Treatment of hepatitis

Abstract

Provided herein are methods of treating hepatitis B and / or hepatitis D using a Na + / taurocholic acid co-transporter polypeptide (NTCP) inhibitor (e.g., a compound of Formula (I) or a pharmaceutically acceptable salt thereof, or a compound of Formula (II) or a pharmaceutically acceptable salt thereof). Such methods may include reducing the concentration of hepatitis B DNA, reducing the concentration of hepatitis D DNA, reducing hepatitis B surface antigen, and reducing hepatitis B core antigen (HBcAg).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 350,693, filed on June 9, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to methods for treating hepatitis B and / or hepatitis D using one or more NTCP inhibitors (e.g., 1,5-benzothiazepine and 1,2,5-benzothiadiazepine derivatives or pharmaceutically acceptable salts thereof). The present disclosure also relates to methods for reducing hepatitis B and / or hepatitis D replication in hepatocytes and reducing hepatitis B virus particles and / or hepatitis D virus particles from entering hepatocytes using such inhibitors. Background Art

[0004] Hepatitis B virus (HBV) infection is a major global public health problem (see, e.g., World Health Organization Hepatitis B Fact Sheet, 2021, available at: who.int / news-room / fact-sheets / detail / hepatitis-b). Globally, an estimated 296 million people are infected with HBV, while HDV infects an estimated 48 to 60 million people. HBV causes nearly 1 million deaths each year. HBV and HDV infect liver cells, and chronic HBV and HDV infection can lead to severe liver disease. HDV depends on HBV for replication and can therefore only be transmitted when co-infected with HBV.

[0005] Certain 1,5-benzothiazepine and 1,2,5-benzothiadiazepine derivatives are potent inhibitors of the apical sodium-dependent bile acid transporter (ASBT) and / or the Na+ / taurocholic acid cotransporting polypeptide (NTCP; also known as the hepatic bile acid transporter (LBAT)). Na+-taurocholic acid cotransporting polypeptide (NTCP) is a bile acid (BA) transporter on the sinusoidal membrane of hepatocytes. NTCP mediates the entry of bile acids into hepatocytes. NTCP also acts as a host receptor for hepatitis B and hepatitis D viruses (HBV / HDV). Pharmacological inhibition of NTCP may be a method to prevent HBV and HDV infection. Summary of the invention

[0006] Provided herein are methods for treating hepatitis B (HBV) in a subject in need thereof, the methods comprising orally administering to the subject a therapeutically effective amount of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

[0007] Also provided herein is a method for preventing or reducing the entry of hepatitis B virus particles into hepatocytes in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

[0008] Also provided herein is a method of reducing hepatitis B virus replication in hepatocytes in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

[0009] In some embodiments, the subject has hepatitis D.

[0010] Also provided herein is a method for preventing hepatitis D infection in a subject having hepatitis B, the method comprising orally administering to the subject a therapeutically effective amount of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

[0011] Also provided herein is a method for treating hepatitis delta (HDV) in a subject in need thereof, the method comprising orally administering to the subject a therapeutically effective amount of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

[0012] Also provided herein is a method for preventing or reducing the entry of hepatitis delta virus particles into hepatocytes in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

[0013] Also provided herein is a method of reducing hepatitis delta virus replication in hepatocytes in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

[0014] In some embodiments, the method further comprises administering an additional antiviral agent.

[0015] Also provided herein is a method for treating HBV in a subject in need thereof, the method comprising orally administering to the subject a therapeutically effective amount of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof, and an additional antiviral agent.

[0016] Also provided herein is a method of treating HDV in a subject in need thereof, comprising orally administering to the subject a therapeutically effective amount of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof, and an additional antiviral agent.

[0017] In some embodiments, the additional antiviral agent is selected from: entecavir, tenofovir, tenofovir disoproxil, tenofovir alafenamide, lamivudine, adefovir, adefovir dipivoxil, telbivudine, bulvir peptide, interferon and combinations thereof. In some embodiments, the interferon is pegylated interferon, interferon alpha or a combination thereof. In some embodiments, the additional antiviral agent is tenofovir disoproxil.

[0018] In some embodiments, the subject has hepatitis B. In some embodiments, the subject has chronic hepatitis B. In some embodiments, the subject has chronic hepatitis D.

[0019] In some embodiments, after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof, the concentration of one or more biomarkers selected from HBV DNA, hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), hepatitis B e antigen (HBeAg), HDV DNA and hepatitis D antigen (HDAg) in the subject's serum is reduced.

[0020] In some embodiments, the concentration of HBV DNA in the serum of a subject is reduced after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of HBV DNA is measured in a serum sample of a subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of HBV DNA in a serum sample of a subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is reduced compared to a reference concentration of HBV DNA. In some embodiments, the reference concentration of HBV DNA is the level of HBV DNA in a serum sample of a subject obtained before administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

[0021] In some embodiments, the concentration of HBV DNA in the serum of a subject is reduced by about 10% to about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of HBV DNA in the serum of a subject is reduced by about 5%, about 10%, about 25%, about 50%, about 75%, or about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of HBV DNA in the serum of a subject is undetectable after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

[0022] In some embodiments, the concentration of hepatitis B surface antigen (HBsAg) in the serum of a subject is reduced after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of HBsAg is measured in a serum sample of a subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of HBsAg in a serum sample of a subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is reduced compared to a reference concentration of HBsAg. In some embodiments, the reference concentration of HBsAg is the concentration of HBsAg in a serum sample of a subject obtained before administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

[0023] In some embodiments, the concentration of HBsAg in the serum of a subject is reduced by about 10% to about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of HBsAg in the serum of a subject is reduced by about 5%, about 10%, about 25%, about 50%, about 75%, or about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of HBsAg in the serum of a subject is undetectable after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

[0024] In some embodiments, the concentration of hepatitis B core antigen (HBcAg) in the serum of a subject is reduced after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of HBcAg is measured in a sample obtained from a subject after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of HBcAg in a serum sample of a subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is reduced compared to a reference concentration of HBcAg. In some embodiments, the reference concentration of HBcAg is the concentration of HBcAg in a serum sample of a subject obtained before administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

[0025] In some embodiments, the concentration of HBcAg in the serum of a subject is reduced by about 10% to about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of HBcAg in the serum of a subject is reduced by about 5%, about 10%, about 25%, about 50%, about 75%, or about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of HBcAg in the serum of a subject is undetectable after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

[0026] In some embodiments, the concentration of hepatitis B e antigen (HBeAg) in the serum of a subject is reduced after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of HBeAg is measured in a serum sample of a subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of HBeAg in a serum sample of a subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is reduced compared to a reference concentration of HBeAg. In some embodiments, the reference concentration of HBeAg is the concentration of HBeAg in a serum sample of a subject obtained before administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

[0027] In some embodiments, the concentration of HBeAg in the serum of a subject is reduced by about 10% to about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of HBeAg in the serum of a subject is reduced by about 5%, about 10%, about 25%, about 50%, about 75%, or about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of HBeAg in the serum of a subject is undetectable after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

[0028] In some embodiments, the concentration of HDV DNA in the serum of a subject is reduced after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of HDV DNA is measured in a serum sample of a subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of HDV DNA in a serum sample of a subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is reduced compared to a reference concentration of HDV DNA. In some embodiments, the reference concentration of HDV DNA is the concentration of HDV DNA in a serum sample of a subject obtained before administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

[0029] In some embodiments, the concentration of HDV DNA in the serum of a subject is reduced by about 10% to about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of HDV DNA in the serum of a subject is reduced by about 5%, about 10%, about 25%, about 50%, about 75%, or about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of HDV DNA in the serum of a subject is undetectable after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

[0030] In some embodiments, the concentration of hepatitis delta antigen (HDAg) in the serum of a subject is reduced after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of HDAg is measured in a serum sample of a subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of HDAg in a serum sample of a subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is reduced compared to a reference concentration of HDAg. In some embodiments, the reference concentration of HDAg is the concentration of HDAg in a serum sample of a subject obtained before administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

[0031] In some embodiments, the concentration of HDAg in the serum of a subject is reduced by about 10% to about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of HDAg in the serum of a subject is reduced by about 5%, about 10%, about 25%, about 50%, about 75%, or about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of HDAg in the serum of a subject is undetectable after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

[0032] In some embodiments, (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is administered to the subject within 18 hours of exposure to hepatitis B. In some embodiments, (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is administered to the subject within 18 hours of exposure to hepatitis D.

[0033] In some embodiments, a therapeutically effective amount of (R)-(Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is administered to a subject.

[0034] In some embodiments, a therapeutically effective amount of (S)-(Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is administered to a subject.

[0035] Details of one or more embodiments of the present disclosure are set forth in the accompanying drawings and the following description. Other features, objects, and advantages of the present invention will be apparent from the description and drawings, as well as from the claims. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Each patent, application, published application, and other publication mentioned in the specification and the attached appendix are incorporated herein by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a schematic diagram depicting the enterohepatic circulation and NTCP in the HBV / HDV entry receptor.

[0038] Figure 2A It is K showing NTCP i Value graph.

[0039] Figure 2B It is K showing ASBT i Value graph.

[0040] Figure 3A is a graph showing the inhibition of NTCP after washout with Myrcludex B. Mean (SD) values ​​(n=3) are depicted.

[0041] Figure 3B is a graph showing the inhibition of NTCP after washout with Compound 1. Mean (SD) values ​​(n=3) are depicted.

[0042] Figure 4A and 4B is a graph showing inhibition of bile acid transport in HEK293 cells expressing NTCP. Mean (SD) values ​​of four determinations of a representative experiment are depicted. *P<0.01 vs control by Student t-test.

[0043] Figure 4C and 4D is a graph showing inhibition of preS1 peptide binding in HEK293 cells expressing NTCP. Means (SD) of four determinations of a representative experiment are shown. *P<0.01 vs control by Student t-test.

[0044] Figure 5A and 5B Figure 2 is a graph showing NTCP inhibition in cynomolgus monkeys.

[0045] Figure 6 is a graph showing the cytotoxicity of compound 1 in HepG2 cells expressing NTCP.

[0046] Fig. 7AGraph showing the inhibition of HBV by compound 1 in HepG2 cells expressing NTCP.

[0047] Figure 7B Graph showing the inhibition of HDV by compound 1 in HepG2 cells expressing NTCP.

[0048] Figure 7C Graph showing the inhibition of HBV by compound 1 in HepG2 cells expressing NTCP.

[0049] Fig.7D and 7E Graph showing inhibition of in vitro HBV infection by secreted HBeAg by compound 1. Data represent mean ± SD of three replicates; 50%: 50% control (w / o inhibitor); significantly different from uninhibited control, p>0.05 (ns), p≤0.05 (*), p≤0.01 (**), p≤0.001 (***), p≤0.0001 (****).

[0050] Figure 7F and 7G Graph showing inhibition of in vitro HBV infection by HBc-positive cells by compound 1. Data represent mean ± SD of triplicate experiments; 50%: 50% control (w / o inhibitor); significantly different from uninhibited control, p>0.05 (ns), p≤0.05 (*), p≤0.01 (**), p≤0.001 (***), p≤0.0001 (****).

[0051] Fig. 8A This is a graph showing the inhibition of HBV infection in primary human hepatocytes when Compound 1 or tenofovir disoproxil fumarate (TDF) was administered 18 hours before HBV infection. The average values ​​(n=3) are shown.

[0052] Figure 8B This is a graph showing the inhibition of HBV infection in primary human hepatocytes when Compound 1 or tenofovir disoproxil fumarate (TDF) was administered 18 hours after HBV infection. The average values ​​(n=3) are shown.

[0053] Figure 8C is a graph showing the cytotoxicity of Compound 1 or tenofovir disoproxil fumarate (TDF) in primary human hepatocytes. Mean values ​​(n=3) are shown.

[0054] Fig.8D is a graph showing the inhibition of HBV infection by different concentrations of Compound 1 when combined with TDF.

[0055] Fig. 9is a graph showing a summary of antiviral data for Compound 1. Mean values, n=3. Lines represent nonlinear regression fits to pooled data from three different studies. Data points at concentrations of 10-11 M in Study #01 were excluded for curve fitting purposes.

[0056] Fig.10 is a graph showing the pharmacokinetics of compound 1 in humanized mice. Mean (SEM) values ​​(n=3), oral administration, are depicted.

[0057] Fig.11A is a graph showing serum HBV DNA concentrations in humanized mice during and after treatment with Compound 1.

[0058] Fig. 11B is a graph showing serum HBV DNA concentration in humanized mice during treatment with Compound 1.

[0059] Fig. 12A is a graph showing serum HBsAg concentrations in humanized mice during and after treatment with Compound 1.

[0060] Fig. 12B is a graph showing serum HBsAg concentrations in humanized mice during treatment with Compound 1.

[0061] Fig. 12C is a graph showing serum HBeAg concentrations in humanized mice during treatment with Compound 1. DETAILED DESCRIPTION OF THE INVENTION

[0063] HBV and HDV use the Na+-taurocholate cotransporting polypeptide (NTCP, also known as the hepatic bile acid transporter (LBAT); gene symbol SLC10A1) to enter and infect human hepatocytes. See Yan H et al., eLife. 2012;1:e00049. NTCP and other members of the SLC10 family of solute carrier proteins, such as the apical sodium-dependent bile acid transporter (ASBT, also known as the ileal bile acid transporter (IBAT), ISBT, ABAT, or NTCP2; gene symbol SLC10A2), control the transport of bile acids in the human body. In the liver, bile acids are efficiently extracted from the portal blood by the hepatic bile acid transporter (LBAT) and re-secreted across the canalicular membrane by the bile salt export pump (BSEP; gene symbol ABCB11). Reabsorption of bile acids in the ileum is handled by the apical sodium-dependent bile acid transporter (ASBT), commonly referred to as the ileal bile acid transporter (IBAT). Both NTCP and ASBT function as electrogenic sodium solute cotransporters, moving two or more Na+ ions per solute molecule.

[0064] LBAT also acts as a cellular receptor for viral entry of hepatitis B virus (HBV) and hepatitis D virus (HDV), which are in turn the main causes of liver disease and hepatocellular carcinoma. NTCP interacts with a key region in the pre-S1 domain of the HBV envelope L protein. Yan H, et al. eLife. 2012; 1: e00049. It has been shown that residues 157 to 165 of NTCP are important for binding to the receptor binding region of the preS1 domain of the L protein of HBV, and that these residues contribute to NTCP-mediated HBV and HDV infection. Yan H et al. eLife. 2012; 1: e00049.

[0065] Provided herein are methods for treating hepatitis B (HBV) and / or hepatitis D (HDV) in a subject in need thereof, comprising administering one or more NTCP inhibitors (e.g., any NTCP inhibitors described herein) to the subject. Also provided herein are methods for preventing or reducing hepatitis B virus particles and / or hepatitis D virus particles from entering the hepatocytes of a subject in need thereof, comprising administering one or more NTCP inhibitors (e.g., any NTCP inhibitors described herein) to the subject. Also provided herein are methods for reducing hepatitis B and / or hepatitis D virus replication in the hepatocytes of a subject in need thereof, comprising administering one or more NTCP inhibitors (e.g., any NTCP inhibitors described herein) to the subject.

[0066] As used herein, "NTCP inhibitors" include any compound that exhibits NTCP inactivation activity (e.g., inhibition or reduction). In some embodiments, NTCP inhibitors reduce NTCP activity. In some embodiments, NTCP inhibitors prevent or reduce the production and / or function of NTCP. In some embodiments, NTCP inhibitors are dual inhibitors, that is, they inhibit both NTCP and IBAT. In some embodiments, NTCP inhibitors are more selective for NTCP than other members of the solute carrier protein SLC10 family, such as IBAT. In some embodiments, the molecular weight of the NTCP inhibitor is less than about 1,000 g / mol.

[0067] The ability of a compound as an NTCP inhibitor can be demonstrated by assays known in the art. The activity of compounds and compositions provided herein as NTCP inhibitors can be measured in vitro, in vivo or in a cell line. In vitro assays include assays to determine the inhibition of bile salt transport by NTCP. Assays may include, for example, the assays described in U.S. Patent No. 11,180,465.

