Methods for prevention, delay of progression or treatment of cholestasis and / or fibrosis associated with cholestasis

Abstract

The present invention relates to a method of prevention, delay of progression or treatment of cholestasis and / or fibrosis associated with cholestasis.

Description

Technical Field

[0001] The present invention relates to a method for preventing, delaying the progression of or treating cholestasis and / or fibrosis associated with cholestasis. Background Art

[0002] Cholestasis is characterised by a reduction or stagnation of bile flow in the liver. This can be caused by obstruction of the extrahepatic bile ducts or defects in bile synthesis or circulation within the liver. Causes include drug intoxication, alcoholic or viral hepatitis, biliary atresia, gallstones and genetic disorders. Cholestatic liver disease is a significant personal and economic burden for patients and society; liver dysfunction due to cholestasis accounts for approximately 10% of all liver transplants in Europe. 1 The reason for this unusually high number is the sad lack of effective treatment options for cholestatic diseases like primary sclerosing cholangitis (PSC) or primary biliary cholangitis (PBC). Current first-line treatment involves administration of the immunomodulatory and bicarbonate secretion stimulating drug ursodeoxycholic acid (UDCA), which improves transplant-free survival in approximately 60% of PBC patients, with limited efficacy in PSC patients. 2 New therapeutic approaches focus on immune-modulatory strategies 3-5 , changes in the microbiome 6-7 Or activate FGF19 / FXR signaling to produce anti-fibrotic effects and improve bile acid excretion and detoxification 8-10 Therefore, some research groups have suggested the use of PPAR agonists to treat cholestasis. 11-13 , to increase the efflux of toxic bile acids. Currently, treatment of chronic cholestatic diseases is difficult and often ineffective. New treatments for cholestasis and / or cholestasis-related fibrosis are needed to meet the high medical needs. Summary of the invention

[0003] The inventors of the present application have now unexpectedly discovered that Claudin-3 inhibition is a therapeutic method that can be used to treat cholestasis and / or fibrosis associated with cholestasis. The use of siRNA targeting Claudin-3 is expected to achieve improvement in cholestatic liver damage. In view of these unexpected findings, the inventors hereby provide the following aspects of the present invention.

[0004] In a first aspect, the present invention provides an agent for inhibiting the expression and / or activity of Claudin-3, for use in a method for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis.

[0005] In a second aspect, the present invention provides a composition comprising an agent that inhibits the expression and / or activity of claudin-3 and a pharmaceutically acceptable carrier, wherein the composition is used for a method for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis.

[0006] In a third aspect, the present invention provides a dosage form for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis, the dosage form comprising an agent that inhibits the expression and / or activity of claudin-3 or a composition comprising the agent and a pharmaceutically acceptable carrier.

[0007] In a fourth aspect, the present invention provides a siRNA targeting Claudin-3. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 :Knockout of claudin-3 leads to impaired bile acid metabolism, bile dilution, and impaired uptake of the fluorescent tracer FITC-dextran. (A) Metascape analysis. (B) Comparison with wild-type Cldn3 + / + and Cldn3 - / - Differential analysis of gene expression in liver tissue (n=3). (C) RT-qPCR of genes involved in bile acid synthesis (n=4 to 6, Student's t-test). (D) Quantification of total bile acid levels in gallbladder bile (n=3, Student's t-test). (E) Quantification of bile autofluorescence (excitation wavelength 489nm / emission wavelength 534nm, n=4 / 5, Student's t-test). (F) Schematic diagram showing the setup for quantifying bile flow over time and blood bile passage of FITC dextran (40kDa) (IV, intra-vital) (schema contains modified vector images from smart.servier.com, under a Creative Commons Attribution 3.0 Unported License). (G) Quantification of average bile flow over 20 minutes (n=4, Student's t-test). (H) Quantification of blood bile passage over time after intravenous injection of FITC-dextran (40 kDa) (0 to 30 min: n = 3-5 per time point; 33 min to 40 min: n = 1, Student's t-test, *P < 0.05, **P < 0.01, ns: not significant). (I) Images of mice injected with FITC-dextran (40 kDa), 20 min after IV injection. Fluorescent FITC-dextran (40 kDa) signal (white) is expressed in Cldn3 - / - Less obvious in the liver. Time series images acquired with the Spectrum system (n=1). (J) Quantification of FITC dextran (40 kDa) signal in the time series. (K) Orthogonal slice images (25 um thick) from a mouse liver z-stack, 20 minutes after IV injection of FITC dextran (40 kDa, FITC signal in light gray). (L) Quantification of FITC dextran signal in the liver z-stack from (K) (n=3-4, Student's t-test). (FD-40: FITC dextran (40 kDa); RFU: relative fluorescence units).

[0009] Figure 2 : Loss of tight junction protein-3 protects against obstructive cholestatic liver injury. (A) Photos of the liver before and after bile duct ligation. (B) Hematoxylin and eosin-stained liver tissue before and after bile duct ligation. The circled area highlights the necrotic area. (C) Quantification of tissue necrosis (n=5 at control group, 2 days, 7 days, n=3 / 4 at 12 days, Student's t-test, **P<0.01). (D) Serum ALT level. (E) Serum AST level. (F) Serum ALP level. (Serum analysis: n=5 at control group, 2 days, 7 days, n=3-4 at 12 days, Student's t-test). (G) Bacterial translocation quantified as colony forming units (CFU) per gram of liver tissue (n=9-7 at 2 days, n=4 at 7 days, Student's t-test, + / +=Cldn3 + / + ,- / -=Cldn3 - / - (H) FACS analysis of immune cell populations 7 days after bile duct ligation (n=3-4, Student's t-test).

[0010] Figure 3 : Bile acid concentrations in liver and gallbladder bile are reduced after common bile duct ligation (BDL). (A) Photographs of bile collected from the gallbladder 7 days after BDL (representative images from n = 5). (B) Quantification of TBA in bile after BDL. (C) Quantification of TBA in liver bile extracts after BDL. (D) Quantification of TBA in serum after BDL (n = 5 at 7 days, n = 3-4 at 12 days, Student's t test). (E) Quantification of total bilirubin levels in serum after BDL (n = 5 at 2 and 7 days, n = 3-4 at 12 days, Student's t test). (F) Quantification of bile embolism in kidney tissue 7 days after BDL. Quantification is based on hematoxylin and eosin-stained kidney sections, in which bile embolism appears as yellow casts. Values ​​reported are the integrated density of yellow per square millimeter of tissue (n = 3 / 4, Student's t test). For details, please refer to Materials and Methods.

[0011] Figure 4 : Cldn3 before and after BDL + / + and Cldn3 - / -Bile acid quantification and composition in liver tissue and serum. (A) LC-MS / MS quantification of bile acids in liver tissue of control group and 7 days after BDL (n=12 in control group, n=8 in 7 days after BDL, Student's t-test). (B) LC-MS / MS quantification of bile acid isoforms in liver of control group. Data are reported as mean percentage of the entire bile acid pool. (n=12, Student's t-test comparing Cldn3 + / + and Cldn3 - / - ). (C) LC-MS / MS quantification of bile acid isoforms in the liver 7 days after BDL. Data are reported as the mean percentage of the entire bile acid pool. (n=8, Student's t-test). (D) LC-MS / MS quantification of serum bile acids in the control group and 7 days after BDL (n=11 in the control group, n=5 in 7 days after BDL, Student's t-test). (E) LC-MS / MS quantification of bile acid isoforms in control serum. Data are reported as the mean percentage of the entire bile acid pool. (n=11, Student's t-test). (F) LC-MS / MS quantification of bile acid isoforms in serum 7 days after BDL. Data are reported as the mean percentage of the entire bile acid pool. (n=5, Student's t-test).

[0012] Figure 5 :Cholestatic Cldn3 - / - Expression of BA synthesis and import genes is low in the liver. (A) Gene expression differential analysis of liver tissue 2 days after BDL (n=3, annotated genes P<0.05). (B) RT-qPCR analysis of genes involved in bile acid synthesis. (C) Quantification of serum cholesterol levels. (D) RT-qPCR analysis of genes involved in bile acid import. (E) RT-qPCR analysis of genes involved in bile component export. (For BE, n=5 at 2 and 7 days, n=3-4 at 12 days, Student's t-test).

[0013] Figure 6 :Cldn3 - / - Increased periductal edema in mice. Histopathological analysis of the periportal area after BDL. (A) Hematoxylin and eosin staining shows the periportal area at the indicated times after BDL. (B) Periductal edema scores. (n=6 / 4 in control group, n=5 at 2 and 7 days after BDL, 4 / 3 at 12 days after BDL.) Box plots show mean ± SD. Student's t test (PV, portal vein; BD, bile duct; BI, bile infarct, HA, hepatic artery).

[0014] Figure 7 :Cldn12 - / -Animals are not protected from cholestatic liver injury. (A) Representative liver photographs and hematoxylin and eosin stained tissues 2 days after BDL. (B) Quantification of tissue necrosis 2 days after BDL (n=5, circled areas highlight tissue necrosis, Student's t-test, ns: not significant). (C) Quantification of total bile acids in liver bile extracts. (D) Quantification of total bile acids in serum. (For C and D, n=3 / 4 in control group, n=4 in 2 days after BDL, Student's t-test).

[0015] Figure 8 : Absence of claudin-3 prevents intrahepatic cholestasis. (A) Acute two-day ANIT challenge model scheme. (B) Representative photographs of livers 2 days after ANIT challenge. (C) Representative images of hematoxylin and eosin-stained liver sections 2 days after ANIT challenge (n=6, circled areas highlight tissue necrosis). (D) Quantification of necrotic tissue area 2 days after ANIT challenge (n=6, Student's t-test). (E) Serum ALT, AST, ALP, and bilirubin levels before and two days after ANIT challenge (n=6, Student's t-test, bd: below detection). (F) Liver and serum total bile acid levels 2 days after ANIT challenge (n=6, Student's t-test). (G) RT-qPCR expression analysis of Gsta1, Cyp4a14, and Cyp7b1 before and two days after ANIT challenge (n=6, Student's t-test). (H) 10-day ANIT challenge model scheme. (I) Masson trichrome staining after 10 days of ANIT gavage (the circled area highlights high collagen staining, BI: bile infarction). (J) Quantification of collagen levels in (I) (n=8-9, Student's t-test). (K) 0.1% ANIT feeding model regimen. (L) Representative images of hematoxylin and eosin-stained liver sections after 4 weeks of corn oil or ANIT feeding (the circled area highlights tissue necrosis). (M) Quantification of tissue necrosis. (N) Quantification of ALP, ALT, and AST levels in the ANIT feeding model. (O) Serum total bilirubin and cholesterol quantification. (P) Sirius red-stained liver tissue of the ANIT feeding model. Arrows indicate example areas of positively stained tissue, which appear darker in the grayscale image. (Q) Quantification of Sirius red staining (n=5 / 6 for corn oil and n=7 for 0.1% ANIT for Figure LQ, Student's t-test).

[0016] Fig. 9: GalNAc siRNA targeting claudin-3 can achieve moderate improvement in cholestatic injury. (A) Scheme shows the process of in vivo GalNAc siRNA BDL experiment. (B) Western blot shows the protein level of claudin-3 in control group or inhibition GalNAc siRNA treated mice before and after BDL (C: control group, A: AHSA1 siRNA). (C) Quantification results of claudin-3 mRNA and protein in control group or GalNAc siRNA treated mice before or after BDL (mRNA, protein: n=4, control group n=2, after BDL n=4). (D) Serum markers of cholestasis and liver injury after BDL and anti-claudin-3 GalNAc siRNA injection. P values ​​were calculated by comparing the corresponding conditions with the BDL control group (Student's t test). (E) Hematoxylin and eosin stained liver sections 2 days after BDL (with or without claudin-3 GalAc siRNA injection). (F) Scheme showing the in vivo GalNAc siRNA ANIT experimental process. (G) ALP serum markers of cholestasis and liver damage after anti-claudin-3 GalNAc siRNA injection and ANIT gavage. P values ​​were calculated by comparing the corresponding conditions with the ANIT AHSA1 control siRNA group (Student's t test). (H) Hematoxylin and eosin-stained liver sections 2 days after ANIT (with or without claudin-3 GalAc siRNA injection). (For D and E, n=3 for the control group, n=3 for the BDL+control surgery group, and n=4 for the BDL+GalNAc siRNA group; for G and H, n=3 for the AHSA1 group, n=5 for the siRNA1 group, and n=4 for the no ANIT control group. Student's t test).

[0017] Fig.10 : Binding of UB-VS003 anti-Claudin-3 antibody to SNU449, Hepa1-6, and human and mouse hepatocytes (h-Heps or m-Heps, respectively) at the indicated dilutions was determined by FACS.

[0018] Fig.11 : UB-VS003-GalNAc induced knockdown of human claudin-3 in human hepatocytes as determined by Western blotting. DETAILED DESCRIPTION

[0019] As described above, the present invention relates to a method for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis.

[0020] Therefore, the first aspect of the present invention provides an agent for inhibiting the expression and / or activity of Claudin-3, which is used in a method for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis.

[0021] For the purpose of interpreting this specification, the following definitions shall apply, and where appropriate, singular terms shall include the plural and vice versa. It should be understood that the terms used herein are only used to describe specific embodiments and are not intended to be limiting. Unless otherwise indicated, the terms "including", "having" and "comprising" shall be understood as open terms (i.e., meaning "including but not limited to").

[0022] Features, integers, characteristics, agents, compounds, chemical molecules or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless inconsistent therewith. All features disclosed in this specification (including any accompanying claims, abstract and drawings) and / or all steps of any disclosed method or process may be combined in any combination, except combinations in which at least some of the features and / or steps are mutually exclusive. The invention is not limited to the details of any foregoing embodiments. The invention covers any novel feature or any combination of novel features disclosed in this specification (including any accompanying claims, abstract and drawings), or any novel feature or any novel combination of steps of any disclosed method or process.

[0023] The term "cholestasis" as used herein refers to any condition in which the release of bile from the liver is blocked. The blockage can occur in the liver (intrahepatic cholestasis) or in the bile duct (extrahepatic cholestasis). Physiologically, "cholestasis" refers to the obstruction of bile flow and the inability to secrete inorganic and organic components in bile. In particular, cholestasis is caused by molecular and ultrastructural changes that hinder the entry of small organic molecules, inorganic salts, proteins and ultimately water into the bile duct space. Clinically, signs such as jaundice and pruritus are the main ones, accompanied by an increase in serum concentrations of bilirubin, bile salts and alkaline phosphatase (ALP). Cholestasis can be caused by extrahepatic problems, such as gallstones or tumors blocking the flow of bile outside the liver. But it may also have intrahepatic causes, such as viral diseases, hereditary diseases and bile duct stenosis. Bile is the main pathway for eliminating bilirubin, excess cholesterol (free cholesterol and bile salts) and foreign substances that are insufficiently water soluble and cannot be discharged into the urine. The basic driver of bile formation is the secretion of bile salts by hepatocytes into the canalicular space, which in turn drives the secretion of phosphatidylcholine and cholesterol by hepatocytes. Fluid secretion by hepatocytes and downstream cholangiocytes lining the biliary tree together contribute to the several liters of bile secreted by the human liver each day. Bile facilitates the digestion and absorption of lipids in the intestine. Because bile formation requires well-functioning hepatocytes and an intact biliary tree, the process can be easily interrupted.

