Treatment of atherosclerosis, primary biliary cirrhosis and NRLP3 inflammasome-associated disease by NTCP inhibitors

Abstract

To provide treatment of atherosclerosis, primary biliary cirrhosis and NRLP3 inflammasome-associated disease by NTCP inhibitors.SOLUTION: The present invention relates to novel therapy of atherosclerosis, primary biliary cirrhosis and NRLP3 inflammasome-associated disease. The invention provides an inhibitor of Na+-taurocholate cotransporting polypeptide (NTCP) for use in a method of treatment of primary biliary cirrhosis, atherosclerosis, or an NRLP3 inflammasome-associated disease in a subject.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a novel method for treating atherosclerosis, primary biliary cirrhosis, or NRLP3 inflammasome-related diseases.In particular, the present invention provides an NTCP inhibitor, preferably a pre-S1 peptide inhibitor, and a composition comprising the same for treating said diseases.A method for treating said diseases with an NTCP inhibitor, preferably a pre-S1 peptide inhibitor, is also provided. [Background technology]

[0002] [Atherosclerosis] Atherosclerosis, also known as arteriosclerotic vascular disease or ASVD, is a disease associated with the infiltration and accumulation of leukocytes, particularly macrophages, and subsequent intimal proliferation and the appearance of plaque.

[0003] Atherosclerosis is initiated by the accumulation of low-density lipoprotein (LDL) in the intima of arteries, and elevated LDL cholesterol is a major risk factor for cardiovascular disease. HMGCoA reductase inhibitors (statins) are widely used to lower plasma LDL cholesterol levels and reduce the incidence of major vascular events and cardiovascular death. In addition to statins, research over the past decades has led to the development of many drugs that affect cholesterol homeostasis. For example, ezetimibe inhibits the action of Niemann-Pick C1-like 1 (NPC1L1), a protein on the intestinal brush border membrane that plays a key role in cholesterol absorption. Bile acid sequestrants inhibit cholesterol absorption as well as bile acid reabsorption. On the other hand, high-density lipoprotein (HDL) cholesterol levels are inversely correlated with disease risk, and HDL mediates reverse cholesterol transport and exhibits antioxidant activity. Pharmacological therapies attempting to raise HDL cholesterol levels or improve HDL function have been investigated, but so far with limited success (Shihet al., 2013).

[0004] Despite the availability of therapeutic options, there remains an unmet medical need in the treatment of atherosclerosis, especially the reduction of existing plaque burden in patients with advanced symptomatic atherosclerosis remains an unsolved problem.

[0005] [Primary biliary cirrhosis] Primary biliary cirrhosis, also known as primary cholangitis (PBC), is a chronic cholestatic liver disease of unknown etiology. Progressive bile duct damage due to portal and periportal inflammation can lead to progressive fibrosis and ultimately cirrhosis. Evidence to date suggests that immunological and genetic factors play a role in the disease. Affected individuals are typically middle-aged women with asymptomatic elevations in serum liver biochemical variables. Initial symptoms of fatigue, pruritus, or unexplained hyperlipidemia may suggest a diagnosis of primary biliary cirrhosis.

[0006] Current evidence supports the concept of primary biliary cirrhosis being an immune-mediated disease. Both cellular and humoral abnormalities have been noted. Immunohistochemical staining of T lymphocytes in portal and periportal areas shows CD4- and CD8-positive T cells. Furthermore, abnormal suppressor T cell activity has been reported in asymptomatic first-degree relatives of patients. Intracellular adhesion molecules (e.g., ICAM-1), expressed in areas of epithelial cell damage, may also be involved in the pathogenesis of primary biliary cirrhosis (Talwalkar & Lindor, 2003). It has also been observed that interleukin-1β (IL-1β) plays a role in the pathogenesis of PBC by contributing to altered immune function and fibrosis (Mueller et al, 1995).

[0007] The only currently approved treatment for PBC is bile acid therapy with ursodeoxycholic acid (UDSA) and obeticholic acid (OCA). The mechanism of action of both drugs in PBC is associated with their ability to activate FXR and TGFR-5, thus leading to the exertion of anti-inflammatory effects. UDCA was approved by the FDA for the treatment of PBC in 1997. However, approximately 40% of patients treated with UDCA do not achieve an adequate biochemical response.

[0008] For the recently approved OCA, 90% of patients enrolled in the pivotal trial had early PBC with baseline normal total bilirubin (TB) and normal albumin values, but the majority of patients enrolled in the trial were treated with UDCA. The primary endpoint was prespecified as a reduction in both alkaline phosphatase (ALP) and TB, but due to the nature of the enrolled population, the primary endpoint was determined to be ALP alone. In a 12-month phase 3 clinical trial, a total of 46% of patients in the OCA titration group and 47% of patients in the OCA 10 mg group achieved the primary endpoint (mainly due to a reduction in ALP) compared with 10% of patients in the placebo group.

[0009] Thus, there is a significant medical need in the treatment of PBC, particularly for patients with advanced disease.

[0010] [NRLP3 inflammasome-related diseases and NASH] Inflammasomes are cytosolic multimeric protein complexes consisting of nucleotide-binding domains and leucine-rich repeat-containing proteins (NLRs) or AIM2, the adaptor protein ASC, and caspase-1. They play an important role in host defense against pathogens and inflammation (Davis et al., 2011; Martinon et al., 2006). ). Activation of inflammasomes is involved in the pathogenesis of several inflammatory disorders, including type 2 diabetes, atherosclerosis, gout, and Alzheimer's disease. In particular, accumulating data strongly indicate that type 2 diabetes is an inflammatory disease. The influx of macrophages and other immune cells due to chronic caloric excess and the subsequent amplified proinflammatory environment play a key role in B cell dysfunction and insulin resistance. Clinically, elevated circulating IL-1β is a risk factor for the development of type 2 diabetes in humans. Mouse models and human clinical trials suggest that antagonism of IL-1β is a promising treatment for type 2 diabetes (Schroder,etal., 2010; Guo et al., 2016).

[0011] It is well known that bile acids bind to and activate several nuclear receptors, such as the farnesoid X receptor (FXR). FXR is highly expressed in the liver and intestine and regulates the transcription of specific target genes involved in bile acid, lipid and glucose metabolism. Recent studies have revealed that bile acids also serve as ligands for the G protein-coupled receptor TGR5 (or G protein-coupled bile acid receptor 1). TGR5 is expressed in various tissues and cell types, with relatively high expression in the gallbladder, intestine, placenta and spleen. Previous in vitro studies have shown that activation of TGR5 suppresses proinflammatory cytokine production and phagocytosis of monocytes / macrophages. In vivo studies have shown that the immunosuppressive properties of TGR5 also include a protective role in metabolic diseases such as atherosclerosis and diabetes, where tissue infiltration and activation of immune cells are hallmarks of their initiation and development. The anti-inflammatory and anti-fibrotic properties of FXR and TGR5 signaling can be successfully used in the treatment of non-alcoholic steatohepatitis (NASH). Summary of the Invention

[0012] The present invention provides an NTCP inhibitor for use in a method for treating primary biliary cirrhosis (PBC) in a patient. Also provided is a method for treating primary biliary cirrhosis in a patient by providing an effective amount of an NTCP inhibitor to a patient in need thereof. It is envisaged that administration of an NTCP inhibitor will result in improvement of primary biliary cirrhosis, including normalization of biochemical parameters and reduction of inflammation and fibrosis progression. The NTCP inhibitors used according to the present invention can be used individually or together and / or as a combination of drugs for the treatment of primary biliary cirrhosis. It can be administered to the patient in combination with other drugs.

[0013] The present invention further provides an NTCP inhibitor for use in a method for treating atherosclerosis in a patient.Also provided is a method for treating atherosclerosis in a patient by providing an effective amount of an NTCP inhibitor to a patient in need thereof.It is assumed that administration of an NTCP inhibitor leads to improvement of atherosclerotic condition, including reduction, resorption or elimination of atherosclerotic plaque.The NTCP inhibitor used according to the present invention can be administered to a patient individually or together and / or in combination with other drugs for treating atherosclerosis.

[0014] The present invention further provides NTCP inhibitors for use in the method of treating other NRLP3 inflammasome-related diseases in patients.Specifically, the treatment of type 2 diabetes, atherosclerosis, gout, Alzheimer's disease and NASH is provided.Also provided is a method of treating the above-defined NRLP3 inflammasome-related diseases in patients by providing an effective amount of NTCP inhibitor to a patient in need thereof.It is assumed that administration of NTCP inhibitors leads to improvement of NRLP3 inflammasome-related diseases, including normalization of biochemical parameters and improvement of disease progression.The NTCP inhibitors used according to the present invention can be administered to patients individually or together and / or in combination with other drugs for the treatment of NRLP3 inflammasome-related diseases.

[0015] In one embodiment, the NTCP inhibitor of the present invention may be a pre-S1 peptide inhibitor, the pre-S1 peptide inhibitor comprising a peptide consisting of the amino acid sequence NPLGFX0P (SEQ ID NO:15), where X0 is any amino acid, preferably F or L, more preferably F. The pre-S1 peptide inhibitor may further comprise an N-terminal sequence of at least 4 amino acids at the N-terminus of NPLGFX0P (SEQ ID NO:15). The N-terminal sequence may be any length greater than 4 amino acids, but preferably not more than 19 amino acids. It is preferred that at least one amino acid in the N-terminal sequence has an amino group in the side chain. Thus, the pre-S1 peptide inhibitor may comprise a peptide consisting of the amino acid sequence NX1SX2X3 (SEQ ID NO:15), where X0 is any amino acid, preferably F or L, more preferably F. ID NO:16), where X1, X2 and X3 are any amino acid and are preferably directly linked at the N-terminus to NPLGFX0P (SEQ ID NO:15). Preferably, X1 is L, I or Q, preferably L, X2 is T, V, A or absent, preferably T or V, more preferably T, and / or X3 is P, S, T or F, preferably P or S, more preferably S. Thus, a pre-S1 peptide inhibitor may comprise the amino acid sequence NX1SX2X3NPLGFX0P (SEQ ID NO:17), where X0 is any amino acid, X1 is L, I or Q, preferably L, X2 is T, V, A or absent, preferably T or V, more preferably T, and X3 is P, S, T or F, preferably P or S, more preferably S.

[0016] The pre-S1 peptide inhibitor may further comprise a C-terminal sequence of at least one amino acid at the N-terminus of NPLGFX0P (SEQ ID NO:15). The C-terminal sequence may be of any length, preferably 93 amino acids or less, 35 amino acids or less, 20 amino acids or less, or 10 amino acids or less. The pre-S1 peptide inhibitor may comprise a C-terminal amino acid sequence X4HQLDP (SEQ ID NO:18), where X4 is any amino acid. Preferably, X4 is D, E or S, preferably D or E, more preferably D. The C-terminal sequence is preferably directly linked to NPLGFX0P (SEQ ID NO:15). Thus, the pre-S1 peptide inhibitor may comprise an amino acid sequence NPLGFX0PX4HQLDP (SEQ ID NO:19), where X0 is any amino acid, and X4 is D, E or S, preferably D or E, more preferably D.

[0017] The pre-S1 peptide inhibitor may comprise the amino acid sequence NX1SX2X3NPLGFX0PX4HQLDP (SEQ ID NO:20), where X0 is any amino acid, X1 is L, I or Q, preferably L, X2 is T, V, A or absent, preferably T or V, more preferably T, and X3 is P, S, T or F, preferably or P or S, more preferably S; and X4 is D, E or S, preferably D or E, more preferably D.

[0018] The pre-S1 peptide inhibitors may be any length between 7 and 119 amino acids, between 15 and 47 amino acids, or between 15 and 25 amino acids. The pre-S1 peptide inhibitors may be 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 74, 80, 85, 90, 95, 100, 110, etc. amino acids in length.

[0019] The amino acid sequence NPLGFX0P (SEQ ID NO:15) contained in the pre-S1 peptide inhibitor corresponds to amino acid positions 9-15 of the pre-S1 peptide of the HBV virus according to the standard pre-S peptide numbering. The amino acid sequences NX1SX2X3 (SEQ ID NO:16) and X4HQLDP (SEQ ID NO:18) correspond to amino acid positions 4-8 and 16-21, respectively, of the pre-S1 peptide of the HBV virus.

[0020] Therefore, the pre-S1 peptide inhibitor may comprise the pre-S1 peptide of HBV virus, or its functional fragment.The functionality of the fragment can be evaluated based on, for example, its ability to bind to NTCP, its ability to inhibit NTCP, or its ability to reduce NTCP activity.

[0021] The pre-S1 peptide inhibitor may comprise at least amino acids 9-15 of the pre-S1 peptide of the HBV virus according to standard pre-S peptide numbering. Alternatively, the pre-S1 peptide inhibitor may further comprise amino acids 4-8, 3-8, 2-8 of the pre-S1 peptide, and / or amino acids 16, 16-17, 16-18, 16-19, 16-20, or 16- of the pre-S1 peptide of the HBV virus. The pre-S1 peptide inhibitor may comprise or consist of amino acids 2-48 or 2-21 of the pre-S1 peptide of the HBV virus. Also contemplated are pre-S1 peptide inhibitors having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with any of the above sequences.

[0022] The HBV virus may be any HBV virus, such as HBV strain alpha 1, HBV strain LSH, woodchuck HBV, woolly monkey HBV (WMHBV), orangutan HBV, chimpanzee HBV, gorilla HBV, human HBV, HBV subtypes AD, ADR, ADW, ADYW, AR or AYW, or HBV genotypes A, B, C, D, E, F, G or H.

[0023] Representative pre-S1 peptide inhibitors consist of the amino acid sequence between positions 2 and 48 of the HBV pre-S1 consensus sequence. GTNL SVPNP LGFFP DHQLD PAFRA NSNNP DWDFN PNKDH WPEAN KVG(SEQ ID NO:12), or Amino acid sequence between positions 2 and 48 of the HBV pre-S1 genotype C sequence GTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDH WPEAN QVG(SEQ ID NO:14), or The amino acid sequence between positions 2 and 48 of the HBV pre-S1 genotype C sequence, which contains an amino acid substitution at position 46 (Gln(Q)→Lys(K)) GTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDH WPEAN KVG(SEQ ID NO:13), or Amino acid sequence between positions 2 and 48 of the HBV pre-S1 genotype D sequence GQNL STSNP LGFF DHQLD PAFRA NTANP DWDFN PNKDT WPDAN KVG(SEQ ID NO:5) Includes.

[0024] Also contemplated are fragments of the above amino acid sequences that are at least 15 amino acids in length, as well as sequences that have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the above representative sequences, and maintain the activity of the pre-S1 peptide inhibitors. Activity includes either NTCP binding, NTCP inhibition, or reduction of NTCP activity. Activity may also be prevention or reduction of HBV binding to NTCP.

