CN108938600B

CN108938600B - Microspheres for intravitreal injection and double protection of antibody drugs and preparation method thereof

Info

Publication number: CN108938600B
Application number: CN201810757645.8A
Authority: CN
Inventors: 李又欣; 刘佳欣; 孙凤英; 李爽; 滕乐生
Original assignee: Nanjing Rui Li Shi Biotechnology Co ltd
Current assignee: Nanjing Rui Li Shi Biotechnology Co ltd
Priority date: 2018-07-11
Filing date: 2018-07-11
Publication date: 2021-10-08
Anticipated expiration: 2038-07-11
Also published as: CN108938600A

Abstract

The invention provides microspheres for intravitreal injection and double protection of antibody drugs and a preparation method thereof. The denaturation and aggregation of the antibody caused by the acidic microenvironment of polylactic acid-glycolic acid in the organic phase/water phase interface and the degradation process improve the drug-loading rate of the microsphere and reduce the burst release phenomenon in the release process of the microsphere, thereby realizing the double protection of the monoclonal antibody drug in the microsphere; the polyketal improves the drug loading of the microspheres, reduces the stimulation of acidic degradation products of polylactic acid-glycolic acid to the eye environment, reduces the side effects of endophthalmitis and the like, the microspheres can slowly release monoclonal antibodies for not less than 28 days in an in vitro environment, and can reduce the drug administration frequency for not less than 2 months in eyes, thereby relieving the pain and economic burden of patients and improving the compliance of the patients.

Description

Microspheres for intravitreal injection and double protection of antibody drugs and preparation method thereof

Technical Field

The invention relates to a microsphere for intravitreal injection and double protection of antibody drugs, further provides a preparation method of the microsphere, and belongs to the technical field of medicines.

Background

Retinal disease has become the second leading cause of blindness in our country after cataract, with age-related macular degeneration (AMD) being one of the most difficult recognized eye diseases to treat, and Choroidal Neovascularization (CNV) being the most important cause of vision loss. Among them, Vascular Endothelial Growth Factor (VEGF) and its receptors play an important role in the proliferation and repair of pathological neovasculature. Abnormal angiogenesis can cause vascular leakage in ocular vascular diseases. anti-VEGF drugs are therefore key to the treatment of wet age-related macular degeneration.

Abirapsis (Eylea) is the first fully humanized fusion protein in the world, is a soluble decoy receptor capable of being combined with VEGF-A, PIGF and can inhibit the combination and activation of endogenous VEGF receptor with VEGF-A and PIGF, thereby inhibiting the formation of new blood vessels and reducing the permeability of the blood vessels. The half-life of the aflibercept not bound with VEGF is 1-3 d, while the half-life of the aflibercept bound with VECF is 18d, so that frequent intravitreal injection administration is required, the pain and economic burden of patients are increased, and most importantly, the probability of complications in the repeated injection process is greatly increased, such as vitreous hemorrhage, retinal detachment, endophthalmitis, cataract and the like. Similar antibody drugs for treating diseases of the posterior retina also comprise ranibizumab (Lucentis), Bevacizumab (Avastin) and Corbescept, etc.

The sustained-release microsphere preparation for injection is widely used in sustained-release drug delivery systems of protein and antibody drugs, can effectively widen drug delivery routes and reduce drug delivery times and drug dosage, and is more and more widely applied. The most currently used microsphere formulations are those based on PLGA, a biodegradable material. This material has been approved by the FDA in the united states for use as a slow release drug carrier and other devices for human implantation. However, the main disadvantage of PLGA systems is the accumulation of acidic degradation products inside the degraded material, which can lead to adsorption or polymerization of the antibody drug, resulting in loss of activity or immunogenicity, and incomplete protein release. In addition, in the aspect of intraocular administration, due to the special physiological structure characteristics of eyes, the generation of acidic substances can cause irritation to eyes, easily cause side effects such as endophthalmitis and the like, and cause irreversible damage to eyes. The microspheres can be prepared by various methods, such as solvent evaporation, co-emulsification, spray drying, ultrasonic-assisted atomization, and microfluidic methods. For antibody drugs having high water solubility, the most common method for encapsulating antibody drugs in microsphere formulations is the water-in-oil-in-water emulsion solvent evaporation method. However, the drug has large molecular weight and fragile structure, and the denaturation and aggregation of antibodies are easily caused by an organic phase/aqueous phase interface generated in the preparation process, so that the activity of the drug is reduced. And the medicament in the inner water phase is easy to diffuse into the outer water phase due to hydrophilicity, so that the medicament loading rate and the encapsulation efficiency are reduced.

Disclosure of Invention

The invention aims to provide a microsphere which is injected in a vitreous body and has double protection to antibody drugs and a preparation method thereof, in particular to a preparation method of a sustained release microsphere which is loaded with monoclonal antibodies and has double protection and is used for treating age-related macular degeneration. The method comprises the steps of firstly preparing dextran-protected monoclonal antibody drug particles by a water phase-water phase emulsification method, then preparing sustained-release microspheres wrapping and carrying the dextran-monoclonal antibody drug particles by an emulsification solvent volatilization method of water phase oil phase-in-water phase solid phase (S/O/W) by taking biodegradable material polylactic acid-glycolic acid copolymer and polyketal with degradation products as neutral as carrier materials.