[0024] The term "cholestasis-associated fibrosis" refers to any fibrotic liver disease associated with cholestasis and / or initially caused by hepatic cholestasis.

[0025] The term "agent that inhibits the expression and / or activity of Claudin-3" as used herein refers to any biological or chemical agent that can inhibit the expression and / or activity of Claudin-3, for example, by reducing or destroying the interaction of Claudin-3 or its gene with other biological molecules, such as but not limited to protein-protein interactions, ligand-receptor interactions, or protein-nucleic acid interactions. Such agents include, but are not limited to, antibodies, protein binders, nucleic acid molecules, small molecules, recombinant proteins, peptides, aptamers, avimers, and protein-binding derivatives or fragments thereof. For example, the activity of Claudin-3 can be inhibited by an antibody that binds to Claudin-3 (e.g., an antibody that binds to at least one extracellular domain of Claudin-3) or a toxin that binds to Claudin-3. For example, the expression of Claudin-3 can be inhibited by a DNA targeting agent (e.g., a CRISPR system, a TALE, a zinc finger protein) or an RNA targeting agent (e.g., an inhibitory nucleic acid molecule). Inhibition of the expression of Claudin-3 includes, in the presence of the agent, a decrease in expression of at least 10%, preferably at least 40%, compared to the expression of Claudin-3 in the absence of the agent. Inhibition of the activity of Claudin-3 includes, in the presence of the agent, a decrease in activity of at least 10%, preferably at least 40%, compared to the activity of Claudin-3 in the absence of the agent.

[0026] The tight junction proteins mentioned in this article are a family of intramembrane proteins that make up TJs, which are the main intercellular junctions that act as permeability barriers and confer polarity to epithelial cells by dividing the upper and lower regions of the membrane. At present, the mammalian tight junction protein family contains 27 proteins, and a variety of alternatively spliced ​​tight junction proteins are expressed in various tissues. In the past decade, the crystal structures of this protein family have been gradually elucidated. Claudin is a tetraspanin protein, including four transmembrane domains (TM1-4), intracellular N-terminus and C-terminus, and two extracellular loops (ECL1 and ECL2). ECL1 contains four β-strands and an extracellular helix (ECH), while ECL2 contains β-strands and a transmembrane 3 domain exposed to the cell surface. ECL participates in the formation of interactions between tight junction protein chains and determines the gating function of tight junction protein-based TJs through two variable regions. Claudin-3 was originally called rat ventral prostate 1 protein (RVP1) and Clostridium perfringens enterotoxin receptor 2 (CPETR2). Based on its cDNA similarity to claudin-1 and claudin-2 and antibody studies showing that it is expressed at tight junctions, claudin-3 was reclassified as claudin-3. As used herein, the term "claudin-3" refers to mammalian claudin-3, preferably human claudin-3, whose Uniprot (www.uniprot.org) identifier is Uniprot.O15551, and the human sequence is shown in SEQ ID NO:4: MSMGLEITGTALAVLGWLGT IVCCALPMWR VSAFIGSNII TSQNIWEGLW MNCVVQSTGQMQCKVYDSLLALPQDLQAAR ALIVVAILLA AFGLLVALVGAQCTNCVQDD TAKAKITIVAGVLFLLAALL TLVPVSWSANTIIRDFYNPVVPEAQKREMGAGLYVGWAAAALQLLGGALLCCSCPPREKKYTATKVVYSA PRSTGPGASLGTGYDRKDYV.

[0027] As used herein, the term "toxin that binds to Claudin-3" refers to a peptide that binds to Claudin-3, such as CPE (Clostridium perfringens enterotoxin) or a fragment or variant thereof, such as the C-terminal fragment of Clostridium perfringens enterotoxin (cCPE), for example as described in doi: 10.1074 / jbc.M111.312165As shown. "Its fragment or variant" related to CPE refers to a fragment or variant (such as a cCPE analog, derivative or mutant) that is able to bind to the extracellular domain of tight junction protein-3, thereby inhibiting the binding of tight junction protein-3 to another protein. Such variants include naturally occurring allelic variants and non-naturally occurring variants. CPE is the main virulence determinant of Clostridium perfringens.

[0028] "Antibody" is also synonymously referred to as "immunoglobulin" (Ig), usually comprising four polypeptide chains, two heavy chains (H) and two light chains (L), and is therefore a multimeric protein or its equivalent Ig homologue (e.g., a camelid nanobody comprising only a heavy chain, a single domain antibody (dAb) that can be derived from a heavy chain or a light chain); including full-length functional mutants, variants or derivatives thereof (including but not limited to mouse antibodies, chimeric antibodies, humanized antibodies and fully human antibodies that retain the basic epitope binding characteristics of Ig molecules, and including dual specific, bispecific, multispecific and dual variable domain immunoglobulins); immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) and allotype. The antibody used in the present invention is preferably a monoclonal antibody or a fragment thereof, including modified antibody forms and antibody mimetics, particularly human antibodies or humanized antibodies.

[0029] As used herein, "antibody fragment" refers to a molecule comprising at least one polypeptide chain derived from a non-full-length antibody, including but not limited to: (i) a Fab fragment, which is a monovalent fragment consisting of a variable light chain (VL), a variable heavy chain (VH), a constant light chain (CL), and a constant heavy chain 1 (CH1) domain; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments connected by a disulfide bond at the hinge region; (iii) the heavy chain portion of a Fab (Fa) fragment, which consists of a VH domain and a CH1 domain; (iv) a variable fragment (Fv) fragment, which consists of a VL domain and a VH domain of a single arm of an antibody; (v) a domain antibody (dAb) fragment, which comprises a single variable domain; (v i) isolated complementarity determining regions (CDRs); (vii) single-chain Fv fragments (scFv); (viii) diabodies, which are bivalent, bispecific antibodies in which the VH domain and VL domain are expressed on a single polypeptide chain, but a linker that is too short to allow pairing between the two domains on the same chain is used, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites; and (ix) linear antibodies, which comprise a pair of tandem Fv fragments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form a pair of antigen-binding regions; and (x) other non-full-length portions of immunoglobulin heavy and / or light chains, alone or in any combination, or mutants, variants or derivatives thereof.

[0030] The term "modified antibody format" as used herein encompasses antibody-drug conjugates, polyalkylene oxide-modified scFvs, monobodies, diabodies, camelid antibodies, domain antibodies, bispecific antibodies or trispecific antibodies, IgA or two IgG structures connected by a J chain and a secretory component, shark antibodies, New World primate frameworks + non-New World primate CDRs, IgG4 antibodies with the hinge region removed, IgG designed with two additional binding sites in the CH3 domain, antibodies with altered Fc regions to enhance affinity for Fcγ receptors, dimerization constructs comprising CH3+VL+VH, and the like.

[0031] As used herein, the term "antibody mimics" refers to proteins that do not belong to the immunoglobulin family, or even non-proteins such as aptamers or synthetic polymers. Some types have a β-sheet structure similar to antibodies. The potential advantages of "antibody mimics" or "alternative scaffolds" over antibodies are better solubility, higher tissue penetration, higher stability to heat and enzymes, and relatively low production costs.

[0032] The term "coupling" or "coupled" as used herein refers to the covalent or non-covalent binding of one molecule to another molecule. Covalent binding includes the formation of covalent bonds. Non-covalent binding includes pp (aromatic) interactions, van der Waals interactions, H-bond interactions and ionic interactions. The conjugate comprising covalent binding of the present invention is, for example, an N-acetylgalactosamine (GalNAc) siRNA conjugate, wherein the siRNA (e.g., siRNA targeting tight junction protein-3) is covalently bound to N-acetylgalactosamine (GalNAc), preferably covalently bound to one to five N-acetylgalactosamine (GalNAc) moieties, more preferably covalently bound to three N-acetylgalactosamine (GalNAc) moieties. The conjugate comprising non-covalent binding of the present invention is, for example, a lipid nanoparticle, a liposome or an adenovirus comprising a silencing RNA targeting tight junction protein-3.

[0033] The terms "nucleic acid", "nucleic acid sequence", "nucleic acid molecule", "polynucleic acid sequence", "nucleotide sequence" and "nucleotide sequence" are used interchangeably herein, have the same meaning herein, and preferably refer to DNA or RNA. In some embodiments, a nucleic acid sequence is a polymer comprising or consisting of nucleotide monomers, which are covalently linked to each other by phosphodiester bonds of a sugar / phosphate backbone. The term "nucleic acid sequence" also encompasses modified nucleic acid sequences (e.g., base-modified, sugar-modified, or backbone-modified, etc.), DNA or RNA.

[0034] As used herein, the term "nucleic acid targeting a gene or mRNA" refers to at least one nucleic acid sequence encoding or comprising a nucleic acid such as a small interfering RNA (siRNA), a short or small hairpin RNA (shRNA), a microRNA (miRNA), a piwi-interacting RNA (piRNA), and a long non-coding RNA (IncRNA). Small interfering RNA (siRNA) is capable of binding to a target gene or a target messenger RNA (mRNA). In some embodiments, the siRNA used herein may be processed from a dsRNA or shRNA. Therefore, the term "siRNA" used herein may encompass siRNA and molecules according to the present invention, particularly dsRNA molecules, from which siRNA according to the present invention may be produced in mammalian cells through an RNA interference pathway. RNA may be prepared by synthetic chemistry and enzymatic methods known to those of ordinary skill in the art, or by using recombinant technology, or may be isolated from natural resources, or by a combination thereof. RNA may optionally contain non-natural and naturally occurring nucleoside modifications known in the art, such as N1-methylpseudouridine, also known as methylpseudouridine. In some embodiments, the nucleic acid targeting a gene or mRNA of the present invention comprises siRNA that can target multiple copies of an mRNA.

[0035] The term "siRNA that binds to Claudin-3" as used herein relates to a small interfering RNA (siRNA) that is capable of binding to a target messenger RNA (mRNA) of Claudin-3. The siRNA used herein may comprise a double-stranded RNA (dsRNA) region, a hairpin structure, a loop structure, or any combination thereof. In some embodiments, the siRNA may comprise at least one shRNA, at least one dsRNA region, or at least one loop structure. In some embodiments, the siRNA may be processed from a dsRNA or shRNA. In some embodiments, the siRNA may be processed or cut by an endogenous protein (e.g., DICER) from the shRNA. In some embodiments, the hairpin structure or loop structure may be cut or removed from the siRNA. For example, the hairpin structure or loop structure of the shRNA may be cut or removed. In some embodiments, the RNA described herein may be prepared by synthetic, chemical, or enzymatic methods known to those of ordinary skill in the art, prepared by recombinant techniques known to those of ordinary skill in the art, or isolated from a natural source, or prepared by any combination thereof. The RNA may comprise modified or unmodified nucleotides or mixtures thereof, for example, the RNA may optionally comprise chemical and naturally occurring nucleoside modifications known in the art. In some embodiments, the siRNA may include a nucleic acid sequence containing a sense siRNA strand. In some embodiments, the siRNA may include a nucleic acid sequence containing an antisense siRNA strand. In some embodiments, the siRNA may include a nucleic acid sequence containing a sense siRNA strand and a nucleic acid sequence containing an antisense siRNA strand.

[0036] The term "compound that promotes delivery of an agent to the liver" or "compound that promotes delivery of siRNA to the liver" as used herein refers to, for example, sugars, lipid nanoparticles, liposomes or adenoviruses. Compounds that promote delivery of siRNA to the liver are, for example, N-acetylgalactosamine (GalNAc), which is preferred.

[0037] The terms "individual", "subject" or "patient" are used interchangeably herein. In certain embodiments, the subject is a mammal. Mammals include, but are not limited to, primates (including humans and non-human primates). In preferred embodiments, the subject is a human.

[0038] The term "pharmaceutically acceptable carrier" as used herein refers to a carrier that is suitable for use in humans and / or animals without undue adverse side effects (e.g., toxicity, irritation, and allergic reactions) and has a reasonable benefit / risk ratio. A "carrier" can be a solvent, suspending agent, or excipient for delivering an immediate medicament to a subject.

[0039] As used herein, the term "about" refers to + / - 10% of a given measurement.

[0040] Therefore, the first aspect of the present invention provides an agent for inhibiting the expression and / or activity of Claudin-3, which is used in a method for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis.

[0041] Agents that inhibit the expression and / or activity of claudin-3

[0042] In one embodiment, the agent that inhibits the expression and / or activity of Claudin-3 is siRNA targeting Claudin-3 or an antibody or a fragment thereof that binds to Claudin-3.

[0043] In one embodiment, the agent that inhibits the expression and / or activity of Claudin-3 is an agent that inhibits the expression of Claudin-3.

[0044] In other embodiments, the agent that inhibits the expression of Claudin-3 is a nucleic acid that targets a gene or mRNA encoding Claudin-3.

[0045] The nucleic acid targeting the gene encoding Claudin-3 or targeting the mRNA encoding Claudin-3 may be, for example, at least one nucleic acid sequence encoding or comprising a nucleic acid such as a small interfering RNA (siRNA), a short or small hairpin RNA (shRNA), a microRNA (miRNA), a piwi-interacting RNA (piRNA), and a long non-coding RNA (IncRNA). Preferably, the agent for inhibiting the expression of Claudin-3 is a small interfering RNA (siRNA) targeting Claudin-3, i.e., a siRNA capable of binding to the gene encoding Claudin-3 or a target messenger RNA (mRNA) encoding Claudin-3, more preferably a small interfering RNA (siRNA) capable of binding to a target messenger RNA (mRNA) encoding Claudin-3. Preferably, the siRNA targeting Claudin-3 comprises 10 to 50 nucleotides, more preferably, 15 to 40 nucleotides, and even more preferably, 17 to 24 nucleotides. In certain embodiments, an siRNA according to the invention comprises 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides.

[0046] In a more preferred embodiment, the siRNA targeting Claudin-3 comprises the sequence shown in SEQ ID NO: 1 (sense strand), an siRNA comprising the sequence shown in SEQ ID NO: 2 (sense strand), an siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity to the siRNA comprising the sequence shown in SEQ ID NO: 1, and an siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity to the siRNA comprising the sequence shown in SEQ ID NO: 2.

[0047] In a preferred embodiment, the siRNA targeting Claudin-3 is characterized in that it has a sequence that is reverse complementary to SEQ ID NO: 1, or has a sequence that is reverse complementary to SEQ ID NO: 2. Therefore, in one embodiment, the sequence that is reverse complementary to SEQ ID NO: 1 is the sequence shown in SEQ ID NO: 5 (antisense strand), and the sequence that is reverse complementary to SEQ ID NO: 2 is the sequence shown in SEQ ID NO: 6 (antisense strand).

[0048] In a particularly preferred embodiment, the siRNA targeting Claudin-3 is selected from the group consisting of an siRNA comprising a sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6 or any one of SEQ ID NO:33 to SEQ ID NO:168, or an siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity with an siRNA comprising a sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6 or any one of SEQ ID NO:33 to SEQ ID NO:168.

[0049] In a more particularly preferred embodiment, the siRNA targeting Claudin-3 is selected from the group consisting of an siRNA comprising a sequence as shown in any one of SEQ ID NO:33 to SEQ ID NO:168 or an siRNA having at least 95% identity with the siRNA comprising a sequence as shown in any one of SEQ ID NO:33 to SEQ ID NO:168, more preferably 96%, 97%, 98%, 99% or 100% identity.

