardiovascular, gastrointestinal, neurological, respiratory, endocrine, or psychiatric disorder that would make it difficult to comply with protocol requirements as determined by the investigator.15. History of significant hypersensitivity, intolerance, or allergy to any drug compound, food, or other substance, unless approved by the investigator.16. History of stomach or intestinal surgery or resection that would potentially alter absorption and / or excretion of orally administered drugs (uncomplicated appendectomy and hernia repair are allowed). No type of bariatric surgery is allowed.17. Positive urine, drug, or breath alcohol test result at screening or Day -1.18. Loss or donation of > 400 mL of blood or blood products within 30 days prior to the first dose of study drug.19. Participation in an investigational new drug trial in the 60 days or five half-lives, whichever is longer, before randomization.20. Hepatic decompensation or impairment defined as presence of any of the following:a. History of esophageal varices, ascites, or hepatic encephalopathy.45NAI-5005346498vlb. Serum albumin < 3.5 g dL-1, except as explained by non-hepatic causes.c. INR > 1.4 unless due to therapeutic anticoagulants or laboratory error. Note: INR may be repeated once to reassess eligibility.d. Total bilirubin > 1.5 x ULN = 1.2 mg dL-1. Note: patients with Gilbert syndrome are eligible with a total bilirubin above 1.5 x ULN if reticulocyte count is within normal limits (typically 0.5-2.5%), hemoglobin is within normal limits unless due to chronic anemia and unrelated to hemolysis (typically 13.5-17.5 g dL1for men; 12.0-15.5 g dL1for women) and direct bilirubin is < 20% of total bilirubin. 21. HbAlc > 9.0%.22. Uncontrolled hypertension (either treated or untreated) defined as systolic blood pressure > 170 mm Hg or a diastolic blood pressure > 100 mm Hg at screening.23. Myocardial infarction, unstable angina, percutaneous coronary intervention, coronary artery bypass graft or stroke within 12 weeks before randomization.24. Use of medications that are metabolized and eliminated by cytochrome P450 (CYP) 2B6, CYP3A4 / 5, CYP2C9, or CYP2C8 within 14 days or five half-lives, whichever is longer, prior to dose administration.

[0181] Inclusion Criteria: PiMZ / MASH Group

[0182] Participants who meet all of the following criteria are eligible to participate in the study:1. Have confirmation of PiMZ genotype at screening and meet one of the following criteria:a. Presumed MASH with liver elastography with kPa > 5.5 and < 10; controlled attenuation parameter > 280 dB m1AND the presence of 2 out of 4 components of metabolic syndrome (obesity, dyslipidemia, elevated blood pressure, elevated fasting glucose) or Type 2 diabetes.b. Biopsy-confirmed MASH with a recent liver biopsy (within past 6 months) documenting MASH with METAVIR fibrosis stage > 1 and < 4 and a nonalcoholic fatty liver disease activity score (NAS) of > 4 with at least a score of 1 in each of the following NAS components (locally read):- Steatosis (scored 0 to 3)- Ballooning degeneration (scored 0 to 2), and- Lobular inflammation (scored 0 to 3).46NAI-5005346498vlNote: Liver elastography does not need to be repeated at screening if conducted at prescreening and / or a historical liver elastography was conducted in the previous 3 months. A historic liver biopsy must not be accompanied by a significant decrease in body weight > 5% since the biopsy or treatment with a new concomitant medication that might affect steatosis.2. PiZZ Inclusion Criteria: 2, 3, 4, 5, 6, 8, 9.3. Participants must have a BMI between 18.0 and 38 kg / m2, inclusive, at screening and a stable body weight between 50 and 120 kg, inclusive, at screening.

[0183] Exclusion Criteria: PiMZ / MASH Group

[0184] Participants who meet any of the following criteria are excluded from participation in the study:1. PiZZ Exclusion Criteria: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24.2. Regular use of drugs historically associated with MASLD, which include, but are not limited, to the following: amiodarone, methotrexate, systemic glucocorticoids at greater than 5 mg / d, tamoxifen, estrogens at doses greater than those used for hormone replacement or contraception, anabolic steroids except testosterone replacement, valproic acid and known hepatotoxins for more than 4 weeks within the last 8 weeks before the initial screening.3. Glucagon-like peptide 1 (GLP-1) agonist unless stable dose for 24 weeks prior to screening.

