TWI457146B - Sustained release systems and preparation method thereof - Google Patents

Description

Drug sustained release system and preparation method thereof

The present invention relates to a drug delivery system, and more particularly to a drug delivery system for coating a hydrophilic drug with β-tricalcium phosphate (β-TCP) on the surface of a biopolymer matrix. The invention also provides a method of preparing a drug delivery system comprising providing a biopolymer matrix coated with a hydrophilic drug and beta-tricalcium phosphate (beta-TCP).

Currently drug delivery systems have been widely used in many places, for example, surgical transplantation, tissue regeneration or dressing of affected areas. In general, a drug carrying system allows a drug or biologically active substance to treat a disease in a particular area. For example, a drug-carrying system can provide antibiotics directly in a certain area to avoid infection at a site, or can be combined with tissue regeneration engineering to provide necessary growth factors.

Therefore, there is a need to develop a safe and good drug carrying system. For example, it produces maximum drug activity with minimal side effects, a stable release rate, and a non-toxic biodegradable material. Biodegradable materials have been widely used in drug delivery systems and are mainly used in situations where continuous administration is required, usually the compound, peptide or protein.

Aliphatic polyesters, for example, poly(lactic acid), PLA, poly(glycolic acid, PGA), poly Lactic-co-Glycolic Acid (PLGA) Or polyanhydride (poly(carprolactone)) and the like are commonly used biodegradable materials. It can be shaped into various shapes, for example, strips, fibers, gels or microspheres, and its shape also affects the physical properties of the drug in muscle or subcutaneous injection. Among them, microspheres are often used, and the release rate of the drug can be easily controlled by the microspheres, and the particle size is about 0.1 to 500 micrometers, which can be directly injected into the living body. Therefore, the current development has a uniform small size and high coating. Rate of particles.

Typical microparticle production methods are emulsion solvent evaporation, phase separation, spray-drying or solvent extraction, atomization-freeze, Salting out, nano-precipitation. The emulsification solvent evapotranspiration is exemplified by dissolving a lipophilic polymer in a water-immiscible organic solvent such as dichloromethane, chloroform, ethyl acetate or the like. Dissolving and suspending the oil-soluble drug in the polymer solution, adding the polymer solution to the aqueous solution, and removing the solvent to obtain a particulate drug release system, but the method is not suitable for water solubility drug.

Another w/o/w secondary emulsification method is applicable to water-soluble drugs. In the method, a biodegradable material is dissolved in a water-insoluble organic solvent to produce a polymer solution, and the first water-soluble drug solution is emulsified in the polymer solution to obtain a w/o emulsion. Then, the emulsion and the second water-soluble solution are emulsified again to obtain a w/o/w secondary emulsification system, and after the solvent is removed, a particulate drug release system coated with the water-soluble drug can be obtained.

In addition, an emulsified form of solid/oil/water (s/o/w) has also been developed, which mainly consists of lyophilization of proteins and other drugs to form a solid, which is then packaged in solid/organic/aqueous form. Coverage, because the protein drug exists in the solid solution in the organic solution, and the freeze-drying procedure is first performed, and the loss of protein drug activity is easily caused in the unprotected condition. Another problem is that the composite solid is not easily dispersed uniformly. An emulsified organic state.

Therefore, there is currently no method or composition that can effectively carry and protect sensitive drugs, particularly peptides, proteins and nucleic acids of hydrophilic drugs. Moreover, when the biodegradable material used is hydrolyzed in a living body, it may cause a decrease in pH in the living body and affect cell growth. In view of this, it is necessary to develop a release system that is stable in pH, can effectively carry, protect, and release controlled release drugs.

Calcium hydrogen phosphate ceramics such as hydroxyapatite (HA-p) and β-tricalcium phosphate are used as bone substitute materials in repairing bone damage. It is worth noting that the high bone conduction effect, simple operation and non-tissue toxicity of β-tricalcium phosphate make it widely used in clinical orthopedic surgery. U.S. Patent No. 6,344,209 discloses the solid component of a biodegradable polymer comprising a pharmaceutical substance and an apatite coating. It also discloses a method of preparing the solid component by immersing a substance comprising a pharmaceutically acceptable substance and apatite-coated biodegradable polymer into a water ion solution capable of forming an apatite A solid component is prepared. U.S. Patent Application No. 2009/0087472 is directed to a drug delivery system for biopharmaceutical growth factors for use in a bioabsorbable material of a bone graft apatite coating.