[0050] In an even more particularly preferred embodiment, the siRNA targeting Claudin-3 is selected from the group consisting of an siRNA comprising a sequence as shown in any one of SEQ ID NO:33 to SEQ ID NO:100 or an siRNA having at least 95% identity with the siRNA comprising a sequence as shown in any one of SEQ ID NO:33 to SEQ ID NO:100, more preferably 96%, 97%, 98%, 99% or 100% identity.

[0051] In other particularly preferred embodiments, the siRNA targeting Claudin-3 is selected from the group consisting of an siRNA comprising a sequence as shown in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:33 to SEQ ID NO:66 or SEQ ID NO:101 to SEQ ID NO:134, or an siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity with an siRNA comprising a sequence as shown in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:33 to SEQ ID NO:66 or SEQ ID NO:101 to SEQ ID NO:134.

[0052] In other more particularly preferred embodiments, the siRNA targeting Claudin-3 is selected from the group consisting of an siRNA comprising a sequence as shown in any one of SEQ ID NO:33 to SEQ ID NO:66 or SEQ ID NO:101 to SEQ ID NO:134, or an siRNA having at least 95% identity with an siRNA comprising a sequence as shown in any one of SEQ ID NO:33 to SEQ ID NO:66 or SEQ ID NO:101 to SEQ ID NO:134, more preferably 96%, 97%, 98%, 99% or 100% identity.

[0053] In other even particularly preferred embodiments, the siRNA targeting Claudin-3 is selected from the group consisting of an siRNA comprising a sequence as shown in any one of SEQ ID NO:33 to SEQ ID NO:66 or an siRNA having at least 95% identity with the siRNA comprising a sequence as shown in any one of SEQ ID NO:33 to SEQ ID NO:66, more preferably 96%, 97%, 98%, 99% or 100% identity.

[0054] In this specification, the terms "sequence identity" and "sequence identity percentage" refer to the values ​​determined by comparing two aligned sequences. Sequence alignment methods for comparison are well known in the art. Sequence alignment for comparison can be performed by local homology algorithm (Smith and Waterman, Adv. Appl. Math. 2: 482 (1981)), global alignment algorithm (Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970)), similarity search method (Pearson and Lipman, Proc. Nat. Acad. Sci. 85: 2444 (1988)) or computer implementation methods of these algorithms (including but not limited to CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA). Software for performing BLAST analysis can be, for example, performed by the National Center for Biotechnology Information ( http: / / blast.ncbi.nlm.nih.gov / ) are publicly available.

[0055] In one embodiment, the agent that inhibits the expression and / or activity of Claudin-3 is an agent that inhibits the activity of Claudin-3.

[0056] In other embodiments, the agent that inhibits the activity of Claudin-3 is selected from the group consisting of an antibody or fragment thereof that binds to Claudin-3 and a toxin that binds to Claudin-3. Preferably, the toxin that binds to Claudin-3 is Clostridium perfringens enterotoxin (CPE) or a fragment or variant thereof.

[0057] In a preferred embodiment, the agent that inhibits the activity of Claudin-3 is an antibody or a fragment thereof that binds to Claudin-3, preferably a monoclonal antibody or a fragment thereof that binds to Claudin-3, and even more preferably an antibody or a fragment thereof that binds to the extracellular domain of Claudin-3, especially a monoclonal antibody or a fragment thereof that binds to the extracellular domain of Claudin-3. In other preferred embodiments, the antibody or a fragment thereof that binds to Claudin-3 is a human antibody or a humanized antibody, more preferably a monoclonal human antibody. In a particularly preferred embodiment, the antibody or a fragment thereof that binds to Claudin-3 comprises a light chain and / or a heavy chain, wherein the light chain comprises the amino acid sequence shown in SEQ ID NO: 169, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 170.

[0058] In a more particularly preferred embodiment, the agent that inhibits the activity of Claudin-3 is an antibody or a fragment thereof, preferably a monoclonal antibody or a fragment thereof that binds to the extracellular loop 1 and / or extracellular loop 2 (ECL1 and / or ECL2) of Claudin-3.

[0059] In one embodiment, the agent that inhibits the expression and / or activity of claudin-3 is coupled to a compound that facilitates delivery of the agent to the liver. The compound that facilitates delivery of the agent to the liver can be, for example, N-acetylgalactosamine (GalNAc), lipid nanoparticles, liposomes, or adenovirus.

[0060] In a preferred embodiment, the antibody or fragment thereof that binds to Claudin-3 is conjugated to a compound that facilitates delivery of the antibody to the liver, and even more preferably, the antibody or fragment thereof that binds to Claudin-3 is an N-acetylgalactosamine (GalNAc) antibody conjugate.

[0061] In a preferred embodiment, the siRNA targeting Claudin-3 is conjugated to a compound that facilitates delivery of the siRNA to the liver. In a more preferred embodiment, the agent is an N-acetylgalactosamine (GalNAc) siRNA conjugate, i.e., a conjugate of the siRNA targeting Claudin-3 and N-acetylgalactosamine (GalNAc). Typically, such a conjugate comprises 1 to 5 GalNAc moieties, preferably 3 GalNAc moieties, covalently bound to one siRNA moiety.

[0062] Composition

[0063] As described above, the present invention also relates to a composition comprising an agent that inhibits the expression and / or activity of Claudin-3 and optionally a pharmaceutically acceptable carrier, wherein the composition is used for a method for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis.

[0064] The term "combination" generally refers to a fixed dose combination (FDC) comprising a compound that inhibits the expression and / or activity of Claudin-3 in a single dosage form, the composition having a predetermined respective dosage combination.

[0065] The composition can also be used as an adjunctive therapy. As used herein, "adjunctive" or "adjunctive therapy" refers to a combination of agents used for treatment, where the subject being treated begins a first treatment regimen of one or more agents and then begins a second treatment regimen of one or more different agents in addition to the first treatment regimen, so that not all agents used for treatment are started at the same time.

[0066] The amount of the agent that inhibits the expression and / or activity of Claudin-3 administered will vary depending on a variety of factors, such as the specific agent, the disease state and its severity, and the specific circumstances of the case including, for example, the specific agent that inhibits the expression and / or activity of Claudin-3 administered, the route of administration, the condition being treated, the target area being treated, and the subject or host being treated.

[0067] In one embodiment, the present invention provides a composition comprising an agent that inhibits the expression and / or activity of Claudin-3, wherein the agent that inhibits the expression and / or activity of Claudin-3 is present in a therapeutically effective amount.

[0068] As used herein, the expression "effective amount" or "therapeutically effective amount" refers to an amount that is capable of causing one or more of the following effects in a subject receiving the composition of the present invention: (i) dilution and / or detoxification of bile, with the bile containing more water than before treatment began; (ii) a decrease in the concentration of bile acids in the liver and / or blood; (iii) a decrease in the level of alkaline phosphatase (ALP), a cholestasis marker in the blood; (iv) a decrease in tissue necrosis in the liver; (v) an improvement in quality of life; (vi) a prolonged symptom-free survival; (vii) a prolonged survival without the need for liver transplantation; (viii) an improvement in cholestatic symptoms, such as jaundice or pruritus; and (ix) an improvement in the survival rate of patients with cholestasis and / or fibrosis associated with cholestasis.

[0069] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0070] In one embodiment, the present invention provides a composition comprising an agent that inhibits the expression and / or activity of Claudin-3, wherein the amount of the agent that inhibits the expression and / or activity of Claudin-3 in the composition is about 0.1 mg to about 10 g.

[0071] Formulation and route of administration

[0072] The composition according to the present invention is preferably suitable for subcutaneous, intravenous or oral administration to a subject, and comprises a therapeutically effective amount of an active ingredient and one or more suitable pharmaceutically acceptable carriers.

[0073] Preferably, the composition (e.g., pharmaceutical composition) can be formulated in a conventional manner using one or more pharmaceutically acceptable inactive ingredients (e.g., pharmaceutical carriers that facilitate processing of the active compound into pharmaceutically usable preparations). Appropriate formulations depend on the selected route of administration, and an overview of pharmaceutical compositions can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999). Unless otherwise indicated, the compositions according to the invention are prepared in a manner known per se, for example by conventional mixing, granulation, coating, dissolution or lyophilization processes. In the preparation of compositions for oral dosage forms, any common pharmaceutical media may be used, for example water, ethylene glycol, oil, alcohol, carriers (e.g. starch, sugar or microcrystalline cellulose), diluents, granulating agents, lubricants, binders, disintegrants, etc.

[0074] In one embodiment, the present invention provides a composition comprising an agent that inhibits the expression and / or activity of Claudin-3 and at least one pharmaceutically acceptable carrier, wherein the composition is a solution.

[0075] In one embodiment, the present invention provides a composition comprising an agent for inhibiting the expression and / or activity of Claudin-3 and at least one pharmaceutically acceptable carrier, wherein the composition is a tablet or a capsule, preferably a tablet.

[0076] Administration regimen

[0077] Exemplary treatment regimens include administration once daily, twice daily, three times daily, once every other day, twice weekly, and once weekly. The compositions of the present invention are generally administered multiple times. The interval between single doses may be, for example, less than one day, once daily, once every other day, twice weekly, or once weekly. The compositions of the present invention may be administered as a continuous, uninterrupted treatment. The compositions of the present invention may also be administered in the following regimen: the subject receives a treatment cycle interrupted by a drug holiday or a non-treatment period. Therefore, the compositions of the present invention may be administered according to the interval selected above, continuously administered for one week or a portion thereof, two weeks, three weeks, four weeks, five weeks, or six weeks, and then stopped for one week or a portion thereof, two weeks, three weeks, four weeks, five weeks, or six weeks. The combination of a treatment interval and a non-treatment interval is referred to as a cycle. The cycle may be repeated one or more times. Two or more different cycles may be used in combination to repeat the treatment one or more times. The interval may also be irregular, as indicated by measuring the blood level of the agent that inhibits the expression and / or activity of tight junction protein-3 in the patient. In a preferred embodiment, the composition according to the present invention is administered once daily. In an exemplary treatment, the agent that inhibits the expression and / or activity of Claudin-3 may be administered at 0.1 mg to 10 g per day.

[0078] Preventing or delaying progression by using an agent or a composition thereof that inhibits the expression and / or activity of claudin-3 or treatment of cholestasis and / or cholestasis-related fibrosis

[0079] The present invention provides an agent or composition thereof for inhibiting the expression and / or activity of Claudin-3 as described herein, which is used in a method for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis in a subject.

[0080] Also provided is use of the agent for inhibiting the expression and / or activity of Claudin-3 or a composition thereof described herein in the manufacture of a medicament for preventing, delaying the progression or treating cholestasis and / or fibrosis associated with cholestasis in a subject.

[0081] Also provided is use of the agent for inhibiting the expression and / or activity of Claudin-3 or a composition thereof described herein for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis in a subject.

[0082] Also provided are methods for preventing, delaying progression, or treating cholestasis and / or fibrosis associated with cholestasis in a subject, comprising administering to the subject a therapeutically effective amount of an agent that inhibits the expression and / or activity of Claudin-3, or a composition thereof, as described herein.

[0083] As used herein, the term "treating" includes: (1) delaying the onset of clinical symptoms of the state, disorder or condition in an animal (particularly a mammal, especially a human) that may have or be susceptible to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; (2) inhibiting the state, disorder or condition (e.g., preventing, alleviating or delaying the development of the disease, or delaying the recurrence of the disease and at least one clinical or subclinical symptom in the case of maintenance treatment); and / or (3) alleviating the condition (i.e., causing regression of the state, disorder or condition or at least one clinical or subclinical symptom). The benefit to the patient receiving the treatment is statistically significant or at least perceptible to the patient or physician. However, it should be recognized that when a drug is administered to a patient to treat a disease, the result may not always be an effective treatment.

[0084] As used herein, "delay of progression" refers to increasing the time from the onset of symptoms of cholestasis and / or fibrosis associated with cholestasis to worsening, and includes reversing or inhibiting disease progression. "Inhibiting" a subject's disease progression or disease complications refers to preventing or reducing a subject's disease progression and / or disease complications.

[0085] Preventive treatment includes predictive treatment. In preventive applications, the composition of the present invention is administered to a subject suspected of having cholestasis and / or cholestasis-related fibrosis or at risk of having such. In therapeutic applications, the composition is administered to a subject, such as a patient already suffering from cholestasis and / or cholestasis-related fibrosis, in an amount sufficient to cure or at least partially suppress the symptoms of the disease. The effective amount for this use will depend on the severity and course of the disease, previous treatments, the subject's health and response to the drug, and the judgment of the attending physician.

[0086] In the event that the subject's condition does not improve, the compositions of the invention may be administered chronically, i.e., for an extended period of time, including throughout the life of the subject, to ameliorate or otherwise control or limit the symptoms of the subject's disease or condition.

[0087] If the subject's condition does improve, the composition may be continuously administered; alternatively, the dose of the administered drug may be temporarily reduced, or administration of the composition may be suspended for a certain period of time (ie, a "drug holiday").

[0088] Once the patient's condition has improved, a maintenance dose of the composition of the present invention is administered as needed. Subsequently, as symptoms change, the dosage or frequency of administration, or both, is optionally reduced to a level that maintains the disease improvement.

[0089] In one embodiment, the cholestasis is intrahepatic cholestasis or extrahepatic cholestasis, preferably intrahepatic cholestasis. In a preferred embodiment, the cholestasis is intrahepatic cholestasis, wherein the intrahepatic cholestasis is acute intrahepatic cholestasis or chronic intrahepatic cholestasis. In an even more preferred embodiment, the cholestasis is chronic intrahepatic cholestasis, preferably chronic intrahepatic cholestasis selected from the group consisting of primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC).

[0090] In one embodiment, cholestasis is a disease selected from the group consisting of obstructive cholestasis, non-obstructive cholestasis, bile duct disease and biliary dysfunction, more preferably bile duct disease, in particular malignant bile duct obstruction.

[0091] In a preferred embodiment, the cholestasis and / or fibrosis associated with cholestasis is selected from the group consisting of chronic intrahepatic cholestasis and bile duct diseases.

[0092] In a more preferred embodiment, the cholestasis and / or fibrosis associated with cholestasis is selected from the group consisting of primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC) and malignant bile duct obstruction.

[0093] Obstructive cholestasis may be associated with or caused by gallstones, pancreatic cancer, biliary adenocarcinoma, common bile duct stricture, biliary atresia (eg, extrahepatic biliary atresia), common bile duct obstruction due to biliary sludge or gallstones, or choledochal cysts with biliary sludge and / or concentrated bile / mucus plugs.

[0094] Non-obstructive cholestasis may be associated with or caused by the following conditions: viral hepatitis (hepatitis B, hepatitis C), toxic effects of drugs or nutrition, paraneoplastic syndromes (such as Hodgkin lymphoma), Wilson disease, familial cholestasis syndrome, intrahepatic cholestasis of pregnancy, infiltrative diseases (such as amyloidosis), metastatic cancer and / or cirrhosis (of any cause), bacterial infection (such as gram-negative enteric bacteremia), syphilis, listeria, toxoplasmosis or sepsis or endotoxemia caused by bacterial infection, viral infection (such as cytomegalovirus, herpes viruses (including herpes simplex virus, herpes zoster virus, parvovirus B19, adenovirus), rubella virus, reovirus, and enterovirus).

