Therapy using HTCP inhibitors

Abstract

The present invention relates to therapies using HTCP inhibitors. An inhibitor of a Na < + >-taurocholic acid co-transporter polypeptide (NTCP) for use in a method of treating atherosclerosis, primary biliary cirrhosis, or a NRLP3 inflammasome-related disease in a subject.

Description

[0001] This application is a divisional application of a patent application with an application date of April 18, 2018, application number 201810351809.7, and name “Therapy using HTCP inhibitors”. Field of the Invention

[0002] The present invention relates to a new therapy for atherosclerosis, primary biliary cirrhosis, or NRLP3 inflammasome-related diseases. Specifically, the present invention provides NTCP inhibitors, preferably pre-S1 peptide inhibitors, and compositions containing the same for treating the diseases. Also provided is a method for treating the diseases using NTCP inhibitors, preferably pre-S1 peptide inhibitors. Background Art

[0003] Atherosclerosis

[0004] Atherosclerosis, also known as arteriosclerotic vascular disease or ASVD, is a disease associated with the invasion and accumulation of blood leukocytes, particularly macrophages, and the subsequent development of intimal hyperplasia and plaques.

[0005] Atherosclerosis is initiated by the accumulation of low-density lipoprotein (LDL) in the arterial intima, and elevated LDL cholesterol is a major risk factor for cardiovascular disease. HMGCoA reductase inhibitors (statins) have been 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 few decades has developed several 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 an important 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. Drug therapies that attempt to increase HDL cholesterol levels or improve HDL function have been studied, but have had limited success so far (Shih et al., 2013).

[0006] Despite the availability of therapeutic options, there remains a large unmet medical need for the treatment of atherosclerosis. In particular, the reduction of existing plaque burden in patients with advanced symptomatic atherosclerosis is an unresolved issue.

[0007] Primary biliary cirrhosis

[0008] Primary biliary cirrhosis, also known as primary biliary cholangitis (PBC), is a chronic cholestatic liver disease of unknown cause. Progressive bile duct damage from portal and periportal inflammation may lead to progressive fibrosis and eventual cirrhosis. Evidence to date suggests that immune and genetic factors play a role in the disease. Affected individuals are typically middle-aged women with asymptomatic elevations in serum liver biochemical parameters. The initial presentation of fatigue, pruritus, or unexplained hyperlipidemia may also suggest the diagnosis of primary biliary cirrhosis.

[0009] Current evidence supports the concept that primary biliary cirrhosis is an immune-mediated disease. Both cellular and humoral abnormalities have been noted. Immunohistochemical staining of T lymphocytes in the portal and periportal regions revealed CD4-positive and CD8-positive T cells. In addition, abnormal suppressive T-cell activity has been reported in asymptomatic first-degree relatives of affected individuals. 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 and Lindor, 2003). It has also been observed that interleukin-1β (IL-1β) may play a role in the pathogenesis of PBC by altering immune function and fibrosis (Mueller et al., 1995).

[0010] The only currently approved treatment for PBC is bile acid therapy using ursodeoxycholic acid (UDSA) and obeticholic acid (OCA). The mechanism of action of both drugs in PBC is related to their ability to activate FXR and TGFR-5, thereby exerting an anti-inflammatory effect. 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.

[0011] For the recently approved OCA, 90% of patients enrolled in the pivotal trial had early PBC with normal total bilirubin (TB) and normal albumin levels at baseline, and the majority of patients included in the study received UDCA treatment. Although the primary endpoint was prespecified as a reduction in alkaline phosphatase (ALP) and TB due to the nature of the enrolled population, the primary endpoint was driven solely by ALP. In the Phase 3 clinical trial at month 12, a total of 46% of patients in the OCA titration group and 47% of patients in the OCA 10mg group achieved the primary endpoint (primarily attributed to a reduction in ALP), compared to 10% of patients in the placebo group.

[0012] Therefore, there is a significant medical need in the treatment of PBC, especially for patients with advanced disease.

[0013] NRLP3 inflammasome-related diseases and NASH

[0014] The inflammasome is a cytoplasmic multimeric protein complex composed of nucleotide binding domains and leucine-rich repeat-containing proteins (NLRs) or AIM2, the adaptor protein ASC, and caspase-1. It plays a key role in host defense against pathogens and inflammation (Davis et al., 2011; Martinon et al., 2006). Activation of the inflammasome has been implicated in the pathogenesis of several inflammatory conditions, including type 2 diabetes, atherosclerosis, gout, and Alzheimer's disease. In particular, increasing data strongly suggest that type 2 diabetes is an inflammatory disease. The influx of macrophages and other immune cells driven by chronic calorie excess and the subsequent amplification of the 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 may be a promising treatment for type 2 diabetes (Schroder et al., 2010; Guo et al., 2016).

[0015] It is well known that bile acids bind to and activate several nuclear receptors, including 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 shown 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 inhibits the production of proinflammatory cytokines and phagocytosis by monocytes / macrophages. In vivo studies have shown that the immunosuppressive properties of TGR5 also include a protective effect against metabolic diseases such as atherosclerosis and diabetes, in which tissue infiltration and immune cell activation are hallmarks of their development and progression. The anti-inflammatory and anti-fibrotic properties of FXR and TGR5 signaling can be successfully used to treat nonalcoholic steatohepatitis (NASH). Summary of the invention

[0016] The present invention provides NTCP inhibitors in methods for treating primary biliary cirrhosis (PBC) of a subject. Also provided is a method for treating primary biliary cirrhosis of a subject by providing an effective amount of an NTCP inhibitor to a subject in need thereof. It is expected that administration of the NTCP inhibitor will result in improvements in primary biliary cirrhosis, including normalization of biochemical parameters and reduction of inflammation and fibrosis progression. The NTCP inhibitors used according to the present invention may be administered to a subject alone or in combination with other drugs for the treatment of primary biliary cirrhosis.

[0017] The present invention also provides NTCP inhibitors in methods for treating atherosclerosis of a subject. Also provided is a method for treating atherosclerosis of a subject by providing an effective amount of an NTCP inhibitor to a subject in need thereof. It is envisioned that administration of an NTCP inhibitor results in an improvement in atherosclerotic conditions, including a reduction, reabsorption or elimination of atherosclerotic plaques. The NTCP inhibitors used according to the present invention can be administered to a subject alone or together with other drugs and / or in combination for the treatment of atherosclerosis.

[0018] The present invention also provides NTCP inhibitors in methods for treating other NRLP3 inflammasome-associated diseases of a subject. Specifically, treatments for type 2 diabetes, atherosclerosis, gout, Alzheimer's disease and NASH are provided. Also provided is a method for treating the above-defined NRLP3 inflammasome-associated diseases of a subject by providing an effective amount of an NTCP inhibitor to a subject in need thereof. It is envisioned that the administration of NTCP inhibitors leads to improvements in NRLP3 inflammasome-associated diseases, including normalization of biochemical parameters and improvement in disease progression. The NTCP inhibitors used according to the present invention can be administered to a subject alone or together with other drugs and / or in combination for the treatment of NRLP3 inflammasome-associated diseases.

[0019] In one aspect, the NTCP inhibitor of the present invention may be a pre-S1 peptide inhibitor, wherein the pre-S1 peptide inhibitor comprises a peptide comprising the amino acid sequence NPLGFX0P (SEQ ID NO: 15), and wherein 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 have any length of 4 amino acids or more, but preferably not more than 19 amino acids. Preferably, at least one amino acid of the N-terminal sequence has an amino group on the side chain. Thus, the pre-S1 peptide inhibitor may comprise the amino acid sequence NX1SX2X3 (SEQ ID NO: 16), wherein X1, X2 and X3 are any amino acids at the N-terminus, preferably directly linked 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, the pre-S1 peptide inhibitor may comprise the amino acid sequence NX1SX2X3NPLGFX0P (SEQ ID NO: 17), wherein 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.

[0020] The pre-S1 peptide inhibitor may further comprise a C-terminal sequence of at least 1 amino acid at the N-terminus of NPLGFX0P (SEQ ID NO: 15). The C-terminal sequence may have any length, preferably no more than 93 amino acids, no more than 35 amino acids, no more than 20 amino acids or no more than 10 amino acids. The pre-S1 peptide inhibitor may comprise a C-terminal 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. The C-terminal sequence is preferably directly linked to NPLGFX0P (SEQ ID NO: 15). Therefore, the pre-S1 peptide inhibitor may comprise the amino acid sequence NPLGFX0PX4HQLDP (SEQ ID NO: 19), wherein X0 is any amino acid; and X4 is D, E or S, preferably D or E, more preferably D.

[0021] The pre-S1 peptide inhibitor may comprise the amino acid sequence NX1SX2X3NPLGFX0PX4HQLDP (SEQ ID NO: 20), wherein 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.

[0022] PreS1 peptide inhibitors can have any length of 7 to 119 amino acids, 15 to 47 amino acids, or 15 to 25 amino acids. PreS1 peptide inhibitors can 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 amino acids in length, etc.

[0023] According to standard pre-S1 peptide numbering, the amino acid sequence NPLGFX0P (SEQ ID NO: 15) contained in the pre-S1 peptide inhibitor corresponds to amino acid positions 9 to 15 of the pre-S1 peptide of the HBV virus. The above amino acid sequences NX1SX2X3 (SEQ ID NO: 16) and X4HQLDP (SEQ ID NO: 18) correspond to amino acid positions 4 to 8 and 16 to 21 of the pre-S1 peptide of the HBV virus, respectively.

[0024] Thus, the pre-S1 peptide inhibitor may comprise the pre-S1 peptide of the HBV virus or a functional fragment thereof. The functionality of the fragment may be assessed, for example, based on its ability to bind to NTCP, inhibit NTCP, or reduce NTCP activity.

[0025] According to the standard pre-S peptide numbering, the pre-S1 peptide inhibitor may comprise at least amino acids 9 to 15 of the pre-S1 peptide of the HBV virus. Alternatively, the pre-S1 peptide inhibitor may further comprise amino acids 16, 16 to 17, 16 to 18, 16 to 19, 16 to 19, 16 to 20 or 16 of the pre-S1 peptide of the HBV virus, and / or amino acids 4 to 8, 3 to 8, 2 to 8 of the pre-S1 peptide. The pre-S1 peptide inhibitor may comprise or consist of amino acids 2 to 48 or 2 to 21 of the pre-S1 peptide of the HBV virus. Pre-S1 peptide inhibitors having sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to any of the above sequences are also contemplated.

[0026] The HBV virus may be any HBV virus, such as HBV strain α1, 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.

[0027] An exemplary pre-S1 peptide inhibitor comprises the amino acid sequence between positions 2 and 48 of the HBV pre-S1 consensus sequence:

[0028] GTNL SVPNP LGFFP DHQLD PAFRA NSNNP DWDFN PNKDH WPEAN KVG (SEQ ID NO: 12), or

[0029] Amino acid sequence from position 2 to 48 of the HBV pre-S1 genotype C sequence:

[0030] GTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDH WPEAN QVG (SEQ ID NO: 14), or

[0031] The amino acid sequence of positions 2 to 48 of the HBV pre-S1 genotype C sequence, wherein position 46 has an amino acid substitution (Gin (Q) → Lys (K)):

[0032] GTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDH WPEAN KVG (SEQ ID NO: 13), or

[0033] Amino acid sequence from position 2 to 48 of HBV pre-S1 genotype D sequence:

[0034] GQNL STSNP LGFFP DHQLD PAFRA NTANP DWDFN PNKDT WPDAN KVG (SEQ ID NO: 5).

[0035] Also contemplated are fragments of the above amino acid sequences of at least 15 amino acids in length, and sequences having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity to the above exemplary sequences, wherein the activity of the pre-S1 peptide inhibitor is maintained. The activity includes any one of NTCP binding, NTCP inhibition, and NTCP activity reduction. The activity may also be the prevention or reduction of HBV binding to NTCP.

[0036] The pre-S1 peptide inhibitor may be modified at or around the N-terminus with one or more hydrophobic moieties. The hydrophobic moiety modification may be by acylation, for example with a myristoyl or stearoyl group.

[0037] A preferred pre-S1 peptide inhibitor has the amino acid sequence of GTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDHWPEAN KVG (SEQ ID NO: 13), wherein the peptide is modified at the N-terminus with a myristoyl group.

[0038] A preferred pre-S1 peptide inhibitor is Myrcludex B having the following formula:

[0039] 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.

[0040] The pre-S1 peptide inhibitor may be further modified at the C-terminus or elsewhere to protect the peptide from degradation. Exemplary protecting moieties useful for this purpose are D-amino acids, cyclic amino acids, modified amino acids, glycans, natural or synthetic polymers such as polyethylene glycol (PEG).

[0041] The pre-S1 peptide inhibitors of the present invention, in particular Myrcludex B, can 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 hepatocytes) is equal to or greater than a Ki of about 1 to 10 nM. Thus, the daily dose of the pre-S1 peptide inhibitor can be between 0.1 mg and 50 mg, 0.5 mg and 20 mg, 1 mg and 15 mg, 1 mg and 10 mg, 1 mg and 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, and most preferably 5 mg. The daily dosage of the pre-S1 peptide inhibitor may also be between about 0.0014 mg / kg body weight to about 0.7 mg / kg body weight, preferably between about 0.014 mg / kg body weight to about 0.28 mg / kg body weight (1 mg to 20 mg).

