CN113491774A

CN113491774A - Liver targeting conjugate for treating hepatitis B, lipid-based pharmaceutical composition, preparation method and application thereof

Info

Publication number: CN113491774A
Application number: CN202010267031.9A
Authority: CN
Inventors: 瞿镕
Original assignee: China Israel Hyde Artificial Intelligence Drug Research And Development Co ltd
Current assignee: China Israel Hyde Artificial Intelligence Drug Research And Development Co ltd
Priority date: 2020-04-03
Filing date: 2020-04-03
Publication date: 2021-10-12

Abstract

The invention provides a liver targeting conjugate for delivering a hepatitis B therapeutic drug across a biological membrane based on a 'molecular motor' and a GalNAc targeting modification technology, a lipid-based carrier-based pharmaceutical composition containing the conjugate, and application of the conjugate and/or the pharmaceutical composition in preparing a drug for treating or preventing hepatitis B.

Description

Liver targeting conjugate for treating hepatitis B, lipid-based pharmaceutical composition, preparation method and application thereof

Technical Field

The invention relates to the technical field of antiviral drugs, in particular to a liver targeting conjugate for treating hepatitis B, a lipid-based pharmaceutical composition, and a preparation method and application thereof.

Background

Human Hepatitis B Virus (HBV) infection is a major public health problem worldwide. After acute hepatitis B virus infection, about 8% of hepatitis B virus still develops into chronic hepatitis B infection, and persistent HBV infection can cause cirrhosis and even liver cancer. China is a big country with hepatitis B, and hepatitis B virus carriers are close to 1.3 hundred million people and account for about 9 percent of the total population. Although the new hepatitis B infection rate is effectively controlled along with the wide popularization of hepatitis B vaccines, the population base of hepatitis B carrying population is large, and the prevention and treatment of hepatitis B become the most important public health problem in China. The hepatitis B transmission pathway is mainly through vertical transmission and horizontal transmission. Vertical transmission refers to mother-to-baby transmission; horizontal transmission is primarily through the blood.

The treatment of hepatitis B is also a long-term process, and the treatment aims to inhibit or eliminate HBV to the maximum extent, relieve inflammation and necrosis of liver cells and liver fibrosis, delay and stop the progress of diseases, reduce and prevent the occurrence of liver decompensation, liver cirrhosis, HCC and complications thereof, thereby improving the quality of life and prolonging the survival time.

There are many hepatitis B therapeutic drugs on the market today, mainly by antiviral treatment with interferon or nucleoside analogues. Wherein the nucleoside analog inhibits HBV production by inhibiting reverse transcriptase activity during HBV replication. Although reverse transcriptase inhibitors can control hepatitis B virus levels in patients, the problems of drug resistance, high medical costs, and serious side effects of drugs are not insignificant. In addition, the lack of liver targeting of reverse transcriptase inhibitors is also a big problem in treatment, for example, 60% -70% of marketed oral anti-hepatitis B drug entecavir is eliminated through the kidney, the long-term use causes the drug side effect of entecavir nephrotoxicity, less than 1% of entecavir is absorbed by liver organs infected with hepatitis B virus, and about 99% of entecavir cannot reach target organs.

In recent years, targeting hepatitis B virus transcripts of small interfering RNA (siRNA) has become a research focus. siRNA binds and inactivates host or viral mRNA, preventing protein translation, resulting in gene silencing. siRNA formulations are currently in preclinical evaluation and/or early clinical trials. Clinical trials in phase II have shown that a single dose of ARC-520 in combination with entecavir results in a significant and persistent reduction in serum HBV DNA levels in patients who are HBeAg positive and HBeAg negative, and a reduction in HBsAg levels in patients who are HBeAg positive, but no significant reduction in HBeAg negative. siRNA can act on the ends of all transcripts from cccDNA, but siRNA has less effect on HBsAg levels in patients who are HBeAg negative. Yet another clinical trial with IIa showed that HBeAg negative patients treated with ETV in combination with 2mg ARC-520 could reduce HBsAg levels by 22% from baseline with no significant adverse effects during the follow-up period. ARC-521 is a second generation siRNA that can silence HBsAg from all sources, including from integrated HBV DNA and cccDNA. But the exact curative effect and the specific clinical application still need to be further explored. In addition, ARB-1467 is a novel interfering RNA, can reduce all HBV transcription products and HBV antigens, and has higher safety and tolerance.

In addition to siRNA, it has also been found that antisense oligonucleotides (ASOs) complementary to RNA can block viral protein expression by steric blocking of protein translation and/or degradation of RNA by RNA cleavage by RNA cleaving enzyme H, thereby acting as gene silencing. Preclinical in vitro and in vivo evaluations have shown their potential to inhibit viral replication and reduce viral antigen load.

However, despite the enormous potential benefits of these approaches in healthcare, the delivery of such macromolecules into cells remains a substantial challenge due to the relatively large and highly charged structure of oligonucleotides (e.g., the average molecular weight of siRNA is 13kDa and it carries about 40 negatively charged phosphate groups). In fact, trans-membrane delivery of oligonucleotides requires overcoming a very large energy barrier. In addition, how to deliver the active agent to the liver cells in a targeted manner is also an urgent technical problem to be solved.

The GalNAc targeted modification technology is a technical means for targeted delivery of an active agent to be delivered to liver cells by using N-acetylgalactosamine which is a liver targeting ligand. Researchers of Alynam Pharmaceuticals company have conducted corresponding research on the mechanism of liver toxic and side effects caused by N-acetylgalactosamine conjugated RNAi, and GIVLAARI developed by the researchers was approved by FDA in 2019 as the first RNAi drug adopting GalNAc targeted modification technology. GalNAc targeted modification technology represents a promising development direction in the field of liver targeting.

Liposomes (liposomes) are an artificial membrane. The hydrophilic head of phospholipid molecule is inserted into water, the hydrophobic tail of liposome extends to air, and spherical liposome with double-layer lipid molecule with diameter of 25-1000nm is formed after stirring. The liposome can be used in transgenic technology or used for preparing medicine, and the medicine is delivered into cells by utilizing the characteristic that the liposome can be fused with cell membranes. In addition, by utilizing the passive targeting of the liver and spleen reticuloendothelial system, the liposome can target the liver, such as the hepatic leishmania drug meglumine antimonate liposome, and the concentration in the liver is improved by 200-700 times compared with that of a common preparation. The liposome is used as a carrier to encapsulate the hepatitis B treatment drug, so that the liver targeting property of the drug can be potentially improved, and the similarity between a lipid bilayer and a lipophilic cell membrane is utilized to assist in loading macromolecules such as hydrophilic drugs and charged oligonucleotides into liver cells. In addition, if the surface of the liposome is modified, such as being connected with targeting ligands such as monoclonal antibodies and the like, the liposome can further have active targeting, so that the curative effect of the medicament is further improved, and the toxic and side effects are reduced.