[0068] The NTCP inhibitor compounds described herein are of formula (I):

[0069]

[0070] in

[0071] M is selected from -CH2- and -NR 7 -;

[0072] R 1 C 1-4 alkyl;

[0073] R 2 are independently selected from hydrogen, halogen, hydroxyl, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, cyano, nitro, amino, N-(C 1-4 alkyl)amino, N,N-di(C 1-4 alkyl)amino, N-(aryl-C 1-4 Alkyl)amino, C 1-6 Alkylcarbonylamino, C 3-6 Cycloalkylcarbonylamino, N-(C 1-4 alkyl)aminocarbonyl, N,N-di(C 1-4 alkyl)aminocarbonyl, C 1-4 Alkyloxycarbonylamino, C 3-6 Cycloalkyloxycarbonylamino, C 1-4 Alkylsulfonamide and C 3-6 Cycloalkylsulfonamide;

[0074] n is an integer 1, 2 or 3;

[0075] R 3 Selected from hydrogen, halogen, cyano, C 1-4 Alkyl, C 3-6 Cycloalkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyloxy, C 1-4 Alkylthio, C 3-6 Cycloalkylthio, amino, N-(C 1-4 Alkyl)amino and N,N-di(C 1-4 Alkyl)amino;

[0076] R 4 and R 5 One of them is a carboxyl group, and R 4 and R 5 The other one of which is selected from hydrogen, fluorine, C 1-4 Alkyl and C 1-4 Haloalkyl;

[0077] R 6 Selected from hydrogen and C 1-4 alkyl; and

[0078] R7 Selected from hydrogen and C 1-4 alkyl;

[0079] or a pharmaceutically acceptable salt thereof.

[0080] In some embodiments, R 1 C 2-4 In some embodiments, R 1 In some embodiments, R 1 It is n-butyl.

[0081] In some embodiments, R 2 is selected from hydrogen, fluorine, chlorine, bromine, hydroxyl, methoxy, amino, methylamino, dimethylamino, isopropylcarbonylamino, tert-butylcarbonylamino, tert-butylaminocarbonyl, tert-butoxycarbonylamino, methylsulfonylamino and cyclopropylsulfonylamino. In some embodiments, n is 1, i.e., the phenyl ring is substituted with only one substituent R 2 In some embodiments, R 2 In counterpoint.

[0082] In some embodiments, R 3 is selected from the group consisting of hydrogen, fluorine, chlorine, bromine, methyl, cyclopropyl, methoxy, ethoxy, methylthio, ethylthio, amino, methylamino and dimethylamino.

[0083] In some embodiments, R 4 is hydrogen or fluorine.

[0084] In some embodiments, R 5 It is a carboxyl group.

[0085] In some embodiments, R 6 For hydrogen.

[0086] In some embodiments, R 7 It is hydrogen or methyl.

[0087] In some embodiments, the compound of formula (I) is a compound of formula (la):

[0088]

[0089] in

[0090] M is selected from -CH2-, -NH- and -NCH3-;

[0091] R 1 C 2-4 alkyl;

[0092] R 2 are independently selected from hydrogen, halogen, hydroxyl, C 1-4 Alkyl, C1-4 Haloalkyl, C 1-4 Alkoxy, amino, N-(C 1-4 alkyl)amino, N,N-di(C 1-4 Alkyl)amino, C 1-6 Alkylcarbonylamino, C 3-6 Cycloalkylcarbonylamino, N-(C 1-4 alkyl)aminocarbonyl, N,N-di(C 1-4 alkyl)aminocarbonyl, C 1-4 Alkyloxycarbonylamino, C 1-4 Alkylsulfonamide and C 3-6 Cycloalkylsulfonamide;

[0093] n is an integer of 1 or 2;

[0094] R 3 Selected from hydrogen, halogen, C 1-4 Alkyl, C 3-6 Cycloalkyl, C 1-4 Alkoxy, C 1-4 Alkylthio, amino, N-(C 1-4 Alkyl)amino and N,N-di(C 1-4 Alkyl)amino;

[0095] R 4 is hydrogen or fluorine;

[0096] or a pharmaceutically acceptable salt thereof.

[0097] In some embodiments, the compound of formula (I) is a compound of formula (Ib):

[0098]

[0099] in

[0100] M is selected from -CH2-, -NH- and -NCH3-;

[0101] R 1 C 2-4 Alkyl, more preferably n-propyl or n-butyl;

[0102] R 2 independently selected from hydrogen, fluorine, chlorine, bromine, hydroxy, methoxy, amino, methylamino, dimethylamino, isopropylcarbonylamino, tert-butylcarbonylamino, tert-butylaminocarbonyl, tert-butoxycarbonylamino, methylsulfonylamino and cyclopropylsulfonylamino;

[0103] R 3 is selected from the group consisting of fluorine, chlorine, bromine, methyl, cyclopropyl, methoxy, ethoxy, methylthio, ethylthio, amino, methylamino and dimethylamino;

[0104] R 4 is hydrogen or fluorine;

[0105] or a pharmaceutically acceptable salt thereof.

[0106] In some embodiments, the compound of formula (I) is a compound of formula (Ib) as defined above, wherein M and R 1 To R 4 As shown in Table 1 below, or a pharmaceutically acceptable salt thereof:

[0107] Table 1

[0108]

[0109]

[0110]

[0111]

[0112]

[0113] In some embodiments, the compound of formula (I) is selected from:

[0114] (E)-3-((3-butyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)acrylic acid;

[0115] (R)-(E)-3-((3-butyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)acrylic acid;

[0116] (S)-(E)-3-((3-butyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)acrylic acid;

[0117] (Z)-3-((3-butyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)-2-fluoroacrylic acid;

[0118] (S)-(Z)-3-((3-butyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)-2-fluoroacrylic acid;

[0119] (R)-(Z)-3-((3-butyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)-2-fluoroacrylic acid;

[0120] (Z)-3-((3-ethyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)-2-fluoroacrylic acid;

[0121] (E)-3-((3-ethyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)acrylic acid;

[0122] (E)-3-((3-butyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)acrylic acid;

[0123] (Z)-3-((3-butyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid;

[0124] (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid;

[0125] (S)-(Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid;

[0126] (R)-(Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid;

[0127] (E)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)acrylic acid;

[0128] (S)-(E)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)acrylic acid;

[0129] (R)-(E)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)acrylic acid;

[0130] (E)-3-((3-butyl-7-(ethylthio)-2-methyl-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)acrylic acid;

[0131] (S)-(E)-3-((3-butyl-7-(ethylthio)-2-methyl-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)acrylic acid;

[0132] (R)-(E)-3-((3-butyl-7-(ethylthio)-2-methyl-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)acrylic acid;

[0133] (E)-3-((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)acrylic acid;

[0134] (S)-(E)-3-((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)acrylic acid;

[0135] (R)-(E)-3-((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)acrylic acid;

[0136] (E)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)acrylic acid;

[0137] (S)-(E)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)acrylic acid;

[0138] (R)-(E)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)acrylic acid;

[0139] (E)-3-((3-butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)acrylic acid;

[0140] (S)-(E)-3-((3-butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)acrylic acid;

[0141] (R)-(E)-3-((3-butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)acrylic acid;

[0142] (E)-3-((3-butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)acrylic acid;

[0143] (E)-3-((3-butyl-7-(ethylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)acrylic acid;

[0144] (E)-3-((3-butyl-7-(ethylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)acrylic acid;

[0145] (E)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)acrylic acid;

[0146] (Z)-3-((3-butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)-2-fluoroacrylic acid;

[0147] (Z)-3-((3-butyl-7-(ethylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)-2-fluoroacrylic acid;

[0148] (Z)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)-2-fluoroacrylic acid;

[0149] (Z)-3-((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid;

[0150] (Z)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid;

[0151] (Z)-3-((3-butyl-7-(ethylthio)-2-methyl-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid;

[0152] (Z)-3-((3-butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid;

[0153] (Z)-3-((3-butyl-7-(ethylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid;

[0154] (Z)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid; and

[0155] (E)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)acrylic acid;

[0156] or a pharmaceutically acceptable salt thereof.

[0157] In some embodiments, the compound of formula (I) is (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid:

[0158]

[0159] Or a pharmaceutically acceptable salt thereof. (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid is also referred to herein as "Compound 1".

[0160] In some embodiments, the compound of formula (I) is (S)-(Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is administered to a subject. In some embodiments, compound 1 is (S)-(Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid.

[0161] In some embodiments, the compound of formula (I) is (R)-(Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is administered to a subject. In some embodiments, compound 1 is (R)-(Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid.

[0162] The compounds of formula (I) can be prepared according to the method described in US Pat. No. 11,180,465.

[0163] The NTCP inhibitors described herein also include compounds of formula (II)

[0164]

[0165] in

[0166] M is selected from -CH2- and -NR 5 -;

[0167] R 1 C 1-4 alkyl;

[0168] R 2 are independently selected from hydrogen, halogen, hydroxyl, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, cyano, nitro, amino, N-(C 1-4 alkyl)amino, N,N-di(C 1-4 Alkyl)amino, C 1-6 Alkylcarbonylamino, C 3-6 Cycloalkylcarbonylamino, N-(C 1-4 alkyl)aminocarbonyl, N,N-di(C 1-4 alkyl)aminocarbonyl, C 1-4 Alkyloxycarbonylamino, C 3-6 Cycloalkyloxycarbonylamino, C 1-4 Alkylsulfonamide and C 3-6 Cycloalkylsulfonamide;

[0169] n is an integer 1, 2 or 3;

[0170] R 3 Selected from hydrogen, halogen, cyano, C 1-4 Alkyl, C 3-6 Cycloalkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyloxy, C 1-4 Alkylthio, C 3-6 Cycloalkylthio, amino, N-(C 1-4 Alkyl)amino and N,N-di(C 1-4 Alkyl)amino;

[0171] R 4A and R 4B are independently selected from hydrogen, halogen, hydroxyl, C 1-4 Alkyl and C 1-4 Alkoxy; or R 4A and R 4B , together with the carbon atom to which they are attached, form a 3- to 5-membered saturated carbocyclic ring;

[0172] R 4C and R 4D are each independently selected from hydrogen and C 1-4 alkyl; and

[0173] R 5 Selected from hydrogen and C 1-4 alkyl;

[0174] or a pharmaceutically acceptable salt thereof.

[0175] In some embodiments, R 1 C 2-4 In some embodiments, R 1 In some embodiments, R 1 It is n-butyl.

[0176] In some embodiments, R 2 is selected from hydrogen, fluorine, chlorine, bromine, hydroxyl, methoxy, amino, methylamino and dimethylamino. In some embodiments, n is 1, that is, the phenyl ring is substituted with only one substituent R 2 In some embodiments, R 2 In counterpoint.

[0177] In some embodiments, R 3 is selected from the group consisting of fluorine, chlorine, bromine, methyl, cyclopropyl, methoxy, ethoxy, methylthio, ethylthio, amino, methylamino and dimethylamino.

[0178] In some embodiments, R 4A and R 4B are independently selected from hydrogen, halogen, hydroxyl, C 1-4 Alkyl and C 1-4 Alkoxy, or R 4A and R 4B , together with the carbon atom to which they are attached, form a cyclopropyl ring. 4A and R 4B Each is independently fluoro, methyl or methoxy, or together with the carbon atom to which they are attached form a cyclopropyl ring.

[0179] In some embodiments, R 4C and R 4D Each is independently hydrogen or methyl. 4C and R 4D Each is hydrogen.

[0180] In some embodiments, R 5 In some embodiments, R 5 It is methyl.

[0181] In some embodiments, the compound of formula (II) is a compound of formula (II-a):

[0182]

[0183] in

[0184] M is selected from -CH2-, -NH- and -NCH3-;

[0185] R 1 C 2-4 alkyl;

[0186] R 2 are independently selected from hydrogen, halogen, hydroxyl, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, amino, N-(C 1-4 alkyl)amino, N,N-di(C 1-4 Alkyl)amino;

[0187] n is an integer of 1 or 2;

[0188] R 3 Selected from halogen, C 1-4 Alkyl, C 3-6 Cycloalkyl, C 1-4 Alkoxy, C 1-4 Alkylthio, amino, N-(C 1-4 Alkyl)amino and N,N-di(C 1-4 Alkyl)amino;

[0189] R 4A and R 4B are independently selected from hydrogen, halogen, hydroxyl, C 1-4 Alkyl and C 1-4 Alkoxy, or R 4A and R 4B , together with the carbon atom to which they are attached, form a cyclopropyl ring;

[0190] or a pharmaceutically acceptable salt thereof.

[0191] In some embodiments, the compound of formula (II) is a compound of formula (II-b):

[0192]

[0193] in

[0194] M is selected from -CH2-, -NH- and -N(CH3)-;

[0195] R 1 C2-4 Alkyl, more preferably n-propyl or n-butyl;

[0196] R 2 independently selected from hydrogen, fluorine, chlorine, bromine, hydroxy, methoxy, amino, methylamino, dimethylamino;

[0197] R 3 is selected from the group consisting of fluorine, chlorine, bromine, methyl, cyclopropyl, methoxy, ethoxy, methylthio, ethylthio, amino, methylamino and dimethylamino;

[0198] R 4A and R 4B are each independently hydrogen, fluorine, methyl, methoxy or ethoxy, or together with the carbon atom to which they are attached form a cyclopropyl ring;

[0199] or a pharmaceutically acceptable salt thereof.

[0200] Compounds of formula (II-b) as defined above also include those wherein M, R 1 , R 2 , R 3 , R 4A and R 4B The compounds shown in Table 2 below, or pharmaceutically acceptable salts thereof:

[0201] Table 2

[0202]

[0203]

[0204]

[0205] In some embodiments, the compound of formula (II) is selected from:

[0206] 3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2,2-dimethylpropanoic acid;

[0207] (S)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2,2-dimethylpropanoic acid;

[0208] (R)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2,2-dimethylpropanoic acid;

[0209] 1-(((3-Butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid;

[0210] (S)-1-(((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid;

[0211] (R)-1-(((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid;

[0212] 3-((3-butyl-7-(ethylthio)-2-methyl-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2,2-dimethylpropanoic acid;

[0213] (S)-3-((3-butyl-7-(ethylthio)-2-methyl-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2,2-dimethylpropanoic acid;

[0214] (R)-3-((3-butyl-7-(ethylthio)-2-methyl-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2,2-dimethylpropanoic acid;

[0215] 1-(((3-Butyl-7-(ethylthio)-2-methyl-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid;

[0216] (S)-1-(((3-butyl-7-(ethylthio)-2-methyl-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid;

[0217] (R)-1-(((3-butyl-7-(ethylthio)-2-methyl-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid;

[0218] 3-((3-butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)-2,2-dimethylpropanoic acid;

[0219] 1-(((3-Butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid;

[0220] 1-(((3-Butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid;

[0221] (S)-1-(((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid;

[0222] (R)-1-(((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid;

[0223] 3-((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2,2-dimethylpropanoic acid;

[0224] (S)-3-((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2,2-dimethylpropanoic acid;

[0225] (R)-3-((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2,2-dimethylpropanoic acid;

[0226] 1-(((3-Butyl-7-(ethylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid;

[0227] 1-(((3-Butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid;

[0228] (S)-1-(((3-butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid;

[0229] (R)-1-(((3-butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid;

[0230] 1-(((3-Butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid;

[0231] (S)-1-(((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid;

[0232] (R)-1-(((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid;

[0233] 3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2,2-difluoropropionic acid;

[0234] (S)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2,2-difluoropropanoic acid;

[0235] (R)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2,2-difluoropropanoic acid;

[0236] 3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-methoxy-2-methylpropanoic acid;

[0237] (S)-3-(((R)-3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-methoxy-2-methylpropanoic acid;

[0238] (S)-3-(((S)-3-Butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-methoxy-2-methylpropanoic acid;

[0239] (R)-3-(((R)-3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-methoxy-2-methylpropanoic acid;

[0240] (R)-3-(((S)-3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-methoxy-2-methylpropanoic acid;

[0241] 3-((3-butyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)propanoic acid;

[0242] (S)-3-((3-butyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)propanoic acid;

[0243] (R)-3-((3-butyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)propanoic acid;

[0244] 3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-ethoxypropanoic acid;

[0245] 3-((3-butyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)-2-hydroxypropanoic acid;

[0246] 3-((3-ethyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)propanoic acid;

[0247] 3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-methoxypropanoic acid;

[0248] 3-(((S)-3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-methoxypropanoic acid;

[0249] 3-(((R)-3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-methoxypropanoic acid;

[0250] (S)-3-(((R)-3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-methoxypropanoic acid;

[0251] (R)-3-(((R)-3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-methoxypropanoic acid;

[0252] (S)-3-(((S)-3-Butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-methoxypropanoic acid;

[0253] (R)-3-(((S)-3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-methoxypropanoic acid; and

[0254] 3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-hydroxypropanoic acid;

[0255] or a pharmaceutically acceptable salt thereof.

[0256] In some embodiments, the compound of formula (II) is 3-((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2,2-dimethylpropanoic acid:

[0257]

[0258] Or a pharmaceutically acceptable salt thereof. (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid is also referred to herein as "Compound 2".