[0042] The pre-S1 peptide inhibitor can 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, 1.8 years, 1.9 years, or 2.0 years, or 3 years, or 4 years or longer.

[0043] The pre-S1 peptide inhibitor may be administered by any suitable route, such as subcutaneously, intravenously, orally, nasally, intramuscularly, transdermally, by inhalation or suppository, preferably intravenously or subcutaneously.

[0044] In another aspect, 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.

[0045] The NTCP inhibitor of the present invention can be administered alone or together. Specifically, the above-mentioned pre-S1 peptide inhibitor can be administered together with another NTCP inhibitor. Two or more NTCP inhibitors can be administered simultaneously / concomitantly. According to the administration schedule, the administration of two or more NTCP inhibitors overlaps in time. The duration of administration of two or more NTCP inhibitors can be the same or substantially the same. For example, the pre-S1 inhibitor and other NTCP inhibitors can be administered for at least one cycle or a course of treatment comprising one or more cycles (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or longer. The duration of a cycle can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. The duration of a course of treatment 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 3.0 years, or 4.0 years. For example, a 24-week course of treatment with the pre-S1 peptide inhibitor Myrcludes B can be administered concurrently with a 24-week course of treatment with another NTCP inhibitor. In this dosing regimen, Myrcludex B can be administered daily, while the NTCP inhibitor can be administered weekly. In this dosing regimen, the pre-S1 peptide inhibitor and the another NTCP inhibitor can be administered simultaneously, for example, substantially simultaneously or in a single composition.

[0046] Depending on the type of component, the pre-S1 peptide inhibitor and the additional NTCP inhibitor can be administered to a subject by various delivery routes. The administration routes include enteral routes (e.g., oral and rectal), parenteral routes (e.g., intravenous, intramuscular, subcutaneous intraperitoneal), topical routes. Preferably, the pre-S1 peptide inhibitor is delivered subcutaneously.

[0047] The NTCP inhibitor used according to the present invention may be in a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient, or adjuvant.

[0048] The present invention is primarily intended for human treatment; however, treatment of any mammal is contemplated. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1. Bile acids inhibit NLRP3 inflammasome-mediated IL-1β secretion. (A) Lipopolysaccharide (LPS)-primed bone marrow-derived macrophages (BMDM) were treated with the indicated bile acids (BA) at 50 mM and then stimulated with nigericin for 45 min. Supernatants were analyzed for IL-1β release by ELISA. (B) and (C) LPS-primed BMDM were treated with different doses of TLCA and then stimulated with nigericin for 45 min. Supernatants were analyzed for IL-1β (B) and IL-18 (C) release by ELISA. From Guo et al., 2016.

[0050] Figure 2 Bile acid species are affected differently by Myrcludex B.

[0051] Figure 3 . TLCA increased in response to Myrcludex B. DETAILED DESCRIPTION

[0052] The present invention is based in part on the discovery that administration of NTCP inhibitors results in elevated blood bile acid levels and increased immunomodulatory signaling mediated by FXR and TGR-5. - Bile acid-mediated signaling of 5 regulates several metabolic pathways, not only bile acid synthesis and enterohepatic recycling, but also lipid, glucose, and energy homeostasis. In addition, FXR and TGR5 agonists display anti-inflammatory and antifibrotic properties, making these drugs interesting candidates for the treatment of several liver and metabolic diseases, including nonalcoholic steatohepatitis (NASH). Specifically, Guo et al., 2016, demonstrated that different bile acids control inflammation and metabolic disorders through NLRP3 inflammasome inhibition. All bile acids tested by the authors exhibited such properties, but lithocholic acid (LCA) and its taurine-conjugated form taurolithocholic acid (TLCA) were particularly effective. For example, inhibition of the formation of the proinflammatory cytokine IL-1β in macrophages was most significant in LCA and TLCA ( Figure 1 The inventors also provide strong evidence that bile acid-mediated TGR5 signaling induces NRLP3 inflammasome ubiquitination and inhibition through an adenylate cyclase- and protein kinase A (PKA)-dependent pathway.

[0053] Human clinical trials completed with the NTCP inhibitory peptide Myrcludex B having the amino acid sequence GTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDH WPEANKVG (SEQ ID NO: 13) induced a significant increase in blood bile acid levels ( Figure 2 ), especially the increase of TLCA level ( Figure 3). Side effects associated with elevated bile acids are not associated with treatment. Therefore, it is expected that treatment of human subjects with the NTCP inhibitors of the present invention will result in an increase in TGR5 signaling and anti-inflammatory, anti-fibrotic and other beneficial effects, leading to clinical benefits and disease improvement of the conditions disclosed in the application.

[0054] In addition, in cholestatic diseases such as PBC and primary sclerosing cholangitis, hepatocellular damage may be partly related to the accumulation of bile acids in hepatocytes. Blocking the reuptake of bile acids by the liver will reduce the intracellular bile acid concentration and relieve the hepatocyte burden, thereby improving liver function.

[0055] The present invention is also based in part on the unexpected discovery that treatment of atherosclerosis with inhibitors of the Na+-taurocholic acid cotransporting polypeptide (NTCP) results in a reduction, reabsorption or elimination of atherosclerotic plaques. It is known that PBC patients have high LDL levels but less evidence of atherosclerosis. The mechanism of how elevated bile acids can reduce atherosclerotic lesions is complex, including activation of reverse cholesterol transport, induction of corresponding genes, and direct cleansing functions of bile acids. Because NTCP is responsible for more than 50% of the reuptake of conjugated bile acids from the bloodstream to the liver; as observed in humans and experimental animals, NTCP blockade results in increased bile acid levels in the blood. As described above, the inventors measured elevated bile acid levels in human clinical trials using the NTCP inhibitor Myrcludex B, without side effects associated with the increase. Therefore, treatment with NTCP inhibitors may result in clinical preservation of elevated bile acid levels in the bloodstream, and thus lead to anti-atherosclerotic effects associated with elevated bile acid levels.

[0056] Therefore, the present invention provides NTCP inhibitors for use in methods for treating atherosclerosis, primary biliary cirrhosis (PBC), NRLP3 inflammasome-related diseases (including type 2 diabetes, atherosclerosis, gout, Alzheimer's disease and NASH) in subjects. Also provided is a method for treating the aforementioned diseases of a subject by administering an effective amount of an NTCP inhibitor to a subject in need thereof. It is conceivable that the administration of an NTCP inhibitor results in improvement, alleviation or irradiation of disease symptoms and effective treatment of the disease.

[0057] NTCP

[0058] The sodium / bile acid co-transporter, also known as sodium / Na+ taurocholate co-transporting polypeptide (NTCP), is a protein encoded by the SLC10A1 (solute carrier family 10 member 1) gene in humans.

[0059] Sodium / bile acid co-transporters are integral membrane glycoproteins involved in the enterohepatic circulation of bile acids. Two homologous transporters are involved in the reabsorption of bile acids, one absorbed from the intestinal lumen, bile ducts and kidneys with an apical localization (SLC10A2) and another sodium-dependent co-transporter present in the basolateral membrane of hepatocytes (SLC10A1).

[0060] The formation of bile is an important function of the liver. Bile salts are the main components of bile and are secreted into bile by hepatocytes and delivered to the small intestine, where they assist in fat digestion. In the liver, hepatocytes absorb bile salts from plasma and secrete them again into bile (mainly through the bile salt export pump (BSEP)) for enterohepatic circulation. Bile salt uptake into hepatocytes occurs mainly by NTCP in a sodium-dependent manner. The transport properties of NTCP have been extensively characterized. It is an electrogenic component of the solute carrier family of transporters (SLC10A1) that primarily transports bile salts and sulfated compounds, but can also mediate the transport of other substrates such as thyroid hormones, drugs and toxins. It is highly regulated under physiological and pathophysiological conditions. Regulation of NTCP responds to changes in the bile salt load of hepatocytes and prevents the entry of cytotoxic amounts of bile salts during liver disease.

[0061] For NTCP, a wide range of substrates can be detected. NTCP transports non-conjugated as well as taurine-conjugated and glycine-conjugated bile acids as well as sulfated bile acids, and unlike the apical sodium-dependent bile acid transporter (ASBT), also transports steroid sulfates and thyroid hormones. Drugs such as rosuvastatin and micafungin have also been shown to have affinity for NTCP. Recent data show that FDA-approved drugs are identified as inhibitors of NTCP. Most of them are antifungal, antihyperlipidemic (simvastatin), antihypertensive, anti-inflammatory or glucocorticoid drugs.

[0062] Examples of bile acids transported into hepatocytes by NTCP are cholates; taurine- or glycine-conjugated bile acids or salts thereof (taurine- or glycine-conjugated dihydroxy and trihydroxy bile salts), such as taurocholate, glycocholate, taurodeoxycholate, tauroneurodeoxycholate, tauroursodeoxycholate, sulfated bile acids and their salts.

[0063] Without being bound by any theory or mode of action, the present application teaches that the blocking of NTCP produces unexpectedly significant favorable effects on atherosclerotic lesions such as plaques, particularly vulnerable plaques with increased rupture potential, by increasing the bile acid levels in the blood. Unlike the treatment of preventive measures that are expected to slow down the development of atherosclerotic lesions by reducing cholesterol levels, particularly LDL levels in the blood, the treatment provided by the present invention targets existing atherosclerotic lesions and causes significant reduction, desorption or complete elimination thereof. Therefore, the therapy provides significant benefits to patients that cannot be achieved using existing therapies.

[0064] Pre-S1 peptide inhibitors

[0065] In one aspect, the inhibitor of Na+-taurocholate cotransporting polypeptide (NTCP) used in the method of treating atherosclerosis in a subject 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), wherein X0 is any amino acid, preferably F or L, more preferably F. It should be understood that the above peptide can be at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 amino acids long, or can be longer than 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 amino acids.

[0066] The pre-S1 peptide inhibitor may further comprise an N-terminal sequence of at least 4 amino acids at the N-terminus of NPLGFXOP (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 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, 4 to 19 amino acids.

[0067] At least one amino acid of the N-terminal sequence may have an amino group on the side chain. At least one amino acid of the N-terminal sequence is selected from lysine, α-aminoglycine, α, γ-diaminobutyric acid, ornithine, α, β-diaminopropionic acid, preferably lysine. At least one amino acid of the N-terminal sequence may be located at the N-terminal of the N-terminal sequence. At least one amino acid of the N-terminal sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids, or wherein at least one amino acid of 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.

[0068] The N-terminal sequence may comprise the amino acid sequence NX1SX2X3 (SEQ ID NO: 16), wherein X1, X2 and X3 are any amino acids. Preferably in NX1SX2X3 (SEQ ID NO: 16):

[0069] X1 is L, I or Q, preferably L;

[0070] X2 is T, V, A or absent, preferably T or V, more preferably T; and / or

[0071] X3 is P, S, T or F, preferably P or S, more preferably S.

[0072] The amino acid sequence NX1SX2X3 (SEQ ID NO: 16) can be directly linked to the N-terminus of NPLGFX0P (SEQ ID NO: 15).

[0073] Thus, the pre-S1 peptide inhibitor may comprise the amino acid sequence NX1SX2X3NPLGFX0P (SEQ ID NO: 17), wherein

[0074] X0 is any amino acid;

[0075] X1 is L, I or Q, preferably L;

[0076] X2 is T, V, A or absent, preferably T or V, more preferably T; and

[0077] X3 is P, S, T or F, preferably P or S, more preferably S.

[0078] The pre-S1 peptide inhibitor may further comprise a C-terminal sequence of at least 1 amino acid at the C-terminus of NPLGFX0P (SEQ ID NO: 15).

[0079] The C-terminal sequence can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 1920, 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 8, 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 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.

[0080] The C-terminal sequence may comprise 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.

[0081] The amino acid sequence X4HQLDP (SEQ ID NO: 18) can be directly linked to the C-terminus of NPLGFX0P (SEQ ID NO: 15).

[0082] Thus, the pre-S1 peptide inhibitor may comprise the amino acid sequence NPLGFX0PX4HQLDP (SEQ ID NO: 19), wherein

[0083] X0 is any amino acid; and

[0084] X4 is D, E or S, preferably D or E, more preferably D.

[0085] The pre-S1 peptide inhibitor may comprise an N-terminal and a C-terminal sequence. Thus, the pre-S1 peptide inhibitor may comprise the amino acid sequence NX1SX2X3NPLGFX0PX4HQLDP (SEQ ID NO: 20), wherein

[0086] X0 is any amino acid;

[0087] X1 is L, I or Q, preferably L;

[0088] X2 is T, V, A or absent, preferably T or V, more preferably T;

[0089] X3 is P, S, T or F, preferably P or S, more preferably S; and

[0090] X4 is D, E or S, preferably D or E, more preferably D.