[0025] The pre-S1 peptide inhibitors may be modified with one or more hydrophobic moieties at or near the N-terminus. The hydrophobic moiety modification can be performed by acylation, for example, with myristoyl or stearoyl.

[0026] Preferred pre-S1 peptide inhibitors are GTNL, SVPNP, LGFFP, DHQLD It has the amino acid sequence PAFGA NSNNP DWDFN PNKDH WPEAN KVG (SEQ ID NO:13), and the peptide is N-terminally modified with myristoyl.

[0027] A preferred pre-S1 peptide inhibitor is Myrcludex B, which has the following chemical formula: N-Myristoyl-glycyl-L-threonyl-L-asparaginyl-L-leucyl-L-seryl-L-valyl-L-prolyl-L-asparaginyl-L-prolyl-L-leucyl-glycyl-L-phenylalanyl-L-phenylalanyl-L-prolyl-L-aspartyl-L-histidyl-L-glutaminyl-L-leucyl-L-aspartyl-L-prolyl-L-alanyl-L-phenylalanyl-glycyl-L-alanyl-L-asparaginyl-L-seryl-L-asparaginyl-L-asparaginyl-L-prolyl-L- Aspartyl-L-tryptophanyl-L-aspartyl-L-phenylalanyl-L-asparaginyl-L-prolyl-L-asparaginyl-L-lysyl-L-aspartyl-L-histidyl-L-tryptophanyl-L-prolyl-L-glutamyl-L-alanyl-L-asparaginyl-L-lysyl-L-valyl-glycinamide, acetate

[0028] The pre-S1 peptide inhibitors may be further modified at the C-terminus or elsewhere to protect against degradation of the peptide form. Exemplary protecting moieties that can be used for this purpose are D-amino acids, cyclic amino acids, modified amino acids, glycans, natural or synthetic polymers such as polyethylene glycol (PEG).

[0029] The pre-S1 peptide inhibitor of the present invention, particularly Myrcludex B, may be administered to a patient at a dose such that the concentration of the pre-S1 peptide inhibitor at the NTCP site, i.e., in the hepatocyte, is equal to or greater than a Ki of about 1-10 nM. Thus, the daily dose of the pre-S1 peptide inhibitor may be 0.1 mg to 50 mg, 0.5 mg to 20 mg, 1 mg to 15 mg, 1 mg to 10 mg, 1 mg to 5 mg, for example 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg, preferably 2 mg, 5 mg, or 10 mg, most preferably 5 mg. The daily dose of the pre-S1 peptide inhibitor may be about 0.0014 mg / kg body weight to about 0.7 mg / kg body weight, preferably about 0.014 mg / kg body weight to about 0.28 mg / kg body weight (1 mg to 20 mg).

[0030] The pre-S1 peptide inhibitor may be administered for 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 12 weeks, 24 weeks, 36 weeks, 48 ​​weeks, 60 weeks, 1 year, 1.1 years, 1.2 years, 1.3 years, 1.4 years, 1.5 years, 1.6 years, 1.7 years, 1.8 years, 1.9 years, or 2.0 years, or 3 years, or 4 years or more.

[0031] The pre-S1 peptide inhibitors may be administered by any suitable route, such as subcutaneous, intravenous, oral, intranasal, intramuscular, transdermal, by inhalation or suppository, preferably intravenously or subcutaneously.

[0032] In another embodiment, the NTCP inhibitor may be a compound or agent other than a pre-S1 peptide inhibitor that prevents or reduces the production and / or function of NTCP.

[0033] The NTCP inhibitors of the present invention may be administered individually or together. In particular, the above pre-S1 peptide inhibitor may be administered together with another NTCP inhibitor. Two or more NTCP inhibitors may be administered simultaneously / concomitantly. According to this administration schedule, the administration of two or more NTCP inhibitors overlaps in time. The administration period of two or more NTCP inhibitors may be the same or essentially the same. For example, the pre-S1 peptide inhibitor and another NTCP inhibitor may be administered in a course that includes at least one cycle, or one or more cycles (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 cycles). The duration of one cycle may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or more. The duration of one cycle may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. The duration of one course may be 12 weeks, 24 weeks, 36 weeks, 48 ​​weeks, 60 weeks, 1 year, 1.1 years, 1.2 years, 1.3 years, 1.4 years, 1.5 years, 1.6 years, 1.7 years, 1.8 years, 1.9 years, or 2.0 years, or 3.0 years, or 4.0 years. For example, a 24-week course of the pre-S1 peptide inhibitor Myrcludex B may be administered simultaneously with a 24-week course of another NTCP inhibitor. In this dosing regimen, Myrcludex B may be administered daily, and another NTCP inhibitor may be administered weekly. In this dosing regimen, the pre-S1 peptide inhibitor and another NTCP inhibitor may be administered simultaneously, for example at essentially the same time or in a single composition.

[0034] Pre-S1 peptide inhibitor and further NTCP inhibitor may be administered to patient by various delivery routes depending on the type of components.Administration routes include topically, enteral route (e.g. oral and rectal), parenteral route (e.g. intravenous, intramuscular, subcutaneous, intraperitoneal).Preferably, pre-S1 peptide inhibitor is delivered subcutaneously.

[0035] The NTCP inhibitors used according to the present invention may be in a pharmaceutical composition comprising pharma- ceutically acceptable carriers, excipients, and adjuvants.

[0036] Although the invention is directed primarily to human treatment, the treatment of any mammal is encompassed. [Brief description of the drawings]

[0037] [Figure 1] Bile acids suppress NLRP3 inflammasome-mediated secretion of IL-1β. (A) Lipopolysaccharide (LPS)-stimulated bone marrow-derived macrophages (BMDMs) were treated with the indicated bile acids (BAs) at 50 mM and then stimulated with nigericin for 45 min. Supernatants were analyzed by ELISA for release of IL-1β. (B) and (C) LPS-stimulated BMDMs were treated with different doses of TLCA and then stimulated with nigericin for 45 min. Supernatants were analyzed by ELISA for release of IL-1β (B) and IL-18 (C). From Guoetal., 2016. [Diagram 2] Bile acid species are differentially affected by Myrcludex B. [Diagram 3] TLCA Elevation in Response to Myrcludex B. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] The present invention is based, in part, on the finding that administration of NTCP inhibitors leads to elevated blood bile acid levels and increased immunomodulatory signaling mediated by FXR and TGR-5. Bile acid-mediated signaling through FXR and TGR-5 regulates several metabolic pathways that regulate not only bile acid synthesis and enterohepatic recirculation, but also lipid, glucose and energy homeostasis. Furthermore, agonists of FXR and TGR5 exhibit anti-inflammatory and antifibrotic properties, making these agents interesting candidates for the treatment of several liver and metabolic diseases, including nonalcoholic steatohepatitis (NASH). In particular, Guo et al., 2016, showed that different bile acids inhibit inflammation via NLRP3 inflammasome inhibition. The authors have demonstrated that bile acids control inflammatory and metabolic disorders. All bile acids tested by the authors exhibit such properties, but lithocholic acid (LCA) and its taurine conjugate taurolithocholic acid (TLCA) were particularly effective. For example, inhibition of the proinflammatory cytokine IL-1β formation in macrophages was most prominent with LCA and TLCA (Figure 1). There is also strong evidence provided by the inventors that bile acid-mediated TGR5 signaling induces ubiquitination of the NRLP3 inflammasome and its inhibition via a pathway dependent on adenylyl cyclase and protein kinase A (PKA).

[0039] A human clinical trial completed with Myrcludex B, an NTCP inhibitor peptide with the amino acid sequence GTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDH WPEAN KVG (SEQ ID NO:13), induced a significant increase in blood bile acid levels (Figure 2), and in particular, an increase in TLCA levels (Figure 3). There were no side effects associated with the increase in bile acids associated with the treatment. Thus, treatment of human patients with the NTCP inhibitors of the present invention is expected to lead to increased TGR5 signaling and anti-inflammatory, anti-fibrotic and other beneficial effects, resulting in clinical benefit and disease modification for the disorders disclosed in this application.

[0040] Moreover, in cholestatic disorders such as PBC and primary sclerosing cholangitis, hepatocellular injury may be partly associated with the accumulation of bile acids in liver cells. Blocking hepatic reuptake of bile acids reduces intracellular bile acid concentrations, unburdens hepatocytes, and thus improves liver function.

[0041] The present invention is further based, in part, on the unexpected finding that treatment of atherosclerosis with inhibitors of Na+-taurocholate cotransporting polypeptide (NTCP) results in the reduction, resorption or elimination of atherosclerotic plaques. PBC patients are known to have high LDL levels, but evidence of atherosclerosis is lacking. The mechanism by which elevated bile acids reduce atherosclerotic lesions is complex and includes activation of reverse cholesterol transport, induction of the respective genes, and direct detergent function of bile acids. Since NTCP is responsible for more than 50% of the reuptake of conjugated bile acids from the bloodstream to the liver, NTCP blockade results in elevated levels of bile acids in the blood, as observed in humans and experimental animals. As mentioned above, in human clinical trials with the NTCP inhibitor Myrcludex B, the inventors measured elevated bile acid concentrations, but no side effects were associated with this increase. Thus, treatment with NTCP inhibitors may clinically suppress elevated levels of bile acids in the bloodstream and, consequently, result in the anti-atherosclerotic effects associated with elevated bile acid concentrations.

[0042] Therefore, the present invention provides NTCP inhibitors for use in the method of treating atherosclerosis, primary biliary cirrhosis (PBC), NRLP3 inflammasome-related diseases in patients, including type 2 diabetes, atherosclerosis, gout, Alzheimer's disease and NASH.Also provided is a method of treating the above diseases in patients by administering an effective amount of NTCP inhibitor to a patient in need thereof.It is assumed that administration of NTCP inhibitors leads to improvement, remission or dissipation of disease symptoms, and effective treatment of disease.

[0043] [NTCP] The sodium / bile acid cotransporter, also known as the sodium / Na+-taurocholate cotransporting polypeptide (NTCP), is a protein that in humans is encoded by the SLC10A1 (solute carrier family 10 member 1) gene.

[0044] The sodium / bile acid cotransporters are integral membrane glycoproteins involved in the enterohepatic circulation of bile acids. Two homologous transporters are involved in the reabsorption of bile acids, one located apically and absorbing them from the intestinal lumen, bile duct and kidney (SLC10A2), while the other sodium-dependent cotransporter is found in the basolateral membrane of hepatocytes (SLC10A1).

[0045] Bile formation is a key function of the liver. Bile salts are the main components of bile and are secreted into the bile by hepatocytes and transported to the small intestine where they aid in fat digestion. In the liver, hepatocytes take up bile salts from plasma and secrete them again into the bile (mainly via the bile salt export pump (BSEP)) for the ongoing enterohepatic circulation. Bile salt uptake into hepatocytes occurs in a sodium-dependent manner, primarily by NTCP. The transport properties of NTCP have been extensively characterized. It is an electrogenic member of the solute transporter family of transporters (SLC10A1), which primarily transports bile salts and sulfated compounds, but can also mediate the transport of additional substrates such as thyroid hormones, drugs and toxins. It is highly regulated under physiological and pathophysiological conditions. Control of NTCP addresses altered bile salt load to hepatocytes and prevents the entry of cytotoxic amounts of bile salts during liver disease.

[0046] For NTCP, a wide range of substrates could be detected. NTCP transports unconjugated bile acids as well as taurine- and glycine-conjugated bile acids, sulfated bile acids, and, in contrast to the apical sodium-dependent bile acid transporter (ASBT), steroid sulfates, and thyroid hormones. Drugs such as rosuvastatin and micafungin have also been shown to have affinity for NTCP. Recent data indicate that FDA-approved drugs have been identified as inhibitors of NTCP. Most of them are antifungal, antihyperlipidemic (simvastatin), antihypertensive, anti-inflammatory, or glucocorticoid drugs.

[0047] Examples of bile acids transported to hepatocytes via NTCP include cholic acid, taurine- or glycine-conjugated bile acids and their salts (dihydroxy and trihydroxy bile acids bound to taurine or glycine), taurocholic acid, glycocholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, tauroursodeoxycholic acid, sulfated bile acids and their salts.

[0048] Without being bound by any theory or mode of action, the present application teaches that blocking NTCP exerts an unexpected and significant beneficial effect on atherosclerotic lesions such as plaques, especially vulnerable plaques with increased rupture potential, by increasing the bile acid level in blood.In contrast to previous therapies that mainly focus on preventive measures that were supposed to slow the progression of atherosclerotic lesions by lowering cholesterol levels, especially LDL levels in blood, the treatment provided by the present invention targets already existing atherosclerotic lesions, resulting in their significant reduction, elimination or complete elimination.Therefore, this therapy provides great benefits to patients who have not been able to achieve treatment with established therapies.

[0049] [Pre-S1 peptide inhibitor] In one embodiment, the inhibitor of Na+-taurocholate cotransporting polypeptide (NTCP) used in the method for treating atherosclerosis in a patient is a pre-S1 peptide inhibitor. The pre-S1 peptide inhibitor comprises / consists of a peptide comprising the amino acid sequence NPLGFX0P (SEQ ID NO:15), where X0 is any amino acid, preferably F or L, more preferably F. It is understood that the peptide may be at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 amino acids in length, or may be longer.

[0050] The pre-S1 peptide inhibitor may further comprise an N-terminal sequence of at least 4 amino acids at the N-terminus of NPLGFX0P (SEQ ID NO:15). The N-terminal sequence may consist of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids. The N-terminal sequence may consist of 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, 4-14, 4-15, 4-16, 4-17, 4-18, or 4-19 amino acids.

[0051] At least one amino acid in the N-terminal sequence may have an amino group in its side chain. The at least one amino acid in the N-terminal sequence is selected from lysine, α-aminoglycine, α,γ-diaminobutyric acid, ornithine, α,β-diaminopropionic acid, preferably lysine. The at least one amino acid in the N-terminal sequence may be located at the N-terminus of the N-terminal sequence. The at least one amino acid in the N-terminal sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids, or the at least one amino acid in the N-terminal sequence is 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9 or 1-10 amino acids.

[0052] The N-terminal sequence may comprise the amino acid sequence NX1SX2X3 (SEQ ID NO:16), where X1, X2 and X3 are any amino acid. Preferably, in the amino acid sequence NX1SX2X3 (SEQ ID NO:16): X1 is L, I or Q, preferably L; X2 is T, V, A or absent, preferably T or V, more preferably T; and / or X3 is P, S, T or F, preferably P or S, more preferably S.

[0053] The amino acid sequence NX1SX2X3 (SEQ ID NO:16) may be directly linked to the N-terminus of NPLGFX0P (SEQ ID NO:15).