The present invention relates to a drug delivery system comprising a biopolymer matrix coated with a mixture of a hydrophilic drug and β-tricalcium phosphate (β-TCP) on the surface. The hydrophilic drug is a small molecule, a protein, a nucleic acid, an antibiotic or a growth factor.

The surface of the biopolymer matrix of the drug sustained release system of the present invention is phospholipid, lecithin, poly(lactic acid), PLA, poly(glycolic acid, PGA), polylactic acid-gan Poly Lactic-co-Glycolic Acid (PLGA), polyglutamic acid (PGA), polycaprolactone (PCL), polyanhydrides, polyamines Polyamino acid, polydioxanone, polyhydroxybutyrate, polyphosphazenes, polyesterurethane, polycarboxyphenoxypropane-co-sebacic acid (polycarbosyphenoxypropane-cosebacic acid) or polyorthoester (polyorthoester) and mixtures thereof.

The surface of the biopolymer matrix of the drug sustained release system of the present invention is a microparticle sponge, fiber or irregular shape. Wherein the surface of the biopolymer matrix has an average particle size of from about 0.1 to 500 microns in the form of a particulate sponge.

The invention also provides a method for preparing a drug sustained release system, comprising: providing a mixture comprising a hydrophilic drug and β-tricalcium phosphate (β-TCP) and a biopolymer matrix; coating the coating on the coating The surface of the biopolymer matrix. Wherein the hydrophilic drug is a small molecule, a protein, a nucleic acid, an antibiotic or a growth factor. The invention provides the preparation of fat, lecithin, poly(lactic acid), PLA, poly(glycolic acid, PGA), polylactic acid-glycolic acid copolymer (Poly Lactic-co- Glycolic Acid, PLGA), polyglutamic acid (PGA), polycaprolactone (PCL), polyanhydrides, polyamino acid, dioxane Polydioxanone, polyhydroxybutyrate, polyphosphazenes, polyesterurethane, polycarbosyphenoxypropane-cosebacic acid or polyorthoic acid Polyorthoester and mixtures thereof.

The surface of the biopolymer matrix of the preparation method of the present invention is a particulate sponge, fiber or irregular shape. Wherein the surface of the biopolymer matrix has an average particle size of from about 0.1 to 500 microns in the form of a particulate sponge.

The method for preparing a sustained release system of the present invention, wherein the surface of the biopolymer matrix is spray-drying or water-in-oil-in-water (w) /o/w), oil-in-water (o/w), solid-in-oil-in-water (s/o/w), electrospinning And prepared by lyophilization and drying. The method for preparing a water-in-oil-in-water (w/o/w), oil-in-water (o/w) type comprises an organic solvent. It is mixed with a second aqueous solution containing a hydrophilic surfactant. The organic solvent is dichloromethane, chloroform, ethyl acetate (EA), 1,4-dioxane, dimethylformamide (DMF), dimethyl sulfoxide (Dimethyl sulfoxide). , DMSO), toluene or tetrahydrofuran DMF, Dimethyl sulfoxide (DMSO), toluene or tetrahydrofuran (THF). Wherein the hydrophilic surfactant is polyvinyl alcohol (PVA), NP-5, Triton x-100, Tween 40, PEG 200, PEG 800, sodium dodecyl sulfate (SDS), alcohol ethoxylates (Alcohol ethoxylates) ), alkylphenol ethoxylates, secondary alcohol ethoxylates, fatty acid esters or alkyl polygylcosides.

The method for preparing a drug sustained release system according to the present invention further comprises coating an excipient on the surface of the biopolymer matrix. Wherein the excipient is dextrin, α,β-trehalose (α,β-Trehalose), D-(+)-trehalose (D-(+)-Trehalose), sucrose, glycerol, cyclodextrin Cyclodextrin, polyhydric alcohols, polyethylene glycol (PEG) or bovine serum albumin (BAS).