[0091] The pre-S1 peptide inhibitor may comprise 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, 66 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119 amino acids.

[0092] The pre-S1 peptide inhibitor may also comprise the pre-S1 peptide of HBV virus or a functional fragment thereof. Preferably, the function is to bind to NTCP, inhibit NTCP or reduce the activity of NTCP.

[0093] Hepatitis B virus (HBV) is a small enveloped DNA virus that replicates its genome by reverse transcription of pregenomic RNA transcripts in the cytoplasm of infected hepatocytes. They are classified into the family Hepadnaviridae and adapted to mammals (primates and rodents) and birds, causing acute and persistent infections. Studies on the HBV susceptible cell line HepaRG and systems based on primary human hepatocytes (PHH) and tree shrew (Tupaia belangeri) primary hepatocytes have shown that viral infectivity can be assigned to different subdomains in two of the three viral envelope proteins. The three HBV envelope proteins are called L (large), M (middle) and S (small). They form a proteinaceous outer viral shell that is embedded in a lipid bilayer derived from the endoplasmic reticulum (ER). Their coding mRNA originates from an open reading frame. Since their three start codons are in phase, they share a C-terminal 226 amino acid long S-domain, which anchors the protein in the lipid bilayer through four putative transmembrane helices (TM domains). The S protein drives particle formation and therefore plays an important role in virus assembly. The S domain has been further shown to be involved in virus invasion. Connected to the N-terminus of the S domain, the M and L proteins have two hydrophilic extensions, one of 55 amino acids and the other of 108 (genotype D), 118 (genotype E and G) or 119 (genotype A, B, C, F, H) amino acids, respectively, depending on the genotype, called pre-S2 (M) and pre-S1 (L). The L proteins of all hepatitis viruses contain a recognition motif for N-myristoyl transferase and therefore undergo this modification. Key elements for virus attachment, specific receptor binding or fusion have been identified within the L, M and S proteins.

[0094] Peptides derived from the HBV pre-S1 polypeptide 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 undergoing clinical trials is a linear 47-amino acid chemically synthesized peptide derived from the pre-S1 domain of the large (L) HBV surface protein (trade name Myrcludex B), which consists of naturally occurring L amino acids, has an N-terminal myristoyl moiety and a C-terminal carboxamide.

[0095] According to the present invention, these pre-S1 derived peptides can be used as NTCP inhibitors, ie as pre-S1 peptide inhibitors.

[0096] Thus, the pre-S1 peptide inhibitor used according to the present invention may be derived from the N-terminal domain of the large (L) HBV surface protein, pre-S1, of any HBV virus, in particular 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 to H of human HBV. According to standard HBV nomenclature, the pre-S1 peptide has amino acid coordinates between -11 and 108. All other genotypes contain an additional 10 (E and G) or 11 (A, B, C, F and H) N-terminal pre-S1 amino acids compared to genotype D. Thus, depending on the viral strain, genotype or subtype, the viral pre-S1 peptide may have different amino acid coordinates and thus different lengths.

[0097] Sequence alignment also showed that the pre-S1 peptides of various genotypes share conserved regions with respect to peptide length. The highly conserved region is located between amino acid positions 9 to 21. In addition, the individual highly conserved amino acids are located between amino acids 2 and 6. These conserved sequences are also present in hepatitis B viruses isolated from chimpanzees, gorillas, and woolly monkeys.

[0098] Experimental evidence suggests that the ability of synthetic HBV pre-S1 derived peptides to interfere with HBV infection depends on the presence of some amino acids within the conserved pre-S1 region. Thus, the region between amino acid positions 9 and 15 of pre-S1 is important for peptide activity, particularly amino acids 11, 12, 13, 14 and / or 15 within this region. In addition, substitution of some amino acids within the region between positions 2 and 9 reduces the HBV inhibitory activity of the pre-S1 peptide, and thus amino acid 9 appears to be essential. Other regions that may contribute to the inhibitory activity of the HBV pre-S1 derived peptides are amino acids 2 to 8, 16 to 20, and to a less significant extent, 34 to 48. These conserved amino acids are also important for the ability of the pre-S1 peptide to inhibit NTCP.

[0099] Thus, a pre-S1 peptide inhibitor may encompass amino acids -11 to 108 of HBV pre-S1 (entire HBV pre-S1) or any portion within this region. Reference will be made to standard HBV amino acid numbering below. In particular, a pre-S1 peptide inhibitor may encompass 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 absent. Thus, a pre-S1 peptide inhibitor may encompass amino acids 1 to 78 of HBV. One or more amino acids exceeding amino acid 48, i.e., amino acids 49 to 108, may be absent. Thus, a pre-S1 peptide inhibitor may encompass 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, a pre-S1 peptide inhibitor may encompass amino acids 1 to 48. Amino acid 1 (Met) may be absent. Various pre-S1 peptide inhibitors may encompass amino acids 2 to 48 and optionally include additional amino acids at the C-terminus. Within the range of 2 to 48, amino acids 2 to 21 and 34 to 48 are more important for the pre-S1 peptide inhibitor function than amino acids 21 to 33. Amino acids 9 to 15, especially 11 to 15, are the most important. Therefore, the pre-S1 peptide inhibitor may consist of amino acids 2 to 48 or a truncated portion thereof. Alternatively, the pre-S1 peptide inhibitor may comprise amino acids 2 to 48 or a truncated portion thereof.

[0100] The truncated portion can be obtained by deleting amino acids from either end of the pre-S1 peptide and has a length of 46 to 7 amino acids. Specifically, the length of the truncated portion can be 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.

[0101] The truncated portion preferably comprises amino acids 9 to 15, 10 to 15 or 11 to 15. The truncated portion preferably consists of amino acids 11 to 15. The truncated portion can be gradually extended by adding amino acids flanking amino acids 11 to 15. For example, the truncated portion can consist of amino acids 2 to 15, 3 to 15, 4 to 15, 5 to 15, 6 to 15, 7 to 15, 8 to 15, 9 to 15 or 10 to 15. In this example, any portion of amino acids 16 to 48 can be used to provide further truncated portions.

[0102] In another example, the truncated portion can consist of amino acids 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 21, 2 to 22, 2 to 23, 2 to 24, 2 to 25, 2 to 26, 2 to 27, 2 to 28, 2 to 29, 2 to 30, 2 to 31, 2 to 32, 2 to 33, 2 to 34, 2 to 35, 2 to 36, 2 to 37, 2 to 38, 2 to 39, 2 to 40, 2 to 412 to 42, 2 to 43, 2 to 44, 2 to 45, 2 to 45, 2 to 46, 2 to 47, or 2 to 48. In this example, any of amino acids 3 to 11 can be used to provide a further truncated portion.

[0103] Other examples of truncations are amino acids 9 to 15, 2 to 21, 5 to 21, 2 to 15, 2 to 20, 2 to 25, 2 to 30, 2 to 35, 2 to 40.

[0104] NPLGFX0P (SEQ ID NO: 15) contained in the above-mentioned pre-S1 peptide inhibitor corresponds to amino acid positions 9 to 15 of the pre-S1 peptide of the HBV virus.

[0105] The truncated portion of the preS1 peptide may be a functional fragment of the preS1 peptide that retains the function of the preS1 peptide to bind to and / or inhibit NTCP.

[0106] Thus, the pre-S1 peptide inhibitor used according to the invention may comprise or consist of the following moiety:

[0107] At least amino acids 9 to 15, 8 to 15, 7 to 15, 6 to 15, 5 to 15, 4 to 15, 3 to 15, 2 to 15 of the pre-S1 peptide of the HBV virus;

[0108] at least amino acids 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 9 to 31, 9 to 32, 9 to 33, 9 to 34, 9 to 35, 9 to 36, 9 to 37, 9 to 38, 9 to 39, 9 to 40, 9 to 41, 9 to 42, 9 to 43, 9 to 44, 9 to 45, 9 to 46, 9 to 47, 9 to 48 of the pre-S1 peptide of the HBV virus;

[0109] At least amino acids 8 to 16, 7 to 17, 6 to 18, 5 to 19, 4 to 20, 3 to 21, or 2 to 22 of the pre-S1 peptide of HBV virus;

[0110] at least amino acids 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 21, 2 to 22, 2 to 23, 2 to 24, 2 to 25, 2 to 26, 2 to 27, 2 to 28, 2 to 29, 2 to 30, 2 to 31, 2 to 32, 2 to 33, 2 to 34, 2 to 35, 2 to 36, 2 to 37, 2 to 38, 2 to 39, 2 to 40, 2 to 41, 2 to 42, 2 to 43, 2 to 44, 2 to 45, 2 to 46, 2 to 47, or 2 to 48 of the pre-S1 peptide of HBV virus;

[0111] at least amino acids 9 to 15 and 16 to 20 of the pre-S1 peptide of the HBV virus;

[0112] at least amino acids 2 to 8 and 9 to 15 of the pre-S1 peptide of the HBV virus;

[0113] At least amino acids 2 to 8, 9 to 15 and 16 to 20 of the pre-S1 peptide of the HBV virus;

[0114] at least amino acids 9 to 15 and 34 to 48 of the pre-S1 peptide of the HBV virus;

[0115] At least amino acids 9 to 15, 16 to 20 and 34 to 48 of the pre-S1 peptide of the HBV virus;

[0116] At least amino acids 2 to 8, 9 to 15 and 34 to 48 of the pre-S1 peptide of the HBV virus;

[0117] At least amino acids 2 to 8, 9 to 15, 16 to 20 and 34 to 48 of the pre-S1 peptide of the HBV virus;

[0118] At least amino acids 2 to 48 of the pre-S1 peptide of the HBV virus or a portion of at least 15 amino acids thereof; or

[0119] Amino acids 2 to 48 of the pre-S1 peptide of the HBV virus.

[0120] Pre-S1 peptide inhibitors may also be sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to any of the above sequences.

[0121] Pre-S1 peptide inhibitors can be derived from pre-S1 peptides of various HBV viruses, such as various genotypes, strains or subtypes. In particular, pre-S1 peptide inhibitors can be derived from pre-S1 of genotype A, B, C, D, E, F, G or H or pre-S1 of subtypes AD, ADR, ADW, ADYW, AR and AYW. Pre-S1 peptides from various HBV viruses are generally homologs. Homologs from various species have structural and functional similarities and generally share a common evolutionary ancestor. It is conceivable that pre-S1 homologs of other genotypes, strains or subtypes identified in the future are also suitable as pre-S1 peptide inhibitors. Pre-S1 peptide inhibitors can also be derived from pre-S1 consensus sequences.

[0122] Examples of HBV pre-S1 consensus sequences and resulting sequences between amino acids -11 or -10 or 1 (depending on the genotype) and 48 for various genotypes are shown below:

[0123] HBV pre-S1 consensus sequence (position (-11) to 48)

[0124] (-11)-M GGWSS TPRKG MGTNL SVPNP LGFFP DHQLD PAFRA NSNNP DWDFN PNKDHWPEAN KVG-48(SEQ ID NO: 1)

[0125] HBV pre-S1 genotype A sequence (position (-11) to 48)

[0126] (-11)-M GGWSS KPRKG MGTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PVKDDWPAAN QVG-48(SEQ ID NO: 2)

[0127] HBV pre-S1 genotype B sequence (position (-11) to 48)

[0128] (-11)-M GGWSS KPRKG MGTNL SVPNP LGFFP DHQLD PAFKA NSENP DWDLN PHKDNWPDAN KVG-48(SEQ ID NO: 3)

[0129] HBV pre-S1 genotype C sequence (position (-11) to 48)

[0130] (-11)-M GGWSS KPRQG MGTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDHWPEAN QVG-48(SEQ ID NO: 4)

[0131] HBV pre-S1 genotype D sequence (position (1) to 48)

[0132] 1-MGQNL STSNP LGFFP DHQLD PAFRA NTANP DWDFN PNKDTWPDAN KVG-48 (SEQ IDNO: 5)

[0133] HBV pre-S1 genotype E sequence (position (-10) to 48)

[0134] (-10)-MGLSW TVPLE WGKNI STTNP LGFFP DHQLD PAFRANTRNP DWDHN PNKDHWTEAN KVG-48 SEQ ID NO: 6)

[0135] HBV pre-S1 genotype F sequence (position (-11) to 48)

[0136] (-11)-M GAPLS TTRRG MGQNL SVPNP LGFFP DHQLD PLFRA NSSSP DWDFN TNKDSWPMAN KVG-48 (SEQ ID NO: 7)

[0137] HBV pre-S1 genotype G sequence (position (-10) to 48)

[0138] (-10)-MGLSW TVPLE WGKNL SASNP LGFLP DHQLD PAFRANTNNP DWDFN PKKDPWPEAN KVG-48(SEQ ID NO: 8)

[0139] HBV pre-S1 genotype H sequence (position (-11) to 48)

[0140] (-11)-M GAPLS TARRG MGQNL SVPNP LGFFP DHQLD PLFRA NSSSP DWDFN TNKDNWPMAN KVG-48(SEQ ID NO: 9)

[0141] The pre-S1 sequence between amino acids 1 and 48 from woolly monkey WMHBV is provided below:

[0142] 1-MGLNQ STFNP LGFFP SHQLD PLFKA NAGSA DWDKN PNKDP WPQAH DTA (SEQ IDNO: 10)

[0143] 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 its NTCP inhibitory activity. Preferably, the NTCP inhibitory activity should not be reduced by more than two orders of magnitude. In particular, a reduction of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19 or 20 times is tolerated. The NTCP inhibitory activity can be determined based on the IC 50 or IC 90 Measurement. Amino acid substitutions can be conservative or non-conservative. Amino acid substitutions preferably do not affect amino acids important for the NTCP inhibitory activity of pre-S1 peptide inhibitors. Single amino acids located within the highly conserved portion of pre-S1 spanning amino acids 2 to 21 are more important for activity than amino acids outside this region. Within this region, amino acids 9 to 15 are even more important. Among them, amino acids 9, 11, 12 and 13 are the most important. Therefore, amino acid substitutions are preferably located outside amino acids 11 to 15, 9 to 15, 5 to 15 or 2 to 15. Amino acid substitutions can also be located outside amino acids 2 to 8, 16 to 20 or 34 to 48. Amino acid substitutions can be located within amino acids 20 to 23, 26 to 32.