[0259] In some embodiments, the compound of formula (II) is (S)-3-((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2,2-dimethylpropanoic acid or a pharmaceutically acceptable salt thereof is administered to a subject. In some embodiments, compound 2 is ((S)-3-((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2,2-dimethylpropanoic acid.

[0260] In some embodiments, the compound of formula (II) is (R)-3-((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2,2-dimethylpropanoic acid or a pharmaceutically acceptable salt thereof is administered to a subject. In some embodiments, compound 2 is (R)-3-((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2,2-dimethylpropanoic acid.

[0261] Compounds of formula (II) can be prepared as described in US Application No. 17 / 813,152.

[0262] The NTCP inhibitors used herein may also include compounds disclosed in the following International Publication Nos. WO 93 / 16055, WO 94 / 18183, WO 94 / 18184, WO 96 / 05188, WO 96 / 08484, WO 96 / 16051, WO 97 / 33882, WO 98 / 07449, WO 98 / 40375, WO 99 / 35135, WO 99 / 64409, WO 99 / 64410, WO 00 / 47568, WO 00 / 61568, WO 00 / 38725, WO 00 / 38726, WO 00 / 38727, WO 00 / 38728, WO 00 / 38729, WO 01 / 66533, WO 01 / 68096, WO 02 / 32428, WO 02 / 50051, WO 03 / 020710, WO 03 / 022286, WO 03 / 022825, WO 03 / 022830, WO 03 / 061663, WO 03 / 091232, WO 2004 / 006899, WO2004 / 076430, WO 2007 / 009655, WO 2007 / 009656, WO 2008 / 058628, WO 2008 / 058630, WO2011 / 137135, WO 2019 / 234077, WO 2020 / 161216,WO 2020 / 161217,WO DE 19825804; 3210977.

[0263] In some embodiments, the subject has hepatitis B. In some embodiments, the subject has acute hepatitis B. For example, a subject having acute hepatitis B includes a subject having hepatitis B for less than 6 months. In some embodiments, the subject has chronic hepatitis B. For example, a subject having chronic hepatitis B includes a subject having hepatitis B for six months or longer.

[0264] In some embodiments, the subject has hepatitis D. In some embodiments, the subject has acute hepatitis D. For example, a subject having acute hepatitis D includes a subject who has had hepatitis D for less than 6 months. In some embodiments, the subject has chronic hepatitis D. For example, a subject having chronic hepatitis D includes a subject who has had hepatitis D for six months or longer.

[0265] In some embodiments, the subject has hepatitis B and hepatitis D. In some embodiments, the subject has chronic hepatitis B and hepatitis D. In some embodiments, the subject has chronic hepatitis B and acute hepatitis D. In some embodiments, the subject has chronic hepatitis B and chronic hepatitis D.

[0266] HDV is dependent on HBV for replication and is therefore only transmitted when co-infected with HBV. Lempp FA, Urban S. Viruses. 2017;9:172. Co-infection with HDV and HBV is considered the most severe form of chronic viral hepatitis as it may lead to more rapid progression to hepatocellular carcinoma and liver-related death. World Health Organization Hepatitis D Fact Sheet, 2021, available at: who.int / news-room / fact-sheets / detail / hepatitis-d. Accordingly, also provided herein are methods of preventing hepatitis D infection in a subject having hepatitis B, comprising administering to the patient one or more NTCP inhibitors (e.g., any of the NTCP inhibitors described herein).

[0267] In any of the above embodiments, the NTCP inhibitor (e.g., a compound of formula (I) or a pharmaceutically acceptable salt thereof or a compound of formula (II) or a pharmaceutically acceptable salt thereof) is administered to the subject after exposure to hepatitis B and / or hepatitis D. For example, the NTCP inhibitor (e.g., a compound of formula (I) or a pharmaceutically acceptable salt thereof or a compound of formula (II) or a pharmaceutically acceptable salt thereof) can be administered within about 1 to about 30 hours of exposure to hepatitis B and / or hepatitis D. In some embodiments, the NTCP inhibitor (e.g., a compound of Formula (I) or a pharmaceutically acceptable salt thereof or a compound of Formula (II) or a pharmaceutically acceptable salt thereof) can be administered within about 1 to about 2, about 1 to about 5, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 30, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 30, about 15 to about 20, about 15 to about 25, about 15 to about 30, about 20 to about 25, about 20 to about 30, or about 25 to about 30 hours of exposure to hepatitis B and / or hepatitis D. In some embodiments, the NTCP inhibitor (e.g., a compound of Formula (I) or a pharmaceutically acceptable salt thereof or a compound of Formula (II) or a pharmaceutically acceptable salt thereof) is administered to a subject within about 18 hours of exposure to hepatitis B and / or hepatitis D.

[0268] In some embodiments of any method described herein, the presence of hepatitis B and / or hepatitis D in a subject can be determined by one or more biomarkers. Non-limiting examples of such biomarkers of hepatitis B include HBV DNA concentration, hepatitis B surface antigen (HBsAg) concentration, hepatitis B core antigen (HBcAg) concentration, and hepatitis B e antigen (HBeAg) concentration. Non-limiting examples of such biomarkers of hepatitis D include HDV DNA concentration and hepatitis D antigen (HDAg) concentration. See, e.g., Coffin et al. New and Old Biomarkers for Diagnosis and Management of Chronic Hepatitis B Virus Infection. Gastroenterology. 2019 Jan; 156 (2): 355-368. e3. Such biomarkers can be detected in a sample (e.g., serum or biopsy sample) of a subject.

[0269] In certain embodiments of the methods described herein, the assay for determining whether a subject has hepatitis B and / or hepatitis D using a sample from the subject may include, but is not limited to, next generation sequencing, immunohistochemistry, Southern blot, Western blot, Northern blot, and PCR-based amplification (e.g., RT-PCR and quantitative real-time RT-PCR). As is well known in the art, immunoassays can be used to detect hepatitis antigens, such as HBsAg, HBcAg, HBeAg, and HDAg. Non-limiting examples of such assays include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), electrochemiluminescent immunoassay (ECLIA), microparticle enzyme immunoassay (MEIA), and chemiluminescent microparticle immunoassay (CMIA). See, for example, Tyas et al. Recent Advances of Hepatitis B Detection towards Paper-Based Analytical Devices. Scientific World Journal. 2021 Feb 26; 2021: 6643573. HBV DNA and / or HDV DNA can be detected using, for example, PCR-based amplification (e.g., RT-PCR and quantitative real-time RT-PCR). See, e.g., Coffin et al. Gastroenterology 2019 Jan; 156(2):355-368.e3.

[0270] In some embodiments, the biomarkers described herein (e.g., HBV DNA, HBsAg, HBcAg, HBeAg, HDV DNA, or HDAg) can be used to monitor the responsiveness of a subject to a particular therapy (e.g., an NTCP inhibitor, such as a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a compound of formula (II) or a pharmaceutically acceptable salt thereof). For example, before starting treatment with an NTCP inhibitor described herein (e.g., a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a compound of formula (II) or a pharmaceutically acceptable salt thereof), a sample can be obtained from the subject and the level of the biomarker in the sample can be determined. The sample can be considered a reference sample. Then, one or more doses of an NTCP inhibitor described herein (e.g., a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a compound of formula (II) or a pharmaceutically acceptable salt thereof) can be administered to the subject, and the level of the biomarker can be monitored after administration. For example, the level of the biomarker can be monitored after the first dose, the second dose, the third dose, etc., or after one week, two weeks, three weeks, four weeks, etc. If the concentration of the biomarker decreases (i.e., decreases) relative to the reference sample, it indicates a response to the treatment. In some embodiments, the level of a biomarker is decreased relative to a reference sample by about 1% to about 99%, about 1% to about 95%, about 1% to about 90%, about 1% to about 85%, about 1% to about 80%, about 1% to about 75%, about 1% to about 70%, about 1% to about 65%, about 1% to about 60%, about 1% to about 55%, about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 5% to about 99%, about 10% to about 99%, about 15% to about 99%, about 20% to about 99%, about 25% to about 99%. From about 99%, about 30% to about 99%, about 35% to about 99%, about 40% to about 99%, about 45% to about 99%, about 50% to about 99%, about 55% to about 99%, about 60% to about 99%, about 65% to about 99%, about 70% to about 99%, about 75% to about 95%, about 80% to about 99%, about 90% to about 99%, about 95% to about 99%, about 5% to about 10%, about 5% to about 25%, about 10% to about 30%, about 20% to about 40%, about 25% to about 50%, about 35% to about 55%, about 40% to about 60%, about 50% to about 75%, about 60% to about 80%, about 65% to about 85%, about 70% to about 90%, or about 75% to about 95%. In some embodiments, the concentration of the biomarker is reduced to below the detection limit of the instrument, ie, the level of the biomarker is "undetectable."

[0271] In some embodiments, the severity of hepatitis B and / or hepatitis D is determined via one or more biomarkers or scoring systems that indicate one or more of liver damage, inflammation, liver fibrosis, and / or cirrhosis. The concentration of the biomarker can be determined, for example, by measuring, quantifying, and monitoring the expression of genes or mRNA encoding the biomarker and / or the peptides or proteins of the biomarker. Non-limiting examples of biomarkers and / or scoring systems that indicate one or more of liver damage, inflammation, liver fibrosis, and / or cirrhosis include aspartate aminotransferase (AST) to platelet ratio index (APRI); aspartate aminotransferase (AST) to, alanine aminotransferase (ALT) ratio (AAR); FIB-4 score, which is based on APRI, alanine aminotransferase (ALT) content and subject age (see, e.g., McPherson et al., Gut 2010, Vol. 59(9), pp. 1265-9); hyaluronic acid; proinflammatory cytokines; a panel of biomarkers consisting of α2-macroglobulin, haptoglobin, apolipoprotein A1, bilirubin, gamma-glutamyl transferase (GGT) in combination with the age and sex of the subject to produce a measure of fibrotic and necroinflammatory activity in the liver (e.g., see, e.g., A panel of biomarkers consisting of bilirubin, gamma-glutamyl transferase, hyaluronic acid, α2-macroglobulin in combination with the age and sex of the subject (e.g., see, e.g., A Dams et al., Clin. Chem. 2005, Vol. 51(10), pp. 1867-1873), and a panel of biomarkers consisting of tissue inhibitor of metalloproteinase-1, hyaluronic acid and α2-macroglobulin (for example); a panel of biomarkers consisting of tissue inhibitor of metalloproteinase-1 (TIMP-1), amino-terminal propeptide of type III procollagen (PIIINP) and hyaluronic acid (HA) (for example, enhanced liver fibrosis (ELF) score, see, for example, Lichtinghagen R et al., J Hepatol. 2013 Aug; 59(2): 236-42).In some embodiments, the presence of fibrosis in a subject with hepatitis B and / or hepatitis D is assessed via the FIB-4 score, a panel of biomarkers consisting of α2-macroglobulin, haptoglobin, apolipoprotein A1, bilirubin, gamma glutamyl transpeptidase (GGT) in combination with the age and sex of the subject to produce a measure of fibrotic and necroinflammatory activity in the liver (e.g., a panel of biomarkers consisting of bilirubin, gamma glutamyl transferase, hyaluronic acid, α2-macroglobulin (GGT) in combination with the age and sex of the subject. For example, see, e.g., Adams et al., Clin. Chem. 2005, Vol. 51(10), pp. 1867-1873), and a panel of biomarkers consisting of tissue inhibitor of metalloproteinase-1, hyaluronic acid, and α2-macroglobulin (for example); and a panel of biomarkers consisting of tissue inhibitor of metalloproteinase-1 (TIMP-1), amino-terminal propeptide of type III procollagen (PIIINP), and hyaluronic acid (HA), such as the enhanced liver fibrosis (ELF) score.

[0272] In some embodiments, after administration of an NTCP inhibitor as described herein (e.g., a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a compound of formula (II) or a pharmaceutically acceptable salt thereof), the concentration of aspartate aminotransferase (AST) in a sample from a subject is reduced or not increased. In some embodiments, after administration of an NTCP inhibitor as described herein (e.g., a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a compound of formula (II) or a pharmaceutically acceptable salt thereof), the concentration of alanine aminotransferase (ALT) in a sample from a subject is reduced or not increased.

[0273] In some embodiments, during or after administration of an NTCP inhibitor described herein (e.g., a compound of Formula (I) or a pharmaceutically acceptable salt thereof, or a compound of Formula (II) or a pharmaceutically acceptable salt thereof), the concentration of one or more biomarkers indicative of one or more of liver damage, inflammation, liver fibrosis, and / or liver cirrhosis is reduced compared to before administration of the NTCP inhibitor, indicating that hepatitis B and / or hepatitis D is treated. For example, during or after administration of an NTCP inhibitor described herein (e.g., a compound of Formula (I) or a pharmaceutically acceptable salt thereof, or a compound of Formula (II) or a pharmaceutically acceptable salt thereof), the concentration of one or more biomarkers indicative of one or more of liver damage, inflammation, liver fibrosis, and / or liver cirrhosis is reduced compared to before administration of the NTCP inhibitor, indicating that hepatitis B and / or hepatitis D is treated. For example, a decrease in the concentration of one or more biomarkers indicative of one or more of liver damage, inflammation, fibrosis and / or cirrhosis by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% indicates that hepatitis B and / or hepatitis D has been treated. In some embodiments, the concentration of one or more biomarkers indicative of one or more of liver damage, inflammation, liver fibrosis and / or cirrhosis is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% after administration of an NTCP inhibitor.

[0274] In some embodiments, the "concentration" of an enzyme or antigen refers to the concentration of the enzyme in, for example, blood or serum. For example, the level of AST or ALT can be expressed as units / L.

[0275] Samples can be obtained from subjects at multiple time points during diagnosis, monitoring and / or treatment (e.g., using NTCP inhibitors described herein) to determine one or more clinically relevant parameters, including but not limited to the progression and treatment effects of hepatitis B and / or hepatitis D. For example, a first sample can be obtained at a first time point during diagnosis, monitoring and / or treatment, and a second sample can be obtained at a second time point. In some embodiments, the first time point can be a time point before the subject is diagnosed with hepatitis B and / or hepatitis D (e.g., when the subject is healthy), and the second time point can be a time point after the subject develops hepatitis B and / or hepatitis D (e.g., the second time point can be used to diagnose the subject with the disease). In some embodiments, the first time point can be a time point before the subject is diagnosed with hepatitis B and / or hepatitis D (e.g., when the subject is healthy), after which the subject is monitored, and the second time point can be a time point after the subject is monitored. In some embodiments, the first time point may be a time point after the subject is diagnosed with hepatitis B and / or hepatitis D, after which the subject is administered an NTCP inhibitor (e.g., a compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a compound of Formula (II), or a pharmaceutically acceptable salt thereof), and the second time point may be a time point after the administration of the NTCP inhibitor (e.g., a compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a compound of Formula (II), or a pharmaceutically acceptable salt thereof); in this case, the second time point can be used to evaluate the efficacy of the NTCP inhibitor (e.g., if the concentration of one or more biomarkers detected at the second time point is reduced or undetectable compared to the first time point).

[0276] In some embodiments, the time difference between the first time point and the second time point is from about 1 day to about 1 year, from about 1 day to about 11 months, from about 1 day to about 10 months, from about 1 day to about 9 months, from about 1 day to about 8 months, from about 1 day to about 7 months, from about 1 day to about 6 months, from about 1 day to about 5 months, from about 1 day to about 4 months, from about 1 day to about 3 months, from about 1 day to about 10 weeks, from about 1 day to about 2 months, from about 1 day to about 6 weeks, from about 1 day to about 1 month, from about 1 day to about 25 days, from about 1 day to about 20 days, from about 1 day to about 15 days, from about 1 day to about 10 days, from about 1 day to about 5 days, from about 2 days to about 1 year, from about 5 days to about 1 year, from about 10 days to about 1 year, from about 15 days to about 1 year, from about 20 days to about 1 year, from about 25 days to about 1 year, about 1 month to about 1 year, about 6 weeks to about 1 year, about 2 months to about 1 year, about 3 months to about 1 year, about 4 months to about 1 year, about 5 months to about 1 year, about 6 months to about 1 year, about 7 months to about 1 year, about 8 months to about 1 year, about 9 months to about 1 year, about 10 months to about 1 year, about 11 months to about 1 year, about 1 day to about 7 days, about 1 day to about 14 days, about 5 days to about 10 days, about 5 days to about 20 days, about 10 days to about 20 days, about 15 days to about 1 month, about 15 days to about 2 months, about 1 week to about 1 month, about 2 weeks to about 1 month, about 1 month to about 3 months, about 3 months to about 6 months, about 4 months to about 6 months, about 5 months to about 8 months, or about 7 months to about 9 months.

