Sodium ion-taurocholic acid cotransporter long-acting inhibitor and application thereof

Abstract

The invention relates to a sodion-taurocholic acid co-transport protein (NTCP) long-acting inhibitor and application thereof, and provides a sodion-taurocholic acid co-transport protein (NTCP) long-acting inhibitor and application of the sodion-taurocholic acid co-transport protein (NTCP) long-acting inhibitor. In particular, provided are long-acting polypeptides that can be used as NTCP inhibitors. The invention also relates to a preparation method and application of the polypeptide, and a pharmaceutical composition containing the polypeptide. The NTCP long-acting inhibitor competitively inhibits the binding between hepatitis virus surface protein and NTCP through binding with NTCP, realizes the hepatitis virus entry inhibition effect, and can be used as a therapeutic or prophylactic drug for blocking hepatitis virus from entering cells, a hepatitis recurrence inhibition drug and a hepatitis functional curing drug; the compound can also be used as a medicine for treating type II diabetes, a medicine for treating obesity and a medicine for treating intestinal autoimmune diseases, biliary cirrhosis and atherosclerosis.

Description

Long-acting inhibitors of sodium ion-taurocholic acid cotransporter and their applications

[0001] This application is based on the application with CN application number 202310990853.3 and application date August 8, 2023, and claims its priority. The disclosed content of the aforementioned CN application is hereby introduced as a whole into this application. Technical Field

[0002] The present invention relates to the field of biomedicine, and specifically to a long-acting inhibitor of sodium / taurocholate cotransporting polypeptide (NTCP) and its use, particularly in treating and / or preventing NTCP-related diseases. The present invention also relates to a method for preparing the inhibitor and a pharmaceutical composition containing the inhibitor. Background Art

[0003] NTCP is a sodium taurocholate cotransporting polypeptide encoded by the SLC10A1 gene. It consists of 349 amino acid residues and has nine transmembrane domains. It is located on the basolateral membrane of hepatocytes [1, 2]. Natural NTCP is glycosylated and has a molecular weight of approximately 55 kD; non-glycosylated NTCP has a molecular weight of approximately 37 kD.

[0004] In recent years, reports have suggested that NTCP is a potential therapeutic target for obesity and type 2 diabetes [3]. The main function of NTCP is to take up conjugated bile acids from plasma into hepatocytes in a sodium-dependent manner, playing an important role in the enterohepatic circulation of bile acids and promoting the body's digestion of lipids. NTCP is used as a target for the treatment of diabetes to control blood sugar, blood lipids and cholesterol levels. NTCP inhibitors can increase the level of bile acids in the blood, and bile acids in the blood can play a hormone-like role. Bile acids downregulate the expression of gluconeogenic enzyme genes and lipogenic enzyme genes by activating the farnesoid X receptor (FXR) [4, 5], and also stimulate the release of glucagon-like peptide 1 (GLP-1) by activating the TGR5 receptor (G-coupled protein receptor specific for bile acids) to promote insulin synthesis, thereby downregulating blood sugar and lipid levels [3]. NTCP can be used as a new therapeutic target for obesity and type 2 diabetes.

[0005] In addition to participating in lipid metabolism, NTCP is one of the key receptors for hepatitis B virus (HBV) infection of human hepatocytes. The HBV preS / S open reading frame, using AUG start codons at different positions, encodes three envelope glycoproteins, including large (L), medium (M), and small (S) surface antigens (HBsAg). Specifically binding of the PreS1 segment of the L envelope protein to the NTCP receptor on the hepatocyte cell membrane is key to HBV infection of hepatocytes. Hepatitis D virus (HDV) cannot synthesize its own envelope protein; instead, it uses the HBV envelope protein as its own envelope protein, and its mechanism of infection of hepatocytes is the same as that of HBV. Therefore, NTCP is a common receptor for HBV and HDV to invade hepatocytes.

[0006] The first NTCP-targeted drug to be marketed, Hepcludex (Myrcludex B, INN name: bulevirtide), has as its active ingredient Bulevirtide, a myristoylated HBV envelope protein large L-surface protein (L protein) preS1 aa 2-48 domain, also known as myr-preS1 aa2-48 lipopeptide. The results of its pivotal Phase III clinical trial for the treatment of chronic HDV infection showed good efficacy and safety. Specifically, compared with subjects who did not receive antiviral treatment during this phase of the study, subjects who received 2mg or 10mg of bulevirtide monotherapy once daily achieved a significantly higher rate of comprehensive virological and biochemical responses, at 45% and 48% (vs 2%), respectively. In the trial, the safety profile of Bulevirtide was consistent with previous reports. No participants experienced adverse events (AEs) leading to discontinuation of the drug, and no serious AEs were reported due to treatment with the drug.

[0007] Although NTCP inhibitors are currently on the market, they have a short half-life and require daily injection, resulting in poor patient compliance.

[0008] In summary, designing NTCP inhibitors that effectively inhibit viral entry, have strong stability, and long half-life, and use them to treat NTCP-related diseases is what patients who take long-term medication are looking forward to.

[0009] Summary of the Invention

[0010] The present invention aims to provide a long-acting inhibitor of sodium / taurocholate cotransporting polypeptide (NTCP). This long-acting NTCP inhibitor competitively inhibits the binding between hepatitis virus surface proteins and NTCP by binding to NTCP, thereby inhibiting the entry of hepatitis viruses. This long-acting NTCP inhibitor can be used as a therapeutic or preventive drug to block hepatitis virus entry into cells, a drug to inhibit hepatitis recurrence, and a drug that functionally cures hepatitis. Furthermore, the long-acting NTCP inhibitor of the present invention can also be used as a drug for treating type 2 diabetes, obesity, intestinal autoimmune diseases, biliary cirrhosis, and atherosclerosis.

[0011] In a first aspect of the present invention, a long-acting inhibitor of sodium-taurocholate cotransporter (NTCP) is provided, comprising a polypeptide represented by the general formula ABR, or an analog thereof, and a pharmaceutically acceptable salt thereof; wherein the amino acid sequence of B is as shown in the following formula I, A is a modification of the N-terminus, R is a modification of the C-terminus,

[0012] Gly-Xaa2-Asn-Xaa4-Ser-Xaa6-Xaa7-Asn-Pro-Leu-Gly-Phe-Xaa13-Pro-Xaa15-His-Gln-Leu-Asp-Pro-Xaa21-Xaa22-Xaa23-Ala-A sn-Xaa26-Xaa27-Asn-Pro-Xaa30-Trp-Asp-Xaa33-Asn-Pro-Xaa36-Xaa37-Asp-Xaa39-Trp-Pro-Xaa42-Ala-Asn-Xaa45-Val-Gly(I);

[0013] Where,

[0014] Xaa2 is Thr, Ser, Lys, modified Lys, Gln or Asn;

[0015] Xaa4 is Leu, Gln, Ile or Phe;

[0016] Xaa6 is Val, Ile, Leu, Met, Phe, Ala, Thr, Trp, Asn, Gln or Gly;

[0017] Xaa7 is Pro, Ala, or Ser;

[0018] Xaa13 is Phe, His, or Leu;

[0019] Xaa15 is Asp or Glu;

[0020] Xaa21 is Ala, Ile, Val, Leu, Glu, Asp, Asn, Cys, Met, Gln, Trp or Pro;

[0021] Xaa22 is Phe or Trp;

[0022] Xaa23 is Gly, Pro, Ala, Arg, Lys, modified Lys, or Glu;

[0023] Xaa26 is Ser, Met, Leu, Thr, Phe, Ile, or Ala;

[0024] Xaa27 is Asn, Gln, His, Lys, modified Lys, Ala, or Arg;

[0025] Xaa30 is Asp or Glu;

[0026] Xaa33 is Phe, Leu, His, Tyr, Arg, Lys, modified Lys, Ala, Trp, Ser, Ile, Val, or Thr;

[0027] Xaa36 is Asn, Gln, Lys, modified Lys, or Leu;

[0028] Xaa37 is Lys or modified Lys;

[0029] Xaa39 is His, Asn, Thr, Phe, Tyr, Gln, Lys, modified Lys, or Arg;

[0030] Xaa42 is Glu, Asp, Val, Ala, Met, Lys, Gln, Thr, Asn, Leu, Arg, Ser, or His;

[0031] Xaa45 is Glu, Asp, Val, Ala, Met, Lys, Gln, Thr, Asn, Leu, Arg, Ser, His, or an amino acid with a modification, specifically Lys or a modified Lys;

[0032] The amino acid sequence of B has a homology of ≥85% with the amino acid sequence of SEQ ID NO: 1, and has the activity of binding to NTCP.

[0033] In another preferred embodiment, Xaa2 is Thr, Ser, Lys, modified Lys or Gln, or Xaa6 is Val, Thr, Ile or Leu, or Xaa7 is Pro or Ser, or Xaa13 is Phe or Leu, or Xaa15 is Asp, or Xaa21 is Ala, Leu, Val, Glu, Ile or Asp; or Xaa23 is Arg, Lys, modified Lys or Glu; or Xaa27 is Asn or Ala, or Xaa33 is Phe, Leu, His or Tyr, or Xaa39 is His, Asn, Thr, Phe or Tyr, or Xaa42 is Glu or Asp;

[0034] Preferably, the amino acid sequence of B is as shown in the following formula II:

[0035] Gly-Xaa2-Asn-Xaa4-Ser-Xaa6-Xaa7-Asn-Pro-Leu-Gly-Phe-Xaa13-Pro-Asp-His-Gln-Leu-Asp-Pro-Xaa21-Xaa22-Xaa23-Ala-As n-Xaa26-Xaa27-Asn-Pro-Xaa30-Trp-Asp-Xaa33-Asn-Pro-Xaa36-Xaa37-Asp-Xaa39-Trp-Pro-Xaa42-Ala-Asn-Xaa45-Val-Gly(II).

[0036] In another preferred embodiment, Xaa2 is Thr or Gln, or Xaa4 is Leu, or Xaa6 is Ile, Val or Thr, or Xaa13 is Phe, or Xaa21 is Ala or Leu, or Xaa23 is Arg, Lys or modified Lys, or Xaa26 is Ser or Thr, or Xaa33 is Phe, Leu or Tyr, or Xaa42 is Glu;

[0037] Preferably, the amino acid sequence of B is as shown in the following formula III:

[0038] Gly-Xaa2-Asn-Leu-Ser-Xaa6-Xaa7-Asn-Pro-Leu-Gly-Phe-Phe-Pro-Asp-His-Gln-Leu-Asp-Pro-Xaa21-Phe-Xaa23-Ala-Asn- Xaa26-Xaa27-Asn-Pro-Xaa30-Trp-Asp-Xaa33-Asn-Pro-Xaa36-Xaa37-Asp-Xaa39-Trp-Pro-Glu-Ala-Asn-Xaa45-Val-Gly (III).

[0039] More preferably, the amino acid sequence of B is as shown in the following formula IV:

[0040] Gly-Xaa2-Asn-Leu-Ser-Xaa6-Xaa7-Asn-Pro-Leu-Gly-Phe-Phe-Pro-Asp-His-Gln-Leu-Asp-Pro-Xaa21-Phe-Arg-Ala-A sn-Xaa26-Xaa27-Asn-Pro-Asp-Trp-Asp-Xaa33-Asn-Pro-Asn-Lys-Asp-Xaa39-Trp-Pro-Glu-Ala-Asn-Lys-Val-Gly(IV),

[0041] Among them, Xaa2 is Thr or Gln, Xaa6 is Ile, Thr, Xaa7 is Pro, Ser, Xaa21 is Leu, Ala; Xaa26 is Ser, Thr, Xaa27 is Asn, Ala; Xaa33 is Leu, Phe, Tyr; Xaa39 is His, Thr.

[0042] In another preferred embodiment, the modified Lys is selected from the following group: Lys-linker1 n1 -Y n2 、Lys-linker1 n1 -Chol, Lys-linker1 n1 -γGlu-C18 diacid or a combination thereof;

[0043] wherein n1 is a positive integer 0 or ≥1, for example, 1-4; n2 is a positive integer ≥1, for example, 1-4; linker1 is AEEA, PEG (polyethylene glycol) or a derivative thereof, and Y is a maleimide-substituted C1-C8 acid; wherein AEEA is 8-amino-3,6-dioxooctanoic acid, and γGlu-C18 diacid is a fatty acid.

[0044] In another preferred embodiment, the maleimide-substituted C1-C8 acid may be 3-maleimidopropionic acid (MPA), 6-maleimidocaproic acid, 5-maleimidopentanoic acid, 4-maleimidobutyric acid, or 4-(N-maleimidomethyl)cyclohexylcarboxylic acid.