[0185] Participants are instructed not use any concomitant medications that are primarily metabolized and eliminated by CYP2B6, CYP3A4 / 5, CYP2C9, or CYP2C8 within 14 days or five half-lives, whichever is longer, prior to administration of Compound 1-A. Examples of restricted medications include, but are not limited to, bupropion, efavirenz, repaglinide, montelukast, pioglitazone, rosiglitazone, resmetirom, celecoxib, glimepiride, phenytoin, tolbutamide, warfarin, alfentanil, alprazolam, aprepitant, atorvastatin, avanafil, budesonide, buspirone, colchicine, conivaptan, darifenacin, darunavir, dasatinib, dronedarone, eletriptan, eliglustat, eplerenone, everolimus, felodipine, ibrutinib, indinavir, isavuconazole, ivabradine, lemborexant, lomitapide, lovastatin, lurasidone, maraviroc, midazolam, mobocertinib, naloxegol, nisoldipine, pimozide, quetiapine, rilpivirine, rivaroxaban, saquinavir, sildenafil, simvastatin, sirolimus, tacrolimus, tadalafil, ticagrelor, tipranavir, tolvaptan, triazolam, vardenafil, and venetoclax.47NAI-5005346498vl

[0186] For PiMZ / MASH participants, the regular use of the drug historically associated with MASLD is prohibited. In addition, GLP-1 agonist is prohibited unless the participant is on a stable dose for 24 weeks prior to screening.

[0187] An AE is any untoward medical occurrence in a clinical study participant, temporally associated with the use of study treatment, whether or not considered related to the study treatment. Assessment of intensity for each AE and SAE reported during the study is assigned one of the following categories (as defined in CTCAE version 5.0):• Grade 1: Mild: asymptomatic or mild symptoms; clinical or diagnostic observations only; intervention not indicated.• Grade 2: Moderate: minimal, local, or noninvasive intervention indicated; limiting age-appropriate instrumental activities of daily living (ADL) (e.g., preparing meals, shopping for groceries or clothes, using the telephone, managing money).• Grade 3 : Severe or medically significant but not immediately life-threatening: hospitalization or prolongation of hospitalization indicated; disabling; limiting self- care ADL (e.g., bathing, dressing / undressing, feeding oneself, using the toilet, taking medications, not bedridden).• Grade 4: Life threatening consequences; urgent intervention indicated.• Grade 5: Death related to AE.

[0188] An SAE is defined as any untoward medical occurrence that, at any dose, meets one or more of the criteria listed:• Results in death• Is life-threatening. The term ‘life-threatening’ in the definition of ‘serious’ refers to an event in which the participant was at risk of death at the time of the event. It does not refer to an event that hypothetically might have caused death if it were more severe.• Requires inpatient hospitalization or prolongation of existing hospitalization.In general, hospitalization signifies that the participant has been admitted to the hospital for inpatient care, either to the inpatient ward or to the emergency room for observation and / or treatment, which would not have been appropriate in the physician’s office or outpatient setting. Complications that occur during hospitalization are AEs. If a complication prolongs hospitalization or fulfills any other serious criteria, the event is serious. When in doubt as to whether “hospitalization” occurred or was necessary, the AE should be considered serious. Hospitalization for48NAI-5005346498vlelective treatment of a pre-existing condition that did not worsen from baseline is not considered an AE.• Results in persistent disability / incapacity. The term disability means a substantial disruption of a person’s ability to conduct normal life functions. This definition is not intended to include experiences of relatively minor medical significance such as uncomplicated headache, nausea, vomiting, diarrhea, influenza, and accidental trauma (e.g., sprained ankle) which may interfere with or prevent everyday life functions but do not constitute a substantial disruption.• Is a congenital anomaly / birth defect in the offspring of participant• Is a suspected transmission of any infectious agent via an authorized medicinal product• Other situations. Medical or scientific judgment is exercised in deciding whether SAE reporting is appropriate in other situations such as important medical events that may not be immediately life-threatening or result in death or hospitalization but may jeopardize the participant or may require medical or surgical treatment to prevent one of the other outcomes listed in the above definition. These events are usually be considered serious.

[0189] For this study, AESIs include clinically significant increases in liver function tests (confirmed with repeat testing), in the absence of alternative explanations, defined as:• levels > 5 x ULN for AST or ALT• levels > 3 x ULN for total bilirubin; or• simultaneous > 3 x ULN for AST or ALT and > 2 x ULN for total bilirubin

[0190] Additional AESI include worsening of pulmonary function tests with a >15% decline in FEV1 (measured post-bronchodilator).

[0191] In dose escalation, there is an interval of at least 14 days between doses, to allow for review of cumulative safety data through Day 15. Dose escalation is paused if a participant experiences any of the following DLT criteria up until the date of the last protocol-specified visit / assessment:• any serious adverse events• any severe adverse events (excluding National Cancer Institute Common Terminology Criteria for Adverse Events Grade 3 or 4 laboratory elevations which are not clinically significant)49NAI-5005346498vl• clinically significant increases in liver function tests (confirmed with repeat testing), in the absence of alternative explanations, defined as > 5 * ULN for AST or ALT, > 3 x ULN for total bilirubin, or the combination of levels > 3 * ULN for AST or ALT and > 2 x ULN for total bilirubin; or• decline in forced expiratory volume in 1 second (FEV1) of > 15% (must be confirmed on repeat measurement post-bronchodilator) with symptoms of breathlessness or decline of FEV1 of >20% (must be confirmed on repeat measurement post bronchodilator) regardless of concurrent symptoms

[0192] Pulmonary function testing is performed at screening, and at the follow up visits on Day 8 and Day 36. Testing is performed post-bronchodilator using spirometry, and the absolute values of forced vital capacity (FVC) and FEV1 is also reported as percentages of predicted values.