[0144] According to standard HBV nomenclature, an example of a pre-S1 sequence from genotype C containing an amino acid substitution at position 46 (Gin(Q)→Lys(K)) is as follows:

[0145] (-11)-M GGWSS KPRQG MGTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDHWPEAN KVG-48 (SEQ ID NO: 11).

[0146] Preferred pre-S1 peptide inhibitors are pre-S1 peptides between positions 2 and 48 based on the amino acid sequence of the HBV pre-S1 consensus sequence (according to standard HBV nomenclature):

[0147] GTNL SVPNP LGFFP DHQLD PAFRA NSNNP DWDFN PNKDHWPEAN KVG (SEQ ID NO: 12).

[0148] Another preferred pre-S1 peptide inhibitor is based on the pre-S1 peptide between positions 2 and 48 of the amino acid sequence of HBV pre-S1 genotype C (according to standard HBV nomenclature):

[0149] GTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDHWPEAN QVG(SEQ ID NO: 14)

[0150] The most preferred peptide inhibitors are based on the pre-S1 peptide between positions 2 and 48 of the amino acid sequence of genotype C containing an amino acid substitution at position 46 (Gin(Q)→Lys(K)) (according to standard HBV nomenclature):

[0151] GTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDH WPEAN KVG (SEQ ID NO: 13).

[0152] The pre-S1 peptide inhibitor may be an N-terminal or C-terminal truncated portion of the pre-S1 inhibitor described above, which is 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. The portion retains its function as an NTCP inhibitor or NTCP binding protein.

[0153] The pre-S1 peptide inhibitor may be a sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to any of the above sequences.

[0154] Pre-S1 peptide inhibitors are preferably hydrophobic. They may be modified by at least one hydrophobic moiety. Hydrophobic modification by acylation is preferred. Acylation can be carried out with carboxylic acids, fatty acids or amino acids with lipophilic side chains. Alternatively, pre-S1 peptide inhibitors can be modified by cholesterol, cholesterol derivatives, phospholipids, glycolipids, glycerides, steroids, ceramides, isoprene derivatives, adamantane, farnesol, aliphatic groups or polyaromatic compounds. Fatty acids can be saturated or unsaturated fatty acids, branched or unbranched fatty acids, preferably having 8 to 22 carbon atoms. Examples of suitable fatty acids for acylation are myristic acid (D14), stearic acid (C18), palmitic acid (C16). Acylation with myristoyl is preferred. Variations in the hydrophobic moiety allow the regulation of specific pre-S1 peptide inhibitor activity.

[0155] The hydrophobic moiety is preferably attached to the N-terminus of the pre-S1 peptide inhibitor or 1, 2, 3 or 4 amino acids adjacent to the N-terminus. Thus, the hydrophobic moiety can 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, such as amino acid -5, -4, -3, -2, -1, 1, 2, 3, 4 or 5. More than one hydrophobic moiety can be used to modify the pre-S1 peptide inhibitor. The hydrophobic moieties can be the same or different. The attachment of the hydrophobic moiety is preferably by covalent bonding, which can be achieved by carbamate, amide, ether, disulfide or any other connection within the skill of a person skilled in the art.

[0156] A preferred peptide inhibitor is a pre-S1 peptide having an amino acid sequence of GTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDHWPEAN KVG (SEQ ID NO: 13), wherein the peptide is modified at the N-terminus with a myristoyl or stearoyl group.

[0157] The chemical name of the pre-S1 peptide inhibitor Myrcludex 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.

[0158] The abbreviated chemical name of Myrcludex 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-Ala-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.

[0159] The pre-S1 peptide inhibitor may be further modified in a conventional manner to improve the stability of the peptide, for example, stability against degradation. Such modifications may occur at 1, 2, 3 or 4 amino acids at or near the C-terminus. Such modifications may include modifications with, for example, amides, D-amino acids, modified amino acids, cyclic amino acids, natural polymers, synthetic polymers or polysaccharides. Modification with PEG is an example. Preferably, the protecting moiety is an amide. The protecting moiety may be attached to the amino acid of the pre-S1 peptide inhibitor via a linker. The linker may be polyalanine, polyglycine, a carbohydrate or (CH2) n A group wherein n is at least 1.

[0160] Preferably, the pre-S1 peptide inhibitor consists of amino acid sequence 2 to 48 of the genotype C pre-S1 peptide, which is modified by myristoylation at the N-terminus and further modified by amide at the C-terminus.

[0161] A preferred pre-S1 peptide inhibitor for use according to the invention is Myrcludex B

[0162] Myr-GTNLSVNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANKVG-amide.

[0163] According to the present invention, the pre-S1 peptide inhibitor may be administered to a subject, preferably a human, at the following dosage:

[0164] The dosage is such that the concentration of the pre-S1 peptide inhibitor at the NTCP site, i.e., in the hepatocytes, is equal to or greater than a Ki of about 1 to 10 nM;

[0165] In a dosage 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

[0166] 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).

[0167] The unit dose of the pre-S1 peptide inhibitor, such as Myrcludex B, in the combination or composition may be 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 or 20 mg, preferably 2 mg, 5 mg or 10 mg.

[0168] Pre-S1 peptide inhibitors can be administered to subjects (preferably humans) 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, 1.8 years, 1.9 years, or 2.0 years, or 3 years, or 4 years or longer. The duration of administration will depend on the progress of the treatment and side effects.

[0169] The pre-S1 peptide inhibitor can be administered to a subject, preferably a human subject, subcutaneously, intravenously, orally, nasally, intramuscularly, transdermally, by inhalation or by suppository, preferably intravenously or subcutaneously, most preferably subcutaneously.

[0170] Pre-S1 peptide inhibitors can be administered to a subject, preferably a human, at various doses, treatment schedules, and administration routes as described above. For example, the present invention provides the following treatment regimens for Myrcludex B:

[0171] 2 mg of Myrcludex B is administered daily for 24 weeks. This course of treatment may be followed by 2 mg of Myrcludex B administered daily for another 24 weeks. Myrcludex B may be delivered subcutaneously. In this example, the daily Myrcludex B dose may also be 5 mg or 10 mg.

[0172] 2 mg of Myrcludex B is given daily. Treatment can last for 48 weeks. In this example, the daily Myrcludex B dose can also be 5 mg or 10 mg.

[0173] 2 mg of Myrcludex B is given daily. Treatment may continue for 2 years or longer (indefinitely). In this example, the daily Myrcludex B dose may also be 5 mg or 10 mg.

[0174] 2 mg of Myrcludex B may be administered in combination daily. Treatment may continue for at least 48 weeks. In this example, the daily Myrcludex B dose may be 5 mg or 10 mg.

[0175] Myrcludex B 5 mg may be given daily for 24 weeks or 48 weeks.

[0176] Other NTCP inhibitors

[0177] For the 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.

[0178] The NTCP inhibitor can be selected from bendrofluazide, ezetimibe, simvastatin, nitrendipine, rosuvastatin, nefazodone, indomethacin, nifedipine, tioconazole, methylprednisolone, prochlorperazine, nateglinide, irbesartan, losartan, olmesartan, fenofibrate, candesartan, cyclosporin A, cyclosporin B, a non-immunosuppressive cyclosporin A analog, progesterone, propranolol, bosentan, or a combination thereof.

[0179] The NTCP inhibitor may further comprise an siRNA targeting a gene encoding NTCP, wherein the siRNA comprises siRNA-1 to siRNA-4 targeting the tree shrew SLC10A1 gene, or siRNA-11, siRNA-405, siRNA-406, siRNA-set (4), siRNA-set (5), siRNA-set (6) or siRNA-set (7) targeting the human SLC10A1 gene. The anti-NTCP agent comprises an antisense RNA targeting a gene encoding NTCP.

[0180] Sometimes, the NTCP inhibitor inhibits or modifies a nuclear factor that controls the transcription of NTCP. For example, the agent can be TTNPB, a panRAR agonist, all-trans retinoic acid (ATRA), 9-cis retinoic acid (9CRA), and the like.

[0181] Alternatively, NTCP inhibitors inhibit or modify histone or genomic DNA modifications that control NTCP transcription. DNA methylation inhibitors, including nucleoside analogs of cytosine, such as 5-azacytidine, 5-aza-2′-deoxycytidine (5-azadC); MG98 (an antisense oligodeoxynucleotide directed to the 3′ untranslated region of the DNA methyltransferase-1 enzyme mRNA); histone deacetylase inhibitors (HDAC inhibitors, HDIs), such as vorinostat (suberoylanilide hydroxamic acid; SAHA) and moxidectin (MGCD0103)) are all considered NTCP inhibitors for use according to the present invention.

[0182] NTCP inhibitors can inhibit or modify the phosphorylation and / or glycosylation of NTCP, such as Tunicamysin, which is a mixture of homologous nucleoside antibiotics that blocks all N-glycosylation of proteins. In some cases, the agent is a PI3K inhibitor, such as LY294002, wortmannin, etc.

[0183] NTCP inhibitors may include antibodies that specifically bind to NTCP. The antibody may specifically bind to a portion of NTCP that interacts with HBV and / or HDV, such as an epitope in the extracellular domain. In some embodiments, the extracellular domain includes amino acids 17-27, 73-89, 142-152, 207-217 or 275-278 of NTCP.

[0184] The NTCP inhibitor may comprise a variant form of NTCP, wherein the variant form of NTCP is selected from mutant NTCP, an NTCP fragment and a soluble NTCP polypeptide. The fragment may comprise an extracellular domain of NTCP.

[0185] The NTCP inhibitor may comprise an NTCP substrate or an NTCP substrate derivative or analog. Preferably, the NTCP substrate comprises a bile acid, wherein the bile acid is selected from taurolithocholic acid, cholic acid, chenodeoxycholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, lithocholic acid, ursodeoxycholic acid and hyodeoxycholic acid.

[0186] The anti-NTCP inhibitor may also be a member of the pyrrolotriazine-based class of insulin-like growth factor type 1 receptor (IGF-1R) kinase inhibitors defined in U.S. Pat. No. 7,534,792 as Formula I, particularly BMS-754807. This 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.