[0054] Thus, the pre-S1 peptide inhibitor may comprise the amino acid sequence NX1SX2X3NPLGFX0P (SEQ ID NO:17), X0 is any amino acid, X1 is L, I or Q, preferably L; X2 is T, V, A or absent, preferably T or V, more preferably T; and X3 is P, S, T or F, preferably P or S, more preferably S.

[0055] The pre-S1 peptide inhibitor may further comprise a C-terminal sequence of at least one amino acid at the C-terminus of NPLGFX0P (SEQ ID NO:15).

[0056] The C-terminal sequences are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 11 It may consist of 0, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 amino acids. The C-terminal sequence may consist of 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 29, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, or 1 to 35 amino acids.

[0057] The C-terminal sequence may comprise the amino acid sequence X4HQLDP (SEQ ID NO:18), where X4 is any amino acid. Preferably, X4 is D, E or S, preferably D or E, more preferably D.

[0058] The amino acid sequence X4HQLDP (SEQ ID NO:18) may be directly linked to the C-terminus of NPLGFX0P (SEQ ID NO:15).

[0059] Thus, the pre-S1 peptide inhibitor may comprise the amino acid sequence NPLGFX0PX4HQLDP (SEQ ID NO:19), X0 is any amino acid, and X4 is D, E or S, preferably D or E, more preferably D.

[0060] The pre-S1 peptide inhibitor may comprise both N-terminal and C-terminal sequences. Thus, the pre-S1 peptide inhibitor may comprise the amino acid sequence NX1SX2X3NPLGFX0PX4HQLDP (SEQ ID NO:20); X0 is any amino acid, X1 is L, I or Q, preferably L; X2 is T, V, A or absent, preferably T or V, more preferably T; X3 is P, S, T or F, preferably P or S, more preferably S, and X4 is D, E or S, preferably D or E, more preferably D.

[0061] Pre-S1 peptide inhibitors have been shown to be effective in treating 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68 , 69, 70, 71, 72, 73, 74, 75, 76, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118 or 119 amino acids.

[0062] The pre-S1 peptide inhibitor may also comprise the pre-S1 peptide of the HBV virus or a functional fragment thereof. Preferably, the function is binding to NTCP, inhibiting NTCP, or reducing NTCP activity.

[0063] Hepatitis B viruses (HBV) are small enveloped DNA viruses that replicate their genomes via reverse transcription of pregenomic RNA transcripts in the cytoplasm of infected hepatocytes. They are classified in the Hepadnaviridae family and are adapted to mammals (primates and rodents) and birds, causing acute and persistent infections. Studies in the HBV-susceptible cell line HepaRG, and in systems based on primary human hepatocytes (PHH) and primary Tupaia belangeri hepatocytes, revealed that viral infectivity could be assigned to distinct subdomains in two of the three viral envelope proteins. The three HBV envelope proteins are called L (large), M (medium) and S (small). They form a proteinaceous outer viral shell embedded in a lipid bilayer derived from the endoplasmic reticulum (ER). Their coding mRNA is derived from a single open reading frame. Their three initiation codons are coincident and therefore share a C-terminal 226 amino acid long S domain that anchors the protein through four putative transmembrane helices (TM domains) in the lipid bilayer. The S protein promotes particle formation and thereby plays a key function in virus assembly. Furthermore, the S domain has been shown to be involved in virus entry. The M and L proteins linked to the N-terminus of the S domain have two hydrophilic extensions, one of 55 amino acids and, depending on the genotype, one of 108 (genotype D), 118 (genotypes E and G) or 119 (genotypes A, B, C, F, H) amino acids, called pre-S2 (M) and pre-S1 (L), respectively. The L proteins of all hepadnaviruses contain a recognition motif for N-myristoyltransferase and therefore undergo this modification. Key elements for virus attachment, binding of specific receptors, or fusion have been identified within the L, M and S proteins.

[0064] Peptides derived from the pre-S1 polypeptide of HBV have previously been identified as inhibitors of viral entry into cells (hepatocytes), i.e. HBV / HDV entry inhibitors. An example of an HBV / HDV entry inhibitor currently in clinical trials is a linear 47 amino acid chemically synthesized peptide (known under the trade name Myrcludex B), which is derived from the pre-S1 domain of the large (L) HBV surface protein, composed of naturally occurring L-amino acids and with an N-terminal myristoyl moiety and a C-terminal carboxamide.

[0065] According to the invention, these pre-S1 derived peptides can be used as NTCP inhibitors, ie as pre-S1 peptide inhibitors.

[0066] Thus, the pre-S1 peptide inhibitors used according to the invention may be derived from the N-terminal domain of the large (L) HBV surface protein, pre-S1, of any HBV virus, in particular of any HBV strain, genotype or subtype. Examples of HBV are human HBV, gorilla HBV, chimpanzee HBV, woodchuck HBV, woolly monkey HBV (WMHBV). Examples of HBV strains are HBV strain α1 and HBV strain LSH (chimpanzee isolate). Examples of HBV subtypes are AD, ADR, ADW, ADYW, AR and AYW. Examples of HBV genotypes are genotypes A-H of human HBV. According to standard HBV nomenclature, the pre-S1 peptide has amino acid coordinates of 11-108. Compared to genotype D, all other genotypes contain an additional 10 (E and G) or 11 (A, B, C, F and H) N-terminal pre-S1 amino acids. Thus, the viral pre-S1 peptide may have different amino acid coordinates and therefore different lengths depending on the viral strain, genotype or subtype.

[0067] Sequence alignment also shows that the pre-S1 peptides of various genotypes share conserved regions along the peptide length. The highly conserved region is located between amino acid positions 9 and 21. In addition, individual highly conserved amino acids are located at amino acids 2 to 6. These conserved sequences are also present in hepatitis B viruses isolated from chimpanzees, gorillas, and woolly monkeys.

[0068] Experimental evidence indicates that the ability of synthetic HBV pre-S1 derived peptides to hinder HBV infection depends on the presence of several amino acids within the conserved pre-S-1 region. Thus, it is the region between amino acid positions 9 and 15 of pre-S1 that is critical for peptide activity, particularly amino acids 11, 12, 13, 14 and / or 15 within this region. Furthermore, substitution of several amino acids within the region between positions 2 and 9 reduces the HBV inhibitory activity of pre-S1 peptides, according to which amino acid 9 appears to be essential. Further regions that may contribute to the inhibitory activity of HBV pre-S1 derived peptides are amino acids 2-8, 16-20 and, to a less pronounced extent, 34-48. These conserved amino acids are also important for the ability of pre-S1 peptides to inhibit NTCP.

[0069] Thus, the pre-S1 peptide inhibitor may span amino acids -11 to 108 of HBV pre-S1 (the entire HBV pre-S1) or any part within this region. In the following, reference is made to standard HBV amino acid numbering. In particular, the pre-S1 peptide inhibitor may span amino acids -11 to 78. In some genotypes, the first 10 (genotypes E and G) or 11 (genotypes A, B, C, F and H) amino acids are missing. Thus, the pre-S1 peptide inhibitor may span amino acids 1 to 78 of HBV. One or more amino acids beyond amino acid 48, i.e., amino acids 49 to 108, may be missing. Thus, the pre-S1 peptide inhibitor may span amino acids -11 to 48. Within the range of -11 to 48, the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids may be omitted. Thus, the pre-S1 peptide inhibitor may span amino acids 1 to 48. Amino acid 1 (Met) may be absent. Each pre-S1 peptide inhibitor may span amino acids 2-48, optionally including additional amino acids at the C-terminus. Within the range of 2-48, amino acids 2-21 and 34-48 are more important for the function of the pre-S1 peptide inhibitor than amino acids 21-33. Amino acids 9-15, particularly 11-15, are most important. Thus, the pre-S1 peptide inhibitor may consist of amino acids 2-48 or a truncated portion thereof. Alternatively, the pre-S1 peptide inhibitor may comprise amino acids 2-48 or a truncated portion thereof.

[0070] The truncated portion may be obtained by amino acid deletion from either end of the pre-S1 peptide and may have a length of 46 to 7 amino acids. In particular, the truncated portion may have a length of 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, or 7 amino acids.

[0071] The truncated portion preferably comprises amino acids 9-15, 10-15, or 11-15. The truncated portion preferably consists of amino acids 11-15. The truncated portion may be gradually extended by the addition of amino acids adjacent to amino acids 11-15. For example, the truncated portion may consist of amino acids 2-15, 3-15, 4-15, 5-15, 6-15, 7-15, 8-15, 9-15 or 10-15. In this embodiment, any of amino acids 16-48 may be used to provide a further truncated portion.

[0072] In another example, the truncated portion may consist of amino acids 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-21, 2-22, 2-23, 2-24, 2-25, 2-26, 2-27, 2-28, 2-29, 2-30, 2-32, 2-32, 2-27, 2-28, 2-29, 2-30, 2-31, 2-32, 2-33, 2-34, 2-35, 2-36, 2-37, 2-38, 2-39, 2-40, 2-41, 2-42, 2-43, 2-44, 2-45, 2-47 or 2-48. In this example, any of the amino acids 3-11 may be used to provide a further truncated portion.

[0073] Further examples of truncated portions are amino acids 9-15, 2-21, 5-21, 2-15, 2-20, 2-25, 2-30, 2-35, 2-40.

[0074] The above-mentioned pre-S1 peptide inhibitor includes NPLGFX0P (SEQ ID NO:15), which corresponds to amino acid positions 9 to 15 of the pre-S1 peptide of the HBV virus.

[0075] The truncated portion of the pre-S1 peptide may be a functional fragment of the pre-S1 peptide that retains the function of the pre-S1 peptide in binding to and / or inhibiting NTCP.

[0076] Thus, the pre-S1 peptide inhibitors used in the present invention include At least amino acids 9-15, 8-15, 7-15, 6-15, 5-15, 4-15, 3-15, 2-15 of the pre-S1 peptide of the HBV virus; At least amino acids 9-16, 9-17, 9-18, 9-19, 9-20, 9-21, 9-22, 9-23, 9-24, 9-25, 9-26, 9-27, 9-28, 9-29, 9-30, 9-31, 9-32, 9-33, 9-34, 9-35, 9-36, 9-37, 9-38, 9-39, 9-40, 9-41, 9-42, 9-43, 9-44, 9-45, 9-46, 9-47, 9-48, at least amino acids 8-16, 7-17, 6-18, 5-19, 4-20, 3-21, or 2-22 of the pre-S1 peptide of the HBV virus; HBV virus pre-S1 peptides at least 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-21, 2-22, 2-23, 2-24, 2-25, 2-26, 2-27, 2-28, 2-29, 2-30, 2-31, 2-32, 2-33, 2-34, 2-35, 2-36, 2-37, 2-38, 2-39, 2-40, 2-41, 2-42, 2-43, 2-44, 2-45, 2-46, 2-47, or 2-48; at least amino acids 9-15 and 16-20 of the pre-S1 peptide of the HBV virus; at least amino acids 2-8 and 9-15 of the pre-S1 peptide of the HBV virus; at least amino acids 2-8, 9-15, and 16-20 of the pre-S1 peptide of the HBV virus; at least amino acids 9-15 and 34-48 of the pre-S1 peptide of the HBV virus; at least amino acids 9-15, 16-20, and 34-48 of the pre-S1 peptide of the HBV virus; at least amino acids 2-8, 9-15, and 34-48 of the pre-S1 peptide of the HBV virus; at least amino acids 2-8, 9-15, 16-20, and 34-48 of the pre-S1 peptide of the HBV virus; a portion of the pre-S1 peptide of the HBV virus consisting of at least amino acids 2 to 48 or at least 15 amino acids, or Amino acids 2-48 of the HBV pre-S1 peptide It may comprise or consist of:

[0077] The pre-S1 peptide inhibitor may also be a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to any of the above sequences.

[0078] The pre-S1 peptide inhibitor may be derived from the pre-S1 peptide of various HBV viruses, such as various genotypes, strains or subtypes. In particular, the pre-S1 peptide inhibitor may be derived from the pre-S1 of genotypes A, B, C, D, E, F, G or H, or subtypes AD, ADR, ADW, ADYW, AR and AYW. The pre-S1 peptides from various HBV viruses are usually homologs. Homologs from various species have structural and functional similarities and usually share a common evolutionary ancestor. It is envisioned that pre-S1 homologs from additional genotypes, strains or subtypes identified in the future will be similarly suitable as pre-S1 peptide inhibitors. The pre-S1 peptide inhibitor may also be derived from the pre-S1 consensus sequence.

[0079] Examples of HBV pre-S1 sequences between amino acids -11 or -10 or 1 (depending on the genotype) and 48 for various genotypes and consensus sequences are shown below. HBV pre-S1 consensus sequence (positions (-11) to 48) (-11)-M GGWSS TPRKG MGTNL SVPNP LGFFP DHQLD PAFRA NSNNP DWDFN PNKDH WPEAN KVG-48 (SEQ ID NO:1) HBV pre-S1 genotype A sequence (position (-11) to 48) (-11)-M GGWSS KPRKG MGTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PVKDD WPAAN QVG-48 (SEQ ID NO:2) HBV pre-S1 genotype B sequence (position (-11) to 48) (-11)-M GGWSS KPRKG MGTNL SVPNP LGFFP DHQLD PAFKA NSENP DWDLN PHKDN WPDAN KVG-48 (SEQ ID NO:3) HBV pre-S1 genotype C sequence (position (-11) to 48) (-11)-M GGWSS KPRQG MGTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDH WPEAN QVG-48 (SEQ ID NO:4) HBV pre-S1 genotype D sequence (positions 1-48) 1-MGQNL STSNP LGFFP DHQLD PAFRA NTANP DWDFN PNKDT WPDAN KVG-48 (SEQ ID NO:5) HBV pre-S1 genotype E sequence (position (-10) to 48) (-10)-MGLSW TVPLE WGKNI STTNP LGFP DHQLD PAFRA NTRNP DWDHN PNKDH WTEAN KVG-48 (SEQ ID NO:6) HBV pre-S1 genotype F sequence (position (-11) to 48) (-11)-M GAPLS TTRRG MGQNL SVPNP LGFFP DHQLD PLFRA NSSSP DWDFN TNKDS WPMAN KVG-48 (SEQ ID NO:7) HBV pre-S1 genotype G sequence (position (-10) to 48) (-10)-MGLSW TVPLE WGKNL SASNP LGFLP DHQLD PAFRA NTNNP DWDFN PKKDP WPEAN KVG-48 (SEQ ID NO:8) HBV pre-S1 genotype H sequence (position (-11) to 48) (-11)-M GAPLS TARRG MGQNL SVPNP LGFFP DHQLD PLFRA NSSSP DWDFN TNKDN WPMAN KVG-48 (SEQ ID NO:9)

[0080] The pre-S1 sequence of amino acids 1 to 48 from woolly monkey WMHBV is shown below. 1-MGLNQ STFNP LGFFP SHQLD PLFKA NAGSA DWDKN PNKDP WPQAH DTA (SEQ ID NO:10)

[0081] The pre-S1 peptide inhibitor may contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions that do not substantially reduce NTCP inhibitor activity. Preferably, NTCP inhibitor activity should not be reduced by more than two orders of magnitude. In particular, reductions of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 fold are acceptable. NTCP inhibitor activity is measured by the IC 50 or IC 90 The amino acid substitutions may be measured over a period of time. The amino acid substitutions may be conservative or non-conservative. The amino acid substitutions preferably do not affect amino acids important for the NTCP inhibitory activity of the pre-S1 peptide inhibitor. Individual amino acids located within the highly conserved portion of pre-S1 spanning amino acids 2-21 are more important for activity than amino acids outside this region. Within this region, amino acids 9-15 are even more important. Of these, amino acids 9, 11, 12 and 13 are the most important. Thus, the amino acid substitutions are preferably located outside amino acids 11-15, 9-15, 5-15, or 2-15. The amino acid substitutions may also be located outside amino acids 2-8, 16-20, or 34-48. The amino acid substitutions may also be located inside amino acids 20-23, 26-32.