[0193] Although administration of Compound 1-A at 250 mg is expected to saturate plasma AAT binding in PiZZ / PiMZ participants, resulting in near complete AAT activity inhibition, this is not anticipated to have an impact on lung disease development for at least the following reasons:i) Lung disease development takes decades to manifest clinically, even in the presence of severe AAT deficiency;ii) Only a single dose is administered to participants; andiii) Z-AAT depletion in severely deficient PiZZ individuals with Fazirsiran was not associated with major pulmonary adverse events or forced expiratory volume in 1 second (FEV1) decline over 24 to 48 weeks of treatment (see, e.g., ClinicalTrial.gov identifier NCT 03946449).

[0194] Twelve-lead ECGs are performed at screening, pre-dose, and 1-, 2-, 4-, 8-, and 24-hours post-dose, and at the follow-up visits on Day 8 and Day 36. All 12-lead ECGs are performed after the participant has been in the supine position for at least 5 minutes.Ambulatory ECG monitoring through use of a Holter recorder or cardiac telemetry are performed starting 1 hour pre-dose on Day 1 and continue for 8 hours after study drug administration. During this time, the 12-lead ECGs are obtained from the continuous 12-lead ECG system monitor, unless a 12-lead ECG in the standard limb lead position is required.

[0195] Blood samples are collected for measurement of PK and PD parameters. PD assessments include measurement of plasma total AAT protein levels, plasma Z-AAT protein50NAI-5005346498vllevels, proportion of Compound 1-A bound to Z-AAT / M-AAT protein in plasma, activity of circulating AAT against neutrophil elastase, and AAT polymer levels in plasma.

[0196] Blood and urine samples are also collected to evaluate biochemical, molecular, cellular, and genetic / genomic aspects relevant to AATD. Biomarkers of particular interest include, e.g., liver health biomarkers such as Pro-C3, PDCD6, APOCI, APOC3, M30, M65, and FVII.Example 4: A Phase 2, Randomized, Double-Blind, Placebo-Controlled, Multiple Ascending Dose Study Evaluating the Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of Compound 1-A in PiZZ and PiMZ / MASH Adult Participants

[0197] This Phase 2, randomized, double-blind, placebo-controlled, multiple ascending dose study evaluates the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of Compound 1-A in adult participants: (a) with alpha- 1 antitrypsin (AAT) deficiency (AATD) associated liver disease resulting from homozygosity for the Z mutation of the SERPINA1 gene (hereafter referred to as protease inhibitor [Pi] ZZ [PiZZ]); and (b) with alpha- 1 antitrypsin (AAT) deficiency (AATD) associated liver disease resulting from the heterozygosity for the Z mutation of the SERPINA1 gene (hereafter referred to as protease inhibitor [Pi] MZ [PiMZ]) with presumed or biopsy confirmed metabolic dysfunction-associated steatohepatitis (MASH).

[0198] The primary and secondary objectives and corresponding endpoints are presented in Table 3 below.51NAI-5005346498vl

[0199] Doses to be evaluated are informed by findings in healthy participants from single ascending dose and multiple ascending dose studies of Compound 1-A and from the PiZZ and PiMZ / MASH participants from Example 3. Doses and dosing regimen are selected such that expected total systemic steady-state exposures achieved in PiZZ and PiMZ / MASH participants do not exceed exposures shown to be safe and well tolerated in the multiple ascending dose part of the study in healthy participants. The extent of accumulation of drug with repeat dosing and PK / PD differences between healthy participants and PiZZ and PiMZ / MASH participants (from Example 3) are taken into account to determine the anticipated total systemic steady-state exposures. Dose escalations are not greater than 3 -fold between groups.