In view of the foregoing, there is an urgent need in the art to develop a drug with transmembrane properties and/or liver targeting for the treatment of hepatitis b.

Disclosure of Invention

The present invention solves the above problems by devising a liver targeting conjugate for the delivery of therapeutic drugs for hepatitis b across biological membranes based on "molecular motor" and GalNAc targeted modification technology, a lipid-based carrier based pharmaceutical composition comprising said conjugate.

In a first aspect, the present invention provides a liver targeting conjugate of the structure according to formula (I) for delivery of a therapeutic agent for hepatitis b across a biological membrane:

(G) y-D- (E) z formula (I)

Or pharmaceutically acceptable salts, solvates and metal chelates thereof, wherein:

d is a therapeutic agent for hepatitis b to be delivered across a biological membrane selected from small molecule drugs, peptides, proteins and natural or modified single or double stranded DNA or RNA, siRNA or antisense oligonucleotides (ASO) for the treatment of hepatitis b; y and z are each independently selected from integers of 0, 1, 2, 3, 4, 5 or 6, provided that at least one of y or z is not 0; g is a liver-targeting ligand moiety having one or more GalNAc (N-acetylgalactosamine) modules and optionally a linker module; e is a compound having the structure shown in the general formula (II):

(A)a-B-Q-L

(formula II)

Wherein a is an integer of 1, 2, 3 or 4, and wherein A is selected from the group consisting of structures shown as formulas III, IV and V

Wherein M is selected from-O-or-CH₂-; and g and h are independently integers selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16; and wherein

B is a saturated or partially saturated linear, branched or cyclic C₁、C₂、C₃、C₄、C₅、C₆、C₇、C₈、C₉、C₁₀、C₁₁、C₁₂、C₁₃、C₁₄、C₁₅、C₁₆、C₁₇、C₁₈、C₁₉、C₂₀、C₂₁、C₂₂、C₂₃、C₂₄、C₂₅、C₂₆、C₂₇、C₂₈、C₂₉、C₃₀、C₃₁、C₃₂、C₃₃、C₃₄、C₃₅、C₃₆、C₃₇、C₃₈、C₃₉、C₄₀、C₄₁、C₄₂Alkyl, alkylene, heteroalkylene, aryl, heteroaryl; a steroid or a combination thereof;

q is absent or selected from the group consisting of esters, thioesters, amides, carbamates, disulfide [ - (S-S) - ], ethers [ -O- ], pH sensitive groups and redox sensitive groups;

l is absent or is optionally substituted linear, cyclic or branched saturated, unsaturated or partially saturated C₁、C₂、C₃、C₄、C₅、C₆、C₇、C₈、C₉、C₁₀、C₁₁、C₁₂、C₁₃、C₁₄、C₁₅、C₁₆、C₁₇、C₁₈、C₁₉、C₂₀、C₂₁、C₂₂、C₂₃、C₂₄、C₂₅、C₂₆、C₂₇、C₂₈、C₂₉、C₃₀、C₃₁、C₃₂、C₃₃、C₃₄、C₃₅、C₃₆、C₃₇、C₃₈、C₃₉、C₄₀、C₄₁、C₄₂Alkyl, alkylene, heteroalkylene, aryl, heteroaryl; steroids or- (O-CH)₂-CH₂)_u-, where u is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; or a combination thereof.

In a preferred embodiment, the liver targeting ligand moiety comprises the structure:

in a preferred embodiment, the linker moiety of the liver targeting ligand moiety is selected from a cleavable or non-cleavable linking group. In a further preferred embodiment, the linking group is selected from a redox cleavable linking group, a phosphate based cleavable linking group, an acid cleavable linking group, an ester based cleavable linking group, a peptide based cleavable linking group.

In a preferred embodiment, E can be represented by formula (VI):

wherein n and m are integers each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; k is an integer selected from 2, 3, 4, 5, 6, 7; and z is absent or-S-S-; and Q is the point of attachment to a therapeutic agent for hepatitis b.

In a preferred embodiment, E can be as shown in formula VII:

in a preferred embodiment, E can be according to formula VIIa:

in a preferred embodiment, E can be represented by formula VIII:

wherein n and m are integers each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; z is absent or-S-S-; and Q is the point of attachment to a therapeutic agent for hepatitis b.

In a preferred embodiment, E can be represented by formula IX:

in a preferred embodiment, E can be according to formula IXa:

in a preferred embodiment, the therapeutic agent for hepatitis b is selected from the group consisting of siRNA, ASO and a therapeutic protein.

In a preferred embodiment, the siRNA or ASO is selected from the group consisting of: ARC-520, ARC-521, ARB-1467, ARB-1740, RG6004, ARO-HBV, ALN-HBV, Hepbarna and Lunar-HBV.

In a preferred embodiment, wherein the therapeutic protein is selected from CRISPR proteins or antibodies; more preferably selected from the group consisting of Cas9 protein, EBT106 and GC 1102.

In a second aspect, the present invention provides a pharmaceutical composition for the treatment of hepatitis b comprising at least one lipid-based carrier, a conjugate as described in any of the embodiments above, and optionally a label and/or a liver-targeting ligand.

In a preferred embodiment, the at least one lipid-based carrier is a liposome. In a further preferred embodiment, the liposome is a unilamellar liposome or a multilamellar liposome.

In a preferred embodiment, the liposomes comprise 40-70% mol of liposome forming lipids, 0-50% mol of cholesterol and/or 0-8% mol of PEG-lipids.

In a preferred embodiment, the therapeutic agent for hepatitis b in the conjugate is selected from the group consisting of siRNA, ASO and a therapeutic protein.

In a preferred embodiment, the label is selected from the group consisting of: fluorophores, chromophores, chemiluminescent molecules, magnetic particles, dyes, metals, rare earth metals, and radioisotopes.

In a preferred embodiment, the liver targeting ligand is selected from the group consisting of proteins, antibodies, peptides, small molecules, aptamers and/or carbohydrates with liver targeting efficacy.