[0187] The compound of formula I is as follows:

[0188]

[0189] in

[0190] Q 1 is aryl, substituted aryl, heteroaryl or substituted heteroaryl;

[0191] X is C=O, C=S, C=NR 9 , or CH2;

[0192] R 1 , R 2 , and R 3are 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, urea, cyano, sulfonylamino, substituted sulfonylamino, alkylsulfone, nitro, thio, thioalkyl, alkylthio, disubstituted amino, alkylsulfonyl, alkylsulfinyl, carboxy, alkoxycarbonyl, alkylcarbonyloxy, carbamoyl, substituted carbamoyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, or alkylcarbonyl;

[0193] R 4 is hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, halogen, haloalkyl, haloalkoxy, oxo, aryloxy, arylalkyl, arylalkoxy, alkanoyl, substituted alkanoyl, alkanoyloxy, amino, substituted amino, aminoalkyl, substituted aminoalkyl, alkylamino, substituted alkylamino, hydroxyalkyl, substituted amino, amide, substituted amide, carbamate, substituted carbamate, urea, cyano, sulfonamide, substituted sulfonamide, alkylsulfone, heterocycloalkyl, substituted heterocycloalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, cycloalkylalkyl, cycloalkylalkoxy, nitro, thio, thioalkyl, alkylthio, alkylsulfonyl, alkyl alkyl, arylsulfinyl, carboxyl, alkoxycarbonyl, alkylcarbonyloxy, carbamoyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heteroaryloxy, arylheteroaryl, arylalkoxycarbonyl, heteroarylalkyl, heteroarylalkoxy, aryloxyalkyl, aryloxyaryl, heterocycle, substituted heterocycle, alkylcarbonyl, substituted heteroalkyl, heteroalkenyl, substituted heteroalkenyl, heteroalkynyl, substituted heteroalkynyl, arylamino, arylalkylamino, alkanoylamino, aroylamino, arylalkanoylamino, arylthio, arylalkylthio, arylsulfonyl, arylalkylsulfonyl, alkylsulfonyl, arylcarbonylamino, or alkylaminocarbonyl;

[0194] R 5 is hydrogen, halogen, cyano, alkyl, or substituted alkyl;

[0195] R 6and alkyl. are independently hydrogen, alkyl, substituted alkyl, alkylene, substituted alkylene, hydroxy, alkoxy, halogen, haloalkyl, haloalkoxy, oxo, aryloxy, arylalkyl, arylalkoxy, alkanoyl, substituted alkanoyl, alkanoyloxy, amino, substituted amino, aminoalkyl, substituted aminoalkyl, alkylamino, substituted alkylamino, hydroxyalkyl, substituted amino, amide, substituted amide, carbamate, substituted carbamate, urea, cyano, sulfonamide, substituted sulfonamide, alkylsulfone, heterocycloalkyl, substituted heterocycloalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, cycloalkylalkyl, cycloalkylalkoxy, nitro, thio, thioalkyl, alkyl thio, alkylsulfonyl, carboxyl, alkoxycarbonyl, alkylcarbonyloxy, carbamoyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heteroaryloxy, arylheteroaryl, arylalkoxycarbonyl, heteroarylalkyl, heteroarylalkoxy, aryloxyalkyl, aryloxyaryl, heterocycle, substituted heterocycle, alkylcarbonyl, substituted heteroalkyl, heteroalkenyl, substituted heteroalkenyl, heteroalkynyl, substituted heteroalkynyl, arylamino, arylalkylamino, alkanoylamino, aroylamino, arylalkanoylamino, arylthio, arylalkylthio, arylsulfonyl, arylalkylsulfonyl, alkylsulfonyl, arylcarbonylamino, or alkylaminocarbonyl;

[0196] n is 0, 1, 2, 3, 4, 5, or 6; or when n is 2 and R 6 When they are geminal substituents, they may together form an optionally substituted 3-6 membered saturated or unsaturated carbocyclic or heterocyclic ring; or when n is 2 and R 6 When n is 2 and R is a 1,2-cis substituent, they may together form an optionally substituted 3-6 membered fused saturated carbocyclic or heterocyclic ring; or when n is 2 and R 6 When there are 1,3-cis substituents, they can be taken together to form an optionally substituted 1-4 membered alkyl or heteroalkyl bridge; or when there are two R on the same carbon 6 ', they can form a carbonyl group (C=O) or an alkylene group (C=CHR 9 );

[0197] R 7 and R 8 R 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 4-8 membered saturated or unsaturated carbocyclic or heterocyclic ring, or an optionally substituted bicyclic 7-12 membered saturated or unsaturated carbocyclic or heterocyclic ring;

[0198] R 9 is hydrogen or lower alkyl;

[0199] or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0200] In another embodiment, the compounds of Formula I are those wherein, independently,

[0201] R 1 , R 2 , R 3 and R 4 Each is H;

[0202] Q 1 is aryl or heteroaryl;

[0203] R 5 , R 6 , R 7 , R 8 and R 9 are each independently H or lower alkyl; and

[0204] X is CH2, C=O, or C=NR 9 ;

[0205] or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0206] "Alkyl" refers to a straight or branched chain unsubstituted hydrocarbon group having 1 to 20 carbon atoms, preferably 1 to 7 carbon atoms.

[0207] "Lower alkyl" refers to an unsubstituted alkyl group containing 1 to 4 carbon atoms.

[0208] "Substituted alkyl" refers to an alkyl group substituted with 1 to 4 substituents, such as halogen, hydroxy, alkoxy, oxo, alkanoyl, aryloxy, alkanoyloxy, amino, alkylamino, arylamino, arylalkylamino, disubstituted amino wherein the two substituents are selected from alkyl, aryl or arylalkyl, alkanoylamino, aroylamino, aralkanoylamino, substituted alkanoylamino, substituted arylamino, substituted aralkanoylamino, mercapto, alkylthio, arylthio, arylalkylthio, alkylthiocarbonyl, arylthio, arylalkylthio, alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, thio Sulfonylamino, substituted sulfonylamino, nitro, cyano, carboxyl, carbamoyl, substituted carbamoyl (e.g., CONHalkyl, CONHaryl, CONHarylalkyl or the case where there are two substituents selected from alkyl, aryl or arylalkyl on nitrogen), alkoxycarbonyl, aryl, substituted aryl, guanidino, heterocyclic group (e.g., indolyl, imidazolyl, furyl, thienyl, thiazolyl, pyrrolidinyl, pyridyl, pyrimidinyl, pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, homopiperazinyl, etc.), and substituted heterocyclic group. When the substituent is further substituted, it may be substituted by alkyl, alkoxy, aryl or arylalkyl.

[0209] "Halogen" or "halo" refers to fluorine, chlorine, bromine and iodine.

[0210] The "aryl group" refers to a monocyclic or bicyclic aromatic hydrocarbon group having 6 to 12 carbon atoms in the ring, for example, phenyl, naphthyl, biphenyl and diphenyl, each of which may be substituted.

[0211] "Aryloxy", "arylamino", "arylalkylamino", "arylthio", "arylalkanoylamino", "arylsulfonyl", "arylalkoxy", "arylsulfinyl", "arylheteroaryl", "arylalkylthio", "arylcarbonyl", "arylalkenyl", and "arylalkylsulfonyl" refer to aryl or substituted aryl groups bonded to oxygen; amino; alkylamino; thio; alkanoylamino; sulfonyl; alkoxy; sulfinyl; heteroaryl or substituted heteroaryl; alkylthio; carbonyl; alkenyl; or alkylsulfonyl.

[0212] The "arylsulfonylaminocarbonyl group" refers to an arylsulfonyl group bonded to an aminocarbonyl group.

[0213] "Aryloxyalkyl", "aryloxycarbonyl", or "aryloxyaryl" refers to an aryloxy group bonded to an alkyl or substituted alkyl group; a carbonyl group; or an aryl or substituted aryl group.

[0214] "Arylalkyl" refers to an alkyl or substituted alkyl group in which at least one of the hydrogen atoms bonded to at least one of the carbon atoms is replaced by an aryl or substituted aryl group. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethyl-1-yl, 2-phenylvinyl-1-yl, naphthylmethyl, 2-naphthylethyl-1-yl, 2-naphthylmethyl-1-yl, naphthylmethyl, and 2-naphtholethyl-1-yl.

[0215] "Arylalkoxy" refers to an arylalkyl group bonded through an oxygen linkage (-O-arylalkyl).

[0216] The "substituted aryl" refers to an aryl group substituted with, for example, 1 to 4 substituents such as alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, arylalkyl, halogen, trifluoromethoxy, trifluoromethyl, hydroxy, alkoxy, alkanoyl, alkanoyloxy, aryloxy, arylalkoxy, amino, alkylamino, arylamino, arylalkylamino, dialkylamino, alkanoylamino, thiol, alkylthio, ureido, nitro, cyano, carboxyl, carboxyalkyl, carbamoyl, alkoxycarbonyl, alkylthiocarbonyl, arylthiocarbonyl, arylsulfonamide, sulfonic acid, alkylsulfonyl, sulfonamido, aryloxy, etc. The substituent may be further substituted with hydroxy, halogen, alkyl, alkoxy, alkenyl, alkynyl, aryl or arylalkyl.

[0217] "Heteroaryl" refers to an optionally substituted aromatic group which is a 4- to 7-membered monocyclic, 7- to 11-membered bicyclic, or 10- to 15-membered tricyclic ring system having at least one heteroatom and at least one carbon-containing ring, e.g., pyridine, tetrazole, indazole.

[0218] "Alkenyl" 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 double bonds.

[0219] "Substituted alkenyl" refers to alkenyl substituted with one or two substituents such as halogen, hydroxy, alkoxy, alkanoyl, alkanoyloxy, amino, alkylamino, dialkylamino, alkanoylamino, mercapto, alkylthio, alkylthiocarbonyl, alkylsulfonyl, sulfonamido, nitro, cyano, carboxy, carbamoyl, substituted carbamoyl, guanidinyl, indolyl, imidazolyl, furanyl, thiophenyl, thiazolyl, pyrrolidinyl, pyridinyl, pyrimidinyl, and the like.

[0220] "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.

[0221] "Substituted alkynyl" refers to alkynyl substituted with halogen, hydroxy, alkoxy, alkanoyl, alkanoyloxy, amino, alkylamino, dialkylamino, alkanoylamino, mercapto, alkylthio, alkylthiocarbonyl, alkylsulfonyl, sulfonamido, nitro, cyano, carboxy, carbamoyl, substituted carbamoyl, guanidinyl and heterocycle, for example, imidazolyl, furanyl, thiophenyl, thiazolyl, pyrrolidinyl, pyridinyl, pyrimidinyl and the like.

[0222] "Alkylene" 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 in the definition of "substituted alkyl".

[0223] "Cycloalkyl" refers to an optionally substituted saturated cyclic hydrocarbon ring system, preferably containing 1 to 3 rings and 3 to 7 carbons per ring, which may be further fused to an unsaturated C3-C7 carbocyclic ring. Exemplary groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl and adamantyl. Exemplary substituents include one or more alkyl groups or one or more groups described herein as alkyl substituents.

[0224] "Heterocycle", "heterocyclic" and "heterocyclyl" refer to an optionally substituted, fully saturated or unsaturated aromatic or non-aromatic cyclic group that is a 4- to 7-membered monocyclic ring, a 7- to 11-membered bicyclic ring, or a 10- to 15-membered tricyclic ring system having at least one heteroatom in at least one carbon atom-containing ring. Each ring of the heterocyclic group containing a heteroatom may have 1, 2 or 3 heteroatoms selected from nitrogen, oxygen and sulfur, wherein the nitrogen and sulfur heteroatoms may also be optionally oxidized, and the nitrogen heteroatom may also be optionally quaternized. The heterocyclic ring may be attached at any heteroatom or carbon atom.

[0225] Exemplary monocyclic heterocyclic groups include pyrrolidinyl, pyrrolyl, indolyl, pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl, imidazolinyl, imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl, isoxazolyl, thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, furanyl, tetrahydrofuranyl, thienyl, oxadiazolyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, homopiperazinyl, 2-oxohomopiperazinyl, 2-oxopyrrolidinyl, 2-oxoazepine Base, Aza 4-Piperidinyl, pyridinyl, N-oxo-pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, thiomorpholinyl, 1,3-dioxolane, tetrahydro-1,1-dioxothiphenyl, dioxanyl, isothiazolidinyl, thietanyl, thienyl, triazinyl and triazolyl, etc.

[0226] Exemplary bicyclic heterocyclic groups include 2,3-dihydro-2-oxo-1H-indolyl, benzothiazolyl, benzoxazolyl, benzothiophenyl, quinuclidinyl, quinolinyl, quinolinyl-N-oxide, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, chromonyl, coumaroyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridinyl, furopyridinyl (e.g., furano[2,3-c]pyridinyl, furano[3,1-b]pyridinyl, furano[2,3-b]benzothiazolyl), dihydroisoindolyl, dihydroquinazolinyl (e.g., 3,4-dihydro-4-oxo-quinazolinyl), benzisothiazolyl, benzisoxazolyl, benzodiazinyl, benzofurazanyl, benzothiopyranyl, benzotriazolyl, benzopyrazolyl, 1,3-benzodioxolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone, dihydrobenzopyranyl, indolinyl, indazolyl, isochromanyl, isoindolinyl, naphthyridinyl, phthalazinyl, piperonyl, purinyl, pyridopyridinyl, pyrrolotriazinyl, quinazolinyl, tetrahydroquinolinyl, thienofuranyl, thienopyridinyl, thienothiphenyl and the like. Exemplary 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 heterocyclic groups, such as epoxides and aziridines.

[0227] "Carbocycle" or "carbocyclyl" refers to a stable, saturated, partially saturated or unsaturated monocyclic or bicyclic hydrocarbon ring containing 3-12 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, dihydroindanyl and tetrahydronaphthyl.

[0228] When "optionally substituted" is used to refer to "carbocycle" or "carbocyclyl", it means that the carbocycle can be substituted at one or more substitutable ring positions by one or more substituents independently selected from alkyl (preferably lower alkyl), alkoxy (preferably lower alkoxy), nitro, monoalkylamino (preferably lower alkylamino), dialkylamino (preferably di[lower]alkylamino), cyano, halogen, 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), the aryl being optionally substituted with halogen, lower alkyl and lower alkoxy.

[0229] "Heteroatoms" include oxygen, sulfur and nitrogen.

[0230] "Alkyl sulfone" refers to -R k S(=O)2Rk , where R k It is an alkyl group or a substituted alkyl group.

[0231] "Oxo" means a divalent radical =0.

[0232] "Carbamate" refers to -OC(=O)NH2.

[0233] "Amide" refers to -OC(=O)NH2.

[0234] "Sulfonamide" refers to -SO2NH2.