[0082] An example of a pre-S1 sequence derived from a genotype C pre-S1 sequence and containing an amino acid substitution at position 46 (Gln(Q)→Lys(K)) is provided below, according to standard HBV nomenclature. (-11)-M GGWSS KPRQG MGTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDH WPEAN KVG-48 (SEQ ID NO:11)

[0083] A preferred pre-S1 peptide inhibitor is a pre-S1 peptide between amino acid positions 2 and 48 (according to standard HBV nomenclature) of the HBV pre-S1 consensus sequence. GTNL SVPNP LGFFP DHQLD PAFRA NSNNP DWDFN PNKDH WPEAN KVG (SEQ ID NO:12) It is based on.

[0084] Another preferred pre-S1 peptide inhibitor is a pre-S1 peptide inhibitor comprising the pre-S1 peptide between positions 2 and 48 (according to standard HBV nomenclature) of the amino acid sequence of HBV pre-S1 genotype C. GTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDH WPEAN QVG (SEQ ID NO:14) It is based on.

[0085] The most preferred peptide inhibitor is a pre-S1 peptide having an amino acid substitution at position 46 (Gln(Q)→Lys(K)) between positions 2 and 48 (according to standard HBV nomenclature) of the genotype C amino acid sequence. GTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDH WPEAN KVG (SEQ ID NO:13) It is based on.

[0086] The pre-S1 peptide inhibitor may be an N-terminally or C-terminally truncated portion of said pre-S1 inhibitor of at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46 amino acids, which portion retains its function as an NTCP inhibitor or NTCP binding protein.

[0087] The pre-S1 peptide inhibitor may be a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to any of the above sequences.

[0088] The pre-S1 peptide inhibitors are preferably hydrophobic. They may be modified by at least one hydrophobic moiety. Hydrophobic modification by acylation is preferred. Acylation may be with carboxylic acids, fatty acids, or amino acids with lipophilic side chains. Alternatively, the pre-S1 peptide inhibitors may be modified by cholesterol, derivatives of cholesterol, phospholipids, glycolipids, glycerol esters, steroids, ceramides, isoprene derivatives, adamantane, farnesol, aliphatic groups, or polyaromatic compounds. The fatty acids may be saturated or unsaturated fatty acids, branched or unbranched fatty acids, preferably having 8 to 22 carbon atoms. Examples of fatty acids suitable for acylation are myristic acid (C14), stearic acid (C18), palmitic acid (C16). Acylation with myristoyl is preferred. The change in the hydrophobic moiety allows the modulation of the specific pre-S1 peptide inhibitor activity.

[0089] The hydrophobic moiety is preferably attached to the N-terminus or the 1st, 2nd, 3rd or 4th amino acid adjacent to the N-terminus of the pre-S1 peptide inhibitor. Thus, the hydrophobic moiety may be attached to the N-terminal amino acid of the pre-S1 peptide inhibitor or to an amino acid very close to the N-terminus, for example, amino acid -5, -4, -3, -2, -1, 1, 2, 3, 4 or 5. Two or more hydrophobic moieties may be used to modify the pre-S1 peptide inhibitor. The hydrophobic moieties may be the same or different. The attachment of the hydrophobic moiety is preferably by a covalent bond, which may be achieved via a carbamate, amide, ether, disulfide or any other bond within the skill of the art.

[0090] A preferred peptide inhibitor has the amino acid sequence GTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDH WPEAN KVG(SEQ ID NO:13) The pre-S1 peptide of is N-terminally modified with myristoyl or stearoyl.

[0091] The chemical name of the pre-S1 peptide inhibitor Myrcurdex B is N-Myristoyl-glycyl-L-threonyl-L-asparaginyl-L-leucyl-L-seryl-L-valyl-L-prolyl-L-asparaginyl-L-prolyl-L-leucyl-glycyl-L-phenylalanyl-L-phenylalanyl-L-prolyl-L-aspartyl-L-histidyl-L-glutaminyl-L-leucyl-L-aspartyl-L-prolyl-L-alanyl-L-phenylalanyl-glycyl-L-alanyl-L- Asparaginyl-L-seryl-L-asparaginyl-L-asparaginyl-L-prolyl-L-aspartyl-L-tryptophanyl-L-aspartyl-L-phenylalanyl-L-asparaginyl-L-prolyl-L-asparaginyl-L-lysyl-L-aspartyl-L-histidyl-L-tryptophanyl-L-prolyl-L-glutamyl-L-alanyl-L-asparaginyl-L-lysyl-L-valyl-glycinamide, acetate It is.

[0092] The abbreviated chemical name for Myrculdex B is Myristoyl-Gly-Thr-Asn-Leu-Ser-Val-Pro-Asn-Pro-Leu-GLy-Phe-Phe-Pro-Asp-His-Gln-Leu-Asp-Pro-Ala-Phe-Gly-Al a-Asn-Ser-Asn-Asn-Pro-Asp-Trp-Asp-Phe-Asn-Pro-Asn-Lys-Asp-His-Trp-Pro-Glu-Ala-Asn-Lys-Val-Gly-NH2, Acetate It is.

[0093] The pre-S1 peptide inhibitors may be further modified in conventional ways to improve the stability of the peptide, e.g., stability against degradation. Such modifications may be made at the C-terminus or at the 1, 2, 3 or 4 amino acids proximal to the C-terminus. Modifications may include modification with a protecting moiety, such as an amide, a D-amino acid, a modified amino acid, a cyclic amino acid, a natural polymer, a synthetic polymer, or a glycan. Modification with PEG is an example. The protecting moiety is preferably an amide. The protecting moiety may be linked to an amino acid of the pre-S1 peptide inhibitor via a linker. The linker may be a polyalanine, a polyglycine, a carbohydrate, or a (CH2) n group, where n is at least 1.

[0094] The pre-S1 peptide inhibitor preferably consists of the amino acid sequence 2 to 48 of the genotype C pre-S1 peptide, N-terminally modified by myristoylation and further C-terminally modified with an amide.

[0095] A preferred pre-S1 inhibitor for use according to the present invention is Myrcurdex B. Myristoyl-GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANKVG-amide It is.

[0096] According to the invention, a pre-S1 peptide inhibitor is administered to a patient, preferably a human, At a dose such that the concentration of pre-S1 peptide inhibitor at the NTCP site, i.e., in the hepatocyte, is equal to or greater than a Ki of approximately 1-10 nM. at a daily dose of 0.1 mg to 50 mg, 0.5 mg to 20 mg, 1 mg to 15 mg, 1 mg to 10 mg, 1 mg to 5 mg per day, preferably 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg or 10 mg per day, or A daily dose of about 0.0014 mg / kg body weight to about 0.7 mg / kg body weight, preferably about 0.014 mg / kg body weight to about 0.28 mg / kg body weight (1 mg to 20 mg) may be administered.

[0097] The unit dose of the pre-S1 peptide inhibitor, such as myrcudex B, in the combination or composition may be from 0.5 mg to 20 mg, for example 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 It may be 5 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg, preferably 2 mg, 5 mg, or 10 mg.

[0098] The pre-S1 peptide inhibitors may be administered to a patient, preferably a human, for 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 12 weeks, 24 weeks, 36 weeks, 48 ​​weeks, 60 weeks, 1 year, 1.1 years, 1.2 years, 1.3 years, 1.4 years, 1.5 years, 1.6 years, 1.7 years, 1.8 years, 1.9 years, or 2.0 years, or 3 years, or 4 years or more. The duration of administration depends on the progress of the treatment and side effects.

[0099] The pre-S1 peptide inhibitors may be administered to a patient, preferably a human, subcutaneously, intravenously, orally, intranasally, intramuscularly, transdermally, by inhalation or by suppository, preferably intravenously or subcutaneously, most preferably subcutaneously.

[0100] The pre-S1 peptide inhibitors may be administered to patients, preferably humans, in various doses, treatment schedules and routes of administration, as described above. For example, the present invention provides the following treatment regimen for Myrcurdex B: 2 mg of Myrrudex B administered daily for 24 weeks. This course may be followed by 2 mg of Myrrudex B administered daily for an additional 24 weeks. Myrrudex B may be delivered subcutaneously. In this example, the daily dose of Myrrudex B may be 5 mg or 10 mg. Daily administration of 2 mg of Myrrudex B. Treatment may continue for 48 weeks. In this example, the daily dose of Myrrudex B may be 5 mg or 10 mg. Daily administration of 2 mg of Myrrudex B. Treatment may continue for 2 years or more (indefinitely). Again, in this example, the daily dose of Myrrudex B may be 5 mg or 10 mg. 2 mg of Myrcludex B may be administered in combination daily. Treatment may continue for at least 48 weeks or more. In this example, the daily Myrcludex B dose may be 5 mg or 10 mg. 5 mg of Myrcludex B may be administered daily for 24 or 48 weeks.

[0101] [Other NTCP inhibitors] For purposes of the present invention, an NTCP inhibitor may be an agent or compound other than the pre-S1 peptide that is capable of inhibiting the activity or production of NTCP.

[0102] The NTCP inhibitor may be selected from bendroflumethiazide, ezetimibe, simvastatin, nitrendipine, rosuvastatin, nefazodone, indomethacin, nifedipine, tioconazole, methylprednisolone, prochlorperazine, nateglinide, irbesartan, losartan, olmesartan, fenofibrate, candesartan, cyclosporine A, cyclosporine B, non-immunosuppressive cyclosporine A analogs, progesterone, propranolol, bosentan, or combinations thereof.

[0103] The NTCP inhibitor may further comprise an siRNA targeting the gene encoding NTCP, the siRNA comprising the nucleotide sequence shown in siRNA-1 to siRNA-4 targeting the tree shrew SLC10A1 gene, or siRNA-11, siRNA-405, siRNA-406 targeting the human SLC10A1 gene, siRNA pool (4), siRNA pool (5), siRNA pool (6) or siRNA pool (7). The anti-NTCP agent comprises an antisense RNA targeting the gene encoding NTCP.

[0104] Sometimes, the NTCP inhibitor inhibits or modifies nuclear factors that control NTCP transcription. For example, the agent may be TTNPB, panRAR agonist, all-trans retinoic acid (ATRA), 9-cis retinoic acid (9CRA), etc.

[0105] Alternatively, NTCP inhibitors inhibit or modify the modification of histones or genomic DNA that controls NTCP transcription.DNA methylation inhibitors, including nucleoside analogues of cytosine such as 5-azacytidine, 5-aza-2'-deoxycytidine (5-azadC), histone deacetylase inhibitors (HDAC inhibitors, HDIs) such as MG98 (antisense oligodeoxynucleotide against the 3' untranslated region of DNA methyltransferase-1 enzyme mRNA), vorinostat (suberoylanilide hydroxamic acid, SAHA) and mocetinitostat (MGCD0103) are all intended as NTCP inhibitors used according to the present invention.

[0106] The NTCP inhibitor may inhibit or alter the phosphorylation and / or glycosylation of NTCP, such as tunicamycin, a mixture of homologous nucleoside antibiotics that block all N-glycosylation of proteins. In some cases, the agent is a PI3K inhibitor, such as LY294002, wortmannin, etc.

[0107] The NTCP inhibitor may comprise an antibody that specifically binds to NTCP. The antibody may specifically bind to a portion of NTCP that interacts with HBV and / or HDV, such as an epitope of the extracellular domain. In some embodiments, the extracellular domain comprises amino acids 17-27, 73-89, 142-152, 207-277, or 275-278 of NTCP.

[0108] NTCP inhibitor can comprise mutant form of NTCP, and mutant form of NTCP is selected from the group consisting of mutant NTCP, fragment of NTCP and soluble NTCP polypeptide.Fragment can comprise extracellular domain of NTCP.

[0109] The NTCP inhibitor may comprise an NTCP substrate or a derivative or analogue of an NTCP substrate. The NTCP substrate preferably comprises a bile acid, and the bile acid is selected from the group consisting of taurolithocholate, cholic acid, chenodeoxycholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, lithocholic acid, ursodeoxycholic acid and hyodeoxycholic acid.

[0110] Additionally, the anti-NTCP inhibitor may be a member of the pyrrolotriazinyl class of insulin-like growth factor type 1 receptor (IGF-1R) kinase inhibitors as defined by formula I of U.S. Patent No. 7,534,792, particularly BMS-754807. The preferred compound is (S)-1-(4-(5-cyclopropyl-1H-pyrazol-3-ylamino)pyrrolo[1,2-f][1,2,4]triazin-2-yl)-N-(6-fluoropyridin-3-yl)-2-methylpyrrolidine-2-carboxamide.