[0045] In another preferred embodiment, the maleimide-substituted C1-C8 acid is MPA.

[0046] In another preferred embodiment, the modification of the modified Lys is selected from:

[0047] In another preferred embodiment, the modification of the N-terminus of the polypeptide is selected from the group consisting of myristic acid (Myr) modification, cholesterol (Chol) or its derivative modification, γGlu-C18 diacid modification, hydrophobic modification, acylation modification or a combination thereof;

[0048] Preferably, the acylation modification is selected from myristoyl (CH3 (CH2) 12 CO-), palmitoyl (C16, i.e. CH3 (CH2) 14 CO-) or stearoyl (C18, i.e. CH3(CH2) 16 CO) acylation modification.

[0049] In another preferred embodiment, the modification of the N-terminus of the polypeptide is Myr modification.

[0050] In another preferred embodiment, the modification of the N-terminus of the polypeptide is Chol-PEG4-COOH modification.

[0051] In another preferred embodiment, the modification of the N-terminus of the polypeptide is myristoyl (CH3(CH2) 12 CO-) modification.

[0052] In another preferred embodiment, the modification at the N-terminus of the polypeptide is γGlu-C18 diacid modification.

[0053] In another preferred embodiment, the modification of the C-terminus of the polypeptide is selected from the following group: linker2 n3 -X n3 modification, Chol-PEG4-COOH modification, D-amino acid modification, cyclic amino acid modification, PEG modification and glycan modification or a combination thereof,

[0054] Wherein, linker2 is a diamine, PEG or a derivative thereof, and the diamine is preferably ethylenediamine, propylenediamine, butanediamine or 1,8-diamino-3,6-dioxaoctane (AEEEA); X is a maleimide-substituted C1-C8 acid, a γGlu-C18 diacid modification or MPA amide; and n3 is a positive integer ≥1, for example, 1-4.

[0055] In another preferred embodiment, the maleimide-substituted C1-C8 acid may be 3-maleimidopropionic acid (MPA), 6-maleimidocaproic acid, 5-maleimidopentanoic acid, 4-maleimidobutyric acid, or 4-(N-maleimidomethyl)cyclohexylcarboxylic acid.

[0056] In another preferred embodiment, the maleimide-substituted C1-C8 acid is MPA.

[0057] In another preferred embodiment, the modification of the C-terminus of the polypeptide is AEEEA-MPA modification.

[0058] In another preferred embodiment, the modification of the C-terminus of the polypeptide is

[0059] In another preferred embodiment, the polypeptide is artificially synthesized.

[0060] In another preferred embodiment, the amino acid sequence of B in the polypeptide is selected from the following group:

[0061] (a) a polypeptide having the amino acid sequence shown in SEQ ID NO: 1;

[0062] (b) A polypeptide derived from (a) having the function of blocking hepatitis virus infection, formed by substituting, deleting or adding 1-5 (preferably 1-3, more preferably 1-2) amino acid residues of the amino acid sequence shown in SEQ ID NO: 1.

[0063] In another preferred embodiment, the hepatitis virus is selected from the group consisting of hepatitis B virus (HBV), hepatitis D virus (HDV), or a combination thereof.

[0064] In another preferred embodiment, the amino acid sequence of B in the polypeptide is the polypeptide shown in SEQ ID NO: 1 with 1-3, preferably 1-2, more preferably 1 amino acid substitution or deletion; and / or

[0065] It is formed by adding 1-5, preferably 1-4, more preferably 1-3, and most preferably 1-2 amino acids.

[0066] In another preferred embodiment, the analog retains ≥70% of the activity of the polypeptide of SEQ ID NO: 1 in blocking hepatitis virus infection.

[0067] In another preferred embodiment, the amino acid sequence of the analog has a homology of ≥85% with that of SEQ ID NO: 1, preferably ≥90%; more preferably ≥95%.

[0068] In another preferred embodiment, the amino acid sequence of B in the long-acting inhibitor is shown in SEQ ID NO: 2-13.

[0069] In another preferred embodiment, the amino acid sequence of B in the long-acting inhibitor is as shown in SEQ ID NO: 2, 4 or 6.

[0070] In some preferred embodiments, in the sodium-taurocholate cotransporter long-acting inhibitor,

[0071] The amino acid sequence of B in the polypeptide is as shown in any one of SEQ ID NOs: 2-13; preferably, the amino acid sequence of B in the polypeptide is selected from SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6;

[0072] The modification of the N-terminus of the polypeptide is selected from myristic acid modification and acylation modification; preferably, the acylation modification is selected from myristoyl (CH3 (CH2) 12 CO-) acylation modification;

[0073] And the modified Lys is selected from: Lys-linker1 n1 -Y n2 ,

[0074] wherein n1 is 1, n2 is 1, linker 1 is AEEA, PEG or a derivative thereof, preferably AEEA, and Y is a maleimide-substituted C1-C8 acid; wherein AEEA is 8-amino-3,6-dioxooctanoic acid;

[0075] Preferably, the maleimide-substituted C1-C8 acid is 3-maleimidopropionic acid (MPA), 6-maleimidocaproic acid, 5-maleimidopentanoic acid, 4-maleimidobutyric acid, or 4-(N-maleimidomethyl)cyclohexylcarboxylic acid, and the maleimide-substituted C1-C8 acid is preferably MPA.

[0076] In some preferred embodiments, in the sodium-taurocholate cotransporter long-acting inhibitor,

[0077] The amino acid sequence of B in the polypeptide is as shown in any one of SEQ ID NOs: 2-13; preferably, the amino acid sequence of B in the polypeptide is selected from SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6;

[0078] The modification of the N-terminus of the polypeptide is selected from myristic acid modification and acylation modification; preferably, the acylation modification is selected from myristoyl (CH3 (CH2) 12 CO-) acylation modification;

[0079] The modification of the C-terminus of the polypeptide is selected from: linker2 n3 -X n3 Modification,

[0080] Wherein, linker2 is a diamine, PEG or a derivative thereof, wherein the diamine is preferably ethylenediamine, propylenediamine, butylenediamine or 1,8-diamino-3,6-dioxaoctane, more preferably 1,8-diamino-3,6-dioxaoctane; X is a maleimide-substituted C1-C8 acid, a γGlu-C18 diacid modification or MPA amide; and n3 is 1;

[0081] Preferably, the maleimide-substituted C1-C8 acid is 3-maleimidopropionic acid (MPA), 6-maleimidocaproic acid, 5-maleimidopentanoic acid, 4-maleimidobutyric acid, or 4-(N-maleimidomethyl)cyclohexylcarboxylic acid, and the maleimide-substituted C1-C8 acid is preferably MPA.

[0082] In some preferred embodiments, the sodium-taurocholate co-transporter long-acting inhibitor is selected from the following:

[0083] Among them, (Myr') represents

[0084] (AEEEA'-MPA')

[0085] (AEEA'-MPA')

[0086] The second aspect of the present invention provides a polypeptide-HSA conjugate selected from the following:

[0087] 12M1-HSA, 12M2-HSA, 28M1-HSA, 28M2-HSA, 34M1-HSA, 34M2-HSA,

[0088] Wherein, the structures of 12M1, 12M2, 28M1, 28M2, 34M1, and 34M2 are as defined above, HSA is human serum albumin, and the polypeptide-HSA conjugate is a covalent conjugate of the sulfhydryl group on HSA and the side chain MPA of 12M1, 12M2, 28M1, 28M2, 34M1, and 34M2.

[0089] The third aspect of the present invention provides an isolated nucleic acid molecule encoding the long-acting inhibitor of sodium-taurocholate cotransporter (NTCP) according to the first aspect of the present invention or the polypeptide-HSA conjugate according to the second aspect.

[0090] In some preferred examples, the nucleic acid molecule encodes the polypeptide whose amino acid sequence is shown in SEQ ID NO: 2-13 according to the first aspect of the present invention.

[0091] A fourth aspect of the present invention provides a pharmaceutical composition comprising:

[0092] (a) a therapeutically effective amount of the long-acting inhibitor of sodium-taurocholate cotransporter (NTCP) according to the first aspect of the present invention, or the polypeptide-HSA conjugate according to the second aspect of the present invention, or the nucleic acid molecule according to the third aspect of the present invention; and

[0093] (b) a pharmaceutically acceptable carrier.

[0094] In another preferred embodiment, the pharmaceutical composition is used to treat, inhibit and / or prevent diseases related to sodium-taurocholate cotransporter (NTCP).

[0095] In another preferred embodiment, the NTCP-related diseases include hepatitis virus infection-related diseases, type 2 diabetes, obesity, intestinal autoimmune diseases, biliary cirrhosis and atherosclerosis.

[0096] In another preferred embodiment, the pharmaceutical composition is used to competitively bind to sodium-taurocholic acid co-transporter (NTCP) to inhibit hepatitis virus infection, and the hepatitis virus includes hepatitis B virus and / or hepatitis D virus.

[0097] In a fifth aspect, the present invention provides a long-acting inhibitor of sodium-taurocholate cotransporter (NTCP) according to the first aspect of the present invention, or the polypeptide-HSA conjugate according to the second aspect of the present invention, or the nucleic acid molecule according to the third aspect of the present invention for the preparation of a medicament for treating, inhibiting and / or preventing NTCP-related diseases.

[0098] In another preferred embodiment, the NTCP-related diseases include hepatitis virus infection-related diseases, type 2 diabetes, obesity, intestinal autoimmune diseases, biliary cirrhosis and atherosclerosis.

[0099] In another preferred embodiment, the hepatitis virus infection-related disease is hepatitis B and / or hepatitis D.

[0100] In another preferred embodiment, the drug is administered via the following routes: subcutaneous, intravenous, intramuscular, inhalation, injection or suppository.

[0101] In a sixth aspect, the present invention provides a method for preventing hepatitis virus from infecting host cells, comprising administering to the host cell culture system the long-acting inhibitor of sodium-taurocholate cotransporter (NTCP) described in the first aspect of the present invention, or the polypeptide-HSA conjugate described in the second aspect of the present invention, or the nucleic acid molecule described in the third aspect of the present invention, or the pharmaceutical composition described in the fourth aspect of the present invention.

[0102] In another preferred embodiment, the host cell is derived from humans or non-human mammals.

[0103] In another preferred embodiment, the method is an in vitro method.

[0104] In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

[0105] The seventh aspect of the present invention provides a method for preparing the long-acting inhibitor of sodium-taurocholate cotransporter (NTCP) according to the first aspect of the present invention, the method comprising the steps of:

[0106] (i) performing mutation and modification design based on the PreS1 sequences of different hepatitis B viruses to obtain a designed polypeptide sequence;

[0107] (ii) synthesizing a main chain sequence according to the polypeptide sequence designed in step (i), and then modifying the synthesized main chain sequence to obtain the sodium ion-taurocholic acid cotransporter (NTCP) long-acting inhibitor.

[0108] In another preferred embodiment, in step (ii), the main chain sequence is synthesized by a method comprising the following steps: using peptide resin Resin as a solid phase carrier, gradually coupling amino acids according to the main chain sequence, and using a suitable condensation system such as HBTU / 6-Cl-HOBt / DIEA.

[0109] In another preferred embodiment, in step (ii), the C-terminus of the synthesized main chain is subjected to a long-term modification, or the long-term modification is carried out in a liquid phase system.

[0110] In another preferred embodiment, the long-acting modification is selected from the following group: linker3 n3 -X n3modification, Chol-PEG4-COOH modification, D-amino acid modification, cyclic amino acid modification, PEG modification and glycan modification or a combination thereof,

[0111] Wherein, linker3 is a diamine, PEG or a derivative thereof, and the diamine is preferably ethylenediamine, propylenediamine, butanediamine or 1,8-diamino-3,6-dioxaoctane; X is a maleimide-substituted C1-C8 acid, a γGlu-C18 diacid modification or MPA amide; and n3 is a positive integer ≥1, for example, 1-4.

[0112] In another preferred embodiment, the maleimide-substituted C1-C8 acid may be 3-maleimidopropionic acid (MPA), 6-maleimidocaproic acid, 5-maleimidopentanoic acid, 4-maleimidobutyric acid, or 4-(N-maleimidomethyl)cyclohexylcarboxylic acid.

[0113] In another preferred embodiment, the maleimide-substituted C1-C8 acid is MPA.

[0114] In another preferred embodiment, the modification of the C-terminus of the polypeptide is AEEEA-MPA modification.

[0115] In an eighth aspect, the present invention provides a method for treating an NTCP-related disease, comprising administering to a subject in need thereof the long-acting inhibitor of the sodium-taurocholic acid cotransporter (NTCP) described in the first aspect of the present invention, or the polypeptide-HSA conjugate described in the second aspect of the present invention, or the nucleic acid molecule described in the third aspect, or the pharmaceutical composition described in the fourth aspect of the present invention.