[0095] Biliary disease may be associated with or caused by primary biliary cholangitis, primary sclerosing cholangitis, graft-versus-host disease (acute and chronic), transplant rejection (acute and chronic), transplant: bile duct obstruction secondary to hepatic artery obstruction, vanishing bile duct syndrome due to toxic effects of drugs such as ibuprofen or chlorpromazine.

[0096] Defective biliary function may be associated with or caused by alpha-1-antitrypsin storage disease, cystic fibrosis, galactosemia, tyrosinemia, fatty acid oxidation defects, lipid storage disorders, glycogen storage disorders, peroxisomal disorders, bile acid biosynthesis defects, PFIC1, PFIC2, PFIC3 (PFIC, progressive familial intrahepatic cholestasis), bile duct deficiency disorders (eg, Alagille syndrome), or nonsyndromic bile duct deficiency syndromes.

[0097] In a preferred embodiment, cholestasis is a disease selected from the group consisting of: obstructive cholestasis associated with or caused by common bile duct stenosis, biliary atresia such as extrahepatic biliary atresia and / or common bile duct obstruction caused by bile sludge or gallstones, preferably obstructive cholestasis associated with or caused by common bile duct stenosis; non-obstructive cholestasis associated with or caused by drug toxic effects, Wilson's disease and / or familial cholestasis syndrome; bile duct diseases associated with or caused by primary biliary cholangitis, primary sclerosing cholangitis and / or vanishing bile duct syndrome caused by the toxic effects of drugs such as ibuprofen or chlorpromazine; and biliary function defects associated with or caused by bile acid biosynthesis defects, PFIC1, PFIC2, PFIC3.

[0098] In a more preferred embodiment, the cholestasis is obstructive cholestasis. In an even more preferred embodiment, the cholestasis is obstructive cholestasis associated with or caused by common bile duct stenosis or biliary atresia (such as extrahepatic biliary atresia).

[0099] In an embodiment, cholestasis-associated fibrosis refers to a disease in which fibrosis is caused at least in part by unresolved cholestasis, preferably, cholestasis-associated fibrosis refers to a liver disease in which fibrosis is caused at least in part by unresolved cholestasis.

[0100] The fibrosis associated with cholestasis is generally selected from a disease or condition selected from the group consisting of: fibrosis caused by hepatitis A virus infection associated with cholestasis, fibrosis caused by hepatitis B virus infection associated with cholestasis, fibrosis caused by hepatitis C virus infection associated with cholestasis, fibrosis caused by Epstein-Barr virus infection associated with cholestasis, fibrosis caused by hemochromatosis associated with cholestasis, fibrosis caused by autoimmune hepatitis associated with cholestasis, fibrosis caused by secondary biliary cirrhosis associated with cholestasis, fibrosis caused by alcoholic liver disease associated with cholestasis and / or clinical jaundice, fibrosis caused by drug-induced liver disease associated with cholestasis. Fibrosis, fibrosis caused by metabolic syndrome (e.g., impaired glucose metabolism, fat accumulation, or impaired lipid metabolism) associated with cholestasis, liver fibrosis caused by non-alcoholic fatty liver disease (NAFLD) associated with cholestasis, liver fibrosis caused by iron and copper homeostasis disorders associated with cholestasis, liver fibrosis caused by non-alcoholic fatty liver disease associated with cholestasis, liver fibrosis caused by non-alcoholic steatohepatitis (NASH) associated with cholestasis, fibrosis caused by radiation liver disease associated with cholestasis, fibrosis caused by Wilson's disease associated with cholestasis, fibrosis caused by α1-antitrypsin deficiency associated with cholestasis, and fibrosis caused by sinusoidal obstruction syndrome associated with cholestasis. Preferably, the present invention provides a method for treating fibrosis caused by metabolic diseases such as NAFLD or NASH using an agent as described herein, particularly a method for treating fibrosis caused by liver disease.

[0101] In another aspect, the present invention provides a dosage form for preventing, delaying the progression or treating cholestasis and / or fibrosis associated with cholestasis, comprising an agent that inhibits the expression or activity of claudin-3 or a composition comprising the agent and optionally a pharmaceutically acceptable carrier.

[0102] In another aspect, the present invention provides siRNA targeting Claudin-3. In a preferred embodiment, the present invention provides siRNA targeting Claudin-3, the siRNA being selected from the group consisting of siRNA comprising the sequence as shown in SEQ ID NO: 1, siRNA comprising the sequence as shown in SEQ ID NO: 2, siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity to siRNA comprising the sequence as shown in SEQ ID NO: 1, and siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity to siRNA comprising the sequence as shown in SEQ ID NO: 2.

[0103] In another embodiment, the present invention provides an siRNA targeting Claudin-3, which siRNA is selected from the group consisting of an siRNA comprising the sequence as shown in SEQ ID NO:5, an siRNA comprising the sequence as shown in SEQ ID NO:6, an siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity to the siRNA comprising the sequence as shown in SEQ ID NO:5, and an siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity to the siRNA comprising the sequence as shown in SEQ ID NO:6.

[0104] In a preferred embodiment, the present invention provides an siRNA targeting claudin-3, which is selected from the group consisting of an siRNA comprising a sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6 or any one of SEQ ID NO:33 to SEQ ID NO:168, or an siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity with an siRNA comprising a sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6 or any one of SEQ ID NO:33 to SEQ ID NO:168.

[0105] In a more preferred embodiment, the present invention provides a siRNA targeting Claudin-3, which siRNA is selected from the group consisting of an siRNA comprising a sequence as shown in any one of SEQ ID NO:33 to SEQ ID NO:168 or an siRNA having at least 95% identity with the siRNA comprising a sequence as shown in any one of SEQ ID NO:33 to SEQ ID NO:168, more preferably 96%, 97%, 98%, 99% or 100% identity.

[0106] In an even more preferred embodiment, the present invention provides an siRNA targeting Claudin-3, which siRNA is selected from the group consisting of an siRNA comprising a sequence as shown in any one of SEQ ID NO:33 to SEQ ID NO:100 or an siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity with an siRNA comprising a sequence as shown in any one of SEQ ID NO:33 to SEQ ID NO:100.

[0107] In a particular embodiment, the present invention provides an siRNA targeting claudin-3, which is selected from the group consisting of an siRNA comprising a sequence as shown in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:33 to SEQ ID NO:66 or SEQ ID NO:101 to SEQ ID NO:134, or an siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity with an siRNA comprising a sequence as shown in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:33 to SEQ ID NO:66 or SEQ ID NO:101 to SEQ ID NO:134.

[0108] In a more specific embodiment, the present invention provides an siRNA targeting Claudin-3, which siRNA is selected from the group consisting of an siRNA comprising a sequence as shown in any one of SEQ ID NO:33 to SEQ ID NO:66 or SEQ ID NO:101 to SEQ ID NO:134, or an siRNA having at least 95% identity with an siRNA comprising a sequence as shown in any one of SEQ ID NO:33 to SEQ ID NO:66 or SEQ ID NO:101 to SEQ ID NO:134, more preferably having 96%, 97%, 98%, 99% or 100% identity.

[0109] In a more specific embodiment, the present invention provides an siRNA targeting Claudin-3, which siRNA is selected from the group consisting of an siRNA comprising a sequence as shown in any one of SEQ ID NO:33 to SEQ ID NO:66 or an siRNA having at least 95% identity with the siRNA comprising a sequence as shown in any one of SEQ ID NO:33 to SEQ ID NO:66, more preferably 96%, 97%, 98%, 99% or 100% identity.

[0110] On the other hand, the present invention provides an antibody or fragment thereof that binds to tight junction protein-3. In one embodiment, the antibody or fragment thereof that binds to tight junction protein-3 is a human antibody. In a preferred embodiment, the antibody or fragment thereof that binds to tight junction protein-3 comprises a light chain and / or a heavy chain, the light chain comprising an amino acid sequence as shown in SEQ ID NO: 169, and the heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 170. In other embodiments, the antibody or fragment thereof that binds to tight junction protein-3 is coupled to a compound that promotes delivery of the antibody to the liver. In other preferred embodiments, the antibody or fragment thereof that binds to tight junction protein-3 is an N-acetylgalactosamine (GalNAc) antibody conjugate. Particularly preferred antibodies are human antibodies that bind to tight junction protein-3, more particularly human antibodies that bind to tight junction protein-3, comprise a light chain and / or a heavy chain, and are N-acetylgalactosamine (GalNAc) antibody conjugates, the light chain comprising an amino acid sequence as shown in SEQ ID NO: 169, and the heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 170.

[0111] Example

[0112] The present embodiment is intended to explain the present invention rather than to limit the present invention.

[0113] Materials and methods

[0114] Animal care and ethical approval

[0115] 12- to 18-week-old C57BL / 6J background Cldn3 + / + or CLDN3 - / - Mice (approximately 18 to 22 g) were housed in a room controlled by a 12-h light cycle and fed a standard laboratory diet. All mouse experiments were performed with the approval of the Veterinary Office of the Canton of Bern (license number BE37 / 20), in accordance with the guidelines for good animal practice set by the Office for Laboratory Animal Welfare, and in compliance with the standards of the National Center for the Replacement, Improvement and Reduction of Animals in Research Guidelines ( https: / / www.nc3rs.org.uk / arrive-guidelines ).

[0116] Bile flow and FITC-dextran throughput into bile

[0117] The integrity of the paracellular barrier of hepatocyte tight junctions was determined as previously described. 14. The passage of FITC-dextran (40 kDa) from blood to bile was quantified. In anesthetized mice, the common bile duct was ligated and the cystic duct was cannulated. Bile was then collected for 20 minutes to determine the bile flow rate. Then, 400 μl of FITC-dextran (40 kDa, 25 mg / ml in saline) was injected into the inferior vena cava. Bile was collected from the cannula every 3 minutes for 40 minutes. The liver was surgically removed and weighed. Bile was diluted 1 / 50 in water and analyzed using a Tecan FITC fluorescence was measured in a plate reader at 489 nm excitation / 534 nm emission. Data were reported as relative fluorescence units (RFU) per microliter of bile per minute per gram of liver.

[0118] Quantification of FITC-dextran uptake

[0119] In anesthetized mice, the common bile duct was ligated and 300 μl of FITC-dextran (40 kDa, 6.25 mg / ml in saline) was injected via the tail vein. FITC fluorescence was acquired by time-lapse imaging using the Spectrum system (Perkin Elmer). Images were acquired every 30 seconds for a total of 45 minutes. Regions of interest with equal area sizes were drawn around the liver. The FITC signal intensity in each image was quantified using the instrument software. Data are reported as total radiant efficiency [p / s] / [μW / cm 2 ]. Using the same experimental setup, mouse livers were harvested 20 minutes after injection of FITC dextran (40 kDa) and frozen sections (25 μm) were made. The sections were stained with DAPI (D9542, 1:2000 dilution; Sigma-Aldrich) for 15 minutes and then mounted with fluorescent mounting medium (H-1000; Vectorlabs, Burlin-game, CA). Confocal Z-stack images were acquired in the periportal liver area to observe the uptake of FITC dextran (LSM 710; Zeiss, 20x objective, Oberkochen, Germany). FITC signals were quantified in complete Z-stacks of the same area and thickness using Image J software (version 1.48; National Institutes of Health, Bethesda, Maryland). Data are reported as total signal intensity (integrated density).

[0120] Bile duct ligation

[0121] The BDL operation was performed as previously described 15 Briefly, buprenorphine (0.1 ug / g body weight, ) for analgesia, and a transverse laparotomy was performed. The common bile duct was exposed and sutured with 7-0 silk suture (Sofsilk TM , Covidien / Medtronic, Dublin, Ireland) double ligature and cut between the ligatures. The laparotomy was closed with sutures (Ethicon, Bridgewater, NJ, USA). Buprenorphine analgesia was repeated if necessary based on the animal health score.

[0122] Hematoxylin and eosin staining

[0123] Liver paraffin sections were dewaxed and stained with hematoxylin (Merck, Cat. # HX43078349) for six minutes and differentiated by three dips in HCL-ALC (1:1). Slides were incubated in eosin (Fluka Chemical Corp, Cat. # 45240) for 3 minutes, then dehydrated and (Kindler) mounting.

[0124] Sirius red staining and quantification

[0125] The slides were dewaxed, hydrated and placed in preheated Brunner's fixative (Sigma-Aldrich, Cat. No. HT-10-1-32) at 54°C for 20 minutes. After rinsing in running water, the slides were stained with Weigert iron hematoxylin (Sigma-Aldrich, Cat. No. HAT 10-79) for 5 minutes. After rinsing in running tap water for 5 minutes, the slides were destained with hydrochloric acid-ethanol (1 liter of 70% ethanol containing 5 ml of 37% hydrochloric acid) and rinsed in water for 5 minutes. The slides were then stained with Picosirius red (0.5 g dissolved in 1L Brunner's solution, Sigma-Aldrich Cat. No. 365548) for 5 minutes. After rinsing twice with 0.5% acetic acid in water for 5 minutes each time, the slides were dehydrated and mounted with Eukitt (Kindler). Images were acquired using a slide scanner (Panoramic 250Flash III, 3DHISTECH), and staining was quantified using Image J (v1.52n, National Institutes of Health, Bethesda, MD). Data analysis was performed in a blinded manner. For each mouse liver, 12 randomly selected image areas of equal size at 10x magnification were quantified. Only unmodified images were used in the analysis, and all five liver lobes were covered. The Sirius red signal was separated from the other channels using the deconvolution setting "Azon-Mallory". In the red channel, a threshold was set. After converting the threshold-adjusted images to 8-bit images, the signal intensity was measured as integrated density. The results are expressed as the average integrated density per square millimeter.

[0126] Masson's trichrome staining and quantification

[0127] Masson trichrome staining. Paraffin-embedded liver tissue was dewaxed and placed in Brunner's fixative (HT10-1-32; Sigma-Aldrich) at 56°C for 10 minutes. After washing the slides with tap water and distilled water, stain them with hematoxylin (HT10-79; Sigma-Aldrich) for 5 minutes. After washing the slides with running tap water and distilled water, decolorize them once with hydrochloric acid-ethanol (1:1) and rinse them once with distilled water. Next, place the slides in Biebrich scarlet-acid fuchsin (HT151-250ML; Sigma-Aldrich), diluted in 1% acetic acid (K45741563425; Dr. Grogg Chemie, Stettlen, Switzerland) at a ratio of 1:2 for 1 minute. The slides were rinsed and stained with phosphomolybdic acid-phosphotungstic acid (HT153-250ML and HT152-250ML; Sigma) at a ratio of 1:1 for 5 min. The slides were then stained with aniline blue (HT154-250ML; Sigma) for 20 min. After the last rinse, the slides were dehydrated in 0.75% acetic acid and mounted with Eukitt (Kindler). Images were acquired using a slide scanner (Panoramic250Flash III, 3DHISTECH), and staining was quantified using Image J (v1.52n, National Institutes of Health, Bethesda, MD). For each mouse liver, 6 randomly selected areas of the same size at 7x magnification were quantified. Only unmodified images were used. The blue collagen signal was separated from the other channels using the deconvolution setting “Azon-Mallory”. In the blue channel, a threshold was set. After converting the threshold-adjusted images into 8-bit images, the signal intensity was measured as integrated density. Results are expressed as mean integrated density.

