Her-2 antibody-polyethylene glycol-reverse phospholipid, gemcitabine reverse phospholipid liposome preparation, and preparation method and application thereof

Abstract

The application provides a Her-2 antibody-polyethylene glycol-reverse phospholipid, a gemcitabine reverse phospholipid liposome preparation and a preparation method and application thereof, and belongs to the technical field of biological medicines. The Her-2 antibody-polyethylene glycol-reverse phospholipid provided by the application has a structure shown in formula I. The Her-2 antibody-polyethylene glycol-reverse phospholipid is used to prepare a gemcitabine reverse phospholipid liposome coupled with a Her-2 antibody by simultaneously compounding a reverse phospholipid as a main auxiliary material, so that the gemcitabine can be accurately targeted to Her-2 positive breast cancer cells and kill the Her-2 positive breast cancer cells, and the gemcitabine has excellent Her-2 positive breast cancer treatment effect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to Her-2 antibody-polyethylene glycol-phospholipid, gemcitabine-phospholipid liposome formulations, their preparation methods and applications. Background Technology

[0002] Gemcitabine is a cell cycle-specific antimetabolite belonging to the pyrimidine class of drugs. It does not possess antitumor activity on its own; it requires a three-step phosphorylation process within cells to exert its antitumor effect. Recent studies have confirmed that gemcitabine, used alone or in combination with other anticancer drugs, is significantly effective in treating various solid tumors, including non-small cell lung cancer, pancreatic cancer, and breast cancer. However, gemcitabine has a relatively short intracellular half-life, necessitating continuous intravenous administration of high doses to maintain effective drug concentrations. This continuous administration also leads to significant toxic side effects, clinically manifested as edema, bone marrow suppression, nephrotoxicity, gastrointestinal reactions, flu-like reactions, and allergic reactions. This dose-limiting toxicity greatly affects its clinical efficacy.

[0003] Encapsulating gemcitabine in liposomes to create nanomedicines can effectively enhance its targeting and reduce its toxic side effects on organs. However, existing gemcitabine liposome formulations are passively targeted nanomedicines, achieving their targeting effect through the high permeability and retention effect (EPR effect) of liposomes in the tumor region. A drawback of passively targeted nanomedicines is the strict requirement for particle size; the nanomedicine particles must be small enough to remain within the irregular blood vessels of the tumor region and then permeate out of the blood vessels to avoid being carried away by the bloodstream. Her-2 (human epidermal growth factor receptor-2) antibody-drug conjugates have an active tumor-targeting effect; however, they lack the nanoscale particle size required to remain in the tumor region, resulting in poor tumor cell killing efficacy. Summary of the Invention

[0004] The purpose of this invention is to provide a Her-2 antibody-polyethylene glycol-phospholipid, gemcitabine-phospholipid liposome formulation, its preparation method, and its application. Using the Her-2 antibody-polyethylene glycol-phospholipid provided by this invention, combined with phospholipid as the main excipient, Her-2 antibody-conjugated gemcitabine-phospholipid liposomes can be prepared, enabling gemcitabine to precisely target and kill Her-2 positive breast cancer cells, resulting in excellent therapeutic effects for Her-2 positive breast cancer.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a Her-2 antibody-polyethylene glycol-reverse phospholipid having the structure shown in Formula I:

[0007]

[0008] The formula described in Formula I This indicates Her-2 antibody.

[0009] This invention provides a method for preparing the Her-2 antibody-polyethylene glycol-antiphospholipid described in the above technical solution, comprising the following steps:

[0010] Hydroxy-reverse phospholipids, carboxyl-terminated polyethylene glycol 2000, 4-dimethylaminopyridine, N,N'-dicyclohexylcarboimide and dichloromethane were mixed and subjected to a first condensation reaction to obtain polyethylene glycol-reverse phospholipids.

[0011] The polyethylene glycol-reverse phospholipid, Her-2 antibody, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were mixed with water and subjected to a second condensation reaction to obtain Her-2 antibody-polyethylene glycol-reverse phospholipid having the structure shown in Formula I.

[0012] Preferably, the temperature of the first condensation reaction and the second condensation reaction are independently 15-35°C, and the time is independently 20-28h.

[0013] This invention provides a Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation, comprising the following raw materials:

[0014] Gemcitabine hydrochloride 0.01–0.5 wt%, antiphospholipid 0.1–5.0 wt%, Her-2 antibody-polyethylene glycol-antiphospholipid 0.01–1.0 wt%, cholesterol 0.01–2.0 wt%, buffer 0.1–10 wt%, balance being water; wherein the Her-2 antibody-polyethylene glycol-antiphospholipid is the Her-2 antibody-polyethylene glycol-antiphospholipid described in the above technical solution or the Her-2 antibody-polyethylene glycol-antiphospholipid prepared by the preparation method described in the above technical solution.