While the present invention has been described in its preferred embodiments, the present invention is not intended to limit the invention, and the present invention may be modified and modified without departing from the spirit and scope of the invention. The scope of protection is subject to the definition of the scope of the patent application.

Example 1 Preparation of a drug sustained release system (1) (secondary emulsification method / alkaline substance / hydrophilic drug)

8 mg of hydroxyapatite (HA-p) powder and 2,500 ng of recombinant human bone morphogenetic protein-2 (rhBMP-2) were added to 250 μl of phosphate solution ( The first aqueous solution (rhBMP-2/HAp/PBS) was formed by shaking in an oscillator for 5 minutes in PBS. 0.25 g of PLGA65/35 was dissolved in 2.5 ml of dichloromethane to become a 10% PLGA solution. The first aqueous solution (rhBMP-2/HAp/PBS) was mixed with a 10% PLGA solution and stirred at 1000 rpm for 15 minutes to form a first emulsion (w/o). The first emulsion was poured into 10 ml of a 0.1% (w/v) polyvinyl alcohol (PVA) second aqueous solution, and stirred at 500 rpm for 5 minutes to form a second emulsion (w/o/w). . After stirring for 4 hours, the second emulsion was allowed to stand for 1 minute and the supernatant liquid was taken. The supernatant was centrifuged at 3,000 rpm for 5 minutes, and then the lower layer was taken, and the original lower layer was washed with 10 ml of secondary water and centrifuged. After the lower layer of liquid for 1 minute. Repeated centrifugation and washing twice, collecting the lower layer and lyophilizing to form hydroxyapatite (HA-p) and recombinant human bone morphogenetic protein-2 (rhBMP-2). Poly Lactic-co-Glycolic Acid (PLGA) microparticles. The PLGA microparticle release curve of the coated HA-p and rhBMP-2 is shown in Figure 1 (Group A). The burst release of rhBMP-2 was effectively reduced by HA-p. The ELISA kit results showed that a very small amount of rhBMP-2 was completely embedded by PLGA microparticles and had a low release rate (1-2 ng/day) in less than 7 days. After 7 days, rhBMP-2 continued to be released.

Example 2

Preparation of drug delivery system (2) (secondary emulsification method / coating / hydrophilic drug)

250 microliters of phosphate solution (PBS) was shaken with an shaker for 5 minutes to form a first aqueous solution of PBS. 0.25 g of PLGA65/35 was dissolved in 2.5 ml of dichloromethane to become a 10% PLGA solution. The PBS first aqueous solution was mixed with a 10% PLGA solution and stirred at 1,000 rpm for 15 minutes to form a first emulsion (w/o). The first emulsion was poured into 10 ml of a 0.1% (w/v) polyvinyl alcohol (PVA) second aqueous solution, and stirred at 500 rpm for 5 minutes to form a second emulsion (w/o/w). After stirring for 4 hours, the second emulsion was allowed to stand for 1 minute and the supernatant liquid was taken. The supernatant liquid was centrifuged at 3000 rpm for 5 minutes, and then the lower layer liquid was taken, and the original lower layer liquid was washed with 10 ml of secondary water and centrifuged. The lower layer of liquid for 1 minute. The mixture was repeatedly centrifuged and washed twice, and the lower layer was collected and freeze-dried to form polylactic acid-co-glycolic acid (PLGA) microparticles. An aqueous solution containing 1,000 ng of rhBMP-2 was coated on the surface of the PLGA microparticles. The PLGA microparticle release curve coated with rhBMP-2 is shown in Figure 1 (Group B). The rhBMP-2 adsorbed on the surface of the PLGA microparticles showed sustained release by diffusion into an in vitro solution. The results of the ELISA kit showed that rhBMP-2 had a high release rate (10-18 ng/day) in the first 3 days and a stable release (1-3 ng/day) was achieved thereafter.