[0116] In another preferred embodiment, the NTCP-related diseases include hepatitis virus infection-related diseases, type 2 diabetes, obesity, intestinal autoimmune diseases, biliary cirrhosis and atherosclerosis.

[0117] In another preferred embodiment, the hepatitis virus infection-related disease is hepatitis B and / or hepatitis D.

[0118] In another preferred embodiment, the subject includes humans and non-human mammals.

[0119] In addition, this application also includes the following technical solutions:

[0120] Technical Solution 1: A long-acting inhibitor of sodium-taurocholate cotransporter, comprising a polypeptide represented by the general formula ABR, or an analog thereof, and a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of B is as shown in the following formula I, A is a modification of the N-terminus, and R is a modification of the C-terminus.

[0121] Gly-Xaa2-Asn-Xaa4-Ser-Xaa6-Xaa7-Asn-Pro-Leu-Gly-Phe-Xaa13-Pro-Xaa15-His-Gln-Leu-Asp-Pro-Xaa21-Xaa22-Xaa23-Ala-A sn-Xaa26-Xaa27-Asn-Pro-Xaa30-Trp-Asp-Xaa33-Asn-Pro-Xaa36-Xaa37-Asp-Xaa39-Trp-Pro-Xaa42-Ala-Asn-Xaa45-Val-Gly(I);

[0122] Where,

[0123] Xaa2 is Thr, Ser, Lys, modified Lys, Gln or Asn;

[0124] Xaa4 is Leu, Gln, Ile or Phe;

[0125] Xaa6 is Val, Ile, Leu, Met, Phe, Ala, Thr, Trp, Asn, Gln or Gly;

[0126] Xaa7 is Pro, Ala, or Ser;

[0127] Xaa13 is Phe, His, or Leu;

[0128] Xaa15 is Asp or Glu;

[0129] Xaa21 is Ala, Ile, Val, Leu, Glu, Asp, Asn, Cys, Met, Gln, Trp or Pro;

[0130] Xaa22 is Phe or Trp;

[0131] Xaa23 is Gly, Pro, Ala, Arg, Lys, modified Lys, or Glu;

[0132] Xaa26 is Ser, Met, Leu, Thr, Phe, Ile, or Ala;

[0133] Xaa27 is Asn, Gln, His, Lys, modified Lys, Ala, or Arg;

[0134] Xaa30 is Asp or Glu;

[0135] Xaa33 is Phe, Leu, His, Tyr, Arg, Lys, modified Lys, Ala, Trp, Ser, Ile, Val, or Thr;

[0136] Xaa36 is Asn, Gln, Lys, modified Lys, or Leu;

[0137] Xaa37 is Lys or modified Lys;

[0138] Xaa39 is His, Asn, Thr, Phe, Tyr, Gln, Lys, modified Lys, or Arg;

[0139] Xaa42 is Glu, Asp, Val, Ala, Met, Lys, Gln, Thr, Asn, Leu, Arg, Ser, or His;

[0140] Xaa45 is Glu, Asp, Val, Ala, Met, Lys, Gln, Thr, Asn, Leu, Arg, Ser, His, or an amino acid with a modification, preferably Lys or a modified Lys;

[0141] The amino acid sequence of B has a homology of ≥85% with the amino acid sequence of SEQ ID NO: 1, and has the activity of binding to NTCP.

[0142] Technical Solution 2: The long-acting inhibitor of the sodium-taurocholate cotransporter according to Technical Solution 1, characterized in that Xaa2 is Thr, Ser, Lys, modified Lys or Gln, or Xaa6 is Val, Thr, Ile or Leu, or Xaa7 is Pro or Ser, or Xaa13 is Phe or Leu, or Xaa15 is Asp, or Xaa21 is Ala, Leu, Val, Glu, Ile or Asp; or Xaa23 is Arg, Lys, modified Lys or Glu; or Xaa27 is Asn or Ala, or Xaa33 is Phe, Leu, His or Tyr, or Xaa39 is His, Asn, Thr, Phe or Tyr, or Xaa42 is Glu or Asp;

[0143] Preferably, the amino acid sequence of B is as shown in the following formula II:

[0144] Gly-Xaa2-Asn-Xaa4-Ser-Xaa6-Xaa7-Asn-Pro-Leu-Gly-Phe-Xaa13-Pro-Asp-His-Gln-Leu-Asp-Pro-Xaa21-Xaa22-Xaa23-Ala-As n-Xaa26-Xaa27-Asn-Pro-Xaa30-Trp-Asp-Xaa33-Asn-Pro-Xaa36-Xaa37-Asp-Xaa39-Trp-Pro-Xaa42-Ala-Asn-Xaa45-Val-Gly(II).

[0145] Technical Solution 3: The long-acting inhibitor of the sodium-taurocholate cotransporter according to Technical Solution 2, characterized in that Xaa2 is Thr or Gln, or Xaa4 is Leu, or Xaa6 is Ile, Val or Thr, or Xaa13 is Phe, or Xaa21 is Ala or Leu, or Xaa23 is Arg, Lys or modified Lys, or Xaa26 is Ser or Thr, or Xaa33 is Phe, Leu or Tyr, or Xaa42 is Glu;

[0146] Preferably, the amino acid sequence of B is as shown in the following formula III:

[0147] Gly-Xaa2-Asn-Leu-Ser-Xaa6-Xaa7-Asn-Pro-Leu-Gly-Phe-Phe-Pro-Asp-His-Gln-Leu-Asp-Pro-Xaa21-Phe-Xaa23-Ala-Asn- Xaa26-Xaa27-Asn-Pro-Xaa30-Trp-Asp-Xaa33-Asn-Pro-Xaa36-Xaa37-Asp-Xaa39-Trp-Pro-Glu-Ala-Asn-Xaa45-Val-Gly (III).

[0148] Technical Solution 4: A long-acting inhibitor of the sodium-taurocholate co-transporter according to any one of Technical Solutions 1 to 3, characterized in that the amino acid sequence of B in the polypeptide is as shown in SEQ ID NO: 2-13.

[0149] Technical solution 5: The long-acting inhibitor of sodium-taurocholate cotransporter according to any one of technical solutions 1 to 4, characterized in that the modified Lys is selected from the following group: Lys-linker 1 n1 -Y n2 、Lys-linker1 n1 -Chol, Lys-linker1n1 -γGlu-C18 diacid or a combination thereof;

[0150] wherein n1 is a positive integer of 0 or ≥1, n2 is a positive integer ≥1, linker1 is AEEA, PEG or a derivative thereof, and Y is a maleimide-substituted C1-C8 acid; wherein AEEA is 8-amino-3,6-dioxooctanoic acid, and γGlu-C18diacid is a fatty acid;

[0151] Preferably, the maleimide-substituted C1-C8 acid is 3-maleimidopropionic acid (MPA), 6-maleimidocaproic acid, 5-maleimidopentanoic acid, 4-maleimidobutyric acid, or 4-(N-maleimidomethyl)cyclohexylcarboxylic acid, and the maleimide-substituted C1-C8 acid is preferably MPA.

[0152] Technical Solution 6: The long-acting inhibitor of the sodium-taurocholate co-transporter according to any one of Technical Solutions 1 to 4, characterized in that the modification of the N-terminus of the polypeptide is selected from the group consisting of myristic acid modification, cholesterol (Chol) or its derivative modification, γGlu-C18 diacid modification, hydrophobic modification, acylation modification, or a combination thereof;

[0153] Preferably, the acylation modification is selected from CO(CH2) 12 Acylation modification with CO-, palmitoyl (C16) or stearoyl (C18).

[0154] Technical Solution 7: The long-acting inhibitor of sodium-taurocholic acid co-transporter according to any one of Technical Solutions 1 to 4, characterized in that the modification of the C-terminus of the polypeptide is selected from the following group: linker2 n3 -X n3 modification, Chol-PEG4-COOH modification, D-amino acid modification, cyclic amino acid modification, PEG modification and glycan modification or a combination thereof,

[0155] Wherein, linker2 is a diamine, PEG or a derivative thereof, wherein the diamine is preferably ethylenediamine, propylenediamine, butanediamine or 1,8-diamino-3,6-dioxaoctane; X is a maleimide-substituted C1-C8 acid, a γGlu-C18 diacid modification or MPA amide; and n3 is a positive integer ≥1;

[0156] Preferably, the maleimide-substituted C1-C8 acid is 3-maleimidopropionic acid (MPA), 6-maleimidocaproic acid, 5-maleimidopentanoic acid, 4-maleimidobutyric acid, or 4-(N-maleimidomethyl)cyclohexylcarboxylic acid, and the maleimide-substituted C1-C8 acid is preferably MPA.

[0157] Technical Solution 8: A nucleic acid molecule encoding the long-acting inhibitor of the sodium-taurocholic acid co-transporter described in any one of Technical Solutions 1 to 7.

[0158] Technical Solution 9: A pharmaceutical composition comprising:

[0159] (a) a therapeutically effective amount of the long-acting inhibitor of sodium-taurocholate cotransporter according to any one of technical solutions 1 to 7, or the nucleic acid molecule according to technical solution 8; and

[0160] (b) a pharmaceutically acceptable carrier.

[0161] Technical Solution 10: Use of the long-acting sodium-taurocholate cotransporter inhibitor according to any one of Technical Solutions 1 to 7 or the nucleic acid molecule according to Technical Solution 8 in the preparation of a drug for treating, inhibiting and / or preventing NTCP-related diseases;

[0162] Preferably, the NTCP-related diseases include hepatitis virus infection-related diseases, type II diabetes, obesity, intestinal autoimmune diseases, biliary cirrhosis and atherosclerosis;

[0163] More preferably, the hepatitis virus infection-related disease is hepatitis B and / or hepatitis D.

[0164] Technical Solution 11: The use as described in Technical Solution 10 is characterized in that the drug is administered through the following routes of administration: subcutaneous, intravenous, intramuscular, inhalation or suppository.

[0165] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0166] FIG1 shows the binding percentage between NTCP inhibitor (polypeptide) and NTCP-positive cells analyzed by flow cytometry in Example 3;

[0167] Figure 2 shows that in Example 4, Western Blot showed:

[0168] The constructed plasmid successfully expressed NTCP-Flag fusion protein;

[0169] NTCP siRNA881 treated cells for 48 hours and significantly knocked down the expression level of NTCP protein;

[0170] FIG3 shows that in Example 4, cell ELISA showed that NTCP siRNA treatment of cells for 48 hours reduced the amount of polypeptide bound to the cells;

[0171] Figure 4 shows that in Example 5, CCK-8 analysis shows that in the experiment of inhibiting HBV infection of hepatocytes, the concentrations of the polypeptide and its conjugate used in treating hepatocytes for 8 or 12 days did not affect the cell survival rate;

[0172] Figure 5 shows that in Example 5, ELISA showed that the inhibitory effects of the polypeptide and its conjugate on HBV multi-subtype infected hepatocytes were stronger than those of Yangshen;

[0173] FIG6 shows that in Example 6, CCK-8 analysis shows that in the experiment of inhibiting HDV infection of hepatocytes, the concentration of 12M2 used for treating hepatocytes for 8 days did not affect the survival rate of hepatocytes;

[0174] FIG7 shows that in Example 6, qPCR analysis showed that 12M2 and Yangshen had similar significant inhibitory effects in inhibiting HDV infection of hepatocytes. DETAILED DESCRIPTION

[0175] The present invention is based on the sequence of PreS1 of different genotypes of HBV virus, and a batch of NTCP long-acting inhibitors are prepared by designing and screening functional non-inactivating mutants. Specifically, the present invention uses biochemical methods to design dozens of candidate sequences based on the structure, properties and point mutations of amino acids and the impact of polypeptide function, and based on homology analysis and biological characteristics analysis; synthesizes them by solid phase method, separates and purifies to obtain high-purity polypeptide compounds; uses flow cytometry to preliminarily screen candidate peptides with strong binding to cell surface hepatitis B virus receptor NTCP; then uses cell ELISA method to detect the EC50 value of binding between polypeptide and NTCP overexpressing cells, and further confirms and screens candidate peptides that show high affinity to NTCP receptor; further, through ELISA, The SA method detects the HBsAg level in the culture supernatant of human primary hepatocytes (PHH) to screen candidate peptides with strong inhibitory ability against hepatitis B virus entering PHH; the amount of hepatitis D virus (HDV) RNA in human primary hepatocytes is also detected by real-time quantitative probe PCR (qPCR) method to determine that the screened polypeptide has a strong inhibitory ability against HDV infection of human primary hepatocytes; Subsequently, the plasma half-life of the NTCP inhibitor was detected by animal in vivo PK experiments, confirming that the NTCP inhibitor provided by the present invention is a long-acting inhibitor, and compared with existing similar competing products, the plasma stability is high and the half-life is significantly increased. Ultimately, a new type of NTCP long-acting inhibitor polypeptide was obtained. On this basis, the present invention was completed.