[0111] The compounds of formula I are as follows:

[0112] [ka]

[0113] Where: Q 1 is aryl, substituted aryl, heteroaryl, or substituted heteroaryl, and X is C=O, C=S, C=NR 9 or CH2, R 1 , R 2 , and R 3 are independently hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, hydroxy, alkoxy, substituted alkoxy, halogen, haloalkyl, haloalkoxy, alkanoyl, substituted alkanoyl, amino, substituted amino, aminoalkyl, substituted aminoalkyl, alkylamino, substituted alkylamino, amido, substituted amido, carbamate, ureido, cyano, sulfonamido, substituted sulfonamido, alkylsulfone, nitro, thio, thioalkyl, alkylthio, disubstituted amino, alkylsulfonyl, alkylsulfinyl, carboxy, alkoxycarbonyl, alkylcarbonyloxy, carbamoyl, substituted carbamoyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, or alkylcarbonyl; R 4is hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, halogen, haloalkyl, haloalkoxy, oxo, aryloxy, arylalkyl, arylalkyloxy, alkanoyl, substituted alkanoyl, alkanoyloxy, amino, substituted amino, aminoalkyl, substituted aminoalkyl, alkylamino, substituted alkylamino, hydroxyalkyl, disubstituted amino, amido, substituted amido, carbamate, substituted carbamate, ureido, cyano, sulfonamido, substituted sulfonamido, alkylsulfone, heterocycloalkyl, substituted heterocycloalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, cycloalkylalkyl, cycloalkylalkoxy, nitro, thio, thioalkyl, alkylthio, alkylsulfonyl, alkylsulfinyl, carboxy, alkoxycarbyl ... aryl, alkylcarbonyloxy, carbamoyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heteroaryloxy, arylheteroaryl, arylalkoxycarbonyl, heteroarylalkyl, heteroarylalkoxy, aryloxyalkyl, aryloxyaryl, heterocyclic, substituted heterocyclic, alkylcarbonyl, substituted heteroalkyl, heteroalkenyl, substituted heteroalkenyl, heteroalkynyl, substituted heteroalkynyl, arylamino, arylalkylamino, alkanoylamino, aroylamino, arylalkanoylamino, arylthio, arylalkylthio, arylsulfonyl, arylalkylsulfonyl, alkylsulfonyl, arylcarbonylamino, or alkylaminocarbonyl; R 5 is hydrogen, halogen, cyano, alkyl, or substituted alkyl; R 6are independently hydrogen, alkyl, substituted alkyl, alkylidene, substituted alkylidene, hydroxy, alkoxy, halogen, haloalkyl, haloalkoxy, oxo, aryloxy, arylalkyl, arylalkyloxy, alkanoyl, substituted alkanoyl, alkanoyloxy, amino, aminoalkyl, substituted aminoalkyl, alkylamino, substituted alkylamino, hydroxyalkyl, disubstituted amino, amido, substituted amido, carbamate, substituted carbamate, ureido, cyano, sulfonamido, substituted sulfonamido, alkylsulfone, heterocycloalkyl, substituted heterocycloalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, cycloalkylalkyl, cycloalkylalkoxy, nitro, thio, thioalkyl, alkylthio, alkylsulfonyl, carboxy, alkoxy carbonyl, alkylcarbonyloxy, carbamoyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heteroaryloxy, arylheteroaryl, arylalkoxycarbonyl, heteroarylalkyl, heteroarylalkoxy, aryloxyalkyl, aryloxyaryl, heterocyclic, substituted heterocyclic, alkylcarbonyl, substituted heteroalkyl, heteroalkenyl, substituted heteroalkenyl, heteroalkynyl, substituted heteroalkynyl, arylamino, arylalkylamino, alkanoylamino, aroylamino, arylalkanoylamino, arylthio, arylalkylthio, arylsulfonyl, arylalkylsulfonyl, alkylsulfonyl, arylcarbonylamino, or alkylaminocarbonyl; n is 0, 1, 2, 3, 4, 5, or 6, or n is 2 and R 6 are geminal substituents, they may together form an optionally substituted 3- to 6-membered saturated or unsaturated carbocyclic or heterocyclic ring, or n is 2 and R 6 are 1,2-cis substituents, they may together form an optionally substituted 3- to 6-membered fused saturated carbocyclic or heterocyclic ring, or n is 2 and R 6When R are 1,3-cis substituents, they may together form an optionally substituted 1-4 membered alkyl or heteroalkyl bridge, or two R on the same carbon. 6 If there is a carbonyl (C=O) or alkylidene group (C=CHR 9 ), R 7 and R 8 is independently hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycloalkyl, substituted heterocycloalkyl, heteroalkyl, substituted heteroalkyl, heteroalkenyl, substituted heteroalkenyl, heteroalkynyl, or substituted heteroalkynyl; or R 7 and R 8 may together form an optionally substituted monocyclic 4- to 8-membered saturated or unsaturated carbocyclic or heterocyclic ring, or an optionally substituted bicyclic 7- to 12-membered saturated or unsaturated carbocyclic or heterocyclic ring, R 9 is hydrogen or lower alkyl, or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof.

[0114] In another embodiment, the compound of formula I is independently R 1 , R 2 , R 3 and R 4 are H, respectively, Q 1 is aryl or heteroaryl, R 5 , R 6 , R 7 , R 8 and R 9 are each independently H or lower alkyl, and X is CH2, C=O, or C=NR 9 and or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof.

[0115] "Alkyl" refers to a straight or branched chain unsubstituted hydrocarbon group of 1 to 20 carbon atoms, preferably 1 to 7 carbon atoms.

[0116] "Lower alkyl" refers to unsubstituted alkyl groups of 1 to 4 carbon atoms.

[0117] "Substituted alkyl" refers to halo, hydroxy, alkoxy, oxo, alkanoyl, aryloxy, alkanoyloxy, amino, alkylamino, arylamino, arylalkylamino, disubstituted amino where two amino substituents are selected from alkyl, aryl, or arylalkyl, alkanoylamino, aroylamino, aralkanoylamino, substituted alkanoylamino, substituted arylamino, substituted aralkanoylamino, thiol, alkylthio, arylthio, arylalkylthio, alkylthiono, arylthiono, arylalkylthiono, alkylsulfonyl, arylsulfonyl, arylalkyl. These include arylsulfonyl, sulfonamido, substituted sulfonamido, nitro, cyano, carboxy, carbamyl, substituted carbamyl (e.g., CONHalkyl, CONHaryl, CONHarylalkyl, and when two substituents are on the nitrogen, selected from alkyl, aryl, or arylalkyl), alkoxycarbonyl, aryl, substituted aryl, guanidino, heterocyclyl (e.g., indolyl, imidazolyl, furyl, thienyl, thiazolyl, pyrrolidyl, pyridyl, pyrimidyl, pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, homopiperazinyl, etc.), and substituted heterocyclyl. When a substituent is further substituted, it may be alkyl, alkoxy, aryl, or arylalkyl.

[0118] "Halogen" or "halo" refers to fluorine, chlorine, bromine, and iodine.

[0119] "Aryl" refers to monocyclic or bicyclic aromatic hydrocarbon groups having 6 to 12 carbon atoms in the ring, such as phenyl, naphthyl, biphenyl, and diphenyl, each of which may be substituted.

[0120] "Aryloxy," "arylamino," "arylalkylamino," "arylthio," "arylalkanoylamino," "arylsulfonyl," "arylalkoxy," "arylsulfinyl," "arylheteroaryl," "arylalkylthio," "arylcarbonyl," "arylalkenyl," and "arylalkylsulfonyl" refer to an aryl or substituted aryl bound to oxygen, amino, alkylamino, thio, alkanoylamino, sulfonyl, alkoxy, sulfinyl, heteroaryl or substituted heteroaryl, alkylthio, carbonyl, alkenyl, or alkylsulfonyl, respectively.

[0121] "Arylsulfonylaminocarbonyl" refers to an arylsulfonyl attached to an aminocarbonyl.

[0122] "Aryloxyalkyl," "aryloxycarbonyl," or "aryloxyaryl" refers to an aryloxy bonded to an alkyl or substituted alkyl, carbonyl, or aryl or substituted aryl, respectively.

[0123] "Arylalkyl" refers to an alkyl or substituted alkyl in which at least one of the hydrogen atoms bonded to at least one of the carbon atoms is replaced with an aryl or substituted aryl. Exemplary arylalkyls include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethene-1-yl, naphthobenzyl, and naphthophenylethan-1-yl.

[0124] "Arylalkyloxy" refers to an arylalkyl attached through an oxygen linkage (-O-arylalkyl).

[0125] The term "substituted aryl" refers to an aryl group substituted with 1 to 4 substituents, such as alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, arylalkyl, halo, trifluoromethoxy, trifluoromethyl, hydroxy, alkoxy, alkanoyl, alkanoyloxy, aryloxy, arylalkyloxy, amino, alkylamino, arylamino, arylalkylamino, dialkylamino, alkanoylamino, thiol, alkylthio, ureido, nitro, cyano, carboxy, carboxyalkyl, carbamyl, alkoxycarbonyl, alkylthiono, arylthiono, arylsulfonylamine, sulfonic acid, alkylsulfonyl, sulfonamide, aryloxy, etc. The substituents may be further substituted with hydroxy, halo, alkyl, alkoxy, alkenyl, alkynyl, aryl, or arylalkyl.

[0126] "Heteroaryl" refers to an optionally substituted aromatic 4- to 7-membered monocyclic, 7- to 11-membered bicyclic, or 10- to 15-membered tricyclic ring system having a ring containing at least one heteroatom and at least one carbon atom, e.g., pyridine, tetrazole, indazole.

[0127] "Alkenyl" refers to a straight or branched chain hydrocarbon group having 1 to 4 double bonds and having 2 to 20 carbon atoms, preferably 2 to 15 carbon atoms, and most preferably 2 to 8 carbon atoms.

[0128] The term "substituted alkenyl" refers to an alkenyl group substituted with 1 to 2 substituents, such as halo, hydroxy, alkoxy, alkanoyl, alkanoyloxy, amino, alkylamino, dialkylamino, alkanoylamino, thiol, alkylthio, alkylthiono, alkylsulfonyl, sulfonamido, nitro, cyano, carboxy, carbamyl, substituted carbamyl, guanidino, indolyl, imidazolyl, furyl, thienyl, thiazolyl, pyrrolidyl, pyridyl, pyrimidyl, or the like.

[0129] "Alkynyl" refers to a straight or branched chain hydrocarbon group of 2 to 20 carbon atoms, preferably 2 to 15 carbon atoms, and most preferably 2 to 8 carbon atoms, having 1 to 4 triple bonds.

[0130] "Substituted alkynyl" refers to alkynyl groups substituted with halo, hydroxy, alkoxy, alkanoyl, alkanoyloxy, amino, alkylamino, dialkylamino, alkanoylamino, thiol, alkylthio, alkylthiono, alkylsulfonyl, sulfonamido, nitro, cyano, carboxy, carbamyl, substituted carbamyl, guanidino, and heterocyclyl, such as imidazolyl, furyl, thienyl, thiazolyl, pyrrolidyl, pyridyl, pyrimidyl, and the like.

[0131] "Alkylidene" refers to an alkylene group consisting of at least two carbon atoms and at least one carbon-carbon double bond. Substituents on this group include those substituents within the definition of "substituted alkyl."

[0132] "Cycloalkyl" refers to an optionally substituted, saturated cyclic hydrocarbon ring system, preferably containing 1-3 rings and 3-7 carbons per ring, which may be further fused to an unsaturated C3-C7 carbocyclic ring. Representative groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl, and adamantyl. Representative substituents include one or more alkyl groups or one or more of the groups described herein as alkyl substituents.

[0133] "Heterocycle", "heterocyclic", and "heterocyclyl" refer to an optionally substituted, fully saturated or unsaturated, aromatic or nonaromatic cyclic group of a 4- to 7-membered monocyclic, 7- to 11-membered bicyclic, or 10- to 15-membered tricyclic ring system having at least one heteroatom in the ring containing at least one carbon atom. Each ring of a heteroatom-containing heterocyclic group may have 1, 2, or 3 heteroatoms selected from nitrogen, oxygen, and sulfur, and the nitrogen and sulfur heteroatoms may also be optionally oxidized and the nitrogen heteroatom may also be optionally quaternized. The heterocyclic group may be attached to any heteroatom or carbon atom.

[0134] Representative monocyclic heterocyclic groups include pyrrolidinyl, pyrrolyl, indolyl, pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl, imidazolinyl, imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl, isoxazolyl, thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolidinyl, furyl, tetrahydrofuryl, thienyl, oxadiazolyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, homopiperazinyl, 2-oxo ... These include isohomopiperazinyl, 2-oxopyrrolidinyl, 2-oxazepinyl, azepinyl, 4-piperidonyl, pyridyl, N-oxo-pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, tetrahydropyranyl, morpholinyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, 1,3-dioxane, tetrahydro-1,1-dioxothienyl, dioxanyl, isothiazolidinyl, thietanyl, thiiranyl, triazinyl, and triazolyl.

[0135] Representative bicyclic heterocyclic groups include 2,3-dihydro-2-oxo-1H-indolyl, benzothiazolyl, benzoxazolyl, benzothienyl, quinuclidinyl, quinolinyl, quinolinyl-N-oxide, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuryl, chromonyl, coumarinyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridyl, furopyridinyl (such as furo[2,3-c]pyridinyl, furo[3,1-b]pyridinyl, furo[2,3-b]pyridinyl), dihydroisoindolyl, dihydroquinazolinyl (3,4-dihydro-4-oxo- quinazolinyl, etc.), benzisothiazolyl, benzisoxazolyl, benzodiazinyl, benzofurazanyl, benzothiopyranyl, benzotriazolyl, benzopyrazolyl, 1,3-benzodioxolyl, dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone, dihydrobenzopyranyl, indolinyl, indazolyl, isochromanyl, isoindolinyl, naphthyridinyl, phthalazinyl, piperonyl, purinyl, pyridopyridyl, pyrrolotriazinyl, quinazolinyl, tetrahydroquinolinyl, thienofuryl, thienopyridyl, thienothienyl, etc. Representative substituents include one or more alkyl or arylalkyl groups as described above, or one or more of the groups described above as alkyl substituents, and smaller heterocyclyls such as epoxides and aziridines.

[0136] "Carbocyclic ring" or "carbocyclyl" refers to a stable, saturated, partially saturated, or unsaturated, monocyclic or bicyclic hydrocarbon ring containing from 3 to 12 atoms. This includes monocyclic rings containing 5 or 6 atoms or bicyclic rings containing 9 or 10 atoms. Suitable values ​​include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, dihydroindenyl, and tetrahydronaphthyl.

[0137] "Optionally substituted" when referring to a "carbocyclic ring" or "carbocyclyl" means that the carbocyclic ring may be substituted at one or more substitutable ring positions with one or more of the group independently selected from alkyl (preferably lower alkyl), alkoxy (preferably lower alkoxy), nitro, monoalkylamino (preferably lower alkylamino), dialkylamino (preferably di(lower)alkylamino), cyano, halo, haloalkyl (preferably trifluoromethyl), alkanoyl, aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl, alkylamido (preferably lower alkylamido), alkoxyalkyl (preferably lower alkoxy(lower)alkyl), alkoxycarbonyl (preferably lower alkoxycarbonyl), alkylcarbonyloxy (preferably lower alkylcarbonyloxy) and aryl (preferably phenyl), wherein said aryl is optionally substituted with halo, lower alkyl, and lower alkoxy groups.

[0138] "Heteroatoms" include oxygen, sulfur, and nitrogen.

[0139] "Alkyl sulfone" means -R k S(=O)2R k R k is alkyl or substituted alkyl.

[0140] "Oxo" refers to the divalent group =O.

[0141] "Carbamate" refers to -OC(=O)NH2.

[0142] "Amide" refers to -C(=O)NH2.