[0015] Preferably, the buffer comprises one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, 4-hydroxyethylpiperazine ethanesulfonic acid, sodium hydroxide, potassium hydroxide, hydrochloric acid, sodium chloride, potassium chloride, sucrose, and glucose.

[0016] Preferably, the liposomes in the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation have a particle size of 85.8–106.5 nm and a particle size distribution index of 0.212–0.312.

[0017] This invention provides a method for preparing the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation described in the above technical solution, comprising the following steps:

[0018] The antiphospholipid, Her-2 antibody-polyethylene glycol-antiphospholipid, cholesterol and alcohol solvent were mixed to obtain an oil phase solution;

[0019] Sulfate is mixed with water to obtain an aqueous solution;

[0020] The oil phase solution and the aqueous phase solution are mixed and then subjected to emulsification and homogenization treatments in sequence to obtain a homogeneous emulsion.

[0021] The alcohol solvent in the homogenized emulsion is removed, and then it is mixed with gemcitabine hydrochloride aqueous solution for incubation treatment. After removing sulfate, the incubation solution is obtained.

[0022] The incubation solution was concentrated and then mixed with a buffer to obtain the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation.

[0023] Preferably, the emulsification process is carried out under ultrasonic conditions, the conditions of which include: ultrasonic power of 100-300W, temperature of 50-60℃, and time of 30-60min.

[0024] The homogenization conditions include: a homogenization speed of 500–1000 rpm, a temperature of 50–60°C, and a time of 30–60 min;

[0025] The incubation conditions include a temperature of 50–60°C and a time of 30–60 minutes.

[0026] This invention provides the application of the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation described in the above-described technical solution or the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation prepared by the preparation method described in the above-described technical solution in the preparation of a drug for treating Her-2 positive breast cancer.

[0027] This invention provides a drug for treating Her-2 positive breast cancer, wherein the active ingredient is a gemcitabine antiphospholipid liposome preparation conjugated with Her-2 antibody as described in the above technical solution or a gemcitabine antiphospholipid liposome preparation conjugated with Her-2 antibody prepared by the preparation method described in the above technical solution.

[0028] This invention provides a Her-2 antibody-polyethylene glycol-phospholipid structure with the structure shown in Formula I. Her-2 antibody-polyethylene glycol-phospholipid provided by this invention is used in combination with phospholipid as the main excipient to prepare Her-2 antibody-conjugated gemcitabine phospholipid liposomes. This enables gemcitabine to precisely target and kill Her-2 positive breast cancer cells, resulting in excellent therapeutic effects for Her-2 positive breast cancer. Specifically, this invention uses polyethylene glycol groups as linking groups to chemically bond Her-2 antibody with antiphospholipids to form Her-2 antibody-polyethylene glycol-antiphospholipid. Based on the Her-2 antibody-polyethylene glycol-antiphospholipid and antiphospholipid as the main excipients, Her-2 antibody-conjugated gemcitabine antiphospholipid liposomes are prepared, which have both active and passive targeting functions. The recognition molecule (i.e., ligand) Her-2 antibody is linked to the gemcitabine antiphospholipid liposomes. The Her-2 antibody specifically binds to the receptor highly expressed on the target cells, and the nanoscale size of the gemcitabine antiphospholipid liposomes is used to retain the drug in the tumor area, thereby improving the drug efficacy. Attached Figure Description

[0029] Figure 1 This is a particle size distribution diagram of liposomes in the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation in Example 2;

[0030] Figure 2 This is a particle size distribution diagram of the liposomes in the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation in Example 3;

[0031] Figure 3 This is a particle size distribution diagram of the liposomes in the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation in Example 4;

[0032] Figure 4 The graph shows the evaluation results of the breast cancer cell-targeting killing ability of the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulations in Examples 2-4. Detailed Implementation

[0033] This invention provides a Her-2 antibody-polyethylene glycol-reverse phospholipid having the structure shown in Formula I:

[0034]

[0035] The formula described in Formula I This indicates Her-2 antibody.

[0036] This invention provides a method for preparing the Her-2 antibody-polyethylene glycol-antiphospholipid described in the above technical solution, comprising the following steps:

[0037] Hydroxy-reverse phospholipids, carboxyl-terminated polyethylene glycol 2000, 4-dimethylaminopyridine, N,N'-dicyclohexylcarboimide and dichloromethane were mixed and subjected to a first condensation reaction to obtain polyethylene glycol-reverse phospholipids.