In a third aspect, the present invention provides a process for preparing a pharmaceutical composition for the treatment of hepatitis b as described in the present invention, comprising the steps of: (a) dissolving lipid material in organic solvent, mixing, and removing organic solvent under reduced pressure to obtain lipid membrane; (b) adding buffer solution and/or pH regulator to regulate pH, shaking, stirring to make lipid membrane completely hydrated, homogenizing and emulsifying at high speed, and filtering with microporous membrane to obtain blank liposome suspension; (c) the conjugate according to any of the embodiments of the present invention is dispersed in water and added to a blank liposome suspension to produce the final liposome.

In a preferred embodiment, the organic solvent is selected from one or more of chloroform, dichloromethane, ethanol, methanol, t-butanol, n-butanol, isopropanol, acetone, diethyl ether, acetonitrile, benzyl alcohol, n-hexane; the buffer solution is selected from one of phosphate buffer solution, citrate buffer solution, acetate buffer solution, borate buffer solution and carbonate buffer solution; the pH regulator is one or more selected from potassium hydroxide, sodium bicarbonate, sodium carbonate, sodium citrate, hydrochloric acid, citric acid and phosphoric acid.

In a fourth aspect, the invention provides the use of a conjugate and/or a pharmaceutical composition according to any of the embodiments of the invention in the manufacture of a medicament for the treatment or prevention of hepatitis b.

The technical scheme of the invention has the following beneficial technical effects:

1. the conjugates of the invention comprise a liver-targeting ligand moiety comprising one or more GalNAc (N-acetylgalactosamine) modules and optionally a linker module, with which effective liver targeting of the conjugate can be achieved.

2. The conjugates of the invention comprise a novel, rationally designed "molecular motor" designed to move from the membrane/water interface to the membrane core within the phospholipid membrane using an internal membrane electric field associated with the membrane dipole potential. The conjugate delivery system can serve to pull the drug toward the membrane core and facilitate movement across the membrane.

3. The conjugate optionally comprises sensitive groups such as pH sensitive groups and redox sensitive groups, and after entering liver cells through a transmembrane, hepatitis B therapeutic drug molecules in the conjugate can be released in time through group hydrolysis and the like, so that the conjugate plays a therapeutic role.

4. The hepatic cell targeting of the liposome carrier is utilized to further carry the conjugate into hepatic cells in a highly efficient targeted manner, and the metabolic damage of the circulating system to the molecules of the conjugate is avoided.

5. The liver targeting ligand is further introduced into the liposome composition to realize the combination of active targeting and passive targeting, so that the liver targeting effect is further improved.

6. The liver cells successfully targeted by the carrier are screened and/or detected by further introducing a marker into the liposome composition, and are further used for detecting the therapeutic activity of the hepatitis B therapeutic drug.

Further embodiments and the full scope of the invention will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Brief Description of Drawings

The present invention will now be described in connection with certain embodiments and implementations in a non-limiting manner with reference to the following illustrative figures so that the invention may be more fully understood. In the drawings:

FIG. 1A is a diagrammatic representation of the principle of asymmetric polarity of the active basis of compounds according to embodiments of the present invention;

FIG. 1B is a schematic depiction of the basic structure of the molecular motor compound in the conjugates of the invention as illustrated by the compound of formula IX;

figure 2 illustrates schematically a possible mechanism of action of a conjugate according to an embodiment of the invention.

Figure 3 illustrates the manner of attachment of liver targeting ligand moieties in conjugates according to embodiments of the invention (exemplified by RNA drugs).

Detailed Description

Conjugates

In one aspect, embodiments of the invention relate to novel conjugates that can be used as liver-targeted delivery systems for therapeutic agents for hepatitis b across biological membranes (e.g., phospholipid cell membranes) into the cytoplasm.

Molecular motor module

The conjugates of the invention comprise a novel, rationally designed "molecular motor" designed to move from the membrane/water interface to the membrane core within the phospholipid membrane using an internal membrane electric field associated with the membrane dipole potential. When linked to a hepatitis b therapeutic drug, the conjugate delivery system serves to pull the drug toward the membrane core and facilitate movement across the membrane. For example, the delivery system is designed to deliver therapeutic macromolecules: proteins or oligonucleotides (single or double stranded DNA or RNA). For example, the conjugate delivery system is designed for delivery of antisense oligonucleotides (ASOs), sirnas, and therapeutic proteins (such as Cas9 protein).

One of the structural principles of the conjugates according to the invention is the principle of "asymmetric polarity" (which relates to the partitioning of hydrophobic, uncharged molecules into biological membranes according to their logP (see FIG. 1A)). The molecules are polar and their local charge is distributed inhomogeneously: the local negative charges are highly localized and concentrated, while the local positive charges are dispersed along the hydrocarbon chain within the molecule. In addition, the local positive charge is also masked by London-type interactions (London dispersion forces) with adjacent hydrocarbon chains within the membrane lipid environment. Thus, as illustrated in fig. 1A, the molecular motor according to the present invention moves in the form of negatively charged molecules within the membrane environment. Since the intra-membrane electric field has a negative pole at the membrane/water interface and a positive pole at the membrane center, the molecules move toward the membrane center in the associated electric field and when they are attached to a hepatitis b therapeutic drug (such as siRNA, ASO, therapeutic protein or other drug) it pulls the drug to the membrane center.

In addition, when an optional cleavable group is included in a molecule according to embodiments of the present invention (e.g., a disulfide group or an oligonucleotide sequence cleavable by Dicer enzyme), the cleavable group can serve to capture a cargo (e.g., siRNA or ASO or other drug) in the cytoplasm of a target cell and also assist in maintaining the concentration gradient of the conjugate across the cell membrane. Thus in the context of the present invention, the term "cleavable group" relates to a chemical group capable of undergoing spontaneous or enzyme-mediated cleavage under certain physiological conditions, such as a change in pH or a change in redox state. Examples of cleavable groups are esters, thioesters, amides, carbamates, disulfide [ - (S-S) - ], ethers (-O-) or thioethers (-S-). Theoretically, the cleavable group may assist in the capture of the drug in its target cell after passage across the membrane, or assist in maintaining a concentration gradient across the biological membrane.

For example, where a conjugate according to the invention comprises siRNA, ASO or therapeutic protein and a disulfide bond group, once inside the cytoplasm, the prevalent surrounding reducing environment will act to reduce the disulfide bond to an-SH group, while releasing the cargo from the delivery conjugate. Without the "molecular motor" (delivery group), the cargo macromolecule would be trapped in the cytoplasm where, for example, in the case of siRNA, it would readily interact with the RNA-induced silencing complex (RISC) to silence the expression of a particular gene.