[0143] "Sulfonamide" refers to -SO2NH2.

[0144] A "substituted amide," "substituted sulfonamide," or "substituted carbamate" refers to an amide, sulfonamide, or carbamate, respectively, that has at least one hydrogen replaced with a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, and substituted cycloalkyl.

[0145] Substituted amide refers to -C(=O)NR''R'', where at least one of R''' or R'' is a substituted moiety, R''' and R'' are independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, and substituted cycloalkyl.

[0146] Substituted sulfonamide is -SONR o R p R refers to the group o or R p is a substituted moiety, then R o and R p is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, and substituted cycloalkyl.

[0147] Substituted carbamates are -OC(=O)NR q R r R q or R r is a substituted moiety, then R q and R r is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, and substituted cycloalkyl.

[0148] "Ureido" refers to the group -NHC(=O)NH2.

[0149] "Cyano" refers to the group -CN.

[0150] "Cycloalkylalkyl" or "cycloalkylalkoxy" refers to a cycloalkyl or substituted cycloalkyl, or alkoxy bound to an alkyl or substituted alkyl, respectively.

[0151] "Nitro" refers to the -N(O) group.

[0152] "Thio" refers to the -SH group.

[0153] "Alkylthio" means -SR s R refers to the group s is alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0154] "Thioalkyl" means -R t S group, R t is alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0155] "Alkylsulfonyl" means -S(=O)R u R refers to the group u is alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0156] "Alkylsulfinyl" means -S(=O)R v R refers to the group v is alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0157] "Carboxy" refers to the group -C(=O)OH.

[0158] "Carboxyalkoxy" or "alkoxycarbonylalkoxy" refers to a carboxy or alkoxycarbonyl, respectively, linked to an alkoxy.

[0159] "Alkoxycarbonyl" means -C(=O)OR w R refers to the groupw is alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl.

[0160] "Arylalkoxycarbonyl" refers to an aryl or substituted aryl bound to an alkoxycarbonyl.

[0161] "Alkylcarbonyloxy" or "arylcarbonyloxy" refers to -OC(=O)R x R refers to the group x is independently alkyl or substituted alkyl, or aryl or substituted aryl.

[0162] "Carbamoyl" means the -OC(=O)NH2 group, -OC(=O)NHR x Group and / or -C(=O)NR y R z R refers to the group y and R z is independently selected from alkyl and substituted alkyl.

[0163] -NR 6 (C=O)R 9 The group is R 6 is selected from hydrogen, lower alkyl and substituted lower alkyl; R 9 is selected from hydrogen, alkyl, substituted alkyl, alkoxy, aminoalkyl, substituted aminoalkyl, alkylamino, substituted alkylamino, aryl, and substituted aryl.

[0164] "Carbonyl" refers to C(=O).

[0165] "Alkylcarbonyl," "aminocarbonyl," "alkylaminocarbonyl," "aminoalkylcarbonyl," or "arylaminocarbonyl" refers to an alkyl, substituted alkyl, amino, alkylamino, substituted alkylamino, aminoalkyl, substituted aminoalkyl, or arylamino, respectively, bonded to a carbonyl.

[0166] An "aminocarbonylaryl" or "aminocarbonylalkyl" refers to an aminocarbonyl bound to an aryl or substituted aryl, or an alkyl or substituted alkyl, respectively.

[0167] "Sulfonyl" refers to the group S(=O).

[0168] "Sulfinyl" refers to the group S(=O).

[0169] "Carboxyalkyl" refers to an alkyl or substituted alkyl linked to a carboxy.

[0170] The compound of formula I may form a salt. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, but other salts are also useful, for example, when isolating or purifying the compound. The compound of formula I may form a salt with alkali metals such as sodium, potassium and lithium, alkaline earth metals such as calcium and magnesium, organic bases such as dicyclohexylamine, tributylamine, pyridine, and amino acids such as arginine and lysine. Such salts can be formed by methods known to those skilled in the art.

[0171] Examples of pharma- ceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyrate-1,4-diol, hexyrate-1,6-diol, hexyrate-2,7-diol, hexyrate-2,8-diol, hexyrate-2,9-diol, hexyrate-2,10-diol, hexyrate-2,11-diol, hexyrate-2,12-diol, hexyrate-2,13-diol, hexyrate-2,14-diol, hexyrate-2,15-diol, hexyrate-2,16-diol, hexyrate-2,17-diol, hexyrate-2,18-diol, hexyrate-2,20-diol, hexyrate-2,21-diol, hexyrate-2,22-diol, hexyrate-2,3-diol, hexyrate-2,4-diol, hexyrate-2,5-diol, hexyrate-2,6-diol, hexyrate-2,7-diol, hexyrate-2,8-diol, hexyrate-2,19-diol, hexyrate-2,23-diol, hexyrate-2,4-diol, hexyrate-2,5-diol, hexyrate-2,6-diol, hexyrate-2,7-diol, hexyrate-2,8-diol, hex -diacid salts, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, methylsulfonates, propylsulfonates, besylates, xylenesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, gamma-hydroxybutyrates, glycolates, tartrates, and mandelates.

[0172] The compounds of formula I may form salts with a variety of organic and inorganic acids. Such salts include those formed with hydrochloride, hydrobromide, methanesulfonic acid, sulfuric acid, acetic acid, trifluoroacetic acid, oxalic acid, maleic acid, benzenesulfonic acid, toluenesulfonic acid, and various others (e.g., nitrates, phosphates, borates, tartrates, citrates, succinates, benzoates, ascorbates, salicylates, etc.). Such salts may be formed by methods known to those skilled in the art. Additionally, zwitterions ("inner salts") may be formed.

[0173] Also, all stereoisomers of the compound are contemplated as anti-NTCP agents, either in mixture or in pure or substantially pure form.The definition of the compound therefore embraces all possible stereoisomers and their mixtures, in particular racemates and isolated optical isomers with the specified activity.The racemates can be resolved by physical methods, such as fractional recrystallization, separation or crystallization of diastereomeric derivatives, or separation by chiral column chromatography.The individual optical isomers can be obtained from the racemates by conventional methods, such as, for example, crystallization after salt formation with an optically active acid.

[0174] The compounds of formula I may also be administered in the form of prodrugs. "Prodrug" is intended to include any covalently bonded carrier that releases an active parent drug in vivo when the prodrug is administered to a mammalian subject. Prodrugs are prepared by modifying functional groups present in the compound such that the modification is cleaved, either by routine manipulation or in vivo, to the parent compound. Prodrugs include the compounds of the present invention in which a hydroxy, amino, or sulfhydryl group is bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxyl, free amino, or free sulfhydryl group, respectively. Examples of prodrugs include acetate, formate, and benzoate derivatives of alcohol and amine functional groups in the compounds of the present invention. Various forms of prodrugs are well known in the art. For example, see Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and Methods in E. nzymology, Vol.112, pp. 309-396, edited by K. Widder, et al. (Academic Press, 1 See 985).

[0175] Also included as anti-NTCP agents are solvates (eg, hydrates) of the compounds of formula I. Methods of solvation are known in the art.

[0176] The compounds of formula I may be administered in a dose range of about 0.05 to 200 mg / kg / day, preferably less than 100 mg / kg / day, in a single dose or in 2 to 4 divided doses.

[0177] [Combination of NTCP inhibitors] According to the present invention, one or more NTCP inhibitors may be administered to a subject. One of the NTCP inhibitors may be a pre-S1 peptide inhibitor. Two or more NTCP inhibitors may be administered sequentially. For example, one NTCP inhibitor, such as a pre-S1 peptide inhibitor, may be administered in a course that includes at least one cycle or one or more cycles (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles), followed by a next NTCP inhibitor in a course that includes at least one cycle or one or more cycles (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles). The duration of one cycle may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. The duration of one cycle may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. The duration of a course may be 12 weeks, 24 weeks, 36 weeks, 48 ​​weeks, 60 weeks, 1 year, 1.1 years, 1.2 years, 1.3 years, 1.4 years, 1.5 years, 1.6 years, 1.7 years, 1.8 years, 1.8 years, 1.9 years, or 2.0 years, or 3 years, or 4 years or more.

[0178] In the use and method of the present invention, two or more NTCP inhibitors may be administered concomitantly / simultaneously. According to this administration schedule, the administration of two or more NTCP inhibitors overlaps in time. The duration of administration of two or more NTCP inhibitors may be the same or essentially the same. For example, two NTCP inhibitors, one of which is a pre-S1 peptide inhibitor, may be administered in a course that includes at least one cycle or one or more cycles (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles). The duration of one cycle may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. The duration of one cycle may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. The duration of one course may be 12 weeks, 24 weeks, 36 weeks, 48 ​​weeks, 60 weeks, 1 year, 1.1 years, 1.2 years, 1.3 years, 1.4 years, 1.5 years, 1.6 years, 1.7 years, 1.8 years, 1.8 years, 1.9 years, or 2.0 years, or 3 years, or 4 years or more. For example, a 24-week course of myrrhodex B (e.g., 5 mg daily) may be administered simultaneously with a 24-week course of another NTCP inhibitor, such as a compound of formula I or a modified bile salt. In this dosing regimen, myrrhodex B may be administered daily, while the other NTCP inhibitor may be administered weekly. In this dosing schedule, the two NTCP inhibitors may be administered simultaneously, e.g., essentially simultaneously, or in a single composition.

[0179] In the use and method of the present invention, NTCP inhibitor may be delivered by various delivery routes depending on the type of component.Administration routes include enteral route (e.g. oral and rectal), parenteral route (e.g. intravenous, intramuscular, subcutaneous, intraperitoneal) and topical.Preferably, pre-S1 peptide inhibitor is delivered subcutaneously.

[0180] [Pharmaceutical composition] The NTCP inhibitors used according to the present invention may be included in pharmaceutical compositions.The pharmaceutical compositions used according to the present invention may be in a form suitable for oral use, such as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs.

[0181] The composition intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and may contain one or more agents selected from the group consisting of sweeteners, flavorings, colorings and preservatives to provide pharma-ceutical elegant and palatable preparations.The preparation for oral use may be provided as a hard gelatin capsule in which the active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate or kaolin, or as a soft gelatin capsule in which the active ingredient is mixed with a water-soluble carrier, such as polyethylene glycol, or an oil medium, such as peanut oil, liquid paraffin, or olive oil.

[0182] The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution and isotonic sodium chloride solution. The sterile injectable preparation may also be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oily phase. For example, the active ingredient may be first dissolved in a mixture of soybean oil and lecithin. The oil solution is then introduced into a mixture of water and glycerol and processed to form a microemulsion.

[0183] The injectable solution or microemulsion may be introduced into the patient's bloodstream by local bolus injection. Alternatively, it may be advantageous to administer the solution or microemulsion in such a way as to maintain a constant circulating concentration of the compound currently present. In order to maintain such a constant concentration, a continuous intravenous delivery device may be utilized.

[0184] The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension for intramuscular and subcutaneous administration, which suspension may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.

[0185] [Diseases to be treated] The present invention relates to the treatment of primary biliary cirrhosis, atherosclerosis, and NRLP3 inflammasome-related diseases, including but not limited to type 2 diabetes, atherosclerosis, gout, Alzheimer's disease, and NASH.

[0186] [Treatment regimen] When the NTCP inhibitor used according to the present invention is administered to human subjects, the daily dosage is usually determined by the prescribing physician, with the dosage generally varying according to the age, weight, sex and response of individual patient, and the severity of the patient's condition.The representative dosage of some NTCP inhibitors used according to the present invention is described herein.

[0187] Within the scope of the use of the present invention, NTCP inhibitors may be provided in therapeutically effective amounts.The term "therapeutically effective amount" or "effective amount" generally refers to the amount of therapeutic agent that is effective when administered alone or in combination with additional therapeutic agent to cell, tissue, or subject to prevent or improve infection, or prevent or improve the progression of infection or the progression of associated disease associated with infection.Therapeutically effective dose further refers to the amount of therapeutic agent that is sufficient to improve symptoms, for example, treat, cure, prevent or improve associated medical condition, or increase the rate of treatment, cure, prevention or improvement of that condition.

[0188] When applied to an individual active ingredient administered alone, the therapeutically effective dose refers to that ingredient alone. When applied to a combination, the therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, sequentially or simultaneously. In particular, an effective amount is an amount that inhibits or reduces viral entry into cells.

[0189] In the uses and methods of the present invention, the dosage of each active ingredient may be adjusted depending on the progress of the treatment and / or on any side effects the patient experiences during treatment.

[0190] The present invention expressly includes the specific active ingredients (as NTCP inhibitors) and dosing schedules set forth in the Examples of this application.

[0191] The invention will now be described with reference to the following non-limiting examples.

[0192] [Specific item] This application discloses, among other things, certain items.

[0193] 1. An inhibitor of Na+-taurocholate cotransporting polypeptide (NTCP) for use in a method for treating atherosclerosis, primary biliary cirrhosis, or an NRLP3 inflammasome-associated disease in a subject.

[0194] 2. The NTCP inhibitor for use in item 1, wherein the NTCP inhibitor is a pre-S1 peptide inhibitor, the pre-S1 peptide inhibitor comprising a peptide comprising the amino acid sequence NPLGFX0P (SEQ ID NO:15), wherein X0 is any amino acid, preferably F or L, more preferably F.

[0195] 3. The NTCP inhibitor for use in item 2, wherein the pre-S1 peptide inhibitor further comprises an N-terminal sequence of at least 4 amino acids at the N-terminus of NPLGFX0P (SEQ ID NO:15).

[0196] 4. An NTCP inhibitor for use in item 3, wherein the N-terminal sequence consists of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids, or the N-terminal sequence consists of 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 4 to 11, 4 to 12, 4 to 13, 4 to 14, 4 to 15, 4 to 16, 4 to 17, 4 to 18, or 4 to 19 amino acids.

[0197] 5. An NTCP inhibitor for use in item 3 or 4, wherein at least one amino acid in the N-terminal sequence has an amino group in the side chain.

[0198] 6. An NTCP inhibitor for use in item 5, wherein at least one amino acid in the N-terminal sequence is selected from lysine, α-aminoglycine, α,γ-diaminobutyric acid, ornithine, α,β-diaminopropionic acid, preferably lysine.

[0199] 7. An NTCP inhibitor for use in item 5 or 6, wherein at least one amino acid of the N-terminal sequence is located at the N-terminus of the N-terminal sequence.

[0200] 8. NTCP inhibitors for use according to items 5 to 7, wherein at least one amino acid in the N-terminal sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids, or at least one amino acid in the N-terminal sequence is 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10 amino acids.

[0201] 9. An NTCP inhibitor for use in items 3 to 8, wherein the N-terminal sequence comprises the amino acid sequence NX1SX2X3 (SEQ ID NO:16), where X1, X2 and X3 are any amino acid.