The invention relates to a preparation method of a slow release microsphere for intravitreal injection and double protection of antibody drugs, which comprises the following steps:

1) weighing 450-1500mg dextran and 6000mg PEG, dissolving in 60mL deionized water, adding 150mg monoclonal antibody drug, performing vortex treatment for 5min, and freeze-drying. Centrifugally washing the freeze-dried powder with dichloromethane for 3 times to remove PEG dispersed phase, and vacuum drying to obtain monoclonal antibody-dextran microparticles;

2) weighing 100mg of polylactic acid-glycolic acid copolymer and polyketone, dissolving in 2mL of dichloromethane, adding 20-50mg of particles prepared in the step 1), performing ultrasonic dispersion at 0-4 ℃ to prepare an S/O emulsion, supplementing 10-20 mL of 1.0-2.0% PVA aqueous solution, homogenizing at room temperature, volatilizing for 3h to remove an organic phase, centrifuging to collect microspheres, and performing freeze drying to obtain the sustained-release microsphere drug delivery system loaded with the monoclonal antibody drug.

The monoclonal antibody of the invention is selected from: bevacizumab or ranibizumab or aflibercept or combaiccept.

The polylactic acid-glycolic acid copolymer is carboxyl-terminated and has the model of PLGA 75254A or PLGA 75255A.

The polyketals of the invention include PCADK or PK 3.

The mass ratio of the polylactic acid-glycolic acid copolymer to the polyketone is 8:2-5: 5.

The invention has the positive effects that:

1) according to the invention, an S/O/W emulsion solvent volatilization method and polyketal with neutral degradation products are used as carrier materials, so that the denaturation and aggregation of antibodies caused by an organic phase/water phase interface in a preparation process and an acidic microenvironment of polylactic acid-glycolic acid in a degradation process can be improved, the drug loading capacity of microspheres is improved, the burst release phenomenon in the release process of the microspheres is reduced, and the double protection of monoclonal antibody drugs in the microspheres is realized;

2) the microspheres prepared by the invention are used for vitreous injection administration, the addition of the polyketal can not only protect monoclonal antibody medicaments from degradation and improve the drug-loading rate of the microspheres, but also reduce the stimulation of acidic degradation products of polylactic acid-glycolic acid to the ocular environment, thereby greatly reducing the risk of side effects such as endophthalmitis and the like, the microspheres can slowly release monoclonal antibodies for not less than 28 days in an in vitro environment, and can reduce administration frequency for not less than 2 months in eyes, thereby relieving the pain and economic burden of patients, improving the compliance of patients and having good application prospect.

Drawings

FIG. 1 is an SEM image of particles and microspheres of the present invention;

figure 2 is an in vitro release profile of the microspheres of the invention;

FIG. 3 is an in vivo pharmacokinetics of the microspheres of the present invention;

FIG. 4 illustrates the biocompatibility of the microspheres of the present invention;

FIG. 5 is a graph showing the in vitro release profile of the aflibercept loaded microspheres prepared by the method of the present invention;

FIG. 6 shows that the conventional administration form is a direct injection of an aqueous solution of aflibercept;

FIG. 7 is an in vitro release profile of ranibizumab-loaded microspheres prepared by the methods of the invention;

FIG. 8 shows a conventional administration form of direct injection of aqueous ranibizumab solution;

FIG. 9 is a graph showing the in vitro release profile of microspheres loaded with combretastatin-cypress prepared by the method of the present invention;

FIG. 10 shows that the conventional administration form is direct injection of Corbina occipital aqueous solution.

Detailed description of the preferred embodiments

The invention will be described in detail with reference to the following examples, but the scope of the invention is not limited thereto:

example 1

Note: the experimental group Bev-PPsm is PLGA/PCADK microspheres loaded with bevacizumab prepared by S/O/W method; the control group Bev-PPwm is PLGA/PCADK microspheres loaded with bevacizumab prepared by W/O/W method; the control group Bev-Pwm is PLGA microspheres loaded with bevacizumab prepared by W/O/W method;

Bev-PPsm prescription

And (3) S phase: bevacizumab-dextran microparticles (1: 3), 50 mg;

and (3) phase O: PLGA 75254A, 80 mg; PCADK, 20 mg; dichloromethane, 2 mL;

phase W: 1.0% PVA aqueous solution, 10 mL;

Bev-PPsm preparation process

1) Weighing bevacizumab, dextran and PEG (1: 3: 40), dissolving in deionized water, performing vortex treatment, and freeze-drying; centrifugally washing the freeze-dried powder with dichloromethane to remove PEG dispersed phase, and vacuum drying to obtain bevacizumab-dextran microparticles;