As exemplified below, embodiments of the invention include conjugates comprising one or more "molecular motors" and the hepatitis B therapeutic drug.

The conjugates according to the invention generally comprise hydrophobic (octanol/water partition coefficient (logP > 1)), dipolar, uncharged chemical groups, designed according to the asymmetric polarity principle (as explained above). As discussed, when the unique structure of a molecule (which is hydrophobic, neutral, but contains a concentrated local negative charge and a dispersed local positive charge) is placed into the force field of the membrane (from the membrane/water interface to the center of the membrane by its molecular movement in the phospholipid membrane), it creates a vector system. When attached to a drug, the molecule will pull the drug to the center of the membrane, respectively.

As illustrated in fig. 1B, conjugates according to the invention typically comprise a "molecular motor", which is typically a combination of the following structural elements:

(i) negative electrode (a): generally containing at least 1 electronegative atom selected from halogens (e.g., fluorine atoms) and oxygen; wherein in the case where the pole comprises a plurality of electronegative atoms, they are arranged in a concentrated, spherical (or near spherical) arrangement. The negative electrode of the compound is an electron rich focus due to the electron withdrawing properties of the atoms and their spatial arrangement.

(ii) Positive electrode (B): comprises a relatively electropositive atom selected from the group consisting of carbon, silicon, boron, phosphorus and sulfur; arranged in such a way as to enable maximum interaction with the adjacent hydrocarbon chains when placed in a phospholipid membrane, preferably by being arranged in the form of aliphatic or aromatic structures with linear, branched or cyclic chains, or combinations thereof. In one embodiment of the invention, the positive electrode comprises a linear saturated hydrocarbon chain, or a steroid group, such as cholesterol, bile acid, estradiol, estriol or combinations thereof.

In addition to the "molecular motor", the conjugate according to the invention may comprise one or more linkers (L) and cleavable groups (Q) as further described below. The molecular motor compound may be conjugated or linked to the drug (D) through a linker or cleavable group.

The conjugates according to the invention may be beneficial in improving the delivery of siRNA, ASO or therapeutic protein for the treatment of hepatitis b through the cell membrane, thereby improving the performance of the siRNA or ASO in one or more aspects (e.g. efficacy, toxicity or pharmacokinetics).

As described above, as a non-limiting possible mechanism of action (MOA), when a conjugate comprising a therapeutic drug for hepatitis b (such as siRNA or a therapeutic protein) according to an embodiment of the present invention is located within a phospholipid membrane, the "molecular motor" acts to pull the drug toward the center of the membrane. Theoretically, lateral movement of the phospholipid headgroup and creation of a transient membrane pore through which transmembrane passage of the drug can occur, followed by local destabilization of the membrane. This possible MOA is summarized graphically in fig. 2.

In one embodiment, as presented graphically in fig. 2, the conjugate comprises a hepatitis b therapeutic drug (which is an siRNA, ASO, or therapeutic protein), and disulfide bond groups for capturing the drug in the cytoplasm. In the first stage (a), the "motor" moves from the membrane surface to the membrane center, energized by the internal membrane electric field, due to the principle of asymmetric polarity.

In the second stage (B), the macromolecules attached to the "motor" are concentrated close to the membrane surface, disturbing the hydrated outer layer. Thus, lateral movement of the phospholipid headgroup occurs and transient membrane pores are formed through which macromolecules are delivered into the cell. The subsequent closing of the transient pore is thermodynamically favourable (C).

Liver targeting ligand module

The conjugates of the invention also comprise a novel, rationally designed GalNAc (N-acetylgalactosamine) -based liver-targeting ligand module. The GalNAc conjugate targets the therapeutic agent for hepatitis b to the liver cells. The connection mode of the medicine and the hepatitis B therapeutic medicine can be seen in figure 3. In fig. 3, an exemplary therapeutic agent for hepatitis b is RNA.

In a preferred embodiment, the therapeutic agent for hepatitis b is selected from the group consisting of siRNA, ASO and therapeutic proteins, which are delivered across the cell membrane to exert a beneficial therapeutic effect on the anti-hepatitis b virus target within the hepatocytes.

In a preferred embodiment, wherein the therapeutic protein is selected from CRISPR proteins or antibodies; more preferably selected from the group consisting of Cas9 protein, EBT106 and GC1102 (monoclonal antibody).

In summary, the present invention provides a liver-targeting conjugate of the structure as described in formula (I) for delivery of a therapeutic agent for hepatitis b across a biological membrane:

(G) y-D- (E) z formula (I)

(A)a-B-Q-L

(formula II)

B is a saturated or partially saturated linear, branched or cyclic C₁、C₂、C₃、C₄、C₅、C₆、C₇、C₈、C₉、C₁₀、C₁₁、C₁₂、C₁₃、C₁₄、C₁₅、C₁₆、C₁₇、C₁₈、C₁₉、C₂₀、C₂₁、C₂₂、C₂₃、C₂₄、C₂₅、C₂₆、C₂₇、C₂₈、C₂₉、C₃₀、C₃₁、C₃₂、C₃₃、C₃₄、C₃₅、C₃₆、C₃₇、C₃₈、C₃₉、C₄₀、C₄₁、C₄₂Alkyl, alkyleneAlkyl, heteroalkylene, aryl, heteroaryl; a steroid or a combination thereof;

In a preferred embodiment, E may be as shown in formula (VI):

In a preferred embodiment, E can be as shown in formula VII:

in a preferred embodiment, E can be according to formula VIIa:

in a preferred embodiment, E can be represented by formula VIII:

In a preferred embodiment, E can be represented by formula IX:

in a preferred embodiment, E can be according to formula IXa:

Liposomes

Carrier

In some embodiments, the at least one lipid-based carrier is a liposome.

In one embodiment, the carrier (e.g., liposome) has a diameter of less than 500nm to facilitate its entry into cells through the extracellular matrix. In one embodiment, the carrier (e.g., liposome) has a diameter of less than 400nm to facilitate its entry into cells through the extracellular matrix.

In another embodiment, the carrier (e.g., liposome) has a diameter of at least 1nm, at least 5nm, at least 10nm, at least 20nm, at least 30nm, at least 40nm, at least 50nm, at least 60nm, at least 70nm, at least 80nm, at least 90nm, at least 100nm, at least 1500nm, at least 200nm, at least 250nm, or at least 300 nm. Each possibility represents a separate embodiment of the invention.