[0202] 10.NX1SX2X3 (SEQ ID NO:16), X1 is L, I or Q, preferably L; X2 is T, V, A or absent, preferably T or V, more preferably T; and / or X3 is P, S, T or F, preferably P or S, more preferably S NTCP inhibitors for use in item 9.

[0203] 11. The NTCP inhibitor for use in item 9 or 10, in which the amino acid sequence NX1SX2X3 (SEQ ID NO: 16) is directly linked to the N-terminus of NPLGFX0P (SEQ ID NO: 15).

[0204] 12. The pre-S1 peptide inhibitor comprises the amino acid sequence NX1SX2X3NPLGFX0P (SEQ ID NO: 17); X0 is any amino acid, X1 is L, I or Q, preferably L; X2 is T, V, A or absent, preferably T or V, more preferably T; and X3 is P, S, T or F, preferably P or S, more preferably S NTCP inhibitors for use in items 2 to 11.

[0205] 13. The NTCP inhibitor for use in items 2 to 12, wherein the pre-S1 peptide inhibitor further comprises a C-terminal sequence of at least one amino acid at the C-terminus of NPLGFX0P (SEQ ID NO:15).

[0206] 14. The C-terminal sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122 14. The NTCP inhibitor for use in accordance with item 13, which consists of 2, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 amino acids, or the C-terminal sequence consists of 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-26, 1-27, 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, 1-34 or 1-35 amino acids.

[0207] 15. The NTCP inhibitor for use in item 13 or 14, wherein the C-terminal sequence comprises the amino acid sequence X4HQLDP (SEQ ID NO:18), where X4 is any amino acid.

[0208] 16. The NTCP inhibitor for use of item 15, wherein X4 is D, E or S, preferably D or E, more preferably D.

[0209] 17. The NTCP inhibitor for use in item 15 or 16, in which the amino acid sequence X4HQLDP (SEQ ID NO: 18) is directly linked to the C-terminus of NPLGFX0P (SEQ ID NO: 15).

[0210] 18. The pre-S1 peptide inhibitor comprises the amino acid sequence NPLGFX0PX4HQLDP (SEQ ID NO: 19); X0 is any amino acid, and X4 is D, E or S, preferably D or E, more preferably D NTCP inhibitors for use in items 2 to 17.

[0211] 19. The pre-S1 peptide inhibitor comprises the amino acid sequence NX1SX2X3NPLGFX0PX4HQLDP (SEQ ID NO: 20); X0 is any amino acid, preferably F or L, more preferably F; X1 is L, I or Q, preferably L; X2 is T, V, A or absent, preferably T or V, more preferably T; X3 is P, S, T or F, preferably P or S, more preferably S, and X4 is D, E or S, preferably D or E, more preferably D. NTCP inhibitors for use in items 2 to 18.

[0212] 20. Pre-S1 peptide inhibitors 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 19. An NTCP inhibitor for use in any one of items 2 to 19, comprising or consisting of 0, 71, 72, 73, 74, 75, 76, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118 or 119 amino acid.

[0213] 21. An NTCP inhibitor for use according to items 2 to 20, wherein the pre-S1 peptide inhibitor comprises a pre-S1 peptide of the HBV virus or a functional fragment thereof, the function of which is preferably binding to NTCP, inhibiting NTCP or reducing NTCP activity.

[0214] 22. Pre-S1 peptide inhibitors At least amino acids 9-15, 8-15, 7-15, 6-15, 5-15, 4-15, 3-15, 2-15 of the pre-S1 peptide of the HBV virus; At least amino acids 9-16, 9-17, 9-18, 9-19, 9-20, 9-21, 9-22, 9-23, 9-24, 9-25, 9-26, 9-27, 9-28, 9-29, 9-30, 9-31, 9-32, 9-33, 9-34, 9-35, 9-36, 9-37, 9-38, 9-39, 9-40, 9-41, 9-42, 9-43, 9-44, 9-45, 9-46, 9-47, 9-48, at least amino acids 8-16, 7-17, 6-18, 5-19, 4-20, 3-21, or 2-22 of the pre-S1 peptide of the HBV virus; at least amino acids 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-21, 2-22, 2-23, 2-24, 2-25, 2-26, 2-27, 2-28, 2-29, 2-30, 2-31, 2-32, 2-33, 2-34, 2-35, 2-36, 2-37, 2-38, 2-39, 2-40, 2-41, 2-42, 2-43, 2-44, 2-45, 2-46, 2-47, or 2-48 of the pre-S1 peptide of the HBV virus; at least amino acids 9-15 and 16-20 of the pre-S1 peptide of the HBV virus; at least amino acids 2-8 and 9-15 of the pre-S1 peptide of the HBV virus; at least amino acids 2-8, 9-15, and 16-20 of the pre-S1 peptide of the HBV virus; at least amino acids 9-15 and 34-48 of the pre-S1 peptide of the HBV virus; at least amino acids 9-15, 16-20, and 34-48 of the pre-S1 peptide of the HBV virus; at least amino acids 2-8, 9-15, and 34-48 of the pre-S1 peptide of the HBV virus; at least amino acids 2-8, 9-15, 16-20, and 34-48 of the pre-S1 peptide of the HBV virus; a portion of the pre-S1 peptide of the HBV virus consisting of at least amino acids 2 to 48 or at least 15 amino acids; Amino acids 2 to 48 of the pre-S1 peptide of the HBV virus, or A sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to any of the above sequences. 22. The NTCP inhibitor for use according to item 21, comprising or consisting of:

[0215] 23. The NTCP inhibitor for use in accordance with item 21 or 22, wherein the HBV virus is HBV strain alpha 1, HBV strain LSH, woodchuck HBV, woolly monkey HBV (WMHBV), orangutan HBV, chimpanzee HBV, gorilla HBV, human HBV, HBV subtype AD, ADR, ADW, ADYW, AR or AYW, or HBV genotype A, B, C, D, E, F, G or H.

[0216] 24. Pre-S1 peptide inhibitors have the amino acid sequence (-11)-M GGWSS TPRKG MGTNL SVPNP LGFFP DHQLD PAFRA NSNNP DWDFN PNKDH WPEAN KVG-48 (SEQ ID NO:1) (-11)-M GGWSS KPRKG MGTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PVKDD WPAAN QVG-48 (SEQ ID NO:2) (-11)-M GGWSS KPRKG MGTNL SVPNP LGFFP DHQLD PAFKA NSENP DWDLN PHKDN WPDAN KVG-48 (SEQ ID NO:3) (-11)-M GGWSS KPRQG MGTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDH WPEAN QVG-48 (SEQ ID NO:4) 1-MGQNL STSNP LGFFP DHQLD PAFRA NTANP DWDFN PNKDT WPDAN KVG-48 (SEQ ID NO:5) (-10)-MGLSW TVPLE WGKNI STTNP LGFP DHQLD PAFRA NTRNP DWDHN PNKDH WTEAN KVG-48 (SEQ ID NO:6) (-11)-M GAPLS TTRRG MGQNL SVPNP LGFFP DHQLD PLFRA NSSSP DWDFN TNKDS WPMAN KVG-48 (SEQ ID NO:7) (-10)-MGLSW TVPLE WGKNL SASNP LGFLP DHQLD PAFRA NTNNP DWDFN PKKDP WPEAN KVG-48 (SEQ ID NO:8) (-11)-M GAPLS TARRG MGQNL SVPNP LGFFP DHQLD PLFRA NSSSP DWDFN TNKDN WPMAN KVG-48 (SEQ ID NO:9) 1-MGLNQ STFNP LGFFP SHQLD PLFKA NAGSA DWDKN PNKDP WPQAH DTA (SEQ ID NO:10) (-11)-M GGWSS KPRQG MGTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDH WPEAN KVG-48 (SEQ ID NO:11), or a fragment thereof of at least 15 amino acids, or A sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to any of the above sequences. 24. An NTCP inhibitor for use according to items 2 to 23, comprising or consisting of:

[0217] 24. Pre-S1 peptide inhibitors have the amino acid sequence A sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to any of the above sequences. 24. An NTCP inhibitor for use according to items 2 to 23, comprising or consisting of:

[0218] 25. NTCP inhibitors have the amino acid sequence GTNL SVPNP LGFFP DHQLD PAFRA NSNNP DWDFN PNKDH WPEAN KVG (SEQ ID NO:12), GTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDH WPEAN KVG (SEQ ID NO:13), GTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDH WPEAN QVG (SEQ ID NO:14), or A sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to any of the above sequences. 25. An NTCP inhibitor for use according to items 2 to 24, comprising or consisting of:

[0219] 26. An NTCP inhibitor for use in items 2 to 25, wherein the pre-S1 peptide inhibitor is modified by at least one hydrophobic moiety at the N-terminus or the 1st, 2nd, 3rd or 4th amino acid proximal to the N-terminus.

[0220] 27. The NTCP inhibitor for use in item 26, wherein the hydrophobic moiety modification is acylation.

[0221] 28. The NTCP inhibitor for use according to item 27, wherein the acylation is with a carboxylic acid, a fatty acid or an amino acid having a lipophilic side chain.

[0222] 29. NTCP inhibitor for use in item 28, wherein the fatty acid is a saturated or unsaturated fatty acid, a branched or unbranched fatty acid, preferably having 8 to 22 carbon atoms (C8 to C22).

[0223] 30. An NTCP inhibitor for use in items 27 to 29, wherein the acylation is with myristoyl (C14), palmitoyl (C16), or stearoyl (C18).

[0224] 31. An NTCP inhibitor for use in accordance with items 27 to 30, wherein acylation is acylation with myristoyl.

[0225] 32. The NTCP inhibitor for use in accordance with item 26, wherein the hydrophobic moiety modification is the addition of a hydrophobic moiety.

[0226] 33. The NTCP inhibitor for use according to item 32, wherein the hydrophobic moiety is cholesterol, a derivative of cholesterol, a phospholipid, a glycolipid, a glycerol ester, a steroid, a ceramide, an isoprene derivative, an adamantane, farnesol, an aliphatic group, a polyaromatic compound, oleic acid, a bile salt or a bile salt conjugate, preferably oleic acid, cholesterol, a bile salt or a bile salt conjugate.

[0227] 34. An NTCP inhibitor for use in accordance with item 32 or 33, in which the hydrophobic moiety is attached by a covalent bond, preferably via a carbamate, amide, ether, or disulfide.

[0228] 35. An NTCP inhibitor for use in items 2 to 34, wherein the pre-S1 peptide inhibitor is modified with at least one protecting moiety at the C-terminus or the 1st, 2nd, 3rd or 4th amino acid proximal to the C-terminus, and the protecting moiety protects the pre-S1 peptide inhibitor from degradation.

[0229] 36. The NTCP inhibitor for use in accordance with item 35, wherein the protective moiety is an amide, a D-amino acid, a modified amino acid, a cyclic amino acid, an albumin, a glycan, or a natural or synthetic polymer, preferably PEG.

[0230] 37. An NTCP inhibitor for use in item 35 or 36, wherein the protective moiety is an amide.

[0231] 38. An NTCP inhibitor for use in items 35 to 37, in which the protective moiety is attached via a linker.

[0232] 39. The linker is polyalanine, polyglycine, carbohydrate, or (CH2) n 39. The NTCP inhibitor for use in accordance with item 38, wherein n is 1 or more.

[0233] 40. An NTCP inhibitor for use in items 2 to 39, wherein the pre-S1 peptide inhibitor consists of amino acid sequence 2 to 48 of the pre-S1 peptide of genotype C, modified at the N-terminus by myristoylation and further modified at the C-terminus with an amide.

[0234] 41. An NTCP inhibitor for use in accordance with items 2 to 40, wherein the pre-S1 peptide inhibitor is myristoyl-GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANKVG-amide.

[0235] 42. Pre-S1 peptide inhibitor is N-myristoyl-glycyl-L-threonyl-L-asparaginyl-L-leucyl-L-seryl-L-valyl-L-prolyl-L-asparaginyl-L-prolyl-L-leucyl-glycyl-L-phenylalanyl-L-phenylalanyl-L-prolyl-L-aspartyl-L-histidyl-L-glutaminyl-L-leucyl-L-aspartyl-L-prolyl-L-alanyl-L-phenylalanyl-glycyl-L-alanyl-L-asparaginyl 41. The NTCP inhibitor for use of items 2 to 40 which is -L-seryl-L-asparaginyl-L-asparaginyl-L-prolyl-L-aspartyl-L-tryptophanyl-L-aspartyl-L-phenylalanyl-L-asparaginyl-L-prolyl-L-asparaginyl-L-lysyl-L-aspartyl-L-histidyl-L-tryptophanyl-L-prolyl-L-glutamyl-L-alanyl-L-asparaginyl-L-lysyl-L-valyl-glycinamide, or an acetate salt thereof.

[0236] 43. The method comprises administering a pre-S1 peptide inhibitor At a dose such that the concentration of pre-S1 peptide inhibitor at the NTCP site, i.e., in the hepatocyte, is equal to or greater than a Ki of approximately 1-10 nM. at a daily dose of 0.1 mg to 50 mg, 0.5 mg to 20 mg, 1 mg to 15 mg, 1 mg to 10 mg, 1 mg to 5 mg, preferably 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg or 10 mg per day, or A daily dose of about 0.0014 mg / kg body weight to about 0.7 mg / kg body weight, preferably about 0.014 mg / kg body weight to about 0.28 mg / kg body weight (1 mg to 20 mg) 43. The NTCP inhibitor for use in accordance with items 2 to 42, comprising administering

[0237] 44. An NTCP inhibitor for use of items 2 to 43, wherein the method comprises administering the pre-S1 peptide inhibitor for 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 12 weeks, 24 weeks, 36 weeks, 48 ​​weeks, 60 weeks, 1 year, 1.1 years, 1.2 years, 1.3 years, 1.4 years, 1.5 years, 1.6 years, 1.7 years, 1.8 years, 1.9 years, or 2.0 years, or 3 years, or 4 years or more.

[0238] 45. An NTCP inhibitor for use in accordance with items 2 to 44, wherein the method comprises administering a pre-S1 peptide inhibitor at a dose of 2 mg daily or 5 mg daily for 24 weeks or 48 weeks.

[0239] 46. ​​An NTCP inhibitor for use of items 2 to 45, the method comprising administering the pre-S1 peptide inhibitor subcutaneously, intravenously, orally, intranasally, intramuscularly, transdermally, by inhalation or suppository, preferably intravenously or subcutaneously.

[0240] 47. An NTCP inhibitor for use in accordance with items 2 to 46, wherein the method comprises administering a pre-S1 inhibitor and a further NTCP inhibitor.

[0241] 48. An NTCP inhibitor for use in accordance with items 1 to 47, wherein the subject is a human.

[0242] 49. NTCP inhibitors for use in items 1 to 48, where atherosclerosis is at the stage of unstable atherosclerotic plaques.