[0200] Inclusion Criteria

[0201] Participants who meet all of the following criteria are eligible to participate in the study:1. Have confirmation ofa. PiZZ genotype at screening and METAVIR fibrosis stage < 4 defined by:• Liver elastography with kPa < 1052NAI-5005346498vl• Recent liver biopsy (within past 6 months) documenting METAVIR fibrosis stage < 4 (locally read); orb. PiMZ genotype at screening and meet 1 of the following criteria:• Presumed MASH with liver elastography with kPa > 7 and < 12; controlled attenuation parameter > 280 dB m-1 and the presence of 2 out of 4 components of metabolic syndrome (obesity, dyslipidemia, elevated blood pressure, elevated fasting glucose) or Type 2 diabetes.• Biopsy-confirmed MASH with a recent liver biopsy (within past 6 months) documenting MASH with METAVIR fibrosis stage > 1 and < 4 and a nonalcoholic fatty liver disease activity score (NAS) of > 4 with at least a score of 1 in each of the following NAS components (locally read):o Steatosis (scored 0 to 3)o Ballooning degeneration (scored 0 to 2), ando Lobular inflammation (scored 0 to 3).2. Females and males, of any race, 18 to 70 years of age, inclusive, at screening3. Female participants of childbearing potential ( / .<?., ovulating, premenopausal, and not permanently surgically sterile) with male partners and all male participants with female partners of childbearing potential who agree to use a highly effective contraceptive regimen during their participation in the study and for 30 days after the last administration of study drug.4. Female participants of childbearing potential with a negative serum pregnancy test result at screening and a negative urine pregnancy test pre dose (Day -1) and not pregnant, lactating, or planning to become pregnant during their participation in the study.5. Female participants of nonchildbearing potential who meet the following definition of nonchildbearing potential: are either surgically sterile (hysterectomy, bilateral salpingectomy, and / or bilateral oophorectomy at least 26 weeks before screening) or postmenopausal, defined as spontaneous amenorrhea for at least 1 year with follicle stimulating hormone in the postmenopausal range at screening, based on the central laboratory’s ranges.6. Agree to abstain from sperm or egg donation through 90 days after administration of the last dose of study drug.53NAI-5005346498vl7. Have a body mass index between 18.0 and 32.0 kg / m2, inclusive, at screening and body weight between 50 and 110 kg, inclusive, at screening.8. Comprehend and sign an Informed Consent Form in their language and abide by the study restrictions.9. Are nonsmokers, defined as not using tobacco or nicotine-containing products for at least 6 months prior to screening. A history of daily cigarette use for < 24 months is permitted. A cotinine test is administered at the screening visit and subsequently on a weekly basis throughout the treatment period to monitor and assess smoking status.

[0202] Exclusion Criteria

[0203] Participants are excluded from the study if any of the following criteria apply:1. International normalized ratio (INR) > 1.22. ALT and AST levels > 125 U / L at screening for PiZZ subjects or > 150 U / L for PiMZ / MASH subjects.3. Total bilirubin (TB) >ULN (normal range 0.2 to 1.2 mg / dL). Participants with Gilbert’s syndrome are allowed if they have a known history of Gilbert’s syndrome with a normal direct bilirubin value and normal reticulocyte count, and upon review by the medical monitor.4. Vibration-controlled transient elastography ( / .<?., FibroScan®) score > 10 kPa for PiZZ subjects or presence of cirrhosis on liver biopsy or > 12 kPa for PiMZ / MASH subjects or presence of cirrhosis on liver biopsy.5. Estimated glomerular filtration rate <60 mL / min / 1.73 m2 based on Chronic Kidney Disease Epidemiology Collaboration.6. Current use of AAT augmentation therapy or recent use of AAT augmentation therapy defined as less than 5 half-lives from last AAT augmentation therapy.7. Abnormal spirometry according to 2021 European Respiratory Society / American Thoracic Society criteria. Abnormal spirometry is defined as post bronchodilation forced expiratory volume in 1 second (FEV1) % predicted value below the lower limit of normal (< 5th centile corresponding to a z-score of < -1.645) based on Global Lung Function Initiative reference equations for spirometry. For PiMZ / MASH subjects, a FEV1 <90% of predicted for nonsmokers and FEV1 <80% of predicted for active smokers or previous smokers with a history of heavy smoking (history of daily cigarette use for > 24 months) at screening.8. Recent (last 3 months) diagnosis of pneumonia.54NAI-5005346498vl9. History of significant alcohol consumption for a period of more than 3 consecutive months within 1 year before screening. Significant alcohol consumption is defined by alcohol consumption of > 14 units per week for males and > 7 units per week for females. One unit of alcohol equals 12 oz (360 mL) beer, 1.5 oz (45 mL) liquor, or 5 oz (150 mL) wine.10. Human immunodeficiency virus (HIV) infection, as shown by the presence of antiHIV antibody (seropositive).11. Any active hepatitis including those seropositive for hepatitis B virus (hepatitis B surface antigen positive at screening) or hepatitis C virus (HCV) (detectable HCV antibody at screening).12. Positive test result for severe acute respiratory syndrome coronavirus 2 at Day -1 13. Clinically significant electrocardiogram abnormalities or heart rate-corrected QT interval using Fridericia’s formula > 450 ms for males and > 460 ms for females at either screening or baseline, or any prior history of QT abnormality.14. Significant history or clinical manifestation of any allergic, dermatological, renal, hematological, cardiovascular, gastrointestinal, neurological, respiratory, endocrine, or psychiatric disorder that would make it difficult to comply with protocol requirements as determined by the investigator.15. History of significant hypersensitivity, intolerance, or allergy to any drug compound, food, or other substance, unless approved by the investigator.16. History of stomach or intestinal surgery or resection that would potentially alter absorption and / or excretion of orally administered drugs (uncomplicated appendectomy and hernia repair are allowed). No type of bariatric surgery is allowed.17. Positive urine drug or breath alcohol test result at screening or Day -1.18. Loss or donation of > 400 mL of blood or blood products within 30 days prior to the first dose of study drug.19. Participation in an investigational new drug trial in the 60 days or 5 half-lives, whichever is longer, before randomization.20. Hepatic decompensation or impairment defined as presence of any of the following:a. History of esophageal varices, ascites, or hepatic encephalopathyb. Serum albumin < 3.5 g dL-1, except as explained by non-hepatic causes.c. INR > 1.2 unless due to therapeutic anticoagulants or laboratory error. Note: INR may be repeated once to reassess eligibility.55NAI-5005346498vld. TB > ULN (normal range 0.2 to 1.2 mg / dL). Participants with Gilbert’s syndrome are allowed if they have a known history of Gilbert’s syndrome with a normal direct bilirubin value and normal reticulocyte count, and upon review by the medical monitor.21. A hemoglobin Ale > 9.0%.22. Uncontrolled hypertension (either treated or untreated) defined as systolic blood pressure > 140 mm Hg or a diastolic blood pressure > 90 mm Hg at screening.23. Myocardial infarction, unstable angina, percutaneous coronary intervention, coronary artery bypass graft or stroke within 12 weeks before randomization.24. For PiMZ / MASH subjects: Regular use of drugs historically associated with metabolic dysfunction associated steatotic liver disease, which include, but are not limited, to the following: amiodarone, methotrexate, systemic glucocorticoids at greater than 5 mg / d, tamoxifen, estrogens at doses greater than those used for hormone replacement or contraception, anabolic steroids except testosterone replacement, valproic acid and known hepatotoxins for more than 4 weeks within the last 8 weeks before the initial screening.25. Use of drugs that are primarily metabolized and eliminated by cytochrome P450 (CYP) 2B6, CYP3 A4 / 5, CYP2C9, or CYP2C8 within 14 days prior to dose administration.26. For PiMZ / MASH subjects: Use of glucagon-like peptide 1 agonist unless stable dose for 24 weeks prior to screening.

