CN113116821A

CN113116821A - Medicine composition for treating hepatitis B and preparation method and application thereof

Info

Publication number: CN113116821A
Application number: CN202010025502.5A
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: 2020-01-10
Filing date: 2020-01-10
Publication date: 2021-07-16

Abstract

The present invention provides a pharmaceutical composition for the treatment of hepatitis b comprising at least one lipid-based carrier, one or more active drugs for the treatment of hepatitis b, and optionally a label and/or a liver-targeting ligand. The invention also provides a preparation method of the pharmaceutical composition and application of the pharmaceutical composition in preparing a medicament for treating or preventing hepatitis B.

Description

Medicine composition for treating hepatitis B and preparation method and application thereof

Technical Field

The invention relates to the technical field of antiviral drugs, in particular to a pharmaceutical composition for treating hepatitis B 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. Therefore, there is an urgent need in the art to provide a therapeutic agent for hepatitis b that can effectively reach a target site of the liver.

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 hydrophilic drugs, charged DNA and the like 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 above, there is an urgent need in the art to develop a liver-targeted drug for treating hepatitis b.

Disclosure of Invention

The present invention solves the above mentioned problems by devising a pharmaceutical composition based on a lipid-based carrier.

In one aspect, the present invention provides a pharmaceutical composition for the treatment of hepatitis b comprising at least one lipid-based carrier, one or more active drugs for the treatment of hepatitis b, and optionally a labeling and/or liver targeting ligand.

In some embodiments, the at least one lipid-based carrier is a liposome, such as a unilamellar liposome or a multilamellar liposome.

In some embodiments, the liposome comprises 40-70% mol of liposome-forming lipids, 0-50% mol of cholesterol, and/or 0-8% mol of PEG-lipids.

In some embodiments, the one or more active agents for treating hepatitis b are 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 some embodiments, the optional label (e.g., tracer) is selected from the group consisting of a fluorophore, a chromophore, a chemiluminescent molecule, a magnetic particle or dye, a metal, a rare earth, and a radioisotope. In some embodiments, the marker is used to screen and/or detect hepatocytes that are successfully targeted by the vector. In some embodiments, the label is further used to detect the activity of the therapeutic agent for hepatitis b.

In some embodiments, the optional liver targeting ligand is used to assist in targeting the pharmaceutical composition to a liver target, the liver targeting ligand comprising proteins, antibodies, peptides, small molecule substances, aptamers, and/or carbohydrates, and the like, with liver targeting efficacy.

In some embodiments, the composition is formulated for systemic administration.

In another aspect, there is provided a process for preparing the pharmaceutical composition for the treatment of hepatitis b according to claim 1, 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 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 a third aspect, there is provided the use of a pharmaceutical composition provided herein 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 liver cell targeting of the liposome carrier is utilized to efficiently target and load the active drug into liver cells, and the metabolic damage of the circulating system to the molecules is avoided.

2. By introducing one or more different active drugs into the composition, the co-action of the multiple different active drugs can be realized, the therapeutic dose and drug resistance of a single active drug can be potentially reduced, and the therapeutic effects of synergy/addition and the like can be potentially realized.

3. The combination of active targeting and passive targeting is realized by further introducing a liver targeting ligand into the composition, so that the liver targeting effect is further improved.

4. The liver cells successfully targeted by the carrier are screened and/or detected by further introducing a marker into the 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.

Detailed Description

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, 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 vector is selected to enhance the stability of the hepatitis b active agent and optionally the labeled liver targeting ligand contained 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 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.

Active pharmaceutical

Entecavir (Entecavir) is chemically known as 2-amino-1, 9-dihydro-9- [ (1S, 3R, 4S) -4-hydroxy-3- (hydroxymethyl) -2-methylenecyclopentane ] -6H-purin-6-one and has the following structural formula:

US patent US5206244 discloses entecavir and its use for the treatment of hepatitis b virus; a novel synthesis of entecavir is disclosed in WO 9809964; WO0164421 discloses low dose entecavir solid formulations.

Entecavir is a highly effective antiviral agent, developed by schrobo corporation in the 90 s of the 20 th century, and has a strong anti-HBV effect. It can be phosphorylated to active triphosphate, which has a half-life in cells of 15 h. Entecavir triphosphate inhibits all three activities of the viral polymerase (reverse transcriptase) by competing with deoxyguanosine triphosphate, the natural substrate of HBV polymerase: (1) the start of HBV polymerase; (2) formation of a reverse transcribed negative strand of a pregenomic mRNA; (3) synthesis of HBV DNA plus strand.

Tenofovir disoproxil fumarate (the name of England: (TDF); (R) - [ [2- (6-amino-9H-purin-9-yl) -1-methylethoxy ] methyl ] phosphonic acid diisopropoxycarbonylmethyl ester fumarate) is an ester precursor of Tenofovir, belongs to a novel nucleotide reverse transcriptase inhibitor, and has the activity of inhibiting HBV viruses.

TDF is another novel open-ring nucleoside phosphonate successfully developed by Gilidard company in the United states following Adefovir dipivoxil, is first marketed in the United states in 10 months in 2001, and is currently marketed in countries such as Europe, Australia, and Canada.