[0038] The polyethylene glycol-reverse phospholipid, Her-2 antibody, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were mixed with water and subjected to a second condensation reaction to obtain Her-2 antibody-polyethylene glycol-reverse phospholipid having the structure shown in Formula I.

[0039] In this invention, unless otherwise specified, all raw materials used are commercially available products well known to those skilled in the art or prepared using methods well known to those skilled in the art.

[0040] In this invention, hydroxyl-reverse phospholipids (the preparation method of which will be described in detail below), carboxyl-terminated polyethylene glycol 2000, 4-dimethylaminopyridine, N,N'-dicyclohexylcarboimide and dichloromethane are mixed and subjected to a first condensation reaction to obtain polyethylene glycol-reverse phospholipids. In this invention, the preferred ratio of hydroxyl-antiphospholipid, carboxyl-terminated polyethylene glycol 2000, 4-dimethylaminopyridine, N,N'-dicyclohexylcarboimide, and dichloromethane is 10g:20-25g:0.08-0.12g:1.8-2.2g:450-550mL, more preferably 10g:23g:0.1g:2g:500mL; the 4-dimethylaminopyridine is a catalyst, the N,N'-dicyclohexylcarboimide is a dehydrating agent, and the carboxyl-terminated polyethylene glycol 2000 specifically refers to carboxyl-terminated polyethylene glycol with a weight-average molecular weight (Mw) of 2000. In this invention, the preferred temperature for the first condensation reaction is 15-35°C, specifically at room temperature, which is 25°C; the preferred time for the first condensation reaction is 20-28 hours, more preferably 24 hours. After the first condensation reaction, the present invention preferably removes the excess terminal carboxyl polyethylene glycol 2000 and 4-dimethylaminopyridine by washing with tetrahydrofuran to obtain the polyethylene glycol-reverse phospholipid.

[0041] After obtaining polyethylene glycol-antiphospholipid, the present invention mixes the polyethylene glycol-antiphospholipid, Her-2 antibody, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide with water to carry out a second condensation reaction to obtain the Her-2 antibody-polyethylene glycol-antiphospholipid. In the present invention, the preferred ratio of polyethylene glycol-antiphospholipid, Her-2 antibody, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to water is 100mg:8-12mg:8-12mg:8-12mg:4-6mL, more preferably 100mg:10mg:10mg:10mg:5mL; the water is preferably deionized water, wherein the N-hydroxysuccinimide is a catalyst, and the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is a dehydrating agent. In this invention, the temperature of the second condensation reaction is preferably 15–35°C, specifically, the second condensation reaction can be carried out at room temperature; the time of the second condensation reaction is preferably 20–28 h, more preferably 24 h. After the second condensation reaction, the resulting product system is preferably purified by dialysis to obtain Her-2 antibody-polyethylene glycol-antiphospholipid having the structure shown in Formula I.

[0042] This invention provides a Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation, comprising the following raw materials:

[0043] Gemcitabine hydrochloride 0.01–0.5 wt%, antiphospholipid 0.1–5.0 wt%, Her-2 antibody-polyethylene glycol-antiphospholipid 0.01–1.0 wt%, cholesterol 0.01–2.0 wt%, buffer 0.1–10 wt%, balance being water; wherein the Her-2 antibody-polyethylene glycol-antiphospholipid is the Her-2 antibody-polyethylene glycol-antiphospholipid described in the above technical solution or the Her-2 antibody-polyethylene glycol-antiphospholipid prepared by the preparation method described in the above technical solution.

[0044] The raw materials for preparing the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation of the present invention include 0.01-0.5 wt% gemcitabine hydrochloride, preferably 0.1-0.4 wt%, and more preferably 0.2-0.3 wt%.

[0045] The raw materials for preparing the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation of the present invention include antiphospholipids (also known as choline phospholipids, the preparation method of which is described in detail below) in 0.1–5.0 wt%, preferably 0.5–4.0 wt%, more preferably 1–3 wt%, and even more preferably 1.5–2.0 wt%. In the present invention, the antiphospholipids can achieve effective protection and efficient transport of the drug.

[0046] The raw materials for preparing the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation of the present invention include Her-2 antibody-polyethylene glycol-antiphospholipid at 0.01-1.0 wt%, preferably 0.05-0.8 wt%, more preferably 0.1-0.5 wt%, and even more preferably 0.2-0.4 wt%. In the present invention, the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome can provide effective drug protection and achieve efficient active targeting.

[0047] The raw materials for preparing the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation of the present invention include 0.01–2.0 wt% cholesterol, preferably 0.05–1.5 wt%, more preferably 0.1–1.0 wt%, and even more preferably 0.3–0.5 wt%. In the present invention, the cholesterol can enhance the stability of the liposomes.