[0277] In some embodiments provided herein, the biomarkers described herein (e.g., HBV DNA, HBsAg, HBcAg, HBeAg, HDV DNA, or HDAg) are detected in a serum sample of a subject. In some embodiments, the biomarker levels in the serum sample are compared with the biomarker levels in a reference sample, e.g., a sample obtained from a subject before starting treatment with an NTCP inhibitor as described herein. In some embodiments, the reference sample is a serum sample from a subject (i.e., a serum sample obtained from a subject before starting treatment with an NTCP inhibitor).

[0278] Some embodiments of the methods described herein also include administering one or more additional antiviral agents. Therefore, the present invention also provides a method for treating hepatitis B (HBV) and / or hepatitis D (HDV) in a subject in need thereof, the method comprising administering one or more NTCP inhibitors (e.g., any NTCP inhibitor described herein) and one or more additional antiviral agents to the subject. One or more NTCP inhibitors (e.g., a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a compound of formula (II) or a pharmaceutically acceptable salt thereof) and one or more additional antiviral agents can be administered simultaneously, sequentially or separately.

[0279] Non-limiting examples of additional antiviral agents include nucleoside reverse transcriptase inhibitors, bulvir peptide (also known as hepcludex or Myrcludex-B), and interferon. Non-limiting examples of nucleoside reverse transcriptase inhibitors include tenofovir (e.g., ), tenofovir alafenamide (e.g., ), tenofovir disoproxil, lamivudine (e.g., or ), entecavir (e.g., ), abacavir (e.g., ), stavudine (e.g., ), didanosine (e.g., ), telbivudine (e.g., or ), zidovudine (e.g., ), zalcitabine (e.g., ), adefovir (e.g., ), adefovir dipivoxil and emtricitabine (e.g., ). Non-limiting examples of interferons include pegylated interferon (e.g., ) and interferon alpha (e.g., Intron A).

[0280] Also provided herein are methods for treating hepatitis B and / or hepatitis D, comprising administering to a corresponding subject a compound of Formula I or a pharmaceutically acceptable salt thereof, and an additional antiviral agent (e.g., any additional antiviral agent described herein), wherein the amount of the compound of Formula I or a pharmaceutically acceptable salt thereof and the additional antiviral agent together is effective for treating hepatitis.

[0281] Also provided herein are methods for treating hepatitis B and / or hepatitis D, comprising administering to a corresponding subject a compound of Formula II or a pharmaceutically acceptable salt thereof, and an additional antiviral agent (e.g., any additional antiviral agent described herein), wherein the amount of the compound of Formula I or a pharmaceutically acceptable salt thereof and the additional antiviral agent together is effective for treating hepatitis.

[0282] In some embodiments, the additional antiviral agent is tenofovir disoproxil.

[0283] Also provided herein is a method for treating hepatitis B and / or hepatitis D, comprising administering to a corresponding subject a compound of formula I or a pharmaceutically acceptable salt thereof and tenofovir disoproxil, wherein the amount of the compound of formula I or a pharmaceutically acceptable salt thereof and tenofovir disoproxil together is effective for treating hepatitis.

[0284] Also provided herein is a method for treating hepatitis B and / or hepatitis D, comprising administering to a corresponding subject a compound of formula II or a pharmaceutically acceptable salt thereof and tenofovir disoproxil, wherein the amount of the compound of formula II or a pharmaceutically acceptable salt thereof and tenofovir disoproxil together is effective for treating hepatitis.

[0285] Also provided herein is an NTCP inhibitor (such as a compound of formula (I) or a pharmaceutically acceptable salt thereof or a compound of formula (II) or a pharmaceutically acceptable salt thereof) for use in treating hepatitis B (HBV) and / or hepatitis D (HDV).

[0286] Also provided herein is an NTCP inhibitor (such as a compound of formula (I) or a pharmaceutically acceptable salt thereof or a compound of formula (II) or a pharmaceutically acceptable salt thereof) for preventing or reducing the entry of hepatitis B virus particles and / or hepatitis D virus particles into hepatocytes.

[0287] Also provided herein are NTCP inhibitors (such as compounds of formula (I) or pharmaceutically acceptable salts thereof or compounds of formula (II) or pharmaceutically acceptable salts thereof) for use in reducing hepatitis B and / or hepatitis D virus replication in hepatocytes.

[0288] Also provided herein is the use of an NTCP inhibitor (such as a compound of formula (I) or a pharmaceutically acceptable salt thereof or a compound of formula (II) or a pharmaceutically acceptable salt thereof) in the preparation of a medicament for treating hepatitis B (HBV) and / or hepatitis D (HDV).

[0289] Also provided herein is the use of an NTCP inhibitor (such as a compound of formula (I) or a pharmaceutically acceptable salt thereof or a compound of formula (II) or a pharmaceutically acceptable salt thereof) in the preparation of a medicament for preventing or reducing the entry of hepatitis B virus particles and / or hepatitis D virus particles into liver cells.

[0290] Also provided herein is the use of an NTCP inhibitor (such as a compound of formula (I) or a pharmaceutically acceptable salt thereof or a compound of formula (II) or a pharmaceutically acceptable salt thereof) in the preparation of a medicament for reducing hepatitis B and / or hepatitis D virus replication in hepatocytes.

[0291] In some embodiments, the methods described herein include administering an NTCP inhibitor described herein (e.g., a compound of formula (I) or a pharmaceutically acceptable salt thereof or a compound of formula (II) or a pharmaceutically acceptable salt thereof) as a pharmaceutical composition comprising an NTCP inhibitor and one or more pharmaceutically acceptable excipients, and optionally one or more other therapeutic agents described herein. In some embodiments, the methods include orally administering an NTCP inhibitor (e.g., a compound of formula (I) or a pharmaceutically acceptable salt thereof or a compound of formula (II) or a pharmaceutically acceptable salt thereof) as a pharmaceutical composition.

[0292] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an NTCP inhibitor as described herein (e.g., a compound of formula (I) or a pharmaceutically acceptable salt thereof or a compound of formula (II) or a pharmaceutically acceptable salt thereof), and one or more pharmaceutically acceptable excipients. Excipients may include, but are not limited to, fillers, binders, disintegrants, glidants, and lubricants. Typically, pharmaceutical compositions can be prepared in a conventional manner using conventional excipients.

[0293] Examples of suitable fillers include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose (e.g., lactose monohydrate), sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, dry starch, hydrolyzed starch, and pregelatinized starch. In certain embodiments, the filler is mannitol and / or microcrystalline cellulose.

[0294] Examples of suitable binders include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (e.g., sucrose, glucose, dextrose, lactose, and sorbitol), polyethylene glycol, waxes, natural and synthetic gums (e.g., gum arabic and gum valine), sodium alginate, cellulose derivatives (e.g., hydroxypropyl methylcellulose (or hypromellose), hydroxypropyl cellulose, and ethyl cellulose), and synthetic polymers (e.g., copolymers of acrylic acid and methacrylic acid, copolymers of methacrylic acid, copolymers of methyl methacrylate, aminoalkyl methacrylate copolymers, copolymers of polyacrylic acid / polymethacrylic acid, and polyvinyl pyrrolidone (povidone). In certain embodiments, the binder is hydroxypropyl methylcellulose (hypromellose).

[0295] Examples of suitable disintegrants include, but are not limited to, dry starch, modified starches (e.g., (partially) pregelatinized starch, sodium starch glycolate, and sodium carboxymethyl starch), alginic acid, cellulose derivatives (e.g., sodium carboxymethyl cellulose, hydroxypropyl cellulose, and low-substituted hydroxypropyl cellulose (L-HPC)), and cross-linked polymers (e.g., carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, and cross-linked PVP (crospovidone)). In certain embodiments, the disintegrant is cross-linked sodium carboxymethyl cellulose.

[0296] Examples of suitable glidants and lubricants include, but are not limited to, talc, magnesium stearate, calcium stearate, stearic acid, behenic acid glyceryl, colloidal silicon dioxide, aqueous silicon dioxide dispersion, synthetic magnesium silicate, finely divided silicon oxide, starch, sodium lauryl sulfate, boric acid, magnesium oxide, wax (such as carnauba wax), hydrogenated oil, polyethylene glycol, sodium benzoate, polyethylene glycol and mineral oil. In certain embodiments, the glidant or lubricant is magnesium stearate or colloidal silicon dioxide.

[0297] The pharmaceutical composition of the present invention can be conventionally coated with one or more coating layers. The present invention also includes an enteric coating layer or a coating layer for delayed or targeted release of the compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof. The coating layer may contain one or more coating agents and may optionally contain a plasticizer and / or a pigment (or colorant).

[0298] Examples of suitable coating agents include, but are not limited to, cellulose-based polymers (e.g., ethylcellulose, hydroxypropylmethylcellulose (or hypromellose), hydroxypropylcellulose, cellulose acetate phthalate, cellulose acetate succinate, hydroxypropylmethylcellulose acetate succinate, and hydroxypropylmethylcellulose phthalate), vinyl-based polymers (e.g., polyvinyl alcohol), and polymers based on acrylic acid and its derivatives (e.g., acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, methacrylic acid aminoalkyl ester copolymers, polyacrylic acid / polymethacrylic acid copolymers). In certain embodiments, the coating agent is hydroxypropylmethylcellulose. In other embodiments, the coating agent is polyvinyl alcohol.

[0299] Examples of suitable plasticizers include, but are not limited to, triethyl citrate, triacetin, tributyl citrate, diethyl phthalate, acetyl tributyl citrate, dibutyl phthalate, dibutyl sebacate, and polyethylene glycol. In certain embodiments, the plasticizer is polyethylene glycol.

[0300] Examples of suitable pigments include, but are not limited to, titanium dioxide, iron oxide (eg, yellow, brown, red or black iron oxide), and barium sulfate.

[0301] The pharmaceutical composition can be in a form suitable for oral administration, parenteral injection (including intravenous injection, subcutaneous injection, intramuscular injection and intravascular injection), topical administration or rectal administration. In some embodiments, the pharmaceutical composition is in a form suitable for oral administration. Pharmaceutical compositions formulated for oral administration can include, for example, tablets and capsules.

[0302] The dosage required for therapeutic or prophylactic treatment will depend on the route of administration, the severity of the disease, the age and weight of the patient, and other factors normally considered by the attending physician in determining the appropriate regimen and dosage level for a particular patient.

[0303] The amount of NTCP inhibitor (e.g., a compound of formula (I) or a pharmaceutically acceptable salt thereof or a compound of formula (II) or a pharmaceutically acceptable salt thereof) administered will vary depending on the patient being treated and may range from about 1 μg / kg body weight to about 50 mg / kg body weight per day. Unit dosage forms (e.g., tablets or capsules) typically contain about 1 to about 250 mg of active ingredient (e.g., about 1 to about 100 mg, or such as about 1 to about 50 mg, or such as about 1 to about 20 mg, such as about 2.5 mg, or about 5 mg, or about 10 mg, or about 15 mg). The daily dose may be administered as a single dose or may be divided into one, two, three or more unit doses. The oral daily dose of the bile acid regulator is preferably within about 0.1 to about 250 mg, more preferably within about 1 to about 100 mg, such as within about 1 to about 5 mg, such as within about 1 to about 10 mg, such as within about 1 to about 15 mg, or such as within about 1 to about 20 mg.

[0304] Some compounds of formula (I) or formula (II) or pharmaceutically acceptable salts thereof may show a higher free fraction in plasma. In some embodiments, the free fraction is greater than about 0.2%, for example, greater than about 0.4%, for example, greater than about 0.6%, for example, greater than about 0.8%, for example, greater than about 1.0%, for example, greater than about 1.25%, for example, greater than about 1.5%, for example, greater than about 1.75%, for example, greater than about 2.0%, for example, greater than about 2.5%, for example, greater than about 3%, for example, greater than about 4%, for example, greater than about 5%, for example, greater than about 7.5%, for example, greater than about 10% or for example, greater than about 20%.

[0305] Some compounds of formula (I) or formula (II) or pharmaceutically acceptable salts thereof may be excreted through urine. In some embodiments, the proportion of compounds excreted through urine is greater than about 0.2%, such as greater than about 0.4%, such as greater than about 0.6%, such as greater than about 0.8%, such as greater than about 1.0%, such as greater than about 2%, such as greater than about 3%, such as greater than about 5%, such as greater than about 7.5%, such as greater than about 10%, such as greater than about 15%, such as greater than about 20%, such as greater than about 30%, or such as greater than about 50%.

[0306] After absorption from the intestine, some compounds of Formula (I) or Formula (II) or their pharmaceutically acceptable salts may circulate through the enterohepatic circulation. In some embodiments, the proportion of compounds circulating through the enterohepatic circulation is greater than about 0.1%, such as greater than about 0.2%, such as greater than about 0.3%, such as greater than about 0.5%, such as greater than about 1.0%, such as greater than about 1.5%, such as greater than about 2%, such as greater than about 3%, such as greater than about 5%, such as greater than about 7%, such as greater than about 10%, such as greater than about 15%, such as greater than about 20%, such as greater than about 30%, or such as greater than about 50%.

[0307] Some compounds of formula (I) or formula (II) or pharmaceutically acceptable salts thereof may result in excretion of bile salts via the kidneys. In some embodiments, the fraction of circulating bile acids excreted via the kidneys is greater than about 1%, such as greater than about 2%, such as greater than about 5%, such as greater than about 7%, such as greater than about 10%, such as greater than about 15%, such as greater than about 20%, or such as greater than about 25%.

[0308] Some compounds of Formula (I) or Formula (II) or pharmaceutically acceptable salts thereof may exhibit improved or optimal permeability. Permeability can be measured in Caco2 cells and given as Papp (apparent permeability) values ​​in cm / s. In some embodiments, the permeability is greater than at least about 0.1 x 10 -6 cm / s, for example, greater than about 0.2 x 10 -6 cm / s, for example, greater than about 0.4x10 -6 cm / s, for example, greater than about 0.7 x 10 -6 cm / s, for example, greater than about 1.0 x 10 -6 cm / s, for example greater than about 2 x 10 -6 cm / s, for example greater than about 3 x 10 -6 cm / s, for example greater than about 5 x 10 -6 cm / s, for example greater than about 7 x 10 -6 cm / s, for example, greater than about 10x10 -6 cm / s, for example greater than about 15 x 10 -6 cm / s.

[0309] Some compounds of formula (I) or formula (II) or their pharmaceutically acceptable salts may show improved or optimal bioavailability. In some embodiments, the oral bioavailability is greater than about 5%, for example, greater than about 7%, for example, greater than about 10%, for example, greater than about 15%, for example, greater than about 20%, for example, greater than about 30%, for example, greater than about 40%, for example, greater than about 50%, for example, greater than about 60%, for example, greater than about 70% or for example, greater than about 80%. In other embodiments, the oral bioavailability is about 10 to about 90%, such as about 20 to about 80%, such as about 30 to about 70% or such as about 40 to about 60%.

[0310] Some compounds of Formula (I) or Formula (II) or pharmaceutically acceptable salts thereof may be substrates for relevant transport proteins in the kidney.

[0311] Also provided herein is a method for preventing or reducing hepatitis B virus particles and / or hepatitis D virus particles from entering a cell, the method comprising contacting the cell with an NTCP inhibitor as described herein (e.g., a compound of formula (I) or a pharmaceutically acceptable salt thereof or a compound of formula (II) or a pharmaceutically acceptable salt thereof). Also provided herein is a method for reducing hepatitis B and / or hepatitis D virus replication in a cell, the method comprising contacting the cell with an NTCP inhibitor as described herein (e.g., a compound of formula (I) or a pharmaceutically acceptable salt thereof or a compound of formula (II) or a pharmaceutically acceptable salt thereof). Also provided herein is a method for preventing the preS1 domain of the L protein of HBV from binding to NTCP in a cell, the method comprising contacting the cell with an NTCP inhibitor as described herein (e.g., a compound of formula (I) or a pharmaceutically acceptable salt thereof or a compound of formula (II) or a pharmaceutically acceptable salt thereof). In some embodiments, the contact is in vitro. In some embodiments, the contact is in vivo. In some embodiments, the cell is a hepatocyte. In some embodiments, the contacting is in vivo, wherein the method comprises administering to the subject's hepatocytes an effective amount of an NTCP inhibitor described herein (e.g., a compound of Formula (I) or a pharmaceutically acceptable salt thereof or a compound of Formula (II) or a pharmaceutically acceptable salt thereof).

[0312] definition

[0313] As used herein, the term "halogen" refers to fluorine, chlorine, bromine and iodine.