[0204] Dose escalation is paused if a participant experiences any of the following dose-limiting toxicity (DLT) criteria within the 28-days treatment period and a further 14-day observation period:• Any serious adverse events• Any severe adverse events (excluding CTCAE Grade 3 or 4 laboratory elevations that are not clinically significant).• Clinically significant increases in serum creatinine, defined asi. For PiZZ subjects: > 1.5 x participant’s baseline value which is known or presumed to have occurred within the prior 7 days (confirmed with repeat testing), in 2 or more participants in a group;ii. For PiMZ / MASH subjects: an increase of 0.5 mg / dL over baseline level (BL) or values > 1.7 mg / dL at any timepoint in 2 or more56NAI-5005346498vlparticipants in a dose group. Creatinine values should be confirmed with repeat testing within 24 to 48 hours of identifying the first abnormal value.• Clinically significant increases in liver function tests (confirmed with repeat testing), in the absence of alternative explanations, defined:i. For subjects with normal baseline liver tests:1. Isolated ALT or AST levels > 5 * ULN or > 300 U / L whichever occurs first;2. Isolated alkaline phosphatase (ALP) levels > 3 * ULN or > 500 IU / L, whichever occurs first;3. ALT or AST levels > 3 * ULN or > 300 U / L and meet either of the following:a. TB > 2 * ULN value; orb. INR>ULN; and4. ALP levels > 2.5 x ULN or > 450 U / L and meet either of the following:a. TB > 2 x ULN; orb. INR> 1.5.5. If the subject presents with jaundice (i.e., TB > 2 x ULN) AND symptoms consistent with clinical hepatitis, regardless of AST or ALT levelsii. For subjects with elevated liver tests at baseline:1. Isolated ALT or AST levels > 3 x baseline (BL) and > 300 U / L whichever occurs first;2. Isolated ALP levels > 3 x BL or > 500 IU / L, whichever occurs first; 3. ALT or AST levels > 2 x BL or > 300 U / L and meet either of the following:a. TB > 2 x ULN; orb. INR > 1.5; and4. ALP levels > 2 x BL or > 450 U / L and meet either of the following:a. TB > 2 x ULN; orb. INR> 1.5.57NAI-5005346498vl5. If the subject presents with jaundice ( / .<?., TB > 2 * ULN) and symptoms consistent with clinical hepatitis, regardless of AST or ALT levels• Decline in FEV1 of > 15% for PiZZ subjects and (must be confirmed on repeat measurement post-bronchodilator) with symptoms of breathlessness or decline of FEV1 of > 20% (must be confirmed on repeat measurement post-bronchodilator) regardless of concurrent symptoms; or > for PiMZ / MASH subjects.