2) dissolving PLGA and PCADK in dichloromethane, adding the prepared particles, preparing an O/W emulsion by ultrasonic dispersion at 0-4 ℃, supplementing a PVA aqueous solution, stirring at low speed at room temperature, volatilizing to remove an organic phase, centrifugally collecting microspheres, and freeze-drying to obtain a bevacizumab-loaded sustained-release microsphere drug delivery system;

Bev-PPwm prescription

Phase W: 25 mg/mL bevacizumab aqueous solution, 500. mu.L;

and (3) phase O: PLGA 75254A, 80 mg; PCADK, 20 mg; dichloromethane, 2 mL;

phase W: 1.0% PVA aqueous solution, 10 mL;

Bev-PPwm preparation process:

dissolving PLGA and PCADK in dichloromethane, adding a bevacizumab aqueous solution, performing ultrasonic dispersion at 0-4 ℃ to prepare an O/W emulsion, supplementing a PVA aqueous solution, stirring at low speed at room temperature, volatilizing to remove an organic phase, centrifuging to collect microspheres, and performing freeze drying to obtain a bevacizumab-loaded sustained-release microsphere drug delivery system;

Bev-Pwm prescription

Phase W: 25 mg/mL bevacizumab aqueous solution, 500. mu.L;

and (3) phase O: PLGA 75254A, 100 mg; dichloromethane, 2 mL;

phase W: 1.0% PVA aqueous solution, 10 mL;

Bev-Pwm preparation process:

dissolving PLGA in dichloromethane, adding a bevacizumab aqueous solution, performing ultrasonic dispersion at 0-4 ℃ to prepare an O/W emulsion, supplementing a PVA aqueous solution, stirring at low speed at room temperature, volatilizing to remove an organic phase, centrifuging to collect microspheres, and performing freeze drying to obtain the bevacizumab-loaded sustained-release microsphere drug delivery system.

Example 2

Note: the experimental group Afl-PPsm is PLGA/PCADK microspheres loaded with Abirascip prepared by an S/O/W method; the Afl-PPwm of the control group is PLGA/PCADK microspheres loaded with aflibercept prepared by a W/O/W method; the control group Afl-Pwm is PLGA microspheres which are prepared by a W/O/W method and are loaded with aflibercept;

Afl-PPsm prescription

And (3) S phase: aflibercept-dextran microparticles (1: 4), 40 mg;

and (3) phase O: PLGA 75254A, 70 mg; PCADK, 30 mg; dichloromethane, 2 mL;

phase W: 2.0% PVA aqueous solution, 15 mL;

Afl-PPsm preparation process

1) Weighing aflibercept, dextran and PEG (1: 4: 40) and dissolving in deionized water, performing vortex treatment and freeze-drying; centrifugally washing the freeze-dried powder with dichloromethane to remove PEG dispersed phase, and vacuum drying to obtain aflibercept-dextran microparticles;

2) dissolving PLGA and PCADK in dichloromethane, adding the prepared particles, preparing an O/W emulsion by ultrasonic dispersion at 0-4 ℃, supplementing a PVA aqueous solution, stirring at low speed at room temperature, volatilizing to remove an organic phase, centrifugally collecting microspheres, and freeze-drying to obtain the sustained-release microsphere drug delivery system carrying the aflibercept;

Afl-PPwm prescription

Phase W: 2 μ L of 25 mg/mL aflibercept in water;

and (3) phase O: PLGA 75254A, 70 mg; PCADK, 30 mg; dichloromethane, 2 mL;

phase W: 2.0% PVA aqueous solution, 15 mL;

the preparation process of Afl-PPwm:

dissolving PLGA and PCADK in dichloromethane, adding a bevacizumab aqueous solution, performing ultrasonic dispersion at 0-4 ℃ to prepare an O/W emulsion, supplementing a PVA aqueous solution, stirring at low speed at room temperature, volatilizing to remove an organic phase, centrifuging to collect microspheres, and performing freeze drying to obtain the sustained-release microsphere drug delivery system carrying the aflibercept;

Afl-Pwm prescription

Phase W: 2 μ L of 25 mg/mL aflibercept in water;

and (3) phase O: PLGA 75254A, 100 mg; dichloromethane, 2 mL;

phase W: 2.0% PVA aqueous solution, 15 mL;

the preparation process of Afl-Pwm:

dissolving PLGA in dichloromethane, adding an aflibercept aqueous solution, performing ultrasonic dispersion at 0-4 ℃ to prepare an O/W emulsion, supplementing a PVA aqueous solution, stirring at low speed at room temperature, volatilizing to remove an organic phase, centrifuging to collect microspheres, and performing freeze drying to obtain the aflibercept-loaded sustained-release microsphere drug delivery system.