[0048] The raw materials for preparing the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation of the present invention include 0.1–10 wt% buffer, preferably 0.5–8 wt%, more preferably 1–6 wt%, and even more preferably 2–4 wt%. In the present invention, the buffer preferably includes one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, 4-hydroxyethylpiperazine ethanesulfonic acid, sodium hydroxide, potassium hydroxide, hydrochloric acid, sodium chloride, potassium chloride, sucrose, and glucose, more preferably one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium hydroxide, sodium chloride, potassium chloride, and hydrochloric acid.

[0049] The raw materials for preparing the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation of the present invention include the remainder water, preferably water for injection.

[0050] The preferred particle size of the liposomes in the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation of the present invention is 85.8–106.5 nm, and the preferred particle size distribution index is 0.212–0.312. The preferred pH value of the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation of the present invention is 7.0–7.4.

[0051] This invention provides a method for preparing the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation described in the above technical solution, comprising the following steps:

[0052] The antiphospholipid, Her-2 antibody-polyethylene glycol-antiphospholipid, cholesterol and alcohol solvent were mixed to obtain an oil phase solution;

[0053] Sulfate is mixed with water to obtain an aqueous solution;

[0054] The oil phase solution and the aqueous phase solution are mixed and then subjected to emulsification and homogenization treatments in sequence to obtain a homogeneous emulsion.

[0055] The alcohol solvent in the homogenized emulsion is removed, and then it is mixed with gemcitabine hydrochloride aqueous solution for incubation treatment. After removing sulfate, the incubation solution is obtained.

[0056] The incubation solution was concentrated and then mixed with a buffer to obtain the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation.

[0057] This invention involves mixing antiphospholipids, Her-2 antibody-polyethylene glycol-antiphospholipids, cholesterol, and an alcohol solvent to obtain an oil phase solution. In this invention, the alcohol solvent is preferably ethanol, and the preferred ratio of antiphospholipids to the alcohol solvent is 5g:1-10mL, more preferably 5g:2-5mL. In this invention, the mixing is preferably carried out under heating conditions, and the heating temperature is preferably 45-60℃, more preferably 45-50℃.

[0058] This invention involves mixing sulfate with water to obtain an aqueous solution. In this invention, the sulfate is preferably ammonium sulfate, which provides an ion gradient to ensure efficient drug loading; the water is preferably water for injection; the concentration of the aqueous solution is preferably 100–300 mM, more preferably 200 mM. In this invention, the mixing is preferably carried out at 20–30°C, but can specifically be done at room temperature.

[0059] After obtaining the oil phase solution and the aqueous phase solution, the present invention mixes the oil phase solution and the aqueous phase solution, and then performs emulsification and homogenization treatments sequentially to obtain a homogeneous emulsion. In the present invention, the mixing is preferably carried out under heating conditions, and the heating temperature is preferably 55-65°C, more preferably 60°C. In the present invention, the emulsification treatment is preferably carried out under ultrasonic conditions, and the emulsification treatment conditions include: ultrasonic power preferably 100-300W, more preferably 200-300W; temperature preferably 50-60°C, more preferably 55-60°C; and time preferably 30-60 min, more preferably 30-40 min. During the emulsification treatment process of the present invention, vesicles with a bilayer structure are formed in the system. In the present invention, the homogenization treatment conditions include: homogenization speed preferably 500-1000 rpm, more preferably 800 rpm; temperature preferably 50-60°C, more preferably 55-60°C; and time preferably 30-60 min, more preferably 30-40 min. In this invention, the homogenization process is preferably carried out in a high-pressure homogenizer. Preferably, the homogenization process results in liposomes in the homogenized emulsion having a particle size of 50–150 nm, more preferably 80–120 nm.

[0060] After obtaining a homogenized emulsion, the present invention removes the alcohol solvent from the homogenized emulsion, then mixes it with an aqueous gemcitabine hydrochloride solution for incubation treatment, followed by sulfate removal to obtain an incubation solution. Preferably, the homogenized emulsion is subjected to rotary evaporation under reduced pressure to remove the alcohol solvent. In the present invention, the solvent in the aqueous gemcitabine hydrochloride solution is preferably water for injection, and the concentration of the aqueous gemcitabine hydrochloride solution is preferably 5-10 mg / mL, more preferably 8-10 mg / mL. In the present invention, the incubation treatment conditions include: a temperature preferably 50-60°C, more preferably 55-60°C; and a time preferably 30-60 min, more preferably 50-60 min. In the present invention, during the incubation treatment, the drug enters the cavity of the liposomes. After the incubation treatment, the resulting solution is passed through an ultrafiltration system to remove sulfate, obtaining an incubation solution; during the sulfate removal process using the ultrafiltration system, unencapsulated gemcitabine hydrochloride in the system is also removed.