[0205] All dose administrations are halted to perform additional safety analyses and review, including causality assessments, should any of the following occur:• One (1) participant develops a CTCAE Grade 5 AE• Two (2) participants develop any CTCAE Grade 4 AEs (excluding Grade 3 or 4 laboratory elevations that are not clinically significant).• Three (3) participants develop the same CTCAE Grade 3 AEs (excluding Grade 3 or 4 laboratory elevations that are not clinically significant).• One (1) participant experiences liver dysfunction, failure, or injury defined by any of the following criteria (criteria only apply to ALT / AST elevations of hepatic etiology):i. ALT or AST > 8 * ULN if baseline was normal, or ALT or AST > 8 * BL if baseline was abnormal.ii. ALT or AST > 5 * ULN for more than 2 weeks if baseline was normal, or ALT or AST > 5 * BL for more than 2 weeks if baseline was abnormal.iii. ALT or AST > 3 * ULN if baseline was normal, or ALT or AST >3 x BL if baseline was abnormal with the appearance of clinical signs of acute hepatitis (right upper quadrant pain or tenderness, fever, rash, and / or eosinophilia [eosinophil differential > 5%]).• Two (2) participants experience a decline in FEV1 of > 15% (must be confirmed on repeat measurement) with symptoms of breathlessness or decline of FEV1 of > 20% (must be confirmed on repeat measurement) regardless of concurrent symptoms.• Any two (2) participants have a clinically significant finding or arrhythmia on electrocardiogram that indicates abnormal cardiac function or conduction or prolongation of heart rate-corrected QT interval using Fridericia’s formula > 500 ms.58NAI-5005346498vl• The incidence or severity of AEs in this study indicates a potential health hazard to participants.

[0206] Intensity of AEs are the same as described in Example 3.

[0207] Pulmonary function testing is performed at screening; Days 8 and 28; and on Day 64 / ET visit. Testing is performed post bronchodilator using spirometry, and the absolute values of FVC and FEV1 are also reported as percentages of predicted values. Additional unscheduled, symptom-directed pulmonary function tests are performed if required based on daily AE and pulmonary symptom assessments.

[0208] Twelve-lead ECGs are performed at screening; on Day 1 predose, and at 1, 2, 4, 8, and 24 hours postdose; on Day 8, 15, and 22 predose; on Day 28 predose and 24 hours postdose (Day 29); and on Day 64 / ET visit. All 12-lead ECGs are performed in triplicate after the participant has been in the supine position for at least 5 minutes.

[0209] Blood samples for PK analysis are collected on Day 1 predose, and at 1, 2, 4, 6, 8, 12, and 24 hours postdose; on Days 3, 8, 15, and 22 predose; on Day 28 predose, and at 1, 2, 4, 6, 8, 12, 24, 32, 48, and 96 hours postdose; and at all follow up visits. Urine samples for PK analysis are collected on Day 1 predose, and at 0 to 4, 4 to 8, 8 to 12, and 12 to 24 hours postdose; and on Day 28 predose, and at 0 to 4, 4 to 8, 8 to 12, and 12 to 24 hours postdose. Blood samples for plasma total AAT, plasma Z-AAT, and plasma polymer AAT are collected at screening; at baseline (Day -1), on Days 1, 2, 3, 8, 15, 22, and 28 predose; on Days 29, 30, and 32; and at all follow-up visits. Blood samples for analysis of Compound 1-A bound to AAT protein in plasma are collected on Day 1 predose, and at 2, 4, 6, 8, 12, and 24 hours postdose; on Days 3, 8, 15, and 22 predose; on Day 28 predose and at 2, 4, 6, 8, and 12 hours postdose; on Days 29, 30, and 32; and at all follow up visits. Blood samples for analysis of AAT functional activity in plasma (activity against neutrophil elastase) is collected at screening; at baseline (Day -1); on Days 1, 2, 3, 8, 15, 22, and 28 predose; on Days 29, 30, and 32; and at all follow-up visits.

[0210] Blood samples for exploratory biomarker analysis (e.g., Pro-C3, PDCD6, APOCI, APOC3, M30, M65, and FVII, SomaSignal NASH tests, proteomic profiling using the SomaScan Assay) are collected at baseline (Day -1), Day 30, and Day 57. Genomic analysis includes testing genetic risk variants known to be associated with liver disease incidence and progression.59NAI-5005346498vlExample 5: Human Subjects with AATD Liver Disease Exhibiting Altered Protein Expression Levels of Biomarkers

[0211] Human subjects included in the studies described in Examples 3 and 4 exhibiting altered protein expression levels of biomarkers, such as FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3 are orally administered 25 mg to 250 mg (e.g., 25 mg, 50 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, or 250 mg, e.g., in a tablet, daily. Subjects are expected to have one or more of the following improvements: (1) a normalization of biomarker protein expression levels; (2) a decrease in liver fibrosis by at least one stage after administration, e.g., for 3 months, 6 months, 9 months, 12 months, 24 months, 36 months, 48 months, or 60 months; (3) a stabilization in fibrosis (e.g., the fibrosis does not worsen) after administration, e.g., for 3 months, 6 months, 9 months, 12 months, 24 months, 36 months, 48 months, or 60 months; (4) an improved neutrophil elastase inhibition as evidence of presence of circulating (not polymerized) Z-AAT / M-AAT; and (5) an improvement in ELF score, FIB4 score, PROC3 score, MRE scan, and / or FibroScan after administration, e.g., for 3 months, 6 months, 9 months, 12 months, 24 months, 36 months, 48 months, or 60 months.