Example 3

Note: the experiment group Luc-PPsm is PLGA/PCADK microspheres loaded with ranibizumab and prepared by an S/O/W method; the control group Luc-PPwm is PLGA/PCADK microspheres which are prepared by a W/O/W method and are loaded with the ranibizumab; the control group Luc-Pwm is PLGA microspheres which are prepared by a W/O/W method and are loaded with ranibizumab;

Luc-PPsm prescription

And (3) S phase: ranibizumab-dextran microparticles (1: 5), 48 mg;

and (3) phase O: PLGA 75255 a, 80 mg; PCADK, 20 mg; dichloromethane, 2 mL;

phase W: 1.0% PVA aqueous solution, 20 mL;

Luc-PPsm preparation process

1) Weighing ranibizumab, dextran and PEG (1: 5: 40) and dissolving in deionized water, performing vortex treatment and freeze drying; centrifugally washing the freeze-dried powder with dichloromethane to remove PEG dispersed phase, and vacuum drying to obtain ranibizumab-dextran microparticles;

2) dissolving PLGA and PCADK in dichloromethane, adding the prepared particles, preparing an O/W emulsion by ultrasonic dispersion at 0-4 ℃, supplementing a PVA aqueous solution, stirring at low speed at room temperature, volatilizing to remove an organic phase, centrifugally collecting microspheres, and freeze-drying to obtain a sustained-release microsphere drug delivery system carrying the ranibizumab;

Luc-PPwm prescription

Phase W: 25 mg/mL ranibizumab aqueous solution, 320 uL;

and (3) phase O: PLGA 75255 a, 80 mg; PCADK, 20 mg; dichloromethane, 2 mL;

phase W: 1.0% PVA aqueous solution, 20 mL;

the Luc-PPwm preparation process comprises the following steps:

dissolving PLGA and PCADK in dichloromethane, adding a ranibizumab aqueous solution, performing ultrasonic dispersion at 0-4 ℃ to prepare an O/W emulsion, supplementing a PVA aqueous solution, stirring at low speed at room temperature, volatilizing to remove an organic phase, centrifuging, collecting microspheres, and performing freeze drying to obtain a sustained-release microsphere drug delivery system loaded with the ranibizumab;

Luc-Pwm prescription

Phase W: 25 mg/mL ranibizumab aqueous solution, 320 uL;

and (3) phase O: PLGA 75255 a, 100 mg; dichloromethane, 2 mL;

phase W: 1.0% PVA aqueous solution, 20 mL;

the Luc-Pwm preparation process comprises the following steps:

dissolving PLGA in dichloromethane, adding a ranibizumab aqueous solution, preparing an O/W emulsion by ultrasonic dispersion at 0-4 ℃, supplementing a PVA aqueous solution, stirring at low speed at room temperature, volatilizing to remove an organic phase, centrifugally collecting microspheres, and freeze-drying to obtain the sustained-release microsphere drug delivery system loaded with the ranibizumab.

Example 4

Note: the experimental group Con-PPsm is PLGA/PK 3 microspheres loaded with Corbeset cypress prepared by an S/O/W method; the control group Con-PPwm is PLGA/PK 3 microspheres loaded with Corbeset cypress prepared by the W/O/W method; the control group Con-Pwm is PLGA microspheres loaded with Corbina cypress Western prepared by the W/O/W method;

Con-PPsm prescription

And (3) S phase: combavacypress cypress-dextran microparticles (1: 6), 42 mg;

and (3) phase O: PLGA 75255 a, 60 mg; PK3, 40 mg; dichloromethane, 2 mL;

phase W: 10 mL of 2.0% PVA aqueous solution;

Con-PPsm preparation process

1) Weighing combretastatin, dextran and PEG (1: 6: 40) and dissolving in deionized water, performing vortex treatment and freeze drying; centrifugally washing the freeze-dried powder with dichloromethane to remove PEG dispersed phase, and vacuum drying to obtain Corbina cypress-dextran microparticles;

2) dissolving PLGA and PK3 in dichloromethane, adding the prepared particles, preparing an O/W emulsion by ultrasonic dispersion at 0-4 ℃, supplementing a PVA aqueous solution, stirring at low speed at room temperature, volatilizing to remove an organic phase, centrifuging to collect microspheres, and freeze-drying to obtain a sustained-release microsphere drug delivery system loaded with combretacept;

Con-PPwm prescription

Phase W: 25 mg/mL bevacizumab aqueous solution, 240. mu.L;

and (3) phase O: PLGA 75255 a, 60 mg; PK3, 40 mg; dichloromethane, 2 mL;

phase W: 10 mL of 2.0% PVA aqueous solution;

the Con-PPwm preparation process comprises the following steps:

dissolving PLGA and PK3 in dichloromethane, adding a Corbinapacap aqueous solution, preparing an O/W emulsion by ultrasonic dispersion at 0-4 ℃, supplementing a PVA aqueous solution, stirring at low speed at room temperature, volatilizing to remove an organic phase, centrifuging to collect microspheres, and freeze-drying to obtain a sustained-release microsphere drug delivery system loaded with the Corbinapacap;

Con-Pwm prescription

Phase W: 25 mg/mL combavancil in water, 240 μ L;

and (3) phase O: PLGA 75255 a, 100 mg; dichloromethane, 2 mL;

phase W: 10 mL of 2.0% PVA aqueous solution;

the Con-Pwm preparation process comprises the following steps:

dissolving PLGA in dichloromethane, adding a Corbina cypress aqueous solution, preparing an O/W emulsion by ultrasonic dispersion at 0-4 ℃, supplementing a PVA aqueous solution, stirring at low speed at room temperature, volatilizing to remove an organic phase, centrifuging to collect microspheres, and freeze-drying to obtain the slow-release microsphere drug delivery system loaded with the Corbina cypress.