[0061] After obtaining the incubation solution, the present invention concentrates the incubation solution and mixes it with a buffer to obtain the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation. The present invention does not have specific limitations on the concentration, as long as the concentration requirements of each component in the final Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation are met. After mixing, the present invention preferably sterilizes the obtained solution to obtain the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation; the preferred sterilization method is filtration using a 0.22 μm microporous membrane.

[0062] This invention uses a solvent dispersion method to prepare the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation. The operation is simple, and the prepared Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation has excellent targeted killing effect on breast cancer cells.

[0063] This invention provides the application of the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation described in the above-described technical solutions, or the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation prepared by the preparation method described in the above-described technical solutions, in the preparation of drugs for treating Her-2 positive breast cancer. Currently, approximately one-fifth of female breast cancer patients have cancer cells containing excessive amounts of growth-promoting Her-2 / neu (or Her-2 only) proteins; this type of cancer is called Her-2 positive breast cancer, and it grows and spreads more rapidly. The Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation provided by this invention can be used to treat Her-2 positive breast cancer, exhibiting high tumor targeting ability and low toxicity, and can achieve precise killing of breast cancer cells. This invention does not specifically limit the specific method of application; any method well known to those skilled in the art can be used.

[0064] This invention provides a medicament for treating Her-2 positive breast cancer. The active ingredient is a gemcitabine antiphospholipid liposome formulation conjugated with a Her-2 antibody as described in the above-described technical solution, or a gemcitabine antiphospholipid liposome formulation conjugated with a Her-2 antibody prepared by the method described in the above-described technical solution. This invention does not specifically limit the content of the active ingredient in the medicament for treating Her-2 positive breast cancer. The medicament for treating Her-2 positive breast cancer of this invention also contains pharmaceutically acceptable excipients. This invention does not specifically limit the specific types of pharmaceutically acceptable excipients; any pharmaceutically acceptable excipient well known to those skilled in the art can be used.

[0065] The preparation methods of the antiphospholipids and hydroxy-antiphospholipids described in this invention will be described below.

[0066] In this invention, the method for preparing the antiphospholipid preferably includes the following steps:

[0067] A substitution reaction was carried out by mixing 2-chloro-2-oxo-dioxophosphazene, ethanol, triethylamine, and tetrahydrofuran to give compound E:

[0068] The compound E, 3-dimethylamino-1,2-propanediol, and acetonitrile were mixed and subjected to a ring-opening reaction to obtain compound D.

[0069] The compound D, octadecanoic acid, N,N'-diisopropylcarbodiimide, 4-dimethylaminopyridine, and dichloromethane were mixed and subjected to an esterification reaction to obtain the antiphospholipid.

[0070] Compound E has the structure shown in Formula E, compound D has the structure shown in Formula D, and the antiphospholipid has the structure shown in Formula C.

[0071]

[0072]

[0073] This invention involves mixing 2-chloro-2-oxo-dioxophosphanecyclopentane, ethanol, triethylamine, and tetrahydrofuran to undergo a substitution reaction, yielding compound E. In this invention, the molar ratio of 2-chloro-2-oxo-dioxophosphanecyclopentane, ethanol, and triethylamine is preferably 0.5:0.48–0.52:0.48–0.52, more preferably 0.5:0.5:0.51; the ethanol is preferably anhydrous ethanol. In this invention, the tetrahydrofuran is preferably anhydrous tetrahydrofuran. This invention does not have a specific limitation on the amount of tetrahydrofuran used; it serves as a solvent to ensure the smooth progress of the reaction. Preferably, this invention involves mixing triethylamine, ethanol, and tetrahydrofuran, and then adding 2-chloro-2-oxo-dioxophosphanecyclopentane dropwise to the resulting mixture under nitrogen protection to carry out the substitution reaction. In this invention, the temperature of the substitution reaction is preferably -2 to 2°C, more preferably carried out under ice-water bath conditions (0°C); the time of the substitution reaction is preferably 1.5 to 2.5 h, more preferably 2 h, and the time of the substitution reaction begins from the time when 2-chloro-2-oxo-dioxophosphazenecyclopentane is completely added; the substitution reaction is preferably carried out under nitrogen protection. After the substitution reaction, the resulting product system is preferably filtered to remove tetrahydrofuran from the filtrate to obtain compound E.