[0176] The long-acting NTCP inhibitor of the present invention can not only prevent hepatitis B virus and hepatitis D virus from infecting liver cells, but also the NTCP signaling pathway is closely related to obesity and type 2 diabetes, intestinal autoimmune diseases, biliary cirrhosis and atherosclerosis. Therefore, the long-acting NTCP inhibitor can be used as a therapeutic drug for other NTCP-related diseases.

[0177] The inhibitors of the present invention can be used to treat NTCP-related diseases, including hepatitis virus inhibitory and therapeutic drugs, as well as NTCP-related diseases such as type 2 diabetes, obesity, intestinal autoimmune diseases, biliary cirrhosis, and atherosclerosis. For example, they inhibit HBV infection of hepatocytes by inhibiting the binding of the PreS1 segment to host NTCP.

[0178] On this basis, the present invention was completed.

[0179] Polypeptide of the present invention

[0180] In the present invention, the terms "long-acting inhibitor of sodium-taurocholate cotransporter", "long-acting inhibitor of NTCP", "long-acting inhibitor of NTCP polypeptide" and "polypeptide of the present invention" can be used interchangeably, all referring to the polypeptides prepared by the present invention having the amino acid sequences shown in SEQ ID NO: 1-13 and their variant forms.

[0181] Furthermore, the term also encompasses variants of SEQ ID NOs: 1-13 that function as long-acting NTCP inhibitors. These variants include (but are not limited to): deletions, insertions, and / or substitutions of 1-5 (generally 1-4, preferably 1-3, more preferably 1-2, and most preferably 1) amino acids, as well as additions or deletions of one or more (generally 5 or fewer, preferably 3 or fewer, and more preferably 2 or fewer) amino acids at the C-terminus and / or N-terminus. For example, substitutions with amino acids having similar or similar properties generally do not alter protein function. For another example, additions or deletions of one or more amino acids at the C-terminus and / or N-terminus generally do not alter protein structure and function. Furthermore, the term encompasses both monomeric and multimeric forms of the polypeptides of the present invention. The term also encompasses linear and nonlinear polypeptides (e.g., cyclic peptides).

[0182] The present invention obtains a long-acting NTCP inhibitor (modified mutant polypeptide) by mutating and modifying the consensus sequence of PreS1 of various HBV virus subtypes. The long-acting inhibitor can improve the affinity between NTCP and enhance its ability to inhibit the entry of HBV / HDV, thereby improving its solubility and plasma stability. The long-acting modification also prolongs its half-life, thereby reducing the frequency of administration.

[0183] The NTCP long-acting inhibitor allows it to react with groups (particularly available sulfhydryl groups) on blood components to form stable covalent conjugates. Preferred NTCP receptor long-acting inhibitors are designed to react specifically with sulfhydryl groups on flowing blood proteins. This reaction is established by covalent bonding of peptides with long-acting modifications (e.g., MPA modifications, or other modifications containing compounds with maleimide ring structures; wherein, compounds containing maleimide ring structures include, for example, 6-maleimidohexanoic acid, 5-maleimidovaleric acid, 4-maleimidobutyric acid, 4-(N-maleimidomethyl)cyclohexylcarboxylic acid, etc.) connectors to sulfhydryl groups on flowing blood proteins (e.g., serum albumin or immunoglobulin (Ig)). Therefore, one embodiment of the present invention includes modified peptides covalently linked to blood proteins, and blood proteins include endogenous flowing blood proteins. Particularly preferred embodiments of the present invention include covalent bonding of modified modified peptides to serum albumin.

[0184] The present invention also includes active fragments and analogs of long-acting inhibitory polypeptides of the sodium-taurocholic acid cotransporter (NTCP). As used herein, the terms "fragment" and "analog" refer to polypeptides that substantially retain the activity of long-acting inhibitory polypeptides of the sodium-taurocholic acid cotransporter (NTCP). The polypeptide fragments or analogs of the present invention can be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) polypeptides having a substituent group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a long-acting inhibitory polypeptide of the sodium-taurocholic acid cotransporter (NTCP) with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of additional amino acid sequences to this polypeptide sequence (proteins formed by fusion with a leader sequence, secretory sequence, or tag sequence such as 6His). Based on the teachings herein, these fragments and analogs are well known to those skilled in the art.

[0185] A preferred class of active analogs refers to polypeptides formed by replacing at most 6, preferably at most 3, more preferably at most 2, and most preferably 1 amino acid with similar or similar properties compared to the amino acid sequence of Formula I.

[0186] The present invention also provides analogs of long-acting inhibitory polypeptides of the sodium-taurocholate cotransporter (NTCP). The differences between these analogs and the polypeptide shown in SEQ ID NO: 1 may be differences in the amino acid sequence, or differences in the form of modifications that do not affect the sequence, or both. Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0187] Some commonly used unnatural amino acids are listed in Table A below.

[0188] Table A

[0189] Modifications (generally without altering the primary structure) include chemical derivatization of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those resulting from polypeptide synthesis and processing or further processing steps. Such modifications can be accomplished by exposing the polypeptide to glycosylation enzymes (e.g., mammalian glycosylases or deglycosylases). Modifications also include sequences containing phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, and phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or optimize their solubility.

[0190] In a preferred embodiment, the present invention particularly provides a C 10 -C 24 Acyl modified polypeptides, and long-acting preparations containing the modified polypeptides of the present invention. It should be understood that the acyl groups that can be used for acylation modification of the present invention are not particularly limited and can be acyl groups having 10-24 carbon atoms, including substituted or unsubstituted, and / or saturated or unsaturated acyl groups. Preferably, acyl groups having 10-24 carbon atoms that can be used in the present invention include acyl groups containing 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 carbon atoms.

[0191] The long-acting sodium-taurocholate cotransporter (NTCP) inhibitors of the present invention can also be used or formulated in the form of salts derived from pharmaceutically or physiologically acceptable acids or bases. These salts include, but are not limited to, salts formed with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid. Other salts include salts formed with alkali metals or alkaline earth metals (such as sodium, potassium, calcium, or magnesium), as well as in the form of esters, carbamates, or other conventional "prodrugs."

[0192] In the present invention, the terms "positive control" and "P3" refer to the sequence of the synthetic sodium-taurocholate cotransporter (NTCP) polypeptide-Myrclude B in the prior art.

[0193] In the present invention, the peptide with Myr coupled to its N-terminus is recorded as compound X, the peptide with MPA modification at its C-terminus and Myr coupled to its N-terminus is recorded as compound XM1, and the peptide with MPA modification at position 45 (counting from the N-terminus) of compound X and Myr coupled to its N-terminus is recorded as compound XM2: for example, compound 12, the peptide with MPA modification at its C-terminus and Myr coupled to its N-terminus, and the polypeptide with MPA modification at position 45 of compound 12 and Myr coupled to its N-terminus are recorded as compound 12, compound 12M1, and compound 12M2, respectively.

[0194] Unless otherwise specified, "12M1", "12M2", "28M1", "28M2", "34M1" and "34M2" herein refer to "Compound 12M1", "Compound 12M2", "Compound 28M1", "Compound 28M2", "Compound 34M1" and "Compound 34M2" respectively.

[0195] Coding sequence

[0196] The present invention also relates to a polynucleotide encoding the polypeptide of the present invention. A preferred polynucleotide encodes the polypeptide shown in SEQ ID NO: 1-13.

[0197] The polynucleotides of the present invention may be in the form of DNA or RNA. The DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical or a degenerate variant. As used herein, "degenerate variant" refers to a nucleic acid sequence encoding a polypeptide having the amino acid sequence set forth in any one of SEQ ID NOs: 1-13, but having a different coding region sequence.

[0198] The full-length nucleotide sequence of the present invention or its fragments can generally be obtained by PCR amplification or artificial synthesis. Currently, DNA sequences encoding the polypeptides of the present invention (or fragments thereof, or analogs thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be inserted into various existing DNA molecules (or vectors) known in the art or introduced into cells.

[0199] The present invention also relates to vectors comprising the polynucleotides of the present invention, and host cells produced by genetic engineering using the vectors or polypeptide encoding sequences of the present invention.

[0200] In the context of two nucleic acids or polypeptides, when compared and aligned for maximum correspondence, the term "substantially identical" refers to two or more sequences or subsequences that have at least about 80%, for example, at least about 85%, about 90%, about 95%, about 98%, or about 99% of the nucleotide or amino acid residues homologous to a specified reference sequence, as determined by sequence analysis using relevant software and / or by visual analysis.

[0201] Preparation method

[0202] The polypeptides of the present invention may be recombinant polypeptides or synthetic polypeptides. The polypeptides of the present invention may be chemically synthesized or recombinant. Accordingly, the polypeptides of the present invention may be synthesized artificially using conventional chemical methods or produced using recombinant methods.

[0203] A preferred method is to use solid-phase synthesis technology or a solid-liquid combination method, wherein the solid-phase synthesis method adopts the Boc method, the Fmoc method or a combination of the two methods. Solid-phase synthesis can quickly prepare and obtain samples, and an appropriate resin carrier and synthesis system can be selected according to the sequence characteristics of the target peptide. For example, a preferred solid-phase carrier in the Fmoc system is Wang resin connected to the C-terminal amino acid in the peptide, treated at room temperature for 20 minutes to remove the Fmoc protecting group, and extended from the C-terminus to the N-terminus one by one according to the given amino acid sequence. After the synthesis is completed, it is cut from the resin and the protecting group is removed, and the crude peptide is precipitated and filtered to obtain the crude peptide. After purification by chromatography, the desired polypeptide raw material is obtained by freeze-drying, nanofiltration or precipitation.

[0204] In a preferred embodiment, the polypeptide of the present invention is prepared according to its sequence by solid phase synthesis, purified by high performance liquid chromatography to obtain a high purity target peptide lyophilized powder, which is refrigerated or frozen for storage.

[0205] Another method is to produce the polypeptide of the present invention using recombinant technology. The polynucleotide sequences of the present invention can be used to express or produce recombinant hepatitis virus entry inhibitory long-acting polypeptides using conventional recombinant DNA technology. Generally, the following steps are involved:

[0206] (1) Using molecular cloning technology, the coding sequence (or variant) of the NTCP long-acting inhibitor polypeptide is cloned into a prokaryotic or eukaryotic expression vector to construct a polypeptide expression plasmid;

[0207] (2) Transforming the polypeptide expression plasmid into the engineered cell line;

[0208] (3) Cultivating engineered cells using appropriate culture medium;

[0209] (4) Collect cells or culture fluid for polypeptide separation and purification.

[0210] Recombinant polypeptides can be expressed intracellularly or on the cell membrane or secreted extracellularly. If desired, the polypeptides can be isolated and purified by various separation methods utilizing their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, collecting the supernatant or collecting and disrupting the cells, obtaining total protein by treatment with a protein precipitant (e.g., salting out), or a combination of affinity chromatography, molecular sieve chromatography (gel filtration), ion exchange chromatography, hydrophobic chromatography, and high performance liquid chromatography (HPLC).

[0211] Pharmaceutical compositions and methods of administration

[0212] In another aspect, the present invention also provides a pharmaceutical composition comprising (a) a safe and effective amount of a long-acting inhibitor of the sodium-taurocholate cotransporter or its analogues, and pharmaceutically acceptable salts thereof, as described herein; and (b) pharmaceutically acceptable excipients, including excipients. The amount of the polypeptide of the present invention is generally 10 μg to 100 mg per dose, preferably 100 to 1000 μg per dose.

[0213] To achieve the purpose of the present invention, an effective dose is about 0.01 to 50 mg / kg, preferably 0.05 to 10 mg / kg, of the polypeptide of the present invention administered to an individual. Furthermore, the polypeptide of the present invention can be used alone or in combination with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).

[0214] The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier used in a formulation. The term refers to pharmaceutical carriers that do not themselves induce the production of antibodies harmful to the individual receiving the composition and that have no significant toxicity after administration. These carriers are well known to those of ordinary skill in the art. A comprehensive discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991). Such carriers include (but are not limited to): acidic substances (hydrochloric acid, carbonic acid, acetic acid, phosphoric acid, citric acid, tartaric acid, maleic acid, malic acid and acidic amino acids), alkaline substances (sodium hydroxide, sodium bicarbonate, ammonia, organic amines and basic amino acids) and buffer salts (sodium acetate, phosphate, carbonate, etc.), various salts (such as phosphate, carbonate, acetate, citrate, tartrate and maleate, etc.), glucose, water, glycerol, ethanol, PEG, adjuvants and combinations thereof.