In some embodiments, the carrier for intravenous administration has a diameter of 5-250 nm. In some embodiments, wherein the carrier for intravenous administration is a liposome, the diameter of the carrier is 40-250 nm.

In one embodiment, the vector may be selected and/or prepared to optimize the delivery of the therapeutic drug to the target cell. For example, where the target cell of the invention is a hepatocyte, the properties of the transfer vector (e.g., size, charge and/or pH) can be optimized to efficiently deliver such vector to the target cell.

The liposome-incorporated therapeutic drug/conjugate, label, and/or liver targeting ligand may be located entirely or partially within the interior space of the liposome, within the bilayer membrane of the liposome, or conjugated to the liposomal lipid material. Incorporation of the agent into the liposome is also referred to herein as "encapsulation," wherein the agent is completely contained within the interior space of the liposome. The purpose of incorporating an agent into a carrier, such as a liposome, is often to protect the agent from the environment, which may contain enzymes or chemical agents that degrade the agent and/or systems or receptors that cause rapid excretion of the agent. Thus, in a preferred embodiment of the invention, the transfer vehicle is selected to be capable of enhancing the stability of the conjugate and optionally the labelled liver targeting ligand comprised therein. Liposomes can allow the encapsulated agent to reach the target cell and/or can preferentially allow the encapsulated agent to reach the target cell, or alternatively limit delivery of the agent to other undesired target sites or cells.

In another embodiment, the at least one carrier is a nanoliposome. Nanoliposomes are capable of enhancing the solubility and bioavailability of bioactive agents, stability in vitro and in vivo, and avoiding their undesirable interactions with other molecules. Another advantage of nanoliposomes is cell-specific targeting, which is a prerequisite for obtaining optimal therapeutic effect in the target cell while minimizing the concentration of drug required for side effects on healthy cells and tissues.

The liposome carrier used in the composition of the present invention can be prepared by various techniques currently known in the art. For example, the selected lipid is deposited on the inner wall of a suitable container or vessel by dissolving the lipid in a suitable solvent and then evaporating the solvent to leave a film on the interior of the vessel or by spray drying. The aqueous phase may then be added to the vessel using a swirling motion, which may result in the formation of multi-layered vesicles (MLVs). Monolayer vesicles (ULV) can then be formed by homogenizing, sonicating or extruding multiple layer vesicles. Alternatively, the monolayer of vesicles may be formed by detergent removal techniques.

In one embodiment, the preparation of liposomes comprises the steps of: (a) dissolving lipid material in organic solvent, mixing, and removing organic solvent under reduced pressure to obtain lipid membrane; (b) adding buffer solution and/or pH regulator to regulate pH, shaking, stirring to make lipid membrane completely hydrated, homogenizing and emulsifying at high speed, and filtering with microporous membrane to obtain blank liposome suspension; (c) the active drug is dispersed in water and added to the blank liposome suspension to make the final liposome.

In some embodiments, the organic solvent is selected from one or more of chloroform, dichloromethane, ethanol, methanol, t-butanol, n-butanol, isopropanol, acetone, diethyl ether, acetonitrile, benzyl alcohol, n-hexane; the buffer solution is selected from one of phosphate buffer solution, citrate buffer solution, acetate buffer solution, borate buffer solution and carbonate buffer solution; the pH regulator is one or more selected from potassium hydroxide, sodium bicarbonate, sodium carbonate, sodium citrate, hydrochloric acid, citric acid and phosphoric acid.

In certain embodiments, the compositions of the invention may be loaded with diagnostic radionuclides, fluorescent materials, or other materials detectable in both in vitro and in vivo applications.

In some embodiments, the vectors of the present invention comprise 40-70% mol of liposome-forming lipids. In some embodiments, the vectors of the present invention comprise 0 to 50% mol cholesterol. In some embodiments, the carrier of the present invention comprises 0-8% mol PEG-lipid. In some embodiments, the vectors of the present invention also optionally include 0-3% mol of a functional lipid (e.g., a cationic lipid or a lipid having a targeting moiety). According to a specific embodiment, nanoparticles comprising 40-70% mol of liposome forming lipids, 0-50% mol of cholesterol, 0-8% mol of PEG-lipids and 0-3% mol of functional lipids are suitable for intravenous administration.

In some embodiments, the lipid is a naturally occurring phospholipid. Examples of phospholipids include, but are not limited to, glycerophospholipids, Phosphatidylglycerols (PGs), which include dimyristoyl phospholipid phosphatidylglycerol; phosphatidylcholines (PCs), including egg yolk phosphatidylcholine, dimyristoyl phosphatidylcholine (DMPC), 1-hexadecanoyl-2-oleoyl phosphatidylcholine (POPC), Hydrogenated Soybean Phosphatidylcholine (HSPC), distearoyl phosphatidylcholine (DSPC); phosphatidic Acid (PA); phosphatidylinositol (PI); phosphatidylserine (PS).

Cholesterol is known to have an effect on the tissue structure properties of lipids (lipid assembly) and may be used for stabilization, for influencing surface charge, membrane fluidity and/or to aid in loading of active drugs into lipid structures. Thus, in some embodiments, cholesterol is employed in order to control the fluidity of the lipid structure. Cholesterol: the greater the ratio of lipids (structure of the formed lipids), the more rigid the lipid structure.

Examples of the cationic lipid may include, for example, 1, 2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1, 2-dioleoyloxy-3- (trimethylamino) propane (DOTAP), N- [1- (2, 3, -bistetradecyloxy) propyl ] -N, N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N- [1- (2, 3, -dioleoyloxy) propyl ] -N, N-dimethyl-N-hydroxyethylammonium bromide (DORIE), N- [1- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium chloride (DOTMA), 3 β [ N- (N ', N' -dimethylaminoethane) carbamoylcholesterol (DC-cholesterol) ], Dimethyl-dioctadecylammonium (DDAB), N- [2- [ [2, 5-bis [ 3-aminopropyl) amino ] -1-oxopentyl ] amino ] ethyl ] -N, N-dimethyl-2, 3-bis [ (1-oxo-9-octadecenyl) oxo ] -1-propanaminium (DOSPA) and Ceramide Carbamoyl Spermine (CCS), or the neutral lipid Dioleoylphosphatidylethanolamine (DOPE) derivatized with polylysine to form a cationic lipopolymer.

The liposome may be any of the following: unilamellar liposomes (SLV), Multilamellar Liposomes (MLV), multivesicular vesicles (MVV), Small Unilamellar Vesicles (SUV), Large Unilamellar Vesicles (LUV), or large multivesicular vesicles (LMVV). In some embodiments, the liposome is a unilamellar liposome or a multilamellar liposome.