[0074] After obtaining compound E, the present invention mixes compound E, 3-dimethylamino-1,2-propanediol, and acetonitrile to carry out a ring-opening reaction to obtain compound D. In the present invention, the molar ratio of compound E to 3-dimethylamino-1,2-propanediol is preferably 1:1.0 to 1.5, more preferably 1:1.2. In the present invention, the acetonitrile is preferably anhydrous acetonitrile; the present invention does not have a special limitation on the amount of acetonitrile used, as long as it serves as a solvent to ensure the smooth progress of the reaction. The present invention does not have a special limitation on the mixing method of compound E, 3-dimethylamino-1,2-propanediol, and acetonitrile, as long as the components are thoroughly mixed. In the present invention, the temperature of the ring-opening reaction is preferably 65 to 75°C, more preferably 70°C; the time is preferably 40 to 55 hours, more preferably 48 hours. After the ring-opening reaction, the present invention preferably precipitates the obtained product system in tetrahydrofuran, and the precipitate obtained is compound D.

[0075] After obtaining compound D, the present invention mixes compound D, octadecanoic acid, N,N'-diisopropylcarbodiimide, 4-dimethylaminopyridine, and dichloromethane to carry out an esterification reaction to obtain the antiphospholipid. In the present invention, the molar ratio of compound D, octadecanoic acid, N,N'-diisopropylcarbodiimide, and 4-dimethylaminopyridine is preferably 0.10:0.20-0.25:0.20-0.25:0.04-0.06, more preferably 0.10:0.22:0.22:0.05. The present invention does not have a specific limitation on the amount of dichloromethane used; it is used as a solvent to ensure the smooth progress of the reaction. Preferably, the present invention adds compound D and octadecanoic acid to dichloromethane, stirs thoroughly, and then adds N,N'-diisopropylcarbodiimide and 4-dimethylaminopyridine to carry out the esterification reaction. In this invention, the esterification reaction temperature is preferably 15–35°C, specifically, the esterification reaction can be carried out at room temperature; the esterification reaction time is preferably 20–28 h, more preferably 24 h. After the esterification reaction, the resulting product system is preferably filtered, the filtrate is concentrated and then recrystallized for purification to obtain the reverse phospholipid.

[0076] The present invention preferably prepares the hydroxy-antiphospholipid using the same method as for preparing antiphospholipids, except that ethanol is replaced with ethylene glycol in the preparation of compound E; the hydroxy-antiphospholipid has the structure shown in formula B:

[0077]

[0078] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0079] Preparation Example 1

[0080] The steps for preparing reverse phospholipids are as follows:

[0081] (1) Triethylamine (51 g, 0.51 mol) and anhydrous ethanol (22 g, 0.5 mol) were added to a side-mouth flask containing 300 mL of anhydrous tetrahydrofuran. Under nitrogen protection, 2-chloro-2-oxo-dioxophosphazenecyclopentane (71 g, 0.5 mol) was added dropwise from a constant pressure dropping funnel. After the addition was completed, the reaction was carried out in an ice-water bath for 2 h. After the reaction was completed, the resulting product system was filtered to remove tetrahydrofuran from the filtrate, and compound E was obtained.

[0082] (2) Compound E (4.56 g, 30 mmol) and 3-dimethylamino-1,2-propanediol (4.28 g, 36 mmol) were added to a flask containing 50 mL of anhydrous acetonitrile and heated to 70 °C for 48 h. After the reaction was completed, the resulting product system was precipitated three times in tetrahydrofuran to obtain compound D.

[0083] (3) Add 62 g, 0.22 mol of stearic acid and compound D (27 g, 0.1 mol) to 300 mL of dichloromethane, stir thoroughly, and then add N,N'-diisopropylcarbodiimide (DIC, 27.8 g, 0.22 mol) and 4-dimethylaminopyridine (DMAP, 6.1 g, 0.05 mol). Stir thoroughly at room temperature for 24 h. After the reaction is complete, filter the obtained product system, concentrate the filtrate and recrystallize it three times to obtain antiphospholipid.

[0084] Preparation Example 2

[0085] In step (1) of Preparation Example 1, anhydrous ethanol was replaced with ethylene glycol, and the other operations were the same as in Preparation Example 1, finally yielding hydroxy-reverse phospholipids.

[0086] Example 1

[0087] (1) 10g of hydroxyl-antiphospholipid, 23g of carboxyl-terminated polyethylene glycol 2000, 0.1g of catalyst 4-dimethylaminopyridine (DMAP), 2g of dehydrating agent N,N'-dicyclohexylcarboimide (DCC) and 500mL of dichloromethane were mixed and reacted at room temperature for 24h. After the reaction was completed, the excess carboxyl-terminated polyethylene glycol 2000 and catalyst were washed away with tetrahydrofuran to obtain polyethylene glycol-antiphospholipid.