[0314] The term "C 1-6 "Alkyl" refers to a straight or branched chain alkyl group having 1 to 6 carbon atoms. 1-4 "Alkyl" refers to a straight or branched chain alkyl group having 1 to 4 carbon atoms. 1-4 Examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

[0315] The term "C 1-4 "Haloalkyl" refers to a straight or branched chain C 1-4 Alkyl in which one or more hydrogen atoms have been replaced by halogen. 1-4 Examples of the haloalkyl group include chloromethyl, fluoroethyl and trifluoromethyl.

[0316] The term "C 1-4 Alkoxy" and "C 1-4 "Alkylthio" refers to a straight or branched C 1-4 alkyl.

[0317] The term "C 3-6 "Cycloalkyl" refers to a monocyclic saturated hydrocarbon ring having 3 to 6 carbon atoms. 3-6 Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0318] The term "aryl" refers to an aromatic monocyclic ring consisting of 6 carbon atoms or an aromatic bicyclic ring system consisting of 10 carbon atoms. Examples of aryl groups include phenyl, naphthyl and azulenyl.

[0319] The term "amino" refers to a -NH2 group. As used herein, the term "N-(C 1-4 Alkyl)amino" and "N,N-di(C 1-4 "Alkyl)amino" means a group in which one or two hydrogen atoms are replaced by a straight chain or branched C 1-4 Alkyl substituted amino. N-(C 1-4 Examples of N,N-di(C 1-4 Examples of the alkyl)amino group include dimethylamino and diethylamino.

[0320] As used herein, the term "N-(aryl-C 1-4 "alkyl)amino" refers to a group in which a hydrogen atom is replaced by an aryl-C 1-4 Alkyl substituted amino. N-(aryl-C 1-4 Examples of the amino group include benzylamino and phenylethylamino. 1-6 "Alkylcarbonylamino" means a group in which one of the hydrogen atoms is replaced by a C 1-6 Alkylcarbonyl substituted amino group. 1-6 Examples of alkanoylamino groups include acetylamino and tert-butylcarbonylamino. 1-4 "Alkoxycarbonylamino" refers to a group in which the hydrogen atom is replaced by a C 1-4 Alkoxycarbonyl substituted amino group. 1-4 An example of an alkoxycarbonylamino group is tert-butyloxycarbonylamino. 1-4Alkylsulfonamide" and "C 3-6 "Cycloalkylsulfonyl" refers to a cycloalkylsulfonyl group in which the hydrogen atoms are replaced by C 1-4 Alkylsulfonyl or C 3-6 Amine substituted with cycloalkylsulfonyl.

[0321] Some compounds of formula (I) or formula (II) or their pharmaceutically acceptable salts may have chiral centers and / or geometric isomer centers (E and Z isomers). It should be understood that the present invention encompasses all such optical isomers, diastereomers and geometric isomers having ASBT and / or LBAT inhibitory activity. The present invention also encompasses any and all tautomeric forms of compounds of formula (I) or formula (II) or their pharmaceutically acceptable salts having ASBT and / or LBAT inhibitory activity. Certain compounds of formula (I) or formula (II) or their pharmaceutically acceptable salts may exist in unsolvated and solvated forms, such as hydrated forms. It should be understood that the present invention encompasses all such solvated forms having ASBT and / or LBAT inhibitory activity.

[0322] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for human pharmaceutical use and are generally safe, non-toxic, and have no adverse effects, biologically or otherwise.

[0323] Suitable pharmaceutically acceptable salts of the compounds disclosed herein are, for example, base addition salts of the compounds with sufficient acidity, such as alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., calcium or magnesium salts), ammonium salts, or salts with organic bases providing physiologically acceptable cations, such as salts with methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine.

[0324] As used herein, the terms "treat", "treatment" and "treatment" refer to reversing, alleviating, delaying the onset of a disease or disorder or one or more symptoms thereof, or inhibiting its progression, as described herein. In some embodiments, the NTCP inhibitor is administered after the onset of one or more symptoms. In other embodiments, the NTCP inhibitor may be administered in the absence of symptoms. For example, the NTCP inhibitor may be administered to susceptible individuals before the onset of symptoms (e.g., based on a history of symptoms and / or based on genetic or other susceptibility factors). After the symptoms subside, the NTCP inhibitor may also continue to be administered, for example, to prevent or delay its recurrence.

[0325] As used herein, the terms "subject," "individual," or "patient" are used interchangeably and refer to any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, primates, and humans. In some embodiments, the subject is a human. In some embodiments, the subject has experienced and / or exhibits at least one symptom of hepatitis B and / or hepatitis D.

[0326] As used herein, the term "contacting" refers to bringing the indicated moieties together in an in vitro system or an in vivo system. For example, "contacting" a compound provided herein with NTCP includes administering a compound provided herein to a subject (e.g., a human) having NTCP, and, for example, introducing a compound provided herein into a sample containing cells or purified preparations containing NTCP.

[0327] The term "about" as used herein refers to a value or parameter herein, which includes (and describes) embodiments for the value or parameter itself. For example, a description referring to "about 20" includes a description of "20". Numerical ranges include numbers that define ranges. In general, the term "about" refers to the indicated value of a variable and all values ​​of the variable that are within the experimental error range of the indicated value (e.g., within a 95% confidence interval of the mean) or within 10% of the indicated value, whichever is greater. Example

[0328] Example 1. In vitro inhibition of NTCP and IBAT.

[0329] The effect of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid (Compound 1) on NTCP- and IBAT-mediated bile acid transport was evaluated using an overexpression cell line system.

[0330] method

[0331] NTCP detection procedure

[0332] 20,000 cells (human and mouse NTCP overexpressing cells) were seeded into 96-well plates in 100 μL of MEM-alpha medium supplemented with 10% FBS and containing geneticin (1 mg / mL) and incubated for 24 hours at 37°C and 5% CO2. After incubation, the medium was poured out from the wells and the cells were washed twice with 250 μL of basic MEM-alpha medium (without FBS). After each wash, the dish was patted with a paper towel to ensure maximum removal of residual medium. For human NTCP (hNTCP), the incubation mixture was prepared by rinsing in 100 μL of MEM-alpha medium containing 0.3 μM 3 Test inhibitor dilutions (3-fold serial dilutions in DMSO, 10 concentrations) were added to MEM-alpha (without FBS) containing 0.3 μM H-taurocholic acid and 7.5 μM cold taurocholic acid (maintaining a final concentration of 0.2% DMSO). For mouse NTCP, the incubation mixture was prepared by adding test inhibitor dilutions (3-fold serial dilutions in DMSO, 10 concentrations) to MEM-alpha (without FBS) containing 0.3 μM 3 H-taurocholic acid and 25 μM cold taurocholic acid (maintaining a final DMSO concentration of 0.2%) in MEM-alpha (without FBS). Then 50 μL of the incubation mixture containing the test inhibitor was added to the wells (in duplicate) and the plates were incubated in a CO2 incubator at 37°C for 20 minutes. The wells were washed twice with 250 μL of frozen unlabeled 1 mM taurocholic acid (dissolved in HEPES buffer (10 mM) HBSS (pH 7.4)).

[0333] After each wash, the plate was tapped with a paper towel to ensure maximum removal of the blocking buffer. 100 μL MicroScint-20 was added to the wells and left overnight at room temperature before reading the plates in a PerkinElmer MicroBeta2 liquid scintillation counter according to the 3H Test of the I / L-BAT protocol (set to 120 seconds per well read time, plate orientation normal).

[0334] IBAT testing procedures.

[0335] 10,000 cells (human and mouse IBAT overexpressing cells) were seeded into 100 μL of MEM-alpha medium in a 96-well plate supplemented with 10% FBS containing puromycin (10 μg / mL) and incubated for 48 hours at 37°C and 5% CO2. After pouring the incubation medium from the wells, the cells were washed twice with 250 μL of basic MEM-alpha medium (without FBS). After each wash, the plate was tapped on a paper towel to ensure maximum removal of residual medium. Dilutions of the test inhibitor prepared in DMSO (highest tested concentration was 10 μM, 3-fold serial dilutions, 10 concentrations) containing 0.25 μM were added to the incubation mixture (maintaining a final DMSO concentration of 0.2%). 3 H-taurocholic acid and 5 μM cold taurocholic acid. 50 μL of the incubation mixture containing the test inhibitor was then added to the wells (repeated twice) and the plates were incubated in a CO2 incubator at 37°C for 20 minutes.

[0336] After incubation, the plate was placed in an ice-water mixture for 2-3 minutes to stop the reaction, and the incubation mixture was completely aspirated from the wells. The wells were washed twice with 250 μL of chilled unlabeled 1 mM taurocholic acid (dissolved in HEPES-buffered (10 mM) HBSS (pH 7.4)). After each wash, the plate was tapped on a paper towel to ensure maximum removal of the blocking buffer. 100 μL of MicroScint-20 was added to the wells and left overnight at room temperature, and then counted using a PerkinElmer MicroBeta2 liquid scintillation counter according to the I / L-BAT protocol. 3 Read the plate using the H Test (set to 120 seconds read time per well, normal plate orientation).

[0337] Statistical analysis

[0338] The percentage of inhibition was calculated relative to the experimental control. Further data analysis was performed using validated statistical software (GraphPad Prism) to calculate the IC of the test compound. 50 value.

[0339] result

[0340] IC of compound 1 in human NTCP 50 The value was 0.53±0.081 nM. The positive control myrcludex B (MyrB) performed as expected.

[0341] IC of compound 1 in mouse NTCP 50 The value was 1.82±0.25 nM. The positive control MyrB performed as expected.

[0342] IC of compound 1 in human IBAT50 The value was 535 ± 83.3 nM. The performance of the positive control AS0075 was in line with expectations.

[0343] IC of compound 1 in mouse IBAT 50 The value was 40.8±4.6 nM. The positive control AS0075 performed as expected.

[0344] Example 2. In vitro inhibition of NTCP and HBV.

[0345] The in vitro potency of compound 1 in inhibiting bile acid transport was evaluated.

[0346] method

[0347] Assay procedure in CHO cells.

[0348] CHO cells stably transfected with hNTCP were seeded into 24-well plates. After overnight incubation, CHO cells were treated with 10 mM sodium butyrate to induce NTCP transporter expression. Twenty-four hours later, the medium was aspirated and the cells were washed with warm Hanks balanced salt solution (HBSS) containing sodium (+Na). 3 Cells were incubated with increasing doses of compound 1 (e.g., 0, 10 nM, 100 nM, 1 μM, 10 μM) at 37°C for <10 min at a tracer dose of 100 μM H]taurocholate. Cells were irradiated. Apparent K was determined by complementary Dixon / Cornish-Bowden plot analysis and Lineweaver-Burk plot analysis. i and type of kinetic inhibition (competitive, noncompetitive, uncompetitive, mixed).

[0349] In vitro assessment of the dissociation time course.

[0350] This study used [ 3 H] Taurocholic Acid Uptake Assay Protocol. Stably transfected CHO cells expressing human NTCP (hNTCP) were seeded into 24-well plates at a density of 1.5-2.0 x 10 5Cells / well, the culture medium is CHO cell culture medium (DMEM / F12 medium, supplemented with 10% FBS, penicillin / streptomycin) plus geneticin (G418) to maintain culture selection. After overnight incubation, the culture medium is changed and CHO cells are treated with 10mM sodium butyrate to induce transporter expression (final concentration = 10μM). After 24 hours, the culture medium is removed and the cells are washed with warm (37°C) Hanks balanced salt solution (HBSS) containing sodium (Na+). The cells are then incubated with 1μM test inhibitor or vehicle (DMSO) in CHO cell culture medium at 37°C for 15 minutes. After a 15-minute inhibitor incubation period, the culture medium is removed and the cells are washed again with warm Hanks balanced salt solution (HBSS) containing sodium (Na+). New CHO cell culture medium (without compound) is added to the cells to start the inhibitor "washout" period, and then the plate is returned to the tissue culture incubator.

[0351] for[ 3 [H] Taurocholic acid uptake assay: At the end of each washout period, the medium was aspirated and the cells were washed three times with warm Hanks balanced salt solution (HBSS) containing sodium (Na+). The cells were then incubated with 5 μM [ 3 H] taurocholate in HBSS and incubate at 37 °C on a slide warmer for 10 min. For the 0 min time point, 3 The test inhibitor was present at 1 μM during the taurocholic acid uptake period. The cells were then moved to ice and the [ 3 H] taurocholic acid plus a mixture of inhibitors. Cells were washed with ice-cold HBSS + choline (instead of Na +) and lysed with 0.1N NaOH. 100 μL replicates of the lysate were transferred to vials containing scintillation fluid and counted after neutralization and overnight quenching. Cell-associated proteins were measured in duplicate using the BCA protein assay, and cell-associated radioactivity was normalized to protein content.

[0352] result

[0353] Compound 1 acts as a competitive inhibitor of transporter activity and has a significant effect on human NTCP ( Figure 2A ) and ASBT( Figure 2B ) of the inhibition constant (K i ) were 15nmol / L and 2410nmol / L, respectively. In vitro experiments showed that compound 1 showed persistent inhibition of human NTCPBA transport activity (inhibition rate >50% after 6 hours of elution) ( Figure 3A (Myrcludex B) and 3B (Compound 1)).

[0354] Example 3. HBV / HDV derived preS1 peptides bound to NTCP.

[0355] The myristoylated preS1 domain, which comprises amino acids 2-48 of the large HBV envelope, is required for viral binding to NTCP and can be used as a surrogate parameter for HBV / HDV viral binding to NTCP. In this in vitro study, the potency and selectivity of compound 1 in inhibiting taurocholic acid transport via human NTCP and cynomolgus monkey NTCP bound to human NTCP, HBV / HDV preS1-peptide, and in vitro HDV and HBV infection of NTCP-HepG2 cells were investigated.

[0356] method

[0357] NTCP-HEK293 and NTCP-HepG2 cell lines.

[0358] Human embryonic kidney (HEK293) cells were stably transfected with human NTCP and C-terminally tagged with a FLAG epitope (referred to here as NTCP-HEK293 cells). Cells were cultured in DMEM / F-12 medium (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C, 5% CO2, and 95% humidity with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 4 mM L-glutamine, and penicillin / streptomycin. HepG2 cells stably transfected with NTCP-FLAG (referred to here as NTCP-HepG2) were cultured under the same conditions in DMEM with all the supplements mentioned above (except L-glutamine). To induce the transgene, 1 μg / mL tetracycline was added to the culture medium in NTCP-HEK293 cells and 2 μg / mL doxycycline was added to the culture medium in NTCP-HepG2 cells. See also A et al (2014) Kinetics of the bile acid transporter and hepatitis B virus receptor Na+ / taurocholate cotransporting polypeptide (NTCP) in hepatocytes. Journal of Hepatology 61:867-875; Kirstgen M et al (2021) Hepatitis D Virus Entry Inhibitors Based on Repurposing Intestinal Bile Acid Reabsorption Inhibitors. Viruses 13(4):666; Kirstgen M et al (2021) Identification of novelHBV / HDVentry inhibitors by pharmaceutical- and QSAR-guided virtual screening. Viruses 13:1489; and Grosser G et al (2021) Substrate Specificities and Inhibition Pattern of the Solute CarrierFamily 10MembersNTCP, ASBT and SOAT. Front Mol Biosci 8:689757.

[0359] HEK293 cells transfected with cynomolgus monkey NTCP.

[0360] GripTite 293MSR cells (referred to here as HEK293 cells, Invitrogen) are a modified HEK293 cell line that expresses the human macrophage scavenger receptor for enhanced adhesion and were maintained at 37°C, 5% CO2, and 95% humidity in DMEM / F-12 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Sigma), 4mM L-glutamine (PAA), and penicillin / streptomycin. HEK293 cells were transiently transfected with cynomolgus monkey NTCP cDNA constructs for transport and peptide binding assays. Transfections were performed using Lipofectamine 2000 (Thermo Fisher Scientific). See also Müller SF et al (2018) Characterization of the hepatitis B virus cross-species transmission pattern via Na+ / taurocholate co-transporting polypeptides from 11 New World and Old World primate species. PLoSOne 13(6):e019920.

[0361] [ 3 H]TC transport and 3 H]preS1 binding inhibitory concentration (IC 50 ).