Example 3

Preparation of drug delivery system (3) (secondary emulsification method / coating / alkaline substance / hydrophilic drug)

250 microliters of phosphate solution (PBS) was shaken with an shaker for 5 minutes to form a first aqueous solution of PBS. 0.25 g of PLGA65/35 was dissolved in 2.5 ml of dichloromethane to become a 10% PLGA solution. The PBS first aqueous solution was mixed with a 10% PLGA solution and stirred at 1,000 rpm for 15 minutes to form a first emulsion (w/o). The first emulsion was poured into 10 ml of a 0.1% (w/v) polyvinyl alcohol second aqueous solution, and stirred at 500 rpm for 5 minutes to form a second emulsion (w/o/w). After stirring for 4 hours, the second emulsion was allowed to stand for 1 minute and the supernatant liquid was taken. The supernatant liquid was centrifuged at 3000 rpm for 5 minutes, and then the lower layer liquid was taken, and the original lower layer liquid was washed with 10 ml of secondary water and centrifuged. The lower layer of liquid for 1 minute. The mixture was repeatedly centrifuged and washed twice, and the lower layer was collected and lyophilized to form polylactic acid and polylactic acid copolymer (PLGA) microparticles. An aqueous solution containing 2 mg of HAp and 2,000 ng of rhBMP-2 was coated on the surface of the PLGA microparticles. The PLGA microparticle release curve of the coated HAp and rhBMP-2 is shown in Figure 1 (Group C). The rhBMP-2 adsorbed on the surface of the PLGA microparticles showed sustained release by diffusion into an in vitro solution and hydrolysis of PLGA. The ELISA kit showed that rhBMP-2 had a high release rate (20-50 ng/day) in the first 3 days and a stable release (1-3 ng/day) thereafter.

Example 4

Type and diameter analysis of drug delivery system (secondary emulsification method / alkaline substance)

An appropriate amount of PLGA 50/50 and 0.2% by weight of span 83 (Sorbitan sesquioleate sorbitan sesquioleate, a surfactant) were dissolved in dichloromethane to give a 10% oil solution. The PBS was mixed to form an aqueous solution. The aqueous solution was added to the oil solution and emulsified with a Vortex mixer to form an emulsion. After the dichloromethane is removed from the emulsion by an extraction method, PLGA microparticles can be obtained. 2A and 2B show the pattern and diameter distribution of PLGA microparticles under an electron microscope. The PLGA microparticles are spherical and have a diameter distribution from 20 to 70 nm.

Example 5 Preparation, Coating Efficiency and Diameter Analysis of Drug Delivery System (Single Emulsification Method)

0.5 ml of methanol was mixed with 84.2 mg of PLGA in 2 ml of dichloromethane, and shaken with an ultrasonic shaker for 10 minutes to form a single organic solvent (oil phase). 10 ml of a 0.1% (w/v) aqueous solution of polyvinyl alcohol (aqueous phase) was prepared. The oil phase was slowly dropped into an aqueous phase containing 0.1% polyvinyl alcohol deionized water solution in an ice bath and stirring at 800 rpm to form oil/water ratios of 1:5, 1:8 and 1:10, respectively. The emulsion. The emulsion was stirred at room temperature for 24 hours to evaporate the solvent therein, after which solidified PLGA microparticles were formed. The PLGA microparticle embedding efficiency was 97%. The PLGA microparticles show two distinct diameter distribution peaks ranging from 100 nm to 400 nm and from 900 nm to 2,000 nm. Figure 3 shows the morphology of PLGA microparticles under (A) optical microscopy and (B) fluorescence microscopy. Due to the light scattering of the lipophilic fluorescent drug, a green light source is used to obtain the best optical resolution. Table 1 shows various oil/water ratios of PLGA microparticles and their diameters. When the oil/water ratio is 1:5, the average diameter is 412 nm; when the oil/water ratio is 1:8, the average diameter is 379 nm; when the oil/water ratio is 1:10, the average diameter is 282 nm. .

Example 6 Preparation, Type and Diameter Analysis (Spray-drying/Basic Substance) of Drug Delivery System (6)

2 g of PLGA was dissolved in 20 ml of dichloromethane (DCM) to give a 10% solution of PLGA/DCM. The HAp powder was dispersed in water to form an aqueous solution. The aqueous solution was mixed with a PLGA/DCM/SIM (simvastatin) solution and stirred with a magnetic stirrer for 30 minutes to form an emulsion. The emulsion is placed in a pelletizer for granulation to form PLGA microparticles. Table 2 shows the conditions of the granulation process. 4A is a view showing a form of pure PLGA microparticles prepared by a spray drying method under an electron microscope according to an embodiment of the present invention; and FIG. 4B shows pure PLGA microparticles prepared by a spray drying method according to an embodiment of the present invention. The diameter distribution under an electron microscope. The PLGA microparticles are spherical and have a diameter distribution from 10 to 30 nm. However, dynamic light scattering (DLS) measurements indicate that the diameter distribution therein is from 10 to 100 nm due to the aggregation effect caused by spray granulation.