[0088] (2) 100 mg of polyethylene glycol-antiphospholipid, 10 mg of Her-2 antibody, 10 mg of catalyst N-hydroxysuccinimide (NHS) and 10 mg of dehydrating agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) were added to 5 mL of deionized water and reacted at room temperature for 24 h. After the reaction was completed, the resulting product system was dialyzed to remove the catalyst, and Her-2 antibody-polyethylene glycol-antiphospholipid was obtained.

[0089] Example 2

[0090] (1) Mix 5g of antiphospholipid, 0.4g of Her-2 antibody-polyethylene glycol-antiphospholipid, 1.5g of cholesterol and 2mL of ethanol at 45℃ to obtain an oil phase solution;

[0091] (2) Ammonium sulfate was mixed and dissolved with 100 mL of water for injection at room temperature to obtain an aqueous solution with an ammonium sulfate concentration of 200 mM.

[0092] (3) The oil phase solution and the aqueous phase solution are mixed at 60°C, and emulsified for 30 min at an ultrasonic power of 300 W and a temperature of 60°C. Then, a high-pressure homogenizer is used to homogenize the emulsion for 30 min at a speed of 800 rpm and a temperature of 60°C, so that the particle size of the liposomes in the resulting homogenized emulsion is 80-120 nm.

[0093] (4) The homogenized emulsion was subjected to rotary vacuum evaporation to remove ethanol, and then mixed with 1 mL of gemcitabine hydrochloride aqueous solution with a concentration of 10 mg / mL. The mixture was incubated at 60°C for 60 min. The resulting product system was passed through an ultrafiltration system to remove unencapsulated gemcitabine hydrochloride and ammonium sulfate. The resulting solution was then concentrated, and 5 mL of buffer solution (water for injection) was added to the residue until the concentration of gemcitabine hydrochloride was 2 mg / mL. The resulting solution was then filtered through a 0.22 μm microporous membrane for sterilization to obtain Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation (pH 7.4).

[0094] Figure 1 The image shows the particle size distribution of liposomes in the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation prepared in Example 2. The results show that the particle size of the liposomes in the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation is 106.5 nm, and the PDI is 0.312.

[0095] Example 3

[0096] Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation was prepared according to the method of Example 2, except that the amount of Her-2 antibody-polyethylene glycol-antiphospholipid used in this example was 0.6g.

[0097] Figure 2 The image shows the particle size distribution of liposomes in the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation prepared in Example 3. The results show that the particle size of the liposomes in the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation is 104.0 nm, and the PDI is 0.286.

[0098] Example 4

[0099] Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation was prepared according to the method of Example 2, except that the amount of Her-2 antibody-polyethylene glycol-antiphospholipid used in this example was 0.8g.

[0100] Figure 3The image shows the particle size distribution of liposomes in the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation prepared in Example 4. The results show that the particle size of the liposomes in the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation is 85.8 nm, and the PDI is 0.212.

[0101] Comparative Example 1

[0102] Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation was prepared according to the method in Example 4, the only difference being that the Her-2 antibody-polyethylene glycol-antiphospholipid was omitted, and the antiphospholipid was replaced with lecithin.

[0103] Test Example 1

[0104] The following method is used to evaluate the targeting efficacy of Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulations:

[0105] Normal human breast epithelial cells (MDA-kb2 cell line) and human Her-2 positive breast cancer cells (MDA-MB-453 cell line) were incubated in culture dishes for 12 h. Then, Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulations prepared in Examples 2-4 were added to the culture dishes of the two cell lines, with the gemcitabine concentration at 10 μg / mL. After incubation for 24 h, Celltiter-Blue reagent was added to each well, and the cells were read using a microplate reader (λ). ex =560nm, λ em Cell viability was detected at 590 nm.

[0106] Figure 4 The figure shows the evaluation results of the targeted killing ability of the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulations in Examples 2-4 for breast cancer cells. The results show that the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulations in Examples 2-4 all exhibited strong targeting ability for Her-2 positive breast cancer cells. Among them, the survival rate of MDA-kb2 cells using the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation in Example 4 was 87.3%, and the survival rate of MDA-MB-453 cells was 23.5%, showing the greatest difference in killing ability and the best targeting effect.

[0107] Test Example 2

[0108] Comparison of the targeting effects of Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulations and gemcitabine liposome formulations without Her-2 antibodies:

[0109] Human Her-2 positive breast cancer cells (MDA-MB-453 cell line) were incubated in culture dishes for 12 h. Gemcitabine liposome formulations prepared in Example 4 and Comparative Example 1 were then added to the culture dishes to achieve a gemcitabine concentration of 10 μg / mL. After incubation for 24 h, Celltiter-Blue reagent was added to each well, and the cells were then analyzed using a microplate reader (λ). ex =560nm, λ em Cell viability was detected at 590 nm.