[0362] Tritium-labeled taurocholic acid (referred to herein as [ 3 Bile acid transport measurements were performed in NTCP-HEK293 cells using tritium-labeled myr-preS12-48 lipopeptide-HBV subgenotype D3- (referred to herein as [ 3Peptide binding experiments were performed using 50 μL DMEM (120 Ci / mmol, 1 mCi / mL, Cardiff, UK). Briefly, cells were seeded onto poly-lysine-coated 96-well plates, induced with 1 μg / mL tetracycline, and grown to confluence at 37°C for 72 h. Cells were then washed once with thermoregulated phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 7.3 mM Na2HPO4, pH 7.4) at 37°C and preincubated with 80 μL DMEM for 5 min at 37°C. The medium was replaced with 80 μL DMEM containing the corresponding inhibitor concentration or solvent only (100% uptake / binding control), and cells were incubated for an additional 5 min at 37°C. After preincubation, the cells were incubated with 5 μM [ 3 The bile acid transport experiment was started by adding 20 μL DMEM containing 25 nM [ 3 H]preS1 (final concentration: 5 nM) in DMEM to start 3 H] preS1 binding. The experiment was stopped after 10 min by washing twice with ice-cold PBS. For the 0% uptake / binding control, NTCP-HEK293 cells were not induced with tetracycline (-tet). [ 3 H]TC or [3 The cell-associated radioactivity of H]preS1 was determined in counts per minute (cpm). 3 H]preS1 binding. Subtract the mean of 0% control and net [ 3 H]TC transport rate and net [ 3 H]preS1 binding rates were expressed as percentages of the control. 50 Values ​​were calculated from four determinations using GraphPad Prism 6 (GraphPad, San Diego, CA, USA). See also Kirstgen et al. 2020, 2021, Grosser et al. 2021.

[0363] MTT cytotoxicity assay.

[0364] The cytotoxicity of the test substance was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay using an in vitro toxicology test kit (Sigma-Aldrich) according to the manufacturer's protocol. In brief, NTCP-HepG2 cells were incubated at 37°C for 6 hours with 100 μL of the test substance at the indicated concentration (dissolved in hepatocyte growth medium (HGM)). After 6 hours, the culture medium was replaced with HGM without inhibitors, and the cells were cultured for another 24 hours. The culture medium was then removed, 100 μL of DMEM containing 0.5 mg / mL MTT was added, and the cells were incubated at 37°C for 1 hour. Finally, the culture medium was replaced with 100 μL of isopropanol (Sigma-Aldrich), and the samples were measured with the Glo-Max-Multi detection system (Promega, Madison, WI, USA). See also Lowjaga KAA et al. (2020). Long-term trans-inhibition of the hepatitis B and D virus receptor NTCP by taurolithocholic acid. Am J Physiol Gastrointest Liver Physiol 320(1):G66-G80; and Kirstgen et al. 2021.

[0365] HDV infection in vitro.

[0366] HDV (HDV genotype 1, coated with HBV surface proteins of genotype D, see HBV below) was produced in vitro as described in the literature (Rasche A, et al. (2019) Highly diversified shrew hepatitis B viruses corroborate ancient origins and divergent infection patterns of mammalian hepadnaviruses. Proc Natl Acad Sci US A 116(34): 17007-17012; DeCarvalho Dominguez Souza BF, et al. (2018) A novel hepatitis B virus species discovered in capuchin monkeys sheds new light on the evolution of primate hepadnaviruses. Journal of Hepatology 68: 1114-1122). RT-qPCR was performed to determine the HDV RNA genome equivalent. NTCP-HepG2 cells were pre-incubated with compound 1 in HGM for 5 minutes at concentrations of 100nM, 200nM and 400nM, respectively. Infection experiments were performed in NTCP-HepG2 cells. Cell cultures were cultured in HGM in 48-well plates, and HGM consisted of William's E medium (Thermo Fisher Scientific), which contained 2% bovine serum albumin (BSA, Roth), 2mM L-glutamine, 100μg / mL gentamicin, 10nM dexamethasone, 1mM sodium pyruvate, 1X insulin transferrin selenium, 2% DMSO, 4% polyethylene glycol and 2μg / mL doxycycline. HDV stock solution was diluted in HGM (HDV: 2x 109GE / well) and added to cells for 6 hours. Subsequently, cells were washed with DMEM and cultured in HGM containing 2% DMSO, 2% BSA and 2μg / mL doxycycline. The medium was changed every three days until day 9 after infection. Cells were fixed with 3% paraformaldehyde in PBS for 30 minutes at room temperature (RT). Cells were permeabilized with 0.2% Triton X 100 in PBS for 30 minutes at room temperature and blocked by incubation with 5% bovine serum albumin in PBS for 30 minutes at room temperature. Cells were then immunostained with purified human anti-HDV positive serum for 1 hour at 37°C (1:400 dilution).Goat anti-human IgG secondary antibody conjugated with Alexa Fluor fluorophore (1:400 dilution, Thermo Fisher Scientific) was added and incubated for 1 hour at 37°C to detect hepatitis D antigen (HDAg). Cell nuclei were stained with Hoechst33342 (1 μg / mL, Thermo Fisher Scientific). The number of infected cells in each well was determined by fluorescence microscopy.

[0367] In vitro HBV infection.

[0368] In the first study, NTCP-HepG2 cells were seeded for 16 h with similar HBV genome equivalents per cell. HBV (genotype D) was generated in vitro as described ( et al., 2014). Infections were performed in HGM supplemented with 2% DMSO and 4% polyethylene glycol (5 x 10 9 GE / well HBV). Thereafter, the cells were washed twice with HGM and cultured in HGM supplemented with 2% DMSO and 2% FCS until day 10 after infection. 10 days after infection, fixation was performed with 3.7% formaldehyde and 1% methanol at 4°C for 30 minutes. The cells were permeabilized with 0.2% Triton X100 in PBS for 20 minutes at room temperature. Nonspecific binding epitopes were blocked by incubation with 10% fetal bovine serum in PBS at 37°C for 45 minutes. To detect the expression of HBV core (HBc) protein, cells were incubated in PBS with polyclonal rabbit anti-HBcAg antiserum (1:500 dilution, Dako, Hamburg, Germany) at 37°C for 2 hours and then incubated with anti-rabbit IgG AlexaFluor594 (1:200 dilution in PBS, ImmunoJackson) at 37°C for 1 hour. The nuclei were stained with DAPI (10 μg / mL) in PBS. The number of infected cells in each well was determined by fluorescence microscopy. In addition, HBeAg was quantified in the supernatants from day 7 to day 11 post-infection.

[0369] In the second study, recombinant HBV (genotype D) was produced in vitro as described ( et al., 2014). For infection experiments, NTCP-HepG2 cells (2x10 5 Cells) were pre-incubated with compound 1 or Myrcludex B in HGM for 5 min at concentrations of 1, 3, 10, 30, 100, 300, and 1000 nM. Figure 7D-7FThe myr-preS12-48 peptide (500 nM, genotype D) was used as a control inhibitor. All infection experiments were performed in NTCP-HepG2 cells cultured in HGM in the presence of different concentrations of the corresponding compounds, 4% PEG-8000 (Sigma-Aldrich, Darmstadt, Germany), 2% DMSO (Carl Roth, Karlsruhe, Germany) and 100 ng / ml EGF (PeproTech, Hamburg, Germany) as respective final concentrations. Recombinant HBV was diluted in HGM to a final concentration of 2.5 × 10 10 HBV genome copies (7x 10 per well 8 IU) and added to the cells for 6 hours. Subsequently, the cells were washed with HGM and cultured in HGM supplemented with 2% DMSO. The culture medium was replaced every 2-3 days until the cells were fixed at room temperature for 30 minutes with 3.8% paraformaldehyde (Sigma-Aldrich) in PBS on the 9th day after infection. The cells were immunostained at 37°C for 1 hour (1:400 dilution). To detect the expression of HBV core (HBc) protein, polyclonal anti-HBc antiserum from immune guinea pigs (1:500 dilution, Eurogentec, Seraing, Belgium) was used as the primary antibody, and Alexa Fluor 488-conjugated goat anti-guinea pig IgG antibody (1:400 dilution, Thermo Fisher Scientific) was used as the secondary antibody. The nuclei were stained with DAPI (0.5μg / ml, Thermo Fisher Scientific). Immunofluorescence analysis was performed using the ImageXpress Pico automated cell imaging system (Molecular Devices, San José, USA). HBV-infected cells secrete soluble HBeAg, which is a widely accepted marker for quantifying HBV infection in cell culture experiments. In this study, HBeAg secreted from infected cells into the cell culture supernatant was measured using the Architect HBeAg assay, an automated in vitro diagnostic system (Abbott Laboratories, Wiesbaden, Germany), in which HBeAg was expressed in the supernatant from day 5 to day 9 after infection.

[0370] The supernatant was collected on the next day.

[0371] statistics

[0372] IC was determined by nonlinear regression analysis using the equation log(inhibitor) vs. response setting in GraphPad Prism 6.0 software (GraphPad).50 value.[ 3 H]TC transport and 3 The data for H]preS1 binding are presented as the mean ± SD of four determinations. The infection studies were performed in triplicate and the data are presented as the mean ± SD. Statistical analysis of HBV / HDV infection experiments was performed using two-way ANOVA followed by Dunnett's multiple comparison test using GraphPad Prism 9.0, and p < 0.01 was considered statistically significant.

[0373] result

[0374] Inhibition of human NTCP by compound 1.

[0375] Compound 1 inhibited the uptake of [3H]taurocholic acid in a concentration-dependent manner (half maximal inhibitory concentration [IC 50 ], 186nmol / L; Figure 4A )and[ 3 H]preS1 peptide binding (IC 50 ,149nmol / L; Figure 4C 500 nM preS1 peptide was used as a control inhibitor in both assays and was significantly inhibited in both assays, as expected and consistent with literature reports (see Figure 4B and 4D ; et al. 2014, Müller et al. 2018, Lowjaga et al. 2021).

[0376] Inhibition of cynomolgus monkey Ntcp by compound 1.

[0377] In addition, compound 1 was used as an inhibitor of cynomolgus monkey NTCP. The vector was transiently transfected into HEK293 cells and analyzed at increasing concentrations of 10, 25, 63, 158, 398, and 1000 nM for [ 3 H]TC transport inhibition. Figure 5A and 5B As shown in Figure 2, compound 1 effectively inhibited the transport of bile acids in cynomolgus monkey Ntcp in a concentration-dependent manner, with an IC 50 is 200nM.

[0378] Cytotoxicity evaluation of compound 1 in NTCP-HepG2 cells.

[0379] The cytotoxicity of compound 1 was evaluated in HepG2 cells at concentrations ranging from 1 nM to 100 μM. Figure 6 As shown, compound 1 had no cytotoxic effect in HepG2 cells.

[0380] Inhibition of HBV / HDV infection in vitro by compound 1.

[0381] The in vitro inhibitory effect of compound 1 on HBV / HDV infection was analyzed in human NTCP-HepG2 hepatoma cells, which represent a well-established cell culture model for in vitro HBV / HDV infection (see et al., 2014 ).

[0382] In the first study, different concentrations (100 nM, 200 nM, and 400 nM) of compound 1 were used before and during incubation of the cells with the virus inoculum. Compound 1 reduced the number of HBcAg-positive cells by 75%-90% ( Fig. 7A ), and the number of HDAg-positive cells decreased by 40%-60% ( Figure 7B ), reducing HBeAg levels in the supernatant by 83%-87% ( Figure 7C A similar approach using the preS1-peptide (aa2-48, genotype D, 500 nM) almost completely inhibited HDV / HBV infection.

[0383] In the second study, NTCP-HepG2 cells were pre-incubated for 5 min with compound 1 at concentrations of 1, 3, 10, 30, 100, 300, and 1000 nM. In the case of compound A2342, HBeAg secretion was above the cut-off value ( Figure 7D-7G ). Nevertheless, it is still possible to calculate the IC 50 Compound 1 inhibits HBV infection in vitro, IC 50 The value is 19.1nM (HBeAg) ( Fig.7D and 7E ) and 16.1nM(HBc)( Figure 7F and 7G ).

[0384] discuss

[0385] This embodiment describes the dual [ 3 H]TC transport and 3 H]preS1 peptide binding assays have been used to characterize the inhibitory potency and selectivity of human NTCP inhibitors for viral binding inhibition (see Kirstgen et al., 2020, 2021). Since the same assay with the same parameters was used in this Example y, the measured IC values ​​of compound 1 can be used as 50 The values ​​were directly compared with published data. Compound 1 was equivalent to the viral preS1 peptide as an inhibitor and was more potent than the well-established NTCP inhibitor troglitazone.

[0386] Compound 1 showed strong inhibitory effects on HBV and HDV infection at selected concentrations. Compound 1 effectively blocked the entry of HBV and HDV viral particles into NTCP-HepG2 cells in the nanomolar range.

[0387] Example 4. Effect of Compound 1 on HBV infection in human hepatocytes in vitro.

[0388] The antiviral effect of compound 1 was evaluated using HBV-infected human hepatocytes in vitro.

[0389] method

[0390] Hepatocyte incubation.

[0391] According to the protocol provided by the hepatocyte supplier (BiolVT), one vial of primary human hepatocytes (PHH) was seeded in a 48-well collagen-coated plate. The total volume per well was 200 μL, and the number of cells seeded was 0.14 x 10 6 Cells were incubated overnight at 37°C in a 5% CO2 atmosphere using InVitroGro HI medium and Torpedo antibiotic cocktail. Unless otherwise stated, diluted compounds were added to cells 18 hours prior to infection (standard conditions).

[0392] The multiplicity of infection (MOI) is defined as the number of HBV genome equivalents added per cell in the corresponding growth form as determined by qPCR quantification. Overnight medium was removed from the cells and replaced with 190 μL of new serum-free HI medium / 4% PEG8000 and 10 μL of diluted virus stock (HepG2 AD38 genotype D) at an MOI of 500. Medium was added only to cells containing compounds for parallel cytotoxicity assessments. After 18 hours of viral infection, cells were gently washed five times with DPBS, and then new 200 μL HI medium containing compounds was added on days 1, 4, and 7 after infection. In one set of experiments (study #01), the effect of administering compounds 18 hours before infection versus 18 hours after infection was investigated. At each change of medium, supernatants removed from the cytotoxicity plates were stored at -20°C for shipment to customers. Supernatants were collected 10 days after infection and stored at -20°C for quantification of total extracellular HBV DNA copies by qPCR. On day 10, cell viability was measured on cytotoxicity plates using XTT tetrazolium dye.

[0393] Quantitative PCR detection of total HBV DNA in supernatants.

[0394] Ten microliters (10 μL) of cell culture supernatant collected on day 10 was diluted into 90 μL of buffer consisting of 10 mM Tris, 40 μg / mL sheared salmon sperm DNA, and boiled for 15 minutes. Quantitative PCR (qPCR) was performed in 384-well plates using the Bio-Rad CFX384 Touch Real-Time PCR Detection System and the accompanying CFX Manager Software. Five microliters (5 μL) of diluted and boiled cell culture supernatant and serial 10-fold dilutions of the quantitative DNA standards were subjected to real-time qPCR using Platinum Quantitative PCR SuperMix-UDG (Invitrogen) and specific DNA oligonucleotide primers (IDT, Coralville, ID) HBV-AD38-qF1 (5′-CCG TCT GTG CCT TCT CAT CTG-3′), HBV-AD38-qR1 (5′-AGT CCA AGA GTY CTC TTA TRY AAG ACC TT-3′), and HBV-AD38-qP1 (5′-FAM-CCG TGT GCA / ZEN / CTT CGC TTC ACC TCT GC-3′BHQ1) with a final concentration of 0.2 μmol / L for each primer in a total reaction volume of 25 μL. The amount of HBV DNA in each sample was estimated based on the standard curve, and the data were imported into an Excel spreadsheet for analysis.

[0395] Cytotoxicity.

[0396] After 10 days of culture in a 37°C 5% CO2 incubator, the test plates were stained with the tetrazolium dye XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl}-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide). XTT-tetrazolium is metabolized by mitochondrial enzymes of metabolically active cells to a soluble formazan product. A 1 mg / mL XTT solution was prepared in RPMI1640 as a stock solution every day. A 0.15 mg / mL phenazine methosulfate (PMS) solution was prepared in PBS and stored at -20°C in the dark.

[0397] Prepare the XTT / PMS stock solution immediately before use by adding 40 μL of PMS per mL of XTT solution. Add 50 μL of XTT / PMS to each well of the plate and re-incubate the plate at 37°C for 4 hours. Seal the plate with an adhesive plate sealer, gently shake or invert several times to mix the soluble formazan product, and then measure the plate spectrophotometrically at 450 / 650 nm using a Molecular Devices Vmax plate reader.

[0398] Experimental design.

[0399] The experimental design is shown in Table 3.

[0400] Table 3.

[0401]

[0402] Data analysis.

[0403] Each concentration of inhibitor and control was analyzed in triplicate for HBV DNA copy number and cytotoxicity. The average of these triplicates was calculated from the control wells without inhibitor, and the HBV DNA average was set as 100% virus control and the XTT average was set as 100% cell viability. The effects of the compounds were then expressed as a percentage of virus control and a percentage of cell viability, respectively.