The positive effects of the invention are further demonstrated by the following tests:

test example 1

And (3) determining the drug loading and encapsulation efficiency of the microspheres:

20mg of the microspheres prepared in example 1 were placed in a test tube, 1ml of dichloromethane was added, 1ml of PBS was added after the macromolecule was completely dissolved, vortex shaking and centrifugation were performed, and PBS containing the monoclonal antibody drug was collected and analyzed by High Performance Liquid Chromatography (HPLC). Drug loading was 100/microsphere weight of the actual drug loaded encapsulated. Encapsulation efficiency is the experimental determination of drug loading 100/theoretical drug loading;

the drug loading and encapsulation efficiencies are shown in Table 1 below, where the encapsulation efficiency of Bev-PPsm is about 23%, Bev-PPwm is about 11%, and Bev-Pwm is only about 7%. This shows that the addition of polyketal can effectively maintain the stability of the drug due to its neutral degradation product, thereby significantly improving the drug loading, while the preparation of S/O/W of this experiment is higher and further has higher encapsulation efficiency and drug loading; the medicine carrying amount is obviously improved, so that the injection amount can be reduced, the pain of a patient is relieved, and the compliance of the patient is enhanced;

TABLE 1 Bev microsphere drug loading and encapsulation efficiency

	Drug loading (%)	Encapsulation efficiency (%)
			Bev-PPsm	1.98±0.02	23.86±0.17
Bev-PPwm	1.24±0.05	11.16±0.33
			Bev-Pwm	0.80±0.03	7.27±0.18

Test example 2

Microsphere morphology experiments:

the particle size and surface morphology of the bevacizumab-dextran microparticles and microspheres in example 1 were observed using a Scanning Electron Microscope (SEM). FIG. 1d shows Bev-Pwm microsphere as a control, which has a less uniform particle size and a smooth and round surface; FIG. 1c shows Bev-PPwm microsphere of control group, which has particle size below 20 μm and a few voids on the surface, because the mechanical strength of polyketal is poor and the microsphere morphology is greatly influenced by the diffusion of internal and external aqueous phases during the preparation process. FIG. 1b shows Bev-PPsm microsphere of the present invention, which has particle size below 20 μm, size suitable for intraocular injection, rounded spherical shape, smooth surface and no pore.

And (4) conclusion: the reason is that the preparation method of S/O/W avoids the diffusion phenomenon of internal and external water phases, so that the microsphere form is not influenced even if the polyketal is added, and the polyketal has a protective effect on antibody drugs in subsequent experiments. In FIG. 1a, the particle size of the bevacizumab-dextran microparticles is about 0.5-2 μm, and the bevacizumab-dextran microparticles are suitable for being microencapsulated by microspheres to obtain Bev-PPsm microspheres.

Test example 3

Microsphere in vitro release experiments:

30mg of the microspheres prepared in example 1 were put in a test tube, and 2ml of PBS (pH7.4) was added thereto. This mixture was shaken at a constant temperature of 100rpm at 37 ℃. At intervals, 100. mu.1 of the supernatant was collected by centrifugation and analyzed by High Performance Liquid Chromatography (HPLC). The tubes were simultaneously replenished with 100. mu.1 fresh PBS to keep the total volume constant. The microspheres were then resuspended. The suspension is again brought to 37 ℃ with constant shaking at 100 rpm. FIG. 2 shows the in vitro release curve of the microspheres loaded with bevacizumab prepared by the method of the present invention, Bev-PPsm microspheres can release not less than 28 days in vitro, the cumulative release amount exceeds 70%, the cumulative release amount far exceeds less than 60% of the drug in Bev-PPwm microspheres and Bev-Pwm microspheres, and the burst release amount of the drug in Bev-PPsm microspheres for 24h is 17%, and is far less than the burst release of Bev-PPwm microspheres and Bev-Pwm microspheres. The microsphere prepared by the method has small burst release and slower and more stable release behavior. The Bev-PPsm microsphere prepared by the method is milder in preparation method, the neutral degradation product of the polyketal can well protect the antibody, and the damage to the structural stability of the antibody is small, so that the drug release is more complete. The microspheres prepared by the method can be slowly released for not less than 28 days in vitro, and the medicament can be slowly released for a longer time due to the special composition of the vitreous humor in the eyes.