[0235] "Substituted amide," "substituted sulfonamide," or "substituted carbamate" refers to an amide, sulfonamide, or carbamate, respectively, in which at least one hydrogen is replaced by a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, and substituted cycloalkyl.

[0236] Substituted amide refers to -C(=O)NR"'R", wherein R"' and R" are independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl and substituted cycloalkyl, provided that at least one of R"' and R" is a substituted moiety.

[0237] Substituted sulfonamide refers to the group -SO2NR o R p , where R o and R p are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, and substituted cycloalkyl, provided that R o and R p At least one of is a substituted moiety.

[0238] Substituted carbamate refers to -OC(=O)NR q R r , where R q and R r are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, and substituted cycloalkyl, provided that R q and R r At least one of is a substituted moiety.

[0239] "Urea" refers to the group -NHC(=O)NH2.

[0240] "Cyano" refers to the radical -CN.

[0241] "Cycloalkylalkyl" or "cycloalkylalkoxy" refers to a cycloalkyl or substituted cycloalkyl group bonded to an alkyl or substituted alkyl group; or an alkoxy group, respectively.

[0242] "Nitro" refers to the group -N(O)2.

[0243] "Mercapto" refers to the group -SH.

[0244] "Alkylthio" refers to the group -SR s , where R s is alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0245] "Thioalkyl" refers to the group -R t S, where R t is alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0246] "Alkylsulfonyl" refers to the group -S(=O)2R u , where R u is alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0247] "Alkylsulfinyl" refers to the group -S(=O)R v , where R v is alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0248] "Carboxy" refers to the group -C(=O)OH.

[0249] "Carboxyalkoxy" or "alkoxycarbonylalkoxy" refers to a carboxyl group or an alkoxycarbonyl group, respectively, bonded to an alkoxy group.

[0250] "Alkoxycarbonyl" refers to the group -C(=O)OR w , where R w is alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl.

[0251] The "arylalkoxycarbonyl group" refers to an aryl or substituted aryl group bonded to an alkoxycarbonyl group.

[0252] "Alkylcarbonyloxy" or "arylcarbonyloxy" refers to the group -OC(=O)R x , where R x are alkyl or substituted alkyl, or aryl or substituted aryl, respectively.

[0253] "Carbamoyl" refers to the group -OC(=O)NH2, -OC(=O)NHR x and / or -C(=O)NR y R z , where R y and R z are independently selected from alkyl and substituted alkyl.

[0254] Group-NR 6 (C=O)R9 R 6 is selected from hydrogen, lower alkyl and substituted lower alkyl, and R 9 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkoxy, aminoalkyl, substituted aminoalkyl, alkylamino, substituted alkylamino, aryl and substituted aryl.

[0255] "Carbonyl" refers to C(=O).

[0256] "Alkylcarbonyl", "aminocarbonyl", "alkylaminocarbonyl", "aminoalkylcarbonyl" or "arylaminocarbonyl" refers to an alkyl, substituted alkyl, amino, alkylamino, substituted alkylamino, aminoalkyl, substituted aminoalkyl or arylamino group, respectively, bonded to a carbonyl group.

[0257] "Aminocarbonylaryl" or "aminocarbonylalkyl" refers to an aminocarbonyl group bonded to an aryl or substituted aryl group; or an alkyl or substituted alkyl group, respectively.

[0258] "Sulfonyl" refers to the group S(=O)2.

[0259] "Sulfinyl" refers to the group S(=O).

[0260] "Carboxyalkyl" refers to an alkyl or substituted alkyl group bonded to a carboxyl group.

[0261] The compounds of formula I can form salts. For example, in the separation or purification of compounds, pharmaceutically acceptable salts (e.g., non-toxic, physiologically acceptable) are preferred, although other salts may also be used. The compounds of formula I can form salts with alkali metals such as sodium, potassium and lithium, with alkaline earth metals such as calcium and magnesium, with organic bases such as dicyclohexylamine, tributylamine, pyridine and amino acids such as arginine, lysine, etc. Such salts can be formed by methods known to those skilled in the art.

[0262] Examples of pharmaceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, octanoate, acrylate, formate, isobutyrate, hexanoate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, 1,4-butynedioate, 1,6-hexynedioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, methylsulfonate, propylsulfonate, benzenesulfonate, xylenesulfonate, naphthalene 1-sulfonate, naphthalene-2-sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, and mandelate.

[0263] The compounds of formula I can form salts with various organic and inorganic acids. These salts include salts formed with hydrogen chloride, hydrogen bromide, methanesulfonic acid, sulfuric acid, acetic acid, trifluoroacetic acid, oxalic acid, maleic acid, benzenesulfonic acid, toluenesulfonic acid and various other acids (e.g., nitrates, phosphates, borates, tartrates, citrates, succinates, benzoates, ascorbates, salicylates, etc.). Such salts can be formed by methods known to those skilled in the art. In addition, zwitterions ("inner salts") can be formed.

[0264] All stereoisomers of the compound are also considered to be anti-NTCP agents, in the form of a mixture or in a pure or substantially pure form. Therefore, the definition of the compound includes all possible stereoisomers and mixtures thereof, in particular racemic forms and separated optical isomers with a specific activity. The racemic form can be resolved by physical methods, such as fractional crystallization, separation or crystallization of diastereomeric derivatives, or separation by chiral column chromatography. A single optical isomer can be obtained from the racemate by conventional methods, such as forming a salt with an optically active acid, and then crystallizing.

[0265] The compounds of formula I can also be administered in the form of prodrugs. "Prodrugs" are intended to include any covalently bonded carriers that release active parent drugs in vivo when the prodrug is administered to a mammalian subject. Prodrugs are prepared by modifying the functional groups present in the compound so that the modifications are cleaved into parent compounds in routine operations or in vivo. Prodrugs include compounds of the present invention in which hydroxyl, amino or sulfhydryl groups are bonded to any group, and when the prodrug of the present invention is administered to a mammalian subject, its cleavage forms free hydroxyl, free amino or free sulfhydryl groups, 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. See, for example, Design of Prodrugs, ed. H. Bundgaard (Elsevier, 1985) and Methods in Enzymology, ed. K. Widder et al., Vol. 112, pp. 309-396 (Academic Press, 1985).

[0266] Also included as anti-NTCP agents are solvates (eg, hydrates) of compounds of Formula I. Methods of solvation are known in the art.

[0267] The compound of formula I may be administered in a dosage 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.

[0268] Combinations of NTCP inhibitors

[0269] According to the present invention, one or more NTCP inhibitors can be administered to a subject. One of the NTCP inhibitors can be a pre-S1 peptide inhibitor. Two or more NTCP inhibitors can be administered in sequence. For example, a NTCP inhibitor such as a pre-S1 peptide inhibitor can be administered for a course of treatment that lasts at least one cycle or includes one or more cycles (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 cycles), and then other NTCP inhibitors are administered for a course of treatment that lasts at least one cycle or includes one or more cycles (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 cycles). The duration of a cycle can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. The duration of a cycle can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. The duration of a course of treatment 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 longer.

[0270] In the purposes and methods of the present invention, two or more NTCP inhibitors can be administered simultaneously / concomitantly. According to the administration schedule, the administration of two or more NTCP inhibitors overlaps in time. The administration duration of two or more NTCP inhibitors can be identical or substantially identical. For example, two NTCP inhibitors, one of which is a pre-S1 inhibitor and other NTCP inhibitors can be administered for at least one cycle or a course of treatment comprising one or more cycles (e.g., 2,3,4,5,6,7,8,9 or 10 cycles). The duration of a cycle can be 1,2,3,4,5,6,7,8,9,10,11, or 12 weeks. The duration of a cycle can be at least 1,2,3,4,5,6,7,8,9,10,11, or 12 weeks. The duration of a course of treatment 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. For example, a 24-week course of Myrcludex B (e.g., 5 mg per day) can 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, Myrcludex B can be administered daily, and the NTCP inhibitor can be administered weekly. In this dosing regimen, 2 inhibitors can be administered simultaneously, for example, at substantially the same time or in a single composition.

[0271] In the methods and uses of the present invention, NTCP inhibitors can be delivered by various delivery routes, depending on the type of component. Administration routes include enteral routes (e.g., oral and rectal), parenteral routes (e.g., intravenous, intramuscular, subcutaneous intraperitoneal) and topical routes. Preferably, the pre-S1 peptide inhibitor is delivered subcutaneously.

[0272] Pharmaceutical composition

[0273] The NTCP inhibitor used according to the present invention may be included in a pharmaceutical composition. The pharmaceutical composition used according to the present invention may be in a form suitable for oral use, such as tablets, lozenges, pastilles, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs.

[0274] The composition for oral use can be prepared according to any method known to the pharmaceutical composition manufacturer, and the composition may contain one or more agents selected from: sweeteners, flavoring agents, colorants and preservatives to provide pharmaceutically elegant and palatable preparations. The oral preparation can also be prepared 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 a soft gelatin capsule in which the active ingredient is mixed with a water-soluble carrier such as polyethylene glycol or an oily medium such as peanut oil, liquid paraffin or olive oil.

[0275] The pharmaceutical composition can be in the form of a sterile injectable aqueous solution. Acceptable carriers and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. The sterile injectable preparation can also be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oil phase. For example, the active ingredient can first be dissolved in a mixture of soybean oil and lecithin. The oil solution is then introduced into a water and glycerol mixture and processed to form a microemulsion.

[0276] The injectable solution or microemulsion can be introduced into the patient's blood stream by local bolus injection. Alternatively, it may be advantageous to administer the solution or microemulsion in a manner that maintains a constant circulating concentration of the compound of the invention. In order to maintain such a constant concentration, a continuous intravenous delivery device can be used.

[0277] The pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension for intramuscular and subcutaneous administration. The suspension may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.

[0278] Disease to be treated

[0279] The present invention relates to the treatment of primary biliary cirrhosis, atherosclerosis and NRLP3 inflammasome-related diseases. NRLP3 inflammasome-related diseases include but are not limited to type 2 diabetes, atherosclerosis, gout, Alzheimer's disease and NASH.

[0280] Treatment options

[0281] When the NTCP inhibitor used according to the present invention is administered to a human subject, the daily dosage is generally determined by the prescribing physician, and the dosage is generally varied according to the age, weight, sex and response of the individual patient, as well as the severity of the patient's symptoms. Exemplary dosages of some NTCP inhibitors used according to the present invention are described herein.

[0282] In the use of the present invention, NTCP inhibitors can be provided in a therapeutically effective amount. The term "therapeutically effective amount" or "effective amount" generally refers to the amount of a therapeutic agent that effectively prevents or improves infection or the progression of infection or a related disease associated with infection when administered to a cell, tissue or object alone or in combination with another therapeutic agent. A therapeutically effective dose further refers to an amount of a therapeutic agent that is sufficient to cause symptom improvement, such as treatment, recovery, prevention or improvement of a related medical condition, or increase the treatment, recovery, prevention or improvement rate of such a condition.

[0283] When referring to a single active ingredient administered alone, the therapeutically effective dose refers only to that ingredient. When referring to a combination, the therapeutically effective dose refers to the total amount of active ingredients that can produce a therapeutic effect when administered in combination, either sequentially or simultaneously. Specifically, the effective amount is the amount that inhibits or reduces viral entry into cells.

[0284] In the methods and uses of the present invention, the dosage of each active ingredient may be adjusted according to the therapeutic progress and / or side effects developed by the patient during the treatment.

[0285] The present invention specifically encompasses the specific active ingredients (as NTCP inhibitors) and dosing regimens as described in the Examples of the application.

[0286] The invention will be illustrated by reference to the following non-limiting examples.

[0287] Specific Projects

[0288] This application discloses but is not limited to specific projects:

[0289] 1. An inhibitor of Na+-taurocholate cotransporting polypeptide (NTCP) for use in a method of treating atherosclerosis, primary biliary cirrhosis, or NRLP3 inflammasome-associated disease in a subject.

[0290] 2. The NTCP inhibitor for use as described in item 1, wherein the NTCP inhibitor is a pre-S1 peptide inhibitor, wherein the pre-S1 peptide inhibitor comprises a peptide comprising the amino acid sequence NPLGFX0P (SEQ ID NO: 15), and wherein X0 is any amino acid, preferably F or L, more preferably F.

[0291] 3. The NTCP inhibitor for use as described 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).

[0292] 4. The NTCP inhibitor for use as described 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 wherein 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, 4 to 19 amino acids.

[0293] 5. The NTCP inhibitor for use as described in item 3 or 4, wherein at least one amino acid of the N-terminal sequence has an amino group in the side chain.

[0294] 6. The NTCP inhibitor for use as described in item 5, wherein at least one amino acid of the N-terminal sequence is selected from lysine, α-aminoglycine, α, γ-diaminobutyric acid, ornithine, α, β-diaminopropionic acid, preferably lysine.

[0295] 7. The NTCP inhibitor for use as described in item 5 or 6, wherein at least one amino acid of the N-terminal sequence is located at the N-terminal of the N-terminal sequence.

[0296] 8. The NTCP inhibitor for use as described in item 5-7, wherein at least one amino acid of the N-terminal sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids, or wherein at least one amino acid of 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.