[0128] Quantification of tissue necrosis

[0129] Liver tissue sections before and after BDL were stained with hematoxylin / eosin and images were taken using a bright field microscope (Panoramic 250Flash III, 3DHISTECH). The sections used for quantification covered an average of 75 square millimeters per individual. Necrotic parenchymal areas were manually circled. Data were reported as the percentage of necrotic areas to the entire section.

[0130] Determination of ALT, AST, ALP, cholesterol and total bilirubin in serum

[0131] Liver injury markers ALT and AST were measured on a Cobas 8000 modular analyzer using module C502 (Roche, Switzerland). Similarly, ALP and total bilirubin were measured on a Cobas 8000 using module C702 (Roche, Switzerland). All measurements were performed according to the manufacturer's instructions.

[0132] Quantification of renal bile embolism

[0133] The kidneys of 7 days after BDL were all sliced ​​and the tissues were stained with hematoxylin / eosin. Images were taken using a bright field microscope (Panoramic 250Flash III, 3DHISTECH). Image J software was used to deconvolute yellow bile embolism from pink hematoxylin / eosin staining. Yellow was converted to black and white 8-bit images, and signal intensity was measured as integral density. At least 10 different images were taken for each mouse, and each image covered 3034 square millimeters of kidney tissue, and the results were averaged. Data are reported as the integral density of each square millimeter of kidney tissue.

[0134] Scoring of periductal edema of the liver

[0135] Periductal edema was scored on whole hematoxylin and eosin-stained liver sections based on an arbitrary scale from 0 (no edema) to 10 (maximum amount of edema observed). Only the periportal area was considered. Periductal edema was defined as prominent cuff-like structures around the bile duct with swelling of the connective tissue and enlargement of the extracellular matrix.

[0136] Assessment of bacterial translocation

[0137] The liver and spleen were harvested under sterile conditions two or seven days after BDL. After tissue weighing, sterile PBS and stainless steel beads were added to the samples, followed by homogenization of the tissues at 30 Hz for 5 minutes using a Tissuelyser (Quiagen). The samples were then placed on lysogeny broth (LB) agar plates and plated as previously described. 16 The incubation was carried out under aerobic conditions for 48 h at 37 °C. At the end of the incubation, bacterial colonies were manually counted and normalized to organ weight to obtain results in colony forming units. For data visualization, the obtained CFU / g were normalized using a logarithmic transformation.

[0138] Flow cytometry

[0139] The antibodies used for fluorescence activated cell sorting are listed below. The liver was placed in Roswell Park Memorial Institute (RPMI) 1640 medium, supplemented with GlutaMAX, and digested with collagenase D (Merck), 0.05% collagenase IV (Worthington Biochemical Company), DNaseI (Sigma-Aldrich Company) and Dispase at 37°C for 25 minutes. The digested liver tissue was poured into a 100μm cell strainer (BD Falcon) and washed with 50ml RPMI at 4°C. After centrifugation at 300g for 5 minutes, the supernatant was discarded and 30ml RPMI was added again. Repeat the centrifugation at 300g for 5 minutes, discard the supernatant, add 5ml red blood cell (RBC) lysis buffer to each sample, filter through a 40μm cell strainer, and incubate at room temperature. After incubation, the cell suspension was spun and washed with 5ml PBS (Gibco) containing 3% fetal bovine serum and 2mM EDTA and Hepes (staining buffer). The isolated non-parenchymal cells were then incubated with purified anti-CD16 / CD32 and viability dye eFluor 506 (Thermo Fisher Scientific) diluted in PBS for 20 minutes at 4°C in the dark to block nonspecific binding antibodies and exclude dead cells. After the first staining and washing of the cells, the samples were stained with the primary antibodies (Table 1) diluted in staining buffer for 20 minutes at 4°C in the dark. After the final washing step (300g for 5 minutes), the samples were resuspended in staining buffer and fixed by adding IC fixation buffer (eBioscience). Fixed samples were evaluated using a flow cytometer (BD LSR Fortessa; BD Pharmingen, Inc, San Diego, CA) and the corresponding BD FACSDiva software. Data analysis was performed using FlowJo software (Treestar, Inc, Ashland, OR).

[0140] FACS Antibodies

[0141]

[0142] Determination of total bile acids in serum and liver tissue samples

[0143] Total bile acid levels were determined using a test kit purchased from Crystal Chem (Cat. #80470). Bile, serum, and total liver tissue samples were processed according to the manufacturer's instructions. Final absorbance levels were measured by Tecan The assay was performed using a plate reader.

[0144] Bile Acid Quantification Using LC-MS / MS

[0145] The method applied was recently described 17 Briefly, for the quantification of bile acids, 25 μL of serum samples were diluted with water in a ratio of 1:4, and the calibrants were protein precipitated by the addition of 900 μL of 2-propanol and a mixture of deuterated internal standards. The extraction was performed at 4 °C for 30 min with constant shaking, followed by centrifugation at 16,000 g for 10 min. The supernatant was transferred to a new tube, evaporated to dryness, and reconstituted with 100 μL of methanol:water (1:1, v / v). For the extraction of liver samples, 900 μL of chloroform:methanol:water (1:3:1, v / v / v) and 100 μL of the internal standard mixture were added to a Precellys tube containing beads and 30 ± 5 mg of liver tissue. The samples were homogenized using a Precellys tissue homogenizer and centrifuged at 16,000 g for 10 min at 20 °C. The supernatant was transferred to a new tube, and the process was repeated by adding 800 μL of the extraction solvent. After evaporation to dryness, the samples were resuspended in 200 μL methanol: water (1:1, v / v). The injection volume in both cases was 3 μL. LC-MS / MS was performed by Agilent 1290UPLC coupled to an Agilent 6490 triple quadrupole mass spectrometer (Agilent Technologies, Basel, Switzerland) equipped with an electrospray ionization source. Chromatographic separation of bile acids was achieved using a reverse phase column (ACQUITY UPLC BEH C18, 1.7 mm, 2.1 μm, 150 mm, Waters, Wexford, Ireland).

[0146] ANIT poisoning

[0147] The α-naphthyl isothiocyanate (ANIT) poisoning protocol was derived from a previous publication. 19-20 . In the acute intoxication model, ANIT (Sigma-Aldrich, St. Louis, Missouri, US) was dissolved in corn oil and administered orally at 60 mg / kg body weight. The same volume of corn oil was administered to the control animals. For the two-day or ten-day models, a single or double dose was given. In the chronic injury model, a customized diet containing 0.1% ANIT (Granovit, Lucens, Switzerland) was freely available. The control group was fed a customized diet containing the same amount of corn oil. Each group was fed the customized diet for four weeks.

[0148] Immunohistochemistry

[0149] Paraffin-embedded liver tissue was sliced ​​into 6 μm thick slices for routine imaging or 30 μm thick slices for confocal z-stack imaging. Slides were dewaxed and hydrated in a series of xylene and ethanol. Antigen retrieval was performed by heat-induced epitope retrieval, boiling the slides at 95°C for 10 minutes in citrate buffer (pH 6.0, Sigma-Aldrich, Cat.#C9999). Nonspecific antibody binding was blocked for one hour at room temperature using protein blocking solution (Dako, Cat.#X0909). Antibodies were prepared in antibody diluent (Dako, Cat.#S3022) at the following dilutions. Primary antibody: Cytokeratin 7 (Novus Biologicals, Cat.#NBP1-88080), 1:200. Secondary antibody: polyclonal rabbit anti-goat immunoglobulin / HRP (Dako, Cat.#P0449). For immunohistochemical staining, streptavidin peroxidase (BioConcept, Cat. #71-00-38) and DAB (Sigma-Aldrich, Cat. #D4293-50SET) were used.

[0150] The primary antibody was incubated overnight at 4°C in a humidified chamber with gentle agitation. The slides were washed in PBS-Tween-20 [0,5%] (Sigma-Aldrich, Cat. # P1379) for 20 minutes and incubated in the dark with the secondary antibody and DAPI (Sigma-Aldrich, Cat. # D9542, dilution 1:2000) for 90 minutes. After a final wash in PBS-Tween 20 [0.5%], the slides were mounted. Before the primary antibody incubation, the erythrocytes were quenched in 5% H2O2 for 10 minutes and after the application of the secondary antibody, the staining was developed by incubation with streptavidin-peroxidase for 30 minutes and with DAB for 1 minute.

[0151] Western blotting

[0152] Total protein was extracted from liver tissue or cultured cells using RIPA lysis buffer and TissueLyser (Qiagen, TissueLyserII). The lysate was centrifuged at 20000g for 15 minutes and the supernatant was aliquoted. Protein concentration was quantified by Bradford assay (Bio-Rad, Cat.#5000006) and microplate reader. Equal amounts of protein in each sample were separated by SDS-PAGE under reducing conditions using precast gels (Bio-Rad, Cat.#456-1094). The protein was then transferred to a nitrocellulose membrane (Biorad, Cat.#170-4158). The membrane was blocked for 1 hour with 5% w / v skim milk in PBS at room temperature. The first antibody was diluted in blocking medium and incubated overnight at 4°C. Primary antibody: CLDN3, (Novus Biologicals, Cat.#NBP1-35668), 1:1000; anti-β-actin peroxidase, (Sigma-Aldrich, Cat.#A3854), 1:50000; secondary antibody: anti-rabbit HRP (Dako, Cat.#P0448), 1:2000.

[0153] After incubation with the primary antibody, the membrane was washed three times in PBS-Tween-20 [0.1%] for 5 minutes each time. The secondary antibody was diluted with 5% w / v skim milk in PBS and the membrane was incubated for one hour at room temperature, followed by three washing steps for a total of 30 minutes. Enhanced chemiluminescence solution (Perkin Elmer, Cat. # NEL105001EA) was added for one minute to generate the signal. Film was used in combination with a developer (AGFA, CURIX 60) to visualize the bands. The correct band size was estimated with the aid of a standard protein ladder (Biorad, Cat. # 161-0374).

[0154] Human siRNA candidate screening

[0155] Candidate siRNAs targeting human claudin-3 mRNA were first identified by bioinformatics methods (human reference sequence used: NM_001306.4). All possible siRNAs were created and then scored for specificity, cross-reactivity, activity, and off-target (Axolabs, Kulmbach, Germany). The best candidate was selected and synthesized, which contained stabilizing modifications with 2'-fluoro- or 2'O-methyl modifications and contained a phosphorothioate linker at the specified position (see Table 6). The synthesized candidate siRNAs were then transfected into the human PLC hepatocellular carcinoma cell line (ATCC Cat. No CRL-8024) using lipofectamine-3000. The final concentration of siRNA used was 50 nM. Two days after transfection, RNA was isolated from cell cultures and the remaining human claudin-3 mRNA levels were quantified using RT-qPCR according to the following protocol. 34 siRNAs with a knockdown efficiency of 50% or higher were selected for further testing (Table 6). Table 7 shows the same identified human Claudin-3 siRNA sequences but without chemical stabilization modifications.

[0156] RT-qPCR mRNA expression analysis

[0157] RNA was extracted from snap-frozen tissues or cell cultures using NucleoZOL (Macherey-Nagel, Cat. #740404.200). cDNA was prepared from 500 μg of tissue RNA using the Omniscript Reverse Transcriptase Kit (Qiagen, Cat. #205113). cDNA was analyzed using QuantStudio according to the manufacturer's instructions. TM Real-time qPCR was performed using SYBR-green or Taqman-based assays on a 7Flex thermal cycler (Applied Biosystems, Foster City, CA). Primer sequences are shown below.

[0158] Primer sequences (m: mouse; F: forward; R: reverse)

[0159]

[0160]

[0161] Antibody generation and sequences

[0162] Recombinant mouse IgG2A Kappa anti-Claudin-3 antibody was produced using Expi293 cells. The antibody consists of a heavy chain (SEQ ID NO: 170) and a light chain (SEQ ID NO: 169), as shown below.

[0163] After Expi293 cells were transfected with the expression vector, the cells efficiently produced and secreted the recombinant antibody. The cell supernatant was then purified using protein A resin (Repligen, #CA-PRI-0005).

[0164] Light chain (SEQ ID NO: 169):

[0165] DIVMSQSPSSLAVSVGEKVTMSCKSSPSLLYSNNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSFPYTFGGGT KLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNEC

[0166] Heavy chain (SEQ ID NO: 170):

[0167] EVQLQQSGPELVKPGGSMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYNDGTNYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCSKKGGGYGGTWFAYWG QGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTI KPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIER TISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0168] Primary Cell and Cell Line Culture Methods

[0169] Liver samples were obtained from patients undergoing liver resection at the University Hospitals of Basel and Bern, Switzerland. Liver perfusion was performed using a two-step enzymatic perfusion protocol (doi: 10.1172 / JCI115207 ) Human hepatocytes isolated from liver specimens and from C57BL / 6 and C57BL / 6Cldn3 - / - Mouse hepatocytes from mice. The viability of isolated hepatocytes was determined by trypan blue exclusion, and only preparations with viability exceeding 90% were used. Hepatocytes were seeded onto tissue culture plastic coated with rat tail collagen in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, allowed to adhere for 1 to 2 hours, and then washed twice with phosphate buffered saline (PBS) to remove any residual non-viable cells in the culture. Hepatocytes were cultured for 24 hours in Williams E medium without arginine, supplemented with insulin (0.015 IU / mL), hydrocortisone (5 μmol / L), penicillin (100 IU / mL), streptomycin (100 μg / mL), glutamine (2 mmol / L), and ornithine (0.4 mmol / L) before use.

[0170] SNU-449 cells (ATCC#2234) were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, 100 mg / mL streptomycin. Hepa1-6 (ATCC#CRL-1830) were grown in DMEM supplemented with 10% fetal bovine serum, 100 U / mL penicillin, 100 mg / mL streptomycin.

[0171] Flow cytometry for human claudin-3 antibody testing

[0172] The anti-claudin-3 antibody stock solution (0.5 mg / ml) was serially diluted in FACS buffer and exposed to 5.10 5 The binding capacity of Cldn3 antibody was evaluated by 4C for 30 minutes in human hepatocytes or SNU449 cells. The cells were then rinsed with PBS and exposed to cyanine5-conjugated secondary anti-mouse IgG (Life technologies #A10524) at 1:200 in a dark box at 4C for 30 minutes and then rinsed with PBS. Finally, the cell data were acquired on SORP LSRII (BD Pharmingen Inc., San Diego, CA). Flow cytometric analysis was performed using FlowJo software (Treestar, Inc., Ashland, OR).

[0173] Evaluation of the binding capacity of Cldn3 antibodies

[0174] To determine the binding capacity of anti-Claudin-3 antibodies, a series of dilutions of the stock solution (0.5 mg / ml) were prepared in FACS buffer. The diluted antibody solutions were then exposed to 5.10 5 Human hepatocytes or SNU449 cells for 30 min.

[0175] After the incubation period, the cells were washed with PBS to remove unbound antibodies. Subsequently, secondary FITC-anti-mouse IgG (ref) was applied to the cells at a dilution of 1:200. The secondary antibody was allowed to bind for 30 minutes at 4°C in the dark. Subsequently, the cells were washed with PBS to eliminate excess secondary antibody.