Medicine for treating hepatitis B

In a preferred embodiment, the therapeutic agent for hepatitis B is selected from the group consisting of siRNA, ASO and therapeutic proteins.

In some other embodiments, the hepatitis b therapeutic agent is selected from one or more of entecavir, tenofovir disoproxil fumarate, and tenofovir alafenamide, for example, one selected from entecavir, tenofovir disoproxil fumarate, and tenofovir alafenamide or at least two selected from entecavir, tenofovir disoproxil fumarate, and tenofovir alafenamide.

In addition to the above active agents, the pharmaceutical compositions described herein may optionally further comprise one or more additional other agents useful in the treatment of HBV, such as, but not limited to, 3-dioxygenase (IDO) inhibitors, antisense oligonucleotides targeted to viral mRNA, apolipoprotein a1 modulators, arginase inhibitors, B-and T-lymphocyte attenuating agent inhibitors, Bruton's Tyrosine Kinase (BTK) inhibitors, CCR2 chemokine antagonists, CD137 inhibitors, CD160 inhibitors, CD305 inhibitors, CD4 agonists and modulators, compounds targeted to HBcAg, compounds targeted to hepatitis B core antigen (HBcAg), covalently closed circular DNA cccdna (cccdna) inhibitors, cyclophilin inhibitors, cytokines, cytotoxic T-lymphocyte-associated protein 4(ipi4) inhibitors, DNA polymerase inhibitors, endonuclease modulators, epigenetic modifiers, farnesol X receptor agonists, gene modifiers or editors, HBsAg inhibitors, HBsAg secretion or assembly inhibitors, HBV antibodies, HBV DNA polymerase inhibitors, HBV replication inhibitors, HBV RNase inhibitors, HBV vaccines, HBV viral entry inhibitors, HBx inhibitors, hepatitis B large envelope protein modulators, hepatitis B large envelope protein stimulators, hepatitis B structural protein modulators, hepatitis B surface antigen (HBsAg) inhibitors, hepatitis B surface antigen (HBsAg) secretion or assembly inhibitors, hepatitis B virus E antigen inhibitors, hepatitis B virus replication inhibitors, hepatitis virus structural protein inhibitors, HIV-1 reverse transcriptase inhibitors, hyaluronidase inhibitors, IAP inhibitors, IL-2 agonists, IL-7 agonists, immunoglobulin G modulators, immunomodulators, indoleamine-2, ribonucleotide reductase inhibitors, interferon agonists, interferon alpha 1 ligands, interferon alpha 2 ligands, interferon alpha 5 ligand modulators, interferon alpha ligands, interferon alpha ligand modulators, interferon alpha receptor ligands, interferon beta ligands, interferon receptor modulators, interleukin-2 ligands, ipi4 inhibitors, lysine demethylase inhibitors, histone demethylase inhibitors, KDM5 inhibitors, KDM1 inhibitors, lectin-like receptor subfamily G member 1 inhibitors, lymphocyte activation gene 3 inhibitors, lymphotoxin beta receptor activators, microRNA (miRNA) gene therapy agents, Axl modulators, B7-H3 modulators, B7-H4 modulators, CD160 modulators, CD161 modulators, CD27 modulators, CD47 modulators, CD70 modulators, GITR modulators, HEVEM modulators, ICOS modulators, Mer modulators, NKG2A modulators, NKG2D modulators, OX40 modulators, SIRPa modulators, TIGIT modulators, Tim-4 modulators, Tyro modulators, Na + -taurate cotransporter polypeptide (NTCP) inhibitors, natural killer cell receptor 2B4 inhibitors, NOD2 gene stimulators, nucleoprotein inhibitors, nucleoprotein modulators, PD-1 inhibitors, PD-L1 inhibitors, PEG-interferon lambda, prolyl isomerase inhibitors, phosphatidylinositol-3 kinase (PI3K) inhibitors, recombinant Scavenger Receptor A (SRA) proteins, recombinant thymosin alpha-1, retinoic acid inducible gene 1 stimulators, reverse transcriptase inhibitors, ribonuclease inhibitors, RNA DNA polymerase inhibitors, short interfering RNA (siRNA), short interfering RNA synthetic hairpin SLC (sshRNA), (ssh)) SLC10A1 gene inhibitors, SMAC mimetics, src tyrosine kinase inhibitors, interferon gene Stimulator (STING) agonists, NOD1 stimulators, T cell surface glycoprotein CD28 inhibitors, T cell surface glycoprotein CD8 modulators, thymosin agonists, thymosin alpha 1 ligands, Tim-3 inhibitors, TLR-3 agonists, TLR-7 agonists, TLR-9 agonists, TLR9 gene stimulators, toll-like receptor (TLR) modulators, viral ribonucleotide reductase inhibitors, zinc finger nucleases or synthetic nucleases (TALENs), and combinations thereof.

As used herein, "therapeutically effective amount" or "effective amount" refers to an amount that is effective at a dose and for a period of time required to achieve a desired therapeutic result. A therapeutically effective amount of a therapeutic agent for hepatitis b will depend on the nature of the disorder or condition and on the particular agent, and can be determined by standard clinical techniques known to those skilled in the art.

The therapeutic outcome may be, for example, alleviation of symptoms, prolongation of survival, increased mobility, and the like. The therapeutic result need not be a "cure". The therapeutic outcome may also be prophylactic.

Marking

In another embodiment, one or more vectors of the invention optionally comprise at least one label or detectable moiety.

Labels that may be used in the compositions and methods of the present invention include, but are not limited to, fluorophores, chromophores, chemiluminescent molecules, radioactive labels, metals, rare earth elements, magnetic particles, or dyes.

In some embodiments, the label or detectable moiety is a label useful in an assay, including but not limited to immunoassays, such as ELISA, bead-based, chip-based, or plate-based multiplex immunoassays, mass spectrometry, electrophoresis, immunoturbidimetry, enzymatic assays, colorimetric or fluorescent assays, as evaluable by photometers, and fluorescence-related cell sorting (FACS) -based assays or by other clinically established assays. All these methods are known to the person skilled in the art and are described in the literature.

Generally, the amount of label will depend on the assay to be performed and can be determined by and well within the abilities of those skilled in the art. In some embodiments, the vector comprises one molecule or a plurality of molecules of the label.