Example 7

Preparation of drug delivery system (2) (secondary emulsification method / coating / hydrophilic drug)

250 microliters of phosphate solution (PBS) was shaken with an shaker for 5 minutes to form a first aqueous solution of PBS. 0.25 g of PLGA65/35 was dissolved in 2.5 ml of dichloromethane to become a 10% PLGA solution. The PBS first aqueous solution was mixed with a 10% PLGA solution and stirred at 1,000 rpm for 15 minutes to form a first emulsion (w/o). The first emulsion was poured into 30 ml of a 0.1% (w/v) polyvinyl alcohol second aqueous solution, and stirred at 600 rpm for 5 minutes to form a second emulsion (w/o/w). After stirring for 4 hours, the second emulsion was allowed to stand for 1 minute and the supernatant liquid was taken. The supernatant liquid was centrifuged at 3000 rpm for 5 minutes, and then the lower layer liquid was taken, and the original lower layer liquid was washed with 10 ml of secondary water and centrifuged. The lower layer of liquid for 1 minute. The mixture was repeatedly centrifuged and washed twice, and the lower layer was collected and lyophilized to form polylactic acid and polylactic acid copolymer (PLGA) microparticles. Each of them is coated with an aqueous solution containing 1,000 ng of rhBMP-2 and 2 mg of HAp, 2 mg of β-tricalcium phosphate (β-TCP), 4 mg of β-TCP or 6 mg of β-TCP in PLGA microparticles. Subsurface. The PLGA microparticles are lyophilized to form the final product.

Figure 5 shows the release profile of rhBMP-2 activity measured by four samples (50 mg) in PBS by ELISA detection. 50 mg of the microspheres rhBMP-2 composition was added to 5 ml of PBS for release. HAp+rhBMP-2 or β-TCP+rhBMP-2 adsorbed on the surface of PLGA microparticles showed sustained release by diffusion into an in vitro solution.

The ELISA kit showed that 2 mg of HAp+rhBMP-2 had a high release rate (20-45 ng/day) in the first 5 days, and then reached a stable release condition (10-3 ng/day); 2 mg β-TCP+rhBMP-2 had a high release rate (20-35 ng/day) in the first 4 days, and then reached a stable release condition (7-1 ng/day); 4 mg β-TCP +rhBMP-2 had a high release rate (25-48 ng/day) in the first 2 days, and then reached a stable release condition (5-1 ng/day); 6 mg β-TCP+rhBMP-2 The first day had a high release rate (35 ng/day) and a stable release (15-1 ng/day) was achieved thereafter.

Figure 1 shows polylactic acid and polyglycolic acid copolymer (Poly) coated with hydroxyapatite (HA-p) and human recombinant human bone morphogenetic protein-2 (rhBMP-2). Lactic-co-Glycolic Acid (PLGA) microparticle release curve, coated with rhBMP-2 and HAp and rhBMP-2 according to an embodiment of the invention.

2A is a view showing the form of pure PLGA microparticles prepared by the w/o/w method under an electron microscope according to an embodiment of the present invention; and FIG. 2B showing the w/o/w according to an embodiment of the present invention. The diameter distribution of pure PLGA microparticles prepared by the method under an electron microscope.

Figure 3 shows the morphology of PLGA microparticles under (A) optical microscopy and (B) fluorescence microscopy.

4A is a view showing a form of pure PLGA microparticles prepared by a spray drying method under an electron microscope according to an embodiment of the present invention; and FIG. 4B is a view showing a pure PLGA prepared by a spray drying method according to an embodiment of the present invention. The diameter distribution of microparticles under an electron microscope.