TDF inhibits viral polymerase in vivo by competitively binding to the natural deoxyribose substrate and terminates DNA strand synthesis by insertion into DNA. The main action mechanism is that the tenofovir is hydrolyzed into tenofovir after being orally taken, the tenofovir is phosphorylated by cell kinase to generate a metabolite tenofovir diphosphate with pharmacological activity, the tenofovir diphosphate competes with 5 '-triphosphate deoxyadenosine monophosphate to participate in the synthesis of virus DNA, and after entering the virus DNA, the DNA is prevented from being prolonged due to the lack of 3' -OH groups, so that the replication of the virus is blocked. Clinical application shows that TDF has obvious curative effect on HBV virus and less toxic side effect, so that TDF has wide clinical application foreground.

Tenofovir Alafenamide (Tenofovir Alafenamide), a prodrug of the new Nucleoside Reverse Transcriptase Inhibitor (NRTI) Tenofovir (Tenofovir) developed by Gilidard scientific, USA. Compared with the prior generation of similar anti-hepatitis B medicine tenofovir disoproxil TDF, the antiviral activity of tenofovir alafenamide is 10 times, the stability in blood plasma is 200 times, and the half-life period is improved by 225 times. Compared with TDF, the tenofovir alafenamide only needs one tenth of TDF administration dosage to achieve the same antiviral curative effect as TDF. Therefore, the tenofovir alafenamide is used for preventing or/and treating Hepatitis B Virus (HBV) infection and has better curative effect, higher safety and lower drug resistance.

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 construction of humanized hepatitis B murine model

The humanized hepatitis B mouse model is prepared by the following specific steps:

first, obtaining human stem cell

1. Isolated culture of human stem cells

1) Purified human stem cells were obtained.

2) And (4) culturing stem cells and carrying out passage.

3) Culturing at 20-40 deg.C and 2-10% CO₂In an incubator.

2. Commercial isolated or cryopreserved human stem cells or cell lines are obtained.

Second, the mice with liver injury transplanted with stem cells

1. Obtaining experimental mice of different strains, wherein the experimental mice comprise normal mice, immunodeficient mice, normal rats and immunodeficient rats.

2. Liver damage drugs are applied by means of intraperitoneal, intramuscular and peripheral intravenous injection, oral administration or intragastric administration, or surgical partial hepatectomy is applied, and a liver damage mouse model is established.

3. Transplantation of 1X 10 by peripheral vein, portal vein, spleen or liver injection^4-8A stem cell.

Third, HBV infects human mouse

Each mouse was injected with hepatitis b virus via peripheral vein, subcutaneous, intramuscular, or intraperitoneal injection.

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 (Alabaster, Alabama, USA) and cholesterol (catalog number: C8667-500MG) was obtained from Sigma (Rehovot, Israel).

Synthesis of liposomes carrying active agents: referring to the above blank liposome synthesis method, the difference lies in that serial diluents with different concentrations of the hepatitis B therapeutic agent are introduced into the ethanol dissolving process, or the hepatitis B therapeutic agent is dispersed in water and then added into the blank liposome suspension to prepare 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: adopting Sephadex 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 break emulsion of liposome, measuring content by High Performance Liquid Chromatography (HPLC), and calculating liposome encapsulation efficiency and loading rateThe dosage.

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 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 pharmaceutical composition for the treatment of hepatitis b comprising at least one lipid-based carrier, one or more active drugs for the treatment of hepatitis b, and optionally a label and/or a liver-targeting ligand.

2. The pharmaceutical composition of claim 1, wherein the at least one lipid-based carrier is a liposome.

3. The pharmaceutical composition of claim 2, wherein the liposome is a unilamellar liposome or a multilamellar liposome.

4. The pharmaceutical composition according to claim 2, wherein the liposomes comprise 40-70% mol of liposome forming lipids, 0-50% mol of cholesterol and/or 0-8% mol of PEG-lipids.

5. The pharmaceutical composition according to claim 1, wherein the one or more active agents for the treatment of hepatitis b are selected from one or more of entecavir, tenofovir disoproxil fumarate and tenofovir alafenamide.

6. The pharmaceutical composition according to claim 5, wherein the one or more active agents for the treatment of hepatitis B are selected from at least two of entecavir, tenofovir disoproxil fumarate, and tenofovir alafenamide.

7. The pharmaceutical composition of claim 1, wherein the label is selected from the group consisting of: fluorophores, chromophores, chemiluminescent molecules, magnetic particles, dyes, metals, rare earth metals, and radioisotopes.

8. The pharmaceutical composition of claim 1, 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.

9. The pharmaceutical composition of claim 1, formulated for systemic administration.

10. A process for preparing a pharmaceutical composition for the treatment of hepatitis b according to claim 1, 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 active drug is dispersed in water and added to the blank liposome suspension to make the final liposome.

11. The method of claim 10, 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.

12. Use of a composition according to any one of claims 1 to 9 in the manufacture of a medicament for the treatment or prevention of hepatitis b.