Test example 4

In-microsphere pharmacokinetic experiments:

1.5kg of male New Zealand white rabbits were subjected to intravitreal injection of the microspheres of example 1 and a vitreous injection of bevacizumab solution as a control group, 5 rabbits per group were sacrificed at specific time points (1 d, 4d, 7d, 14d, 28d, 42d, 56 d), the eyeballs were extracted, the vitreous humor was collected and the bevacizumab content was quantitatively measured using ELISA method. The results are shown in fig. 3, the conventional administration form is direct injection of bevacizumab aqueous solution, and the experimental results show that bevacizumab has almost completely disappeared at 14d in the vitreous humor, and bevacizumab in Bev-PPsm microspheres prepared according to the invention in example 1 and Bev-PPwm microspheres and Bev-Pwm microspheres can stay in the eyes for 56d, but the microsphere eyes prepared by the method of the invention have higher bevacizumab content and have significant difference.

Test example 5

Microsphere biocompatibility experiment:

1.5kg of male New Zealand white rabbits were injected with the left eye vitreous body of the rabbits, and a control group without drug administration, a normal saline group, and a microsphere group in example 1 were set, each group was parallel to 5 rabbits, the rabbits were sacrificed at 3d, the eyeballs were extracted, fixed with 10% paraformaldehyde, paraffin-embedded, sectioned at the retinal location, and observed with a microscope. As shown in FIG. 4, the solutions, Bev-PPsm microspheres and Bev-PPwm microspheres, were observed in rabbit eyes without inflammation, pus, etc. According to the HE dyeing result, only Bev-Pwm microsphere group has a plurality of inflammatory cells, and the result shows that acidic degradation products of polylactic acid-glycolic acid can stimulate eye cells to generate inflammatory reaction, and after polyketal is added into the microsphere carrier, the stimulation of microsphere materials to eyes is avoided, so that the pain of patients can be relieved to a great extent, and the generation of side effects is avoided.

Test example 6

20mg of the microspheres prepared in example 2 were placed in a test tube, 1mL of dichloromethane was added, 1mL of PBS was added after the macromolecule was completely dissolved, the mixture was vortexed and centrifuged, and PBS containing the monoclonal antibody drug was collected and analyzed by High Performance Liquid Chromatography (HPLC). Drug loading was 100/microsphere weight of the actual drug loaded encapsulated. Encapsulation efficiency is the experimental determination of drug loading 100/theoretical drug loading;

the drug loading and encapsulation efficiency are shown in Table 2 below, and the encapsulation efficiency of Afl-PPsm is about 31%, that of Afl-PPwm is about 13%, and that of Afl-Pwm is only about 10%. This shows that the addition of polyketal can effectively maintain the stability of the drug due to its neutral degradation product, thereby significantly improving the drug loading, while the preparation of S/O/W of this experiment is higher and further has higher encapsulation efficiency and drug loading; the medicine carrying amount is obviously improved, so that the injection amount can be reduced, the pain of a patient is relieved, and the compliance of the patient is enhanced;

TABLE 2 Afl microsphere drug loading and encapsulation efficiency

	Drug loading (%)	Encapsulation efficiency (%)
			Afl-PPsm	1.78±0.04	31.17±0.07
Afl-PPwm	1.03±0.03	13.90±0.23
			Afl-Pwm	0.81±0.04	10.93±0.38

Test example 7

Microsphere in vitro release experiments:

30mg of the microspheres prepared in example 2 were put in a test tube, and 2ml of PBS (pH7.4) was added thereto. This mixture was shaken at a constant temperature of 100rpm at 37 ℃. At intervals, 100. mu.1 of the supernatant was collected by centrifugation and analyzed by High Performance Liquid Chromatography (HPLC). The tubes were simultaneously replenished with 100. mu.1 fresh PBS to keep the total volume constant. The microspheres were then resuspended. The suspension is again brought to 37 ℃ with constant shaking at 100 rpm. FIG. 5 shows the in vitro release curve of the microsphere loaded with aflibercept prepared by the method of the invention, the Afl-PPsm microsphere can be released in vitro for not less than 28 days, the cumulative release amount exceeds 75 percent, the cumulative release amount is far more than 56 percent of the drug in the Afl-PPwm microsphere and the Afl-Pwm microsphere, and the burst release amount of the drug in 24h of the Afl-PPsm microsphere is 17 percent and is far less than that of the Afl-PPwm microsphere and the Afl-Pwm microsphere. The microsphere prepared by the method has small burst release and slower and more stable release behavior. The Afl-PPsm microsphere prepared by the method is milder in preparation method, the neutral degradation product of the polyketal can well protect the antibody, and the damage to the structural stability of the antibody is small, so that the drug release is more complete. The microspheres prepared by the method can be slowly released for not less than 28 days in vitro, and the medicament can be slowly released for a longer time due to the special composition of the vitreous humor in the eyes.