[0176] Cell data were acquired using a SORP LSRII flow cytometer (BD Pharmingen Inc., San Diego, CA). The acquired data were analyzed using FlowJo software for flow cytometry analysis (Treestar, Inc., Ashland, OR).

[0177] GalNAc-anti-Cldn3 antibody functionalization

[0178] The antibody buffer was first exchanged into PBS using a 7K Zeba size exclusion column, which effectively removed any unwanted buffer components and ensured compatibility with subsequent reactions.

[0179] To functionalize the antibody, it was reacted with 25 equivalents of DBCO-PEG4-NHS (BroadPharm #BP-22288). The antibody solution in PBS was incubated overnight at room temperature to achieve efficient coupling between the antibody and DBCO-PEG4-NHS.

[0180] After the coupling reaction, the functionalized antibody was purified using a 7K Zeba size exclusion column equilibrated in PBS. This purification step helps separate the coupled antibody from any unreacted DBCO-PEG4-NHS and other impurities, resulting in a purified functionalized antibody preparation.

[0181] Next, the purified functionalized antibody was reacted with 100 equivalents of Trebler GalNAc-Azide (primetech #0079). This reaction facilitated the attachment of the galactose N-acetyl (GalNAc) moiety to the functionalized antibody, enabling targeted delivery to the GalNAc receptor.

[0182] After the conjugation reaction with GalNAc-Azide, the antibody is purified again, this time using a 40K Zeba size exclusion column equilibrated in PBS. This purification step further separates the conjugated antibody from any unreacted GalNAc-Azide and other residual impurities, resulting in a purified and functionalized antibody that can be used in downstream applications.

[0183] Cldn3 KD using GalNAc-anti-Cldn3 antibody

[0184] Human hepatocytes in 12-well plates were treated with different concentrations of GalNAc-anti-Cldn3 antibodies. Specifically, cells were treated with 0nM, 1nM, 10nM, and 100nM concentrations of the antibody for 24 hours. After the 24-hour treatment period, proteins were extracted from the treated cells using 50ul RIPA lysis buffer.

[0185] The extracted proteins were quantified using the Bio-Rad protein analysis system (Bio-Rad Laboratory, Melville, NY).

[0186] Western blotting to test the efficacy of human claudin-3 inhibitory antibodies

[0187] Protein lysates were boiled in Laemmli buffer, run on a BIO-RAD Mini-Protean TGXTM (10–20% Ready Gel Tris-HCl gel system, 12-well comb #456–1095 and 10-well comb #456–1094) at 120 volts for 90 min, and then transferred to BIO-RAD Trans-Blot Turbo Mini or Midi PVDF membranes (Mini PVDF TransferPacks #170–4156 and Midi PVDF Transfer Packs #170–4159) by semi-dry transfer (Trans-Blot Turbo Transfer System BIO-RAD). Proteins were detected using the following primary antibodies: claudin-3 (1:300) (Novus Biologicals, Cat. #NBP1-35668) and beta actin conjugated to HRP (1:5000) (Sigma-Aldrich, #2228, RRID:AB_476697). Antibodies were diluted in 5% milk and incubated overnight at 4°C. The following secondary antibodies were used: HRP-conjugated anti-rabbit (1:2000) (Dako, #P0448, RRID:AB_2617138). The membranes were then washed and protein expression was analyzed by chemiluminescence (Western LightningPlus-ECL Perkin Elmer) using Fusion-FX (Vilber).

[0188] Bile autofluorescence assay

[0189] Bile obtained from the gallbladder was diluted 1:50 with water. The plate reader measured autofluorescence at 489 nm excitation / 534 nm emission.

[0190] Bulk RNA sequencing of tissue samples

[0191] Total RNA was extracted from liver using NucleoZOL and quantified by Bioanalyzer. The settings for the sequencing run included TruSeq Stranded mRNA with paired read ends. The read length was set to 50 and the multiplex level was 1.

[0192] RNA sequence alignment

[0193] The fastq files were aligned to the mouse reference genome mm10 using hisat2 and converted to bam files using samTOOLS. The read count matrix was generated from the bam files using the featurecounts function of the R package Rsubread.

[0194] Dimensionality reduction

[0195] Principal component analysis: The read count matrix was normalized to reads per million (RPM) and log-transformed, f(x) = (1 + log(x)). Principal components were calculated using the R function prcomp. Data were displayed using ggplot2. Heatmap: The read count matrix was normalized to RPM. When comparing gene expression under different conditions, each gene was normalized from 0 to 1 f(x_i) = (x_i - min(x_i)) / (max(x_i) - min(x_i). Where max(x_i) is the maximum value of gene i across all conditions (same for min(x_i)). When comparing gene expression across different cell types, each cell type was normalized from 0 to 1 f(x_i) = (x_i - min(x_i)) / (max(x_i) - min(x_i). Where max(x_i) is the maximum value of cell i among all genes.

[0196] RNA-seq differential expression

[0197] The differentially expressed genes were calculated using the R software package DESeq2. Genes with false discovery rate-adjusted p values ​​lower than 0.05 were considered statistically significant and were available for further analysis.

[0198] Enrichment analysis

[0199] Metascape was used to identify pathways associated with genes. When there were more than 2000 genes in the list, the top 2000 were ranked by fold change.

[0200] GalNAc siRNA construction and GalNAc siRNA injection

[0201] The following siRNA sequences were generated (synthetic terms: n = 2'OMe-RNA, Nf = 2'-fluoro-RNA, s = phosphorothioate):

[0202] siRNA targeting claudin-31:

[0203] ·Sense strand sequence (5′-3′):

[0204] csusAfcCfaGfcAfgUfcGfaUfgAfaAf(SEQ ID NO:1)

[0205] Antisense strand sequence (5′-3′):

[0206] UfsUfsuCfaUfcGfaCfuGfcUfgGfuAfgsusu(SEQ ID NO:5)

[0207] siRNA targeting claudin-32:

[0208] ·Sense strand sequence (5′-3′):

[0209] gsasAfaCfgGfgCfcAfuUfuCfaUfaAf(SEQ ID NO:2)

[0210] Antisense strand sequence (5′-3′):

[0211] UfsUfsaUfgAfaAfuGfgCfcCfgUfuUfcsusu(SEQ ID NO:6)

[0212] AHSA1 control siRNA

[0213] ·Sense strand sequence (5′-3′):

[0214] uscsUfcGfuGfgCfcUfuAfaUfgAfaAf(SEQ ID NO:3)

[0215] The above siRNAs were synthesized by Axolab, Kulmbach, Germany. All siRNAs were created from the mouse Cldn3 mRNA sequence (NM_009902.4). The bases within the siRNA were modified using 2′ fluorine and / or 2′ O-methyl residues and thiophosphate bonds to improve chemical stability. siRNA1 targeting tight junction protein-3 (sense strand sequence), siRNA2 targeting tight junction protein-3 (sense strand sequence) and AHSA1 control siRNA (sense strand sequence) were all coupled to a ternary GalNAc cluster (CPG, Primetech). siRNA was HPLC purified and lyophilized. For in vivo use, GalNAc siRNA was diluted with physiological saline solution. siRNA was injected subcutaneously at a concentration of 10 mg / KG body weight. Mice were pre-treated with siRNA two or four days before the start of the experiment.

[0216] result

[0217] Claudin-3 knockout resulted in impaired bile acid metabolism, dilution of bile, increased bile flow rate, and impaired uptake of the tracer FITC-dextran.

[0218] By analyzing published comparative Cldn3+ / + and Cldn3 - / - Using an RNAseq dataset of mice, we found that bile acid and bile salt metabolism was one of the top gene pathways with low expression levels in the absence of claudin-3. Figure 1 A). Differential gene expression analysis revealed that genes like Cyp27a1, which plays a key role in BA biosynthesis, were less expressed ( Figure 1 B) RT-qPCR confirmed the results for Cyp27a1 and showed Cldn3 + / + Cyp7b1 expression in the liver was also low ( Figure 1 C). It was wondered whether inhibited BA synthesis would affect the total bile acid (TBA) content in bile. + / + The average bile TBA level of mice was 4194 μmol / L, while Cldn3 - / - Bile only contained 3932μmol / L (P<0.01) ( Figure 1 D) Bile has been described to have the property of autofluorescence, which can be mainly attributed to the abundant presence of bilirubin. Bile autofluorescence was measured under the conditions of excitation wavelength of 489nm and emission wavelength of 534nm. Cldn3 + / + The relative fluorescence unit (RFU) of bile was 499.7 RFU / μl bile / g liver, while Cldn3 - / - The autofluorescence of bile was surprisingly reduced to only 14.4 RFU / μl bile / g liver (P=0.0079) ( Figure 1 E), which indicates that the bilirubin in the bile is diluted.

[0219] Next, we aimed to identify Cldn3 - / - Bile flow rate and uptake and paracellular permeability of the blood-bile barrier in mice. The experimental setup described previously was used. Briefly, common bile duct ligation (BDL) was performed and the gallbladder was cannulated ( Figure 1 F). Collect bile over a twenty-minute period to determine bile flow rate ( Figure 1 G) Discovery of Cldn3 - / - The bile flow rate of mice was faster, 3.7 μl / min / g liver, while Cldn3 + / + The bile flow rate of mice was only 0.8 μl / min / g liver (P=0.0036) ( Figure 1 G).

[0220] In the same setting, the paracellular barrier ( Figure 1H) It was found that from 3 minutes onwards, FITC-dextran was increasingly taken up and secreted into bile, however, in Cldn3 - / - In mice, the abundance of FITC-dextran was significantly lower at all times ( Figure 1 H). This suggests that the paracellular barrier of hepatocytes is not significantly impaired for the passage of FITC-dextran.

[0221] Next, we further demonstrated that Cldn3 - / - Hypothesis of impaired uptake of FITC-dextran (40 kDa) in mice. BDL was performed and FITC-dextran (40 kDa, 6.25 mg / ml, 300 μl volume) was injected via the tail vein. After laparotomy in anesthetized mice, FITC-dextran fluorescence was imaged continuously for 45 min using the Spectrum in vivo imaging system (PerkinElmer). To analyze FITC-dextran intensity in the liver, regions of interest were selected in the same area and FITC signal was determined using the instrument software. - / - Lower intensity FITC signals were observed in the liver ( Figure 1 I). Quantification of FITC signal over time showed that FITC-dextran was taken up by Cldn3. + / + The liver takes up rapidly, but Cldn3 - / - The liver takes up slowly ( Figure 1 J). Using the same experimental setup, livers were harvested 20 min after injection and 25 μm thick cryosections were prepared. Cryosections were stained with Dapi for 15 min and then imaged in confocal Z stacks (LSM 710; Zeiss, 20× objective, Oberkochen, Germany). Quantification of FITC fluorescence throughout the Z stack showed that Cldn3 was expressed 20 min after injection. - / - The uptake of FITC-dextran in the liver was significantly reduced (P=0.0049, n=3 / 4, Student's t-test).

[0222] Collectively, the data suggest that loss of claudin-3 impairs bile acid synthesis and results in dilution of total bile acid and bilirubin in bile. - / - Bile flow was faster in mice, and paracellular permeability between hepatocytes was not impaired when FITC-dextran was passed. - / - Hepatic uptake and bile secretion of FITC-dextran (40 kDa) were reduced.

[0223] Deficiency of claudin-3 protects against obstructive cholestatic liver injury

[0224] Since bile acids are considered to be the main cause of the pathobiology of cholestatic liver diseases 29 Next, we will discuss Cldn3 - / - Effect of bile dilution on cholestasis. First, an extrahepatic obstructive cholestasis model was used. + / + and Cldn3 - / - Mice were treated with BDL for two, seven, or twelve days. As expected, macroscopic photographs showed no differences from the control group. However, significant differences in the appearance of the liver were observed after BDL. Wild-type animals showed the expected BDL phenotype, including dark green bile and visible necrotic plaques in liver tissue ( Figure 2 A) Cldn3 - / - These features were absent in the β-catenin group, indicating a different bile composition and significantly improved necrosis.

[0225] Histological analysis of liver tissue showed no difference between the control groups, and the liver morphology was normal ( Figure 2 B). After BDL, wild-type animals developed tissue necrosis, a typical feature of obstructive liver injury ( Figure 2 B and Figure 2 C), and as cholestasis progressed, the severity of tissue necrosis increased. Surprisingly, mice lacking claudin-3 showed almost no necrosis ( Figure 2 B and Figure 2 C). Thus, in the absence of claudin-3, levels of clinical liver injury markers were significantly reduced. Seven days after BDL, Cldn3 + / + The average alanine aminotransferase (ALT) level of mice was 702 U / L, while Cldn3 - / - The alanine aminotransferase (ALT) level of mice was only 176U / L ( Figure 2 D) 7 days after BDL, Cldn3 + / + The aspartate aminotransferase (AST) level of mice was 882 U / L, while Cldn3 - / - The aspartate aminotransferase (AST) level of mice was only 242U / L ( Figure 2 E) 12 days after BDL, the levels of alkaline phosphatase (ALP) were significantly different, and Cldn3 + / + The average level in mice was 822.5 U / L, while Cldn3 - / - The average value for mice was 452.0 U / L ( Figure 2 F).

[0226] BDL has been previously described to cause bacterial translocation to the liver, an indicator of tissue damage. Liver tissue was homogenized after BDL, and the number of colony-forming units (CFU) was counted. Quantification results showed that Cldn3- / - The number of bacteria transferred to the liver was significantly lower than that in the wild-type liver (P = 0.01) ( Figure 2 G). 7 days after BDL, the number of CFU was similar in each group.

[0227] As a final indicator of inflammation and injury, the frequency of immune cells was quantified 7 days after BDL using fluorescence-activated cell sorting (FACS). The frequency of inflammatory monocytes was significantly lower; however, in cholestatic Cldn3 - / - The frequencies of cytoprotective and inflammation-resolving T cells were higher in the liver ( Figure 2 H). Cldn3 - / - The frequency of neutrophils in the liver also trended toward a decrease, with no differences observed in the other analyzed populations, including B cells and Kupffer cells.

[0228] Collectively, the results demonstrate that loss of claudin-3 leads to significant amelioration of inflammation and necrosis and protects mouse livers from injury in an obstructive cholestasis model.

[0229] Bile acid concentrations in liver and gallbladder bile are reduced after BDL

[0230] Next, we further explored the protective mechanism of claudin-3 deficiency. - / - The bile acid composition of mice was slightly altered. We hypothesized that a significant reduction in bile acid levels could explain the improvement in injury. Therefore, we analyzed the concentration and composition of hepatic bile acids in the BDL model.

[0231] 7 days after BDL, Cldn3 + / + Bile extracted from mouse gallbladder is yellow or dark green, while Cldn3 - / - The bile of mice is yellow ( Figure 3 A, Cldn3 - / - Bile from mice appears darker in the grayscale image. TBA levels in gallbladder bile collected 7 days after BDL were significantly increased in Cldn3 + / + In Cldn3 - / - Only 2611μmol / L (P<0.01) ( Figure 3 B) TBA in liver 2 days after BDL in Cldn3 + / + In liver, the average was 308.3 nmol / g, while in Cldn3 - / - In the liver, it was only 191.8 nmol / g (p<0.01) ( Figure 3 C). Accompanied by worsening liver damage ( Figure 2 ), after 7 days, liver TBA levels increased, Cldn3 + / + The average value of Cldn3 in mice was 447.2 nmol / g.- / - The average value of mice was 226.2 nmol / g (p<0.05) ( Figure 3 C).