[0404] In two sets of experiments, the effect of compound 1 in combination with tenofovir was studied. The effect of the drug combination was calculated based on the activity of the two compounds when tested individually. The expected additional antiviral protection (based on the dose-response curves of the individual drugs used alone) was subtracted from the experimentally determined antiviral activity at each combination concentration to obtain a positive value (synergy), a negative value (antagonism), or zero (additive effect). The data were analyzed with the most rigorous statistical methods by assuming that the compounds inhibited HBV replication by acting at the same site (mutually exclusive). The results of the combination assay were expressed as synergy volume μM2% calculated using the MacSynergy II template with a 95% confidence interval.

[0405] In these studies, synergy was defined as a drug combination producing a synergistic effect greater than 50 μM 2%. Mild synergy and high synergy were defined as producing a synergistic effect between 50 and 10 μM 2% and >100 μM 2%, respectively.

[0406] Synergistic amounts between -50 and 50 μM2% were considered additive, whereas synergistic amounts less than -50 μM2% were considered antagonistic.

[0407] result

[0408] Study #01.

[0409] EC values ​​of tenofovir and compound 1 were 50 The values ​​are summarized in Table 4 and are Fig. 8A Tenofovir showed expected potency (EC 50 :43nmol / L). myrcludexB (EC 50 :0.4nmol / L) and compound 1 (EC 50: 4nmol / L) were more effective than tenofovir. These compounds did not induce cytotoxicity at any concentration tested (highest concentration: 1μmol / L). Figure 8C .

[0410] When tenofovir, compound 1, and myrcludex B were administered 18 h after infection, the EC 50 The values ​​are summarized in Table 4 and are Figure 8B As shown. The efficacy of tenofovir was maintained when the compounds were given 18 hours after administration, consistent with inhibition of HBV replication. In contrast, antiviral activity was lost when compound 1 and myrcludex B were given 18 hours after infection, consistent with an anti-entry mechanism.

[0411] Study #02.

[0412] The effect of compound 1 in combination with tenofovir was also studied. The EC values ​​of tenofovir and compound 1 50 The values ​​are summarized in Table 4. Tenofovir showed expected potency (EC 50 : 64nmol / L). Compound 1 is more potent than tenofovir, and the exact EC 50 , as there was 58% inhibition at the lowest tested concentration (6.25 nmol / L). None of the compounds induced cytotoxicity at any concentration tested (highest concentration: 0.1-0.2 μmol / L). The combination of the two drugs provided a very potent antiviral effect. Because compound 1 was very potent on its own, it was challenging to judge whether the combination effect was additive or synergistic; an additional study was performed.

[0413] Study #03.

[0414] The effect of combining compound 1 with tenofovir at concentrations lower than those in study #02 was investigated. The EC 50 The values ​​are summarized in Table 4 and are Fig.8D Tenofovir showed expected potency (EC 50 :59nmol / L). However, in contrast to previous experiments, the maximum inhibitory effect of tenofovir was ~60%. The EC of compound 1 50 The maximum inhibitory effect of compound 1 was also ∼60%. When used in combination, the data showed that compound 1 and tenofovir exerted an additive effect.

[0415] Table 4. Data summary

[0416]

[0417] Compound 1 continuously inhibited HBV infection of human primary hepatocytes in a concentration-dependent manner. Compound 1 did not affect cell viability at any tested concentration (up to 100 μmol / L). Compound 1 produced an additive antiviral effect when used in combination with tenofovir disoproxil fumarate.

[0418] Fig. 9 The antiviral activity of compound 1 in study #01, study #02, and study #03 was summarized. The data were compared with the nonlinear four-parameter regression curve (R 2 :0.93) fits well. EC can be determined from the fitting curve 50 It is 4.9nmol / L.

[0419] Example 5. Effect of Compound 1 on HBV-infected PXB mice

[0420] The plasma exposure of compound 1 after oral administration to humanized, uninfected PXB mice and the prophylactic anti-HBV effect of compound 1 after oral administration to PXB mice before HBV infection were evaluated.

[0421] method

[0422] Study design for the pharmacokinetic phase.

[0423] This study was conducted on compound 1, and the experimental design is shown in Table 5.

[0424] Table 5.

[0425]

[0426] Research design of the main study

[0427] The study was conducted according to the experimental design shown in Table 6.

[0428] Table 6.

[0429]

[0430] *The administration time on day 0 was 60 minutes before HBV vaccination (0.1 mL HBV C inoculum (108 copies / mL) intravenous injection).

[0431] Blood sampling pharmacokinetic phase

[0432] Thirty microliters (30 μL) of blood were collected from all animals under isoflurane anesthesia via the retro-orbital plexus / sinus using a calibrated pipette coated with sodium heparin according to the collection schedule shown in Table 7.

[0433] Table 7.

[0434]

[0435] Individual blood samples from animals were transferred to labeled microtubes and kept under refrigeration until plasma was obtained by centrifugation at 3500×g for 10 min at 5°C. Individual plasma samples were transferred to individually labeled microtubes and snap-frozen in liquid nitrogen.

[0436] Blood sampling for main study

[0437] At each time point, Intramedic TM A targeted amount of blood was collected from all surviving animals under isoflurane anesthesia via a polyethylene tube through the retro-orbital plexus / sinus. Two microliters (2 μL) of the collected blood was drawn for human albumin measurement. The remaining blood was centrifuged to separate serum for analysis of the indicated biomarkers. Blood samples were collected according to Table 8.

[0438] Table 8.

[0439]

[0440] *Terminal Collection

[0441] A single blood sample from each animal was left to clot at room temperature for at least 5 minutes and then centrifuged at 13,200 × g for 3 minutes at 4°C to obtain serum. The serum was aliquoted into individually labeled microtubes and stored at -80°C until analysis.

[0442] Autopsy and tissue sampling.

[0443] The entire liver of all animals was harvested and weighed at necropsy. The left liver samples were immersed in RNAlater Incubate overnight and store at -80 °C until use.

[0444] Bioanalysis.

[0445] The plasma exposure of compound 1 during the pharmacokinetic phase was measured by LC / MS-MS. The lower limit of quantification was 1 ng / mL.

[0446] Blood human albumin concentration.

[0447] Blood human albumin concentration was measured by latex agglutination immunoturbidimetry using a clinical chemistry analyzer (BioMajesty™ series JCA-BM6050, JEOL Ltd., Tokyo, Japan).

[0448] Serum HBV DNA.

[0449] The KUBIX HBV qPCR Kit (KUBIX Inc.) and CFX96 Touch TMSerum HBV DNA concentration was measured by real-time PCR detection using the Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Ten microliters (10 μL) of HBV 2× PCR solution was added to 10 μL of heated sample. Initial activation was performed at 95°C for 2 min. Subsequent PCR amplification consisted of 45 cycles, each of which consisted of denaturation at 95°C for 5 s, annealing and extension at 54°C for 30 s, performed on a CFX96Touch TM The real-time PCR detection system was used. The average serum HBV DNA level was calculated based on the values ​​of two independent wells. The primers and probes are shown in Table 9.

[0450] Table 9.

[0451]

[0452] The minimum limit of quantification of this assay is 4 x 10 4 copies / mL serum.

[0453] Serum HBsAg.

[0454] Serum HBsAg concentration was measured using a chemiluminescent enzyme immunoassay (CLEIA) developed by SRL, Inc. (Tokyo, Japan) based on the Fujirebio assay (LUMIPULSE HBsAg-HQ, PrestoⅡ) method. The dilution multiples are 30 and 10, and the measurement range of this test method is 0.005-150 IU / mL. For samples diluted 30 times, the measurement range is adjusted to 0.15-4500 IU / mL. For samples diluted 10 times, the measurement range is adjusted to 0.05-1500 IU / mL. The minimum quantitative limit of this test method is 0.15 IU / mL serum.

[0455] Serum HBeAg.

[0456] Serum HBeAg concentration was measured using the chemiluminescent enzyme immunoassay (CLEIA) developed by SRL, Inc. based on Fujirebio (LUMIPULSE HBeAg, PrestoⅡ). The dilution factor is 30 and 10. The measurement range of this test method is 0.1 to 1590C.OI. For the 30-fold diluted sample, the measurement range is adjusted to 3 to 47700C.OI. For the 10-fold diluted sample, the measurement range is adjusted to 1 to 15900C.OI. The minimum limit of quantification of this test method is 3.0COI.

[0457] Liver HBV DNA.

[0458] use Blood and tissue kits (Qiagen KK, Tokyo, Japan) were prepared from frozen HBV DNA was extracted from preserved liver tissue using TaqMan Fast Advanced Master Mix and CFX96 Touch TM Liver HBV DNA concentration (expressed as copies / 100 ng DNA) was determined using a real-time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). DNA was dissolved in 200 μL of nuclease-free water and then used The concentration of the DNA solution was determined by ABS Plus microplate reader. The concentration of the DNA solution was adjusted to 20 ng / μL using nuclease-free water.

[0459] Liver HBV DNA quantitative standard was extracted from 5 μL of serum using SMITEST EX-R&D Nucleic Acid Extraction Kit (MEDICAL&BIOLOGICAL LABORATORIES CO., LTD., Nagoya, Japan). DNA was dissolved in 20 μL of nuclease-free water.

[0460] Using TaqMan Fast Advanced Master Mix and CFX96 Touch TM Liver HBV DNA concentration (expressed as copies / 100 ng DNA) was determined using a real-time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The PCR reaction mixture was added to 5 μL of extracted DNA. The PCR reaction mixture was added to 5 μL of extracted DNA. Uracil-N-glycosylase was initially activated at 50°C for 2 min, followed by polymerase activation at 95°C for 20 s. Subsequent PCR amplification included a CFX96 Touch TM 53 cycles were performed in the real-time PCR detection system, with each cycle consisting of 3 s of denaturation at 95°C and 32 s of annealing and extension at 60°C. The average hepatitis HBV DNA level was calculated based on the values ​​of two independent wells.

[0461] The minimum quantification limit of the assay is 50 copies / 100 ng DNA in the extracted DNA solution.

[0462] Liver cccDNA.

[0463] Using TaqMan Fast Advanced Master Mix and CFX96 Touch TMLiver HBV cccDNA concentration was measured by real-time PCR using a real-time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The PCR reaction mixture was added to 5 μL of the extracted DNA. The PCR reaction was performed according to Takkenberg conditions. Uracil-N-glycosylase was initially activated at 50°C for 2 minutes, followed by polymerase activation at 95°C for 20 seconds. TM 55 PCR amplification cycles were performed in the real-time PCR detection system, each cycle was performed at 95°C for 3 seconds, annealing and extension at 60°C for 32 seconds. The average HBV cccDNA level was calculated based on the values ​​of two independent wells. The primers and probes are shown in Table 10.

[0464] Table 10.

[0465]

[0466] The minimum quantification limit of the assay was 100 copies / 100 ng DNA in the extracted DNA solution.

[0467] result

[0468] Exposure of uninfected PXB mice following oral administration of compound 1.

[0469] Table 11 summarizes the data for mice dosed orally at 10 mg / kg, while Table 12 summarizes the data for mice dosed orally at 30 mg / kg. Fig.10 The relationship between plasma and time is also described. In both groups, compound 1 was detected in plasma during the 24-hour period. As the dose increased, the plasma exposure of compound 1 increased.

[0470] Table 11. Individual and mean exposures of compound 1 in uninfected PXB mice after administration of 10 mg / kg (oral) during the pharmacokinetic phase

[0471]

[0472] Table 12. Individual and mean exposures of compound 1 in uninfected PXB mice after administration at 30 mg / kg (oral) during the pharmacokinetic phase

[0473]

[0474] Effects of compound 1 on HBV-infected PXB mice

[0475] Animal No. 102 and Animal No. 104 were found dead on Day 16 and Day 23, respectively. Both were from the vehicle-treated group. The cause of death was believed to be intestinal bleeding. Animal No. 203 from the 10 mg / kg Compound 1 group was found dead on Day 33. The cause of death was believed to be metastatic thymoma, which is reported to be a spontaneous lesion in SCID mice. No noteworthy observations were found related to Compound 1.

[0476] weight

[0477] Body weight remained constant throughout the study. No differences were found between the groups.

[0478] Human serum albumin concentration

[0479] During the treatment period, the blood concentration of human albumin in the animals treated with compound 1 was stable (10 mg / kg compound 1, baseline: 10.8 ± 1.0 mg / mL, day 14: 10.8 ± 0.9 mg / mL; 30 mg / kg compound 1, baseline: 10.6 ± 1.5 mg / mL, day 14: 11.3 ± 1.2 mg / mL), while the blood concentration of human albumin in the vehicle-treated group tended to decrease (baseline: 10.7 ± 0.9 mg / mL, day 14: 8.1 ± 2.6 mg / mL). The decrease in the vehicle group was mainly due to the subsequent death of two animals found on days 16 and 23.

[0480] Serum HBV DNA

[0481] Fig.11A and 11B A summary of HBV DNA concentrations is shown. HBV DNA was detected on day 7 and was lower in mice treated with 30 mg / kg Compound 1 compared to the control group. Although treatment was stopped as planned on day 14, HBV DNA levels induced by Compound 1 remained low throughout the study until planned termination on day 42.

[0482] Serum HBsAg

[0483] Fig. 12A and 12B A summary of HBsAg concentrations is shown. HBsAg was detected on day 7, and mice treated with 10 mg / kg and 30 mg / kg of compound 1 had lower HBsAg concentrations compared to the control group. Although treatment was stopped as planned on day 14, HBsAg levels in response to both doses of compound 1 remained low throughout the study until planned termination on day 42.

[0484] Serum HBeAg

[0485] Fig. 12CA summary of HBeAg concentrations is shown. HBeAg was detected on day 14 (the first measurement time point), and mice treated with 10 mg / kg and 30 mg / kg of compound 1 had lower HBeAg concentrations compared to the control group. Although treatment was stopped as planned on day 14, HBeAg levels in response to both doses of compound 1 remained low throughout the study until planned termination on day 42.

[0486] Liver HBVDN and HBVcccDNA levels

[0487] Table 13 summarizes liver HBV DNA and HBV cccDNA levels at planned discontinuation of treatment (Day 42). There were no differences between treatment groups.

[0488] Table 13. Effect of Compound 1 on serum HBV DNA levels.

[0489]

[0490]

[0491] discuss

[0492] In uninfected PXB mice, compound 1 was detectable in plasma over a 24-hour period when administered at 10 and 30 mg / kg (oral) doses. Plasma exposure to compound 1 increased with increasing doses. Compound 1 had a preventive effect against HBV genotype C infection in PXB mice. Serum HBsAg ( Fig. 12A and 12B )、HBeAg( Fig. 12C ) and HBV DNA( Fig.11A and 11B ) levels were lower, especially during the 14-day treatment period. After treatment, HBV infection markers remained lower in mice treated with 30 mg / kg Compound 1 compared with 10 mg / kg Compound 1 and vehicle controls.

[0493] Example 6. Characterization of Compound 2

[0494] Na+-taurocholate cotransporting polypeptide (NTCP) is a hepatocyte sinusoidal bile acid (BA) transporter. In addition to playing a major role in BA clearance from hepatocytes, hepatitis B virus and hepatitis D virus (HBV, HDV) utilize NTCP to enter and infect human hepatocytes. The effect of compound 2 on inhibiting BA uptake and HBV entry was evaluated.

[0495] method

[0496] NTCP and the related BA transporter apical sodium-dependent BA transporter (ASBT) were derived from humans and expressed in cell lines. 3 The inhibitory effect of compound 2 was studied using [H]taurocholic acid as a substrate. Cryopreserved human hepatocytes infected with HBV clinical isolates were used for infection experiments. Two female non-human primates (NHPs) were administered compound 2 by oral gavage at dose levels between 3-30 mg / kg once daily for 5 days. Total serum, urine, and fecal BAs were analyzed enzymatically, and plasma concentrations of compound 2 were assessed by liquid chromatography-tandem mass spectrometry. The oral pharmacokinetics of compound 2 in mice, rats, and dogs were also studied.

[0497] result

[0498] The half maximal inhibitory concentration (IC) of compound 2 on human NTCP and ASBT 50 ) values ​​were 3.4 and 193 nmol / L, respectively. Compound 2 can prevent HBV from infecting human hepatocytes, IC 50 The concentration of BA in NHP was 13.5 nmol / L, and it did not affect cell viability. In NHP, the plasma exposure of compound 2 increased with the dose, and serum BA increased in a dose-dependent manner after oral administration of compound 2 on days 1 and 5. Doses of 10 and 30 mg / kg of compound 2 caused an increase in serum BA on days 1 and 5, which lasted for at least 8 hours. After taking 10 mg / kg A7387, serum BA returned to baseline 24 hours after administration, while it remained elevated after taking 30 mg / kg of compound 2. Compared with the vehicle, the urinary BA excretion of compound 2 tended to increase, while the fecal BA levels did not change. After repeated administration for 5 days, the level of BA synthesis biomarker 7-α-hydroxy-4-cholesten-3-one (C4) was not changed by compound 2. Pharmacokinetic studies showed good oral bioavailability in all species. Compound 2 is a highly effective, selective, and orally available NTCP inhibitor with the potential to treat HBV / HDV infection and cholestatic diseases.