[0212] Throughout this application, various publications, patents, patent applications and other documents have been referenced. The disclosures of these publications, patents, patent applications and other documents in their entireties are hereby incorporated by reference in this application for all purposes, including in order to more fully describe the state of the art to which the subject matter disclosed herein pertains. Although the disclosed subject matter has been described with reference to the examples provided above, it should be understood that various modifications could be made without departing from the spirit of the disclosed subject matter. Many variations will become apparent to those skilled in the art upon review of this specification.60NAI-5005346498vl

Claims

CLAIMSWhat is claimed is:

1. A method of treating a subject having alpha- 1 antitrypsin deficiency (AATD) liver disease, the method comprising:a. identifying a subject as having an altered blood protein expression level of one or more of Coagulation Factor VII (FVII), Programmed Cell Death 6 (PDCD6), apolipoprotein C-l (APOCI), apolipoprotein C-III (APOC3), and N-terminal pro-peptide of type III collagen (PRO-C3), relative to a control; andb. administering to the subject a therapeutically effective amount of Compound 1-A:

2. A method of treating a subject having alpha- 1 antitrypsin deficiency (AATD) liver disease, the method comprising administering to the subject a therapeutically effective amount of Compound 1-A:wherein the subject has been identified as having an altered blood protein expression level of one or more of Coagulation Factor VII (FVII), Programmed Cell Death 6 (PDCD6), apolipoprotein C-l (APOCI), apolipoprotein C-III (APOC3), and N- terminal pro-peptide of type III collagen (PRO-C3), relative to a control.

3. A method of treating a subject having alpha- 1 antitrypsin deficiency (AATD) liver disease, the method comprising:61NAI-5005346498vla. selecting a subject who has been determined as having an altered blood protein expression level of one or more of Coagulation Factor VII (FVII), Programmed Cell Death 6 (PDCD6), apolipoprotein C-l (APOCI), apolipoprotein C-III (APOC3), and N-terminal pro-peptide of type III collagen (PRO-C3), relative to a control; andb. administering to the subject a therapeutically effective amount of Compound 1-A:

4. A method of treating a subject having alpha- 1 antitrypsin deficiency (AATD) liver disease, the method comprising administering a therapeutically effective amount of Compound 1-A:wherein the subject has been selected based on an altered blood protein expression level of one or more of Coagulation Factor VII (FVII), Programmed Cell Death 6 (PDCD6), apolipoprotein C-l (APOCI), apolipoprotein C-III (APOC3), and N-terminal propeptide of type III collagen (PRO-C3), relative to a control.

5. A method of treating a subject identified as having alpha- 1 antitrypsin deficiency (AATD) liver disease, the method comprising:a. determining a blood protein expression level of one or more of Coagulation Factor VII (FVII), Programmed Cell Death 6 (PDCD6), apolipoprotein C-l (APOCI), apolipoprotein C-III (APOC3), and N-terminal pro-peptide of type III collagen (PRO-C3) in a sample obtained from the subject;62NAI-5005346498vlb. comparing the blood protein expression level of the one or more of FVII, PDCD6, APOCI, APOC3, and PRO-C3 to a control, wherein an alteration in the blood protein expression level of the one or more of FVII, PDCD6, APOCI, APOC3, and PRO-C3 in the subject sample relative to the control identifies the subject as having the AATD liver disease; andc. administering to the subject a therapeutically effective amount of Compound 1-A:

6. A method of treating a subject having alpha- 1 antitrypsin deficiency (AATD) liver disease, the method comprising administering to the subject a therapeutically effective amount of Compound 1-A:wherein the subject has been determined to have an altered blood protein expression level of one or more of Coagulation Factor VII (FVII), Programmed Cell Death 6 (PDCD6), apolipoprotein C-l (APOCI), apolipoprotein C-III (APOC3), and N- terminal pro-peptide of type III collagen (PRO-C3) compared to the blood protein expression level of the one or more of FVII, PDCD6, APOCI, APOC3, and PRO-C3 in a control.

7. A method for identifying a subject having alpha-1 antitrypsin deficiency (AATD) liver disease who is (a) likely to, or (b) less likely to, respond to treatment with Compound 1-A:63NAI-5005346498vlwherein the method comprises determining whether the subject has (a) an altered blood protein expression level or (b) an unaltered blood protein expression level of one or more of Coagulation Factor VII (FVII), Programmed Cell Death 6 (PDCD6), apolipoprotein C- 1 (APOCI), apolipoprotein C-III (APOC3), and PRO-C3, as compared to a control, wherein the altered blood protein expression level identifies that the subject is (a) likely to respond to treatment with Compound 1-A, and the unaltered blood protein expression level indicates that the subject is (b) less likely to respond to treatment with Compound 1-A.

8. The method of any one of the preceding claims, wherein the blood protein expression level of FVII, PDC6, AP0C1, and AP0C3 is measured using shotgun liquid chromatography -tandem mass spectrometry and wherein the blood protein expression level of PR0-C3 is measured by enzyme-linked immunosorbent assay (ELISA).