Liver targeting ligands

In some embodiments, the optionally included liver targeting ligands, including proteins, antibodies, peptides, small molecule substances, aptamers, and/or carbohydrates, and the like, with liver targeting efficacy, are used to assist in targeting the pharmaceutical composition to a liver target.

In one embodiment, the liver targeting ligand can be attached to the outer ends of polyethylene glycol (PEG) chains on stealth liposomes, while PEG can reduce MPS clearance and provide long circulation in the liposomes. The liver cell surface highly expresses various vectors and receptors for endocytosis, and the liver targeting ligand modified liposome realizes active targeting action through receptor-ligand interaction and the like by positioning at a specific site on a cell membrane in the liver.

In one embodiment, examples of liver targeting ligands include, but are not limited to, the envelope protein preS1 peptide, glycyrrhetinic acid, asialoglycoprotein receptors (ASGPR), aptamers, apolipoprotein a1, arginine-glycine-aspartic acid (RGD), bile salts, NK4 protein molecules, and the like.

As used herein, the term "about" when combined with a value refers to plus or minus 10% of the referenced value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000nm + -100 nm.

Additional objects, advantages and novel features of the present invention will become apparent to one of ordinary skill in the art upon examination of the following examples.

Examples

Example 1 general method for synthesizing conjugates comprising oligonucleotides according to embodiments of the invention

First, the gene to be silenced is selected based on its role in disease etiology or pathogenesis. Then, the nucleotide sequence of each siRNA of the DNA sequence of the ASO (typically 19-21 base pairs for RISC substrates and 25-29 for Dicer substrates) was determined based on bioinformatics methodologies known in the art.

The synthesis proceeds in the 3 'to 5' direction. Solid phase synthesis is applied using phosphoramidite building blocks derived from protected 2' -deoxynucleosides (dA, dC, dG and T), ribonucleosides (A, C, G and U) or chemically modified nucleosides, such as LNA (locked nucleic acid) or BNA (bridge nucleic acid). The building block sequence (in the order required by the sequence of the desired siRNA or ASO) is coupled to the growing oligonucleotide strand.

After construction of the oligonucleotide, the compound according to the invention is added as one of the building blocks of the oligonucleotide. The compound may optionally be added in the form of its precursors as described above. For addition of the compound at the 5' -end of the oligonucleotide, the compound in its precursor form may comprise a phosphoramidite group. For addition of compounds at the 3' -end of the oligonucleotide, the compounds in their precursor form may contain acetylene or azide groups. Typically, the process is fully automated. Once assembly of the strand is complete, the product is released from the solid phase into solution, deprotected and collected. The desired conjugate is then collected and isolated by High Performance Liquid Chromatography (HPLC) to obtain the desired oligonucleotide in high purity. In the case of siRNA, each complementary RNA strand is separately synthesized, and then annealing of both strands is performed to obtain the desired siRNA double-stranded RNA.

Example 2 Liposome Synthesis/preparation/characterization

Liposomes are prepared using methods known in the art. HSPC, PEG-DSPE and cholesterol 58%, 2%, 40% were dissolved in pure ethanol and heated at 65 ℃ until complete dissolution.

Synthesis of blank liposomes: the liposome comprises the following lipid composition: 58% mol Hydrogenated Soy Phosphatidylcholine (HSPC), Mw 762.1; 2 mol% polyethylene glycol distearoyl-phosphoethanolamine (m2000PEG DSPE) -its effect was to reduce aggregation/fusion of liposomes due to steric effects, Mw 2805.54; and 40 mol% cholesterol Mw 386.65. The working concentration of total lipid in the solution was 50 mM. The medium was 10% PBS/5% glucose in deionized water. First, the lipids were dissolved in pure ethanol, warmed to 65 ℃ and added to 1ml of medium (also warmed to the same temperature). After direct injection and pipetting, more medium was added to reach the final lipid concentration. Hydrogenated soy phosphatidylcholine is contributed by Lipoid (Ludwigshafen, Germany); m2000PEG-DSPE was purchased from Avanti (AIabaster, Alabama, USA) and cholesterol (catalog number: C8667-500MG) was obtained from Sigma (Rehovot, Israel).

Synthesis of conjugate-loaded liposomes: referring to the above synthesis method of blank liposome, the difference is that serial dilutions of the conjugate with different concentrations are introduced together during ethanol dissolution, or the conjugate is dispersed in water and then added to the blank liposome suspension to obtain the final liposome.

Extrusion process: the lipid solution was extruded 3 times through 400nm and 200nm membranes. The extruder temperature was set to 65 degrees celsius (c).

Measurement of Liposome encapsulation efficiency and drug-loading capacity: by usingSephadex column chromatography, collecting liposome suspension 0.5mL, loading on Sephadex G-50 Sephadex column, eluting with HEPES buffer solution (pH6.8), collecting, separating liposome and free drug, adding methanol to demulsify liposome, measuring content by High Performance Liquid Chromatography (HPLC), and calculating liposome encapsulation efficiency and drug loading rate.

Particle size distribution: the particle size distribution of the prepared liposome is detected by using a Malvern ZEN1690 type laser particle size analyzer. Resuspending the prepared lyophilized liposome product with 1mL sterile water for injection, filtering with 0.22 μm polycarbonate membrane filter to remove impurities, collecting 100 μ L liposome solution, diluting with PBS (pH6.5, 0.1mM) to 2mL, and stirring thoroughly; 1.2mL of the above sample was added to the sample container for detection.

Examination of in vitro Release: taking 3 batches of liposome solution, respectively sucking 1mL of the liposome solution, transferring the liposome solution into a treated dialysis bag, fastening two ends of the dialysis bag, placing the dialysis bag into 200mL of PBS (phosphate buffer solution) with the pH value of 7.4, stirring at constant temperature (37 +/-1) DEG C and constant speed (100r/min), respectively taking 1mL of release solution at 0.5, 1, 2, 4, 6, 8, 12, 24, 48 and 72 hours, and simultaneously adding release medium with the same volume and temperature. The sample was acidified with 10 μ L glacial acetic acid for 2h, the solution was filtered through a 0.22 μm microporous membrane, the content was measured by HPLC, and the average cumulative release percentage was calculated.

EXAMPLE 3 therapeutic Effect of conjugate-carrying liposomes in patients with chronic viral hepatitis B

In this example, the liposome prepared in example 2 was used to treat patients with chronic viral hepatitis b, and the therapeutic effect and effect thereof on chronic viral hepatitis b were investigated.

(1) Selection of subjects: patients with chronic viral hepatitis b were selected as subjects.