[0110] The results showed that the survival rate of breast cancer cells using the gemcitabine antiphospholipid liposome formulation conjugated with the Her-2 antibody in Example 4 was 25.3%, while the survival rate of breast cancer cells using the gemcitabine liposome formulation in Comparative Example 1 was 51.2%. This indicates that the addition of Her-2 antibody-polyethylene glycol-antiphospholipid and antiphospholipid in this invention significantly improves the targeted therapeutic effect of the liposome formulation. Compared with gemcitabine liposome formulations prepared with lecithin but without Her-2 antibody-polyethylene glycol-antiphospholipid and antiphospholipid, as well as Her-2 antibody drug conjugates, it has stronger active targeting of Her-2 positive breast cancer cells and lower toxicity, achieving precise killing of Her-2 positive breast cancer cells.

[0111] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A Her-2 antibody-polyethylene glycol-antiphospholipid, having the structure shown in Formula I: The formula described in Formula I This indicates Her-2 antibody.

2. The preparation method of Her-2 antibody-polyethylene glycol-antiphospholipid according to claim 1, comprising the following steps: Hydroxy-reverse phospholipids, carboxyl-terminated polyethylene glycol 2000, 4-dimethylaminopyridine, N,N'-dicyclohexylcarboimide and dichloromethane were mixed and subjected to a first condensation reaction to obtain polyethylene glycol-reverse phospholipids. The polyethylene glycol-reverse phospholipid, Her-2 antibody, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were mixed with water and subjected to a second condensation reaction to obtain Her-2 antibody-polyethylene glycol-reverse phospholipid having the structure shown in Formula I.

3. The preparation method according to claim 2, characterized in that, The temperature of the first condensation reaction and the second condensation reaction are independently 15–35°C, and the time is independently 20–28 h.

4. A Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation, characterized in that, The following raw materials are included in the preparation: Gemcitabine hydrochloride 0.01–0.5 wt%, antiphospholipid 0.1–5.0 wt%, Her-2 antibody-polyethylene glycol-antiphospholipid 0.01–1.0 wt%, cholesterol 0.01–2.0 wt%, buffer 0.1–10 wt%, balance being water; wherein the Her-2 antibody-polyethylene glycol-antiphospholipid is the Her-2 antibody-polyethylene glycol-antiphospholipid described in claim 1 or the Her-2 antibody-polyethylene glycol-antiphospholipid prepared by the preparation method described in claim 2 or 3.

5. The Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation according to claim 4, characterized in that, The buffer comprises one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, 4-hydroxyethylpiperazine ethanesulfonic acid, sodium hydroxide, potassium hydroxide, hydrochloric acid, sodium chloride, potassium chloride, sucrose, and glucose.

6. The Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation according to claim 4 or 5, characterized in that, The Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation has a liposome particle size of 85.8–106.5 nm and a particle size distribution index of 0.212–0.

312.

7. A method for preparing the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation according to any one of claims 4 to 6, comprising the following steps: The antiphospholipid, Her-2 antibody-polyethylene glycol-antiphospholipid, cholesterol and alcohol solvent were mixed to obtain an oil phase solution; Sulfate is mixed with water to obtain an aqueous solution; The oil phase solution and the aqueous phase solution are mixed and then subjected to emulsification and homogenization treatments in sequence to obtain a homogeneous emulsion. The alcohol solvent in the homogenized emulsion is removed, and then it is mixed with gemcitabine hydrochloride aqueous solution for incubation treatment. After removing sulfate, the incubation solution is obtained. The incubation solution was concentrated and then mixed with a buffer to obtain the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation.

8. The preparation method according to claim 7, characterized in that, The emulsification process is carried out under ultrasonic conditions, which include: ultrasonic power of 100-300W, temperature of 50-60℃, and time of 30-60min. The homogenization conditions include: a homogenization speed of 500–1000 rpm, a temperature of 50–60°C, and a time of 30–60 min; The incubation conditions include a temperature of 50–60°C and a time of 30–60 minutes.

9. The use of the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation according to any one of claims 4 to 6, or the Her-2 antibody-conjugated gemcitabine antiphospholipid liposome formulation prepared by the preparation method according to claim 7 or 8, in the preparation of a medicament for treating Her-2 positive breast cancer.

10. A medicament for treating Her-2 positive breast cancer, wherein the active ingredient is a gemcitabine antiphospholipid liposome preparation conjugated with Her-2 antibody as described in any one of claims 4 to 6, or a gemcitabine antiphospholipid liposome preparation conjugated with Her-2 antibody prepared by the preparation method described in claim 7 or 8.

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