[0243] 50. An NTCP inhibitor for use in accordance with items 1 to 49, wherein the method results in the reduction, resolution or elimination of atherosclerotic plaque.

[0244] 51. An NTCP inhibitor for use in items 1 to 48, wherein the method results in the reduction or elimination of symptoms of primary biliary cirrhosis.

[0245] 52. NTCP inhibitors for use in accordance with item 51, the method resulting in normalization of biochemical parameters and a reduction in the progression of inflammation and fibrosis.

[0246] 53. An NTCP inhibitor for use in items 1 to 48, wherein the NRLP3 inflammasome-associated disease is type 2 diabetes, atherosclerosis, gout, Alzheimer's disease, and NASH.

[0247] 54. NTCP inhibitors for use in item 53, the method of which results in normalization of biochemical parameters and improvement of disease beliefs.

[0248] 55. A method for treating atherosclerosis, primary biliary cirrhosis, or an NRLP3 inflammasome-associated disease in a subject, comprising administering to the subject an NTCP inhibitor.

[0249] 56. The method of item 55, wherein the NTCP inhibitor is a pre-S1 inhibitor as defined in items 2 to 42.

[0250] 57. The method of item 55 or 56, wherein the method is defined in items 43 to 47.

[0251] 58. The method of items 55 to 57, wherein the subject is a human.

[0252] 59. Methods according to items 55-58 in which the disease is defined in items 49-54. EXAMPLES

[0253] [Example 1: Examination of HTCP inhibitors in APO-E knockout mice] Objective: The aim of this study was to evaluate the effect of the HTCP inhibitor Milcurdex B at various doses on physiological and atherosclerotic lesion development in APO-E knockout mice, an animal model closely resembling the human atherosclerotic condition.

[0254] Methodology: Exam weeks 1-8: Randomization and assignment to the four study arms:

[0255] [Table 1]

[0256] Administration: daily subcutaneous injection Special Containment Conditions:

[0257] [Table 2]

[0258] Parameters to be determined:

[0259] [Table 3]

[0260] RESULTS: Myrcludex B was well tolerated.

[0261] [Example 2: Examination of HTCP inhibitors in LDL receptor knockout mice] Objective: The aim of this 23-week study was to evaluate the effect of the HTCP inhibitor Milcurdex B at various doses on physiological and atherosclerotic lesion development in LDL receptor knockout mice (LDLRKO), an animal model closely resembling the human atherosclerotic condition.

[0262] Methodology: 16 male and 6 female LDLRKO mice 8 weeks on a high calorie diet 4 male and 4 female victims The remaining animals were randomized into four groups. Daily subcutaneous Myrcurdex B treatment was initiated at week 15 and week 9 on a standard diet. Species / strain: Mouse LDLR knockout / B6.129S7-Ldlrtm1Her / J Supplier:The Jackson Laboratory.USA Exam weeks 1-8:

[0263] [Table 4]

[0264] [Table 5]

[0265] Parameters to be determined:

[0266] [Table 6]

[0267] Exam weeks 9-23: Randomization and assignment to the four study arms:

[0268] [Table 7]

[0269] Treatment: Daily subcutaneous injections starting at week 9 Special Containment Conditions:

[0270] [Table 8]

[0271] Parameters to be determined:

[0272] [Table 9]

[0273] RESULTS: Myrcludex B was well tolerated.

[0274] [Example 3: Pilot clinical trial of HTCP inhibitor Myrcludex B in patients with dyslipidemia] Objective: The objective of the clinical trial was to evaluate the safety, tolerability and efficacy of Myrcludex B in patients with dyslipidemia.

[0275] Methodology: 20 patients with dyslipidemia Myrcludex B, 10 mg daily for 8 weeks Endothelial function tests, lipid panels and additional biomarkers: Assays for macrophage cholesterol efflux and the ATP-binding cassette transporter genes ABCA1 and ABCG1 RESULTS: Myrcludex B was well tolerated.

[0276] [Example 4: Phase 2 clinical trial of PBC treatment with HTCP inhibitor Milcludex B] Objective: The objective of the clinical trial was to evaluate the safety and tolerability as well as efficacy of Myrcludex B in patients with primary biliary cirrhosis.

[0277] [Table 10-1]

[0278] [Table 10-2]

[0279] [References] Shih, DM, Shaposhnik, Z., Meng, Y., Rosales, M., Wang, X., Wu, J., … Lusis, AJ (2013).Hyodeoxycholic acid improves HDL function and inhibits atheroscler otic lesion formationin LDLR-knockout mice. FASEB Journal, 27(9), 3805-3817. ht tp: / / doi.org / 10.1096 / fj.12-223008 Talwalkar, J.A., & Lindor, K. D. (2003). Primary biliary cirrhosis. Lancet,362 (9377), 53-61.http: / / doi.org / S0140-6736(03)13808-1 [pii]\r10.1016 / S0140-6736(03 )13808-1 Davis, B.K.,Wen, H., and Ting, J.P. (2011). The inflammasome NLRs in immunity, inflammation,and associated diseases. Annu. Rev. Immunol. 29, 707-735 Martinon,F.,Petrilli, V., Mayor, A., Tardivel, A., and Tschopp, J. (2006). Gou t-associateduric acid crystals activate the NALP3 inflammasome. Nature 440, 237 -241 Schroder, K.,Zhou, R., and Tschopp, J. (2010). The NLRP3 inflammasome: a sensor formetabolic danger? Science 327, 296-300 Miller LC etal, (1995) Synthesis of interleukin-1β in primary biliary cirrhosi s: Relationship to treatment with methotrexate or colchicine and disease progres sion HepatologyVolume 22, Issue 2, 518-524 The present invention provides, for example, the following items. (Item 1) An inhibitor of Na+-taurocholate cotransporting polypeptide (NTCP) for use in a method for treating primary biliary cirrhosis, atherosclerosis, or an NRLP3 inflammasome-associated disease in a subject. (Item 2) The NTCP inhibitor for use in item 1, wherein the NTCP inhibitor is a pre-S1 peptide inhibitor, the pre-S1 peptide inhibitor comprising a peptide comprising the amino acid sequence NPLGFX0P (SEQ ID NO:15), wherein X0 is any amino acid, preferably F or L, more preferably F. (Item 3) The pre-S1 peptide inhibitor further comprises an N-terminal sequence of at least four amino acids at the N-terminus of NPLGFXOP (SEQ ID NO:15), preferably the N-terminal sequence comprises the amino acid sequence NX1SX2X3 (SEQ ID NO:16), where X1, X2 and X3 are any amino acid, preferably X1 is L, I or Q, preferably L; X2 is T, V, A or absent, preferably T or V, more preferably T; and / or X3 is P, S, T or F, preferably P or S, more preferably S NTCP inhibitors for use in item 2. (Item 4) The NTCP inhibitor for use in item 2 or 3, wherein the pre-S1 peptide inhibitor further comprises a C-terminal sequence of at least one amino acid at the C-terminus of NPLGFXOP (SEQ ID NO:15), preferably the C-terminal sequence comprises the amino acid sequence X4HQLDP (SEQ ID NO:18), wherein X4 is any amino acid, preferably X4 is D, E or S, preferably D or E, more preferably D. (Item 5) Pre-S1 peptide inhibitors have been shown to be effective in treating 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 1 5. An NTCP inhibitor for use in any of items 2 to 4, comprising or consisting of 1, 72, 73, 74, 75, 76, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118 or 119 amino acid. (Item 6) The pre-S1 peptide inhibitor comprises a pre-S1 peptide of the HBV virus or a functional fragment thereof, the function of which is preferably to bind to NTCP, inhibit NTCP, or reduce NTCP activity, and preferably the pre-S1 peptide inhibitor comprises at least amino acids 9-15, 8-15, 7-15, 6-15, 5-15, 4-15, 3-15, 2-15, 3-20, 4-25, 5-30, 6-30, 7-40, 8-40, 9-50, 10-15, 11-15, 12-15, 13-15, 14-15, 16-16, 17-17, 18-18, 19-20, 21-22, 22-24, 23-25, 24-26, 25-27, 26-28, 27-29, 28-30, 29-31, 32-33, 34-35, 36-37, 37-38, 38-39, 39-40, 39-41, 39-42, 39-43, 39-44, 39-45, 39-56, 39-57, 39-58, 39-60, 39-61, 39-70, 39-71, 39-82, 39-83, 39-90, 39-91, 39-92, 39-93, 39-94, 39-95, 39-106, 39-107, 39-108, 39-109, 39-109, 39-1109, 3 At least amino acids 9-16, 9-17, 9-18, 9-19, 9-20, 9-21, 9-22, 9-23, 9-24, 9-25, 9-26, 9-27, 9-28, 9-29, 9-30, 9-31, 9-32, 9-33, 9-34, 9-35, 9-36, 9-37, 9-38, 9-39, 9-40, 9-41, 9-42, 9-43, 9-44, 9-45, 9-46, 9-47, 9-48, at least amino acids 8-16, 7-17, 6-18, 5-19, 4-20, 3-21, or 2-22 of the pre-S1 peptide of the HBV virus; at least amino acids 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-21, 2-22, 2-23, 2-24, 2-25, 2-26, 2-27, 2-28, 2-29, 2-30, 2-31, 2-32, 2-33, 2-34, 2-35, 2-36, 2-37, 2-38, 2-39, 2-40, 2-41, 2-42, 2-43, 2-44, 2-45, 2-46, 2-47, or 2-48 of the pre-S1 peptide of the HBV virus; at least amino acids 9-15 and 16-20 of the pre-S1 peptide of the HBV virus; at least amino acids 2-8 and 9-15 of the pre-S1 peptide of the HBV virus; at least amino acids 2-8, 9-15, and 16-20 of the pre-S1 peptide of the HBV virus; at least amino acids 9-15 and 34-48 of the pre-S1 peptide of the HBV virus; at least amino acids 9-15, 16-20, and 34-48 of the pre-S1 peptide of the HBV virus; at least amino acids 2-8, 9-15, and 34-48 of the pre-S1 peptide of the HBV virus; at least amino acids 2-8, 9-15, 16-20, and 34-48 of the pre-S1 peptide of the HBV virus; a portion of the pre-S1 peptide of the HBV virus consisting of at least amino acids 2 to 48 or at least 15 amino acids; Amino acids 2 to 48 of the pre-S1 peptide of the HBV virus, or A sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to any of the above sequences. An NTCP inhibitor for use in any of items 2 to 5, comprising or consisting of: (Item 7) 7. The NTCP inhibitor for use in accordance with item 6, wherein the HBV virus is HBV strain alpha 1, HBV strain LSH, woodchuck HBV, woolly monkey HBV (WMHBV), orangutan HBV, chimpanzee HBV, gorilla HBV, human HBV, HBV subtype AD, ADR, ADW, ADYW, AR or AYW, or HBV genotype A, B, C, D, E, F, G or H. (Item 8) Pre-S1 peptide inhibitors have the amino acid sequence (-11)-M GGWSS TPRKG MGTNL SVPNP LGFFP DHQLD PAFRA NSNNP DWDFN PNKDH WPEAN KVG-48 (SEQ ID NO:1) (-11)-M GGWSS KPRKG MGTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PVKDD WPAAN QVG-48 (SEQ ID NO:2) (-11)-M GGWSS KPRKG MGTNL SVPNP LGFFP DHQLD PAFKA NSENP DWDLN PHKDN WPDAN KVG-48 (SEQ ID NO:3) (-11)-M GGWSS KPRQG MGTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDH WPEAN QVG-48 (SEQ ID NO:4) 1-MGQNL STSNP LGFFP DHQLD PAFRA NTANP DWDFN PNKDT WPDAN KVG-48 (SEQ ID NO:5) (-10)-MGLSW TVPLE WGKNI STTNP LGFP DHQLD PAFRA NTRNP DWDHN PNKDH WTEAN KVG-48 (SEQ ID NO:6) (-11)-M GAPLS TTRRG MGQNL SVPNP LGFFP DHQLD PLFRA NSSSP DWDFN TNKDS WPMAN KVG-48 (SEQ ID NO:7) (-10)-MGLSW TVPLE WGKNL SASNP LGFLP DHQLD PAFRA NTNNP DWDFN PKKDP WPEAN KVG-48 (SEQ ID NO:8) (-11)-M GAPLS TARRG MGQNL SVPNP LGFFP DHQLD PLFRA NSSSP DWDFN TNKDN WPMAN KVG-48 (SEQ ID NO:9) 1-MGLNQ STFNP LGFFP SHQLD PLFKA NAGSA DWDKN PNKDP WPQAH DTA (SEQ ID NO:10) (-11)-M GGWSS KPRQG MGTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDH WPEAN KVG-48 (SEQ ID NO:11), or a fragment thereof of at least 15 amino acids, or A sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to any of the above sequences. comprising or consisting of Preferably NTCP inhibitors have the amino acid sequence GTNL SVPNP LGFFP DHQLD PAFRA NSNNP DWDFN PNKDH WPEAN KVG (SEQ ID NO:12), GTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDH WPEAN KVG (SEQ ID NO:13), GTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDH WPEAN QVG (SEQ ID NO:14), or A sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to any of the above sequences. An NTCP inhibitor for use in any of items 2 to 7, comprising or consisting of: (Item 9) 9. An NTCP inhibitor for use according to any of items 2 to 8, wherein the pre-S1 peptide inhibitor is modified with at least one hydrophobic moiety at the N-terminus or the 1, 2, 3 or 4 amino acid adjacent to the N-terminus, and the hydrophobic moiety modification is preferably acylation, and the acylation is preferably acylation with myristoyl (C14), palmitoyl (C16), or stearoyl (C18). (Item 10) 10. The NTCP inhibitor for use in any of items 2 to 9, wherein the pre-S1 peptide inhibitor is myristoyl-GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANKVG-amide. (Item 11) 11. The NTCP inhibitor for use according to any of items 2 to 10, wherein the method comprises administering a pre-S1 inhibitor and a further NTCP inhibitor. (Item 12) The NTCP inhibitor for use in any one of items 1 to 11, wherein the subject is a human. (Item 13) 13. An NTCP inhibitor for use according to any of items 1 to 12, wherein the method results in a reduction, or elimination, of the symptoms of primary biliary cirrhosis, preferably wherein the method results in a normalization of biochemical parameters and a reduction in the progression of inflammation and fibrosis. (Item 14) 13. The use of any of items 1 to 12, wherein atherosclerosis is a stage of unstable atherosclerotic plaques and the method results in the reduction, resolution or elimination of atherosclerotic plaques. (Item 15) An NTCP inhibitor for use in any of items 1 to 12, wherein the NRLP3 inflammasome-associated disease is type 2 diabetes, atherosclerosis, gout, Alzheimer's disease, and NASH.

Claims

[Claim 1] The invention as depicted in the drawings.

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