[0297] 9. The NTCP inhibitor for use as described in item 3-8, wherein the N-terminal sequence comprises the amino acid sequence NX1SX2X3 (SEQ ID NO: 16), wherein X1, X2 and X3 are any amino acids.

[0298] 10. The NTCP inhibitor for use as described in item 9, wherein in NX1SX2X3 (SEQ ID NO: 16):

[0299] X1 is L, I or Q, preferably L;

[0300] X2 is T, V, A or absent, preferably T or V, more preferably T; and / or

[0301] X3 is P, S, T or F, preferably P or S, more preferably S.

[0302] 11. The NTCP inhibitor for use as described in item 9 or 10, wherein the amino acid sequence NX1SX2X3 (SEQ ID NO: 16) is directly linked to the N-terminus of NPLGFX0P (SEQ ID NO: 15).

[0303] 12. The NTCP inhibitor for use as described in item 2-11, wherein the pre-S1 peptide inhibitor comprises the amino acid sequence NX1SX2X3NPLGFX0P (SEQ ID NO: 17), wherein

[0304] X0 is any amino acid,

[0305] X1 is L, I or Q, preferably L;

[0306] X2 is T, V, A or absent, preferably T or V, more preferably T; and

[0307] X3 is P, S, T or F, preferably P or S, more preferably S.

[0308] 13. The NTCP inhibitor for use as described in item 2-12, wherein the pre-S1 peptide inhibitor further comprises a C-terminal sequence of at least 1 amino acid at the C-terminus of NPLGFX0P (SEQ ID NO: 15).

[0309] 14. The NTCP inhibitor for use as described in item 13, wherein 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 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.

[0310] 15. The NTCP inhibitor for use as described in item 13 or 14, wherein the C-terminal sequence comprises the amino acid sequence X4HQLDP (SEQ ID NO: 18), wherein X4 is any amino acid.

[0311] 16. The NTCP inhibitor for use as described in item 15, wherein X4 is D, E or S, preferably D or E, more preferably D.

[0312] 17. The NTCP inhibitor for use as described in item 15 or 16, wherein the amino acid sequence X4HQLDP (SEQ ID NO: 18) is directly linked to the C-terminus of NPLGFX0P (SEQ ID NO: 15).

[0313] 18. The NTCP inhibitor for use as described in item 2-17, wherein the pre-S1 peptide inhibitor comprises the amino acid sequence NPLGFX0PX4HQLDP (SEQ ID NO: 19), wherein

[0314] X0 is any amino acid; and

[0315] X4 is D, E or S, preferably D or E, more preferably D.

[0316] 19. The NTCP inhibitor for use as described in item 2-18, wherein the pre-S1 peptide inhibitor comprises the amino acid sequence NX1SX2X3NPLGFX0PX4HQLDP (SEQ ID NO: 20), wherein

[0317] X0 is any amino acid, preferably F or L, more preferably F;

[0318] X1 is L, I or Q, preferably L;

[0319] X2 is T, V, A or absent, preferably T or V, more preferably T;

[0320] X3 is P, S, T or F, preferably P or S, more preferably S; and

[0321] X4 is D, E or S, preferably D or E, more preferably D.

[0322] 20. The NTCP inhibitor for use as described in item 2-19, wherein the pre-S1 peptide inhibitor comprises 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, 75 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119 amino acids.

[0323] 21. The NTCP inhibitor for use as described in item 2-20, wherein the pre-S1 peptide inhibitor comprises the pre-S1 peptide of HBV virus, or a functional fragment thereof, wherein the function is preferably to bind to NTCP, inhibit NTCP, or reduce NTCP activity.

[0324] 22. The NTCP inhibitor for use as described in item 21, wherein the pre-S1 peptide inhibitor comprises or consists of:

[0325] At least amino acids 9 to 15, 8 to 15, 7 to 15, 6 to 15, 5 to 15, 4 to 15, 3 to 15, 2 to 15 of the pre-S1 peptide of the HBV virus;

[0326] at least amino acids 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 9 to 31, 9 to 32, 9 to 33, 9 to 34, 9 to 35, 9 to 36, 9 to 37, 9 to 38, 9 to 39, 9 to 40, 9 to 41, 9 to 42, 9 to 43, 9 to 44, 9 to 45, 9 to 46, 9 to 47, 9 to 48 of the pre-S1 peptide of the HBV virus;

[0327] At least amino acids 8 to 16, 7 to 17, 6 to 18, 5 to 19, 4 to 20, 3 to 21, or 2 to 22 of the pre-S1 peptide of HBV virus;

[0328] at least amino acids 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 21, 2 to 22, 2 to 23, 2 to 24, 2 to 25, 2 to 26, 2 to 27, 2 to 28, 2 to 29, 2 to 30, 2 to 31, 2 to 32, 2 to 33, 2 to 34, 2 to 35, 2 to 36, 2 to 37, 2 to 38, 2 to 39, 2 to 40, 2 to 41, 2 to 42, 2 to 43, 2 to 44, 2 to 45, 2 to 46, 2 to 47, or 2 to 48 of the pre-S1 peptide of HBV virus;

[0329] at least amino acids 9 to 16 and 16 to 20 of the pre-S1 peptide of the HBV virus;

[0330] at least amino acids 2 to 8 and 9 to 15 of the pre-S1 peptide of the HBV virus;

[0331] At least amino acids 2 to 8, 9 to 15 and 16 to 20 of the pre-S1 peptide of the HBV virus;

[0332] at least amino acids 9 to 15 and 34 to 48 of the pre-S1 peptide of the HBV virus;

[0333] At least amino acids 9 to 15, 16 to 20 and 34 to 48 of the pre-S1 peptide of the HBV virus;

[0334] At least amino acids 2 to 8, 9 to 15 and 34 to 48 of the pre-S1 peptide of the HBV virus;

[0335] At least amino acids 2 to 8, 9 to 15, 16 to 20 and 34 to 48 of the pre-S1 peptide of the HBV virus;

[0336] At least amino acids 2 to 48 of the pre-S1 peptide of the HBV virus or a portion thereof of at least 15 amino acids;

[0337] Amino acids 2 to 48 of the pre-S1 peptide of HBV virus; or

[0338] Sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to any of the above sequences.

[0339] 23. An NTCP inhibitor for use as described in item 21 or 22, wherein the HBV virus is HBV strain α1, 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.

[0340] 24. The NTCP inhibitor for use as described in item 2-23, wherein the pre-S1 peptide inhibitor comprises or consists of the following amino acid sequence

[0341] (-11)-M GGWSS TPRKG MGTNL SVPNP LGFFP DHQLD PAFRA NSNNP DWDFN PNKDHWPEAN KVG-48(SEQ ID NO: 1)

[0342] (-11)-M GGWSS KPRKG MGTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PVKDDWPAAN QVG-48(SEQ ID NO: 2)

[0343] (-11)-M GGWSS KPRKG MGTNL SVPNP LGFFP DHQLD PAFKA NSENP DWDLN PHKDNWPDAN KVG-48(SEQ ID NO: 3)

[0344] (-11)-M GGWSS KPRQG MGTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDHWPEAN QVG-48(SEQ ID NO: 4)

[0345] 1-MGQNL STSNP LGFFP DHQLD PAFRA NTANP DWDFN PNKDT WPDAN KVG-48 (SEQ IDNO: 5)

[0346] (-10)-MGLSW TVPLE WGKNI STTNP LGFFP DHQLD PAFRA NTRNP DWDHN PNKDHWTEAN KVG-48 SEQ ID NO: 6)

[0347] (-11)-M GAPLS TTRRG MGQNL SVPNP LGFFP DHQLD PLFRA NSSSP DWDFN TNKDSWPMAN KVG-48 (SEQ ID NO: 7)

[0348] (-10)-MGLSW TVPLE WGKNL SASNP LGFLP DHQLD PAFRA NTNNP DWDFN PKKDPWPEAN KVG-48(SEQ ID NO: 8)

[0349] (-11)-M GAPLS TARRG MGQNL SVPNP LGFFP DHQLD PLFRA NSSSP DWDFN TNKDNWPMAN KVG-48(SEQ ID NO: 9)

[0350] 1-MGLNQ STFNP LGFFP SHQLD PLFKA NAGSA DWDKN PNKDP WPQAH DTA (SEQ IDNO: 10)

[0351] (-11)-M GGWSS KPRQG MGTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDHWPEAN KVG-48 (SEQ ID NO: 11), or

[0352] A fragment of at least 15 amino acids thereof, or

[0353] Sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to any of the above sequences.

[0354] 25. The NTCP inhibitor for use as described in item 2-24, wherein the pre-S1 peptide inhibitor comprises or consists of the following amino acid sequence

[0355] GTNL SVPNP LGFFP DHQLD PAFRA NSNNP DWDFN PNKDH WPEAN KVG (SEQ ID NO: 12);

[0356] GTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDH WPEAN KVG (SEQ ID NO: 13);

[0357] GTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDH WPEAN QVG (SEQ ID NO: 14); or

[0358] Sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to any of the above sequences.

[0359] 26. The NTCP inhibitor for use as described in item 2-25, wherein the pre-S1 peptide inhibitor is modified by at least one hydrophobic moiety at the N-terminus or 1, 2, 3 or 4 amino acids adjacent to the N-terminus.

[0360] 27. The NTCP inhibitor for use as described in item 26, wherein the modification of the hydrophobic part is acylation.

[0361] 28. The NTCP inhibitor for use as described in item 27, wherein the acylation is carried out with carboxylic acid, fatty acid and amino acid having a lipophilic side chain.

[0362] 29. The NTCP inhibitor for use as described 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).

[0363] 30. The NTCP inhibitor for use as described in items 27 to 29, wherein the acylation is acylation with a myristoyl (C14), a palmitoyl (C16) or a stearoyl (C18) group.

[0364] 31. The NTCP inhibitor for use as described in item 27-30, wherein the acylation is acylation with a myristoyl group.

[0365] 32. The NTCP inhibitor for use as described in item 26, wherein the modification of the hydrophobic moiety is the addition of a hydrophobic moiety.

[0366] 33. The NTCP inhibitor for use as described in item 32, wherein the hydrophobic part is cholesterol, a cholesterol derivative, a phospholipid, a glycolipid, a glyceride, a steroid, a ceramide, an isoprene derivative, 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.

[0367] 34. The NTCP inhibitor for use as described in item 32 or 33, wherein the hydrophobic moiety is covalently bound, preferably via a carbamate, amide, ether, or disulfide linkage.

[0368] 35. The NTCP inhibitor for use as described in item 2-34, wherein the pre-S1 peptide inhibitor is modified by at least one protecting moiety at the C-terminus or 1, 2, 3 or 4 amino acids adjacent to the C-terminus, wherein the protecting moiety protects the pre-S1 peptide inhibitor from degradation.

[0369] 36. The NTCP inhibitor for use as described in item 35, wherein the protecting moiety is an amide, a D-amino acid, a modified amino acid, a cyclic amino acid, albumin, a polysaccharide, or a natural or synthetic polymer, preferably PEG.

[0370] 37. The NTCP inhibitor for use as described in item 35 or 36, wherein the protecting moiety is an amide.

[0371] 38. The NTCP inhibitor for use as described in items 35-37, wherein the protecting part is connected via a linker.

[0372] 39. The NTCP inhibitor for use as described in item 39, wherein the linker is polyalanine, polyglycine, a carbohydrate, or (CH2) n A group wherein n is 1 or greater.

[0373] 40. The NTCP inhibitor for use as described in item 2-39, wherein the pre-S1 peptide inhibitor consists of amino acid sequence 2 to 48 of genotype C pre-S1 peptide, which is modified by myristoylation at the N-terminus and further modified by amide at the C-terminus.

[0374] 41. The NTCP inhibitor for use as described in item 2-40, wherein the pre-S1 peptide inhibitor is Myr-GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANKVG-amide.

[0375] 42. A composition or combination as described in item 2-40, wherein the 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-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-glycylamide, or its acetate salt.

[0376] 43. The NTCP inhibitor for use as described in item 2-42, wherein the method comprises administering the pre-S1 peptide inhibitor:

[0377] A dose such that the concentration of the pre-S1 peptide inhibitor at the NTCP site, i.e., in hepatocytes, is equal to or greater than a Ki of about 1 to 10 nM;

[0378] 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

[0379] 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).

[0380] 44. An NTCP inhibitor for use as described in item 2-43, 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, 1.8 years, 1.9 years, or 2.0 years, or 3 years, or 4 years or longer.

[0381] 45. The NTCP inhibitor for use as described in item 2-44, wherein the method comprises administering the pre-S1 peptide inhibitor at a dose of 2 mg per day or 5 mg per day for 24 weeks or 48 weeks.

[0382] 46. ​​The NTCP inhibitor for use as described in item 2-45, wherein the method comprises administering the pre-S1 peptide inhibitor subcutaneously, intravenously, orally, nasally, intramuscularly, transdermally, by inhalation or suppository, preferably intravenously or subcutaneously.