Test example 8

In-microsphere pharmacokinetic experiments:

1.5kg of male New Zealand white rabbits were subjected to intravitreal injection of the microspheres prepared in example 2, 5 in parallel groups of vitreally injected Abirascip solution were used as control groups, and at specific time points (1 d, 4d, 7d, 14d, 28d, 42d, 56 d) rabbits were sacrificed, the eyeballs were removed, the vitreous humor was collected and the Abirascip content was quantitatively measured using ELISA. As shown in FIG. 6, the conventional administration form is direct injection of an aqueous solution of aflibercept, and the experimental results show that aflibercept has almost completely disappeared in vitreous humor at 14 days, Afl-PPsm microspheres prepared according to the present invention in example 1 and Afl-PPwm microspheres and Afl-Pwm microspheres in the control group can stay in the eye for 56 days, but the content of aflibercept in the microspheres prepared by the present invention is higher and has significant difference.

Test example 9

20mg of the microspheres prepared in example 3 were placed in a test tube, 1mL of dichloromethane was added, 1mL of PBS was added after the macromolecule was completely dissolved, the mixture was vortexed and centrifuged, and PBS containing the monoclonal antibody drug was collected and analyzed by High Performance Liquid Chromatography (HPLC). Drug loading was 100/microsphere weight of the actual drug loaded encapsulated. Encapsulation efficiency is the experimental determination of drug loading 100/theoretical drug loading;

the drug loading and encapsulation efficiency are shown in Table 3 below, where the encapsulation efficiency of Luc-PPsm is about 33%, the Luc-PPwm is about 16%, and the Luc-Pwm is only about 10%. This shows that the addition of polyketal can effectively maintain the stability of the drug due to its neutral degradation product, thereby significantly improving the drug loading, while the preparation of S/O/W of this experiment is higher and further has higher encapsulation efficiency and drug loading; the medicine carrying amount is obviously improved, so that the injection amount can be reduced, the pain of a patient is relieved, and the compliance of the patient is enhanced;

TABLE 3 Luc microsphere drug loading and encapsulation efficiency

	Drug loading (%)	Encapsulation efficiency (%)
			Luc-PPsm	1.83±0.02	33.83±0.17
Luc-PPwm	1.21±0.03	16.33±0.32
			Luc-Pwm	0.79±0.05	10.66±0.21

Test example 10

Microsphere in vitro release experiments:

30mg of the microspheres prepared in example 3 were put in a test tube, and 2ml of PBS (pH7.4) was added thereto. This mixture was shaken at a constant temperature of 100rpm at 37 ℃. At intervals, 100. mu.1 of the supernatant was collected by centrifugation and analyzed by High Performance Liquid Chromatography (HPLC). The tubes were simultaneously replenished with 100. mu.1 fresh PBS to keep the total volume constant. The microspheres were then resuspended. The suspension is again brought to 37 ℃ with constant shaking at 100 rpm. FIG. 7 shows the in vitro release curve of the microsphere loaded with ranibizumab prepared by the method of the present invention, the Luc-PPsm microsphere can release in vitro for not less than 28 days, the cumulative release exceeds 77%, the cumulative release far exceeds the drug release of less than 57% in the Luc-PPwm microsphere and the Luc-Pwm microsphere, and the burst release of the drug in 24h in the Luc-PPsm microsphere is 18%, and the burst release is far less than that of the Luc-PPwm microsphere and the Luc-Pwm microsphere. The microsphere prepared by the method has small burst release and slower and more stable release behavior. The Luc-PPsm microsphere prepared by the method is milder in preparation method, the neutral degradation product of the polyketal can well protect the antibody, and the damage to the structural stability of the antibody is small, so that the drug release is more complete. The microspheres prepared by the method can be slowly released for not less than 28 days in vitro, and the medicament can be slowly released for a longer time due to the special composition of the vitreous humor in the eyes.

Test example 11

In-microsphere pharmacokinetic experiments:

1.5kg of male New Zealand white rabbits were subjected to intravitreal injection of the microspheres prepared in example 3, 5 parallel rabbits per group were subjected to intravitreal injection of ranibizumab solution as a control group, and the rabbits were sacrificed at specific time points (1 d, 4d, 7d, 14d, 28d, 42d, 56 d), the eyeballs were removed, the vitreous humor was collected, and the ranibizumab content was quantitatively measured using ELISA. The results are shown in fig. 8, the conventional administration form is direct injection of ranibizumab aqueous solution, and the experimental results show that ranibizumab has almost completely disappeared in vitreous humor at 14d, and ranibizumab in the Luc-PPsm microspheres prepared according to the invention example 1 and the Luc-PPwm microspheres and the Luc-Pwm microspheres of the control group can stay in the eyes for 56d, but the content of ranibizumab in the microspheres prepared by the method of the invention is higher and has significant difference.