[0232] Interestingly, the differences in TBA levels were reversed when serum was tested ( Figure 3 D) Two days after BDL, Cldn3 + / + The average serum TBA level in mice was 109.1 μmol / L, while Cldn3 - / - The mean serum TBA level of mice was 125.6 μmol / L (p<0.05) ( Figure 3 D) Serum TBA levels increased within 7 days, and the difference between the groups became larger. After 7 days, Cldn3 + / + The TBA content in mouse serum was 689.2 μmol / L, while Cldn3 - / - The TBA content in mouse serum was significantly higher, at 774.1 μmol / L (p<0.05) ( Figure 3 D). Notably, there were no significant differences in TBA in liver, serum, or bile at the 12-day time point ( Figure 3 B to Figure 3 D) The serum was further analyzed by checking bilirubin levels and found that Cldn3 - / - Mice had lower bilirubin levels 2 days after BDL but not at other time points ( Figure 3 E).

[0233] The effect of slightly elevated serum bile acid levels on Cldn3 - / - Potential negative effects on the kidneys of mice. The number of bile casts (also called "bile casts") in the kidneys was quantified, and no Cldn3 was found seven days after BDL. + / + and Cldn3 - / - There are differences between mice ( Figure 3 F).

[0234] In conclusion, the data suggest that bile is also diluted in claudin-3-deficient animals after induction of cholestasis. Total bile acid levels were found to be significantly lower in the liver and gallbladder, but higher in serum. A reduction in bile acid-associated cytotoxicity in the hepatobiliary system may explain the observed improvement in liver injury.

[0235] Claudin-3 deficiency does not significantly alter the composition of bile acid pools under normal or cholestatic conditions

[0236] During cholestasis, increased bile hydrophobicity may be an important factor in the development of tissue damage, depending on the composition of bile acid (BA) isoforms. We questioned whether claudin-3-deficient mice have different BA pools in the presence or absence of cholestasis. Therefore, we measured the concentrations of individual BA types in the liver and serum of control animals using liquid chromatography-mass spectrometry (LC-MS / MS) 7 days after BDL. Figure 4 and Table 1). It was observed that the conjugated bile acids T-αMCA, T-βMCA, T-ωMCA and taurocholic acid (TCA) were the most abundant bile acid species in the liver bile acid pool ( Figure 4 A to Figure 4 C and Table 1). Bile acid amounts are reported in nanograms per milligram of liver tissue, and the results show that liver bile acid levels increased dramatically after 7 days of BDL ( Figure 4 A). LC-MS / MS analysis further confirmed the previously observed expression of Cldn3 seven days after BDL. - / - Decreased levels of hepatic bile acids in the liver ( Figure 3 C and Figure 4 A).

[0237] Next, we calculated the percentage of bile acid isoforms in the entire liver bile acid pool. + / + and Cldn3 - / - There are only minor differences in the bile acid isoforms, such as in Cldn3 - / - The percentage of T-αMCA in the liver tissue of control mice was slightly lower ( Figure 4 B and Table 1). Seven days after BDL, Cldn3 + / + and Cldn3 - / - The data showed that the diversity of the entire liver bile acid pool was reduced seven days after BDL compared with the control group, and the major bile acid subtypes were more abundant ( Figure 4 B and Figure 4 C and Table 1).

[0238] Next, serum bile acids were analyzed. Bile acids were quantified in μmol / L and the results showed that Cldn3 - / - The level of bile acid in the serum of mice was significantly higher ( Figure 4 D), which confirms previous results ( Figure 3 D) Calculate the percentage of bile acid isoforms in the entire bile acid pool and find that Cldn3 + / + and Cldn3 - / - There were no differences between control sera ( Figure 4 E and Table 1). However, seven days after BDL, Cldn3 was observed - / -Slight differences in serum, such as slightly higher percentages in T-ωMCA and slightly lower percentages in GCA, αMCA, and CDCA ( Figure 4 F and Table 1). Compared with the control group, the diversity of serum bile acid components decreased 7 days after BDL ( Figure 4 E and Figure 4 F). Changes in the diversity of serum bile components were observed, promoting TCA and T-βMCA, as previously seen in liver tissue ( Figure 4 B and Figure 4 C).

[0239] Taken together, analyses of liver and blood bile acids confirmed previous findings that Cldn3 - / - The mice had decreased liver bile acid levels and increased blood bile acid levels. + / + and Cldn3 - / - No major differences in bile acid pool composition were found when comparing liver or serum.

[0240]

[0241]

[0242]

[0243] Table 1 Cldn3 before BDL (control) and after BDL + / + and Cldn3 - / - Bile acid pool composition in the liver.

[0244] Cholestatic Cldn3 - / - Lower expression of BA synthesis and import genes in the liver

[0245] It was observed that before and after BDL, Cldn3 - / - Bile is diluted and contains fewer BAs, raising the question of whether the synthesis and / or transport of BAs is altered. + / + and Cldn3 - / - The transcriptome of liver tissue was sequenced and RT-qPCR was performed for key genes to answer this question. Differential gene expression analysis showed that Cldn3 - / - In the liver, 168 genes were up-regulated and 255 genes were down-regulated (P<0.05) ( Figure 5 A).

[0246] We first performed a Metascape analysis to provide an overview of the Cldn3 + / + and Cldn3 - / -The most significant gene pathways were found in Cldn3 (Table 3 and Table 4). - / - Pathways with higher expression in the liver included those related to metabolism, such as amino acid catabolism, fatty acid transport, and carnitine metabolism. Interestingly, the cholestasis-protective PPAR signaling pathway 2,11,13 In Cldn3 - / - The expression level in the liver was also high (Table 3). Figure 2 B to Figure 2 H), we found that several gene pathways related to inflammation and immune cell response were upregulated in Cldn3 after BDL - / - Expression in the liver was significantly lower. This included leukocyte migration and adhesion, reactive oxygen species biosynthesis, tumor necrosis factor biosynthesis, and neutrophil degranulation, which were less expressed in the absence of claudin-3 (Table 4). Importantly, the analysis showed that bile acid and bile salt metabolism is a Cldn3 - / - One of the top pathways with low expression in mice (Table 4). Key low-expression bile synthesis genes include Cyp7b1, Cyp27a1, Cyp3a11, Akr1b7, Akr1c6, etc. ( Figure 5 A). This finding was confirmed by qPCR of Cyp7b1 and Cyp27a1, which were expressed in Cldn3 in control and 2 days after BDL. - / - The expression level in liver was significantly lower ( Figure 5 B) Cyp7a1 showed a trend of lower expression only 2 days after BDL. Cholesterol, a precursor of bile acid, is expressed in Cldn3 - / - There were no differences in blood levels in mice ( Figure 5 C).

[0247]

[0248] Table 3 Two days after BDL, Cldn3 - / - Gene pathways upregulated in liver

[0249]

[0250]

[0251] Table 4 Two days after BDL, Cldn3 - / - Gene pathways downregulated in liver

[0252] The expression of BA transporters was also examined. As expected, in wild-type animals, the expression of the basolateral BA importers Oatp1a1, Oatp1b2, and Ntcp was downregulated after BDL, whereas Cldn3 was upregulated 2 days after BDL. - / -Oatp1a1 / Oatp1b2 in the liver was significantly reduced ( Figure 5 D) The basolateral BA export protein Ost1-β is expressed in the control Cldn3 - / - The expression of β-catenin in liver was higher, which confirmed our previous findings. 27 The expression of Ost1-β was strongly upregulated after BDL, which was in turn upregulated in Cldn3 + / + and Cldn3 - / - A similar phenomenon occurs in the liver ( Figure 5 E) The apical anion and bile acid exporter Mpr3 is expressed in Cldn3 at 7 days after BDL. + / + Slightly upregulated in liver, but not in Cldn3 - / - No upregulation in liver ( Figure 5 E). Finally, the apical bile phospholipid exporter Mdr2 was not significantly regulated overall after BDL ( Figure 5 E).

[0253] Overall, gene expression analysis confirmed lower inflammation and indicated less expression of BA synthesis genes, consistent with our observations of impaired uptake of bile acid size tracers ( Figure 1 H to Figure 1 L), found Cldn3 - / - Impaired expression of bile acid importin proteins in the liver.

[0254] Cldn3 - / - Increased periductal edema in mice

[0255] To understand Cldn3 - / - The improvement of cholestatic injury in mice was described in more detail in the histological morphology of wild-type and claudin-3-deficient mice after BDL. 27 , and loss of claudin-3 has been previously associated with impaired paracellular water barriers 30-31 Hematoxylin and eosin-stained liver sections were scored for edema in the periductal area. + / + The mean periductal edema score of the animals was 2.8, and Cldn3 - / - The average periductal edema score of the animals was 6 (P < 0.05) ( Figure 6 A. Figure 6 B). It was also observed that ductal edema was more severe in the knockout mice at the seven-day time point. Edema scores were highest at 12 days after BDL, but there were no significant differences between the groups.

[0256] Taken together, histological analysis showed that Cldn3 - / -The liver showed increased periductal edema after BDL, which may be due to water barrier leakage.

[0257] Cldn12 - / - Animals are not immune to cholestatic liver damage

[0258] The next question to ask was whether protection from cholestasis could also be achieved by knocking out another tight junction protein family member. Cell type-specific expression of hepatic tight junction genes was recently described and Cldn12 was found to be expressed in mouse hepatocytes. 27 Therefore, C57BL / 6 mice deficient in global claudin-12 were treated with BDL. + / + and Cldn12 - / - The liver also showed gallbladder enlargement and darkening in color 2 days after BDL ( Figure 7 A). Quantification of the area of ​​tissue necrosis showed that the extent of tissue necrosis was similar in both groups ( Figure 7 A, Figure 7 B) Finally, Cldn12 - / - There were no changes in liver or serum TBA concentrations in mice ( Figure 7 C and Figure 7 D) These results suggest that loss of claudin-12 does not result in alterations in bile acid levels or ameliorate cholestatic liver injury.

[0259] Claudin-3 deficiency prevents intrahepatic cholestasis

[0260] The favorable results in the BDL model raised the question whether loss of claudin-3 would also be beneficial in intrahepatic cholestatic diseases. Therefore, we used the established oral α-naphthalene isothiocyanate (ANIT) model to investigate the expression of Cldn3 - / - Mice were studied.

[0261] First, an acute intrahepatic cholestasis model was tested. Mice were injected with a single dose of ANIT [60 mg / kg body weight], and tissues were analyzed two days later ( Figure 8 A). In wild-type mice, the expected cholestatic liver injury phenotype was induced, manifested by macroscopic and microscopic tissue necrosis ( Figure 8 B to Figure 8 D) high levels of clinical liver injury markers ( Figure 8 E) and elevated levels of bilirubin and bile acids in tissues and serum ( Figure 8 E and Figure 8 F). Strikingly, Cldn3 - / - There is no tissue necrosis in the liver ( Figure 8 B to Figure 8 D) Cldn3- / - Bile in the mouse gallbladder is a normal light yellow color (appears lighter / white in grayscale), whereas Cldn3 + / + The bile of mice is dark green ( Figure 8 B, appears dark / black in grayscale). Cldn3 - / - The blood ALT, ALP and bilirubin levels of the animals were significantly reduced (P<0.05), and the AST level also showed a downward trend ( Figure 8 E) Evidence for the absence of cholestatic injury also includes significantly lower BA levels. + / + The average total BA in the liver was 188 nmol / g, while Cldn3 - / - In the liver, it was only 87 nmol / g (P<0.001) ( Figure 8 F) The differences in serum BA levels were also striking, Cldn3 + / + The average level in mice was 412 μmol / L, while Cldn3 - / - The mice were only 46 μmol / L (P<0.0001) ( Figure 8 F).

[0262] We questioned whether ANIT is involved in the regulation of Cldn3 + / + and Cldn3 - / - As GSH is metabolized in a similar manner in the liver, the expression of genes involved in glutathione metabolism and drug handling, glutathione S-transferase α1 (Gsta1) and Cyp4a12, were examined. As expected, Gsta1 was upregulated 2 days after ANIT, but not in the presence of Cldn3. - / - Less up-regulated in liver ( Figure 8 G). In response to ANIT, Cyp4a14 was similarly downregulated in both groups. As a manifestation of negative feedback to cholestasis, the key BA synthase Cyp7b1 is expressed in Cldn3 + / + However, this was not the case in the absence of claudin-3, indicating less bile acid-induced feedback and showing protection against cholestasis ( Figure 8 G). To examine whether loss of claudin-3 could also prevent fibrosis induced by long-term intrahepatic cholestasis, mice were given two consecutive doses of ANIT for a total of 10 days ( Figure 8 H) Quantification of collagen intensity in Masson's trichrome-stained liver sections showed that Cldn3 - / - The liver contains significantly less collagen than wild-type mice ( Figure 8 I and 8J).

[0263] Finally, the protective effect of claudin-3 deficiency during chronic intrahepatic cholestasis was tested using a 4-week ad libitum model of 0.1% ANIT ( Figure 8 K). The control group of mice was fed a diet containing an equal amount of corn oil. As expected, the control group of mice never developed any tissue necrosis, and clinical markers of injury and bilirubin / cholesterol levels remained normal ( Figure 8 L to Figure 8 O) 4 weeks after ANIT feeding, Cldn3 + / + Mice showed obvious signs of tubular reaction and tissue necrosis, while Cldn3 - / - This did not occur in the organization ( Figure 8 L). Quantification of tissue necrosis showed that Cldn3 + / + The average liver necrosis area was 0.24%, while Cldn3 - / - The liver was only 0.066% (P<0.001) ( Figure 8 M) ALP levels as a clinical marker of cholestatic liver injury in ANIT-fed Cldn3 + / + In mice, it is 448 U / L, but in the corresponding Cldn3 - / - In the group, only 211 U / L (P<0.01) Figure 8 N). There were no differences in AST and ALT levels between the groups fed the ANIT diet ( Figure 8 N). Although there was no difference in bilirubin levels, Cldn3 - / - The serum cholesterol level was significantly lower (P<0.05) ( Figure 8 O).

[0264] Next, the amount of fibrotic liver tissue was quantified by Sirius red staining. Mice fed corn oil showed no fibrosis, as evidenced by minimal Sirius red stained tissue ( Figure 8 P) Cldn3 fed with 0.1% ANIT for 4 weeks + / + Mice developed fibrosis, as evidenced by abundant collagen deposition in Sirius red-stained sections, and Cldn3 - / - This was not the case in mice ( Figure 8 P). Quantification of Sirius Red signal ( Figure 8 Q) confirmed that Cldn3 + / + Compared with mice fed ANIT, Cldn3 - / - The mice did not develop any liver fibrosis as indicated by a significantly lower amount of Sirius Red signal (P = 0.0018, Student's t-test).

[0265] Overall, experiments using acute and chronic intrahepatic cholestasis models suggest that Cldn3- / - Protected from cholestatic liver injury and cholestasis-related fibrosis.