[0215] The pharmaceutically acceptable carrier in the preparation may contain liquids such as water, saline, glycerol and ethanol, etc. In addition, auxiliary substances such as wetting agents or emulsifiers, pH buffers, osmotic pressure regulators, etc. may also be present in these carriers.

[0216] The dosage form can generally be tablets, pills, powders, injections, tinctures, solutions, extracts, ointments, etc. When the pharmaceutical composition of the present invention is used for actual treatment, various dosage forms of the pharmaceutical composition can be adopted according to the use situation. Preferably, lyophilized powder injection or injection can be exemplified.

[0217] The administration of the preparation includes (but is not limited to): injection, such as intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, inhalation administration or suppository administration, etc. The subject to be prevented or treated can be an animal, especially a human.

[0218] These pharmaceutical compositions can be formulated according to conventional methods by mixing or dissolving, and appropriate pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers are occasionally added, and the formulation process can be carried out in a conventional manner according to the dosage form.

[0219] Method of the present invention

[0220] The present invention provides a method for preventing hepatitis viruses from infecting host cells, comprising administering a long-acting inhibitor of the sodium-taurocholate cotransporter described herein to a culture system of the host cells. The inhibitor competitively binds to NTCP on host cells (e.g., hepatocytes), blocking the binding of hepatitis viruses (e.g., HBV and / or HDV) to NTCP, thereby preventing the hepatitis viruses from infecting the host cells.

[0221] The present invention also provides a method for treating, preventing and / or inhibiting NTCP-related diseases (such as hepatitis B and / or hepatitis D, obesity or type 2 diabetes, intestinal autoimmune diseases, biliary cirrhosis and atherosclerosis, etc.), comprising administering the long-acting inhibitor of the sodium-taurocholic acid cotransporter of the present invention to a subject in need.

[0222] Preferably, the polypeptide of the present invention can be used as a long-acting hepatitis virus blocker to prevent HBV and / or HDV from infecting host cells, thereby treating hepatitis B and / or hepatitis D.

[0223] The beneficial effects of the present invention are as follows:

[0224] To address the short half-life of NTCP inhibitors in existing technologies, a series of functional non-inactivating mutants, known as long-acting NTCP inhibitor peptides, were designed and prepared through a process of designing and screening non-inactivating mutations based on the consensus sequence of PreS1 across HBV subtypes. Experimental screening of these non-inactivating mutants revealed that some exhibited binding to NTCP-overexpressing cells exceeding 60% and EC50 values ​​as low as 0.15 μM or even lower. Others exhibited lower binding, ranging from 0.20% to 20%, with EC50 values ​​exceeding 2 μM. Higher binding to NTCP-overexpressing cells indicates stronger affinity for NTCP, greater inhibition of binding between the hepatitis virus surface protein PreS1 and the viral receptor NTCP, and greater inhibition of hepatitis virus infection in liver cells.

[0225] Experiments have also found that long-acting NTCP inhibitor peptides with strong binding ability to NTCP are all water-soluble, and their solubility is significantly higher than that of the existing NTCP inhibitor-P3, about 2-11 times that of P3; experiments also show that compared with existing NTCP inhibitors, these peptides exhibit better HBV entry inhibition ability, which can reach more than 1.5 times, or even more than 5 times, the HBV entry inhibition ability of the existing NTCP inhibitor-P3.

[0226] In addition, after long-term modification of these NTCP long-acting inhibitor polypeptides, such as MPA modification (which can also be replaced with similar compounds such as other compounds containing maleimide ring structures), their half-lives are significantly longer than those of the existing NTCP inhibitor - P3. Among them, some long-term modified NTCP long-acting inhibitor polypeptides have a half-life of more than 5 times that of P3 when administered subcutaneously, and their half-life is about 12 times that of P3 when administered intravenously. Therefore, the plasma stability of the NTCP long-acting inhibitor polypeptide is increased and the drug effect is more lasting, overcoming the technical problem of the short half-life of the existing NTCP inhibitors; the long-term modification can increase its enrichment in hepatocytes and enhance its inhibitory effect on the entry of hepatitis viruses; and it has better solubility and high drugability. This long-acting NTCP inhibitor competitively inhibits the binding between hepatitis virus surface protein and NTCP by binding to NTCP, thereby inhibiting the entry of hepatitis virus. Therefore, it can be used as a therapeutic or preventive drug to block hepatitis virus entry into cells, a drug to inhibit hepatitis recurrence, and a drug for treating hepatitis. In addition, it can achieve a functional cure for hepatitis B by combining it with a hepatitis virus proliferation inhibitor drug.

[0227] Because this peptide is an NTCP inhibitor, it prevents HDV / HBV from infecting hepatocytes by competing with the hepatitis virus envelope protein PreS1 for the HDV and HBV co-receptor NTCP. Since HDV cannot produce its own envelope protein and instead uses the HBV surface antigen protein (HBsAg, including PreS1) as its envelope protein, this long-acting inhibitor is a co-inhibitor of HBV and HDV infecting hepatocytes. Furthermore, inhibiting the NTCP signaling pathway can upregulate insulin secretion, thereby achieving blood sugar and weight loss effects. Furthermore, inhibiting the NTCP signaling pathway can increase circulating levels of modified bile acids, promoting Treg differentiation and inhibiting Th17 cell differentiation, thereby achieving inflammation control. Therefore, this long-acting inhibitor can be used as a drug for the treatment, prevention, and / or inhibition of hepatitis D, hepatitis B, type 2 diabetes, obesity treatment, intestinal autoimmune diseases, biliary cirrhosis, and atherosclerosis.

[0228] Therefore, the polypeptides of the present invention can be developed into drugs for treating, preventing and / or inhibiting diseases related to NTCP binding, including diseases related to hepatitis B virus and / or hepatitis D virus (e.g., hepatitis B, hepatitis D) and type 2 diabetes, obesity, intestinal autoimmune diseases, biliary cirrhosis, and atherosclerosis.

[0229] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0230] Unless otherwise specified, the reagents or materials used in the examples of the present invention are all commercially available products.

[0231] Example 1 Synthesis and Identification of Long-Acting NTCP Inhibitor Peptides

[0232] Sequence design

[0233] Based on the PreS1 sequence of different genotypes of hepatitis B virus, amino acid mutations were performed, resulting in the design of 34 mutant peptides, some of which are shown in Table 1 below. Furthermore, these 34 mutant peptides were modified for long-term efficacy. The sequence of the commercially available NTCP inhibitor peptide Hepcludex (Bulevirtide, Myrcludex B) was synthesized as a yang ginseng, designated P3 in this invention. A polypeptide sequence fragment corresponding to HBV genotype D was also synthesized, designated Compound 26 in this invention.

[0234] Table 1 Basic sequence polypeptide and mutant polypeptide sequences

[0235] Note: Compound X in Table 1 is a peptide coupled to Myr at the N-terminus; for example, compound 21 is a peptide coupled to Myr at the N-terminus based on the free peptide sequence corresponding to compound 21 shown in Table 1, (Myr) represents

[0236] 1.2. Sequence synthesis:

[0237] Backbone sequence synthesis: Using CTC Resin as a solid-phase support, amino acid condensation was performed stepwise along the backbone sequence using a condensation system of HBTU / 6-Cl-HOBt / DIEA. The N- and C-termini were then modified according to specific requirements. The synthesized backbone peptide was N-terminally coupled to Myr, followed by AEEEA and MPA at the C-terminus, or AEEA and MPA at the Lys side chain at position 45, to obtain the crude compound, which was then purified and dried.

[0238] 1.3 Sequence identification

[0239] The synthesized peptide compounds were analyzed for purity by ultra-high performance liquid chromatography (UPLC) and molecular weight by high-resolution time-of-flight (QTOF) mass spectrometry. UPLC analysis showed good purity, and the molecular weights confirmed by high-resolution time-of-flight (QTOF) mass spectrometry were consistent with the theoretical molecular weight. Details are shown in Table 2 below.

[0240] Table 2 MS data of peptide compounds

[0241] 1.4 Preparation of modified peptides and conjugates

[0242] 1.4.1 Preparation of Compound 12M2

[0243] Step 1: Synthesis

[0244] 2Cl-CTC resin is used as a carrier resin and sequentially coupled with protected amino acids corresponding to the polypeptide amino acid sequence to prepare a peptide resin.

[0245] 1. Insert the protected amino acid at position 47 of the main chain

[0246] 1) Take 3 mmol of 2Cl-CTC resin (0.6 mmol / g x 5 g) and swell it with an appropriate amount of DCM for 30 min. After swelling, drain the solvent until almost no liquid flows out.

[0247] 2) Dissolve 1.5 mmol of the 47th protected amino acid Fmoc-Gly-OH in an appropriate amount of DMF:DCM (3:1), add 4.5 mmol of DIEA, and transfer to the resin for coupling of the first amino acid. React for 3 hours. After the reaction is completed, drain the resin, wash it with DMF, and drain it.

[0248] 2. Add other protected amino acids to the main chain

[0249] 1) Remove the Fmoc protecting group of the amino acid at position 47 on the resin with 20% piperidine / DMF, wash with DMF, and drain.

[0250] 2) Dissolve 4.5 mmol of the 46-protected amino acid Fmoc-Val-OH and 4.5 mmol of 6-Cl-HOBt in an appropriate amount of DCM:DMF (1:3) with stirring, then add 4.5 mmol of DIEA. When the temperature drops to 0-10°C, add 4.5 mmol of HBTU, and react at 0-10°C with stirring under nitrogen for 10 min.

[0251] 3) Transfer the amino acid activation solution to the Fmoc-depleted resin and react with stirring at 15-20°C under nitrogen for 1.5 hours. After the reaction, drain the resin. Wash the peptide resin with DMF and drain. A small amount of the peptide resin should be tested with ninhydrin. The result should be colorless. If color is present, re-add the resin under the same conditions as the first condensation.

[0252] According to the Fmoc-Val-OH coupling process of the protected amino acid at position 46, the protected amino acids at positions 45 to 1 and Myr were coupled in sequence, as shown in Table 3 below.

[0253] Table 3: Amino acids coupled accordingly

[0254] 3. Lysine side chain modification

[0255] 1) Dissolve 0.3 mmol of Pd(PPh3)4 in an appropriate amount of DCM:NMP:ACN:DEA (4:2:2:5) and transfer to resin to remove the Alloc side chain protecting group at position 45 of Lys(Alloc). Stir and react for 35 minutes in the dark at 20±5°C. After the reaction, drain the reaction solution, wash with DCM, and drain.

[0256] 2) After Alloc removal, the Lys side chain at position 45 was coupled to Fmoc-AEEA-OH. 4.5 mmol Fmoc-AEEA-OH and 4.5 mmol 6-Cl-HOBt were dissolved in an appropriate amount of DCM:DMF (1:3) with stirring. The mixture was cooled to 0-10°C and 4.5 mmol DIC was added for activation for 5 min. After activation, the mixture was transferred to the resin for coupling reaction. The reaction was stirred at 20-25°C for 3 hours and then drained. The peptide resin was first washed with DMF with stirring and then drained.

[0257] 3) Remove the Fmoc protecting group using 20% ​​piperidine / DMF, wash with DMF, and drain. Dissolve 4.5 mmol of MPA and 4.5 mmol of 6-Cl-HOBt in an appropriate amount of DCM:DMF (1:3) with stirring. Cool to 0-10°C, add 4.5 mmol of DIC, and activate for 5 minutes. After activation, transfer the mixture to the resin for the coupling reaction. Stir at 20-25°C for 3 hours. After completion, drain the mixture. Wash the peptide resin with DMF and DCM with stirring, then drain.

[0258] Step 2: Crude product preparation

[0259] To the peptide resin, add the cleavage reagent (14-16 ml / g resin), mix thoroughly, and react with stirring at 15-20°C for 1.5-2.0 hours to cleave the target peptide from the resin and remove the side chain protecting groups. Collect the filtrate from the reaction mixture, add methyl tert-butyl ether for precipitation, and centrifuge. Wash the filter cake with cold methyl tert-butyl ether three times and drain to obtain an off-white powder, which is the crude peptide. The cleavage reagent composition is as follows: trifluoroacetic acid: water: dithiothreitol = 95:5:1 (vol / vol / weight).