[0499] Other Implementations

[0500] It should be understood that although the disclosure has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims

1. Compound (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof for use in treating hepatitis B (HBV) in a subject.

2. Compound (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof, for preventing or reducing the entry of hepatitis B virus particles into hepatocytes in a subject.

3. Compound (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof for use in reducing hepatitis B virus replication in hepatocytes in a subject.

4. A compound for use according to any one of claims 1 to 3, wherein the subject suffers from hepatitis D.

5. Compound (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof for use in treating hepatitis D (HDV) in a subject.

6. Compound (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof, for use in preventing or reducing the entry of hepatitis D virus particles into hepatocytes in a subject.

7. Compound (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof for use in reducing hepatitis D virus replication in hepatocytes in a subject.

8. A compound for use according to any one of claims 1 to 7, wherein the use further comprises the administration of an additional antiviral agent.

9. Compound (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof, and another antiviral agent for use in treating HBV in a subject.

10. Compound (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof, and another antiviral agent for use in treating HDV in a subject.

11. The compound for use according to any one of claims 8 to 10, wherein the additional antiviral agent is selected from the group consisting of entecavir, tenofovir, tenofovir disoproxil, tenofovir alafenamide, lamivudine, adefovir, adefovir dipivoxil, telbivudine, bulviridine, interferon, and combinations thereof.

12. The compound for use according to claim 11, wherein the interferon is pegylated interferon, interferon alpha or a combination thereof.

13. A compound for use according to any one of claims 8 to 11, wherein the additional antiviral agent is tenofovir disoproxil.

14. A compound for use according to any one of claims 5-8 and 10-13, wherein the subject suffers from hepatitis B.

15. The compound for use according to any one of claims 1-4 and 14, wherein the subject suffers from chronic hepatitis B.

16. A compound for use according to any one of claims 5-8 and 10-15, wherein the subject suffers from chronic hepatitis D.

17. A compound for use according to any one of claims 1 to 16, wherein the concentration of one or more biomarkers selected from HBV DNA, hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), hepatitis B e antigen (HBeAg), HDV DNA and hepatitis D antigen (HDAg) in the serum of the subject is reduced after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

18. The compound for use according to claim 17, wherein the concentration of HBV DNA in the serum of a subject is reduced after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

19. The compound for use according to claim 17, wherein the concentration of HBV DNA is measured in a serum sample of a subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

20. The compound for use according to claim 18, wherein the concentration of HBV DNA in a serum sample of a subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is reduced compared to a reference concentration of HBV DNA.

21. The compound for use according to claim 19, wherein the reference concentration of HBV DNA is the level of HBV DNA in a serum sample obtained from a subject before administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

22. A compound for use according to any one of claims 17 to 20, wherein the concentration of HBV DNA in the serum of a subject is reduced by about 10% to about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

23. A compound for use according to any one of claims 17-21, wherein the concentration of HBV DNA in the serum of a subject is reduced by about 5%, about 10%, about 25%, about 50%, about 75% or about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

24. A compound for use according to any one of claims 17 to 23, wherein the concentration of HBV DNA in the serum of a subject is undetectable after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

25. A compound for use according to any one of claims 17 to 24, wherein the concentration of hepatitis B surface antigen (HBsAg) in the serum of a subject is reduced after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

26. The compound for use according to claim 25, wherein the concentration of HBsAg is measured in a serum sample of a subject after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

27. A compound for use according to claim 26, wherein the concentration of HBsAg in a serum sample of a subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is reduced compared to a reference concentration of HBsAg.

28. The compound for use according to claim 27, wherein the reference concentration of HBsAg is the concentration of HBsAg in a serum sample obtained from a subject before administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

29. A compound for use according to any one of claims 25 to 28, wherein the concentration of HBsAg in the serum of a subject is reduced by about 10% to about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

30. A compound for use according to any one of claims 25-29, wherein the concentration of HBsAg in the serum of a subject is reduced by about 5%, about 10%, about 25%, about 50%, about 75% or about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

31. A compound for use according to any one of claims 25 to 30, wherein the concentration of HBsAg in the serum of a subject is undetectable after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

32. A compound for use according to any one of claims 17 to 31, wherein the concentration of hepatitis B core antigen (HBcAg) in the serum of a subject is reduced after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

33. A compound for use according to claim 32, wherein the HBcAg concentration is measured in a sample obtained from a subject after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

34. A compound for use according to claim 33, wherein the concentration of HBcAg in a serum sample of a subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is reduced compared to a reference concentration of HBcAg.

35. A compound for use according to claim 34, wherein the reference concentration of HBcAg is the concentration of HBcAg in a serum sample obtained from a subject before administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

36. A compound for use according to any one of claims 32-35, wherein the concentration of HBcAg in the serum of a subject is reduced by about 10% to about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

37. A compound for use according to any one of claims 32-36, wherein the concentration of HBcAg in the serum of a subject is reduced by about 5%, about 10%, about 25%, about 50%, about 75% or about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

38. A compound for use according to any one of claims 35-37, wherein the concentration of HBcAg in the serum of a subject is undetectable after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

39. A compound for use according to any one of claims 17 to 38, wherein the concentration of hepatitis B e antigen (HBeAg) in the serum of a subject is reduced after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

40. The compound for use according to claim 39, wherein the concentration of HBeAg is measured in a serum sample of a subject after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

41. A compound for use according to claim 40, wherein the concentration of HBeAg in a serum sample of a subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is reduced compared to a reference concentration of HBeAg.

42. The compound for use according to claim 41, wherein the reference concentration of HBeAg is the concentration of HBeAg in a serum sample obtained from a subject before administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

43. A compound for use according to any one of claims 39-42, wherein the concentration of HBeAg in the serum of a subject is reduced by about 10% to about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

44. A compound for use according to any one of claims 39-43, wherein the concentration of HBeAg in the serum of a subject is reduced by about 5%, about 10%, about 25%, about 50%, about 75% or about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

45. A compound for use according to any one of claims 39-44, wherein the concentration of HBeAg in the serum of a subject is undetectable following administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

46. ​​A compound for use according to any one of claims 17 to 45, wherein the concentration of HDV DNA in the serum of a subject is reduced after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

47. The compound for use according to claim 46, wherein the concentration of HDV DNA is measured in a serum sample of a subject after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

48. The compound for use according to claim 47, wherein the concentration of HDV DNA in a serum sample of a subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is reduced compared to a reference concentration of HDV DNA.

49. The compound for use according to claim 48, wherein the reference concentration of HDV DNA is the concentration of HDV DNA in a serum sample obtained from a subject before administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

50. A compound for use according to any one of claims 46-49, wherein the concentration of HDV DNA in the serum of a subject is reduced by about 10% to about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

51. A compound for use according to any one of claims 46-50, wherein the concentration of HDV DNA in the serum of a subject is reduced by about 5%, about 10%, about 25%, about 50%, about 75% or about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

52. A compound for use according to any one of claims 46-51, wherein the concentration of HDV DNA in the serum of a subject is undetectable after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

53. A compound for use according to any one of claims 17 to 52, wherein the concentration of hepatitis D antigen (HDAg) in the serum of a subject is reduced after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

54. The compound for use according to claim 53, wherein the concentration of HDAg is measured in a serum sample of a subject after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

55. A compound for use according to claim 54, wherein the concentration of HDAg in a serum sample of a subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is reduced compared to a reference concentration of HDAg.

56. The compound for use according to claim 55, wherein the reference concentration of HDAg is the concentration of HDAg in a serum sample obtained from a subject before administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

57. A compound for use according to any one of claims 53-56, wherein the concentration of HDAg in the serum of a subject is reduced by about 10% to about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

58. A compound for use according to any one of claims 53-57, wherein the concentration of HDAg in the serum of a subject is reduced by about 5%, about 10%, about 25%, about 50%, about 75% or about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

59. A compound for use according to any one of claims 53-58, wherein the concentration of HDAg in the serum of a subject is undetectable following administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

60. A compound for use according to any one of claims 1 to 59, wherein (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is administered to a subject within 18 hours of exposure to hepatitis B.

61. A compound for use according to any one of claims 1 to 60, wherein (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is administered to a subject within 18 hours of exposure to hepatitis D.

62. A compound for use according to any one of claims 1 to 61, wherein the compound is (R)-(Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

63. A compound for use according to any one of claims 1 to 61, wherein the compound is (S)-(Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

64. A method for treating hepatitis B (HBV), the method comprising orally administering to a subject a therapeutically effective amount of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

65. A method for preventing or reducing the entry of hepatitis B virus particles into hepatocytes in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

66. A method for reducing hepatitis B virus replication in hepatocytes in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

67. The method of any one of claims 1-3, wherein the subject has hepatitis D.

68. A method for treating hepatitis D (HDV) in a subject in need thereof, the method comprising orally administering to the subject a therapeutically effective amount of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

69. A method for preventing or reducing the entry of hepatitis D virus particles into hepatocytes in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

70. A method for reducing hepatitis D virus replication in hepatocytes in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

71. The method of any one of claims 1-7, wherein the method further comprises administering an additional antiviral agent.

72. A method for treating HBV in a subject in need thereof, the method comprising orally administering to the subject a therapeutically effective amount of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof, and an additional antiviral agent.

73. A method for treating HDV in a subject in need thereof, the method comprising orally administering to the subject a therapeutically effective amount of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof, and an additional antiviral agent.

74. The method of any one of claims 8-10, wherein the additional antiviral agent is selected from the group consisting of entecavir, tenofovir, tenofovir disoproxil, tenofovir alafenamide, lamivudine, adefovir, adefovir dipivoxil, telbivudine, bulviridine, interferon, and combinations thereof.

75. The method of claim 11, wherein the interferon is pegylated interferon, interferon alpha, or a combination thereof.

76. The method of any one of claims 8-11, wherein the additional antiviral agent is tenofovir disoproxil.

77. The method of any one of claims 5-8 and 10-13, wherein the subject has hepatitis B.

78. The method of any one of claims 1-4 and 14, wherein the subject has chronic hepatitis B.

79. The method of any one of claims 5-8 and 10-15, wherein the subject has chronic hepatitis D.

80. The method of any one of claims 1-16, wherein the concentration of one or more biomarkers selected from HBV DNA, hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), hepatitis B e antigen (HBeAg), HDV DNA, and hepatitis D antigen (HDAg) in the subject's serum is reduced after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

81. The method of claim 17, wherein the concentration of HBV DNA in the serum of the subject is reduced after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

82. The method of claim 17, wherein the concentration of HBV DNA is measured in a serum sample of a subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

83. The method of claim 18, wherein the concentration of HBV DNA in a serum sample of a subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is reduced compared to a reference concentration of HBV DNA.

84. The method of claim 19, wherein the reference concentration of HBV DNA is the level of HBV DNA in a serum sample obtained from a subject prior to administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

85. The method of any one of claims 17-20, wherein the concentration of HBV DNA in the serum of the subject is reduced by about 10% to about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

86. The method of any one of claims 17-21, wherein the concentration of HBV DNA in the serum of the subject is reduced by about 5%, about 10%, about 25%, about 50%, about 75% or about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

87. The method of any one of claims 17-23, wherein the concentration of HBV DNA in the subject's serum is undetectable after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

88. The method of any one of claims 17-24, wherein the concentration of hepatitis B surface antigen (HBsAg) in the serum of the subject is reduced after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

89. The method of claim 25, wherein the concentration of HBsAg is measured in a serum sample of a subject after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

90. The method of claim 26, wherein the concentration of HBsAg in a serum sample of a subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is reduced compared to a reference concentration of HBsAg.

91. The method of claim 27, wherein the reference concentration of HBsAg is the concentration of HBsAg in a serum sample obtained from a subject before administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

92. The method of any one of claims 25-28, wherein the concentration of HBsAg in the serum of the subject is reduced by about 10% to about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

93. The method of any one of claims 25-29, wherein the concentration of HBsAg in the serum of the subject is reduced by about 5%, about 10%, about 25%, about 50%, about 75% or about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

94. The method of any one of claims 25-30, wherein the concentration of HBsAg in the subject's serum is undetectable after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

95. The method described in any one of claims 17-31, wherein the concentration of hepatitis B core antigen (HBcAg) in the serum of the subject is reduced after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

96. The method of claim 32, wherein the HBcAg concentration is determined in a sample obtained from a subject after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

97. The method of claim 33, wherein the concentration of HBcAg in a serum sample of a subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is reduced compared to a reference concentration of HBcAg.

98. The method of claim 34, wherein the reference concentration of HBcAg is the concentration of HBcAg in a serum sample obtained from a subject before administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

99. The method of any one of claims 32-35, wherein the concentration of HBcAg in the serum of the subject is reduced by about 10% to about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

100. The method of any one of claims 32-36, wherein the concentration of HBcAg in the serum of a subject is reduced by about 5%, about 10%, about 25%, about 50%, about 75% or about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

101. The method described in any one of claims 35-37, wherein the concentration of HBcAg in the serum of the subject is undetectable after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

102. The method of any one of claims 17-38, wherein the concentration of hepatitis B e antigen (HBeAg) in the serum of the subject is reduced after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

103. The method of claim 39, wherein the concentration of HBeAg is measured in a serum sample of the subject after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

104. The method of claim 40, wherein the concentration of HBeAg in a serum sample of the subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is reduced compared to a reference concentration of HBeAg.

105. The method of claim 41, wherein the reference concentration of HBeAg is the concentration of HBeAg in a serum sample obtained from a subject prior to administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

106. The method of any one of claims 39-42, wherein the concentration of HBeAg in the serum of the subject is reduced by about 10% to about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

107. The method of any one of claims 39-43, wherein the concentration of HBeAg in the serum of the subject is reduced by about 5%, about 10%, about 25%, about 50%, about 75% or about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

108. The method of any one of claims 39-44, wherein the concentration of HBeAg in the serum of the subject is undetectable following administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

109. The method of any one of claims 17-45, wherein the concentration of HDV DNA in the serum of the subject is reduced after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

110. The method of claim 46, wherein the concentration of HDV DNA is measured in a serum sample of the subject after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

111. The method of claim 47, wherein the concentration of HDV DNA in a serum sample of the subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is reduced compared to a reference concentration of HDV DNA.

112. The method of claim 48, wherein the reference concentration of HDV DNA is the concentration of HDV DNA in a serum sample obtained from a subject prior to administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

113. The method of any one of claims 46-49, wherein the concentration of HDV DNA in the serum of the subject is reduced by about 10% to about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

114. The method of any one of claims 46-50, wherein the concentration of HDV DNA in the serum of the subject is reduced by about 5%, about 10%, about 25%, about 50%, about 75%, or about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

115. The method of any one of claims 46-51, wherein the concentration of HDV DNA in the serum of the subject is undetectable after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

116. The method of any one of claims 17-52, wherein the concentration of hepatitis D antigen (HDAg) in the serum of the subject is reduced after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

117. The method of claim 53, wherein the concentration of HDAg is measured in a serum sample of the subject after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

118. The method of claim 54, wherein the concentration of HDAg in a serum sample of the subject obtained after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid, or a pharmaceutically acceptable salt thereof, is reduced compared to a reference concentration of HDAg.

119. The method of claim 55, wherein the reference concentration of HDAg is the concentration of HDAg in a serum sample obtained from a subject prior to administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

120. The method of any one of claims 53-56, wherein the concentration of HDAg in the serum of the subject is reduced by about 10% to about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

121. The method of any one of claims 53-57, wherein the concentration of HDAg in the serum of the subject is reduced by about 5%, about 10%, about 25%, about 50%, about 75%, or about 99% after administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

122. The method of any one of claims 53-58, wherein the concentration of HDAg in the serum of the subject is undetectable following administration of (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof.

123. The method of any one of claims 1-59, wherein (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is administered to the subject within 18 hours of exposure to hepatitis B.

124. The method of any one of claims 1-60, wherein (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is administered to the subject within 18 hours of exposure to hepatitis D.

125. The method of any one of claims 1-61, wherein a therapeutically effective amount of (R)-(Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is administered to the subject.

126. The method of any one of claims 1-61, wherein a therapeutically effective amount of (S)-(Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine-8-yl)oxy)-2-fluoroacrylic acid or a pharmaceutically acceptable salt thereof is administered to the subject.

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