[0266] GalNAc siRNA targeting claudin-3 modestly improves cholestatic injury

[0267] In Cldn3 - / - Promising results obtained in mice suggest that targeting claudin-3 may facilitate the treatment of cholestasis. For proof of concept, claudin-3 was knocked down (KD) using GalNAc siRNA. The advantages of this technology are that it produces a stable target KD, can be administered subcutaneously, targets only liver hepatocytes, and is already used in the clinic. 33 . Twelve candidate siRNAs were first screened in vitro using mouse primary hepatocytes. siRNA targeting heat shock protein ATPase 1 activator (AHSA1) (SEQ ID NO:3, positive strand) was used as a negative control in subsequent experiments. Western blotting showed that candidate compounds 9 and 12 produced strong tight junction protein-3 KD when we treated cells at a concentration of 50 nmol. Therefore, candidate 9 (referred to as "siRNA1" from here on; SEQ ID NO:1, positive strand) and candidate 12 (referred to as "siRNA2" from here on; SEQ ID NO:2, positive strand) continued to be used in the obstructive cholestasis model using BDL technology.

[0268] C57BL / 6 wild-type mice were subcutaneously injected with 10 mg / kg body weight of GalNAc siRNA1 or GalNAc siRNA2. BDL was performed two days after injection. Tissues were then collected two days after surgery and analyzed for tight junction protein-3 levels and cholestatic liver injury markers ( Fig. 9 A). The in vivo efficacy of siRNA for claudin-3 KD is approximately 40% to 50% at both the mRNA and protein levels, but individual efficacy varies widely ( Fig. 9 B and Fig. 9 C). However, claudin-3 KD resulted in a significant decrease in ALP ( Fig. 9 D and Table 5). The ALP level of BDL control mice averaged 1582 U / L, while the average ALP levels of animals receiving siRNA1 or siRNA2 were 713 U / L and 866 U / L, respectively (P<0.01 and P<0.05, respectively) ( Fig. 9 D and Table 5). Similarly, ALT and AST levels were also reduced compared with the control group ( Fig. 9 D and Table 5). Representative H&E-stained liver tissue sections confirmed that tissue damage was improved in the claudin-3 knockout group, but the effect was not significant and the variability between individuals was still high ( Fig. 9 E).

[0269]

[0270] Table 5 Serum markers of cholestasis and liver injury after 2 days of BDL. P values ​​were calculated by comparing the corresponding condition in the first row with the BDL control group (n=3 for BDL control group and non-surgical control group, n=4 for BDL plus siRNA group, Student's t test)

[0271] Next, the most promising siRNA candidate, siRNA1, was tested in the intrahepatic cholestasis model using ANIT. Four days before oral gavage of ANIT [60 mg / kg body weight], control siRNA AHSA1 or siRNA1 targeting claudin-3 ( Fig. 9 F). Two days after ANIT gavage, tissues and serum were collected to quantify liver damage. It was found that the level of ALP, a major clinical blood marker of cholestasis, was significantly reduced in animals treated with anti-claudin-3 siRNA1 ( Fig. 9 G and Table 6). Thus, liver tissue histology of animals receiving anti-claudin-3 siRNA1 showed less necrosis compared to AHSA1 control siRNA ( Fig. 9 H).

[0272]

[0273] Table 6 Serum markers of cholestasis and liver injury after 2 days of ANIT. P values ​​were calculated by comparing the corresponding conditions with the ANIT AHSA1 control siRNA group in the first row (ASHA control n=3, ANIT siRNA1 n=5, no ANIT control n=4, Student's t-test)

[0274] In summary, it was shown that targeted GalNAc siRNA knockdown of claudin-3 could improve extrahepatic or intrahepatic cholestatic liver injury.

[0275] Screening, synthesis and evaluation of siRNA targeting human claudin-3

[0276] As a first step to apply these findings to human patients, the siRNA sequences that can effectively knock out human tight junction protein-3 were screened. In the bioinformatics preselection (Axolabs, Kulmbach, Germany), the target specificity, intra-species and inter-species cross-reactivity and activity of the siRNA were scored. The best siRNA candidates were then synthesized (Axolabs, Kulmbach, Germany). The siRNA sequences contained chemically stable 2'-fluoro- or 2'O-methyl modifications or thiophosphate linkers at the positions shown in Table 6.

[0277] Next, the efficacy of the synthetic siRNA candidates to knock down human claudin-3 was evaluated, which was cultured in the human liver cell line PLC (ATCC Cat.No CRL-8024). Human cell cultures were treated with siRNA at a final concentration of 50nm and RNA was extracted two days after treatment. Finally, RT-qPCR was used to quantify the amount of human claudin-3 mRNA remaining after siRNA treatment. Table 6 lists all human siRNA sequences that knocked down human claudin-3 mRNA by equal to or greater than 50%. Table 7 shows the same identified human claudin-3 siRNA sequences, but without their chemical stabilization modifications.

[0278]

[0279]

[0280] Table 6 siRNA sequences that knock down 50% or more of human claudin-3 mRNA in in vitro cell culture experiments. The experiment was performed twice with comparable results.

[0281]

[0282]

[0283] Table 7 siRNA sequences that knock down 50% or more of human Claudin-3 mRNA, without modification.

[0284] The experiment was performed twice with comparable results. In total, 34 siRNA sequences were identified that knocked down human claudin-3 with an efficacy equal to or greater than 50%. Following the typical drug development process, the best performing siRNAs will be further used for confirmation and risk reduction studies.

[0285] Elimination of human claudin-3 protein using monoclonal antibodies

[0286] To demonstrate that human Claudin-3 inhibition can be achieved by another pharmaceutical approach, human Claudin-3 antibodies conjugated to GalNAc were generated and functionalized (light chain as shown in SEQ ID NO: 169, heavy chain as shown in SEQ ID NO: 170). Our Claudin-3 antibodies were able to effectively bind to human Claudin-3 in a hepatic cell line and inhibit the target in a dose-dependent manner, as described in detail below.

[0287] Binding of anti-claudin-3 antibody UB-VS003 to human and mouse cells

[0288] Fig.10 Binding analysis of the anti-Claudin-3 antibody UB-VS003 against human SNU449 cells and hepatocytes as well as mouse Hepa1-6 cells is shown. The results show a consistent trend of antibody binding across all cell types.

[0289] For human SNU449 cells and hepatocytes, the control samples showed no detectable binding (0%). However, as the antibody concentration increased, a gradual increase in binding was observed. At a dilution of 1 / 400, the binding percentage was approximately 3%. This concentration increased to 7% at a dilution of 1 / 200, 12% at a dilution of 1 / 100, and reached a maximum of 25% at a dilution of 1 / 50.

[0290] For mouse Hepa1-6 cells, no binding was observed in the control sample or at a 1 / 400 dilution. However, the binding rate increased significantly with increasing antibody concentration. At a 1 / 200 dilution, approximately 5% of the cells showed binding, which further increased to 7% at a 1 / 100 dilution and reached a maximum of 10% at a 1 / 50 dilution.

[0291] In addition, hepatocytes from wild-type (WT) mice and Claudin-3 knockout (KO) mice were compared. The background signal (without antibody) was similar between WT and KO hepatocytes, accounting for approximately 2 / 3% of the total cells. After adding anti-Claudin-3 antibody, the background signal in KO hepatocytes increased to 3%, while in WT hepatocytes, the signal increased to 10%.

[0292] Overall, these results indicate that anti-Claudin-3 antibodies bind specifically to both human and mouse cells and that the binding rate increases in a dose-dependent manner. In addition, comparison between WT and KO hepatocytes indicated that UB-VS003 antibodies have Claudin-3-dependent binding.

[0293] GalNAc-functionalized UB-VS003 induces claudin-3 knockdown.

[0294] Fig.11 Western blot analysis is shown depicting the expression levels of Claudin-3 and β-actin as loading controls. The ratio of Claudin-3 to β-actin was quantified to assess the relative abundance of Claudin-3 protein.

[0295] The ratio of claudin-3 to β-actin in the control sample was set to 100%, indicating that claudin-3 is fully expressed under normal conditions. After treatment with GalNAc-anti-claudin-3 antibody for 24 hours, a dose-dependent decrease in the ratio of claudin-3 / β-actin was observed, reflecting the reduction in claudin-3 protein levels.

[0296] When the concentration of GalNAc anti-claudin-3 antibody was 1 nM, the claudin-3 / β-actin ratio decreased to about 45% compared to the control sample. This reduction became more pronounced as the antibody concentration was further increased. At 30 nM, the ratio dropped to about 30%, and at the highest concentration tested, 100 nM, the ratio dropped to about 10%.

[0297] These findings demonstrated that the addition of GalNAc-anti-Claudin-3 antibody resulted in a dose-dependent decrease in the expression level of Claudin-3 protein, suggesting that the functionalized antibody effectively triggered the lysosomal degradation of Claudin-3 in human hepatocytes.

[0298] Taken together, human claudin-3GalNAc siRNA as well as human claudin-3GalNAc antibody UB-VS003 can be effectively used to deplete human claudin-3. Both modalities will now be further applied in preclinical and clinical studies with the aim of treating cholestasis and / or cholestasis-associated fibrosis.

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Claims

1. An agent for inhibiting the expression and / or activity of claudin-3, wherein the agent is used for a method for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis.

2. The medicament for use according to claim 1, wherein: The agent that inhibits the expression and / or activity of Claudin-3 is siRNA targeting Claudin-3 or an antibody or a fragment thereof that binds to Claudin-3.

3. The medicament for use according to claim 1, wherein The agent inhibits the expression of claudin-3.

4. The medicament for use according to claim 3, wherein: The agent that inhibits the expression of Claudin-3 is a nucleic acid that targets a gene or mRNA encoding Claudin-3.

5. The medicament for use according to claim 3, wherein: The agent that inhibits the expression of Claudin-3 is siRNA targeting Claudin-3.

6. The medicament for use according to claim 2 or 5, wherein: The siRNA targeting Claudin-3 is coupled to a compound that facilitates delivery of the siRNA to the liver.

7. The medicament for use according to claim 2 or 5, wherein: The siRNA targeting Claudin-3 is an N-acetylgalactosamine (GalNAc) siRNA conjugate.

8. The medicament for use according to any one of claims 5 to 7, wherein The siRNA is selected from the group consisting of: an siRNA comprising a sequence as shown in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:33 to SEQ ID NO:168, or an siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity to an siRNA comprising a sequence as shown in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:33 to SEQ ID NO:

168.

9. The medicament for use according to any one of claims 5 to 7, wherein: The siRNA is selected from the group consisting of: an siRNA comprising a sequence as shown in any one of SEQ ID NO:33 to SEQ ID NO:168, or an siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity to an siRNA comprising a sequence as shown in any one of SEQ ID NO:33 to SEQ ID NO:

168.

10. The medicament for use according to any one of claims 5 to 7, wherein: The siRNA is selected from the group consisting of: an siRNA comprising a sequence as shown in any one of SEQ ID NO:33 to SEQ ID NO:100, or an siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity to an siRNA comprising a sequence as shown in any one of SEQ ID NO:33 to SEQ ID NO:

100.

11. The medicament for use according to any one of claims 5 to 7, wherein: The siRNA is selected from the group consisting of: an siRNA comprising a sequence as shown in SEQ ID NO:1; an siRNA comprising a sequence as shown in SEQ ID NO:2; an siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity to the siRNA comprising the sequence as shown in SEQ ID NO:1; and an siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity to the siRNA comprising the sequence as shown in SEQ ID NO:

2.

12. The medicament for use according to claim 1, wherein: The agent inhibits the activity of claudin-3.

13. The medicament for use according to claim 12, wherein: The agent that inhibits the activity of Claudin-3 is selected from the group consisting of an antibody or a fragment thereof that binds to Claudin-3 and a toxin that binds to Claudin-3.

14. The medicament for use according to claim 13, wherein: The toxin that binds to Claudin-3 is Clostridium perfringens enterotoxin (CPE) or a fragment or variant thereof.

15. The medicament for use according to claim 12, wherein: The agent that inhibits the activity of Claudin-3 is an antibody or a fragment thereof that binds to Claudin-3.

16. The medicament for use according to claim 15, wherein: The antibody or fragment thereof that binds to Claudin-3 comprises a light chain and / or a heavy chain, wherein the light chain comprises the amino acid sequence shown in SEQ ID NO: 169, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO:

170.

17. The medicament for use according to claim 15 or 16, wherein The antibody or fragment thereof that binds to Claudin-3 is coupled to a compound that facilitates delivery of the antibody to the liver.

18. The medicament for use according to claim 15 or 16, wherein The antibody or fragment thereof that binds to Claudin-3 is an N-acetylgalactosamine (GalNAc) antibody conjugate.

19. The medicament for use according to any one of claims 1 to 18, wherein Cholestasis is a disease selected from the group consisting of obstructive cholestasis, non-obstructive cholestasis, bile duct disease and biliary dysfunction.

20. A composition comprising the agent according to any one of claims 1 to 18 and an optional pharmaceutically acceptable carrier, for use in a method for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis.

21. A dosage form for the prevention, delay of progression or treatment of cholestasis and / or fibrosis associated with cholestasis, the dosage form comprising the agent according to any one of claims 1 to 18 or the composition comprising the agent according to claim 20, and optionally a pharmaceutically acceptable carrier.

22. An siRNA targeting claudin-3.

23. An siRNA targeting tight junction protein-3, wherein the siRNA is selected from the group consisting of: an siRNA comprising a sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6 or any one of SEQ ID NO:33 to SEQ ID NO:168, or an siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity with an siRNA comprising a sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6 or any one of SEQ ID NO:33 to SEQ ID NO:

168.

24. An siRNA targeting tight junction protein-3, wherein the siRNA is selected from the group consisting of: an siRNA comprising a sequence as shown in any one of SEQ ID NO:33 to SEQ ID NO:168, or an siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity with an siRNA comprising a sequence as shown in any one of SEQ ID NO:33 to SEQ ID NO:

168.

25. An siRNA targeting claudin-3, wherein the siRNA is selected from the group consisting of an siRNA comprising a sequence as shown in any one of SEQ ID NO:33 to SEQ ID NO:100 or an siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity with an siRNA comprising a sequence as shown in any one of SEQ ID NO:33 to SEQ ID NO:

100.

26. A siRNA targeting claudin-3, wherein the siRNA is selected from the group consisting of: an siRNA comprising the sequence shown in SEQ ID NO:1; an siRNA comprising the sequence shown in SEQ ID NO:2; an siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity with the siRNA comprising the sequence shown in SEQ ID NO:1; and an siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity with the siRNA comprising the sequence shown in SEQ ID NO:

2.

27. An antibody or fragment thereof that binds to Claudin-3.

28. The antibody or fragment thereof according to claim 27, wherein The antibody or fragment thereof that binds to Claudin-3 is a human antibody.

29. The antibody or fragment thereof according to claim 27 or 28, wherein The antibody or fragment thereof that binds to Claudin-3 comprises a light chain and / or a heavy chain, wherein the light chain comprises the amino acid sequence shown in SEQ ID NO: 169, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO:

170.

30. The antibody or fragment thereof according to any one of claims 27 to 29, wherein The antibody or fragment thereof that binds to Claudin-3 is coupled to a compound that facilitates delivery of the antibody to the liver.

31. The antibody or fragment thereof according to any one of claims 27 to 29, wherein The antibody or fragment thereof that binds to Claudin-3 is an N-acetylgalactosamine (GalNAc) antibody conjugate.

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