Figure 5 shows the release profile of rhBMP-2 activity measured by four samples (50 mg) in PBS by ELISA detection. Sample 1: coated with 1000 ng of rhBMP-2 and 2 mg of HAp on PLGA microparticles; sample 2: coated with 1000 ng of rhBMP-2 and 2 mg of β-tricalcium phosphate (β-TCP) on PLGA microparticles; Sample 3: coated with 1000 ng of rhBMP-2 and 4 mg of β-TCP on PLGA microparticles; Sample 4: coated with 1000 ng of rhBMP-2 and 6 mg of β-TCP on PLGA microparticles.

Claims (14)

A drug sustained release system comprising a biopolymer matrix coated with a mixture of a hydrophilic drug and β-tricalcium phosphate (β-TCP) on a surface, wherein the biopolymer is a phospholipid, lecithin, polylactic acid ( Poly(lactic acid), PLA), poly(glycolic acid, PGA), poly Lactic-co-Glycolic Acid (PLGA), polyglutamic acid , (PGA)), polycaprolactone (PCL), polyanhydride, polyamino acid, polydioxanone, polyhydroxybutyrate, polyphosphorus Polyphosphazenes, polyester urethanes, polycarbosyphenoxypropane-cosebacic acid or polyorthoesters, and mixtures thereof. The drug sustained release system according to claim 1, wherein the hydrophilic drug is a small molecule, a protein, a nucleic acid, an antibiotic or a growth factor. The drug sustained release system according to claim 1, wherein the surface of the biopolymer matrix is a particulate sponge, a fiber or an irregular shape. The drug delivery system of claim 4, wherein the surface of the biopolymer matrix has an average particle size of from 0.1 to 500 microns in the form of a microparticle sponge. A method for preparing a drug delivery system comprising: providing a mixture comprising a hydrophilic drug and β-tricalcium phosphate (β-TCP) and a biopolymer matrix; coating the mixture on the biopolymer matrix Surface, wherein the biopolymer is a phospholipid, Lecithin, poly(lactic acid), PLA, poly(glycolic acid, PGA), poly Lactic-co-Glycolic Acid (PLGA), poly Polyglutamic acid (PGA), polycaprolactone (PCL), polyanhydride, polyamino acid, polydioxanone, polyhydroxybutyrate (polyhydroxybutyrate), polyphosphazenes, polyester urethane, polycarbosyphenoxypropane-cosebacic acid or polyorthoester and mixtures thereof. . The method of claim 6, wherein the hydrophilic drug is a small molecule, a protein, a nucleic acid, an antibiotic or a growth factor. The method of claim 6, wherein the surface of the biopolymer matrix is a particulate sponge, fiber or irregular. The method of claim 9, wherein the surface of the biopolymer matrix has an average particle size of from 0.1 to 500 microns in the form of a microparticle sponge. The method of claim 6, wherein the surface of the biopolymer matrix is spray-drying or water-in-oil-in-water. , w/o/w), oil-in-water (o/w), solid-in-oil-in-water (s/o/w), It is prepared by electrospinning and lyophilization. The method of claim 11, wherein the water-in-oil-in-water type is prepared (water-in-oil-in-water, w/o/w) or oil-in-water (o/w) comprises the method of combining an organic solvent with a hydrophilic surfactant. The second aqueous solution is mixed. The method of claim 12, wherein the organic solvent is dichloromethane, chloroform, ethyl acetate, 1,4-dioxane, dimethylformamide (N, N-Dimethylformamide, DMF). ), Dimethyl sulfoxide (DMSO), toluene or tetrahydrofuran (THF). The method of claim 12, wherein the hydrophilic surfactant is polyvinyl alcohol (PVA), NP-5, Triton x-100, Tween 40, PEG 200, PEG 800, dodecyl sulfate Sodium (SDS), alcohol ethoxylates, alkylphenol ethoxylates, secondary alcohol ethoxylates, fatty acid esters or alkyl glycosides ( Alkyl polygylcosides). The method of claim 6, further comprising coating an excipient on the surface of the biopolymer matrix. The method of claim 15, wherein the excipient is dextrin, α,β-trehalose, D-(+)-trehalose (D-(+)- Trehalose), sucrose, glycerol, cyclodextrin, polyol, polyethylene glycol (PEG) or bovine serum albumin.