[0260] Step 3: Pure product preparation

[0261] The crude peptide was dissolved in 0.1% TFA / 50% ACN / 50% H2O to a concentration of 10±5 mg / ml, stirred until clear, and filtered through a 0.45 μm organic nylon filter. The filtrate was placed at 20-25°C and stirred for decarboxylation for 12 hours. After decarboxylation, it was filtered through a 0.45 μm organic nylon filter. Purification was performed by reverse-phase high-performance liquid chromatography using a nano-C18 (12 μm, 50×250 mm) mobile phase consisting of mobile phase A (0.1% TFA / water solution) and mobile phase B (0.1% TFA / acetonitrile). The mobile phase flow rate was 62 ml per minute. The UV detection wavelength was 214 nm. The crude solution was loaded onto the chromatographic column and gradient eluted to collect the corresponding purified fractions.

[0262] The collected purified fractions were subjected to reverse-phase HPLC using a nano-C18 column (12 μm, 50×250 mm) with a mobile phase consisting of 25% ACN + 10 mM phosphate / water and 70% ACN + 10 mM phosphate / water. TFA salts were exchanged on-column to phosphate salts using the mobile phase of 25% ACN + 10 mM phosphate / water. Following exchange, the fractions were eluted with 70% ACN + 10 mM phosphate / water to collect the salt-exchanged fractions. The mobile phase flow rate was 62 ml / min. UV detection was performed at 214 nm. The salt-exchanged fractions were combined and freeze-dried under vacuum to obtain the pure peptide.

[0263] 1.4.2 Preparation of Compound 12M1

[0264] The synthesis process of compound 12M1 is basically the same as that of compound 12M2. The differences are: the MPA modification site and the linker connecting MPA. The modification site of compound 12M1 is the C-terminus, and MPA is connected through AEEEA; the modification site of compound 12M2 is the 45th Lys side chain, and MPA is connected through AEEA.

[0265] Similarly, compounds 28M1, 28M2, 34M1, and 34M2 were prepared. The specific structures are shown in Table 4 below.

[0266] Table 4 Structures of modified polypeptides

[0267] Among them, (Myr') represents

[0268] (AEEEA'-MPA')

[0269] (AEEA'-MPA')

[0270] The synthesized peptide-modified compounds were analyzed for purity by ultra-high performance liquid chromatography (UPLC) and molecular weight by high-resolution time-of-flight (QTOF) mass spectrometry. UPLC analysis showed good purity, and the molecular weights confirmed by high-resolution time-of-flight (QTOF) mass spectrometry were consistent with the theoretical molecular weight. Details are shown in Table 5 below.

[0271] Table 5 MS data of peptide modified compounds

[0272] 1.4.3 Preparation of Peptide-HSA (Human Serum Albumin) Conjugates

[0273] Compound 12M1 conjugate (12M1-HSA), compound 12M2 conjugate (12M2-HSA), compound 28M1 conjugate (28M1-HSA), compound 28M2 conjugate (28M2-HSA), compound 34M1 conjugate (34M1-HSA), and compound 34M2 conjugate (34M2-HSA) were obtained by the addition reaction of the thiol group on HSA with the side chain MPA of polypeptide-modified compounds 12M1, 12M2, 28M1, 28M2, 34M1, and 34M2. UPLC detection showed that the polypeptide liquid phase peak disappeared and a new conjugate liquid phase peak was generated, indicating that the polypeptide was completely bound to HSA and the polypeptide-HSA conjugate was successfully prepared.

[0274] Example 2 Determination of the solubility of anti-hepatitis polypeptides

[0275] According to data from the prior art, the NTCP inhibitor peptide, Myrcludex B (P3), is water-soluble. To determine the solubility of the anti-hepatitis peptides of the present invention, the test substance was dissolved in the corresponding solvent to saturation, and its solubility was measured. Specifically, the solubility of peptide compounds 12, 28, and 34 was determined. The solubility of these three peptide compounds was measured for free peptide, a modified peptide with MPA at the C-terminus and Myr conjugated to the N-terminus (Compound M1), and a sample of the modified peptide with MPA at the C-terminus and Myr conjugated to HSA (Compound M1 conjugate). The test substances were designated as Compound 12, Compound 12M1, Compound 12M1 conjugate, Compound 28M1, Compound 28M1 conjugate, Compound 34M1, and Compound 34M1 conjugate (these symbols have the same meaning in the following examples). Furthermore, the solubility of a peptide modified at amino acid position 45 of the polypeptide compound with MPA and conjugated to Myr at the N-terminus (e.g., Compound 12M2) and its modified HSA-conjugated sample (e.g., Compound 12M2 conjugate) was determined. P3 and Compound 21 were used as controls. All candidate polypeptides of the present invention were water-soluble, as shown in Table 6 below.

[0276] Table 6

[0277] The above table shows that the solubility of most NTCP inhibitor peptides, such as compound 12, compound 12M1, compound 12M1 conjugate, compound 12M2, compound 12M2 conjugate and compound 28M1 conjugate, is significantly higher than that of P3, approximately 2-11 times that of P3.

[0278] Example 3 Preliminary screening of NTCP long-acting inhibitor polypeptide candidates by detecting the binding affinity of polypeptides to cell surface NTCP

[0279] The HBV infection mechanism is that the hepatocyte surface receptor NTCP specifically binds to the capsid protein PreS1, mediating viral entry into hepatocytes. Accordingly, the compounds designed in this invention compete with HBV PreS1 for binding to NTCP, thereby preventing HBV infection. To identify compounds with HBV entry inhibition potential, affinity testing between peptides and NTCP was performed. Flow cytometry was first used to measure the percentage of cells positive for peptide binding in stably transfected HEK293 cells overexpressing NTCP, using Compound 26 as a positive control.

[0280] The results are shown in Figure 1: Compounds 12, 28, and 34 bind positively to NTCP-overexpressing cells, with the percentage of positive cells being higher than that of the positive control compound 26. Other NTCP inhibitor peptides exhibited low binding rates to NTCP at a concentration of 600 nM, with the highest binding rate being only around 20%. Some, such as compounds 41, 27, 37, and 39, had very low or almost undetectable binding percentages.

[0281] Subsequently, the cell ELISA method was used to further determine the EC50 of the binding of the above candidates to NTCP overexpressing cells, and the results are shown in Table 7. As can be seen from Table 7, the results of the cell ELISA method are basically consistent with the results of flow cytometry, that is, the affinity of compounds 12, 28, and 34 to NTCP cells is stronger than that of the positive control compound 26, and the affinity of these three candidate polypeptides (compounds 12, 28, and 34) to NTCP overexpressing cells is also stronger than that of Yangshen compound P3.

[0282] Table 7

[0283] Example 4 Confirmation of the mechanism of action of long-acting NTCP inhibitors using small interfering RNA (siRNA) technology

[0284] 1. Construction of NTCP overexpression plasmid

[0285] Homo NTCP (hNTCP, Gene ID: 6554) coding region DNA (cDNA) was synthesized with a Flag tag at the 3' end and a Kozak sequence added upstream of the start codon. Using molecular cloning techniques, the NTCP-Flag cDNA was inserted into the pcDNA3.1(+) vector to construct the expression plasmid pcDNA3.1(+)-Homo NTCP-Flag. Sequencing verified the correct insertion sequence.

[0286] 2. Functional Verification of NTCP Overexpression Plasmid

[0287] HEK293T cells (HEK293T; Cell Bank of the Chinese Academy of Sciences, Catalog No. SCSP-502) were seeded in 24-well plates (4E+05 cells / well) and cultured in DMEM containing 10% FBS (Gibco, Catalog No. 10099141C) in a 95% air and 5% CO 2、 The cells were cultured in a humidified 37°C incubator. Eighteen hours after seeding, plasmid (200 ng / well) and siRNA (12 nM) were co-transfected using Lipo2000 transfection reagent (1 μL / well). Forty-eight hours later, cells were harvested and lysed, and total protein was extracted for Western blot analysis. As shown in Figure 2, compared with the untransfected control group (Lane 4), cells transfected with the plasmid pcDNA3.1(+)-hNTCP-Flag showed positive bands detected by the Flag antibody (Zhongshan Jinqiao, TA-05) (Lane 1, 2, and 3). Compared with Lane 3, NTCP small interfering RNA (siRNA881, purchased from GenePharma) significantly knocked down NTCP-Flag expression (Lane 2). The negative siRNA control group (siRNA NC, purchased from GenePharma) had no significant effect on NTCP-Flag expression (Lane 1). The results showed that: first, the constructed pcDNA3.1(+)-hNTCP-Flag plasmid could successfully express NTCP-Flag protein; second, siRNA881 could effectively knock down the expression level of hNTCP mRNA.

[0288] The siRNA sequences used are as follows:

[0289] siRNA NC (siRNA negative control, negative ctrl):

[0290] Sense: 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 15);

[0291] Antisense: 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 16);

[0292] siRNA881:

[0293] Sense: 5'-GUUCCACCAUCCUCAAUGUTT-3' (SEQ ID NO: 17);

[0294] Antisense: 5'-ACAUUGAGGAUGGUGGAACTT-3' (SEQ ID NO: 18).

[0295] 3. Using siRNA technology to reveal the mechanism of action of NTCP inhibitors

[0296] The hNTCP-expressing Huh7D human hepatocellular carcinoma cell line, ordered from Liwo Biotechnology (Shenzhen) Co., Ltd., stably expresses hNTCP. To test whether NTCP peptide inhibitors can bind to NTCP receptors on the surface of hepatocytes, Huh7D cells were treated with siRNA881 for 48 hours, and then analyzed by cell-based ELISA to determine whether the amount of NTCP peptide inhibitors bound to the cell surface was reduced.

[0297] Huh7D cells were seeded in 96-well plates (1.3E+04 cells / well). After 18 hours, siRNA NC and siRNA881 were transfected using RNAiMax (Thermofisher, Catalog No. 13778030), respectively, and cultured for another 48 hours. The old culture medium was discarded, and 100 μL of culture medium containing a certain concentration of the corresponding test sample was added to the cells in each well. After incubation at 37°C for 1.5 hours, the absorbance of each group was detected by ELISA using a microplate reader (Molecular Devices, Catalog No. SpectraMax iD5) (detection wavelength 450 nm, reference wavelength 650 nm) to analyze the changes in the amount of NTCP inhibitor binding to the cells.

[0298] The results are shown in Figure 3. Compared to the siRNA control group, when cells were treated with siRNA881 for 48 hours, the amount of 12M2 bound to the cell surface decreased at all concentrations. This data further demonstrates that 12M2 binds to the hepatocyte surface receptor NTCP.

[0299] The key mechanism of HBV / HDV liver infection lies in binding to the NTCP receptor on the surface of hepatocytes. Furthermore, since the hepatocyte surface receptor NTCP can also bind to 12M2, this suggests that 12M2 competes with HBV / HDV for NTCP receptors, thereby possessing the potential to inhibit HBV / HDV infection of hepatocytes. This inference was subsequently confirmed in Example 5.

[0300] Example 5 Determination of the inhibitory effect of polypeptides on HBV entry into primary hepatocytes

[0301] Primary hepatocytes (PHH, purchased from BIOIVT) were used to evaluate the anti-HBV in vitro infection activity of the compounds. The specific method is as follows:

[0302] Day 0: Resuscitate frozen PHH, adjust the cells to the appropriate density, and plate them into coated 48-well plates (1.32×10 5 cells / well).

[0303] Day 1: Discard cell supernatant and pretreat cells with compound-containing medium for 1 hour. PHH cells were then infected with HBV subtype D and treated with compound simultaneously. Each test compound was diluted 4-fold starting at 50 μM, and the positive control (P3) was diluted 3-fold starting at 500 μM, with 7 concentration points in duplicate. The final DMSO concentration in the culture medium was 2%.

[0304] Day 2: Discard the virus-containing medium and replace with fresh medium without compound. The final DMSO concentration in the fresh medium is 2%. The cell culture medium is DMEM (Gibco, 1965-092) supplemented with 10% fetal bovine serum, 1% penicillin / streptomycin, and other additives.

[0305] Medium replacement: Follow the same steps as on day 2, and replace with fresh medium without compound every other day.

[0306] Sample collection: On day 8 or 12, the supernatant was collected and tested for HBsAg by ELISA. Cell viability was also tested by CCK-8. The remaining cell supernatant was stored at -80°C until further use. During the experiment, P3 was used as a positive control to determine the activity and toxicity of the compound synthesized in Example 1.

[0307] 1. ELISA detection of HBsAg in cell culture supernatant

[0308] Cell culture supernatant was diluted with DPBS and HBsAg content was determined according to the instructions for the HBsAg ELISA kit (Cat. No.: Antu Bio-CL 0310). The method is briefly described as follows: First, the sample was diluted 10-fold (12 μl cell supernatant + 108 μl PBS). Then, 50 μl of each standard, sample, and control was added to the assay plate. Then, 50 μl of enzyme conjugate was added to each well and incubated at 37°C for 60 minutes. The plate was washed with detergent and aspirated dry. Then, 50 μl of premixed luminescent substrate was added and incubated at room temperature in the dark. Finally, luminescence was measured using a microplate reader.