Test example 12

20mg of the microspheres prepared in example 4 were placed in a test tube, 1mL of dichloromethane was added, 1mL of PBS was added after the macromolecule was completely dissolved, the mixture was vortexed and centrifuged, and PBS containing the monoclonal antibody drug was collected and analyzed by High Performance Liquid Chromatography (HPLC). Drug loading was 100/microsphere weight of the actual drug loaded encapsulated. Encapsulation efficiency is the experimental determination of drug loading 100/theoretical drug loading;

the drug loading and encapsulation efficiencies are shown in Table 4 below, with the Con-PPsm encapsulation efficiency being about 38%, the Con-PPwm being about 16%, and the Con-Pwm being only about 9%. This shows that the addition of polyketal can effectively maintain the stability of the drug due to its neutral degradation product, thereby significantly improving the drug loading, while the preparation of S/O/W of this experiment is higher and further has higher encapsulation efficiency and drug loading; the medicine carrying amount is obviously improved, so that the injection amount can be reduced, the pain of a patient is relieved, and the compliance of the patient is enhanced;

TABLE 4 Con microsphere drug loading and encapsulation efficiency

	Drug loading (%)	Encapsulation efficiency (%)
			Con-PPsm	1.64±0.06	38.77±0.13
Con-PPwm	0.93±0.08	16.43±0.34
			Con-Pwm	0.51±0.03	9.01±0.26

Test example 13

Microsphere in vitro release experiments:

30mg of the microspheres prepared in example 4 were put in a test tube, and 2ml of PBS (pH7.4) was added thereto. This mixture was shaken at a constant temperature of 100rpm at 37 ℃. At intervals, 100. mu.1 of the supernatant was collected by centrifugation and analyzed by High Performance Liquid Chromatography (HPLC). The tubes were simultaneously replenished with 100. mu.1 fresh PBS to keep the total volume constant. The microspheres were then resuspended. The suspension is again brought to 37 ℃ with constant shaking at 100 rpm. FIG. 9 shows the in vitro release profile of the microspheres loaded with combretastatin prepared by the method of the present invention, Con-PPsm microspheres can release not less than 28 days in vitro, the cumulative release exceeds 77%, which is far more than 60% of the cumulative release of the drugs in Con-PPwm microspheres and Con-Pwm microspheres, and the burst release of the drugs in Con-PPsm microspheres for 24h is 19%, which is far less than the burst release of Con-PPwm microspheres and Con-Pwm microspheres. The microsphere prepared by the method has small burst release and slower and more stable release behavior. The Con-PPsm microspheres prepared by the method are milder in preparation method, neutral degradation products of the polyketal can well protect the antibody, and damage to the structural stability of the antibody is small, so that the drug release is more complete. The microspheres prepared by the method can be slowly released for not less than 28 days in vitro, and the medicament can be slowly released for a longer time due to the special composition of the vitreous humor in the eyes.

Test example 14

In-microsphere pharmacokinetic experiments:

1.5kg of male New Zealand white rabbits were subjected to intravitreal injection of the microspheres prepared in example 4, and 5 bevacizumab solutions were intravitreally injected into the left eye of the rabbits as a control group, and the rabbits were sacrificed at specific time points (1 d, 4d, 7d, 14d, 28d, 42d, 56 d), the eyeballs were extracted, and the vitreous humor was collected and the content of Corbinacept was quantitatively measured using ELISA. As shown in FIG. 10, the conventional administration form is the direct injection of the aqueous solution of Corbinapact, and the experimental results show that the Corbinapact has almost completely disappeared in the vitreous humor at 7d, and the Con-PPsm microspheres prepared according to the present invention in example 1 and the Con-PPwm microspheres in the control group and the Con-Pwm microspheres can stay in the eyes for 56d, but the content of the Corbinapact in the microspheres prepared by the present invention is higher and has significant difference.

Although specific embodiments of the present invention have been described above, it will be understood by those skilled in the art that this is by way of illustration only, the scope of the present invention being defined by the appended claims, and that various changes or modifications may be made to these embodiments by those skilled in the art without departing from the principle and spirit of the present invention, and these changes and modifications are within the scope of the present invention.

Claims

1. A preparation method of microspheres for intravitreal injection and double protection of antibody drugs comprises the following steps:

1) weighing 450-1500mg dextran and 6000-mgPEG, dissolving in 60mL deionized water, adding 150mg monoclonal antibody drug, performing vortex treatment for 5min, and freeze-drying; centrifugally washing the freeze-dried powder with dichloromethane for 3 times to remove PEG dispersed phase, and vacuum drying to obtain monoclonal antibody-dextran microparticles;

2) weighing 100mg of polylactic acid-glycolic acid copolymer and polyketone, dissolving in 2mL of dichloromethane, adding 20-50mg of particles prepared in the step 1), preparing an S/O emulsion by ultrasonic dispersion at 0-4 ℃, supplementing 10-20 mL of 1.0-2.0% PVA aqueous solution, homogenizing at room temperature, volatilizing for 3h to remove an organic phase, centrifuging to collect microspheres, and freeze-drying to obtain the polylactic acid-glycolic acid copolymer/polyketone microsphere;

the monoclonal antibody is selected from: bevacizumab or ranibizumab, aflibercept, combivicept;

the polylactic acid-glycolic acid copolymer is carboxyl-terminated, and the model is PLGA 75254A or PLGA 75255A;

polyketals include PCADK or PK 3;