9. The method of any of the preceding claims, wherein the altered blood protein expression level is of two or more of FVII, PDCD6, AP0C1, AP0C3, M30, M65, and PR0-C3.

10. The method of any of the preceding claims, wherein the altered blood protein expression level is of three or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3.

11. The method of any of the preceding claims, wherein the altered blood protein expression level is of four or each of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3.

12. The method of any of the preceding claims, wherein the altered blood protein expression level is of five or more of FVII, PDCD6, APOCI, APOC3, M30, M65, and PRO-C3.64NAI-5005346498vl13. The method of any of the preceding claims, wherein the altered blood protein expression level is of six or each of FVII, PDCD6, AP0C1, AP0C3, M30, M65, and PR0-C3.

14. The method of any of the preceding claims, wherein the altered blood protein expression level is of each of FVII, PDCD6, AP0C1, AP0C3, M30, M65, and PROCS.

15. The method of any of the preceding claims, wherein the altered blood protein expression level of one or more of AP0C1, AP0C3, and FVII is a decreased blood protein expression level.

16. The method of claim 13, wherein the blood protein expression level is decreased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5 as compared to the control.

17. The method of any of the preceding claims, wherein the altered blood protein expression level of PDCD6 is an increased blood protein expression level.

18. The method of claim 17, wherein the blood protein expression level is increased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5 as compared to the control.

19. A method of treating a subject having alpha- 1 antitrypsin deficiency (AATD) liver disease, the method comprising administering to the subject a therapeutically effective amount of Compound 1-A,wherein:65NAI-5005346498vla. the blood protein expression level of one or more of apolipoprotein C-l (AP0C1), apolipoprotein C-III (AP0C3), and Coagulation Factor VII (FVII) in the subject is increased following the administering; and / or b. the blood protein expression level of one or both of Programmed Cell Death 6 (PDCD6), M30, M65, and PRO-C3 in the subject is decreased following the administering.

20. The method of claim 19, wherein the blood protein expression level of APOCI is increased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5.

21. The method of claim 19, wherein the blood protein expression level of APOC3 is increased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5.

22. The method of claim 19, wherein the blood protein expression level of FVII is increased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5.

23. The method of claims 19, wherein the blood protein expression level of PDCD6 is decreased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5.

24. The method of claim 18, wherein the blood protein expression level of PRO-C3 is decreased by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5.

25. The method of claim 19, wherein the blood protein expression level of M30 is decreased by a factor of about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, or about 7.5.66NAI-5005346498vl26. The method of claim 19, wherein the blood protein expression level of M65 is decreased by a factor of about 1.5, about 1.75, about 2.0, about 2.25, about 2.5, about 2.75, about 3.0, about 3.25, about 3.5, about 3.75, about 4.0, about 4.25, or about 5.0.

27. The method of any of the preceding claims, wherein the control is a sample having protein expression levels as follows:a PRO-C3 blood protein expression level of 6.1 - 14.7 ng / mL, an APOCI blood protein expression level of 0.45 - 0.75 mg / mL, a APOC3 blood protein expression level of 10 - 15 mg / dL,a FVII blood protein expression level of 0.5 - 8.4 ng / mL, and / or a PDCD6 blood protein expression level of 2.2 pg / L; and / ora M30 blood protein expression level of about 95 ng / mL; and / or a M65 blood protein expression level of about 130 ng / mL.

28. The method of any of the preceding claims, wherein the subject has an altered blood protein expression level of one or more of TIMP-1, CO4A6, IGF2, IGF1R, IGFBP3, IGFBP5, LEPR, ICAM1, VCAM1, APOA5, APOC2, APOC4, APOF, APOL1, APOM, PDCD6IP, CHMP4B, VPS4B, PSMD5, PSMD9, PSDM10, OTUD6B, and PSMG1.

29. The method of any of the preceding claims, wherein the subject has a Pi*Z genotype.

30. The method of any of the preceding claims, wherein the subject has a PiMZ genotype.

31. The method of any of the preceding claims, wherein the subject has metabolic dysfunction-associated steatohepatitis (MASH).

32. The method of any of the preceding claims, wherein the subject has cirrhosis of the liver.

33. The method of any of the preceding claims, wherein the subject has hepatitis.

34. The method of any of the preceding claims, wherein the subject has a PiZZ genotype.

35. The method of any of the preceding claims, wherein the administering of the therapeutically effective amount of Compound 1-A to the subject is by oral administration.67NAI-5005346498vl36. The method of any of the preceding claims, wherein the administering of the therapeutically effective amount of Compound 1-A to the subject is by oral administration of one or more tablets.

37. The method of any of the preceding claims, wherein the administering of the therapeutically effective amount of Compound 1-A to the subject is once per day.

38. The method of any of the preceding claims, wherein the therapeutically effective amount of Compound 1-A is about 25 mg / day to about 750 mg / day of Compound 1- A.

39. The method of any of the preceding claims, wherein the therapeutically effective amount of Compound 1-A is about 25 mg / day to about 250 mg / day of Compound 1- A.68NAI-5005346498vl

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