(2) The administration mode comprises the following steps: the administration is carried out by using an upper arm subcutaneous injection mode, and 900 mug of liposome finished product is administered each time; the liposome finished product is dissolved in 3mL sterile water and is injected subcutaneously for 6 times at 0, 4, 8, 12, 20 and 28 weeks of treatment.

(3) And (3) evaluating the curative effect: after the patients with chronic viral hepatitis B are treated by the liposome of the embodiment 2 of the invention, the peripheral blood of the patients is collected at 12 th, 28 th, 32 th, 40 th, 52 th, 64 th and 76 th weeks respectively, and the HBeAg/anti-HBe conversion rate, the serum HBV virus titer and the serum alanine Aminotransferase (ALT) concentration are respectively detected to evaluate the treatment effect of the liposome.

While the invention has been described with reference to specific embodiments, those skilled in the art will recognize that changes or modifications can be made to the described embodiments without departing from the spirit and scope of the invention, which is defined by the appended claims.

Claims

1. A liver-targeting conjugate of the structure of formula (I) for delivery of a therapeutic agent for hepatitis b across a biological membrane:

(G) y-D- (E) z formula (I)

(A)_a-B-Q-L

(formula II)

B is saturated or partially saturatedLinear, branched or cyclic C₁、C₂、C₃、C₄、C₅、C₆、C₇、C₈、C₉、C₁₀、C₁₁、C₁₂、C₁₃、C₁₄、C₁₅、C₁₆、C₁₇、C₁₈、C₁₉、C₂₀、C₂₁、C₂₂、C₂₃、C₂₄、C₂₅、C₂₆、C₂₇、C₂₈、C₂₉、C₃₀、C₃₁、C₃₂、C₃₃、C₃₄、C₃₅、C₃₆、C₃₇、C₃₈、C₃₉、C₄₀、C₄₁、C₄₂Alkyl, alkylene, heteroalkylene, aryl, heteroaryl; a steroid or a combination thereof;

2. The conjugate of claim 1, wherein the liver-targeting ligand moiety comprises the structure:

3. the conjugate of claim 1, wherein the liver-targeting ligand moiety comprises the structure:

4. the conjugate of claim 1, wherein the linker moiety of the liver-targeting ligand moiety is selected from a cleavable or non-cleavable linking group.

5. The conjugate of claim 4, wherein the linking group is selected from a redox cleavable linking group, a phosphate ester based cleavable linking group, an acid cleavable linking group, an ester based cleavable linking group, a peptide based cleavable linking group.

6. The conjugate of claim 1, wherein E is according to formula (VI):

7. The conjugate of claim 1, wherein E is according to formula VII:

8. the conjugate of claim 1, wherein E can be according to formula vila:

9. the conjugate of claim 1, wherein E is according to formula VIII:

10. The conjugate of claim 1, wherein E is represented by formula IX:

11. the conjugate of claim 1, wherein E is according to formula IXa:

12. the conjugate of any one of claims 1-11, wherein the therapeutic agent for hepatitis b is selected from the group consisting of siRNA, ASO, and a therapeutic protein.

13. The conjugate of claim 12, wherein the siRNA or ASO is selected from the group consisting of: ARC-520, ARC-521, ARB-1467, ARB-1740, RG6004, ARO-HBV, ALN-HBV, Hepbarna and Lunar-HBV.

14. The conjugate of claim 12, wherein the therapeutic protein is selected from a CRISPR protein or an antibody; more preferably selected from the group consisting of Cas9 protein, EBT106 and GC 1102.

15. A pharmaceutical composition for the treatment of hepatitis b comprising at least one lipid-based carrier, a conjugate according to any one of claims 1 to 14, and optionally a label and/or a liver-targeting ligand.

16. The pharmaceutical composition of claim 15, wherein the at least one lipid-based carrier is a liposome.

17. The pharmaceutical composition of claim 16, wherein the liposome is a unilamellar liposome or a multilamellar liposome.

18. The pharmaceutical composition of claim 16, wherein the liposome comprises 40-70% mol of liposome-forming lipids, 0-50% mol of cholesterol and/or 0-8% mol of PEG-lipids.

19. The pharmaceutical composition of claim 15, wherein the therapeutic agent for hepatitis b in the conjugate is selected from the group consisting of siRNA, ASO and a therapeutic protein.

20. The conjugate of claim 19, wherein the siRNA or ASO is selected from the group consisting of: ARC-520, ARC-521, ARB-1467, ARB-1740, RG6004, ARO-HBV, ALN-HBV, Hepbarna and Lunar-HBV.

21. The conjugate of claim 19, wherein the therapeutic protein is selected from a CRISPR protein or an antibody; more preferably selected from the group consisting of Cas9 protein, EBT106 and GC 1102.

22. The pharmaceutical composition of claim 15, wherein the label is selected from the group consisting of: fluorophores, chromophores, chemiluminescent molecules, magnetic particles, dyes, metals, rare earth metals, and radioisotopes.

23. The pharmaceutical composition of claim 15, wherein the liver-targeting ligand is selected from the group consisting of proteins, antibodies, peptides, small molecule substances, aptamers, and/or carbohydrates with liver-targeting efficacy.

24. A process for the preparation of a pharmaceutical composition according to claim 15 for the treatment of hepatitis b comprising the steps of: (a) dissolving lipid material in organic solvent, mixing, and removing organic solvent under reduced pressure to obtain lipid membrane; (b) adding buffer solution and/or pH regulator to regulate pH, shaking, stirring to make lipid membrane completely hydrated, homogenizing and emulsifying at high speed, and filtering with microporous membrane to obtain blank liposome suspension; (c) dispersing the conjugate of any one of claims 1-14 in water and adding to the blank liposome suspension to make the final liposome.

25. The method of claim 24, wherein the organic solvent is selected from one or more of chloroform, dichloromethane, ethanol, methanol, t-butanol, n-butanol, isopropanol, acetone, diethyl ether, acetonitrile, benzyl alcohol, n-hexane; the buffer solution is selected from one of phosphate buffer solution, citrate buffer solution, acetate buffer solution, borate buffer solution and carbonate buffer solution; the pH regulator is one or more selected from potassium hydroxide, sodium bicarbonate, sodium carbonate, sodium citrate, hydrochloric acid, citric acid and phosphoric acid.

26. Use of the conjugate of any one of claims 1 to 14 or the pharmaceutical composition of any one of claims 15 to 23 in the manufacture of a medicament for the treatment or prevention of hepatitis b.