[0309] HBsAg inhibition rate (%) = (1-HBsAg value of compound group / HBsAg value of solvent control group) × 100.

[0310] 2. Cell Counting Kit-8 (CCK-8) assay for cell viability

[0311] Refer to the Cell Counting Kit-8 (Cat. No. Biolite-AC11L057) instructions to assess cell viability. Add 200 μl of CCK-8 working solution (CCK-8 stock solution: cell culture medium = 1:9) to each well, mix thoroughly, and incubate at 37°C in 5% CO2 for 2.5 hours. Measure absorbance using a SpectraMax M2e microplate reader at a detection wavelength of 450 nm and a reference wavelength of -630 nm.

[0312] Cell viability (%) = (OD value of compound sample group - OD value of blank group) / (OD value of solvent control group - OD value of blank group) × 100.

[0313] Experimental Results and Analysis: CCK-8 assay results showed that, at the tested concentrations, the peptide compound and its conjugate had no significant effect on PHH cell viability after treatment for 8 or 12 days (Figure 4). However, at the tested concentrations, the peptide compound and its conjugate exhibited a strong inhibitory effect on the infection of three HBV subtypes (HBV-D, HBV-C, and HBV-B) into human hepatocytes. For example, the EC50 for inhibition of HBV-D subtype entry into hepatocytes ranged from 4.304 nM to 12.92 nM, which was significantly lower than the EC50 of Yangshen P3 (23.02 nM); the EC50 for inhibition of HBV-C subtype entry into hepatocytes ranged from 7.399 nM to 16.24 nM, which was also significantly lower than the EC50 of Yangshen P3 (25.89 nM); the EC50 for inhibition of HBV-B subtype entry ranged from 5.286 nM to 14.66 nM, which was also significantly lower than the EC50 of Yangshen P3 (19.9 nM) (see Tables 8-10, Figure 5).

[0314] Table 8 Inhibitory activity (EC50) of NTCP long-acting inhibitor polypeptides on hepatocyte infection of HBV D subtype (HBV-D)

[0315] Table 9 Inhibitory activity (EC50) of NTCP long-acting inhibitor polypeptides on hepatocyte infection of HBV C subtype (HBV-C)

[0316] Table 10 Inhibitory activity (EC50) of NTCP long-acting inhibitor polypeptides against hepatocyte infection of HBV B subtype (HBV-B)

[0317] Note: M1 indicates that the compound has a C-terminal MPA modification and an N-terminal Myr coupling, M2 indicates that the compound has a 45-position MPA modification and an N-terminal Myr coupling, and the conjugate indicates that the MPA of compound M1 or M2 is coupled to HSA (i.e., the active molecular form of the compound after entering the plasma).

[0318] Example 6 Taking 12M2 as an example, the inhibitory activity of NTCP long-acting inhibitor on HDV infection of human primary hepatocytes was determined

[0319] 1. HDV Preparation

[0320] HDV type I was provided by Shanghai WuXi AppTec. HDV was obtained by co-transfecting the plasmids pAAV-1.2×HDV-ITR 141 and pAAV-HBsAg-141 ITR into the human hepatoma cell line Huh7 cells, and collecting the cell culture fluid. Details are shown in Table 11.

[0321] Table 11. HDV information

[0322] 2. Primary Human Hepatocyte (PHH) Culture

[0323] Primary human hepatocytes (PHH, Lot. RAS) were provided by Shanghai WuXi AppTec. PHH were revived and plated in InvitroGRO CP medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. HDV infection was performed using William's medium E. Subsequently, cells were cultured in complete DMEM supplemented with 10% fetal bovine serum and 2% DMSO.

[0324] 3. Experimental Plan

[0325] On day 0, thaw the frozen PHH and adjust the density (1.32×10 5 After 100 cells / well, the cells were plated into 48-well cell plates and cultured overnight in a 37°C, 5% CO2 incubator.

[0326] Day 1, PHH were infected with type 1 HDV:

[0327] Cells were pretreated 1 hour prior to HDV infection with test compounds and control compounds at seven concentrations each in duplicate wells. HDV infection was then added to the cells, and compound treatment continued throughout the HDV infection period. The final DMSO concentration in the culture medium was 2% (to maintain cell differentiation). Controls containing only 2% DMSO and no compound were also established. The concentrations of test compounds and the control compound, Myrcludex B (P3), are shown in Table 12.

[0328] Table 12. Compound test concentrations

[0329] On days 2, 4, and 6, fresh culture medium without compound was replaced.

[0330] 5. Cell Counting Kit-8 (CCK-8) assay to detect cell viability

[0331] On day 8, cell viability was detected by CCK-8, and then cells were collected and HDV RNA was detected by probe qPCR.

[0332] CCK-8 assay for cell viability: Add CCK-8 working solution (CCK-8 diluted 1:9 with fresh culture medium) to the cell plate wells. Incubate in a 37°C, 5% CO2 incubator for 1-2 hours and measure the OD value using a microplate reader.

[0333] Detection of HDV RNA by qPCR: Extract intracellular RNA using the kit according to the instructions, perform reverse transcription to obtain cDNA, and then quantify the cDNA sample by probe-based qPCR.

[0334] The calculation formula for cytotoxicity test is as follows:

[0335] Cell viability (%) = (OD value of compound sample group - OD value of blank group) / (OD value of solvent control group - OD value of blank group) × 100

[0336] 4. Experimental results:

[0337] Experimental Results and Analysis: At the tested concentrations, the peptide compound and its conjugate had no significant effect on cell viability (see Figure 6 for specific results). However, at the tested concentrations, 12M2 also exhibited a stronger inhibitory effect on HDV infection of hepatocytes compared to Yangshen (see Figure 6).

[0338] The results of Example 5 and Example 6 show that among the NTCP inhibitors provided by the present invention, 12M1 and its conjugates, 12M2 and its conjugates, 34 conjugates, and 28 conjugates exhibited a better inhibitory effect than Yang Shen in terms of multi-subtype HBV infecting hepatocytes; in addition, taking 12M2 as an example, it was found that NTCP inhibitors also exhibited a good antiviral entry effect similar to Yang Shen in inhibiting HDV infection of hepatocytes (Figure 7). This shows that not only does the present invention provide NTCP inhibitors that are significantly stronger than Yang Shen in terms of resisting multi-subtype HBV entry into hepatocytes, but these inhibitors can also effectively inhibit HDV entry into hepatocytes.

[0339] Example 7 Pharmacokinetic (PK) Testing of NTCP Long-Acting Inhibitor Polypeptides

[0340] Based on the above results, compounds 12, 28, and 34 and their conjugates showed good in vitro anti-hepatitis virus entry activity. Subsequently, the plasma stability of the peptides and their conjugates was studied by in vivo pharmacokinetics. In the PK study, compound 12 was used as an example, while P3 was used as a control.

[0341] 1. Rat PK Experiment

[0342] 6-8 week old SPF grade SD rats were used, with 4 rats in each group (2 males and 2 females) for drug treatment. The drug administration methods were subcutaneous injection and intravenous injection, and the dosage was 2.5 mg / kg animal body weight. The blood sample collection time of the test compound (Compound 12M1) group was set as follows: before drug administration (0 min), 5 min, 2 h, 4 h, 6 h, 8 h, 24 h, 48 h, 72 h, and 120 h after drug administration; the blood sampling time points of the P3 control group were set as follows: before drug administration (0 min), 5 min, 2 h, 4 h, 6 h, 8 h, 24 h, and 48 h after drug administration. The anticoagulant was sodium heparin. The whole blood sample was placed in an ice box for no more than 2 hours before centrifugation; at 4 ° C, the blood cells were removed by centrifugation at 1200 × g for 10 minutes to obtain plasma. The samples were aliquoted and frozen at -80 ° C for use in mass spectrometry to detect blood peptide concentrations. The results are shown in Table 13 below.

[0343] Table 13

[0344] From the results in Table 13, it can be seen that regardless of subcutaneous or intravenous administration, the plasma half-life of compound 12M1 (7.84 h subcutaneously, 4.44 h intravenously) is significantly longer than that of P3 (0.52 h subcutaneously, 0.65 h intravenously).

[0345] 2. Sample Preparation for Dog PK Studies: Remove peptide samples from a -20°C freezer and allow to return to room temperature for 25-30 minutes. Dissolve P3 in DPBS at room temperature to a concentration of 5 mg / ml. Dissolve compound 12M1 or 12M2-conjugate in water for injection at room temperature to a concentration of 10 mg / ml. Sterilize by filtration through a 0.22 μm PES filter.

[0346] Administration: P3 and compound 12M1 were administered subcutaneously and intravenously; the dosage was 2.5 mg / kg animal body weight.

[0347] Blood collection: The time points for intravenous and subcutaneous administration of P3 were 0 min before administration and 1 min, 5 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h after administration; the time points for intravenous and subcutaneous administration of compound 12M1 were 0 min before administration and 1 min, 5 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, 72 h, 120 h and 168 h after administration.

[0348] Plasma separation: The blood collected at each time point was centrifuged at room temperature, and the plasma was collected, aliquoted, and frozen at -80°C.

[0349] Peptide concentration detection: Mass spectrometry was used to detect the peptide (characteristic peptide) concentration in plasma and analyze the drug half-life. The results are shown in Table 14 below.

[0350] Table 14

[0351] As can be seen from Table 14 above, when administered intravenously, the plasma half-life of compound 12M1 (33.4 hours) is 11.93 times that of P3 (2.8 hours); when administered subcutaneously, the plasma half-life of compound 12M1 (12.9 hours) is 5.86 times that of P3 (2.2 hours), and in each subcutaneous administration group, the 12M2 conjugate is more stable and has a longer plasma half-life, which is approximately 8.8 times that of P3.

[0352] It can be seen that the stability of the polypeptide compound of the present invention in plasma in vivo is significantly higher than that of Yangshen P.

[0353] All documents mentioned herein are incorporated herein by reference as if each document were individually incorporated by reference. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the appended claims.

[0354] References

[0355] 1.Yayoi Nomura, Kehong Liu, Tomoko Uemura, Yumi Sato, Masatsugu Ono, Masaki Yamamoto, Takeshi Noda, Hideki Shigematsu, David Drew, So Iwata, Toshiyuki Shimizu, Norimichi Nomura, Umeharu Ohto. Structure of the bile acid transporter and HBV receptor NTCP.Nature.2022,606:1021-1026.

[0356] 2.B Stieger,B Hagenbuch,L Landmann,Hchli,Mathias,A Schroeder,PJ Meier.In situ localization of the hepatocytic Na+ / Taurocholate cotransporting polypeptide in rat liver.Gastroenterology.1994,107:1781-1787.

[0357] 3.Ting Fu,Yuwenbin Li,Tae Gyu Oh,Fritz Cayabyab,Nanhai He,Qin Tang,Sally Coulter,Morgan Truitt,Paul Medina,Mingxiao He,Ruth T Yu,Annette Atkins,Ye Zheng,Christopher Liddle,Michael Downes,Ronald M Evans.FXR mediates ILC-intrinsic responses to intestinal inflammation.Proc Natl Acad Sci USA.2022,119:e2213041119.

[0358] 4.Huan Yan,Guocai Zhong,Guangwei Xu,Wenhui He,Zhiyi Jing,Zhenchao Gao,Yi Huang,Yonghe Qi,Bo Peng,Haimin Wang,Liran Fu,Mei Song,Pan Chen,Wenqing Gao,Bijie Ren,Yinyan Sun,Tao Cai,Xiaofeng Feng,Jianhua Sui,Wenhui Li.Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.Elife.2012Nov 13;1:e00049.

[0359] 5.Lili Ding,Li Yang,Zhengtao Wang,Wendong Huang.Bile acid nuclear receptor FXR and digestive system diseases.Review Acta Pharm Sin B.2015,5:135-144.