[0383] 47. The NTCP inhibitor for use as described in item 2-46, wherein the method comprises administering the pro-S1 inhibitor and another NTCP inhibitor.

[0384] 48. The NTCP inhibitor for use as described in item 1-47, wherein the subject is a human.

[0385] 49. The NTCP inhibitor for use as described in item 1-48, wherein the atherosclerosis is at the stage of unstable atherosclerotic plaques.

[0386] 50. An NTCP inhibitor for use as described in item 1-49, wherein the method results in reduction, dissolution or elimination of atherosclerotic plaques.

[0387] 51. An NTCP inhibitor for use as described in item 1-48, wherein the method results in a reduction or elimination of symptoms of primary biliary cirrhosis.

[0388] 52. An NTCP inhibitor for use as described in item 51, wherein the method results in normalization of biochemical parameters and reduction of inflammation and fibrosis progression.

[0389] 53. The NTCP inhibitor for use as described in item 1-48, wherein the NRLP3 inflammasome-related diseases are type 2 diabetes, atherosclerosis, gout, Alzheimer's disease and NASH.

[0390] 54. A NTCP inhibitor for use as described in item 53, wherein the method results in normalization of biochemical parameters and improvement of disease progression.

[0391] 55. A method of treating atherosclerosis, primary biliary cirrhosis, or a NRLP3 inflammasome-associated disease in a subject, comprising administering to the subject an NTCP inhibitor.

[0392] 56. The method of item 55, wherein the NTCP inhibitor is a pre-S1 inhibitor as defined in item 2-42.

[0393] 57. A method as described in item 55 or 56, wherein the method is as defined in items 43 to 47.

[0394] 58. The method of items 55-57, wherein the subject is a human.

[0395] 59. A method as described in items 55-58, wherein the disease is as defined in items 49-54.

[0396] Example

[0397] Example 1: Study of HTCP Inhibitors in APO-E Knockout Mice

[0398] Objective: The aim of this study was to evaluate the effects of the HTCP inhibitor Myrcludex B at different doses on the physiology and development of atherosclerotic lesions in APO-E knockout mice, an animal model that closely resembles human atherosclerotic pathology.

[0399] method:

[0400] Testing Weeks 1-8:

[0401] Randomize and assign to 4 test groups:

[0402]

[0403]

[0404] Dosage: Subcutaneous injection every day

[0405] Specific feeding conditions:

[0406]

[0407] Parameters to be measured:

[0408]

[0409] Results: Myrcludex B was well tolerated.

[0410] Example 2: Study of HTCP Inhibitors in LDL Receptor Knockout Mice

[0411] Objective: The purpose of this 23-week study was to evaluate the effects of the HTCP inhibitor Myrcludex B at different doses on the physiology and development of atherosclerotic lesions in LDL receptor-knockout mice (LDLRKO), an animal model that closely resembles human atherosclerotic disease.

[0412] method:

[0413] 16 male and 6 female LDLRKO mice

[0414] 8 weeks of high-calorie diet

[0415] 4 males and 4 females were killed

[0416] The remaining animals were randomly divided into 4 groups.

[0417] Standard diet for 15 weeks and daily subcutaneous Myrcludex B treatment starting in week 9

[0418] Species / strain: Mouse LDLR knockout / B6.129S7-Ldlrtml Her / J

[0419] Supplier: Jackson Laboratory, USA

[0420] Testing Weeks 1-8:

[0421]

[0422]

[0423] Parameters to be measured:

[0424]

[0425]

[0426] Testing Week 9-23:

[0427] Randomize and assign to 4 test groups:

[0428]

[0429] Dosing: Daily subcutaneous injection starting at week 9 of the test

[0430] Specific feeding conditions:

[0431]

[0432] Parameters to be measured:

[0433]

[0434]

[0435] Results: Myrcludex B was well tolerated.

[0436] Example 3: Pilot clinical trial of HTCP inhibitor Mycludex B in patients with dyslipidemia

[0437] Objective: The purpose of the clinical trial was to evaluate the safety and tolerability and efficacy of Myrcludex B in patients with dyslipidemia.

[0438] method:

[0439] 20 patients with dyslipidemia

[0440] 8 weeks of Myrcludex B, 10 mg per day

[0441] Endothelial function tests, lipidome, and other biomarkers: assays for macrophage cholesterol efflux and the ATP-binding cassette transporter genes ABCA1 and ABCG1.

[0442] Results: Myrcludex B was well tolerated.

[0443] Example 4: Phase II clinical trial of treating PBC with the HTCP inhibitor Myeludex B

[0444] Objective: The purpose of the clinical trial was to evaluate the safety and tolerability and efficacy of Myrcludex B in patients with primary biliary cirrhosis.

[0445]

[0446]

[0447]

[0448]

[0449] Bibliography

[0450] Shih, D. M., Shaposhnik, Z., Meng, Y., Rosales, M., Wang, X., Wu, J., ... Lusis, A. J. (2013). Hydrodeoxycholic acid improves HDL function and inhibits atherosclerotic lesion formation in LDLR-knockout mice. FASEB Journal, 27(9), 3805-3817. http: / / doi.org / 10.1096 / fj.12-223008

[0451] Talwalkar, JA, and Lindor, KD (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

[0452] Davis, BK, Wen, H., and Ting, JP (2011). The inflammasome NLRs in immunity, inflammation, and associated diseases. Annu. Rev. Immunol. 29, 707-735

[0453] Martinon, F., Petrilli, V., Mayor, A., Tardivel, A., and Tschopp, J. (2006). Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 440. 237-241

[0454] Schroder, K., Zhou, R., and Tschopp, J. (2010). The NLRP3 inflammasome: a sensor for metabolic danger? Science 327, 296-300

[0455] Miller LC et al. (1995) Synthesis of interleukin-1β in primary biliary cirrhosis: Relationship to treatment with methotrexate or colchicine and disease progression. Hepatology, Vol. 22, No. 2, 518-524.

Claims

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

2. The NTCP inhibitor for use according to claim 1, wherein the NTCP inhibitor is a pre-S1 peptide inhibitor, wherein the pre-S1 peptide inhibitor comprises a peptide comprising the amino acid sequence NPLGFX0P (SEQ ID NO: 15), and wherein X0 is any amino acid, preferably F or L, more preferably F.

3. The NTCP inhibitor for use as claimed in claim 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), wherein the N-terminal sequence preferably comprises the amino acid sequence NX1SX2X3 (SEQ ID NO: 16), wherein X1, X2 and X3 are any amino acids, wherein 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.

4. The NTCP inhibitor for use as claimed in claims 2-3, wherein the pre-S1 peptide inhibitor further comprises a C-terminal sequence of at least 1 amino acid at the C-terminus of NPLGFX0P (SEQ ID NO: 15), wherein the C-terminal sequence preferably comprises the amino acid sequence X4HQLDP (SEQ ID NO: 18), wherein X4 is any amino acid, wherein preferably, X4 is D, E or S, preferably D or E, more preferably D.

5. The NTCP inhibitor for use as claimed in claims 2-4, wherein the pre-S1 peptide inhibitor comprises 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, 75 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119 amino acids.

6. The NTCP inhibitor for use as claimed in claims 2-5, wherein the pre-S1 peptide inhibitor comprises the pre-S1 peptide of HBV virus, or a functional fragment thereof, wherein the function is preferably binding to NTCP, inhibiting NTCP, or reducing NTCP activity, wherein preferably, the pre-S1 peptide inhibitor comprises or consists of the following: At least amino acids 9 to 15, 8 to 15, 7 to 15, 6 to 15, 5 to 15, 4 to 15, 3 to 15, 2 to 15 of the pre-S1 peptide of the HBV virus; at least amino acids 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 9 to 31, 9 to 32, 9 to 33, 9 to 34, 9 to 35, 9 to 36, 9 to 37, 9 to 38, 9 to 39, 9 to 40, 9 to 41, 9 to 42, 9 to 43, 9 to 44, 9 to 45, 9 to 46, 9 to 47, 9 to 48 of the pre-S1 peptide of the HBV virus; At least amino acids 8 to 16, 7 to 17, 6 to 18, 5 to 19, 4 to 20, 3 to 21, or 2 to 22 of the pre-S1 peptide of HBV virus; at least amino acids 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 21, 2 to 22, 2 to 23, 2 to 24, 2 to 25, 2 to 26, 2 to 27, 2 to 28, 2 to 29, 2 to 30, 2 to 31, 2 to 32, 2 to 33, 2 to 34, 2 to 35, 2 to 36, 2 to 37, 2 to 38, 2 to 39, 2 to 40, 2 to 41, 2 to 42, 2 to 43, 2 to 44, 2 to 45, 2 to 46, 2 to 47, or 2 to 48 of the pre-S1 peptide of HBV virus; at least amino acids 9 to 15 and 16 to 20 of the pre-S1 peptide of the HBV virus; at least amino acids 2 to 8 and 9 to 15 of the pre-S1 peptide of the HBV virus; At least amino acids 2 to 8, 9 to 15 and 16 to 20 of the pre-S1 peptide of the HBV virus; at least amino acids 9 to 15 and 34 to 48 of the pre-S1 peptide of the HBV virus; At least amino acids 9 to 15, 16 to 20 and 34 to 48 of the pre-S1 peptide of the HBV virus; At least amino acids 2 to 8, 9 to 15 and 34 to 48 of the pre-S1 peptide of the HBV virus; At least amino acids 2 to 8, 9 to 15, 16 to 20 and 34 to 48 of the pre-S1 peptide of the HBV virus; At least amino acids 2 to 48 of the pre-S1 peptide of the HBV virus or a portion thereof of at least 15 amino acids; Amino acids 2 to 48 of the pre-S1 peptide of HBV virus; or Sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to any of the above sequences.

7. The NTCP inhibitor for use as claimed in claim 6, wherein the HBV virus is HBV strain α1, 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.

8. The NTCP inhibitor for use as claimed in claims 2-7, wherein the pre-S1 peptide inhibitor comprises or consists of the following amino acid sequence (-11)-M GGWSS TPRKG MGTNL SVPNP LGFFP DHQLD PAFRA NSNNP DWDFN PNKDH WPEANKVG-48(SEQ ID NO: 1) (-11)-M GGWSS KPRKG MGTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PVKDD WPAANQVG-48(SEQ ID NO: 2) (-11)-M GGWSS KPRKG MGTNL SVPNP LGFFP DHQLD PAFKA NSENP DWDLN PHKDN WPDANKVG-48(SEQ ID NO: 3) (-11)-M GGWSS KPRQG MGTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDH WPEANQVG-48(SEQ ID NO: 4) 1-MGQNL STSNP LGFFP DHQLD PAFRA NTANP DWDFN PNKDTWPDAN KVG-48 (SEQ ID NO: 5) (-10)-MGLSW TVPLE WGKNI STTNP LGFFP DHQLD PAFRA NTRNP DWDHN PNKDH WTEANKVG-48SEQ ID NO: 6) (-11)-M GAPLS TTRRG MGQNL SVPNP LGFFP DHQLD PLFRA NSSSP DWDFN TNKDS WPMANKVG-48 (SEQ ID NO: 7) (-10)-MGLSW TVPLE WGKNL SASNP LGFLP DHQLD PAFRA NTNNP DWDFN PKKDP WPEANKVG-48 (SEQ ID NO: 8) (-11)-M GAPLS TARRG MGQNL SVPNP LGFFP DHQLD PLFRA NSSSP DWDFN TNKDN WPMANKVG-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 WPEANKVG-48 (SEQ ID NO: 11), or A fragment of at least 15 amino acids thereof, or A sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to any of the above sequences, Preferably, the pre-S1 peptide inhibitor comprises or consists of the following 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 Sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to any of the above sequences.

9. The NTCP inhibitor for use as claimed in claims 2-8, wherein the pre-S1 peptide inhibitor is modified by at least one hydrophobic moiety at the N-terminus or 1, 2, 3 or 4 amino acids adjacent to the N-terminus, wherein the hydrophobic moiety modification is preferably acylation, and the acylation is preferably acylation with myristoyl (C14), palmitoyl (C16) or stearoyl (C18).

10. The NTCP inhibitor for use as claimed in claims 2-9, wherein the pre-S1 peptide inhibitor is myristoyl-GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANKVG-amide.

11. A NTCP inhibitor for use as claimed in claims 2-10, wherein the method comprises administering the pro-S1 inhibitor and another NTCP inhibitor.

12. The NTCP inhibitor for use as claimed in claims 1-11, wherein the subject is a human.

13. NTCP inhibitor for use according to claims 1-12, wherein said method results in reduction or elimination of symptoms of primary biliary cirrhosis, wherein preferably said method results in normalization of biochemical parameters and reduction of inflammation and fibrosis progression.

14. NTCP inhibitor for use according to claims 1-12, wherein the atherosclerosis is at the stage of unstable atherosclerotic plaques and wherein the method results in reduction, dissolution or elimination of atherosclerotic plaques.

15. The NTCP inhibitor for use as claimed in claims 1-12, wherein the NRLP3 inflammasome-related diseases are type 2 diabetes, atherosclerosis, gout, Alzheimer's disease and NASH.

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