Claims

1. A long-acting inhibitor of sodium ion-taurocholic acid co-transporter, the inhibitor comprising a polypeptide represented by the general formula ABR, or an analog thereof, and a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of B is as shown in the following formula I, A is a modification of the N-terminus, and R is a modification of the C-terminus, Gly-Xaa2-Asn-Xaa4-Ser-Xaa6-Xaa7-Asn-Pro-Leu-Gly-Phe-Xaa13-Pro-Xaa15-His-Gln-Leu-Asp-Pro-Xaa21-Xaa22-Xaa23-Ala-A sn-Xaa26-Xaa27-Asn-Pro-Xaa30-Trp-Asp-Xaa33-Asn-Pro-Xaa36-Xaa37-Asp-Xaa39-Trp-Pro-Xaa42-Ala-Asn-Xaa45-Val-Gly(I); In the formula, Xaa2 is Thr, Ser, Lys, modified Lys, Gln or Asn; Xaa4 is Leu, Gln, Ile or Phe; Xaa6 is Val, Ile, Leu, Met, Phe, Ala, Thr, Trp, Asn, Gln or Gly; Xaa7 is Pro, Ala or Ser; Xaa13 is Phe, His or Leu; Xaa15 is Asp or Glu; Xaa21 is Ala, Ile, Val, Leu, Glu, Asp, Asn, Cys, Met, Gln, Trp or Pro; Xaa22 is Phe or Trp; Xaa23 is Gly, Pro, Ala, Arg, Lys, modified Lys or Glu; Xaa26 is Ser, Met, Leu, Thr, Phe, Ile or Ala; Xaa27 is Asn, Gln, His, Lys, modified Lys, Ala or Arg; Xaa30 is Asp or Glu; Xaa33 is Phe, Leu, His, Tyr, Arg, Lys, modified Lys, Ala, Trp, Ser, Ile, Val or Thr; Xaa36 is Asn, Gln, Lys, modified Lys or Leu; Xaa37 is Lys or modified Lys; Xaa39 is His, Asn, Thr, Phe, Tyr, Gln, Lys, modified Lys or Arg; Xaa42 is Glu, Asp, Val, Ala, Met, Lys, Gln, Thr, Asn, Leu, Arg, Ser or His; Xaa45 is Glu, Asp, Val, Ala, Met, Lys, Gln, Thr, Asn, Leu, Arg, Ser, His or an amino acid with a modification, preferably Lys or a modified Lys; The amino acid sequence of B has a homology of ≥85% with the amino acid sequence of SEQ ID NO: 1, and has the activity of binding to NTCP.

2. The long-acting inhibitor of sodium-taurocholic acid cotransporter according to claim 1, characterized in that The Xaa2 is Thr, Ser, Lys, modified Lys or Gln, or the Xaa6 is Val, Thr, Ile or Leu, or the Xaa7 is Pro or Ser, or Xaa13 is Phe or Leu, or Xaa15 is Asp, or Xaa21 is Ala, Leu, Val, Glu, Ile or Asp; or Xaa23 is Arg, Lys, modified Lys or Glu; or the Xaa27 is Asn or Ala, or Xaa33 is Phe, Leu, His or Tyr, or the Xaa39 is His, Asn, Thr, Phe or Tyr, or the Xaa42 is Glu or Asp; Preferably, the amino acid sequence of B is as shown in the following formula II: Gly-Xaa2-Asn-Xaa4-Ser-Xaa6-Xaa7-Asn-Pro-Leu-Gly-Phe-Xaa13-Pro-Asp-His-Gln-Leu-Asp-Pro-Xaa21-Xaa22-Xaa23-Ala -Asn-Xaa26-Xaa27-Asn-Pro-Xaa30-Trp-Asp-Xaa33-Asn-Pro-Xaa36-Xaa37-Asp-Xaa39-Trp-Pro-Xaa42-Ala-Asn-Xaa45-Val- Gly(Ⅱ).

3. The long-acting inhibitor of sodium-taurocholic acid cotransporter according to claim 2, characterized in that The Xaa2 is Thr or Gln, or the Xaa4 is Leu, or the Xaa6 is Ile, Val or Thr, or the Xaa13 is Phe, or the Xaa21 is Ala or Leu, or the Xaa23 is Arg, Lys or modified Lys, or the Xaa26 is Ser or Thr, the Xaa33 is Phe, Leu or Tyr, or the Xaa42 is Glu; Preferably, the amino acid sequence of B is as shown in the following formula III: Gly-Xaa2-Asn-Leu-Ser-Xaa6-Xaa7-Asn-Pro-Leu-Gly-Phe-Phe-Pro-Asp-His-Gln-Leu-Asp-Pro-Xaa21-Phe-Xaa23-Ala-Asn- Xaa26-Xaa27-Asn-Pro-Xaa30-Trp-Asp-Xaa33-Asn-Pro-Xaa36-Xaa37-Asp-Xaa39-Trp-Pro-Glu-Ala-Asn-Xaa45-Val-Gly(Ⅲ); More preferably, the amino acid sequence of B is as shown in the following formula IV: Gly-Xaa2-Asn-Leu-Ser-Xaa6-Xaa7-Asn-Pro-Leu-Gly-Phe-Phe-Pro-Asp-His-Gln-Leu-Asp-Pro-Xaa21-Phe-Arg-Ala-A sn-Xaa26-Xaa27-Asn-Pro-Asp-Trp-Asp-Xaa33-Asn-Pro-Asn-Lys-Asp-Xaa39-Trp-Pro-Glu-Ala-Asn-Lys-Val-Gly(IV), Among them, Xaa2 is Thr or Gln, Xaa6 is Ile, Thr, Xaa7 is Pro, Ser, Xaa21 is Leu, Ala; Xaa26 is Ser, Thr, Xaa27 is Asn, Ala; Xaa33 is Leu, Phe, Tyr; Xaa39 is His, Thr.

4. The long-acting inhibitor of sodium-taurocholic acid cotransporter according to any one of claims 1 to 3, characterized in that The amino acid sequence of B in the polypeptide is shown in any one of SEQ ID NOs: 2-13; preferably, the amino acid sequence of B in the polypeptide is selected from SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO:

6.

5. The long-acting inhibitor of sodium-taurocholic acid cotransporter according to any one of claims 1 to 4, characterized in that The modified Lys is selected from the following group: Lys-linker1 n1 -Y n2 、Lys-linker1 n1 -Chol, Lys-linker1 n1 -γGlu-C18diacid or a combination thereof; Wherein, n1 is a positive integer of 0 or ≥1, n2 is a positive integer ≥1, linker1 is AEEA, PEG or a derivative thereof, and Y is a C1-C8 acid substituted with maleimide; wherein AEEA is 8-amino-3,6-dioxooctanoic acid, and γGlu-C18diacid is a fatty acid; Preferably, the maleimide-substituted C1-C8 acid is 3-maleimidopropionic acid (MPA), 6-maleimidocaproic acid, 5-maleimidopentanoic acid, 4-maleimidobutyric acid, 4-(N-maleimidomethyl)cyclohexylcarboxylic acid, and the maleimide-substituted C1-C8 acid is preferably MPA; More preferably, the modification of the modified Lys is selected from:

6. The long-acting inhibitor of sodium-taurocholic acid cotransporter according to any one of claims 1 to 5, characterized in that The modification of the N-terminus of the polypeptide is selected from the following group: myristic acid modification, cholesterol (Chol) or its derivative modification, γGlu-C18diacid modification, hydrophobic modification, acylation modification or a combination thereof; Preferably, the acylation modification is selected from myristoyl (CH3(CH2) 12 CO-), palmitoyl (C16, i.e. CH3(CH2) 14 CO-) or stearoyl (C18, i.e. CH3(CH2) 16 CO-) acylation modification.

7. The long-acting inhibitor of sodium-taurocholic acid cotransporter according to any one of claims 1 to 6, characterized in that The modification of the C-terminus of the polypeptide is selected from the group consisting of: linker2 n3 -X n3 modification, Chol-PEG4-COOH modification, D-amino acid modification, cyclic amino acid modification, PEG modification and glycan modification or a combination thereof, Wherein, linker2 is a diamine, PEG or its derivatives, and the diamine is preferably ethylenediamine, propylenediamine, butanediamine, Amine or 1,8-diamino-3,6-dioxaoctane; X is a maleimide-substituted C1-C8 acid, γGlu-C18diacid modification or MPA amide; said n3 is a positive integer ≥1; Preferably, the maleimide-substituted C1-C8 acid is 3-maleimidopropionic acid (MPA), 6-maleimidocaproic acid, 5-maleimidopentanoic acid, 4-maleimidobutyric acid, 4-(N-maleimidomethyl)cyclohexylcarboxylic acid, and the maleimide-substituted C1-C8 acid is preferably MPA; More preferably, the modification of the C-terminus of the polypeptide is selected from:

8. The long-acting inhibitor of sodium-taurocholic acid cotransporter according to any one of claims 1 to 7, characterized in that The amino acid sequence of B in the polypeptide is as shown in any one of SEQ ID NOs: 2-13; preferably, the amino acid sequence of B in the polypeptide is selected from SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6; The modification of the N-terminus of the polypeptide is selected from myristic acid modification and acylation modification; preferably, the acylation modification is selected from myristic acid (CH3 (CH2) 12 CO-) acylation modification; And the modified Lys is selected from: Lys-linker1 n1 -Y n2 , wherein n1 is 1, n2 is 1, linker1 is AEEA, PEG or a derivative thereof, preferably AEEA, and Y is a C1-C8 acid substituted with maleimide; wherein AEEA is 8-amino-3,6-dioxooctanoic acid; Preferably, the maleimide-substituted C1-C8 acid is 3-maleimidopropionic acid (MPA), 6-maleimidocaproic acid, 5-maleimidopentanoic acid, 4-maleimidobutyric acid, 4-(N-maleimidomethyl)cyclohexylcarboxylic acid, and the maleimide-substituted C1-C8 acid is preferably MPA.

9. The long-acting inhibitor of sodium-taurocholic acid cotransporter according to any one of claims 1 to 8, characterized in that The amino acid sequence of B in the polypeptide is as shown in any one of SEQ ID NOs: 2-13; preferably, the amino acid sequence of B in the polypeptide is selected from SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6; The modification of the N-terminus of the polypeptide is selected from myristic acid modification and acylation modification; preferably, the acylation modification is selected from myristic acid (CH3 (CH2) 12 CO-) acylation modification; The modification of the C-terminus of the polypeptide is selected from: linker2 n3 -X n3 Modification, Wherein, linker2 is a diamine, PEG or a derivative thereof, the diamine is preferably ethylenediamine, propylenediamine, butylenediamine or 1,8-diamino-3,6-dioxaoctane, more preferably 1,8-diamino-3,6-dioxaoctane; X is a maleimide-substituted C1-C8 acid, γGlu-C18diacid modification or MPA amide; the n3 is 1; Preferably, the maleimide-substituted C1-C8 acid is 3-maleimidopropionic acid (MPA), 6-maleimidocaproic acid, 5-maleimidopentanoic acid, 4-maleimidobutyric acid, 4-(N-maleimidomethyl)cyclohexylcarboxylic acid, and the maleimide-substituted C1-C8 acid is preferably MPA.

10. The long-acting inhibitor of sodium-taurocholic acid cotransporter according to any one of claims 1 to 9, which is selected from the following: in, (Myr') means (AEEEA'-MPA') means (AEEA'-MPA') display 11. A polypeptide-HSA conjugate selected from the following: 12M1-HSA, 12M2-HSA, 28M1-HSA, 28M2-HSA, 34M1-HSA, 34M2-HSA, in, The structures of 12M1, 12M2, 28M1, 28M2, 34M1, and 34M2 are as defined in claim 10, HSA is human serum albumin, and the polypeptide-HSA conjugate is a covalent conjugate of the sulfhydryl group on HSA and the side chain MPA of 12M1, 12M2, 28M1, 28M2, 34M1, and 34M2.

12. A nucleic acid molecule encoding the long-acting inhibitor of sodium ion-taurocholic acid cotransporter according to any one of claims 1 to 10 or the polypeptide-HSA conjugate according to claim 11, preferably encoding the long-acting inhibitor of sodium ion-taurocholic acid cotransporter according to claim 4.

13. A pharmaceutical composition comprising: (a) a therapeutically effective amount of a long-acting inhibitor of the sodium-taurocholic acid co-transporter according to any one of claims 1 to 10, or a polypeptide-HSA conjugate according to claim 11, or a nucleic acid molecule according to claim 12; and (b) a pharmaceutically acceptable carrier.

14. Use of the long-acting inhibitor of sodium-taurocholic acid cotransporter according to any one of claims 1 to 10, the polypeptide-HSA conjugate according to claim 11, or the nucleic acid molecule according to claim 12 in the preparation of a drug for treating, inhibiting and / or preventing NTCP-related diseases; Preferably, the NTCP-related diseases include hepatitis virus infection-related diseases, type II diabetes, obesity, intestinal autoimmune diseases, biliary cirrhosis and atherosclerosis; More preferably, the hepatitis virus infection-related disease is hepatitis B and / or hepatitis D.

15. The use according to claim 14, characterized in that The drug is administered via the following routes: subcutaneous, intravenous, intramuscular, inhalation or suppository.

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